Failure to detect Chlamydia pneumoniae in the late-onset Alzheimer's brain

Epidemiological studies have yet to identify a single cause for the most common late-onset form of Alzheimer's disease. The common respiratory pathogen Chlamydia pneumoniae recently has been implicated as a risk factor for this form of Alzheimer's disease. Were this true, there would be a dramatic shift in current paradigms of Alzheimer's disease research and treatment. In the absence of published confirmation, we obtained postmortem brain tissue from late-onset Alzheimer's disease patients (n = 15) and representative controls (n = 5) and extracted DNA from up to six separate brain regions in each instance, including those areas particularly relevant to Alzheimer's disease neuropathology. Each sample of DNA (n = 101) was assayed five times or more for the presence of C. pneumoniae DNA using a nested-PCR protocol targeting a species-specific gene sequence coding for the major outer membrane protein of this organism. We were unable unequivocally to detect C. pneumoniae in any of the 101 samples tested by PCR and failed to culture the organism from tissue samples. We conclude that C. pneumoniae is neither strongly nor uniquely associated with the neuropathology seen in late-onset Alzheimer's disease.     

Epithelial Cells Infected with Chlamydophila pneumoniae (Chlamydia pneumoniae) Are Resistant to Apoptosis

The obligate intracellular pathogen Chlamydophila pneumoniae (Chlamydia pneumoniae) initiates infections in humans via the mucosal epithelia of the respiratory tract. Here, we report that epithelial cells infected with C. pneumoniae are resistant to apoptosis induced by treatment with drugs or by death receptor ligation. The induction of protection from apoptosis depended on the infection conditions since only cells containing large inclusions were protected. The underlying mechanism of infection-induced apoptosis resistance probably involves mitochondria, the major integrators of apoptotic signaling. In the infected cells, mitochondria did not respond to apoptotic stimuli by the release of apoptogenic factors required for the activation of caspases. Consequently, active caspase-3 was absent in infected cells. Our data suggest a direct modulation of apoptotic pathways in epithelial cells by C. pneumoniae.     

Chlamydophila (Chlamydia) pneumoniae induces histidine decarboxylase production in the mouse lung

Chlamydophila (Chlamydia) pneumoniae (C. pneumoniae) is the third most common cause of community-acquired pneumonia and is probably involved in the development of certain chronic inflammatory diseases, including atherosclerosis and adult-onset asthma. Histamine, synthesized by histidine decarboxylase (HDC) from L-histidine, plays an essential role in allergic and inflammatory processes and in cell differentiation. The effect of C. pneumoniae infection on the expression of HDC has not been examined. In the present study, normal Balb/c mice and HDC knockouts, and control mice with a CD1 background were infected intranasally with C. pneumoniae. On days 1, 3, 7, 16 and 31 after infection, the normal Balb/c mice were sacrificed and divided into three groups. In the homogenized lungs of the first group, C. pneumoniae titres were determined and demonstrated peak levels on day 7. HDC production was revealed by a Western blot assay throughout the observation period of 1-16 days, and cytokine concentrations were determined by ELISA. The interleukin-3 (IL-3) and interleukin-6 (IL-6) levels were highest on day 1 and on days 1-3, respectively; the interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels reached the maximum on day 7, but the quantity of IL-4 was still three times higher than that in the control group 16 days after infection. The lungs of the mice in the second group were processed for the in situ demonstration of HDC activity, while the lungs in the third group were stained for C. pneumoniae antigen. The HDC activity was increased predominantly in the bronchial epithelial cells, while C. pneumoniae antigens were expressed especially in the interstitial macrophages. The HDC knockout mice exhibited a higher survival rate after C. pneumoniae infection than did the control mice. These results point to a strong association between local histamine production and other inflammatory mediators and are novel in demonstrating the role of histamine in the pathomechanism of C. pneumoniae infections.     

Chlamydophila (Chlamydia) pneumoniae serology and asthma in adults: a longitudinal analysis

BACKGROUND: Many cross-sectional studies have found an association between Chlamydophila pneumoniae infection and asthma, and a possible causative role of C pneumoniae infection in asthma pathogenesis has been proposed. No longitudinal studies have been undertaken to estimate the effect on asthma incidence of previous or chronic infection. OBJECTIVE: We evaluated whether serological findings suggestive for recent or chronic C pneumoniae infection affect asthma risk or lung function during follow-up. METHODS: We followed a population-based adult cohort for 15 years and made a clinical evaluation of persons with new persistent asthma (n = 83) and matched controls (n = 162). Serological testing was performed by microimmunofluorescence and enzyme immunoassay from both baseline and follow-up samples. RESULTS: Subjects with serologically diagnosed recent or chronic C pneumoniae infection did not run a higher risk of new asthma. An increased risk was found in subjects with allergic rhinitis, low lung function, history of smoking, and positive family background of asthma or allergy. However, chronic C pneumoniae infection was found to accelerate the loss of lung function significantly in subjects who contracted new nonatopic asthma (median change in FEV(1), 89.6 vs 55.9 mL/y; P = .032). CONCLUSION: Chronic C pneumoniae infection promotes the development of airflow limitation in adults with nonatopic asthma. However, our results indicate that at the population level, any possible effect of C pneumoniae infection on asthma incidence is of minor significance.     

Apolipoprotein E4 enhances attachment of Chlamydophila (Chlamydia) pneumoniae elementary bodies to host cells

Chlamydophila (Chlamydia) pneumoniae is an intracellular respiratory pathogen known to cause community-acquired pneumonia. Infection with this organism has been associated with atherosclerosis, inflammatory arthritis, and other chronic diseases, many of which also have been associated with possession of the epsilon4 allele at the APOE locus on (human) chromosome 19. An earlier study from this laboratory suggested that some relationship exists between apolipoprotein E4 (apoE4), the product of the epsilon4 allele, and the pathobiology of C. pneumoniae. A standard attachment assay and real time PCR targeting a sequence on the C. pneumoniae chromosome were used to monitor host cell binding of elementary bodies (EB) of that organism. Our data indicate that 3-fold more EB of strain AR-39 attach to an epsilon3 homozygous human cell line transfected with a plasmid expressing the epsilon4 coding sequence than to the same cell line harboring empty vector, vector containing an irrelevant insert sequence, or vector containing the DNA sequence encoding apoE3. The quantitative real time data were confirmed by immunolabeling of chlamydial inclusions in parallel attachment and infection assays. Experiments using Chlamydophila trachomatis EB showed no enhancement of attachment in the presence of the epsilon4 allele in any assays. These observations indicate that apoE4 enhances attachment of C. pneumoniae EB, but not those of C. trachomatis, to target host cells.     

Analysis of children with Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae respiratoryinfections by real-time PCR assay and serological tests

We examined 73 children with respiratory infections for Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae using real-time PCR assay and serological tests. C. pneumoniae and M. pneumoniae infections were found in 11 (15.1%) and 6 (8.2%) cases, respectively. The sensitivities and specificities of real-time PCR versus definite diagnosis of acute infection were 63.6% and 100% for C. pneumoniae, and 100% and 100% for M. pneumoniae, respectively. C. pneumoniae PCR-negative results appeared to be due to poor growth of the organism. The sensitivity and specificity of ImmunoCard tests were 33.3% and 82.1%, respectively, indicating that the efficacy of rapid diagnosis was disputable. The present results suggest that real-time PCR is suitable for rapid diagnosis as a first screening test to determine first-line antibacterial agents to be used against these infectious diseases.     

Choice of antimicrobial drug for infections caused by Chlamydia trachomatis and Chlamydophila pneumoniae

Chlamydia (C.) trachomatis is the most common bacterial cause of sexually transmitted disease in the world. A well documented feature of chlamydial infection is its high rate of recurrence among sexually active populations. However, it is difficult to distinguish whether the high rate of recurrent disease is due to reinfection or to persistent infection with the same organism. Of particular concern in this era of increasing antibiotic resistance is whether persistent infection is the consequence of increasing resistance to standard antimicrobial therapy. Azithromycin and doxycycline are considered by the Centers for Disease Control and Prevention (CDC) as first line drugs for the treatment of chlamydial infections; erythromycin, ofloxacin and levofloxacin are recommended as alternative-regimen. Although C. trachomatis has been historically sensitive to these antibiotics, in vitro resistance is being increasingly reported. However, although in vitro antimicrobial resistance has been described, the clinical significance of these findings is unknown. C. pneumoniae is associated with community-acquired pneumonias, acute exacerbations of chronic bronchitis, otitis media, sinusitis and reactive airway disease. Persistent nasopharyngeal infection with C. pneumoniae has been documented in adults following acute respiratory infection. Chronic infection with C. pneumoniae has also been implicated in the pathogenesis of atherosclerosis, although this is still very controversial. Azithromycin, clarithromycin and quinolones are frequently used for the treatment of C. pneumoniae respiratory infections. Microbiologic failure has been described in C. pneumoniae infections, even after prolonged courses of azithromycin, erythromycin and doxycycline.     

Identification and characterization of secreted effector proteins of Chlamydophila pneumoniae TW183

We report the expression of several chlamydial effector proteins in Chlamydophila pneumoniae, as well as their time-dependent secretion into the inclusion membrane. Localization of the respective genes within type III secretion gene clusters as well as bioinformatic analysis suggest that the identified proteins are type III-secreted effector proteins. Immunocytochemistry with antisera raised against CpMip (C. pneumoniae macrophage infectivity potentiator, Cpn0661), Pkn5 (Cpn0703), Cpn0709, Cpn0712 and Cpn0827 showed secretion of the respective proteins into the inclusion membrane at 20 h postinfection (hpi). CpMip was detected within the inclusion membrane from 20 to 72 hpi, whereas Cpn0324 (CopN) was located in this compartment at 72 hpi only. This was confirmed by co-localization of the respective proteins with IncA, an inclusion membrane marker protein. These data illustrate the fact that different effectors are being expressed and secreted during different time intervals of the infection cycle. Proteins Cpn0706 and Cpn0808 were not secreted by C. pneumoniae. The immunophilin FK506, known to inhibit the activity of Legionella, C. trachomatis and C. psittaci Mip proteins, was shown to interfere with chlamydial infection. Here we report the putatively type III-dependent secretion of CpMip into the inclusion membrane as well as the effect of its inhibition on C. pneumoniae infection of HEp-2 cells.     

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.     

A positive association of peripheral arterial occlusive disease (PAD) and Chlamydophila (Clamydia) pneumoniae

The relationship between Chlamydophila pneumoniae (Cp) infection and peripheral arterial occlusive disease (PAD) was studied by analyzing clinical samples from 95 patients with PAD and 100 controls. The following investigations were conducted: IgG and IgA against lipopolysaccharide (LPS) and against purified Cp-specific antigens from elementary bodies (EB) with ELISA; anti-EB IgG, with MIF; Cp DNA in arterial biopsy and peripheral blood mononuclear cells with heminested PCR; LPS with ELISA; and bacteria culture in HEp-2 cells from arterial biopsy. A significantly higher ratio of anti-EB IgG was detected in patients. There were no significant differences in anti-LPS IgG, anti-LPS IgA and anti-EB IgA between cases and controls. Cp DNA findings in the vascular wall biopsy showed significant differences between cases and controls. We obtained results that significantly involve Cp infection with PAD through the detection of anti-EB IgG from serum and bacterial DNA from arterial biopsy.     

Chlamydia pneumoniae in a Free-Ranging Giant Barred Frog (Mixophyes iteratus) from Australia

The koala biovar of Chlamydia pneumoniae was identified in lung tissue from a sick, free-ranging giant barred frog (Mixophyes iteratus) by using electron microscopy, C. pneumoniae-specific fluorescent-antibody staining, cell culture, and sequencing of the ompA, ompB and 16S rRNA genes. This is the first report of a chlamydial strain infecting both a homeotherm and a poikilotherm and only the fourth host (in addition to humans, koalas, and horses) to be naturally infected with this species of Chlamydia. The frog had severe, chronic, mononuclear pneumonia and nonregenerative anemia and pancytopenia.     

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.     

The load of Chlamydia pneumoniae in the Alzheimer's brain varies with APOE genotype

Studies from this laboratory have indicated that the intracellular eubacterial respiratory pathogen Chlamydophila (Chlamydia) pneumoniae is commonly found in brain regions displaying characteristic neuropathology in patients with late-onset Alzheimer's disease (AD) but not in congruent samples from non-AD control individuals. In later work, we provided evidence suggesting that some relationship exists between the APOE epsilon4 gene product and the pathobiology of this organism. In the present report, in situ hybridization analyses indicated that the number of C. pneumoniae-infected cells in affected brain regions of epsilon4-bearing AD patients was higher overall than that in congruent brain regions from AD patients lacking that allele. Quantitative real-time PCR analyses of AD brain tissue samples demonstrated that actual bacterial burden in those samples varied over several orders of magnitude, but that samples from epsilon4-bearing patients did have significantly higher bacterial loads than did congruent samples from patients without the allele (ANOVA, p<0.05). These results may explain in part the observations that epsilon4-bearing individuals have a higher risk of developing AD, and that such patients progress more rapidly to cognitive dysfunction than do individuals lacking this allele.     

肺炎衣原体对血管内皮细胞的感染及其对ICAM-1表达的影响

目的:观察肺炎衣原体对人脐静脉内皮细胞(HUVECs)的感染及其对细胞分泌和表达细胞间粘附分子1(ICAM-1)的影响,探讨C.pneumoniae感染在动脉粥样硬化形成中的作用及其可能机制。方法:用人喉表皮癌(HEP-2)细胞培养C.pneumoniae,以C.pneumoniae感染HUVE细胞,经透射电镜及PCR检测有无感染。用流式细胞仪检测感染前后HUVE细胞表面ICAM-1蛋白的表达的变化,用荧光定量RT-PCR检测ICAM-1mRNA的变化。结果:C.pneumoniae能感染体外培养的HUVE细胞;感染后12h,细胞表面ICAM-1蛋白的表达即增加,其峰值约在感染后24h;荧光定量RT-PCR结果显示其增加在mRNA水平。结论:C.pneumoniae能感染体外培养的人脐静脉内皮细胞并增加ICAM-1的表达,提示C.pneumoniae感染可能是动脉粥样硬化的始动因子之一,其致动脉粥样硬化机制可能与感染后血管内皮细胞粘附分子表达的增加有关。     

Detection of Chlamydophila (Chlamydia) abortus and Toxoplasma gondii in smears from cases of ovine and caprine abortion by the streptavidin-biotin method

Fetal membranes from 59 cases of ovine and six of caprine abortion from a total of 52 flocks or herds were collected. Immunohistochemical examination of cotyledons fixed in formalin and embedded in paraffin wax detected Chlamydophila abortus in 44 (68%) cases. Immunocytochemical examination of smears made from the surface of fetal membranes detected the organism in 37 (57%) cases. Light microscopical examination of such smears stained by Stamp's method detected C. abortus in 26 (40%) cases. The streptavidin-biotin method described proved to have 100% specificity and 84% sensitivity in the detection of C. abortus in cotyledon smears. Sensitivity could probably be increased still further by the simultaneous examination of smears made from the cut surface of several cotyledons. In five cases Toxoplasma gondii was detected in the cotyledons by immunohistochemical examination. In three of these cases the presence of T. gondii was revealed also by immunocytological examination. In four cases, simultaneous C. abortus and T. gondii infection of the cotyledons was observed. The two pathogens and the lesions caused by them occurred in separate locations.     

Failure to detect Chlamydia pneumoniae in brain sections of Alzheimer's disease patients

A recent North American study detected Chlamydia pneumoniae in 17 of 19 brains of Alzheimer's patients and supposed a C. pneumoniae infection to be a risk factor for Alzheimer's disease (AD). In this study, we analyzed paraffin-embedded tissue samples of 20 AD patients by nested PCR and immunocytochemistry with a panel of antichlamydial antibodies and could detect neither C. pneumoniae-specific DNA nor chlamydial antigens. From our data, the presence of C. pneumoniae in the brains of Alzheimer's patients is not a common phenomenon; an association remains questionable.     

Variation in the LRP-associated protein gene (LRPAP1) is associated with late-onset Alzheimer disease.

The LRP-associated protein is involved in the amount of mature LRP expressed on liver and brain. LRP is the main ApoE receptor and also binds alpha2-macroglobulin (alpha2M), a protein that associates with the beta-amyloid protein (betaA). By binding to alpha2M, LRP is responsible for the clearance of secreted betaA, thus preventing fibril formation. Genetic variation at the APOE, A2M, and LRP genes has been associated with the risk of developing late-onset Alzheimer disease (LOAD). We genotyped 373 patients, 300 controls, and 100 healthy elderly controls for a common DNA-polymorphism at the LRPAP1 gene (Insertion/Deletion, intron 5). Homozygotes for the rare Insertion (I) allele were at a significantly lower frequency in patients compared with controls (P = 0.002; OR = 0.29; 95% CI = 0.13, 0.68), and in patients compared with healthy elderly controls (P = 0.0002; OR = 0.18; 95% CI = 0.07, 0.46). No patient with an age at the onset below 75 years was II (0 of 214) compared with 8 in the group above 75 years (8 of 159) (P = 0.0044), suggesting that this genotype delays the onset of the disease. According to our data, the variation at the LRPAP1 gene is associated with the risk of developing LOAD. This is in agreement with the role of the LRPAP1 protein in the amyloidogenic pathway.