Preclinical models for Chlamydia pneumoniae and cardiovascular disease: hypercholesterolemic mice
1998; Elsevier BV; Volume: 4; Linguagem: Inglês
10.1111/j.1469-0691.1998.tb00700.x
ISSN1469-0691
AutoresLee Ann Campbell, Teresa C. Moazed, C C Kuo, J. Thomas Grayston,
Tópico(s)Urinary and Genital Oncology Studies
ResumoSeroepidemiologic studies have shown an association of Chlamydia pneumoniae antibody with atherosclerosis. Compelling additional evidence has accumulated, in that the organism has been found within atherosclerotic lesions throughout the arterial tree by multiple methods. C. pneumoniae has also been isolated from coronary and carotid atheromatous plaques. Although these studies support a potential role for C. pneumoniae in atherogenesis, confirmation of a causal relationship requires the use of animal models and intervention studies. We have focused on the evaluation of mouse models to address the hypothesis that, following upper respiratory tract infection, lung macrophages are infected, disseminate to the aorta, and alter the onset or progression of atherogenesis. ApoE-deficient knock-out and C57BL/6J mice were used. The apoE-deficient mouse develops atherosclerotic lesions spontaneously on a regular diet in a time- and age-dependent manner. This knock-out strain was developed on the background of the C57BL/6J mouse, which only develops atherosclerosis on a high-fat/high-cholesterol diet. To investigate whether infected macrophages constitute a vehicle for dissemination of C. pneumoniae in vivo, mice were inoculated intranasally or intraperitoneally. The organism was detected in harvested alveolar and peritoneal macrophages at all time points following intranasal and intraperitoneal inoculations, respectively, and in peripheral blood mononuclear cells following inoculation by both routes. In another experiment, alveolar and peritoneal macrophages from intranasally and intraperitoneally inoculated mice were adoptively transferred by intraperitoneal injection to uninfected mice. Subsequently, C. pneumoniae was detected in lung, spleen, abdominal lymph nodes and/or thymus of recipient mice. In control experiments, UV-inactivated C. pneumoniae DNA was not detected in alveolar or peritoneal macrophages beyond 5 min after inoculation in vivo. These cumulative results demonstrate that C. pneumoniae infects macrophages in vivo and that macrophages can serve as a vehicle for dissemination to other sites.To answer the question of whether the organism disseminates to and persists in the aorta, 8-week-old mice were infected intranasally. Following single or multiple inoculations in apoE-deficient mice, C. pneumoniae was detected in the lung and aorta for 20 weeks postinfection. In contrast, in C57BL/6J mice, the organism did not persist in the aorta following a single intranasal inoculation, but could be detected up to 7 weeks postinfection in multiply inoculated mice. Significantly, in apoE-deficient mice with developed atherosclerotic lesions, the organism was found in foam cells within the lesions by immunocytochemical staining. These studies show that persistent C. pneumoniae infection occurs in atherosclerotic lesions in the aorta in the apoE-deficient knock-out mouse model. Infection of the aorta also occurred in C57BL/6J mice but was more transient. Both models should be useful in studying the pathogenic role of C. pneumoniae in atherogenesis and for determining if therapy prevents dissemination of infection.To this end, we have evaluated the susceptibility of C. pneumoniae to roxithromycin, a new macrolide, in vitro in cell culture and in vivo in the apoE-deficient mouse model. In vitro results compare favorably with other new macrolides, quinolines, and tetracyline. Preliminary studies in the apoE model of persistent infection suggest that the organism is detected less frequently in the lung and aorta by PCR following treatment. These promising preliminary studies warrant further investigation. Seroepidemiologic studies have shown an association of Chlamydia pneumoniae antibody with atherosclerosis. Compelling additional evidence has accumulated, in that the organism has been found within atherosclerotic lesions throughout the arterial tree by multiple methods. C. pneumoniae has also been isolated from coronary and carotid atheromatous plaques. Although these studies support a potential role for C. pneumoniae in atherogenesis, confirmation of a causal relationship requires the use of animal models and intervention studies. We have focused on the evaluation of mouse models to address the hypothesis that, following upper respiratory tract infection, lung macrophages are infected, disseminate to the aorta, and alter the onset or progression of atherogenesis. ApoE-deficient knock-out and C57BL/6J mice were used. The apoE-deficient mouse develops atherosclerotic lesions spontaneously on a regular diet in a time- and age-dependent manner. This knock-out strain was developed on the background of the C57BL/6J mouse, which only develops atherosclerosis on a high-fat/high-cholesterol diet. To investigate whether infected macrophages constitute a vehicle for dissemination of C. pneumoniae in vivo, mice were inoculated intranasally or intraperitoneally. The organism was detected in harvested alveolar and peritoneal macrophages at all time points following intranasal and intraperitoneal inoculations, respectively, and in peripheral blood mononuclear cells following inoculation by both routes. In another experiment, alveolar and peritoneal macrophages from intranasally and intraperitoneally inoculated mice were adoptively transferred by intraperitoneal injection to uninfected mice. Subsequently, C. pneumoniae was detected in lung, spleen, abdominal lymph nodes and/or thymus of recipient mice. In control experiments, UV-inactivated C. pneumoniae DNA was not detected in alveolar or peritoneal macrophages beyond 5 min after inoculation in vivo. These cumulative results demonstrate that C. pneumoniae infects macrophages in vivo and that macrophages can serve as a vehicle for dissemination to other sites. To answer the question of whether the organism disseminates to and persists in the aorta, 8-week-old mice were infected intranasally. Following single or multiple inoculations in apoE-deficient mice, C. pneumoniae was detected in the lung and aorta for 20 weeks postinfection. In contrast, in C57BL/6J mice, the organism did not persist in the aorta following a single intranasal inoculation, but could be detected up to 7 weeks postinfection in multiply inoculated mice. Significantly, in apoE-deficient mice with developed atherosclerotic lesions, the organism was found in foam cells within the lesions by immunocytochemical staining. These studies show that persistent C. pneumoniae infection occurs in atherosclerotic lesions in the aorta in the apoE-deficient knock-out mouse model. Infection of the aorta also occurred in C57BL/6J mice but was more transient. Both models should be useful in studying the pathogenic role of C. pneumoniae in atherogenesis and for determining if therapy prevents dissemination of infection. To this end, we have evaluated the susceptibility of C. pneumoniae to roxithromycin, a new macrolide, in vitro in cell culture and in vivo in the apoE-deficient mouse model. In vitro results compare favorably with other new macrolides, quinolines, and tetracyline. Preliminary studies in the apoE model of persistent infection suggest that the organism is detected less frequently in the lung and aorta by PCR following treatment. These promising preliminary studies warrant further investigation. Chlamydia pneumoniae is a human respiratory pathogen. It is an etiologic agent of acute respiratory disease, causing approximately 10% of cases of community-acquired pneumonia and 5% of cases of pharyngitis, bronchitis, and sinusitis [1Kuo C-C Jackson LA Campbell LA Grayston JT Chlamydia pneumoniae.Clin Microbiol Rev. 1995; 8: 451-461Crossref PubMed Google Scholar]. Cumulative seroepidemiologic studies have shown that almost everyone is infected at least once in their lifetime and that reinfection is common [2Saikku P Leinonen M Mattila K Nieminen MS Makela PH Huttunen JK Valtonen V Serological evidence of an association of a novel chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction.Lancet. 1988; 2: 983-986Abstract PubMed Scopus (1393) Google Scholar]. Antibody against C. pneumoniae is rare in children under the age of 5 years, except in underdeveloped and tropical countries. The antibody prevalence increases rapidly between the ages of 5 to 14, reaches 50% at the age of 20, and continues to increase slowly to 70–80% in 60–70 year-old-persons [1Kuo C-C Jackson LA Campbell LA Grayston JT Chlamydia pneumoniae.Clin Microbiol Rev. 1995; 8: 451-461Crossref PubMed Google Scholar]. The expanding clinical spectrum of C. pneumoniae infection has been extended to atherosclerosis and related clinical manifestations such as coronary heart disease, carotid artery stenosis, aortic aneurysm, claudication, and stroke. The first study showing an association of this organism with atherosclerosis was reported by Saikku et al, who showed that patients with coronary artery disease were significantly more likely to have serologic evidence of past infection with C. pneumoniae [2Saikku P Leinonen M Mattila K Nieminen MS Makela PH Huttunen JK Valtonen V Serological evidence of an association of a novel chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction.Lancet. 1988; 2: 983-986Abstract PubMed Scopus (1393) Google Scholar, 3Saikku P Leinonen M Tenkanen L et al.Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study.Ann Intern Med. 1992; 116: 273-278Crossref PubMed Scopus (733) Google Scholar, 4Linnanmäki E Leinonen M Mattila K Nieminen MS Valtonen V Saikku P Chlamydia pneumoniae-specific circulating immune complexes in patients with chronic coronary heart disease.Circulation. 1993; 87: 1130-1134Crossref PubMed Scopus (259) Google Scholar, 5Leinonen M Mattila K Kohlmeier L Saikku P Chlamydia pneumoniae-specific antibodies and immune complexes in German patients with acute myocardial infarction.in: Orfila J Byrne GL Cherneskey MA Chlamydial infections—1994. 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These observations have been confirmed by other investigators and also extended to carotid artery disease and cerebrovascular disease [6Thom DH Grayston JT Siscovick DS Wang S-P Weiss NS Daling JR Association of prior infection with Chlamydia pneumoniae and angiographically demonstrated coronary artery disease.JAMA. 1992; 268: 68-72Crossref PubMed Scopus (412) Google Scholar, 7Thom DH Wang S-P Grayston JT et al.Chlamydia pneumoniae strain TWAR antibody and angiographically demonstrated coronary artery disease.Arterioscler Thromb. 1992; 11: 547-551Crossref Scopus (217) Google Scholar, 8Melnick SL Shahar E Folsom AR et al.Past infection by Chlamydia pneumoniae strain TWAR and asymptomatic carotid atherosclerosis.Am J Med. 1993; 95: 499-504Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 9Dahlén GH Boman J Birgander LS Lindblom B Lp(a) lipoprotein, IgG, IgA and IgM antibodies to Chlamydia pneumoniae and HLA class genotype in early coronary artery disease.Atherosclerosis. 1995; 114: 165-174Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 10Mendall MA Patel P Ballam L Strachan D Northfield TC C reactive protein and its relation to cardiovascular risk factors: a population based cross sectional study.Br Med J. 1996; 312: 1061-1065Crossref PubMed Scopus (777) Google Scholar, 11Wimmer ML Sandmann-Strupp R Saikku P Haberl RL Association of chlamydial infection with cerebrovascular disease.Stroke. 1996; 27: 2207-2210Crossref PubMed Scopus (195) Google Scholar]. Compelling evidence for the association of C. pneumoniae with atherosclerosis has been obtained by the demonstration of C. pneumoniae by PCR, immunocytochemical staining and electron microscopy in atherosclerotic lesions [12Shor A Kuo C-C Patton DL Detection of Chlamydia pneumoniae in coronary arterial fatty streaks and atheromatous plaques.South Afr Med J. 1992; 82: 158-161PubMed Google Scholar,13Kuo C-C Shor A Campbell LA Fukushi H Patton DL Grayston JT Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries.J Infect Dis. 1993; 167: 841-849Crossref PubMed Scopus (670) Google Scholar]. Evidence of the organism has been found in coronary, carotid, aortic, femoral, iliac and popliteal atheromas in both early lesions and developed fibrolipid plaques and in abdominal aneurysms [14Kuo C-C Gown AM Benditt EP Grayston JT Detection of Chlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical stain.Arterioscler Thromb. 1993; 13: 1501-1504Crossref PubMed Scopus (303) Google Scholar, 15Campbell LA O'Brien ER Cappuccio AL et al.Detection of Chlamydia pneumoniae (TWAR) in human coronary atherectomy tissues.J Infect Dis. 1993; 172: 585-588Crossref Scopus (334) Google Scholar, 16Kuo C-C Grayston JT Campbell LA Goo YA Wissler RW Benditt EP Chlamydia pneumoniae (TWAR) in coronary arteries of young (15 to 35 year) adults.Proc Natl Acad Sci USA. 1995; 92: 6911-6914Crossref PubMed Scopus (361) Google Scholar, 17Grayston JT Kuo C-C Coulson AS et al.Chlamydia pneumoniae (TWAR) in atherosclerosis of the carotid artery.Circulation. 1995; 92: 3397-3400Crossref PubMed Scopus (312) Google Scholar, 18Davidson M Kuo C-C Middaugh JP et al.Confirmed previous infection with Chlamydia pneumoniae (TWAR) and its presence in early coronary atherosclerosis.Circulation. 1998; 98: 628-633Crossref PubMed Scopus (157) Google Scholar, 19Kuo C-C Coulson AS Campbell LA et al.Detection of Chlamydia pneumoniae in atherosclerotic plaques in the walls of arteries of lower extremities from patients undergoing bypass operation for arterial obstruction.J Vasc Surg. 1997; 26: 1-3Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Jackson LA Campbell LA Schmidt RA Kuo C-C Cappuccio AL Grayston JT Specificity of detection of Chlamydia pneumoniae in cardiovascular and non-cardiovascular tissues: evaluation of the innocent bystander hypothesis.Am J Pathol. 1997; 150: 1785-1790PubMed Google Scholar, 21Ong G Thomas BJ Mansfield OA Davidson BR Taylor-Robinson D Detection and widespread distribution of Chlamydia pneumoniae in the vascular system and its possible implications.J Clin Pathol. 1996; 49: 102-106Crossref PubMed Scopus (196) Google Scholar, 22Blasi F Denti F Erba M et al.Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms.J Clin Microbiol. 1996; 34: 2766-2769PubMed Google Scholar, 23Ouchi K Fuji B Kanamoto Y Miyazaki H Nakazawa T Detection of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries and large arteries. (Abstract K37). American Society for Microbiology, Washington DC1995: 294Google Scholar, 24Varghese PJ Gaydos CA Arumugham SB Pham DG Quinn TC Tuazon CU Demonstration of Chlamydia pneumoniae in coronary atheroma specimens from young patients with normal cholesterol from the southern part of India. Infectious Disease Society of America, 1995: 30Google Scholar, 25Mühlestein JB Hammond EH Carlquist JF et al.Increased incidence of Chlamydia species within the coronary arteries of patients with symptomatic atherosclerotic versus other forms of cardiovascular disease.J Am Coll Cardiol. 1996; 27: 1555-1561Abstract Full Text PDF PubMed Scopus (382) Google Scholar, 26Juvonen J Juvonen T Laurila A et al.Demonstration of Chlamydia pneumoniae in the walls of abdominal aortic aneurysms.J Vasc Surg. 1997; 25: 499-505Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar]. Finally, C. pneumoniae has been isolated from coronary and carotid atheromatous plaques [27Ramirez J Ahkee S Ganzel BL et al.Isolation of Chlamydia pneumoniae (C pn) from the coronary artery of a patient with coronary atherosclerosis.Ann Intern Med. 1996; 125: 979-982Crossref PubMed Scopus (444) Google Scholar,28Jackson LA Campbell LA Kuo C-C Lee A Grayston JT Isolation of Chlamydia pneumoniae from a carotid endarterectomy specimen.J Infect Dis. 1997; 176: 292-295Crossref PubMed Scopus (284) Google Scholar]. The role of C. pneumoniae in atherosclerosis has yet to be determined. Our working hypothesis is that, following upper respiratory tract infection, C. pneumoniae infects lung macrophages, which disseminate via hematogenous and/or lymphatic routes to the aorta and affect the onset, severity or progression of the disease process. Two mouse models have been developed to study this hypothesis. ApoE-deficient mice develop atherosclerosis on a normal diet in a time-dependent manner, developing lesions showing similar characteristics to human disease [29Piedrahita JA Zhang SH Hagaman JR Oliver PM Maeda N Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.Proc Natl Acad Sci USA. 1992; 189: 4471-4475Crossref Scopus (718) Google Scholar, 30Reddick RL Zhang SH Maeda N Atherosclerosis in mice lacking Apo E. Evaluation of lesional development and progression.Atheroscler Thromb. 1993; 14: 141-147Crossref Scopus (530) Google Scholar, 31Nakashima Y Plump AS Raines EW Breslow JL Ross R ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree.Atheroscler Thromb. 1994; 14: 133-140Crossref PubMed Google Scholar]. C57BL/6J mice, the background strain of apoE-deficient mice, do not develop atherosclerosis on a normal chow diet but will develop atherosclerosis on a high-fat/high-cholesterol diet. This report summarizes our findings using these mouse models for studies on infection, dissemination, and intervention. C. pneumoniae, strain AR-39 [32Grayston JT Campbell LA Kuo C-C et al.A new respiratory tract pathogen: Chlamydia pneumoniae strain TWAR.J Infect Dis. 1990; 161: 618-625Crossref PubMed Scopus (661) Google Scholar], was used for inoculation of animals. The organism was grown in HL cells and purified by density gradient using diatrizoate meglumine centrifugation (Hypaque-76; Winthrop-Breon Laboratories, New York, NY, USA) [33Moazed TC Kuo C-C Grayston JT Campbell LA Murine models of Chlamydia pneumoniae infection and atherosclerosis.J Infect Dis. 1997; 175: 883-890Crossref PubMed Scopus (205) Google Scholar]. The inoculum preparations were resuspended in chlamydia transport media (sucrose phosphate glutamic acid) and frozen at –70°C until use. For controls, UV-inactivated organisms were used in some experiments. Inactivation was done by exposure to UV light at a distance of 13 cm for 30 min at room temperature. Subsequently, organisms were frozen at –70°C. Prior to inoculation, the inoculum was tested for viability by HL cell culture. No inclusions were seen by immunofluorescence after 5 days of incubation. Sixty-eight male homozygous apoE-deficient mice and 122 male and female C57BL/6J mice, aged 6–7 weeks, were obtained from the Jackson Laboratories (Bar Harbor, Maine, USA). As previously described, mice were inoculated intranasally with 3–5×l07 inclusion-forming units (IFUs) with a total volume of 0.03–0.05 mL suspended in SPG medium or inoculated with SPG medium alone at 8 weeks of age [33Moazed TC Kuo C-C Grayston JT Campbell LA Murine models of Chlamydia pneumoniae infection and atherosclerosis.J Infect Dis. 1997; 175: 883-890Crossref PubMed Scopus (205) Google Scholar]. Multiply inoculated apoE-deficient mice were inoculated at 8. 10 and 12 weeks of age. Multiply inoculated C57BL/6J mice were inoculated at 8, 9 and 10 weeks of age. Intraperitoneally inoculated mice received 5×107 IFU per animal in a total volume of 0.5 mL SPG medium. All animals receiving inactivated organisms were given identical doses of inoculum. Control mice were sham-inoculated with 0.03 mL of sterile SPG medium. Mice were heavily sedated, and heparinized whole blood was collected by exsanguination from the femoral arteries. The chest and abdomen were opened and the heart and aorta were perfused with phosphate-buffered saline (PBS). Lung, spleen, heart and ascending and abdominal aorta were removed with a separate set of sterile instruments, placed in sterile glass vials and immediately placed on ice. Tissues were later frozen at –70°C. Heparinized whole blood was diluted with an equal volume of PBS and separated by Ficoll-Paque Plus (Pharmacia, Uppsala, Sweden) by centrifugation at 400g for 30 min at 20°C. The buffy coat was washed twice with PBS and resuspended in 2 mL of Dulbecco's minimum essential medium (DMEM). Plasma was aspirated and filtered through a 0.8-μm filter before culture. Alveolar and peritoneal macrophages and peripheral blood were collected at 3, 5 and 7 days postinoculation from intranasally and intraperitoneally inoculated C56BL/6J mice. Peritoneal macrophages were collected by washing the peritoneal cavity twice with cold Hanks' balanced salt solution (HBSS), pH 7.2, containing 0.2% bovine serum albumin. Alveolar macrophages were collected by removing the lungs to a sterile Petri dish and lavaging through the trachea with cold HBSS. Macrophages were washed twice with cold PBS by centrifugation at 1000 rev/min for 10 min before analysis. To immediately fix macrophages at specific time points, cells were collected by the methods described above with the use of cold HBSS containing 0.02% phosphate-buffered formalin. Tissue samples were thawed and homogenized with a sterile pestle and mortar in 1.0–2.0 mL of SPG medium to make a 10% (w/v) suspension. Tissue homogenates were centrifuged at 500g for 5 min at 4°C to sediment tissue debris and the supernatant was aspirated for inoculation of cultures. Tissue pellets were resuspended in 1.0 mL of lysis buffer (10 mM Tris-C1, pH 8.0, 100 mM EDTA, 0.5% sodium dodecylsulfate (SDS)), and 200 μg/ml proteinase K (Amresco, Solon, OH, USA) was added to each sample. Samples were incubated overnight at 50°C and extracted with hexadecyltrimethyl ammonium bromide (CTAB) according to standard methods [34Preparation of genomic DNA from bacteria.in: Ausubel FM Brent R Kingston RE Current protocols, molecular biology. 1. Wiley Interscience, New York1991Google Scholar]. Lysates were extracted twice with phenol/chloroform/isoamyl alcohol (24:24:1) and once with chloroform/isoamyl alcohol (24:1). DNA was precipitated with 0.7 volumes of 100% isopropanol at room temperature, and this was followed by washing with ice-cold 70% ethanol. DNA pellets were air-dried and resuspended in 50–100 μL of 10 mM Tris-C1, pH 8.0, 1 mM EDTA. An extraction control of lysis buffer alone was included with each set of tissue extractions to detect cross-contamination of samples during the extraction process. Tissue homogenization, DNA extractions and PCR amplification were performed in separate rooms equipped with germicidal lighting. Human line (HL) cell monolayers in shell vials were inoculated with 100 μL of 10% tissue homogenate. Cultures were passed once, 4 days after inoculation. Inclusions were detected by direct fluorescent antibody (DFA) stain using a Chlamydia genus-specific monoclonal antibody (CF-2) conjugated to fluorescein isothiocyanate (FITC) [35Kuo C-C Grayston JT A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR.J Infect Dis. 1990; 162: 755-758Crossref PubMed Scopus (112) Google Scholar]. The infectious titer was assayed by titration of tissue homogenates in HL cells and expressed as log10 IFUs per gram of lung tissue. Alveolar and peritoneal macrophages were washed twice in 10 mL of DMEM containing 1% non-essential amino acids (Gibco BRL, Grand Island, NY, USA), 2 mM glutamine and 100 μg each of vancomycin and streptomycin. Macrophages were counted with a hemocytometer following staining with 0.4% trypan blue dye (Sigma, St Louis, MO, USA). Based on morphology, approximately 70–90% of the cells collected were macrophages. Approximately half of the collected cells were directly plated and the remainder were frozen at –70°C for culture and PCR. Macrophages were plated in 24-well microtiter plates containing 12-mm glass coverslips at a concentration of 2–5×105 cells per well in a total volume of 1 mL of DMEM for up to three coverslips per sample. Cells were incubated at 37°C in 5% CO2 overnight. Coverslips were fixed with acetone and stained with FITC-conjugated Chlamydia genus-specific (CF-2) monoclonal antibody for detection of chlamydial inclusions [35Kuo C-C Grayston JT A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR.J Infect Dis. 1990; 162: 755-758Crossref PubMed Scopus (112) Google Scholar]. DNA samples were amplified using C. pneumoniae-specific primers HL-1 and HR-1 [36Campbell LA Melgosa MP Hamilton DJ Kuo C-C Grayston JT Detection of Chlamydia pneumoniae by polymerase chain reaction.J Clin Microbiol. 1992; 30: 434-439PubMed Google Scholar]. The amplification product, an expected 437-bp C. pneumoniae-specific DNA sequence, was analyzed by gel electrophoresis through a 1.5% agarose gel according to standard methods and transferred by Southern blot to a nylon membrane (Qiagen, Chatsworth, CA, USA). The product was confirmed by hybridization to a digoxigenin–dUTP-labeled probe consisting of a 474-bp PstI fragment which spanned the target sequence [36Campbell LA Melgosa MP Hamilton DJ Kuo C-C Grayston JT Detection of Chlamydia pneumoniae by polymerase chain reaction.J Clin Microbiol. 1992; 30: 434-439PubMed Google Scholar]. DNA probes were labeled using the Genius DNA labeling and detection kit (Boehringer Mannheim Biochemical, Indianapolis, IN, USA). Hybridized amplification products were detected by immuno-chemiluminescence with the use of Lumiphos 530 (Boehringer Mannheim Biochemical, Indianapolis, IN, USA) according to the manufacturer's instructions, followed by autoradiography. Controls for each amplification consisted of serial dilutions of purified C. pneumoniae DNA as positive controls and sterile water in place of sample DNA as a negative control. Positive and negative controls were amplified, analyzed and confirmed concurrently with each set of samples. Abdominal and ascending aorta were fixed in situ prior to removal by perfusion with 10 mL of PBS followed by 10–15 mL of 10% phosphate-buffered formalin (Fisher Scientific, Pittsburgh, PA, USA) injected through the left ventricle of the heart. Lungs were perfused through the trachea with 10% formalin prior to removal. The base of the heart was collected and sectioned at the level of the coronary sinus. Ascending aorta was sampled from the junction of the aorta with the heart at the end of the aortic sinus through the aortic arch and thoracic aorta. Abdominal aorta was sampled from the mesenteric artery branch through the iliac bifurcation. Aorta from singly inoculated and sham-inoculated animals was cut into 2–3-mm pieces, embedded in paraffin, and then cross-sectioned for staining. Aorta from multiply inoculated animals was cut into 6–8-mm pieces, embedded in paraffin and sectioned longitudinally. Tissues for histopathology were stained with hematoxylin and eosin. For detection of chlamydial antigens, tissue sections were reacted with a Chlamydia genus-specific mouse monoclonal antibody (CF-2). Lung sections were stained by the direct avidin–biotin–peroxidase method using CF-2 conjugated to biotin at 1:250 dilution as previously described [23Ouchi K Fuji B Kanamoto Y Miyazaki H Nakazawa T Detection of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries and large arteries. (Abstract K37). American Society for Microbiology, Washington DC1995: 294Google Scholar]. Aorta sections were stained by the indirect method with CF-2 at 1:1000 dilution using the HistoMouse Kit (Zymed Laboratories, San Francisco, CA, USA). Tissue sections were counter-stained with either methyl green or hematoxylin. Duplicate tissue sections and tissues from sham-inoculated mice were reacted with CF-2 and normal mouse ascitic fluid as negative controls. C. pneumoniae infected HL cell pellets were formalin fixed, embedded in paraffin and sectioned for positive controls. Alveolar macrophages were collected from 10 donor mice 3 days after intranasal inoculation. Peritoneal macrophages were collected from 10 donor mice 7 days after intraperitoneal inoculation [37Moazed TC Kuo C-C Grayston JT Campbell LA Evidence of systemic dissemination of Chlamydia pneumoniae via macrophages in the mouse.J Infect Dis. 1998; 177: 1322-1325Crossref PubMed Scopus (224) Google Scholar]. Aliquots of 5×105 cells were analyzed for the presence of C. pneumoniae organisms by direct plating, culture and PCR. Approximately 2×106 cells, either alveolar or peritoneal macrophages, were transferred by intraperitoneal injection to four recipient mice. Three days after transfer, tissues were removed from all recipient mice and tested for the presence of C. pneumoniae by isolation and PCR. In vitro susceptibility of C. pneumoniae to roxithromycin was tested in cell cultures as described previously [38Kuo C-C Grayston JT In vitro drug susceptibility of Chlamydia sp. strain TWAR.Antimicrob Agents Chemother. 1988; 32: 257-258Crossref PubMed Scopus (68) Google Scholar]. The antibiotic was tested at serial two-fold dilutions. Total inhibition of inclusion formation as determined by DFA was used as the endpoint. The MIC was defined as the end point for the first passage containing roxithromycin. The MBC was defined as the endpoint for the second passage containing no roxithromycin. ApoE-deficient mice were intranasally inoculated at 8, 10 and 12 weeks of age. Four weeks after the final inoculation, mice were treated daily for 7 days with either roxithromycin (50 mg/kg) or 0.5% methylcellulose delivered by gastric lavage. To determine if macrophages were infected in vivo, C57BL/6J mice were inoculated intranasally or intraperitoneally and alveolar and peritoneal macrophages, respectively, were harvested. The frequency of detection of C. pneumoniae in macrophages and peripheral blood is shown in Table 1. Staining of macrophages with fluorescein
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