UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Localization of latent adenovirus infection in human lungs and lymph nodes by in situ PCR Behzad, Ali Reza 1998

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1998-0355.pdf [ 13.78MB ]
Metadata
JSON: 831-1.0099302.json
JSON-LD: 831-1.0099302-ld.json
RDF/XML (Pretty): 831-1.0099302-rdf.xml
RDF/JSON: 831-1.0099302-rdf.json
Turtle: 831-1.0099302-turtle.txt
N-Triples: 831-1.0099302-rdf-ntriples.txt
Original Record: 831-1.0099302-source.json
Full Text
831-1.0099302-fulltext.txt
Citation
831-1.0099302.ris

Full Text

L O C A L I Z A T I O N OF L A T E N T A D E N O V I R U S I N F E C T I O N IN H U M A N L U N G S A N D L Y M P H N O D E S B Y IN SITU P C R B Y ALI REZA BEHZAD B.Sc. UNIVERSITY OF BRITISH COLUMBIA, 1993 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF EXPERIMENTAL MEDICINE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1998 © Ali Reza 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. 1 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) , but only 15 to 20% of smokers develop airways obstruction. Respiratory infection caused by group C adenoviruses is a possible independent risk factor for C O P D . Our working hypothesis is that adenoviral E l A D N A persists in airway epithelial cells following respiratory infections and is capable of amplifying cigarette smoked-induced airway inflammation. To develop a protocol that has the potential to detect low copy numbers of adenovirus E l A D N A in histological preparations of human lungs and lymph nodes, we first optimized in situ amplification o f E l A D N A and subsequent detection of the D N A by in situ hybridization (indirect in situ P C R ) on cytospin and paraffin embedded preparations of Graham 293 cells which are known to have 4 to 5 copies of adenoviral E l A gene per cell. Using optimal conditions established for this indirect in situ P C R on paraffin embedded sections of Graham 293 cells, this procedure was performed on paraffin embedded sections of guinea pig lungs 20 days after the resolution of an acute infection with adenovirus 5 when no replicating virus could be recovered from these lungs (Vitalis, et al., 1996) and lungs and lymph nodes from C O P D and non-COPD patients. For successful indirect in situ amplification of E l A D N A in Graham 293 cells, a "hot start" technique with 2 m M M g C b , 1.5 u M E l A primers and 30 cycles of amplification was used. Application of indirect in situ P C R on cytospin preparations of Graham 293 cells after pretreatment with 50 ug/ ml proteinase K for 5 minutes at 37°C resulted in nuclear staining in approximately 60% of the cells, while paraffin embedded 293 cells that had been digested with 1 mg/ ml pepsin in 0.2 N HC1 at room temperature exhibited nuclear staining in approximately 40% of the cells. Staining was not seen in uninfected A549 cells nor in Graham 293 cells hybridized with an irrelevant probe or when Taq polymerase was omitted during amplification. Indirect in situ P C R on paraffin embedded sections of latently infected guinea pig lungs revealed n nuclear staining in bronchiolar and type II alveolar epithelial cells. Nuclear staining was also observed in alveolar epithelial cells when indirect in situ P C R was performed on paraffin embedded sections of lungs from C O P D patients. A s a comparison, direct in situ P C R , where labeled nucleotide is incorporated during amplification, was performed on cytospin and paraffin embedded sections of Graham 293 cells using optimal conditions established for indirect in situ P C R . Our preliminary results from direct in situ P C R revealed significant problems of nonspecific signals. Our findings indicate that indirect in situ P C R allows the detection of 4 to 5 copies of adenovirus E l A D N A in Graham 293 cells. Localization of adenovirus E l A D N A in alveolar epithelial cells could have important implications regarding the regulation of proinflammatory agents that mediate neutrophil migration into the alveolar walls. m TABLE OF CONTENTS A B S T R A C T II T A B L E O F C O N T E N T S IV L I S T O F T A B L E S VII L I S T O F F I G U R E S VIII A C K N O W L E D G M E N T S X I N T R O D U C T I O N 1 M A T E R I A L S A N D M E T H O D S 5 CYTOSPIN PREPARATIONS FOR IN SI TU PCR 5 PARAFFIN EMBEDDED SECTIONS 6 GRAHAM 293 CELLS 6 GUINEA PIG LUNG TISSUE 7 LUNG & LYMPH NODE TISSUES FROM COPD PATIENTS 7 LYMPH NODE TISSUE FROMNON-COPD PATIENTS 8 PCR PRIMERS 8 PROBE PREPARATION 9 TEST OF PROBE SPECIFICITY 10 IV PCR AND HYBRIDIZATION 10 NON-ISOTOPIC DETECTION OF BIOTINYLA TED El A PROBE 10 INDIRECT IN SITU PCR ON CYTOSPIN PREPARATIONS 11 INDIRECT IN SITU PCR ON PARAFFIN EMBEDDED SECTIONS 13 OPTIMAL CONDITIONS FOR INDIRECT IN SITU PCR 14 OPTIMAL PROTEASE CONDITIONS 14 INHIBITIONS OF PCR BY AGAROSE 16 INDIRECT IN SITU PCR ON LUNGS AND LYMPH NODES 16 DIRECT IN SITU PCR 17 RESULTS 18 PROBE SPECIFICITY 18 OPTIMAL PCR CONDITIONS 18 OPTIMAL PROTEASE CONDITIONS 19 CYTOSPIN PREPARA TIONS 19 PARAFFIN EMBEDDED SECTIONS 20 CONTROLS 21 INHIBITION OF PCR BY AGAROSE 21 INDIRECT IN SITU PCR ON LUNGS AND LYMPH NODES 22 V DIRECT IN SITU PCR 24 DISCUSSION 26 R E F E R E N C E S 42 V I LIST OF TABLES Table 1. Optimal permeabilization and P C R conditions for in situ P C R on Graham 293 cells Table 2. Effect of proteinase K permeabilization time on outcome of indirect in situ P C R on cytospin preparations of Graham 293 cells Table 3. Effect of pepsin digestion conditions on outcome of indirect in situ P C R on paraffin embedded sections o Graham 293 cells Table 4. Summary of the detection of amplified E l A D N A by indirect in situ P C R using optimal conditions for permeabilization and P C R Table 5. Comparison of detection of E 1 A D N A in lymph nodes from non-COPD patients by indirect in situ P C R and standard in situ hybridization (ISH) Table 6. Summary of the detection of amplified E l A D N A by direct in situ P C R LIST OF FIGURES Fig. 1. Specificity of biotinylated E l A probe 53 Fig. 2. Indirect in situ P C R on paraffin embedded sections of Graham 293 cells using 2 m M MgCl2 and 40 cycles of in situ amplification showing diffuse background staining 54 Fig. 3. Indirect in situ P C R detection of E l A D N A in paraffin embedded sections of Graham 293 cells under optimal P C R and permeabilization conditions 55 Fig. 4. Effect of proteinase K permeabilization time on the diffusion of amplified E 1 A product from cytospin preparations of Graham 293 cells 56 Fig. 5. Localization of E l A D N A in cytospin preparations of Graham 293 cells by indirect in situ P C R under optimal P C R and permeabilization conditions 57 Fig. 6. Effect of pepsin digestion (1 mg/ ml in 0.2 N HC1) at 37°C for 10 minutes prior to indirect in situ P C R on paraffin embedded sections of Graham 293 cells 58 Fig. 7. Effect of pepsin digestion on the diffusion of amplified E l A product from paraffin embedded sections of Graham 293 cells 59 Fig. 8. Ethidium bromide stained 1% agarose gel electrophoresis of P C R products recovered in the P C R solution after in situ E l A P C R on paraffin embedded sections 60 Fig. 9. Detection of nonspecific binding of the amplified E l A product generated in the solution phase to the paraffin embedded sections 61 Fig. 10. Localization of E l A D N A in paraffin embedded serial sections o f latently infected guinea pig lungs by indirect in situ P C R 62 Fig. 11. Indirect in situ P C R localization of E l A D N A in paraffin embedded serial sections of lung from C O P D patients 63 vm Fig. 12. Localization of E l A D N A in paraffin embedded sections of lymph nodes from non-COPD patients by indirect in situ P C R Fig. 13. Direct in situ P C R localization of E l A D N A in cytospin preparations of Graham 293 cells Fig. 14. Non-isotopic detection of amplified E l A products recovered after direct in situ P C R on cytospin preparations Fig. 15. Direct in situ P C R to detect E l A D N A in paraffin embedded sections of Graham 293 cells Fig. 16. Direct in situ P C R on cytospin preparations of uninfected A549 cells showing artifactual nuclear signals Fig. 17. Direct in situ P C R on paraffin embedded sections o f adenovirus infected A549 cells showing nonspecific staining in the absence of E l A primers Fig. 18. Direct in situ P C R localization of E l A D N A in paraffin embedded sections of latently infected guinea pig lungs Fig. 19. Direct in situ P C R to detect E l A D N A in paraffin embedded sections of lung from C O P D patient ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. James C . Hogg for his continuous help and support. M y sincere thanks go to Dr. Shizu Hayashi for teaching me so much and reading my thesis so many times. I would also like to thank Smart Green for his help with computer imaging. X Introduction Cigarette smoking is a major risk factor for the development of chronic obstructive pulmonary disease (COPD) (Fletcher and Peto, 1977), but only 15 to 20 % of smokers develop C O P D even though all smokers show evidence of airways inflammation (Fletcher et al., 1976). There is considerable evidence suggesting that viral respiratory illness may also predispose patients to chronic airflow disorders (Becroft, 1967, 1971; McFarlane and Somerville, 1957). MacFarlane and Somerville (1957) showed serologic evidence of adenovirus infection in lobectomy specimens removed for bronchiectasis. Adenovirus was also isolated from lung tissue of children with an extensive necrotizing bronchitis and bronchiolitis and in these cases, bronchiolitis was considered to be a direct consequence of necrotizing lesions observed in the acute stage of disease (Becroft, 1967). Group C adenovirus (serotype 1, 2, 5, 6) are of particular interest because following acute infections, virus can persist in lungs, tonsils, and peripheral blood lymphocytes possibly as a form of a latent infection (Evans, 1958; Green et al., 1979; Horvath et al., 1986). Studies from our laboratory (Vitalis et al., 1997) showed that in a guinea pig model of latent adenovirus 5 infection 20 days after the resolution of the an acute infection both viral E l A D N A and the bronchiolitis caused by the virus persisted. Interestingly, the inflammatory response to a single acute exposure to cigarette smoke was enhanced in these latently infected animals (Vitalis et al., 1997). Furthermore, recent studies suggest that I C A M - 1 expression is increased in the airway inflammatory process associated with cigarette smoke induced chronic airway obstruction (Gundel et al., 1992; D i Stefano et al., 1994). The present study is based on the hypothesis that adenovirus E l A D N A persists in airway epithelial cells following viral respiratory infection and is capable of amplifying cigarette smoke induced airway inflammation. Adenovirus D N A has a linear double stranded structure (Younghusband and Bellett, 1971). The genome of adenovirus 2 and 5 is about 36000 base pairs 1 (bp) long (Sussenbach, 1984). The adenovirus genome is subdivided into early (E) and intermediate genes (I) which are expressed before the onset of viral D N A replication and late genes (L) which are transcribed after the replication of the viral D N A has started (Sussenbach, 1984). Electron microscopy in situ hybridization experiments suggest that the replication and transcription of adenovirus D N A takes place within the nucleus of the host cell (Puvion-Dutilleul and Puvion, 1991; Besse and Puvion-Dutilleul, 1994). Following adenovirus infection of permissive cells, the earliest transcripts detected map to the E l A region (early gene) o f the genome (Sussenbach, 1984). The E l A region of adenovirus codes for two proteins of 243 and 280 amino acids that are expressed from two differentially spliced m R N A s of 12S and 13S, respectively (Boyd, et al., 1993). Both proteins can immortalize cells and cooperate with other viral and cellular oncogenes in the transformation of primary cells (Ruley, 1983). In addition, both proteins are involved in transcriptional activation and repression of several viral and cellular promoters (Boyd, et al., 1993). The mechanism by which E l A contributes to the pathogenesis of C O P D is not clear, but E l A could play a role in the amplification of the inflammatory response by regulating activities of several cellular transcription factors that might be responsible for the expression of proinflammatory mediators (Whyte et al., 1988; Bandara and L a Thangue, 1991; Chellappan et al., 1991; Keicho et al., 1997). Previously, we demonstrated that the presence of E l A induces I C A M - 1 and IL-8 expression in the lung epithelial cells after their stimulation with L P S (Keicho et al., 1997a, 1997b). Recent studies also showed that ElA-mediated upregulation of these inflammatory mediators may be due to a common transcription factor, N F - K B , that is activated by E l A protein (Keicho et a l , submitted for publication). Another possible mechanism of ElA-mediated enhancement of inflammation in lungs of C O P D patients may be due to the ability of adenovirus to establish latent infection in lymphoid tissues and peripheral blood lymphocytes. Metcalf (1996) showed that in T-lymphocyte (Jurkat) and monocyte (THP-1) cell 2 l ines transfected w i th an E l A expressing p l asmid , E l A increased the act iv i ty o f tumor necrosis factor ( T N F ) promoter compared to cel ls transfected w i th control p l asmid . P romoter act iv i ty was increased further after P M A st imulat ion o f Jurkat cel ls and L P S st imulat ion o f THP-1 cel ls . Th i s increase was reflected i n an increase i n T N F m R N A product ion after L P S st imulat ion. In other studies, guinea pigs latently infected w i th adenovirus infected a second t ime w i t h E l A deleted adenovirus no longer capable o f repl icat ion resulted i n an in f lammat ion that might be based on a host immune response (V i ta l i s , et a l . , submitted). Furthermore, other studies o f guinea pigs latently infected w i th adenovirus showed that there is a s ignif icant increase i n the C D 4 + lymphocytes i n both the airways and lung parenchyma after an acute exposure to cigarette smoke (V i ta l i s et a l . , 1997 and in press). The authors suggest that in these animals the increase i n C D 4 + cel ls may be due to c lona l expansion triggered by a populat ion o f latently infected antigen presenting cel ls (i.e. dendrit ic cel ls) that are located in the subepi the l ium o f the conduct ing airways and in the alveolar wal ls . A n alternative explanat ion for the accumulat ion o f C D 4 + cel ls at the sites o f chronic in f lammat ion is that they were recruited f r om the per ipheral b l o o d due to the expression o f adhesion receptors and release o f var ious o f cytokines such as IL-1 and T N F - a (Issekutz et a l . , 1994). A l t h o u g h it has been shown that adenovirus persists i n the l y m p h o i d tissues and peripheral b l ood lymphocytes, previous studies f rom our laboratory on human and latently infected guinea p i g lungs indicate that epithel ial cel ls are the major site i n w h i c h adenovirus persist (E l l iot t et a l . , 1995; V i t a l i s et a l . , 1996). Ev idence for this includes the demonstrat ion o f E l A prote in i n a lveolar and a i rway epithel ia l ce l ls us ing immunohis tochemist ry . Howeve r , i n lungs f rom C O P D patients and in latently infected guinea p i g lungs adenovirus D N A cou ld not be detected by standard in situ hybr id izat ion , but the E l A D N A o f adenovirus was readi ly detected by P C R fo l lowed by Southern hybr id izat ion (Matsuse, et a l . , 1992; V i t a l i s et a l . , 1996). 3 The experimental evidence that adenovirus E l A contributes to the pathogenesis o f C O P D in smokers is based on P C R results that the adenovirus E l A D N A was present in greater amounts in lungs of heavy smokers with airway obstruction than in controls with normal lung function matched for age, sex and smoking history (Matsuse, et al., 1992). However, one of the major drawbacks of P C R is that it requires nucleic acid extraction, which destroys tissue morphology, so that a correlation between E l A D N A and histological cell type(s) is not possible. In situ P C R is a technique that allows one to detect nucleic acids present at levels undetectable by standard in situ hybridization and correlate the result with morphology (Nuovo et al., 1991; Komminoth and Long, 1993; Bagasra et al., 1993). Localization of low-copy target D N A can be achieved by two different protocols for in situ P C R . In the direct in situ P C R technique, labeled nucleotides are directly incorporated into the P C R generated amplified products, permitting the non-isotopic detection of in situ amplified sequences whereas, indirect in situ P C R is performed with unlabeled nucleotides and the amplified products are detected by subsequent in situ hybridization using specific labeled probes (Long et a l , 1992). The present study was undertaken to determine the specific cell types (i.e., epithelial cells, dendritic cell and lymphocytes) that harbor low copy numbers of E l A D N A in lungs of C O P D patients using these in situ P C R techniques. In order to develop a protocol which would allow us to detect low copy numbers of adenovirus D N A in histological preparations of human lungs, Graham 293 cells (transformed human embryonic kidney cells) which are known to have 4 to 5 copies of adenovirus E l A D N A per cell (Graham, et al., 1977) and guinea pigs that had been latently infected with adenovirus 5 (Vitalis, et al., 1996) were used as model systems. 4 Materials and Methods Cytospin Preparations for in situ PCR O f all the in situ P C R protocols developed to date it appears that methods using intact cells (cytospins) provide optimal physical conditions for in situ D N A amplification (Long, et al., 1992). Compared to paraffin embedded sections, nucleic acids and membranes are better preserved in whole cells. Except for membrane permeabilization after an initial treatment with proteinase K , the membrane and nucleic acids in cytospins remain intact, whereas in paraffin embedded sections, after embedding and sectioning, both the nucleic acids and membranes are damaged. Also , the fixed whole cells appear to function as amplification sacks with semipermeable membranes that permit the primers, nucleotides, and D N A polymerase to pass into the cell and nucleus, yet sufficiently retard the outward diffusion of the larger P C R product to allow their in situ detection (Komminoth and Long, 1993). For these reasons, although the ultimate goal of this study was to localize the adenovirus E l A D N A in specific cells in paraffin embedded human lung and lymph node tissues, in situ amplification was tested initially on cytospin preparations. Whole cell cytospin preparations of Graham 293 cells (American Type Culture Collection, U S A ) which harbor 4-5 copies of adenovirus E l A D N A were used as a model system to determine whether in situ amplification of target D N A was feasible. Graham 293 cells grown in Eagle's minimal essential medium (Gibco B R L ) , supplemented with horse serum were removed from the culture flask by scraping off the plate and collected in a 15 ml centrifuge tube. A cell pellet was made by centrifugation at 1000 rpm for 5 minutes. The cells were washed in P B S (149 m M N a C l , 12 m M N a 2 H P 0 4 , 4 m M K H 2 P 0 4 ) and then suspended in the same solution (1 x 10 6 cells/ml). A total of 100 pi of cell suspension was cytocentrifuged at 1500 rpm for 4 minutes onto silanized glass slides (1 x 10 5 cells/spot), air dried and stored at -20°C until needed. A549 cells (human lung carcinoma cell line that does not carry adenovirus 5 DNA) obtained from American Type Culture Collection (Rockville, USA) and grown in Eagle's minimal essential medium supplemented with 10 % fetal bovine serum (Hyclone, Logan, UT) were used as a negative control for in situ PCR and in situ hybridization. Cytospins of A549 cells were prepared as described for Graham 293 cells. A549 cells grown to confluence and then infected with adenovirus type 5 (American Type Culture Collection, Rockville, USA) for 24 hours were used as a positive control for in situ hybridization. In situ hybridization with adenovirus genomic probe (Enzo Diagnostics) showed nuclear staining in approximately 90% of these infected cells. To avoid the spread of infected air-borne viral particles during cytocentrifugation in the laboratory, adenovirus 5 infected A549 cells were fixed in 10% buffered formalin for 10 minutes and then cytospin preparations were prepared as described for Graham 293 cells. Paraffin Embedded Sections Graham 293 cells Graham 293 cells were grown to confluence, removed from the culture plate and pelleted as described above. The cell pellet was then fixed in 10% buffered formalin for 24 hours. Following fixation, the cells were washed in PBS to remove formalin and resuspended in PBS. A total of 2 ml (4 xlO 6 cells/ml) of these suspended cells were mixed with an equal volume (1:1) of 2 % low melting agarose and the agarose blocks were embedded in paraffin. Two 4 um-thick serial sections from the paraffin blocks were placed on silanized glass slides and stored at -20° C until needed. Paraffin blocks of uninfected and adenovirus 5 infected A549 cells were prepared as described for the Graham 293 cells. Histological sections of these paraffin embedded cells were prepared as described above. 6 Guinea pig lung tissues Two blocks of paraffin embedded lungs from two latently infected guinea pigs that were previously studied in our laboratory (Vitalis, et al., 1997) were used as an animal model of latent adenovirus infection. D N A extracted from these lungs were positive for E l A by previous P C R and Southern blotting but were negative by standard in situ hybridization (Vitalis et al., 1997). Pairs of 4 urn-thick serial sections from each block were placed on 8 silanized slides. The first 5 slides from each block were used for hybridization with biotinylated E l A probe after E l A in situ amplification. The next 2 slides were used for hybridization with an irrelevant probe after in situ amplification. The remaining 1 slide from one block was used for direct in situ P C R . Two blocks of paraffin embedded lungs from two uninfected guinea pigs that were previously negative for E 1 A D N A by P C R and Southern blotting (Vitalis et al., 1997) were used as the negative controls. Pairs of 4 um-thick serial sections from each block were placed on 3 silanized slides and stored at -20° C until needed. The first 2 slides from each block were used for hybridization with biotinylated E l A probe after E l A in situ amplification. The remaining 1 slide from one block was used for direct in situ P C R . Lung and lymph node tissues from COPD patients Three blocks of paraffin embedded human lungs from three patients with chronic obstructive pulmonary disease who had undergone lung resection for cancer were available from our previous study (Matsuse et al., 1992). The procedure for fixation and embedding was described previously (Matsuse et al., 1992). D N A extracted from the lung tissue of these patients were positive for E l A D N A by previous P C R and Southern blotting, but were negative by in situ hybridization (Matsuse, et al., 1992). Also three blocks of lymph nodes were available from these patients, but these had not been tested for E l A D N A by P C R and Southern blotting. Pairs of 4 um-thick serial sections from each block of lung or lymph node were placed on 4 silane coated 7 glass slides and stored at -20°C until needed. The first 2 slides from each block were used for hybridization with biotinylated E l A probe after E l A in situ amplification. The next slide from each block was used for hybridization with an irrelevant probe after E l A in situ amplification. The remaining slide from one block of lung was used for direct in situ P C R . Lymph node tissue from non-COPD patients Three blocks of paraffin embedded lymph nodes from three non-COPD patients who had lung resection for cancer were also available from a previous study. The procedure for fixation and embedding was described previously (Matsuse, et al., 1992). D N A extracted from the lymph nodes of two patients tested positive for E l A D N A by previous P C R and Southern blotting, while the lymph node from the third patients was negative for E l A D N A . Pairs of 4 um-thick serial sections from each block of lymph node were placed on 3 silane coated glass slides and stored at -20°C until needed. The first 2 slides were used for hybridization with biotinylated E l A probe after E l A in situ amplification. The remaining 1 slide was used for standard in situ hybridization. PCR Primers The P C R primers used to amplify the E l A region of adenovirus 2 and 5 genomes specify 484 and 486 bp (base pair) products, respectively. The selected adenovirus E l A primers have the following sequence: 5' -T A A T G T T G G C G G T G C A G G A A G G - 3 ' 5 ' - T C A G G C T C A G G T T C A G A C A C A G - 3 ' . The primers were tested initially by solution phase P C R on genomic D N A of adenovirus 2. The P C R reaction mixture (50 pi) contained 10 m M Tris buffer (pH 8.4), 50 m M K C 1 , 2 m M M g C l 2 , 0.001% gelatin, 200 u M dNTP, adenovirus 2 D N A (10 5 copies) and 0.5 u M of each 8 primer. After an initial denaturation step at 94°C for 2 minutes, 2 units of Taq polymerase (Gibco BPvL) was added. Then, 40 cycles were performed on the RoboCycler (Stratagene) under the following conditions: denaturation at 94°C for 1 minute, annealing at 63°C for 1 minute and extension at 72°C for 2 minutes. The specificity of amplification was determined both by the size of P C R product on agarose gel electrophoresis and by subsequent Southern hybridization. Probe Preparation The probe for the E l A region was a 756 bp product of a double digest with Pstl and BamHl o f a 742 bp AM fragment from pXC-15 (gift from Dr. Shenk, Princeton) containing the E l A region of adenovirus 2, which was subcloned into Hindi site of pUC13 (Matsuse et al., 1992) and covers the entire sequence of the amplification product. The 756 bp fragment was purified by low-melting agarose gel electrophoresis and labeled with B i o - l l - d U T P (Enzo Diagnostics) by the random priming method (Feinberg and Vogelstein, 1983). The 50 pi reaction mixture contained random priming buffer (50 m M Tris, 5 m M M g C b , 10 m M D T T , 200 m M Hepes p H 6.8), 2 mg/ml bovine serum albumin (BSA) , 72 u M each of d A T P , dGTP, dCTP, 54 u M dTTP, 18 u M Bio -dUTP , 0.4 pg/ul random hexamer, 25 nanogram of purified 756 bp E l A D N A probe, 5 units of Klenow fragment (Gibco, B R L ) and appropriate volume of water. A s a control for non-specific in situ hybridization the 736 bp Taql fragment from pUC13 was also labeled with Bio-11-dUTP by the random priming method. Both probes were then purified from the unincorporated Bio-11-dUTP and recovered in a volume of 100 pi using Sephadex column centrifugation. 9 Test of Probe Specificity PCR and hybridization In order to test the specificity of biotinylated E l A probe, a hybridization experiment was designed in which two different P C R reactions were used to amplify adenovirus 2 E l A and human H L A - D Q a gene (Matsuse et al., 1992). The sizes of the amplified products from E l A and H L A - D Q a genes were 484 and 242 bp, respectively. Purified adenovirus 2 D N A (Gibco, B R L ) (1, 10, and 1000 copies) was used as a template to amplify the E l A D N A . The P C R conditions to amplify E l A D N A were as described above (page 9). Human placental D N A (50 and 500 ng) was used to amplify the H L A - D Q a D N A . The P C R conditions (cycling parameters and primer sequences) to amplify H L A - D Q a D N A were previously described (Matsuse et al., 1992). After P C R amplification of E l A or H L A - D Q a D N A , 35 pi of each 50 pi reaction was subjected to 1 % agarose gel electrophoresis. The D N A was then transferred from the gel to a Hybond N filter (Amersham). The filter was prehybridized for 2 hours at 65° C with 6 X S S C (90 m M sodium citrate, 0.9 M sodium chloride), 0.1 % N a 4 P 2 0 7 , 50 pg/ml heparin, and 0.5 % sodium lauryl sulfate (SDS). After prehybridization, 12.5 nanogram biotinylated E 1 A probe which had been denatured by boiling was added to the prehybridization mixture. The filter was incubated for 16 hours at 65° C. After hybridization, the filter was washed twice for a total of 40 minutes with 2 X SSC, 0.1 % SDS at room temperature followed by two washes 20 minutes each in 0.1 % SSC , 0.1 % SDS at 65° C. Non-isotopic detection of biotinylated El A DNA The labeled E l A probe hybridized to the P C R product was visualized by streptavidin-alkaline phosphatase staining of the labeled D N A . Briefly, the filter was immersed in blocking buffer [phosphate buffered saline (PBS), 0.5 % Triton X , B S A fraction V (50 mg/ml)] at room temperature for 30 minutes with shaking. The filter was then rinsed in P B S and incubated with 10 blocking buffer containing streptavidin-alkaline phosphatase (1 pg/ml) (Gibco B R L ) for 30 minutes at room temperature. Filter was then rinsed in P B S , washed three times for three minutes each in P B S , 0.5 % Triton X , 3 times for three minutes in P B S and three times for three minutes in alkaline phosphate (AP) 9.6 (0.1 M Tris p H 9.6, 0.1 M N a C l , 0.1 M M g C l 2 ) . The filter was then placed in A P 9.6 containing 0.17 mg/ml 5-bromo-4-chloro-3indole phosphate (BCIP), 0.33 mg/ml nitro blue tetrazolium (NBT) and 0.65% dimethyl formamide ( D M F ) and left in the dark for 10 minutes. To stop the color reaction, the filter was then washed for 5 minutes in P B S buffer containing 2 m M E D T A . Indirect in situ PCR on Cytospin Preparations The in situ P C R on cytospin preparations was carried out using a modification of the method of Bagasra et al. (1993). Briefly, slides were air dried then placed on a heating block at 100° C for 2 minutes and then in 1 % paraformaldhyde in P B S for one hour. Then the slides were washed 3 times in 3 X P B S , 3 times in I X P B S . After the cells were digested with proteinase K (50 pg/ml in PBS) for 10 minutes at 37°C, the proteinase K was inactivated by placing the slides on the heating block at 96°C for 2 minutes. Finally, the slides were washed in distilled water. After air drying the slides, a special adhesive frame (Diamed) was placed around each cluster of cytospun cells to allow a 50 pi space for the P C R reaction mixture. The "hot start" technique with TaqStart antibody (Clontech, U S A ) directed against Taq polymerase (equal volume) was used to prevent nonspecific amplification. TaqStart antibody, a neutralizing monoclonal antibody to Taq polymerase, is used to block polymerase activity during set up of the P C R reaction at room temperature (Kellogg, et a l , 1994). During the first denaturation step at 90°C or higher in thermal cycling, the activity of Taq polymerase is restored by denaturation of the thermolabile antibody. The enzyme can then begin to synthesize D N A by extension of 11 primers bound to the specific target sites at elevated temperatures (Kellogg, et al., 1994). The inhibition o f Taq polymerase at ambient temperature and. its reactivation at temperatures above 70°C enhances the specificity and sensitivity of the P C R by preventing nonspecific amplification due to mispriming or primer oligomerization (Chou, et al., 1992). A total of 50 ul of a P C R mixture containing 10 m M Tris buffer (pH 8.4), 2 mm M g C l 2 , 50 m M KC1, 0.01 % gelatin, 200 u M dNTP, 1.5 u M E l A primers, 2.5 units of Taq polymerase mixed with 2.5 units of TaqStart antibody was then placed on each cytospin preparation and sealed with a coverslip (Diamed). The slides were then placed on a PTC-100 thermocycler (M.J . Research) for amplification. Initially, the D N A was denatured at 94°C for 2 minutes. This was followed by an optimal number of cycles as determined below (page 20) of denaturation at 94°C for 1 minute, annealing at 63° C for 1 minute and extension at 72° C for 2 minutes. To ensure complete extension of the P C R product, the last P C R cycle was followed by 7 minutes for primer extension at 72° C. As a negative control, cytospin preparations were covered with the same P C R mixture without Taq polymerase. A s a control to assess the effects of P C R cycling on subsequent in situ hybridization step, cytospins of adenovirus infected A549 cells were incubated with the same P C R mixture containing no primers, dNTP or Taq polymerase. After the P C R , the coverslips were removed by cutting and the P C R solution was recovered for analysis of P C R product diffusion out o f the cells. The cells were then fixed in 4% paraformaldhyde (pH 7.0) for 5 minutes, washed in P B S buffer for 5 minutes, dehydrated in 70% and 95% ethanol and air dried. Visualization of intracellular P C R products was achieved indirectly by in situ hybridization (Hogg et al., 1989) using the biotinylated E l A probe. Briefly, cytospin preparations were covered with 20 pi of hybridization mixture and then coverslipped. The hybridization mixture contained 45 % formamide, 250 pg/ml salmon sperm D N A , 25 m M N a H 2 P 0 4 (pH 6.5), 10 % dextran sulfate and 16.6 pg/u.1 of biotinylated E l A probe. The D N A in both the probe and cells were then denatured 12 by heating the slides at 95° C for 10 minutes in a preheated water bath. The D N A was then allowed to hybridize for 18 hours at 37° C in covered plastic dish sealed in a plastic bag lined with wet towels. After hybridization, the coverslips were rinsed off in 2 X S S C buffer, and the slides were washed twice with 2 X SSC for 5 minutes, twice with 0.1 X S S C for 5 minutes, 2 X S S C for 5 minutes. They were then dehydrated twice in 70% for 10 minutes, once in 95% ethanol for 5 minutes, and air dried. A n in situ P C R on uninfected A549 cells treated in the same manner as the cytospin preparations of Graham 293 was used, in addition to the controls listed above, as a negative control (Table 4). A s a control to assess the quality of detection system, two cytospin preparations of adenovirus-infected A549 cells, which were not subjected to P C R , were also hybridized with the biotinylated E 1 A probe. The biotinylated pUC13 probe was used as a control to monitor non-specific binding of the biotinylated probe. To visualize the probe D N A bound to the in situ P C R product, non-isotopic detection of biotinylated D N A was performed as described above for its detection after Southern hybridization (pages 11 to 12). To stop the color reaction, the slides were then washed for 5 minutes in P B S buffer containing 2 m M E D T A . The slides were then washed in distilled water, air dried, coverslipped with Kaiser glycerol jelly and examined under the light microscope for nuclear staining. Indirect in situ PCR on Paraffin Embedded Sections The in situ P C R on paraffin embedded sections was carried out using a modification of method of Nuovo et al. (1991). Briefly, sections were deparaffinized in Histoclear (Diamed), dehydrated in absolute methanol and air dried. Sections were then digested with 1 mg/ml pepsin in 0.2 N HC1 for 15 minutes at room temperature, washed three times with 2 X S S C buffer followed by dehydration in 70% ethanol twice for 10 minutes and in 95% ethanol for 5 minutes. In situ amplification of E l A D N A was performed as described for cytospin preparations. 13 Cycling parameters and detection of in situ P C R products were also the same as those described for cytospin preparations. Controls for in situ amplification were equivalent as those described for cytospin preparations (pages 12, 13), except that the cells had been fixed in formalin and embedded in paraffin. Optimal Conditions for Indirect in situ PCR To optimize in situ amplification of E l A D N A , indirect in situ P C R was performed on paraffin embedded sections of Graham 293 cells using different MgCh and primers concentrations, and cycling parameters. The optimal MgCh concentration was determined by subjecting paraffin embedded sections of Graham 293 cells to in situ P C R in 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 m M M g C b for 40 cycles each with the other components of the P C R reaction mixture as described above (pages 12 and 13). In further experiments, to determine the optimal primer concentrations, we tested E l A primers at 0.5, 1.0, 1.5, 2.0 and 2.5 u M using 2 m M M g C h for 40 cycles on paraffin embedded sections of Graham 293 cells. Optimal M g C h and primer concentrations that were obtained from indirect in situ P C R on paraffin embedded sections of Graham 293 cells were used for cytospin preparations. P C R cycle number was varied from 20 to 40 cycles for both cytospin preparations and paraffin embedded sections of Graham 293 cells using the above optimized conditions. The other components and conditions o f the P C R reaction were the same as those described above (pages 12 to 13). Optimal Protease Conditions To optimize in situ D N A amplification, it is necessary to optimize protease digestion because either overdigestion or underdigestion can hinder the successful outcome of in situ P C R 14 (Komminoth and Long, 1993). With overdigestion, it is possible that large amounts of P C R products may diffuse into adjacent cells or into the solution phase of the P C R reaction during thermal cycling, and this can lead to false positive results in a mixed cell population. On the other hand, underdigestion can hinder penetration of P C R reagents and result in poor amplification. To maximize the efficiency of in situ E l A D N A amplification and to prevent diffusion of amplified products, cytospins and paraffin embedded sections of Graham 293 cells were subjected to indirect situ P C R after treatment of the preparations with different digestion and permeabilization conditions. Optimal M g C l 2 (2 m M ) and primer (1.5 u M ) concentrations, and cycle number (30) that had been obtained from indirect in situ P C R on paraffin embedded sections of Graham 293 cells (see above) were used for the protease optimization. The other components and conditions of the P C R reactions were the same as those described above (page 12). Cytospin preparations of Graham 293 cells were permeabilized with 50 pg/ml proteinase K at 37°C for 5, 10, 15, 30, 60, and 120 minutes. Paraffin embedded sections were digested with either 1 mg/ml pepsin in 0.2 N HC1 at 37° C or room temperature for 10 or 15 minutes or with 1 to 2 mg/ml pepsin at 37° C or room temperature for 10 to 15 minutes and then subjected to indirect in situ P C R as described above. To test the possibility of diffusion of amplified E l A products, after the P C R , 35 pi of P C R solution was recovered and subjected to 1 % agarose gel electrophoresis and the intensity of E l A band was documented by ethidium bromide staining. The D N A was then transferred from the gel to a Hybond N filter. After hybridization of the D N A on the filter with a radiolabeled E l A probe, the filter was subjected to autoradiography. The radiolabeled E l A probe was made by the random priming technique as described above for the biotinylated E l A probe except that 50 u C i of a - 3 2 P - d C T P (Amersham) was used in place of Bio-11-dUTP, cold dCTP was omitted from the reaction mixture and 72 u M dTTP was used. 15 Inhibition of PCR by Agarose Compared with P C R in the solution phase, in situ amplification of D N A in paraffin embedded sections is less efficient ( Murray, 1993) and this may be partly due to Taq polymerase inhibiting impurities in the agarose gel (Feinberg and Vogelstein, 1983). To determine whether or not the agarose present in sections of paraffin embedded Graham 293 cells as a consequence of the embedding process could inhibit in situ E l A D N A amplification, paraffin embedded sections of these cells were covered with the E l A P C R mixture that was spiked with 10 5 copies of adenovirus 2 D N A and subjected to P C R . The permeabilization step and P C R conditions were the same as those described for indirect in situ P C R on paraffin embedded sections using optimal conditions (see above, pages 15 to 17). A s controls, paraffin embedded sections of uninfected A549 cells, uninfected guinea pig lungs and a slide preparation with no section were also subjected to the same adenovirus 2 DNA-spiked P C R . After completion of the P C R reaction, 35 pi of the P C R solution covering each of the slides was removed and subjected to 1 % gel electrophoresis. The intensity of E l A P C R product was determined by ethidium bromide staining. Furthermore, to study the nonspecific binding of the P C R products generated in the solution phase to the sections, in situ hybridization as described for indirect in situ P C R on paraffin embedded sections (see pages 12 to 15) was performed on the cell preparations after the removal of the P C R solution. Indirect in situ PCR on Lungs and Lymph Nodes Using optimal conditions established for indirect in situ P C R on paraffin embedded sections of Graham 293 cells, indirect in situ P C R was performed on paraffin embedded sections of latently infected guinea pig lungs and lungs and lymph nodes from C O P D and non-COPD patients. Controls for in situ amplification were also the same as those described for paraffin 16 embedded sections of Graham 293 cells (pages 15 to 16). A n additional negative control for the specificity of the amplification step included the use of uninfected guinea pig lungs. Direct in situ PCR Using optimal conditions established for indirect in situ P C R , direct in situ P C R was performed on cytospin preparations of Graham 293 cells and paraffin embedded sections of Graham 293 cells, latently infected guinea pig lungs and lungs from C O P D patients. The process of permeabilization and direct in situ P C R was as described for indirect in situ P C R (pages 15 to 17) with the following exceptions: in the P C R reaction mixture, 33% of dTTP was replaced by Biotin-11-dUTP. The total volume of the reaction mixture was 20 pi . The in situ hybridization step utilizing the biotinylated E l A probe was omitted and instead, P C R generated products that had incorporated the biotin-labeled nucleotide were directly visualized by non-isotopic detection as described above (pages 11 to 12). The slides were then examined under the light microscope for nuclear staining. Controls for specificity of the amplification step included the use of known negative samples (uninfected A549 cells and uninfected guinea pig lungs). A n additional control included the omission of E l A primers from the P C R mixture to detect nonspecific signals generated by mispriming and D N A repair. To examine the possibility of diffusion of amplified labeled product from cytospin preparations, 15 pi of reaction mixture was recovered and subjected to 1 % agarose gel electrophoresis and the intensity of E l A band was documented by ethid.ium bromide staining. The D N A was then transferred from the gel to Hybond N filters and biotin-labeled products were visualized by the streptavidin-alkaline phosphatase method (page 11 to 12). 17 RESULTS Probe Specificity The results obtained from the P C R and Southern hybridization experiments confirmed the expected specificity of the biotinylated E l A probe. E l A probe hybridized exclusively with 484 bp amplification product of the adenovirus 2 E l A D N A ; it did not hybridize with 242 bp amplified product of the human H L A - D Q a gene (Fig. 1). Optimal PCR Conditions Strong nuclear staining was evident when 2 m M M g C l 2 was used to perform in situ P C R on paraffin embedded sections of Graham 293 cells (Fig. 2a). A weak signal was found when the amplifying solution contained 2.5, 3.0, 3.5, 4.0 or 4.5 m M M g C l 2 (data not shown). N o nuclear staining was observed when the M g C l 2 concentration was 1.5 m M . A similar study of primers concentrations on paraffin embedded sections showed that the signal was stronger with 1.5 u M primers compared to 0.5, 1.0, 2.0 and 2.5 u M primers (data not shown). Nuclear staining was first evident after 20 cycles of P C R (data not shown) and was strongest after 30 cycles for both cytospin preparations and paraffin embedded sections of Graham 293 cells (Fig. 3a). After 40 cycles, a more diffuse signal was present in the nucleus and cytoplasm of the cells in paraffin embedded sections (Fig. 2a). In the case of cytospin preparations, an increase in number of cycles from 30 to 40 did not increase the signal, which remained predominantly nuclear. The optimized P C R conditions for cytospin and paraffin embedded preparations of Graham 293 cells are summarized in Table 1. 18 Optimal Protease Conditions Cytospin preparations Permeabilization of cytospin preparations of Graham 293 cells with 50 pg/ml proteinase K for 5 minutes at 37°C resulted in strong nuclear staining (Fig. 4a). The above conditions resulted in 40% cell loss (Table 2). Under these conditions, approximately 60% of the remaining cells after P C R exhibited nuclear staining (Table 4). The background cytoplasmic staining was very low, giving a strong signal to background ratio. Labeled nuclei appeared round and purple. When proteinase K digestion was extended to 10 minutes, 90 % of the cells were lost. After 15 minutes of digestion, more than 90 % of the cells were lost. Compared to 5 minutes of proteinase K digestion, there was no apparent increase in cytoplasmic staining when digestion was extended to 10 or 15 minutes (data not shown). Pretreatment with 50 pg/ml proteinase K at 37° C for 20 minutes or longer prior to indirect in situ P C R resulted in a complete loss of cells. After indirect in situ P C R on cytospin preparations of Graham 293 cells that had been permeabilized with 50 pg/ml proteinase K at 37° C for 15 minutes or more, a strong ethidium bromide stained band corresponding to the 486 bp E l A P C R product was observed when 35 p i of reaction solution was subjected to 1% agarose gel electrophoresis (Fig.5). The intensity of the E l A band observed was the same after 15 minutes of digestion or longer. A weak E l A band was evident when Graham 293 cells were permeabilized for 10 minutes and no E l A band was found after 5 minutes digestion. These results suggest that diffusion of the amplified E l A product occurred during in situ P C R on cytospin preparations of Graham 293 cells. Effects of proteinase K digestion on product diffusion and the outcome of indirect in situ P C R on cytospin preparations of Graham 293 cells are summarized in Table 2. Digestion with proteinase K at 50 pg/ml for 5 minutes at 37°C was considered optimal (Table 1). 19 Paraffin embedded sections In situ P C R on paraffin embedded sections of Graham 293 cells that had been digested with 1 mg/ml pepsin in 0.2 N HC1 for 10 minutes at room temperature resulted in nuclear staining with minor cytoplasmic staining (Fig. 3a). Compared to cytospin preparations, nuclear staining was weaker when in situ P C R was performed on paraffin embedded sections of Graham 293 cells (compare Fig . 4a to 3a) with approximately 40 % of the cells showing nuclear staining (Table 4). Nuclear and cytoplasmic staining was increased when pepsin digestion was extended to 15 minutes (Table 3). On the other hand, digestion at 37°C for 10 minutes resulted in substantial background cytoplasmic staining and extensive damage to cell morphology (Fig. 6a). Digestion with 1 to 2 mg/ml pepsin in the absence of HC1 for 10 to 15 minutes at room temperature or 37°C, resulted in poor nuclear staining (data not shown). Effects of pepsin digestion on the outcome of indirect in situ P C R on paraffin embedded sections of Graham 293 cell is summarized in Table 3. Digestion with 1 mg/ml of pepsin in 0.2 N HC1 for 10 minutes at room temperature was considered optimal (Table 1). When E l A in situ P C R was performed on paraffin embedded sections of Graham 293 cells that had been digested with 1 mg/ml pepsin in 0.2 N HC1 at room temperature or 37°C for 10 or 15 minutes the 486 bp band corresponding to E l A P C R product was not observed on 1 % agarose gel (data not shown). However, after Southern hybridization with a radiolabeled probe the E l A band was detected on the corresponding autoradiogram (Fig. 7). The E l A band intensity was greater after 15 minutes of pepsin digestion at room temperature compared with 10 minutes digestion at the same temperature. The intensity of E l A band did not increase after digestion for 10 minutes at 37°C when compared to the same digestion for 15 minutes at room temperature (data not shown). 20 Controls Using these conditions of optimal permeabilization and P C R (Table 1), negative controls for in situ P C R on cytospin and paraffin embedded preparations of uninfected A549 cells or of Graham 293 cells where Taq polymerase was omitted from the P C R mixture showed no staining (Fig. 3b, 4b, Table 4). Even when the temperature o f pepsin digestion o f paraffin embedded Graham 293 cells was raised to 37°C indirect in situ P C R in the absence of Taq polymerase resulted in no staining (Fig. 6b). The use of irrelevant probe, biotinylated pUC13 , during the in situ hybridization also gave negative results in paraffin embedded sections of Graham 293 cells that had been subjected to E 1 A amplification (data not shown). The pUC13 probe was not tested on cytospin preparations. Indirect in situ P C R on adenovirus infected A549 cells in the absence o f E l A primers, dNTP and Taq polymerase resulted in nuclear staining in 50% of cytospin preparations and 70% of paraffin embedded sections (data not shown). The number of positive cells was slightly higher than that observed by standard in situ hybridization with the same probe alone. Agarose gel analysis of possible P C R products that had diffused into the P C R solution showed that after cytospin preparations of uninfected A549 cells were digested with 50 pg/ m l of proteinase K at 37°C for 5 minutes indirect in situ P C R on these cells did not give a band (Fig. 5, lane g). A n equivalent negative control for paraffin embedded cells, uninfected A549 cells digested with lmg/ml pepsin in 0.2 N HC1 for 10 or 15 minutes at room temperature, also showed no E l A band (Fig. 7, lanes g and k). Inhibition of PCR by Agarose In experiments to determine whether agarose present in sections of paraffin embedded cells inhibits in situ E l A amplification, 10 s copies o f adenovirus 2 D N A were added to the P C R 21 reaction used for amplification. Agarose gel electrophoresis showed a band corresponding to the expected E l A P C R product in the solution recovered from the paraffin embedded sections of Graham 293 cells, uninfected A549 cells and uninfected guinea pig lungs (Fig. 8, lanes, 2, 3, and 4 respectively). The above E l A bands were similar in intensity. Compared to the control slide without a paraffin section, the E l A band observed from paraffin embedded sections was of weaker intensity. However, in situ hybridization with biotinylated E l A probe of paraffin embedded sections of Graham 293 cells (Fig. 9a), uninfected guinea pig lungs (Fig. 9c) and uninfected A549 cells (data not shown) that had been subjected to adenovirus spiked amplification resulted in nuclear and cytoplasmic staining. Since no staining was observed on the adjacent sections where Taq polymerase was omitted from the P C R mixture (Fig. 9b, 9d), the. staining observed on the uninfected guinea pig lung and the A549 cells most likely represents binding of the amplified product of the spiked adenovirus 2 D N A . In the case of the Graham 293 cells, it could be the result of a combination of these false positive staining and genuine amplification in the nuclei of these cells. Indirect in situ PCR on Lungs and Lymph Nodes In situ P C R on paraffin embedded sections of latently infected guinea pig lungs revealed nuclear staining in alveolar and bronchiolar epithelial cells (Fig. 10a, 10c). Examination at higher power to identify E l A positive alveolar epithelial cells type showed that type IJ pneumocytes were the most common positive cell. O f the 10 paraffin embedded sections of lung from the two latently infected guinea pigs, only 4 sections (2 sections per animal) showed evidence of E l A localization in epithelial cells (Table 4). In addition, only one or two cells with nuclear staining were observed on each section. Moreover, nuclear staining in a bronchiolar epithelial cell was observed on only one section from one animal. N o other staining was 22 observed on this section. N o staining was seen on an adjacent section where Taq polymerase was omitted from the P C R mixture (Fig. 10b, lOd, Table 4). The use of irrelevant probe, p U C 13, on adjacent sections after in situ amplification did not show any staining (data not shown). Indirect in situ P C R on uninfected lungs did not show any staining (Table 4). Application of indirect in situ P C R to six paraffin embedded sections of lungs from C O P D patients (2 slides per patient) showed E l A localization in only one o f the two sections from two patients (Fig. 11a). Again only one or two E l A positive cells were found on each section. Examination of these sections at higher magnification showed that E l A D N A was localized to alveolar epithelial cells. N o nuclear staining was observed on an adjacent section where Taq polymerase was omitted from the P C R mixture (Fig. l i b ) . The hybridization of irrelevant probe, pUC13, on the adjacent section after in situ amplification did not result in any staining (data not shown). No nuclear staining was observed when indirect in situ P C R was performed on six paraffin embedded sections (2 sections per patient) of lymph nodes from C O P D patients (data not shown). Large amounts of carbon and other unidentified brown materials, which most likely are due to air pollution, were present on these sections of lymph node (Table 5). Indirect in situ P C R on two E l A positive blocks of lymph nodes from non-COPD patients (2 slides per block) showed evidence of E l A D N A localization in all 4 sections examined. Between 9 to 40 cells with nuclear staining were found on these sections (Tables 4 and 5). Examination of these sections at higher magnification showed that the majority of the nuclear staining was restricted to the follicular mantle and the capsule of the lymph nodes (Fig. 12c, 12d). There was no detectable viral E l A D N A in individual cells in the germinal centers, but E l A D N A was detected in 2 isolated cells around the germinal centers in these sections (Fig. 12a). A l l these positive cells appear to be larger than lymphocytes. Nuclear staining was more 23 frequent in a lymph node that had lower amounts of carbon and other contaminants from air pollution (Table 5, block B) . Nuclear staining was less frequent on the adjacent sections where Taq polymerase was omitted from the P C R mixture where between 3 to 21 positive cells were present (Table 4 and 5), and again these were primarily in the follicular mantle and capsule areas with no nuclear staining in or around the germinal centers. Standard in situ hybridization with biotinylated E l A probe showed evidence of nuclear staining in capsule and follicular mantle areas, but there was no detectable E 1 A D N A staining in or around the germinal centers. Compared to indirect in situ P C R in the absence of Taq polymerase, standard in situ hybridization showed less frequent nuclear staining (Table 5). The use of irrelevant probe, biotinylated pUC13, on adjacent sections did not result in any staining (data not shown). N o nuclear staining was observed when indirect in situ P C R was performed on two sections from an E l A negative block of lymph nodes from a non-COPD patient (Tables 4 and 5). It should be noted that these two sections, like those of the lymph nodes from C O P D patients, which proved to be negative by indirect in situ P C R , also contained high amounts of carbon and other contaminants. Standard in situ hybridization on these sections did not result in any staining (Table 5). Direct in situ PCR Using optimal conditions established for indirect in situ P C R , direct in situ P C R on cytospin preparations of Graham 293 cells yielded strong nuclear signals in approximately 70% of the cells with either no cytoplasmic staining or faint staining (Table 6, F ig . 13a). Labeled nuclei appear round and dark purple. Ethidium bromide staining did not allow detection of an amplified E l A band when solutions recovered after the P C R on cytospins preparations of Graham 293 cells were subjected to 1% agarose gel electrophoresis, but the E l A band was detected after the colorimetric staining of the Hybond N filter after Southern transfer of the D N A 24 on this gel (Fig. 14). Direct in situ P C R on paraffin embedded sections of Graham 293 cells resulted in nuclear staining in approximately 50% of the cells (Table 6, F ig . 15a). Generally, the intensity of cytoplasmic staining on these paraffin embedded sections was lower compared to those subjected to indirect in situ P C R . Omission of primers from the amplifying solution resulted in the detection of false positive nuclear staining in both cytospins and paraffin embedded sections of Graham 293 cells (Table 6, F ig . 13b, 15b). These false positive signals were evident in approximately 20% of cells of cytospin preparations of Graham 293, whereas only about 1% of cells in paraffin embedded sections of Graham 293 showed false positive nuclear staining. Nonspecific signals were also detected in 20% of control experiments using direct in situ P C R on cytospin preparations o f uninfected A549 cells (Table 6, F ig . 16a). Nuclear staining was less frequent when E l A primers were omitted from the amplifying solution (Fig. 16b). N o nuclear staining was observed when direct in situ P C R was performed on paraffin embedded sections of uninfected A549 cells (Table 6). Direct in situ P C R on cytospin and paraffin embedded sections of adenovirus infected A549 cells showed diffuse nuclear and cytoplasmic staining in 100% of the cells (Fig. 17a). When E l A primers were omitted from the P C R mixture, nonspecific false positive signal was observed in 50% of both infected A549 cell preparations (Table 6, F ig . 17b). When E l A primers were included in the P C R mixture, a strong ethidium bromide stained E 1 A band was observed when the supernatant from these infected cells was subjected to 1% agarose gel electrophoresis (data not shown). That this band represented E l A D N A was shown after non-isotopic detection of the biotinylated D N A on the corresponding Hybond N filter (Fig. 14). Direct in situ P C R on one section of paraffin embedded lung from a latently infected guinea pig revealed 4 alveolar epithelial cells with nuclear staining (Table 6). Figure 18a illustrates one of these positive cells. This cellular localization of E l A D N A was similar to that 25 found by indirect in situ P C R . N o nuclear staining was observed on the adjacent section where the primers had been omitted from the P C R mixture (Fig. 18b), or in lung sections from uninfected guinea pigs (Table 6). Application of direct in situ P C R on one section of paraffin embedded human lung revealed one alveolar epithelial cell with nuclear staining (Table 6, F ig . 19a). N o staining was seen on an adjacent section where primers were omitted from the P C R reaction (Fig. 19b). Discussion The purpose of this study was to develop a protocol capable o f detecting low copy numbers of adenovirus E l A D N A in situ in histological preparations of human lungs and lymph nodes. Graham 293 cells and latently infected guinea pigs were used as model systems to develop this procedure. The results show that adenovirus E l A D N A can be demonstrated in situ in the nuclei of Graham 293 cells by indirect in situ P C R both on cytospin and paraffin embedded specimens. These experiments were used to characterize the conditions that optimize the in situ detection of PCR-amplified E l A D N A in Graham 293 cells. Our results also support the findings of others (Long, et al., 1992; Sallstrom, et al., 1993) claiming that indirect in situ P C R which uses the in situ hybridization step for added specificity in the detection of amplified E l A D N A is superior to direct in situ P C R . Our results showed that application of direct in situ P C R on cytospin preparations of Graham 293 and A549 cells yielded a significant number of false positive results. However, compared to cytospin preparations, direct in situ P C R on paraffin embedded sections of Graham 293 and A549 cells showed significantly lower or absent false positive signals, respectively; the reason for that w i l l be discussed in detail below. Application of indirect in situ P C R on guinea pig lungs that had been latently infected with adenovirus 5 revealed nuclear staining in the epithelia of bronchiole and alveolar walls. Nuclear 26 staining was also observed in alveolar epithelial cells when indirect in situ P C R was performed on paraffin embedded sections of human lungs. The protocols used in our studies were based on those of Bagasra et. al. (1993) and Nuovo (1991) but were modified to maximize nuclear staining in cytospin preparations and paraffin embedded sections of Graham 293 cells. A s the success of indirect in situ P C R on Graham 293 cells depends largely on the way these cells have been digested before amplification, protease pretreatment of both cytospin and paraffin embedded preparations were analyzed in detail. In this study, successful in situ amplification and localization of adenovirus E l A D N A was achieved when cytospin preparations and paraffin embedded sections of these cells had been digested with 50 ug/ml proteinase K for 5 minutes at 37°C and with 1 mg/ml pepsin in 0.2 N HC1 for 10 minutes at room temperature, respectively. Inadequate digestion of paraffin embedded sections of Graham 293 cells in the absence of HC1, even when the temperature was elevated to 37°C, resulted in poor nuclear staining. The primary reason for protease digestion is to facilitate entry of P C R reagents into the cell. Therefore, insufficient protease treatment limits the accessibility of nuclear D N A to P C R reagents. In addition, protease removes DNA-histone cross linking, which occurs as a result of formalin fixation (Nuovo, et al., 1991). Such cross linking of histone protein to D N A is likely to prevent the progression of Taq polymerase along the native D N A template (Junqueira, et al., 1977). With insufficient digestion, a large numbers o f these cross links may persist and decrease the efficiency of amplification thus lead to poor nuclear staining. Excessive protease treatment, on the other hand, results in diffusion of amplified E l A products out of the nucleus and consequently poor nuclear staining, damage to cell morphology, and loss of cells or sections from the slide. Diffusion artifacts represented the most significant problem of indirect in situ P C R on paraffin embedded sections of Graham 293 cells. Diffusion of 27 amplified E l A products produced a significant cytoplasmic staining in paraffin embedded sections of Graham 293 cells that had been subjected to more extensive protease treatment. It is possible that excessive digestion may result in total removal of nuclear protein cross links secondary to formalin fixation and this would allow part of amplified E l A products to leak out of the nucleus and cell into the P C R solution. It is hypothesized that P C R products, which have diffused out of the cell, may serve as templates for extracellular amplification, a process that is probably far more efficient than intracellular amplification. When comparing the relative efficiency of extracellular and intracellular amplification after 40 cycles, Ray and coworkers (1995) showed that the band intensity was greater in the supernatant fraction composed of extracellular amplified product consistent with the notion of greater efficiency of P C R on liberated D N A compared with intracellular amplification. These extracellularly amplified D N A s have the potential to adhere to exposed cellular basic proteins resulting in cytoplasmic staining. In contrast to paraffin embedded sections, background cytoplasmic staining was not a problem after indirect in situ P C R in cytospin preparations. One possible explanation for this difference is that in cytospins the almost intact cell membrane may hinder extracellularly amplified E l A products to diffuse back into the cell, whereas after sectioning paraffin embedded cells, most of the cells are left without intact cell membrane and extracellularly generated E l A products can freely bind to cytoplasmic proteins. Results from other studies (Long, et al., 1993; Komminoth, et al., 1992) on nonspecific cellular binding and uptake of extracellularly generated P C R products by cell suspensions support the view that intact cell membrane can prevent back diffusion of large amplified products. Long and coworkers (1993) placed uninfected fixed human fibroblasts into a P C R tube in which viral D N A sequences of different lengths were being tested by amplification extracellularly. After the P C R , the cells were washed and subjected to in situ hybridization using specific oligonucleotide probes. The results indicate that false positive 28 signals were far higher when smaller P C R products less than 150 bp were generated possibly due to back diffusion of the extracellular P C R products into the fixed fibroblasts during thermal cycling. In addition, diffusion artifacts were significantly reduced when biotinylated nucleotides instead of unmodified ones were used to generate bulkier and therefore less diffusible amplified products (Komminoth, et a l , 1992). Moreover, our findings of positive nuclear and cytoplasmic staining after using a P C R reaction spiked with adenovirus D N A during indirect in situ P C R on paraffin embedded sections of Graham 293 cells (Fig. 7a), uninfected A549 cells and uninfected guinea pig lungs (Fig. 7c) support the view that nonspecific cytoplasmic staining in paraffin embedded sections is mainly due to binding of extracellularly produced P C R products onto the sections. Furthermore, the fact that on agarose gel electrophoresis the band intensity of E l A products in the P C R solution recovered from these sections was weaker when compared to controls with no sections (Fig, 8) suggests that some of amplified E l A products must have adhered to the sections. Another explanation is the possibility that in paraffin embedded sections P C R amplification in fixed tissue is less efficient. Adenovirus templates may bind to proteins and becomes less available for amplification leading to reduced amplification efficiency. Therefore, the implication of these results is that in order to minimize cytoplasmic staining in paraffin embedded sections of Graham 293 cells, outward diffusion of amplified E l A product had to be reduced. One approach was to optimize protease digestion in such a way that it would permit penetration of P C R reagents into the nucleus and free D N A from histone to allow successful in situ amplification of target D N A while avoiding outward diffusion of amplified products. Optimal localization of amplified E l A D N A was found when paraffin embedded sections o f Graham 293 cells were digested with 1 mg/ml pepsin in 0.2 N HC1 for 10 min at room temperature. Another approach taken to minimize outward diffusion of amplified product suggested by 29 studies by Komminoth and coworkers (1992) was the use of a relatively low numbers of P C R cycles. When the numbers of P C R cycles was reduced from 40 to 30, there was a significant reduction in cytoplasmic staining on paraffin embedded sections of Graham 293 cells. However, P C R cycle numbers lower than 20 also resulted in poor nuclear staining in both cytospin and paraffin embedded sections of Graham 293 cells. This is consistent with the findings of Haase et. al. (1990) that in situ amplification is less efficient than solution phase P C R and therefore more cycles may be required to achieve the same degree of amplification. Successful in situ amplification of E l A D N A in paraffin embedded preparations of Graham 293 cells was achieved only when 2 m M M g C l 2 was used. The use of either lower (1.5 m M ) or higher concentrations of M g C l 2 (higher than 2 m M ) did not result in nuclear staining. This is contrary to other studies which report the use of much higher concentrations of M g C l 2 (4.0 to 4.5 m M ) for successful in situ amplification (Nuovo et al., 1991; Bagasra et al., 1993). L o w concentrations of M g C l 2 can affect the yield of amplified product. I f M g C l 2 concentration is very low, the extension reaction is impaired as magnesium ions are required as a cofactor for the D N A polymerase activity. Most investigators in this field (Nuovo et al., 1991; Bagasra et al., 1993) believe that it is the sequestration of M g C l 2 ion on cellular proteins and the glass slide that increases the need for higher concentrations of M g C l 2 during in situ P C R . In our study, sequestration of M g C l 2 did not appear to be a significant problem since 2.0 m M M g C l 2 resulted in successful in situ amplification of E l A D N A in Graham 293 cells. It is not clear why higher concentrations of M g C l 2 , which have been reported by others to allow amplification (Nuovo, et al., 1991; Bagasras, et al., 1993), did not result in nuclear staining in our study. In solution phase P C R , a high concentration of M g C l 2 results in the accumulation of non-specific amplification products (Saiki, 1989). Recently, Martinez et al. (1995) showed that the use of high M g C l 2 concentration resulted in nonspecific nuclear staining by direct in situ P C R . Our use of a 30 biotinylated E l A D N A specific probe would not allow the detection o f any nonspecific amplification products and, therefore, it is conceivable that at higher M g C ^ concentrations nonspecific amplification took place but was not detected in our case. The success of indirect in situ P C R on Graham 293 cells also depends on optimal concentrations of E l A primers. Strong nuclear staining was observed when E l A primer concentrations were 1.5 p M . This is three times higher than the concentration used for solution phase P C R . This relatively high concentration of primers probably reflects their partial sequestration on the glass slide and by cellular proteins. However, the use o f primer concentrations higher than 1.5 p M is of particular interest since it resulted in weak nuclear staining. Target specific amplification during P C R suffers from several side reactions that include mispriming and primer oligomerization. One interpretation of our result is that at particularly high concentrations of primers, target specific amplification during P C R may be inhibited by mispriming or primer oligomerization. Another interesting modification that has been used to enhance the in situ P C R is the use of multiple primer pairs (Haase, et al., 1990) which are designed to amplify large P C R products. A s the final P C R product is longer, it is affected less by diffusion and thus increases the power of its subsequent detection. However, the primer multiplicity most likely increases the probability of mispriming and primer oligomerization leading to increased nonspecific amplification (Nuovo, et al., 1991). In addition, Ray et. al. (1994) showed that the length of D N A fragment to be amplified did not affect the final detection of the amplified D N A after in situ hybridization. A t the same time Nuovo and coworkers (1991) showed that the one copy of H P V 16 D N A in the cervical carcinoma cell line (SiHa) was detectable by indirect in situ P C R with a single primer pair that had a target sequence of 450 bp only i f "hot start" modification was employed. We also achieved excellent localization of the amplified E l A product using a single primer pair to amplify a 486 bp target in conjunction 31 with "hot start" P C R . The effect of P C R inhibitors was also considered in this study by checking the suitability of paraffin embedded sections of cultured cells for P C R amplification. The most likely candidate as a cause of inhibition of P C R in these sections was the agarose that had been mixed with the cells before embedding in paraffin. Commercially available agarose are sometimes contaminated with poorly characterized polysaccharides which are potent inhibitors of many of the enzymes commonly used in molecular cloning including Taq polymerase (Sambrook, et al., 1989). In our study, we used special grade low melting agarose that was screened by the manufacturer for the presence of enzyme inhibitors. Although use of low melting agarose has reduced the problem of contamination, there are occasions when enzyme inhibition is seen. Feinberg and Coworkers (1983) showed that contaminants from agarose gel sometimes inhibit nick translation. Our result confirms the findings of Chiu and coworkers (1992) that low melting agarose does not inhibit P C R amplification. However, our results demonstrate that the efficiency of indirect in situ amplification and localization of E l A D N A in paraffin embedded sections of Graham 293 cells was lower when compared to cytospin preparations. Approximately 40% of the Graham 293 cells of paraffin embedded sections showed evidence o f E l A localization when compared to 60% of the cells on cytospin preparations. This is in agreement with other studies in which the efficiency of in situ amplification in paraffin embedded sections was estimated to be significantly lower than that of cytospin preparations (Komminoth, et al., 1992). This lower efficiency may be related to other differences between these two preparations and not necessarily to agarose effects. Reduced amplification efficiency on paraffin embedded sections may relate to D N A damage and loss that occur after embedding and sectioning process. Alternatively, it is possible that reduced retention of amplified products in paraffin embedded preparations due to sectioning through the cell may result in poor signal localization. 32 c The findings from studies conducted here support the view that indirect in situ P C R is a technique that allows very specific detection of target sequences. There was no positive signal in either cytospin preparations or paraffin embedded sections of Graham 293 cells when Taq polymerase was excluded from P C R mixture or when the cells were subjected to in situ hybridization after in situ amplification with an irrelevant probe which in our case was biotinylated pUC13 D N A . Also no positive signal was observed when indirect in situ P C R was performed on uninfected A549 cells. This control is particularly important, since nonspecific amplification of D N A can occur in the presence of Taq polymerase. However, because our E l A probe hybridizes exclusively with 484 bp amplified product of adenovirus E l A D N A , nonspecific amplification, i f it took place, did not result in any staining in uninfected A549 cells and consequently should not compromise the results obtained from Graham 293 cells or lung tissue. In contrast to indirect in situ P C R , nonspecific nuclear staining was frequent and of strong intensity when direct in situ P C R was performed on cytospin preparations of Graham 293 cells when primers were omitted from the P C R reaction or preparations o f uninfected A549 cells where no target D N A is present. One possible explanation for these findings include labeled nucleotide incorporation during D N A repair that occurs as a result o f polymerase activity at sites where formaldehyde fixation has caused single stranded nicks in the D N A (Demkorvicz-Dobrzanski and Castong, 1992). Long et. al. (1992) showed that nonspecific incorporation of labeled nucleotides into damaged D N A undergoing repair by D N A Taq polymerase may occur leading to false positive results. In addition, direct in situ P C R on cytospin preparations of adenovirus infected A549 cells in the absence of primers showed more frequent false positive signals when compared to that of Graham 293 or uninfected A549 cells. One possible explanation for this difference is that virus infected cells induce apoptosis (programmed cell 33 death) through mobilization of apoptosis specific nucleases, fragmenting and degrading D N A of host cells (Sallstrom, et al., 1993). Thus it is possible that the high degree of D N A degradation in adenovirus infected A549 cells may result in an increased D N A repair leading to increased nonspecific signals when compared to Graham 293 or uninfected A549 cells. Many creative approaches have been employed by others to minimize the D N A repair pathway. For example, others have shown that these artifacts can be somewhat reduced by repairing D N A nicks by treatment with T4 ligase before P C R (Koch, et al., 1991), or initial thermal cycling using unlabeled nucleotides (Morital, et al., 1994). However, it has not yet proven possible to eliminate completely these nonspecific pathways which interfere with accurate detection of specific signals. A possible explanation for nonspecific signals observed after direct in situ P C R , even after preliminary treatment to repair nicks in D N A , is mispriming. Mispriming can result in the incorporation of labeled nucleotides into nonspecific amplified products leading to false positive results. Our preliminary results showed that omitting E l A primers from the P C R mixture during direct in situ P C R on cytospin preparations of uninfected A549 cells resulted in reduction o f nonspecific signals. This finding suggests that mispriming is the other probable cause of false positive signals observed in cytospin preparations. Hot Start modification has been used by others to overcome mispriming during direct in situ P C R , but this has not proven to be completely reliable. For instance, while some observers have reported that Hot Start modification of P C R has the potential to reduce mispriming (Erlich, et al., 1991; Nuovo, et al., 1993), we and others (Zehbe, et al., 1992; Komminoth and Long, 1993) have failed to eliminate nonspecific signals by such attempt. The use of labeled primers in place o f labeled nucleotides during direct in situ P C R may have the potential to eliminate nonspecific nuclear staining due to D N A repair pathway, but mispriming could still persist. In this approach, the use of unrelated labeled primers on adjacent sections would allow us to estimate the degree o f nonspecific 34 amplification. Therefore, although in this approach nonspecific signals due to mispriming cannot be eliminated, the frequency of such signals can be monitored using irrelevant labeled primers on adjacent sections. Our preliminary results showed that compared to cytospin preparations, direct in situ P C R on paraffin embedded sections of Graham 293 cells resulted in significantly lower numbers of false positive signals. This is also in contrast to most reports (Sallstrom, et al., 1994; Zehbe, et al., 1994) that showed more frequent false positive signals in paraffin embedded tissue sections when compared to cytospin preparations. One possible explanation for the difference observed between our report and others includes the type of starting materials in paraffin embedded sections. Compared to our model system developed with live cells in culture which allows for precise control of both the duration and nature of fixation used, most o f the tissue samples reported by others include archival paraffin embedded samples that had been subjected to prolonged fixation of uncertain duration. This can have adverse effects on the quality of D N A being amplified. This may also explain the higher frequency of false positive signals in tissue sections (Long et al., 1992), which were likely to have had more D N A damage due to fixation than the cultured cells. Another possible explanation for our finding is that the frequency of nonspecific signal may depend on differences in the efficiency of in situ amplification in paraffin embedded sections compared to cytospin preparations. In agreement with the results of indirect in situ P C R , our preliminary results of direct in situ P C R showed lower in situ E l A amplification signals in paraffin embedded sections of Graham 293 cells when compared to cytospin preparations. Although this suggests reduced amplification efficiency of specific products on paraffin embedded sections, results using direct in situ P C R on cytospin preparations of uninfected A549 cell also indicate that the frequency of nonspecific signals is amplification dependent. The implication of these results is that reduced efficiency of amplification in general 35 may contribute to reduced nonspecific signal in paraffin embedded sections. Moreover, direct in situ P C R , in the presence or absence of E l A primers, on paraffin embedded sections of uninfected A549 cells did not result in any staining. These findings suggest that mispriming and D N A repair mechanism are less important sources o f error in paraffin embedded sections, even though they appear to be the two major contributing factors for nonspecific signals in cytospin preparations. Factors that contribute to low level artifacts in paraffin embedded sections of Graham 293 cells in the absence of primers are not known and require further investigation. However, lack of involvement of mispriming and D N A repair mechanism may partially explain why fewer false positive signals were observed on paraffin embedded sections of Graham 293 cells compared to cytospin preparations. We have successfully transferred the protocol developed on Graham 293 cells to histological sections of lung tissues where our results using indirect in situ P C R on paraffin embedded sections show that the primary target cell was the type II alveolar epithelial cell both in latently infected guinea pig and human lungs. The epithelial cells lining the bronchiole were less frequently positive in guinea pig lungs. The absence o f nuclear staining without Taq polymerase demonstrates the specificity of our indirect in situ P C R protocol, and confirms the relative insensitivity of standard in situ hybridization. The infrequent detection of adenovirus E l A D N A by standard in situ hybridization in C O P D lungs is in agreement with previous studies (Matsuse, et al., 1992) in which adenovirus was demonstrated in only two of 60 blocks of lung tissue from these patients. In contrast, after P C R amplification, two of the six slides prepared from three blocks of lungs from two C O P D patients showed evidence of E l A D N A . Similarly, o f the 10 paraffin embedded sections of latently infected guinea pig lungs examined, 4 sections (2 sections per animal) showed evidence of E l A localization in epithelial cells. Therefore, our study confirms that indirect in situ P C R on paraffin embedded sections of lungs from C O P D patients 36 and latently infected guinea pigs improves the detection rate when E l A D N A is present in low copy numbers. In the present study, E l A D N A was not detected on paraffin embedded sections of lymph nodes from C O P D patients by indirect in situ P C R . The absence of adenovirus E l A D N A in lymph nodes from C O P D patients cannot completely exclude the possibility of latent adenovirus infection in these tissues. This assumption is based on two previous studies in which group C adenovirus D N A was demonstrated in peripheral blood lymphocytes and tonsils from asymptomatic adults by Southern hybridization (Green, et a l , 1979; Horvath, et al., 1986). The Southern hybridization experiments used human tonsils and peripheral blood lymphocytes from asymptomatic adults and showed that between 20 to 100 copies of complete adenovirus genome were present in each cell. In contrast, lymph nodes from non-COPD patients showed E l A D N A localized to non-lymphocytic cells in the capsule and follicular mantle by both indirect in situ P C R and by standard in situ hybridization. The large amounts of carbon present in the lymph nodes in C O P D patients could account for the absence of nuclear staining because these and other contaminants from air pollution may have inhibited in situ amplification of E 1 A D N A . It is also possible that the same materials can absorb the color reagents (BCIP and N B T ) and therefore interfere with the color reaction used to detect the biotinylated probe. This is also consistent with the results of indirect in situ P C R and standard in situ hybridization on lymph nodes from one non-COPD patient in which nuclear staining was absent on sections with deposits from environmental pollution. Alternatively, it is possible that E l A D N A was not present in the limited number of lymph nodes from C O P D patients that were so far examined. Comparison of cellular localization of adenovirus E l A D N A by indirect in situ P C R in human and latently infected guinea pig lungs shows the following similarities. First, the primary target tissue in both cases was alveolar epithelium. Second, this cellular localization was also 37 similar to that of our previous studies (Elliot, et al., 1995; Vitalis , et al., 1996) in which E 1 A protein was detected in the alveolar epithelial cells o f human and latently infected guinea pig lungs. Third, the use of E l A D N A localization in human and guinea pig lungs as a measure of number o f E l A containing cells has yielded results which suggest that latency is typically established in only a minute fraction of the alveolar epithelial cells, from one to two E l A positive cells per paraffin embedded section. However, the use of nuclear staining detected by indirect in situ P C R as a measure of the number of E l A containing cells may underestimate the number of E l A positive cells in these preparations. This underestimation is reflected in the detection of E l A D N A in Graham 293 cells where every cell carries 4 to 5 copies of the target D N A . In cytospin preparations only 60% of the cells were positive and this fell even lower in paraffin embedded preparations, where there were only 40% positive cells. This underestimation may be, in part, related to limitation of assay sensitivity. The indirect in situ P C R was optimized to localize E l A D N A in cytospin preparations and paraffin embedded sections of Graham 293 cells which are known to have four to five copies of adenovirus E l A D N A . It is not known how many copies of E l A D N A are present in each latently infected cell o f human and guinea pig lungs. In this regard, further optimization of in situ E l A D N A amplification on tissue sections may be required. Underestimation, in the case of paraffin embedded sections, may also be related to the absence of E l A D N A in some cells since their nuclei are often bisected by the sectioning. It is also possible that E l A D N A is not evenly distributed within the lung, and detection of E l A positive cell in lung sample reflects this non-random distribution. This is consistent with our previous studies (Matsuse, et al., 1992) in which repeated solution phase P C R on D N A extracted from serial sections of the same blocks o f C O P D and non-COPD lungs confirmed positivity for E l A D N A on 13 of 16 and 8 of 16 D N A samples, respectively. Results of direct in situ P C R on paraffin embedded sections of human and latently 38 infected guinea pig lungs also support the localization of E l A D N A in alveolar epithelial cells. Also , although the number of specimens analyzed was limited to allow statistical comparisons, the level of detection of E l A D N A did not appear to be greater by this method than that found by the indirect method. However, because our preliminary results indicate false positive signals associated with direct in situ P C R on paraffin embedded sections of Graham 293 cells, complete reliance of the direct in situ P C R approach for the cellular localization of E l A D N A in the case of human and latently infected guinea pig lungs should be regarded with caution. The theory regarding the progression of C O P D suggests that neutrophils are important cellular effectors of inflammation (Blue and Janoff, 1978). Neutrophils can cause tissue damage through release of degradative enzymes and oxygen radicals (Weis, S.J., 1984). Recently, Coxson et. al. (in press) showed that there was a significant increase in the number of neutrophils and alveolar macrophages in lungs from C O P D patients when compared to the control group with matched smoking history. Similarly, Thompson et. al. (1989) showed increased number of neutrophils in lavage fluid from patients with C O P D . However, the mechanism by which neutrophils and monocytes are recruited to the lungs in C O P D patients is not clear. We postulate that the presence of E l A D N A and its expression in alveolar epithelial cells may be the driving factor for enhanced recruitment of neutrophils and alveolar macrophages to the lung after exposure to cigarette smoke. In vitro studies showed that expression of E l A D N A in pulmonary epithelial cells can enhance the induction of proinflammatory mediators such as IL-8 and I C A M -1 (Keicho, et al., 1997, 1997). IL-8 is a potent chemoattractant and activator of neutrophils (Oppenheim, et al., 1991), whereas I C A M - 1 serves as a ligand for adhesion receptor on neutrophils (Diamond, et al., 1991). Likewise, in vivo studies suggest that IL-8 and I C A M - 1 expression was significantly higher in C O P D lungs compared to control groups (Keating et al., 1994; D i Stefano, et al., 1996). Taken together with our observations, these results suggest that 39 after exposure to cigarette smoke, expression of E l A D N A in epithelial cells could augment the induction of IL-8 and I C A M - 1 leading to an excessive P M N response to cigarette smoke inhalation. This increased recruitment of neutrophils into' the lung tissue could result in irreversible lung damage. In summary, our data showed that E l A D N A can be detected in cytospin preparations and paraffin embedded sections of Graham 293 cells using indirect in situ P C R technique. The use of cytospin preparations appeared to provide maximal amplification and detection o f E l A D N A in these cells. In this study, we also examined the importance of various conditions such as protease pretreatment, M g C b and primers concentrations, and the number of P C R cycles that would enhance in situ amplification and localization of E l A D N A in Graham 293 cells. In our experience, the most crucial step in obtaining good amplification and localization of E l A D N A was the protease pretreatment. We postulate that protease treatment has a direct effect on amplification efficiency and retention of amplified product. Our experiments revealed significant problems of direct in situ P C R concerning nonspecific positive signals. Based on these findings, we conclude that direct in situ P C R must await for further resolution of its current limitations before it can be used as a reliable technique for in situ amplification. The results obtained using indirect in situ P C R on paraffin embedded sections of human and latently infected guinea pig lungs showed evidence of E l A D N A localization in alveolar epithelial cells. This cellular localization is similar to that found in previous studies (Elliot, et al., 1995; Vital is , et al., 1996) by immunohistochemistry and is consistent with the conclusion reached by Matsuse and coworkers (1992) using in situ hybridization. Localization of adenovirus E l A D N A in alveolar epithelial cells has important implications to the pathogenesis of emphysema because the upregulation of proinflammatory agents in these cells could mediate neutrophil migration into the alveolar walls. This amplification of the inflammatory process by latent adenovirus infection 40 r could provide a partial explanation for the fact that only a small percentage of heavy smokers develop airway obstruction. 41 References Bagasra, O., Seshamma, T., Pomerantz, R.J . Polymerase chain reaction in situ: Intracellular amplification and detection of HIV-1 proviral D N A and other specific genes. J. Immnuological Methods 158: 131-145, 1993. Bandara, L . R . and L a Thangue, N . B . Adenovirus E l A prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature 351: 494-7, 1991. Becroft, D . M . O . Bronchiolitis obliterans, bronchiectasis and other sequel of adenovirus 21 infection in young children. J Clin Pathol 24:72-82, 1971. Becroft, D . M . O . Histopathology of fatal adenovirus infection of the respiratory tract in young children. J Clin Pathol 20:561-9, 1967. Besse, S., Puvion-Dutilleul, F . High resolution localization of replicating viral genome in adenovirus infected Hela cell. European J. Cell Biol. 63,269-279 (1994). Blue, M . L . and Janoff, A . Possible mechanisms of emphysema in cigarette smokers: release of elastase from human polymorphonuclear leukocytes by cigarette smoke condensate in vitro. Am. rev. respir. Dis., 117: 317-25, 1978. Boyd, J., Subarmenian, J., Schaeper, U . , L a regina, M . , Bavely, S., and Chinnadurai, G . A region in the C-terminus of adenovirus 2/5 E l A protein is required for association with a cellular phosphoprotein and important for the negative modulation of T 24- ras mediated transformation, tumorigenesis and metastasis. EMBO J 12(2), 469-78, 1993. Chellappan, S.P., Hiebert, S., Mudyim, M . , Horowitz, J . M . , Nevins, J.R. The E2F transcription factor is a cellular target for the R B protein. Cell 65: 1053-61, 1991. Chiu, K . P . , Cohen, S., Morris, D . , and Jordan, G . C . Intracellular amplification of proviral D N A in tissue sections using the polymerase chain reaction. J. Histochem. and Cytochem 40, 333-341, 1992. Chou, Q., Russell, M . , Birch, D . , Raymond, J., and Bloch, W . Prevention of pre P C R mispriming and primer oligomerization improves low-copy number amplification. Nucleic Acid Res. 20, 1717-1723, 1992. Coxson, H .O. , Whittall, K . P . , Pare, P.D. , Rogers, R . M . , and Hogg, J.C. Quantification of pulmonary emphysema with computed tomography and histology (in press). Demkowics-Dobrzanski, K . , Castonguay, A . Modulation by glutatione of D N A strand breaks by 4-(methylnitrosamine)-l-(3-pyridyl)-l-butanone and its aldehyde methabolites in rat hepatocytes Carcinogenesis 13(8), 1447-54, 1992. Diamond, M . S . , Stauton, D .E . , Marl in , S.D., Spriger, T . A . Binding of integrin Mac-1 (CD1 l b / C D 18) to the third immunoglobulin-like domain of I C A M - 1 (CD-54) and its regulation 42 by glycosylation Cell 65:961-971, 1991. D i Stefano, A . , Maestrelli, P., Roggeri, A . , Tarato, G . , Calabro, S., Potena, A . , Mapp, C E . , Caiccia, A . , Covacer, L . , Fabbri, L M . , and Saetta, M . Upregulation of adhesion molecules in the bronchial mucosa of subjects with chronic obstructive bronchitis. Am JRespir Crit Care Med 149:803-810, 1994. Elliott W . M . , Hayashi, S., Hogg, J.C. Immunodetection of adenoviral E l A proteins in human lung tissue. AmJ. Respir. Cell Mol. Biol. 12: 642-648, 1995). Erlicht, H . A . , Gelfand, D . , Sninsky, J.J. Recent advances in polymerase chain reaction. Science, 252: 1643-1651, 1991. Evans, A . S . Latent adenovirus infections of the human respiratory tract Am JHyg 67: 256-266, 1958. Feinberg, A P . , and Vogelstein B . A technique for radiolabeling D N A restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132:6-13, 1983. Fletcher, C , Peto, R. The natural history of chronic airflow obstruction. Br Med J 1:1645-8, 1977. Fletcher, C , Peto, R., Tinker, C , Speizer, F .E . The natural history of chronic bronchitis and emphysema. A n eight-year study of early chronic obstructive lung disease in working men in London. Oxford University Press. 1976. Graham, F L . , Smiley, J., Russel, W C , Narin, R. Characteristics of human cell line transformed by D N A from human adenovirus type 5. J Gen Virol 36: 59-72, 1977. Green, M . , Wold , W . S . M . , Mackey, K . , Rigden, P. Analysis of human tonsils and cancer D N A ' s and R N A ' s for D N A sequence of group C (serotypes, 1, 2, 5, 6) human adenovirus. Proc. Natl. Acad. Sci. USA 76:6606-6610, 1979. Gundel, R H . , Wegner, C D . , Torcellini, C A . , and Letts, L G . the role of intercellular adhesion molecule-1 in chronic airway inflammation. Clin Exp. Allergy 22:569-575, 1992. Haase, A . T . , Retzel, E .F . , Staskus, S. Amplification and detection of lentiviral D N A inside cells. Proc. Natl. Acad. Sci. USA 871: 4971-4975, 1990. Hogg, J C , Irving, W L . , Porter, M E . , Dunnil l , M S . , Fleming, K . in situ hybridization studies of adenoviral infections of the lung and their relationship to follicular bronchiectasis. Am Rev RespirDis 139: 1531-1535, !989. Horvath, J., Laszlo, P., and Weber, J. Group C adenovirus D N A sequences in human lymphoid cells. J of Virology 59: 189-92, 1986. Junqueira, L . C , Carneikro, J. and Contopoulos, A . Basic Histology Lamge Medical Publications, Ca. , 1977. 43 Issekutz, A . C , Meager, A . , Otterness, I and Issekutz, T .B . The role of tumor necrosis factor-alpha and IL-1 in polymorphonuclear leucocyte and T lymphocyte recruitment to joint inflammation in adjuvant arthritis. Clinical & Experimental Immunology 97(1): 26-32, 1994. Keatings, V . M . , Collins, P.D. , Scott, D . M . , Barnes, P.J. Differences in interleukine 8 and tumor necrosis factor-a in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am. J. Repir. Crit. Care. Med. 153: 530-534, 1994. Keicho, N . , Higashimoto, Y . , Bondy, G.P., Elliot, W . M . , Hogg, J.C. and Hayashi, S. Endotoxin-specific N F - k B activation in pulmonary epithelial cells harbouring adenovirus E l A (submitted for publication). Keicho, N . , Elliot, W M . , Hogg, J C , Hayashi, S. Adenovirus E l A upregulates IL-8 expression induced by endotoxin in pulmonary epithelial cell. Am J Physiol Keicho, N . , Elliot, W M . , Hogg, J C , Hayashi, S. Adenovirus E l A gene disregulates I C A M - 1 expression in pulmonary epithelial cells. Am J Respir Cell Mol Biol 16: 23-30, 1977. Kellogg, D . E . , Rybalkin, I., Mukhamedova, N . , Vlasikt, T., Siebert, P .D. , and Chenchik, A . TaqStart antibody: hot start P C R facilitated by neutralizing monoclonal antibody directed against Taq polymerase. Biotechniques 16: 1134-1137, 1994. Koch , J., Hindkjaer, J., Mogensen, J., Kolraas, S., and Bolen, D . A n improved method for chromosome specific labeling of alpha satellite D N A in situ by using denatured double stranded D N A probe as primers in a primed in situ labeling (PRINS) procedure. Genet. Anal. Tech. Appl. 8, 171-178, 1991. Komminoth, P. and Long, A A . in situ polymerase chain reaction. A n overview of methods, applications and limitations of a new molecular technique. Virchows Archiv B, Cell Pathol 64: 67-73, 1993. Komminoth, P., Adam, V . , Long, A . A . , Roth, A . , Saremaslani, P., Flury, R., Schmiod, M . , Heitz, P. Evaluation of methods for hepatitis C virus ( H C V ) detection in liver biopsies: comparison of histology, immunohistochemistry, in situ hybridization, reverse transcriptase P C R and in situ R T P C R . Pathol. Res. Pract. 190,1017-1025, 1991. Komminoth, P., Long, A . A . , Wolfe, H.J . Comparison on in situ polymerase chain reaction (in situ P C R ) , in situ hybridization (ISH) and polymerase chain reaction (PCR) for the detection of viral infection in fixed tissue. Patologia 25 [suppl]: 253, 1992. Long, A . A . , Komminoth, P., Lee, E . , and Wolf, H.J . Comparison of indirect and direct in situ polymerase chain reaction in cell preparations and tissue sections. Histochemistry 99(2): 151-62, 1992. Martinez, A . , Mi l le r , M . J . , Quinn, K . , Unsworth, E.J . , Ebina, M . , Cuttitta, F . Non-radioactive localization of nucleic acids by direct in situ P C R and in situ R T - P C R in paraffin embedded sections. J. Histochem. Cytochem. 43(8): 739-747. 44 Matsuse, T., Hayashi, S., Kuwan, K . , Keunecke, H . , Jeffries, W . A . , and Hogg, J.C. Latent adenoviral infection in the pathogenesis of chronic airways obstruction. Am rev Respir Dis 146: 177-184, 1992. McFarlane, PS., Somerville, R C . Non-tuberculosis juvenile bronchiectasis; a virus disease. Lancet 1: 770-1, 1957. Metcalf, JP. Adenovirus E l A 13 gene product upregulate tumor necrosis factor gene. Am J Physiol: Lung Cell Mol Physiol 14: L535-40, 1996. Morital , M . , Hachisuk, H . , Sasai Y . Detection of genomic D N A with a high sensitivity in tissue sections using a two step cycling in situ P C R procedure Kurume Med. J. 41,215-220, 1994. Murray, G.I. In Situ P C R . J. Pathology 169,187-188,1993. Nuovo, G . , Gallery, F. , MacConnell , P., Becker, J., Bloch, W . A n improved technique for the in situ detection of D N A after polymerase chain reaction amplification. A J Pathol 139 (6): 1239-1244, 1991. Oppenheim, J.J., Zachariae, C .O.C. , Mukaida, N . , and Matsushima, K . Properties of the novel proinflammatory supergene intercrine cytokine family. Annu, Rev. Immunol 9: 617-684, 1991 Puvion-Dutilleul, F. , Puvion, P. Site of transcription of adenovirus type 5 genome in relation to early viral D N A replication in infected Hela cells, a high resolution in situ hybridization and autoradiographical study. Biol. Cell 71, 135-147, 1991. Ray, R., Smith, M . , Sim, R., Bruce, I., Wahefield, A . in situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymearse chain reaction. J. Virological Methods 52:247-263, 1995. Ruley, E .R. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature 304:602-606, 1983. Saiki, R. the design and optimization of the P C R . in P C R technology, ed. Erl ich, H . A . Stochton press, pp. 7-16, 1989. Sallstrom, J.F., Zehbe, I., A lemi , M . , Wilander, E . Pitfalls of in situ polymerase chain reaction (PCR) using direct incorporation of labeled nucleotides. Anticancer Res. 13: 1153-1154, 1993. Sallstrom, J.F., A lemi , M . , Septs, H . , and Zehbe, I. Nonspecific amplification in in situ P C R by direct incorporation of reporter molecules. Cell Vision 1: 243-251, 1994. Sambrook, J., Fritsch, e.F., Maniatis, T. Agarose gel electrophoresis in Molecular cloning, ed. Cold spring harbor laboratory press, chapter 6, 1989. Suresh, K M . , Middleton, D M . , Stikoo, SK. , Babiuk, L A . Pathogenesis and immunogenecity of bovine adenovirus type 3 in cotton rats. Virol 213: 131-139, 1995. 45 Sussenbach, J.S. The structure of the genome in The adenoviruses. Ginsberg, H.S . ed. Plenum Press, New York, pp. 35-124, 1984. Thompson, A . B . , Daughton, D. , Robbin, R . A . , Ghafouri, M . A . , Oehlerking, M . , Pennard, S.I. Intraluminal airway inflammation in chronic bronchitis. Characterization and correlation with clinical parameters. Am. Rev. respir. Dis. 140:1527-1537, 1989. Vitalis, T .Z. , Keicho, N . , Itabashi, S., Hayashi, S., and Hogg, J.C. A model of latent adenovirus 5 infection in the guinea pig (cavia porcellus). Am. J. Respir. Cell. Mol. Biol. 14: 225-231, 1996. Vitalis , T .Z. , Kern, I., Croome, A . , Behzad, H . , Hayashi, S., and Hogg, J.C. Latent adenovirus 5 infection enhance cigarette smoke induced inflammation. AmJResp Crit Care 155(4) part 2: A468, 1997. Vitalis , T .Z. , Kern, I., Hayashi, S. and Hogg, J.C. Adenovirus 5 E l , E3 deletion mutant causes more inflammation in guinea pigs previously infected with adenovirus 5 wi ld type virus. Pediat Pulmon Suppl 133: 230, 1996. Vitalis , T.Z. , Kern, I., Hegele, R . G . , Elliott, W . M . , Hogg, J . C , and Hayashi, S. Adenovirus gene therapy vector increases inflammation in guinea pigs infected latently with adenovirus 5 (manuscript in preparation). Weiss, S.J. Tissue destruction by neutrophils. TV. Eng. J. Med. 320:365-76, 1989. Whyte, P., Buckovich, K . J . , Horowitz, J . M . Association between an oncogene and antioncogene: the adenovirus E l A protein bind to the retinoblastoma gene product. Nature 334: 124-9, 1988. Younghusband, H . B . , and Bellet, A . J . D . Mature form of D N A from chick embryo lethal orphan virus. J. Virol. 8,265, 1971. Zehbe, I., Hacker, G . , Rylander, E . , Sallstrom, J.F. and Wilander, T. Detection of single H P V copy in S iHa cells by in situ polymerase chain reaction (in situ P C R ) combined with immunoperoxidase and immunogold-silver staining (I-GSS) technique. Anticancer Res. 12, 2165-2168, 1992. Zehbe, I., Sallstrom, J.F., Hacker, G .W. , Hauser Kronberger, C , Rylander, E . , and Wilander, E . Indirect and direct in situ P C R for the detection of human papillomavirus. A n evaluation of two methods and double staining technique. Cell Vision 1:163-167, 1994. 46 Table 1. Optimal permeabilization and PCR conditions for in situ PCR on Graham 293 cells. Conditions Cytospins Paraffin embedded cells Proteinase K 50 pg /ml , 37°C, 5 min n.d. Pepsin n.d. 1 mg/ ml , 0.2 N HC1, R.T. , 10 min M g C l 2 2.0 m M 2.0 m M E1A Pr imers 1.5 u M 1.5 u M Cycle numbers 30 30 (n.d.) not done (R.T.) room temperature 47 Table 2. Effect of proteinase K permeabilization time on outcome of indirect in situ PCR on cytospin preparations of Graham 293 cells Time (min) *Overall nuclear % cell loss Diffusion of staining amplified products 5 +++++ 40 none 10 ++ 90 minor 15 + >90 significant 20 - 100 significant 30 - 100 significant 60 - 100 significant 120 - 100 significant (*) To calculate this, intensity of nuclear staining, % cell loss and amount of diffusion of amplified products were taken into consideration. 48 Table 3. Effect of pepsin digestion conditions on outcome of indirect in situ PCR on paraffin embedded sections o Graham 293 cells Digestion Time (min) 2 mg/ ml pepsin 1 mg/ ml pepsin, 0.2 N H C 1 R.T. 37°C R.T. 37°C 10 negative negative ++ + 15 negative negative + n.d. (R.T.) room temperature (negative) no staining (++) good nuclear staining with minor cytoplasmic background (+) nuclear staining with increased cytoplasmic background (+) nuclear staining with strong cytoplasmic background and damage to cell morphology (n.d.) not done 49 Table 4. Summary of the detection of amplified E 1 A DNA by indirect in situ PCR using optimal conditions for permeabilization and PCR E l A positivity Cells + Taq -Taq Graham 293 cells -cytospin preparations 60% positive negative -paraffin embedded section 40%o positive negative Infected A549 cell* -cytospin preparations n.d. 50% positive -paraffin embedded sections n.d. 70%o positive Uninfected A549 cells -cytospin preparations 0% positive 0%> positive -paraffin embedded section 0% positive 0% positive Tissue Guinea pig lungst 1 or 2 positive cells/ section negative -Animals 2/2 0/2 -Sections 4/10 0/10 COPD lungs 1 or 2 positive cells/ sections negative -Cases 2/3 0/3 -Sections 2/6 0/6 Lymph nodestt negative negative -Cases 0/3 0/3 -Sections 0/6 0/6 Non-COPD lymph nodes m 9 to 40 positive cells/ section 3 to 21 positive cells/section -Cases 2/3 2/3 -Sections 4/6 4/6 Uninfected guinea pig lung negative negative -Animals 0/2 0/2 -Sections 0/4 0/4 (*) A549 cells infected with adenovirus 5 for 24 hours (n.d.) not done (t) Guinea pig latently infected with adenovirus type 5 (tt) from C O P D patients (ttt) see Table 5 for details 50 Table 5. Comparison of detection of E1A DNA in lymph nodes from non-COPD patients by indirect in situ PCR and standard in situ hybridization (ISH) # of cells with nuclear staining indirect in situ PCR standard ISH + Taq - Taq E1A positive (block A) slide 1 12 3 n.d. slide 2 22 5 n.d. slide 3 n.d n.d. 2 E l A positive (block B) slide 1 9 6 n.d slide 2 40. 21 n.d slide 3 n.d n.d. 4 E1A negative (block C) slide 1 0 0 n.d. slide 2 0 0 n.d. slide 3 n.d. n.d. 0 (n.d.) not done Amounts of carbon and other environmental contaminants (block C > block A > block B) Table 6. Summary of the detection of amplified E1A DNA by direct in situ PCR E l A positive cells Cells + primers - primers Graham 293 cells -cytospin preparations (n=2) -paraffin embedded section (n= Infected A549 cells* 2) 70% positive 50%) positive 20% positive 1 % positive -cytospin preparations (n=l) -paraffin embedded sections (n Uninfected A549 cells = 1) 100% positive 100% positive 50% positive 40%> positive -cytospin preparations (n=l) -paraffin embedded sections (n = 1) 20% positive negative 5% positive negative Tissue Guinea pig lungst (n=l) C O P D lung(n=l) C O P D lymph node (n=l) Uninfected guinea pig lung (n= 1) 4 positive cells/ section 1 positive cell/ section n.d negative negative negative n.d. negative (n) is the numbers of slides used (*) A549 cells infected with adenovirus type 5 for 24 hours (t) Guinea pig latently infected with adenovirus type 5 (n.d.) not done 52 242 bp 484 bp a b c d e f a b c d e f Fig. 1. Specificity of biotinylated E l A probe. Adenovirus 2 D N A (Ad 2) and human placental D N A (HPD) were used as templates to amplify E l A and H L A - D Q a D N A by solution phase PCR, respectively. A) Agarose gel of ethidium bromide stained E l A and H L A - D Q a P C R products. A 242 bp H L A -D Q a band is observed when 500 or 50 ng of human placental D N A were used as template for amplification (lanes a,b, and c). A 484 bp E l A band is seen when 103 copies of adenovirus 2 D N A were used as a template for amplification (lane d). B ) Southern blot of E l A and H L A - D Q a P C R products hybridized with biotinylated E l A probe which was detected by streptavidin-alkaline phosphatase staining. Biotinylated E l A probe hybridized exclusively with 484 bp amplification product of adenovirus 2 E l A D N A . The intensity of the E l A band was greater when 103 copies of adenovirus 2 D N A (lane d) were used as template compared to 10 (lane e) or 1 (lane f) copies of templates. E l A probe did not hybridize with 242 bp P C R product of H L A - D Q a gene (lanes, a, b , and c). 53 Fig. 2 . Indirect in situ P C R on paraffin embedded sections of Graham 293 cells using 2 m M M g C l 2 and 40 cycles of in situ E l A amplification showing diffuse background staining. Prior to indirect in situ P C R , paraffin embedded sections were digested with 1 mg/ml pepsin in 0.2 N HC1 at room temperature for 15 minutes. In situ amplification was followed by in situ hybridization with biotinylated E 1 A probe, a) A diffuse signal is in the nuclei (arrows) and cytoplasm of the cells. Note that cytoplasmic signal is evident in all cells, b) No hybridization signal is evident on the adjacent section where Taq polymerase was omitted from the P C R mixture. Scale equals 25 am. 54 a \ 1> mm \ F i g . 3. Indirect in situ P C R detection o f E l A D N A i n paraffin embedded sections o f Graham 293 cells under opt imal P C R and permeabi l izat ion condit ions. Prior to E l A in situ amplification (30 cycles) using 2 m M M g C ^ , paraffin embedded sections had been digested with 1 mg/ml pepsin in 0.2 N HC1 at room temperature for 10 min. a) E l A products in the nuclei (arrows) are detected after in situ hybridization with biotin-labeled E l A probe, b) No hybridization signal is evident on the negative control which is the adjacent section with Taq polymerase omitted from the PCR mixture. Bar equals 10 pm. 55 a • # i • •** b Fig. 4. Localization of E l A D N A in cytospin preparations of Graham 293 cells by indirect in situ PCR under optimal PCR and permeabilization conditions, a) Strong nuclear staining (arrowheads) is evident by in situ hybridization with biotinylated E l A probe after digestion with 50 pg/ml proteinase K for 5 min at 37°C followed by 30 cycles of in situ E1A D N A amplification using 2 m M MgCl? in the presence of Taq polymerase, b) Negative control. In situ hybridization with biotinylated E l A probe after in situ amplification in the absence of Taq polymerase shows no staining. Bar equals 15 um. 486 bp & £ s c ex- <> >$> <p r <p b b 0 , <V JV <V <o T T Qf Qf & O V Fig. 5. Effect of proteinase K permeabilization time on the diffusion of amplified E l A product from cytospin preparations of Graham 293 cells. To demonstrate diffusion of amplified E1A product from cytospin preparations of Graham 293 cells (G 293) into the PCR mixture, the PCR solution overlying the section was recovered and subjected to 1% agarose gel electrophoresis. No E l A band is observed when Graham 293 cells was permeabilized with 50 ng/ml proteinase K at 37° C for 5 min (lane C). A weak E l A band (lane d) is evident when permeabilization was extended to 10 minutes, suggesting minor diffusion of amplified E l A product. A strong E l A band is evident when permeabilization of Graham 293 cells was extended to 15 or 30 minutes (lane e and f, respectively) indicating greater diffusion of amplified E l A product compared to 10 minutes protease treatment (lane d). Amplifying solution recovered from a cytospin preparations of uninfected A549 cells that were permeabilized with 50 ug/ml proteinase K at 37° C for 5 minutes was used as a negative control for E1A DNA contamination of the PCR mixture. No E1A band is evident when amplifying solution from uninfected A549 cells was subjected to gel electrophoresis (lane g). As a positive control, adenovirus 2 DNA was used as a template to amplify E1A DNA by solution phase PCR Strong E l A band (lane a) is evident when 105 copies of Ad2 DNA was used as a template for amplification. Fig. 6. Effects of pepsin digestion (1 mg/ml in 0.2 N HC1) at 37°C for 10 minutes prior to indirect in situ P C R on paraffin embedded sections of Graham 293 cells, a) Strong nuclear and cytoplasmic (arrowheads) staining are evident in all cells after 30 cycles of in situ E l A amplification followed by in situ hybridization with biotinylated E l A probe. Note that strong digestion damaged the cell architecture, b) No staining was observed on adjacent section where Taq polymerase was omitted from the P C R mixture. Bar equals 25 uvm. 4 8 6 b p Fig. 7. Effect of pepsin digestion on the diffusion of amplified E l A product from paraffin embedded sections of Graham 293 cells. Southern hybr id izat ion analysis was used to demonstrate l imi ted d i f fus ion o f ampl i f i ed E l A product f rom Graham 293 cel ls into the P C R solut ion. E 1 A band o f expected size (486 bp) was v isua l ized after hybr id izat ion o f membrane w i th 3 2 P-labeled E l A probe. Pretreatment o f paraff in embedded sections o f Graham 293 cel ls w i th 1 mg/ml pepsin, at room temperature for 15 minutes pr ior to indirect in situ P C R gave a specif ic E l A band on the membrane (lane d, e, and f). Note that the E 1 A band intensity was decreased when pepsin digest ion was reduced to 10 minutes (lanes, h, i, and j). In negative controls, paraff in embedded sections o f uninfected A 5 4 9 cel ls that had been digested w i th 1 mg/ml pepsin in 0.2 N HC1 at room temperature for 10 or 15 minutes pr ior to indirect in situ P C R ampl i f y ing solutions do not show any band (lane g and k, respectively). Strong E l A band is evident when 10 or 1 copies o f adenovirus 2 D N A were used as template to amp l i f y E l A D N A by solut ion phase P C R (lane a and b, respectively). N o E l A band is evident when adenovirus 2 D N A was omitted f rom the ampl i f y ing solut ion o f solut ion phase P C R (lane c). 59 1 2 3 4 5 6 7 484 bp F i g . 8. Ethidium bromide stained 1% agarose gel electrophoresis of P C R products recovered in the P C R solution after in situ E l A P C R on paraffin embedded sections. To test the efficiency of solution phase P C R to amplify E l A D N A on paraffin embedded sections of cell cultures, the P C R solution was spiked with 10 copies of adenovirus 2 D N A . A 484 bp E l A band was evident when amplifying solution from Graham 293 cells (lane 2) was subjected to gel electrophoresis. E 1 A band was also evident when amplifying solutions from uninfected A549 cells (lane 3) and uninfected guinea pig lungs (lane 4) were subjected to gel electrophoresis. The intensity of E l A band was the same in all three cases (lanes 2, 3, and 4). Amplifying solution on a slide preparation with no section that had been spiked with 105 copies of adenovirus 2 D N A showed a corresponding band (lane 1) of greater intensity than that from paraffin embedded sections (lanes, 2,3, and 4). No band was observed when Taq polymerase was omitted from the adenovirus 2 spiked-PCR solution on paraffin embedded sections of Graham 293 cells (lane 5), uninfected A549 cells (lane 6), or uninfected guinea pig lungs (lane 7). 60 Fig. 9 . Detection of nonspecific binding of the amplified E l A product generated in the solution phase to the paraffin embedded sections. In situ hybridization with biotinylated E l A probe after E l A D N A amplification in the presence of 10 5 copies of adenovirus 2 D N A yielded nuclear (arrows) and nonspecific cytoplasmic staining in paraffin embedded sections of Graham 293 cells (panel a) and uninfected guinea pig lungs (panel c ) . As control, the adjacent sections were also subjected to the same adenovirus spiked P C R . No staining was observed on adjacent control sections (panels, b and d) when Taq polymerase was omitted from the P C R mixture. Bar represents 50 pm. 61 Fig. 10. Localization of E l A D N A in paraffin embedded serial sections of latently infected guinea pig lungs by indirect in situ PCR. In situ hybridization with biotinylated E l A probe after in situ E l A D N A amplification reveals nuclear staining (a) in an epithelial cell lining bronchiole (arrowhead) and in (c) a type I I alveolar epithelial cell (arrowhead). Adjacent sections (b and d) to those shown in a and C, respectively, were treated with the same P C R mixture in the absence of Taq polymerase. N o signal is evident after hybridization with biotinylated E l A probe. Bar represents 15 pm. 62 F i g . 1 1 . Indirect in situ PCR localization of E l A D N A in paraffin embedded serial sections of lung from C O P D patients, a) Nuclear staining is evident in alveolar epithelial cell (arrowhead) after in situ E l A D N A amplification followed by in situ hybridization with biotinylated E l A probe, b ) When Taq polymerase was omitted from the P C R mixture, no hybridization signal was evident on the adjacent section. Note that carbon and other unidentified contaminants (arrows) are present on this section. Bar represents 30 pm. 63 Fig. 12. Localization of E l A D N A in paraffin embedded sections of lymph nodes from non-COPD patients by indirect in situ PCR. a) In situ hybridization with a biotinylayted E l A probe after in situ E l A D N A amplification reveals nuclear staining in an isolated cell around the germinal centre (arrowhead), b) When Taq polymerase was omitted from the P C R mixture on the adjacent section, this positive cell was not stained, c) Nuclear staining is evident in a cell near the capsule of lymph node (arrowhead) after in situ E l A D N A amplification followed by in situ hybridization with biotinylated E l A probe, d) H & E staining of the adjacent section to a, b and C showing location of capsule (Cap), germinal centers (GC) and E l A D N A nuclear staining (*) after indirect in situ P C R . Bar equals 15 pm. 64 Fig. 13. Direct in situ PCR localization of E l A D N A in cytospin preparations of Graham 293 cells. Biotin-labeled products were visualized by streptavidin-alkaline phosphatase reaction, a) Strong nuclear staining (arrows) is evident when E l A primers were included in the P C R mixture. Approximately 70% of the cells showed nuclear staining, b) False positive nuclear signals (arrows) are evident when E l A primers were omitted from the P C R mixture. Approximately 20% of the cells show false positive staining. Bar equals 15 pm. 65 a b c d e f g h E 1 A primers + + - - - + + 486 bp F i g . 1 4 . Non-isotopic detection of amplified E l A products recovered after direct in situ P C R on cytospin preparations. Non-isotopic detection of biotinylated E l A D N A on Southern blot was used to demonstrate diffusion of amplified E l A products from Graham 293 and adenovirus infected A549 cells. The P C R products obtained with (+) and without (-) inclusion of E l A primers are shown. A 486 bp E l A band is evident when the recovered amplifying solutions from Graham 293 cells were subjected to streptavidin-alkaline phosphatase (SAAP) staining (lanes a and b). No band is evident when E l A primers were omitted from the P C R mixture (lanes C and d). Strong E l A band is visible when amplifying solution from adenovirus 5 infected A549 cells were subjected to S A A P staining (lane f). In the absence of E l A primers from the P C R mixture on adenovirus infected A549 cells, no E l A band is visible (lane g). A s negative controls, amplifying solutions containing primers (lane h) and without E l A primers (lane e) from uninfected A549 cells do not show any band. Each lane represents P C R solution from a separate preparation. 66 F i g . 1 5 . Direct in situ P C R to detect E l A D N A in paraffin embedded sections of Graham 293 cells. Biotin labeled products are visualized by streptavidin-alkaline phosphatase reaction, a) Nuclear signal is evident in 50% of the cells (arrows) when E l A primers were included in the P C R mixture, b ) False positive signal (arrow) is evident when E l A primers were omitted from the P C R mixture on the adjacent serial section. False positive signal is present in approximately 1% of the cells. Bar equals 25 pm. 67 Fig. 16. Direct in situ P C R on cytospin preparations of uninfected A 5 4 9 cells showing artifactual nuclear signals. Biotin labeled products are visualized by streptavidin-alkaline phosphatase staining, a) Note artifactual nuclear signal in nuclei of uninfected cells (arrowheads). False positive signal is present in approximately 20% of the cells, b) In the absence of the E l A primers from the P C R mixture, false positive signals are less frequent (arrowhead). Approximately 5% of the cells show evidence of nonspecific nuclear staining. Bar equals 15 pm. Fig. 17. Direct in situ PCR on paraffin embedded sections of adenovirus infected A 5 4 9 cells showing nonspecific staining in the absence of E l A primers, a) Strong nuclear (arrowheads) and cytoplasmic staining are evident in all cells when E l A primers were included in the P C R mixture, b) Artifactual nuclear (arrowheads) and cytoplasmic signals are evident when E l A primers were omitted from the P C R mixture. Approximately, 50% of the cells show false positive staining. Bar equals 50 pm. 69 Fig. 18. Direct in situ P C R localization of E l A D N A in paraffin embedded sections of latently infected guinea pig lung. Following in situ E l A amplification, biotin-labeled products were visualized by streptavidin-alkaline phosphatase reaction, a) A nuclear signal is evident in one type II alveolar epithelial cell (arrowhead) when E l A primers were included in the P C R mixture, b) In the absence of E l A primers from the P C R mixture on the adjacent section, no signal is evident. Bar equals 25 pm. 70 a b Fig. 19. Direct /« szYw P C R to detect E l A D N A in paraffin embedded sections of lung from a C O P D patient. After in situ amplification, biotin-labeled products were visualized by streptavidin-alkaline phosphatase staining, a) Nuclear staining is evident in an alveolar epithelial cell (arrowhead) when E l A primers were included in the P C R mixture, b) No signal is evident on an adjacent section when in situ amplification was performed without E l A primers. Bar equals 50 pm. 71 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0099302/manifest

Comment

Related Items