Cpn DNA Research Articles

The Detection of Chlamydia pneumoniae, Helicobacter pylori and Cytomegalovirus in Non-Atherosclerotic Arteries of Patients with Coronary Artery Disease

Atherosclerotic coronary artery disease (ACAD) is a major cause of global morbidity and mortality, characterized as an inflammatory process due to damage to blood vessel walls by risk factors like aging, hyperlipidemia, hypertension, smoking, and diabetes. Infectious agents, including Chlamydia pneumoniae (Cpn), Cytomegalovirus (CMV), and Helicobacter pylori (HP), have been implicated in ACAD’s pathophysiology. A study with 56 subjects undergoing coronary artery bypass grafting (CABG) aimed to detect Cpn, CMV, and HP DNA in unaffected artery segments and explore associations with disease progression and inflammation markers. The study found infectious agents’ DNA in 21.4% of samples, HP in eight samples, and CMV and Cpn in four samples each. Significant correlations were observed between HP and overweight or obese subjects, as well as between the presence of infectious agents and inflammation marker values. An association between HP and renal function was also noted. The findings reaffirm previous discoveries of infectious agents in non-clinically affected arteries used as CABG grafts. Correlations identified between the presence of HP, CMV, and Cpn DNA in grafts and several biomarkers of inflammation and obesity emphasize the potential role of these infectious agents in ACAD pathogenesis.

Quantification of Chlamydia pneumoniae and amyloid plaques within the limbic pathway related to late‐onset dementia/Alzheimer’s

This study aims to quantitate Chlamydia pneumoniae (Cpn) and Aβ amyloid plaques throughout the limbic system in brains from individuals diagnosed with late‐onset dementia/Alzheimer’s disease (AD) and non‐demented conditions. The amount of deposition of amyloid relative to Cpn may further implicate Chlamydia’s role as a trigger for neuroinflammation leading to the pathology observed in late‐onset disease.Sporadic late‐onset Alzheimer’s disease (LOAD) is the most common form of dementia, affecting ~55 million individuals worldwide. Despite the prevalence of the disease the etiology of LOAD remains unknown. In recent years, significant attention has been given to the role of infection in the onset and pathogenesis of late‐onset dementia/AD. The Cpn pathogen has been an organism of particular focus since the seminal study by Balin et. al 1998 which found that Cpn DNA was present in 90% of brain samples taken from patients diagnosed with LOAD. Subsequent immunohistochemical (IHC) studies demonstrated that Cpn antigens were present in the frontal and temporal cortices of LOAD brains and that Aβ amyloid plaques found in those brain regions co‐localize with areas of Cpn immunoreactivity (Hammond et al 2010).Sections of the hippocampus and the pre‐frontal cortex were dissected from the human cadaver brains that had been donated to the Pennsylvania Human Gifts Registry. The brains were paraffin embedded and sectioned for immunostaining. The brains selected were from those whose death certificates indicated dementia/AD or non‐dementia diagnosed with cardiovascular disease. Five sections from the hippocampus and the frontal cortex were immunolabeled with specific antibodies for amyloid plaques and Chlamydial inclusions. The proteins were detected using Abcam’s IHC detection kit and the slides were scanned at 40x magnification. The Cpn inclusions and amyloid plaques were counted in each region using the equivalent total area/section and total count was calculated per mm2.The counts for amyloid deposits in the anterior hippocampal region of the dementia/AD brain revealed on average 84/mm2 and 117/mm2 Chlamydial inclusions. The counts in the frontal cortex of the dementia/AD brain revealed 266/mm2 and 70/mm2 Chlamydial inclusions. The counts in the non‐dementia brain were considerably less for amyloid deposits in the anterior hippocampal region and the frontal cortex averaging between 40/mm2 and 49/mm2, respectively. The Chlamydial inclusion counts were also lower as compared to the dementia/AD brain in the anterior hippocampal region and the frontal cortex averaging 17/mm2 and 14/mm2, respectively.Brain tissues from the Human Gifts registry provide a valuable resource for experimental analysis. This preliminary study yielded data of an association between amyloid pathology and infection with Cpn from both demented and non‐demented aged brains. These data help to support prior work demonstrating a relationship between pathogen association and pathology that may correlate to late‐onset disease.

Systematic Infection of Chlamydia Pneumoniae.

Chlamydia pneumoniae (Cpn) is one of the most common respiratory pathogens in children and adults. It is characterized as an obligate intracellular parasite. Peripheral blood monocytes (PBMC), lymphocytes, and macrophages are involved in spreading chlamydia infection to extrapulmonary organs indicating that Cpn infection can cause systematic symptoms in vivo via blood transmission. This review summarizes the mechanisms of Cpn infection in host cells, the immune response of the body, and the relationship between Cpn infection and some chronic diseases. Cpn participation in extrapulmonary chronic diseases has been proven owing to the presence of Cpn DNA in AS plaque, nerve tissues, and synovium tissues of the joints. Cpn infection is related to the development of chronic diseases such as atherosclerosis, Alzheimer's Disease (AD), and reactive arthritis through in vivo and in vitro experiments.

Cell-mediated immune response associated with Chlamydia pneumoniae infection in atherosclerotic patients

BackgroundChlamydia pneumoniae is an obligate intracellular bacterium that activates cell mediated immune responses; several investigations have demonstrated its strong implication in atherosclerosis. ObjectivesThe main objective of our study was to explore the cell-mediated immune response to C. pneumoniae infection in patients with atherosclerosis by evaluating CD14, CD8 and CD4 expression. MethodsThis investigation involved a total of 27 patients with atherosclerosis and 32 controls, among patients recruited to evaluate the association of C. pneumoniae with atherosclerosis. C. pneumoniae DNA was detected in PBMCs by nested PCR as described in our previous studies. CD4, CD8 and CD14 expression was measured by flow cytometry and data analysis was performed using FlowJo software. ResultsThe results revealed an increase in MFI expression of CD4, CD8 and CD14 in Cpn DNA+ subjects among both patients and healthy subject controls (CD4 Cpn DNA+ = 829.11 vs. CD4 Cpn DNA- = 571.14; CD8 Cpn DNA+ = 1562 vs. CD8 Cpn DNA- = 699; CD14 Cpn DNA+ = 1513.83 vs. CD14 Cpn DNA- = 1170.70), with a statistically significant difference (p < 0.05). Furthermore, the comparison of CD4, CD8 and CD14 expression between Cpn DNA+ patients and Cpn DNA+ healthy subject controls showed a statistically significant increase in expression in the former group (p < 0.05). ConclusionThese data provide incentive to further explore the role of C. pneumoniae in stimulating and changing mechanisms of the cell-mediated immune response induced by C. pneumoniae antigens. This may alter immune cell-mediated responses via increased expression of CD4, CD8 and CD14 during inflammation and the development of thrombosis, leading to fatal atherosclerosis.

An improved method on isolation and serial passage of Chlamydia pneumoniae from human peripheral blood mononuclear cells.

Conventional method for Chlamydia pneumoniae (Cpn) isolation and propagation is technically challenging and time-consuming. Here, we developed a method to improve the isolation and passage of Cpn collected from human peripheral blood mononuclear cells (PBMCs). PBMCs positive with Cpn antigen (Cpn-Ag) were isolated, then centrifuged and cultured with Hep-2 cells after being broken. Cells were broken again and put into new Hep-2 cells to finish totally four passages with isolated and imported Cpn. Microimmunofluorescence method was used to detect Cpn. Inclusion forming unit (IFU) number was counted for each passage. Polymerase chain reaction (PCR) method was used to detect Cpn DNA. Efficiency of different centrifugation modes was compared. Hep-2 cells of the first and second passages were strong positive with Cpn-Ag, the third passage was positive, and the fourth negative. Degeneration appeared in the fourth passage for isolated Cpn and third passage for imported strain. Centrifugation mode of 1,000 rpm for 2 h was the most efficient for Cpn propagation and passage. This simplified method achieved efficient isolation, propagation, and passage of Cpn from PBMCs, and isolated strain was superior to imported strain on propagating ability.

Early atherosclerotic plaques show evidence of infection by Chlamydia pneumoniae

Chlamydia pneumoniae (Cpn) could play an important role in the development of atherosclerosis. Cpn interferes with HIF-1alpha regulation in infected host cells during intracellular replication in hypoxia. We obtained carotid artery specimens with low (n=38), high (n=25) levels of stenosis and 10 middle cerebral arteries. Fifty eight percent of the carotids with low levels of stenosis showed evidence of the viable organism. Ninety one percent of the positive results were derived from pre-atheromatous lesions. Only 12 percent of plaques removed at endarterectomy showed the presence of Cpn DNA. All middle cerebral arteries failed to show evidence of live Chlamydia. Ninety one percent of sera from 22 endarterectomy patients failed to show the presence of Cpn antibodies. Immunohistology of carotid arteries with low levels of stenosis was used to confirm the presence of HIF-1alpha in infected specimens and showed a correlation between the over-expression of HIF-1alpha and Cpn in the plaque (p less than 0.05). Cpn might play an important role in activation and development of the initial stages of atherosclerotic lesions.

Analysis of CPn0308 Immunogenicity in the Infected Patients with CPn

Objectives To analyze CPn0308 immunogenicity in the CPn infected patients with ICVD. Methods ①Clinical blood samples and separate plasma from blood and extract the DNA from PBMC were collected. ②The CPn IgG and CPn IgM were detected by using CPn specified diagnose kit. ③CPn DNA from PBMC was assayed by using PCR.④Imunogenicity of the endogenous CPn0308 was analyzed by Western Blotting. Results①Among the 70 patients with ICVD, the positive rate of CPn IgG was 51.43%, and it was higher than that of control; The positive rate of CPn IgM was 2.86%, and that of control was 8.47%. ②The positive rate of CPn DNA in PBMC from patients with ICVD was 81.08%，and it was higher than that of control. ③After plasma samples were diluted as 1:500, the antibodies to endogenous CPn0308 were still detected by using Western Blotting. The reaction was blocked after the plasma was pre-absorbed with GST-CPn0308 beads ahead, but not pre-absorbed with GST-CPn0186 beads. Conclusion ①There is an increase in the positive rate of CPn IgG in the patients with ICVD. ②The positive rate of CPn DNA in PBMC from the patients with ICVD is higher than that of the control. The positive rate for CPn by PCR is higher than that of EIA, and CPn DNA detection is a more sensitive method. ③The studies preliminarily demonstrate that CPn0308 has immunogenicity in the individual infected with CPn.

Chronic infections and histamine, CRP and IL-6 levels after percutaneous transluminal coronary angioplasty

Our hypothesis was that percutaneous transluminal coronary angioplasty (PTCA) reactivates certain pathogens that contribute to inflammatory processes after the intervention. We determined the levels of antibodies to human Hsp60 and levels of histamine, CRP and IL-6 in sera from 28 patients of unstable angina prior to and on days 4 and 14 after PTCA. We compared the presence of Chlamydophila pneumoniae (Cpn) and human cytomegalovirus (HCMV) DNA in peripheral blood, and levels of antibodies to Cpn, HCMV, herpes simplex virus, Epstein-Barr virus and mycobacterial Hsp65 in the serum. Higher prevalence of Cpn and HCMV DNA was demonstrated after PTCA than before, but titers of antibodies to the pathogens did not increase. Levels of histamine, CRP and IL-6 were enhanced after PTCA. There was no association between the levels of histamine, CRP and IL-6 and the rate of pathogen DNA, or antibody titers to the pathogens, except an association between Cpn IgA and histamine levels before PTCA. Reactivation of Cpn and HCMV and inflammatory change characterized by increased levels of histamine, CRP and IL-6 following PTCA are suggested. An association might exist between Cpn IgA antibody and histamine levels in patients of unstable angina.

Detection of amyloid beta aggregates in the brain of BALB/c mice after Chlamydia pneumoniae infection

Neuroinflammation, initiated by cerebral infection, is increasingly postulated as an aetiological factor in neurodegenerative diseases such as Alzheimer’s disease (AD). We investigated whether Chlamydia pneumoniae (Cpn) infection results in extracellular aggregation of amyloid beta (Aβ) in BALB/c mice. At 1 week post intranasal infection (p.i.), Cpn DNA was detected predominantly in the olfactory bulbs by PCR, whereas brains at 1 and 3 months p.i. were Cpn negative. At 1 and 3 months p.i., extracellular Aβ immunoreactivity was detected in the brain of Cpn-infected mice but also in the brain of mock-infected mice and mice that were neither Cpn infected nor mock infected. However, these extracellular Aβ aggregates showed morphological differences compared to extracellular Aβ aggregates detected in the brain of transgenic APP751SL/PS1M146L mice. These data do not unequivocally support the hypothesis that Cpn infection induces the formation of AD-like Aβ plaques in the brain of BALB/c mice, as suggested before. However, future studies are required to resolve these differences and to investigate whether Cpn is indeed an etiological factor in AD pathogenesis.

Chlamydia Pneumoniae Infection Is Not Associated With Chronic Transplant Dysfunction in a Rat Aortic Allograft Model

Long-term survival of solid-organ transplants is limited as a result of chronic transplant dysfunction (CTD), which is characterized by occlusion of intragraft vascular tissue due to myointimal hyperplasia. Recent studies have shown a role for infections in vascular pathologies. For example, Chlamydia pneumoniae ( Cpn) has been shown to aggravate atherosclerosis, and Cpn immunoglobulin (Ig)G titers correlate with severity of allograft atherosclerosis after cardiac transplantation. In this study, we evaluated the effect of Cpn infection on CTD using a rat aortic allograft model. Orthotopic abdominal aorta transplantations (Tx) were performed with Brown Norway rats as donors and Lewis rats as recipients. Rats were humanely killed at 1 or 8 weeks after surgery. The graft was processed for DNA isolation and histological examination. Influx of macrophages and T cells was assessed using immunohistochemistry. At 1 week after Tx, the perivascular influx of inflammatory cells in the graft was not affected by Cpn infection. Furthermore, only limited numbers of Cpn DNA copies were found in the graft at 1 week after Tx. In addition, Cpn did not alter the severity of myointimal hyperplasia in the rat aortic allograft model at 8 weeks after surgery. Our data suggested that, in the rat aortic allograft model, Cpn infection did not influence the influx of inflammatory cells or the severity of CTD.

Chlamydia pneumoniae infection and the risk of multiple sclerosis: a meta-analysis

We conducted a meta-analysis of studies comparing the presence of Chlamydia pneumoniae (Cpn) between multiple sclerosis (MS) patients and other neurological diseases patients or healthy controls. We identified 26 studies with 1332 MS patients and 1464 controls. Using random-effects methods, MS patients were found more likely to have detectable levels of Cpn DNA (OR = 3.216; 95% CI: 1.204, 8.585) in their cerebrospinal fluid, and intrathecally synthesized immunoglobulins (OR = 3.842; 95% CI: 1.317, 11.212), compared to other patients with neurological diseases. There is no evidence for increased levels of serum immunoglobulins (OR = 1.068; 95% CI: 0.745, 1.530), even though this result is confounded by the presence of studies using normal subjects as controls. Similarly, there is no evidence for association of immunoglobulins against Cpn in the cerebrospinal fluid (OR = 3.815; 95% CI: 0.715, 20.369). Up to 59.7% of the between-studies variability could be explained by the inappropriate matching of cases and controls for gender. In random-effects meta-regressions, adjusting for the confounding effect of gender differences results in stronger and statistically significant associations of MS with detectable levels of Cpn DNA, intrathecally synthesized immunoglobulins and immunoglobulins in the cerebrospinal fluid. Even though the presence of Cpn is clearly more likely in MS patients, these findings are insufficient to establish an etiologic relation.

Chlamydia pneumoniae infection of brain cells: An in vitro study

Inspired by the suggested associations between neurological diseases and infections, we determined the susceptibility of brain cells to Chlamydia pneumoniae (Cpn). Murine astrocyte (C8D1A), neuronal (NB41A3) and microglial (BV-2) cell lines were inoculated with Cpn. Infection was established by immunofluorescence and real-time PCR at various time points. Productive infection was assessed by transferring medium of infected cells to a detection layer. Finally, apoptosis and necrosis post-infection was determined. Our data demonstrate that the neuronal cell line is highly sensitive to Cpn, produces viable progeny and is prone to die after infection by necrosis. Cpn tropism was similar in an astrocyte cell line, apart from the higher production of extracellular Cpn and less pronounced necrosis. In contrast, the microglial cell line is highly resistant to Cpn as the immunohistochemical signs almost completely disappeared after 24 h. Nevertheless, significant Cpn DNA amounts could be detected, suggesting Cpn persistence. Low viable progeny and hardly any necrotic microglial cells were observed. Further research is warranted to determine whether these cell types show the same sensitivity to Cpn in an in vivo setting.

Are Chlamydia pneumoniae and Mycoplasma pneumoniae important etiological agents of respiratory tract infections in a family practice setting?

Rationale A study was conducted over a 4-month winter period within a family practice in Madison WI to identify whether C. pneumoniae (Cpn) and M. pneumoniae (Mpn) are common etiological agents of respiratory tract infections (RTI). Methods Twenty patients diagnosed by clinical criteria to have either acute or chronic RTI and 27 healthy control subjects were tested in this study. Nasopharyngeal swab specimens and sera were obtained from both subject populations and oral washes were obtained only from those patients diagnosed with RTI. DNA extracted from nasopharyngeal swab specimens and oral washes were analyzed with species-specific quantitative real-time PCR (qPCR) assays that previously were validated with both dosed and patient positive nasopharyngeal swabs. The qPCR results were then compared to serological data acquired with commercially available Mpn and Cpn IgM kits. Results All respiratory samples analyzed with qPCR assays were negative for Cpn and Mpn DNA. The prevalence of Cpn- and Mpn-specific IgM antibodies was found to be 1/20 (5%) and 5/20 (25%) for patients diagnosed with RTI and 2/27 (7%) and 1/27 (4%) for healthy control subjects. Conclusions Analysis of the non-invasively obtained respiratory samples with qPCR indicates that active infection with Cpn and Mpn are infrequent within the patient population investigated here. The serological evidence does indicate that the prevalence of Mpn infection may be higher in the clinical cases than in controls. In light of this data, other sampling methods might be necessary for the routine detection of Cpn and Mpn by qPCR especially in cases where antibiotic therapy has been initiated.

Chlamydia pneumoniae infection induces an unstable atherosclerotic plaque phenotype in LDL-receptor, ApoE double knockout mice

Objectives: to study whether Chlamydia pneumoniae (Cpn) infection affects atherosclerotic plaque morphology in atherogenic (LDLr/ApoE−/−) mice. Methods: in mice sacrificed 20 or 40 weeks after Cpn infection aortic arch sections were analysed for lesion and fibrous cap area and the presence of matrix metalloproteinases (MMP)-2 and -9. Results: all infected mice seroconverted, demonstrated Cpn DNA in their aortas on PCR and developed atherosclerotic plaques. Infection was not associated with changes in lesion area or type, but was associated with reduced the fibrous cap area and increased MMP-2 and -9 immunoreactivity. Conclusion: these findings suggest that Cpn infection may predispose to plaque instability.Eur J Vasc Endovasc Surg 26, 88-95 (2003)

Absence of Chlamydia pneumoniae in brain of vascular dementia patients

We recently detected cytomegalovirus (CMV) in brains of 83% of vascular dementia (VaD) patients and 34% of age-matched normal people. Since CMV and also Chlamydia pneumoniae (Cpn) have been found in some studies to be associated with coronary artery disease (which shares several risk factors with VaD), we sought Cpn DNA in VaD brain DNA. We examined brain specimens from 19 VaD patients, 16 elderly normal people and four Alzheimer’s disease (AD) patients for the presence of a sequence in the Cpn gene for rRNA, using polymerase chain reaction (PCR) and taking stringent precautions against contamination. We did not detect Cpn DNA in any of the brain specimens, the sensitivity of detection being 10 copies or fewer bacterial DNA sequences per tube or, in terms of infectious units (IFU), 0.025 IFU. Our results do not support a role for Cpn in the aetiology of VaD, either in the 83% of patients in whose brains we detected CMV, or in the remaining 17% without CMV in brain.

An association between chronic infection with Chlamydia pneumoniae and lung cancer. A prospective 2-year study.

This study assesses a possible relationship between chronic Chlamydia pneumoniae (Cpn) infection and lung cancer (LC). A total of 210 consecutive patients (136 M, 74 F) were diagnosed with LC during a 2-year period. Blood was obtained from 128 M and 70 F patients for Cpn serology. Repeat blood specimens were taken after 3 months. Throat specimens for Cpn DNA analysis by PCR were taken from 110/136 M and 63/74 F Seventy-four cytobrush specimens were taken and also analyzed by polymerase chain reaction (PCR). Fifty (29 M, 21 F) bronchial biopsies and 8 (6 M, 2 F) tumors resected at surgery were analyzed for Cpn by immunohistochemistry (IHC). Males had significantly more often squamous-cell carcinoma (SCC) than females. Other types of LC were more equally distributed between males and females. The difference between males and females regarding smoking history was significant, and male LC patients had significantly higher levels of IgG and/or IgA antibodies than female LC patients. Male and female LC patients had significantly higher prevalences of high antibody titers than controls. A high prevalence of unusually high titers of specific Cpn antibodies was found in male LC patients. This could indicate that LC may be induced by chronic Cpn infection, since stable high titers of Cpn antibodies, especially IgA, are a hallmark of chronic infections.