Analysis of host Toll-like receptor 3 and RIG-I-like receptor gene expression in patients with abdominal aortic aneurysm.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a vascular disease relatively common in the elderly population. Although some events that contribute to the development and progression of AAA are known, there are limited data examining the association of Toll-like receptor 3 (TLR3) and RIG-I-like receptor expression with the pathogenesis of AAAs. In this study, we investigated the gene and protein expression of TLR3 and RIG-I-like receptors (RIG-I and MDA5) in aortic wall and blood of AAA patients and examined the relationship between their expression and immune response. METHODS: Total RNA was extracted from aortic wall tissues and blood samples collected from 20 patients with AAA and blood samples of 17 healthy volunteers without aortic aneurysm. To evaluate the DDX58 (RIG-I), IFIH1 (MDA5), and TLR3 gene expression level, quantitative real-time polymerase chain reaction was used. Extracellular cytokine and pattern recognition receptor levels were quantified by enzyme-linked immunosorbent assays. RESULTS: TLR3, RIG-I, and MDA5 were constitutively expressed in both aortic tissues and blood samples from AAA patients and healthy volunteers. In patients with AAA, higher TLR3 expression in aortic tissues than in blood was found (P = .004). The DDX58 messenger RNA expression was higher in blood of patients with AAA compared with healthy subjects (P = .021). A significantly higher level of plasma interleukin 4 was noticed in patients with AAA than in healthy individuals (P = .008). CONCLUSIONS: This study suggests that RIG-I and TLR3 seem to be important factors in the pathogenesis of AAA.

Murine cytomegalovirus infection leads to increased levels of transplant arteriosclerosis in a murine aortic allograft model.

INTRODUCTION: Cytomegalovirus infection after heart transplantation is considered as risk factor for the development of transplant arteriosclerosis. Therefore, the aim of this study was to investigate the effect of murine cytomegalovirus (MCMV) as a single risk factor on transplant arteriosclerosis in an experimental aortic allograft model. METHODS: Major histocompatibility complex class I-mismatched aortas of C.B10-H2(b)/LilMcdJ donor were transplanted into BALB/c recipients, which were either mock-infected or infected with MCMV (strain Smith) on day 7 and harvested 37 days after transplantation. In one experimental group animals received a daily dose of everolimus to increase the viral load of recipients. Grafts were analyzed by histology, morphometry, and immunofluorescence. Intragraft cytokine mRNA production was analyzed by real-time polymerase chain reaction (PCR), and persistence of cytomegalovirus infection was confirmed by TaqMan PCR. RESULTS: After infection with MCMV, there was significantly more intimal proliferation when compared with uninfected controls (intimal proliferation 83.5%+/-9.6% [MCMV] vs. 43.9%+/-5.1% [MCMV]), indicating that MCMV plays a role in the development of transplant arteriosclerosis. Even after treatment with everolimus, MCMV infection pronounced significantly more intimal proliferation (intimal proliferation 52.5%+/-7.3% [MCMV] vs. 20.2%+/-1.7% [MCMV]). Intragraft mRNA expression showed significantly higher production of CD62E, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 after infection with MCMV. Cellular infiltration revealed significantly more CD4, CD8, and dendritic cells. We could also confirm the presence of MCMV for the duration of the experimental protocol by PCR within the spleen, liver, salivary glands, lung, and the aortic transplant. CONCLUSION: These data suggest that MCMV infection plays an important role in the development of transplant arteriosclerosis.

Detection of cytomegalovirus and Helicobacter pylori DNA in arterial walls with grade III atherosclerosis by PCR.

It has been suggested that some microorganisms may play a role in the etiology or progression of atherosclerotic plaques. The purpose of this study was to assess for the presence of Helicobacter pylori and cytomegalovirus (CMV) DNA using polymerase chain reaction (PCR) technique in vascular-wall specimens obtained during autopsy. Four to 5 mm long samples from 3 different vascular wall specimens (coronary, carotid and abdominal aortas) of 30 patients (23 male, 7 female) were taken for pathologic and microbiologic investigations during autopsy. H. pylori DNA was found in 48.2% atherosclerotic and 19.6% non-atherosclerotic vascular wall specimens, whereas CMV DNA was found in 37.9% atherosclerotic and 32.7% non-atherosclerotic vascular wall specimens. In terms of CMV DNA detection, no statistically significant differences between the atherosclerotic and non-atherosclerotic groups were present (P > 0.05). However, there was a statistically significant difference between the atherosclerosis and non-atherosclerotic groups in terms of H. pylori DNA in coronary and abdominal aorta arteries (p = 0.016 and p = 0.0029 respectively) but not in carotid arteries (p = 1.00). In conclusion, the correlation between H. pylori and atherosclerosis could be suggested. These finding warrant further investigation regarding the role of H. pylori in atherosclerosis.

Prevalence of BK virus and JC virus in peripheral blood leukocytes and normal arterial walls in healthy individuals in China.

Several studies have demonstrated that BK virus (BKV) and JC virus (JCV) establish latent infection in peripheral blood leukocytes (PBLs) of healthy individuals; however, the main populations studied are European. In this study, the prevalence of BKV and JCV DNA in PBLs from healthy adult individuals and umbilical cord blood from newborn children in China was detected by semi-nested polymerase chain reaction (snPCR) followed by restriction enzyme analysis. The results suggest that the healthy adult Chinese population harbors BKV and JCV DNA in peripheral leukocytes. Overall, the prevalence of BKV and JCV DNA in PBLs of healthy adult individuals was 42.1% and 7.8%, respectively. The overall prevalence of BKV DNA was significantly higher than that of JCV DNA. None of the umbilical cord blood samples from newborn children were positive for BKV and JCV DNA. To understand further the target tissues involved in establishment of BKV and JCV latency in healthy individuals, the presence of DNA from both viruses was detected in normal arterial wall samples from 20 young trauma victims by the same method used for leukocyte DNA. BKV DNA was detected alone in 20% of samples tested; JCV DNA was not detected alone in any of the samples. DNA from both viruses was found in 5% of samples. This is the first report to show that normal arterial walls of healthy individuals may be another target site of latency for BKV and JCV.

Expression of secretory group IIA phospholipase A(2) in relation to the presence of microbial agents, macrophage infiltrates, and transcripts of proinflammatory cytokines in human aortic tissues.

Recent seroepidemiological and immunohistochemical studies have demonstrated an association between microbial infections and atherosclerosis. However, the mechanisms underlying this association are widely unknown. In the present study, arterial specimens obtained at autopsy after sudden death were analyzed concerning (1) the presence of Chlamydia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori; (2) the expression of secretory group IIA phospholipase A(2) (sPLA(2)-IIA) and of proinflammatory cytokines; and (3) the stage of atherosclerosis. Genomic DNA of microbial pathogens was determined by the polymerase chain reaction technique. The expression of sPLA(2)-IIA was studied immunohistochemically by using monoclonal antibodies against human sPLA(2)-IIA. Transcripts specific for sPLA(2)-IIA, interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were identified by reverse transcription-polymerase chain reaction. In 18 of 102 analyzed specimens, DNA of microbial pathogens was found. Thirteen sections were positive for C pneumoniae, whereas 2 specimens were positive either for cytomegalovirus or for herpes simplex virus. One section contained genomic DNA of all 3 pathogens simultaneously. None of the analyzed tissues exhibited nucleic acids specific for H pylori. In addition to macrophage infiltrates, the presence of microbial DNA was closely associated with the occurrence of transcripts specific for proinflammatory cytokines and sPLA(2)-IIA. Pathogens as well as sPLA(2)-IIA and cytokines were found to be present not only in advanced but also in early stages of atherosclerosis. In tissues negative for sPLA(2)-IIA and cytokine expression, none of the pathogens could be identified. Because macrophages exposed to phospholipase A(2)-treated lipoproteins are transformed into foam cells in vitro, the results of this study suggest an alternative mechanism by which microbial infections may act in a proatherogenic fashion in vessel walls.

Chlamydia pneumoniae in abdominal aortic aneurysms: abundance of membrane components in the absence of heat shock protein 60 and DNA.

In this article, we describe the results of a comparative study for the detection of Chlamydia pneumoniae in abdominal aortic aneurysm specimens of 19 patients through the use of immunocytochemistry (ICC), in situ hybridization (ISH), and polymerase chain reaction (PCR), along with the detection of cytomegalovirus (CMV) and herpes simplex virus (HSV) by ICC and PCR. C pneumoniae-specific membrane protein was detected in specimens of all 19 (100%; 95% confidence interval [CI] 82% to 100%) and of 15 (79%; 95% CI 54% to 94%) patients with monoclonal antibodies RR-402 and TT-401, respectively. Chlamydial lipopolysaccharide was detected in specimens of 15 (79%; 95% CI 54% to 94%) patients when the results of 4 different monoclonal antibodies were combined. Surprisingly, chlamydial heat shock protein 60 was not detected in any of the specimens by ICC. Furthermore, C pneumoniae DNA was not detected by ISH when a C pneumoniae major outer membrane protein gene fragment was used as probe, nor was it reproducibly detected by PCR on extracted DNA. These results may be explained either by different kinetics of degradation of the different components of C pneumoniae after infection of the vessel wall or by the involvement of other Chlamydia-like microorganisms. Coexistence of C pneumoniae antigens and HSV antigens but not CMV antigens was observed in specimens from 10 of 18 (56%; 95% CI 31% to 78%) patients by ICC. CMV and HSV DNAs were not detected by PCR. In conclusion, we have demonstrated the presence of antigens of C pneumoniae in the absence of specific DNA in abdominal aortic aneurysms, suggesting persistence of the antigens rather than a persistent infection.

Detection of microorganisms in vessel wall specimens of the abdominal aorta: development of a PCR assay in the absence of a gold standard.

A procedure in which the "Invitrogen Easy-DNA" kit was followed by a silica-based method for the isolation of DNA was developed for extraction of PCR-inhibitor-free DNA from up to 300 mg of human vessel wall tissue. Optimally designed PCR assays were developed for the detection of at least one infected cell in this amount of tissue. Details of the procedure are given for the detection of DNA of Chlamydia pneumoniae, cytomegalovirus and herpes simplex virus type 1 and type 2 in human vessel walls. The procedure can serve as a reference method or as a gold standard when a high-performance method is needed.

Aortic endothelium in HIV-1 infection: chronic injury, activation, and increased leukocyte adherence.

Clinical and serological studies provide evidence for a pathogenetically relevant vasculopathy in acquired immune deficiency syndrome (AIDS); however, the morphological status of the endothelium under conditions of human immunodeficiency virus (HIV)-1 infection is only sparsely documented. In this study we adapted an en face preparation technique of endothelium for use in immunohistochemistry and investigated the aortic endothelium of pre-AIDS and AIDS patients (n = 32) in comparison with an HIV-negative group (n = 17). The control group showed a regular pattern of evenly distributed aortic endothelial cells, whereas the endothelial cell pattern in the HIV-1-infected patients was clearly disturbed. Simultaneously, the degree of leukocyte adherence on the aortic endothelium increased significantly. These changes were accompanied by an up-regulation of the vascular cell adhesion molecule-1 (VCAM-1) and E-selectin (ELAM-1). The endothelium turnover increased, and one-half of the HIV-1-infected patients exhibited HLA-DR (major histocompatibility complex class II) antigen in the aortic endothelium. Our results provide evidence for a profound and repeated injury with regeneration and activation of the endothelium in HIV-1 infection. Injury as well as activation of the endothelium impairs its normal regulatory properties. This could have consequences for the maintenance of the blood-brain barrier; it might influence the immunologically important interaction of the endothelium with T cells; and it might trigger Kaposi's sarcoma.