Chlamydia pneumoniae multiplies in human endothelial cells in vitro

The ability of three C. pneumoniae isolates, Kajaani 6, Helsinki 12 and TW-183, to grow in human umbilical vein endothelial cells (HUVEC) and in an immortalized endothelial cell line EA.hy 926 was studied. All C. pneumoniae isolates were capable of multiplying in endothelial cells. EA.hy 926 cells could support the growth of C. pneumoniae better than HUVEC, yet less efficiently than HL and HEp-2 cells that are conventionally used in C. pneumoniae culturing. Although centrifugation of the inoculum greatly increased the inclusion yields, it was not necessary for infectivity. In addition, a persistent infection of C. pneumoniae in EA.hy 926 and HL cells ensued and it was followed up for two months. The fact that endothelial cells can serve as hosts to C. pneumoniae might be a significant contributing factor in the pathogenesis of atherosclerosis, a disease which recent studies show to be associated with chronic C. pneumoniae infection.     

Genetic control of susceptibility to pulmonary infection with Chlamydia pneumoniae in the mouse

A mouse model was used to study the genetic control of differential host response to pulmonary infection with Chlamydia pneumoniae. The A/J and C57BL/6 strains show differential response to intranasal infection with respect to their ability to clear pulmonary bacterial load and the extent of lung pathology developed by 2 weeks post infection. The genetic basis of this interstrain difference was studied by whole-genome scan in an informative [A/J x C57BL/6J] F2 cross using the pulmonary microbial load as a phenotypic readout of host response. We detected a highly significant linkage (LOD score=11.5) on chromosome 17 that overlaps with the major histocompatibility (MHC) locus. This quantitative trait locus (QTL) accounts for approximately 30% of the phenotypic variance with B6 alleles conferring susceptibility and inherited in a recessive fashion. Significant linkage was also detected to chromosome 5 in female mice, while chromosome 6 showed suggestive linkage in male mice, pointing to additional complexity in the genetic control of the difference in susceptibility observed in A/J and C57BL/6J.     

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.     

Chlamydia pneumoniae infection in human monocytes

Chlamydia pneumoniae infection has been associated with cardiovascular diseases in seroepidemiological studies and by demonstration of the pathogen in atherosclerotic lesions. It has the capacity to infect several cell types, including monocyte-derived macrophages, which play an essential role in the development of atherosclerosis. However, the persistence of C. pneumoniae in mononuclear cells is poorly understood. To study the morphology and biological characteristics of the infection, human peripheral blood monocytes were infected with C. pneumoniae. Freshly isolated monocytes resisted the development of infectious progeny, and confocal and transmission electron microscopy showed that the morphology of the inclusions and chlamydial particles was abnormal. Addition of tryptophan or antibodies against gamma interferon did not diminish the inhibition of C. pneumoniae, suggesting that other factors are involved in the chlamydiostatic activity of the monocytes. Chlamydial mRNA was expressed at least 3 days after infection, however, and a capability for infected monocytes to induce a positive lymphocyte proliferative response was detected for up to 7 days, indicating that C. pneumoniae remains metabolically active in the monocytes in vitro. These results are in accordance with the hypothesis that C. pneumoniae may participate in the maintenance of local immunological response and inflammation via infected monocytes and thus enhance atherosclerosis.     

Serological response to Chlamydia pneumoniae infection.

The human serological response was analyzed by using sera from patients who were serologically positive but isolation negative for Chlamydia pneumoniae and from patients with proven C. pneumoniae infection based on serology and isolation. To assess whether seroreactivity to C. pneumoniae proteins had potential diagnostic value, the cross-reactivities of these sera to other Chlamydia species and of sera from patients infected with C. trachomatis and C. psittaci to C. pneumoniae proteins were determined. In all serum samples from patients with proven C. pneumoniae infections, reactivities were seen with 98-, 68-, 60-, 39.5-, and 30-kilodalton proteins. Similar patterns were seen in sera from patients who were serologically positive and isolation negative. The onset of seropositivity for C. pneumoniae was accompanied by reactivities against presumably shared chlamydial antigens and a C. pneumoniae-specific 98-kilodalton protein.     

肺炎衣原体感染对血管平滑肌细胞黏附和迁移的影响

目的:观察肺炎衣原体(C.pn)感染对血管平滑肌细胞(VSMCs)黏附和迁移的影响。方法:C.pn在人喉癌细胞系HEp-2细胞中增殖培养后感染大鼠VSMCs,吖啶橙(AO)荧光染色观察细胞内C.pn包涵体的形态特点;聚合酶链反应(PCR)检测C.pn特异性DNA片段;细胞黏附实验观察C.pn感染对VSMCs黏附能力的影响;wound-healing assay和Transwell assay检测C.pn感染VSMCs后细胞迁移能力的改变。结果:C.pn感染VSMCs后,少数细胞内出现空泡状结构即包涵体,但在多数细胞内以点状感染灶形式存在,包涵体较大,但数量较少。利用PCR可在感染的VSMCs内检测到437 bp的C.pn特异性DNA片段。细胞黏附实验结果显示,C.pn感染VSMCs 2 h后,细胞黏附能力明显增强,其吸光度值明显高于正常对照组(P0.01),细胞黏附率为134.38%。Wound-healing assay结果显示,C.pn感染VSMCs 24 h后,细胞向划痕中央迁移的距离明显大于正常对照组(P0.05)。Transwell assay结果显示,C.pn感染VSMCs 24 h后,C.pn感染组的细胞迁移数明显多于正常对照组(P0.01)。结论:C.pn感染能够显著增强VSMCs的黏附和迁移能力。     

In vitro infection of human epidermal Langerhans' cells with HIV-1.

Epidermal Langerhans' cells (LC) from human immunodeficiency virus type-1 (HIV-1)-infected patients harbour HIV-1 proviral DNA and RNA. In the present study, we investigated whether LC from epidermis of normal, HIV-seronegative subjects could be infected in vitro with HIV-1. Epidermal cells (EC) spontaneously detached from epidermal sheet cultures were enriched for LC (10-25% of CD1a+/CD4+ cells), deprived of contaminating T cells and then incubated with HIV-1IIIB. After 24 hr, purified LC and LC-depleted EC fractions were obtained by immunomagnetic separation. Polymerase chain reaction (PCR) analysis showed the presence of HIV-1 proviral DNA (gag) only in purified LC. In addition, LC-enriched EC, purified LC, LC-depleted EC or the non-permissive cell line, TF-1, the latter having being previously challenged with HIV-1IIIB for the same length of time as the EC, were co-cultivated with C8166 cells, and the co-cultures assessed for the presence of HIV DNA by PCR. Co-cultures of C8166 cells with purified LC or LC-enriched EC previously exposed to HIV-1IIIB exhibited a time-dependent increase in HIV proviral DNA. In contrast, PCR analysis of C8166 cells co-cultured with either LC-depleted EC or TF-1 cells gave negative results. Finally, C8166 cells co-cultured with HIV-infected LC formed syncytia, showed membrane budding and released numerous retroviral particles. The results indicate that LC from normal subjects can be infected in vitro with HIV and can transmit infection to myeloid cells. This in vitro model may help in understanding the regulation of HIV infection of LC.     

Streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro HMPV infection

It remains largely unknown which factors determine the clinical outcome of human metapneumovirus (HMPV) infections. The aim of the present study was to analyse whether exposure to bacterial pathogens can influence HMPV infections. From 57 children, serum samples and colonization data for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pneumoniae were collected at 1.5, 6, 14 and 24 months of age. Seroconversion rates to HMPV were determined and related to bacterial carriage. Frequent nasopharyngeal carriage (≥2 times in the first 2 years of life) of S. pneumoniae, but not of the other three pathogens, was associated with increased seroconversion rates of infants to HMPV at the age of 2 years (frequently vs. less exposed, 93% vs. 59%; p <0.05). Subsequently, the susceptibility of well-differentiated normal human bronchial epithelial cells (wd-NHBE) pre-incubated with bacterial pathogens to in vitro HMPV infection was evaluated. Pre-incubation of wd-NHBE with S. pneumoniae resulted in increased susceptibility to infection with HMPV-enhanced green fluorescent protein (EGFP), as determined by enumeration of EGFP-positive cells. This was not the case for cells pre-incubated with H. influenzae, M. catarrhalis on S. aureus. We conclude that exposure to S. pneumoniae can modulate HMPV infection.     

Asymptomatic respiratory tract infection with Chlamydia pneumoniae TWAR.

Chlamydia pneumoniae is a newly recognized organism associated with respiratory tract infections. Asymptomatic infection with C. pneumoniae, although it has been suggested to occur, has not been previously documented. We describe two asymptomatic individuals infected with this organism; these infections demonstrate that C. pneumoniae is able to establish a subclinical infection.     

Induction of proinflammatory cytokines in human lung epithelial cells during Chlamydia pneumoniae infection

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute respiratory diseases such as pneumonia and bronchitis. Previous studies have established that C. pneumoniae can induce cytokines in mouse and/or human cells, but little information is available on the cytokine response of respiratory epithelial cells, a first line of infection. In this study, heparin treatment of C. pneumoniae significantly reduced its ability to induce interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) mRNA in human lung carcinoma cells, indicating that cytadherence is an important early stimulus for induction of proinflammatory mediators. Although the IL-8, gamma interferon, and TNF-alpha message was consistently induced by infection of A549 cells not treated with heparin, only an elevation of IL-8 protein was detected in A549 supernatants. A549 IL-beta and IL-6 mRNA and supernatant protein profiles were not significantly changed by infection. Heat or UV inactivation of C. pneumoniae only partially reduced the cytokine response, and inhibition of C. pneumoniae protein or DNA synthesis did not affect its ability to induce cytokine gene expression. To prevent stress-induced cytokine release by the A549 cells, centrifugation was not utilized for infection experiments. These experiments establish the importance of cytadherence in cytokine release by cells of respiratory epithelial origin and suggest that further work in the area of cytokine mediators is warranted to gain valuable pathogenic and therapeutic insights.     

Experimental infection with Chlamydia pneumoniae in nonhuman primates.

To serially examine the immunopathogenesis and histopathology of infection with Chlamydia pneumoniae, we inoculated two cynomolgus monkeys in the conjunctival sac, nose, and nasopharynx with C. pneumoniae TWAR. After inoculation, C. pneumoniae was isolated from the inoculation sites and the rectums of both monkeys for a period of 5 weeks. After a second inoculation, C. pneumoniae was recovered from the inoculation sites and the rectums of both monkeys for 20 weeks. A third inoculation with C. pneumoniae caused very little productive infection at any site. Prior C. pneumoniae infection did not prevent subsequent C. trachomatis serovar E (Bour strain) infection. Clinical and histopathologic ocular responses to C. pneumoniae infection were mild compared with those to infection with C. trachomatis serovar E. Rectal infection, demonstrated by culture isolation and immunohistopathology, occurred without direct experimental inoculation. Both immunofluorescent staining of mucosal smears with monoclonal antibodies and tissue culture were able to detect C. pneumoniae infection. Experimental nonhuman primate infection with C. pneumoniae appears to be clinically and histopathologically mild and can occur at extrapulmonary sites.     

Rabbit model for Chlamydia pneumoniae infection.

A rabbit model was established for Chlamydia pneumoniae infection that may be helpful to understand the pathogenesis of disease in humans. Twelve, pathogen-free, 1-month-old New Zealand White rabbits were inoculated with 1.0 x 10(7) to 5.0 x 10(7) CFU of purified C. pneumoniae (ATCC strain VR 1310) via the nasopharynx (1 rabbit died immediately postinoculation, and 11 were available for study). Five controls were inoculated with the carrier buffer. Ten of the 11 study rabbits demonstrated serological evidence of acute infection (immunoglobulin G antibodies, 1:8 to > 1:16), with the weakest response at 7 days and the strongest response at 28 days, whereas none of the controls showed any seroconversion. Study animals were sacrificed in batches of three, on days 7, 14, 21, and 28, but controls were sacrificed on days 7 and 28. Two-thirds of the animals demonstrated evidence of bronchiolitis and pneumonia on days 7 and 14 and resolution by day 21. Two study rabbits demonstrated, on histology, early and intermediate lesions of atherosclerosis: one animal (day 7) showed the accumulation of foamy macrophages (fatty streak) in the arch of the aorta, and the other animal (day 14) showed spindle cell proliferation of smooth muscle cells (intermediate lesion). Focal periaortitis was seen in the same animal (day 7). C. pneumoniae elementary bodies were demonstrated by immunocytochemical stain in the lungs (n = 2), liver (n = 3), spleen (n = 5), and aorta (n = 2), one of which corresponded to the intermediate lesion. C. pneumoniae was cultured from the lungs (n = 2), liver (n = 2), spleen (n = 2), and aortic arch (n = 1). All histopathological, immunocytochemical, and cultural studies were negative in the controls. Hence, the rabbit provides a useful animal model for the study of C. pneumoniae infection and its complications, particularly atherosclerosis.     

Chlamydia pneumoniae infection increases adherence of mouse macrophages to mouse endothelial cells in vitro and to aortas ex vivo

Interactions between monocytes/macrophages and endothelial cells play an important role in the pathogenesis of atherosclerosis, and the adherence of monocytes to the arterial endothelium is one of the early events in atherogenesis. In the present study, peritoneal macrophages harvested from green fluorescent protein (GFP) transgenic mice were used to analyze how Chlamydia pneumoniae infection affects the adherence of GFP-macrophages to mouse endothelial cells in vitro and to the aorta from normolipidemic and hyperlipidemic mice ex vivo. In vitro studies showed that C. pneumoniae-infected GFP-macrophages adhered better than uninfected macrophages to endothelial cells and GFP-macrophages adhered better to infected than uninfected endothelial cells. The ex vivo studies showed that C. pneumoniae-infected macrophages adhered better than uninfected macrophages to aortas from both normolipidemic and hyperlipidemic C57BL/6J mice and apolipoprotein E (ApoE)-deficient mice. In contrast, adherence of C. pneumoniae-infected macrophages to the aortas of intercellular adhesion molecule 1 (ICAM-1) knockout mice was not enhanced, suggesting that ICAM-1 is crucial for activation of the adherence of C. pneumoniae-infected macrophages to the endothelium. In conclusion, the present study defined a homing mechanism by which C. pneumoniae promotes the adherence of mononuclear phagocytes to the endothelium at the site of atherosclerotic lesion formation to promote the progression of atherosclerosis.     

Experimental rabbit models of Chlamydia pneumoniae infection.

Chlamydia pneumoniae (TWAR), a common cause of acute respiratory disease in humans, has recently been associated with coronary and aortic atherosclerosis. In this study, we evaluated rabbit models of chlamydial infection to investigate the pathogenesis of C. pneumoniae infection. New Zealand White rabbits were inoculated intranasally and intratracheally with C. pneumoniae, strain AR-39, and primary and repeated infection were assessed. After a single inoculation, lung pathology was characterized by a moderate self-resolving interstitial pneumonia with bronchiolitis of 21 days in duration. Chlamydial DNA was detected by polymerase chain reaction (PCR) intermittently in the upper respiratory tract and lung tissue through day 21 postinoculation, spleen tissue at day 14, and peripheral blood mononuclear cells at days 3 and 21. After repeated inoculations, chlamydial DNA was detected by PCR in the upper respiratory tract and lung tissue through day 42. Lung lesions consisted of multifocal interstitial mononuclear cell aggregates that persisted up to day 42. Watanabe heritable hyperlipidemic rabbits were less susceptible to C. pneumoniae infection. After multiple inoculations of Watanabe rabbits, C. pneumoniae was detected by PCR and/or immunocytochemistry until day 21. In conclusion, C. pneumoniae induced a moderate respiratory infection in these rabbit models.     

Differential susceptibility of human mononuclear cells to infection with HTLV-I

Infection with human T-cell leukaemia/lymphoma (HTLV-I) preferentially affects T cells of the OKT-4 phenotype. The aim of the present study was to determine whether distinct T-cell subsets exhibit differences in susceptibility to virus infection. T cells from peripheral blood were separated according to cell densities by 7-step Percoll gradients. Separated T-cell subpopulations were infected with HTLV-I, using cocultivation with irradiated virus producer MT-2 cell line. Percentages of HTLV-I-infected cells and their phenotypes were assayed by immunofluorescence assay (IFA), using highly specific mouse monoclonal antibody directed against HTLV-I P-19 core protein, and other surface markers. The results showed that different T-cell subpopulations were susceptible to HTLV-I infection with the exception of large granular lymphocytes (LGL) which exhibit high cell-mediated natural cytotoxicity (CMNC).     

In vitro susceptibility to infection with SIVcpz and HIV-1 is lower in chimpanzee than in human peripheral blood mononuclear cells

This study was undertaken to evaluate and compare the susceptibility of chimpanzee versus human peripheral blood mononuclear cells (PBMCs) to infection with SIVcpz and HIV-1 non-syncitium inducing primary isolates. The results demonstrate clearly that chimpanzee PBMCs have a lower capacity to support viral replication as compared to human PBMCs. There was no experimental evidence that this difference was due to a lower availability of target cells for viral infection (PBMCs positive for CD4 and CCR5 molecules) or to a differential susceptibility to apoptosis (PBMCs positive for CD4 and CD95 molecules). A lower capacity of chimpanzee PBMCs to support SIVcpz and HIV-1 replication in vitro is related to a post-entry barrier to virus replication.     

肺炎衣原体感染致血管平滑肌细胞的改变

肺炎衣原体感染是动脉粥样硬化发生的重要危险因素。肺炎衣原体可以感染血管壁内皮细胞、平滑肌细胞、单核细胞/巨噬细胞,并与这些细胞之间相互作用,影响这些细胞的形态与功能,从而参与动脉粥样硬化发生发展。在动脉粥样硬化发病机制中,平滑肌细胞形态功能的变化与动脉粥样硬化密切相关,肺炎衣原体能够感染平滑肌细胞,影响平滑肌细胞在血管内的生理生化特征,促进动脉粥样硬化的发展过程。