Markers of Chlamydia pneumoniae and human cytomegalovirus infection in patients with chronic peripheral vascular disease and their relation to inflammation, endothelial dysfunction and changes in lipid metabolism

Our aim was to detect markers of Chlamydia pneumoniae (CPN) and human cytomegalovirus (HCMV) infection in patients with peripheral vascular occlusive disease and to follow markers of inflammation, endothelial dysfunction and lipid metabolism alteration in patients with active infection. CPN genome was detected in 9 (47.4 %) patients by at least one PCR method. Serological markers of acute CPN infection were found in 5 (26.3 %) subjects; each of them showed also positivity in at least one of the PCR methods. HCMV DNA were detected in 2 (10.5 %) patients; HCMV-specific antibodies were detected in 14 (73.7 %) subjects, however only in IgG subclass. Subjects with HCMV PCR positivity thus showed no serological markers of active HCMV infection. Laboratory findings of acute CPN infection were associated with increased plasma levels of Lp(a), triacylglycerols, atherogenic index of plasma and E-selectin (p < 0.05). No significant differences were found in the other markers, including plasma levels of total cholesterol, ferritin, homocysteine, oxidized LDL, IL-6, IL-8, IL-18, TNF-α, soluble forms of VCAM-1 and ICAM-1, von Willebrand factor, C-reactive protein, and plasma nitrites & nitrates. Frequent presence of chlamydial DNA in atheromatous plaques from patients with peripheral vascular disease was confirmed. HCMV DNA was detected only sporadically and with positivity in anamnestic anti-HCMV antibodies (IgG) only, indicating a rare presence of latent virus rather than active replication. Patients with laboratory markers of acute CPN infection exhibited more pronounced alterations in lipid metabolism and endothelial dysfunction.     

巨细胞病毒参与动脉粥样硬化致病机制研究新进展

人巨细胞病毒(human cytomegalovirus, HCMV)是β疱疹病毒家族成员,在人群中感染率极高,全球成人中血清阳性率可达40%～100%。研究表明,HCMV感染患者更易患心脑血管疾病。动脉粥样硬化(atherosclerosis,AS)是心血管系统疾病中危害健康的一种常见病。大量流行病学研究证明,在AS组织中可检测出较高的HCMV DNA和抗原、抗体,同时回顾性研究发现AS患者多有HCMV暴露因素,提示HCMV可能参与AS致病。本文就HCMV致AS的依据和机制进行综述, 为研究HCMV在AS病理过程中的作用提供全新视角。     

Prevalence of active infection with<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> and human cytomegalovirus in patients with type II diabetes mellitus

By promoting the inflammatory process in the arterial wall, Chlamydia pneumoniae (CPN) and human cytomegalovirus (CMV) participate in the pathogenesis of cardiovascular disease (CVD). Since patients with diabetes mellitus (DM) are at high risk of CVD, we studied markers of CMV and CPN infection in DM patients as possible predictors of cardiovascular complications. The seroprevalence rates of CMV in 44 DM patients and matched controls were 74 and 88%, respectively. Compared with controls, patients showed lower titers of IgG against CMV (p < 0.001) and higher titers of genus-specific IgA against CPN (p = 0.006). The titers of genus-specific IgG and prevalence rates of type-specific anti-CPN IgA, IgG or IgM were similar in both DM patients and controls. Serological markers of either active or recent CPN infection were detected in 54% of patients and 59% of controls. However, CPN DNA was not detected in the blood of any DM patient. CMV DNA was found in the blood of 1 (2.3%) patient. The results do not indicate an increased rate of CMV or CPN infection in patients with type II DM.     

Transformation of dog embryo kidney cells by human herpesviruses

The infection of dog embryo kidney (DEK) cells with herpes simplex virus type 2 (HSV-2) or human cytomegalovirus (HCMV) led to the development of transformed cell lines. Rapidly dividing DEK cells with unlimited division potential exhibited growth in 2% serum, contained nuclear virus antigens, and formed small (+/- 0.2 mm) colonies in 0.3% agarose. Immortal cell lines showing the same transformation properties were also obtained after transfection with purified HSV-2 or HCMV DNA. These results confirm the transforming capacity of both herpesviruses as well as the usefulness of this different type of mammalian cells in transformation studies.     

Cytokine response and polymerase chain reaction study of peripheral blood mononuclear cells in infants with human cytomegalovirus infection

Abstract We tried to detect human cytomegalovirus (HCMV) DNA in CD4 + and CD8 + T lymphocytes from fourteen infants with HCMV hepatitis using polymerase chain reaction (PCR) assay. HCMV was isolated from their urine and anti-HCMV IgM antibody was detected in their sera. One set of primers were designed from a region — a major immediate early (IE) gene. We detected HCMV IE DNA in the specimens obtained from six infants. HCMV IE DNA was detected from CD4 + cells in two cases and from CD8 + cells in one. In three cases, HCMV IE DNA was detected from both CD4 + and CD8 + cells. We also studied the relationship between HCMV infection and serum levels of cytokines. We determined serum levels of interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α) and soluble interleukin 2 receptor (sIL-2R) which were associated with the activation of T lymphocytes by enzyme immunoassay. In the acute phase of HCMV infection, titers of sIL-2R were correlated with serum levels of liver enzymes in some cases. IL-4 and TNF-α activities were not detected in sera. It is likely that expression of viral genome on T lymphocytes as well as activities of some cytokines are associated with active HCMV infection.     

人巨细胞病毒的生物素DNA探针的制备和应用

本研究用克隆的HCMV AD169株DNA片段,制备了生物素标记的DNA探针,建立了检测临床脐带血、尿标本中HCMV DNA的核酸探针杂交方法。该探针可测出100pg同源DNA,不与人胚肺细胞、Hep-2细胞DNA以及其他疱疹病毒的DNA发生反应。用核酸杂交方法检测了30份脐带血标本,有11例阳性,阳性率为33％。10例孕妇尿标本中,3例阳性,阳性率为30％。检测结果表明：我们建立的生物素标记的HCMV DNA探针的点杂交法,具有高度的特异性、敏感性,比分离病毒法更迅速,可用于HCMV感染的临床标本的病毒核酸检测。     

人巨细胞病毒的分子克隆及其特异性DNA探针的制备

从人巨细胞病毒（ＨＣＭＶ）培养物中提取ＨＣＭＶ并抽提其ＤＮＡ,经限制性内切酶ＢａｍＨＩ完全消化后,与质粒ｐＢｌｕｅｓｃｒｉｐｔ－ＳＫ重组建立了ＨＣＭＶ的ＤＮＡ文库,从此文库。中随机筛选出两个重组质粒（ｐＣＭＶ－１和ｐＣＭＶ－２）,用ＢａｍＨＩ分析证明其中所含的病毒ＤＮＡ片段的大小分别为１．０ｋｂ和７．５ｋｂ,将这两种质粒大量扩增纯化后,用光生物素进行标记作为探针,证明其只与ＨＣＭＶ反应,与正常人细胞ＤＮＡ及Ⅰ型和Ⅱ型单纯疤疹病毒ＤＮＡ无交叉反应。     

Cryomicroscopy of human cytomegalovirus virions reveals more densely packed genomic DNA than in herpes simplex virus type 1

All members of the herpesvirus family have a characteristic virion structure, comprising a DNA containing, icosahedral capsid, embedded in a proteinaceous layer (tegument) and surrounded by a lipid envelope. Human cytomegalovirus (HCMV, the prototypic beta-herpesvirus) has a genome that is significantly larger (>50 %) than that of the alpha-herpesvirus HSV-1. Although the internal volume of the HCMV capsid is approximately 17 % larger than that of HSV-1, this slight increase in volume does not provide adequate space to encapsidate the full length HCMV genome at the same packing density as HSV-1.We have investigated the nature of DNA packing in HCMV and HSV-1 virions by electron-cryomicroscopy and image processing. Radial density profiles calculated from projection images of HCMV and HSV-1 capsids suggest that there is no increase in the volume of the HCMV capsid upon DNA packaging. Packing density of the viral DNA was assessed for both HCMV and HSV-1 by image analysis of both full and empty particles. Our results for packing density in HSV-1 are in good agreement with previously published measurements, showing an average inter-layer spacing of approximately 26 A. Measurements taken from our HCMV images, however, suggest that the viral genomic DNA is more densely packed, with an average inter-layer spacing of approximately 23 A. We propose therefore, that the combination of greater volume in HCMV capsids and increased packing density of viral DNA accounts for its ability to encapsidate a large genome.     

Infection of Vascular Endothelial Cells with Human Cytomegalovirus under Fluid Shear Stress Reveals Preferential Entry and Spread of Virus in Flow Conditions Simulating Atheroprone Regions of the Artery

Atherosclerosis is a major pathogenic factor in cardiovascular diseases, which are the leading cause of mortality in developed countries. While risk factors for atherosclerosis tend to be systemic, the distribution of atherosclerotic plaques within the vasculature is preferentially located at branch points and curves where blood flow is disturbed and shear stress is low. It is now widely accepted that hemodynamic factors can modulate endothelial gene expression and function and influence the pathophysiological changes associated with atherosclerosis. Human cytomegalovirus (HCMV), a ubiquitous pathogen, has long been proposed as a risk factor for atherosclerosis. To date, the role of HCMV in atherogenesis has been explored only in static conditions, and it is not known how HCMV infection is influenced by the physiological context of flow. In this study, we utilized a parallel-plate flow system to simulate the effects of shear stresses in different regions of the vasculature in vitro. We found that endothelial cells cultured under low shear stress, which simulates the flow condition of atheroprone regions in vivo, are more permissive to HCMV infection than cells experiencing high shear stress or static conditions. Cells exposed to low shear stress show increased entry of HCMV compared to cells exposed to high shear stress or static conditions. Viral structural gene expression, viral titers, and viral spread are also enhanced in endothelial cells exposed to low shear stress. These results suggest that hemodynamic factors modulate HCMV infection of endothelial cells, thus providing new insights into the induction/acceleration of atherosclerosis by HCMV.     

肾移植术后巨细胞病毒pp65检测的临床意义

目的探讨肾移植受者术后,巨细胞病毒被膜磷蛋白pp65的检测在活动性巨细胞病毒感染中的意义。方法用间接免疫荧光法检测肾移植受者术后HCMV-pp65,同时用酶联免疫捕获法检测HCMV-IgM抗体,共采集91份血标本。结果 91份血标本中,HCMV-pp65阳性24份（26.4%）,HCMV-IgM抗体阳性4份（4.4%）,在24例HC-MV-pp65抗原血症阳性的患者中,有20例出现HCMV感染症状及HCMV病。结论 HCMV-pp65在活动性巨细胞病毒感染的检测中具有早期、准确的优点,可辅助临床对HCMV感染进行早期诊断与治疗。     

Human cytomegalovirus end-organ disease is associated with high or low systemic viral load in preemptively treated solid-organ transplant recipients

Human cytomegalovirus (HCMV) end-organ disease in solid-organ transplant recipients (SOTR) may be associated with either high or low HCMV load in blood. In transplantation Centers where the preemptive therapy approach is adopted, antiviral therapy of systemic HCMV infections is initiated upon reaching pre-determined cut-off levels of viral DNA in blood, whereas no guidelines are provided for local end-organ infection/disease. In the latter case, clinicians often start antiviral treatment without defining the etiology of local symptoms. Here, we describe 14 cases of SOTR, in which a documented HCMV end-organ disease was observed. Nine patients had a systemic viral load lower than the cut-off for preemptive therapy and were treated based on viral load of local HCMV disease. The remaining five patients had a systemic viral load greater than the preemptive therapy cut-off and were efficiently treated for both the systemic and the local HCMV disease. Thus, HCMV infection in the post-transplant period must be monitored virologically both in blood and locally. End-organ disease in preemptively treated patients, seems to be associated with lack of development (primary HCMV infection) or reconstitution (reactivated infection) of HCMV-specific CD4+ and CD8+ T-cell immunity or with its functional impairment.     

Management of human cytomegalovirus infection in transplantation: validation of virologic cut-offs for preemptive therapy and immunological cut-offs for protection

Human cytomegalovirus (HCMV) still causes major viral complications in the post-transplant period of both solid-organ (SO) and hematopoietic stem cell (HSC) transplant (T) recipients (R). Diagnosis of HCMV infection is mostly made by real-time PCR-based methodologies, which allow quantification of viral DNA in both blood and, if required, organ tissues or local secretions. HCMV infection/disease can be prevented by either universal prophylaxis or preemptive therapy. The latter approach has mostly been used in European Transplantation Centers upon reaching predetermined cut-off levels of viral load, predictive of high risk for HCMV disease. In our Department, these cut-offs are higher for SOTR (3x105 DNA copies/ml whole blood) and lower for HSCTR (3x104 DNA copies/ml). Antiviral therapy is continued until viral DNA disappearance from blood or tissues. However, the authentic long-term control of HCMV infection is achieved when HCMV-specific CD4+ and CD8+ T-cells are detected in blood or tissues. Proposed immunological cut-off levels conferring protection are: one HCMV-specific CD4+ and three CD8+ T-cells/ml blood for HSCTR, and 0.4 HCMV-specific T-cells/ml for both CD4+ and CD8+ in SOTR. However, anti-rejection in SOTR and anti- GvHD in HSCTR steroid therapies make patients susceptible to HCMV infection, even in the presence of protective levels of specific T-cells.     

Use of specific-pathogen-free (SPF) rhesus macaques to better model oral pediatric cytomegalovirus infection

Background Congenital human cytomegalovirus (HCMV) infection can result in lifelong neurological deficits. Seronegative pregnant woman often acquire primary HCMV from clinically asymptomatic, but HCMV‐shedding children. Methods Potential age‐related differences in viral and immune parameters of primary RhCMV infection were examined in an oral rhesus CMV infection model in specific pathogen free macaques. RhCMV shedding was measured by real time PCR in plasma, saliva and urine. Immune parameters, including neutralizing and binding antibodies and RhCMV‐specific T cell responses, were assessed in longitudinally collected blood samples. Results The oral RhCMV infection model in infant SPF rhesus macaques demonstrated that (i) the susceptibility to oral RhCMV infection declines with age, and (ii) infant macaques shed RhCMV more persistently and at higher titers compared to adult macaques. (iii) Conclusions The oral infant RhCMV infection model appears to reflect viral pathogenesis in human HCMV‐infected children. Larger studies are needed to define immune parameters associated with better control of RhCMV in adult compared to young animals.     

Serum and cerebrospinal fluid herpes simplex virus type 1 immunoglobulin G and M titers in four cases of herpes simplex encephalitis

Herpes simplex virus type 1 (HSV-1) IgG and IgM ELISA titers were serially determined in serum and cerebrospinal fluid (CSF) samples from 4 patients with HSV-1 encephalitis during a follow-up period of 1-26 months. In 3 out of 4 patients HSV-1 IgM titers raised in CSF during the acute phase of disease, thus allowing differentiation between primary and reactivated forms of HSV-1 encephalitis. HSV-1 IgG titers showed a sharp elevation earlier in serum than in CSF. Specific IgG index documented a large intrathecal production of HSV-1 IgG and their persistence 2 years following clinical onset. The initial trend of serum and CSF specific IgG titer represents a reliable tool for a retrospective diagnosis of HSV-1 encephalitis.     

Chronic infections and genetic factors in the development of ischemic stroke

The aim of this study was to examine whether chronic infections and genetic factors of the host play roles in the pathophysiology of acute noncardioembolic ischemic stroke. Blood samples from 59 subjects with ischemic stroke and 52 control patients were investigated by nested PCR for the presence of C. pneumoniae DNA, HCMV DNA and enterovirus RNA, by ELISA for the levels of antibodies to C. pneumoniae, HCMV, HSV, HHV-6, EBV and the inflammatory chemokine IL-8, and by PCR for promoter polymorphism of the IL-8 and CD14 host genes. Associations of stroke with the HCMV IgG and HSV-1 IgA antibody levels were observed. No association of stroke was detected with the presence of C. pneumoniae, HCMV or enterovirus nucleic acids in the peripheral blood, C. pneumoniae IgM, IgG and IgA, the HSV IgG, the EBV IgG, or HHV-6 IgG antibody levels, the pathogen burden, the IL-8 or CD14 promoter polymorphisms, or with the serum levels of IL-8 in the overall study population. These results are consistent with the hypothesis that certain pathogens are involved in the development of ischemic stroke.     

Anti-cytomegalovirus IgG antibody titer is positively associated with advanced T cell differentiation and coronary artery disease in end-stage renal disease

Background

Accumulating evidence indicates that persistent human cytomegalovirus (HCMV) infection is associated with several health-related adverse outcomes including atherosclerosis and premature mortality in individuals with normal renal function. Patients with end-stage renal disease (ESRD) exhibit impaired immune function and thus may face higher risk of HCMV-related adverse outcomes. Whether the level of anti-HCMV immune response may be associated with the prognosis of hemodialysis patients is unknown.

Results

Among 412 of the immunity in ESRD study (iESRD study) participants, 408 were HCMV seropositive and were analyzed. Compared to 57 healthy individuals, ESRD patients had higher levels of anti-HCMV IgG. In a multivariate-adjusted logistic regression model, the log level of anti-HCMV IgG was independently associated with prevalent coronary artery disease (OR?=?1.93, 95% CI?=?1.2~?3.2, p =?0.01) after adjusting for age, sex, hemoglobin, diabetes, calcium phosphate product and high sensitivity C-reactive protein. Levels of anti-HCMV IgG also positively correlated with both the percentage and absolute number of terminally differentiated CD8+ and CD4+?CD45RA+?CCR7- T_EMRA cells, indicating that immunosenescence may participate in the development of coronary artery disease.

Conclusion

This is the first study showing that the magnitude of anti-HCMV humoral immune response positively correlates with T cell immunosenescence and coronary artery disease in ESRD patients. The impact of persistent HCMV infection should be further investigated in this special patient population.

     

Inhibition of calmodulin-dependent kinase kinase blocks human cytomegalovirus-induced glycolytic activation and severely attenuates production of viral progeny

Viruses depend on the host cell to provide the energy and biomolecular subunits necessary for production of viral progeny. We have previously reported that human cytomegalovirus (HCMV) infection induces dramatic changes to central carbon metabolism, including glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and nucleotide biosynthesis. Here, we explore the mechanisms involved in HCMV-mediated glycolytic activation. We find that HCMV virion binding and tegument protein delivery are insufficient for HCMV-mediated activation of glycolysis. Viral DNA replication and late-gene expression, however, are not required. To narrow down the list of cellular pathways important for HCMV-medicated activation of glycolysis, we utilized pharmaceutical inhibitors to block pathways reported to be both involved in metabolic control and activated by HCMV infection. We find that inhibition of calmodulin-dependent kinase kinase (CaMKK), but not calmodulin-dependent kinase II (CaMKII) or protein kinase A (PKA), blocks HCMV-mediated activation of glycolysis. HCMV infection was also found to target calmodulin-dependent kinase kinase 1 (CaMKK1) expression, increasing the levels of CaMKK1 mRNA and protein. Our results indicate that inhibition of CaMKK has a negligible impact on immediate-early-protein accumulation yet severely attenuates production of HCMV viral progeny, reduces expression of at least one early gene, and blocks viral DNA replication. Inhibition of CaMKK did not affect the glycolytic activation induced by another herpes virus, herpes simplex virus type 1 (HSV-1). Furthermore, inhibition of CaMKK had a much smaller impact on HSV-1 replication than on that of HCMV. These data suggest that the role of CaMKK during the viral life cycle is, in this regard, HCMV specific. Taken together, our results suggest that CaMKK is an important factor for HCMV replication and HCMV-mediated glycolytic activation.     

Human cytomegalovirus downregulates expression of receptors for platelet-derived growth factor by smooth muscle cells

Infection by human cytomegalovirus (HCMV) is associated with the development of vascular diseases and may cause severe brain damage in infected fetuses. Platelet-derived growth factor receptors alpha and beta (PDGFR-α and -β) control important cellular processes associated with atherosclerosis and fetal development. In the present investigation, our goal was to determine whether infection by HCMV can influence the expression of PDGFR-α and -β in human smooth muscle cells (SMCs). In connection with HCMV infection in vitro the levels of PDGFR-α and -β at the cell surface and in the total cellular protein of SMCs were reduced in parallel with decreases in the levels of the corresponding mRNAs. These effects were dependent on immediate-early (IE) or early (E) HCMV gene products, since inhibition of late genes did not prevent HCMV from affecting the expression of PDGFR-α and -β. The downregulation of PDGFR caused by HCMV was dose dependent. Furthermore, confocal microscopy revealed that the localization of PDGFR-β was altered in HCMV-infected cells, in which this protein colocalized with proteins associated with endosomes (Rab4 and -5) and lysosomes (Lamp1 and -2), indicating entrance into pathways for protein degradation. Altogether these observations indicate that an IE and/or E HCMV protein(s) downregulates the expression of PDGFR-α and -β in SMCs. This phenomenon may disrupt cellular processes of importance in connection with cellular differentiation, migration, and/or proliferation. These observations may explain why congenital infection with HCMV can cause fetal brain damage.     

人类巨细胞病毒感染与急性冠脉综合症患者血清炎症介质的相关性研究

目的:分析人类巨细胞病毒(HCMV)感染与急性冠脉综合症(ACS)患者炎症介质的相关性,探讨HCMV感染在ACS发生、发展过程中的作用。方法:选取我院2017年5月~2019年5月收治的冠心病患者118例,根据病情将其分为ACS组(n=81)和稳定型心绞痛(SAP)组(n=37),另选取同时期在我院进行健康检查的健康志愿者40例作为对照组。检测所有受试者血清特异性HCMV-Ig G、HCMV-Ig M,比较所有受试者血清sP-选择素(sP-selectin)、肿瘤坏死因子-α(TNF-α)及超敏C-反应蛋白(hs-CRP)水平。分析ACS组患者血清sP-selectin、TNF-α、hs-CRP水平与HCMV-Ig M抗体滴度的相关性。结果:ACS组、SAP组的HCMV-Ig G阳性率分别为81.48%、78.38%,均明显高于对照组的45.00%,差异有统计学意义(P<0.05)。ACS组的HCMV-Ig M阳性率为40.74%,明显高于SAP组的10.81%和对照组的5.00%,差异有统计学意义(P<0.05)。ACS组患者血清sP-selectin、TNF-α及hs-CRP水平均明显高于SAP组和对照组,差异有统计学意义(P<0.05)。ACS组HCMV-Ig M阳性患者血清sP-selectin、TNF-α及hs-CRP水平均明显高于HCMV-Ig M阴性患者,差异有统计学意义(P<0.05)。ACS组患者血清sP-selectin、TNF-α、hs-CRP水平与HCMV-Ig M抗体滴度均呈正相关(P<0.05)。结论:慢性HCMV感染可能在动脉粥样硬化的发生及发展中起着重要作用,而急性HCMV感染可能通过上调机体sP-selectin、TNF-α、hs-CRP等炎症因子水平,进一步促进ACS的发生发展。