Chlamydia pneumoniae Infection in Polarized Epithelial Cell Lines

We set up a polarized cell culture model to study the pathogenicity of a common respiratory tract pathogen, Chlamydia pneumoniae. Immunofluorescence staining of ZO-1 (a tight junction protein) and Na⁺K⁺ ATPase (a protein pump localized at the basolateral membrane in the polarized epithelial cells), as well as TER measurements, suggested that the filter-grown Calu-3 cells, but not the A549 cells, were polarized when grown on collagen-coated membranes. Both the flat and the filter-grown cultures were infected with C. pneumoniae. Infection in the polarized Calu-3 cultures produced more C. pneumoniae genome equivalents than infection in the flat cultures. However, this progeny was not as infective as that in the flat cultures. The maximum amount of C. pneumoniae was detected at 6 days postinfection in the filter-grown A549 cells, indicating a slower developmental cycle than that observed in the flat A549 cultures. The effect of cycloheximide on the growth of C. pneumoniae in the polarized cells was negligible. Furthermore, the infection in the polarized Calu-3 cells was resistant to doxycycline, and several cytokines were released mainly on the apical side of the polarized cells in response to C. pneumoniae infection. These findings indicate that the growth of chlamydiae was altered in the filter-grown epithelial culture system. The diminished production of infective progeny of C. pneumoniae, together with the resistance to doxycycline and polarized secretion of cytokines from the infected Calu-3 cells, suggests that this model is useful for examining epithelial cell responses to C. pneumoniae infection, and it might better resemble in vivo infection in respiratory epithelial cells.Chlamydia pneumoniae is a common respiratory tract bacterium that infects most people at some stage of their life. The clinical symptoms of C. pneumoniae infection can be negligible, or they may include respiratory tract diseases ranging from mild flu to severe pneumonia, the latter especially in elderly or immunocompromised patients (12). Although C. pneumoniae was identified as its own bacterial species within the genus Chlamydia 20 years ago (13), much of its pathogenicity still remains to be revealed.The epithelium of the respiratory tract constitutes a physical barrier and the first line of host defense that C. pneumoniae encounters. It consists of ciliated pseudostratified/simple columnar epithelial cells and mucus-producing goblet cells. The alveoli are lined by squamous type 1 pneumocytes. Surfactant-producing type II pneumocytes are typically found at the alveolar-septal junctions, and macrophages are present in the alveoli. During murine experimental intranasal infection, C. pneumoniae can be cultured from lung homogenates (19, 30), and immunohistochemistry has revealed chlamydial inclusions in bronchial epithelial cells and macrophages (26, 43). Also in humans, the respiratory epithelial cells, as well as the alveolar macrophages, represent the primary target cells of C. pneumoniae, and yet the bacterium is able to invade and infect several different cell types (8, 11, 21).Epithelial cells lining the respiratory tract are architecturally structured in a polarized orientation with distinct apical and basal faces. Lateral contacts between the epithelial cells, together with adhesion to the extracellular matrix, lead to a reorganization of the cell cytoskeleton and a distinct distribution of both apical and basolateral proteins on different cell membranes (44). Formation of a proper functional barrier between the apical and basal compartments of the epithelial cell layer is also crucial. In polarized epithelial cells, a tight junction serves as a specialized intercellular junctional complex that mediates adhesion between the cells and regulates diffusion through the intercellular space (33). Various cell culture models have been extensively used to study the characteristics and pathogenesis of chlamydial infections, but most published papers with epithelial cell lines describe infection in cell cultures grown on impermeable plastic or glass supports, in which case the membrane asymmetry of a polarized cell and proper cell-cell contacts that form the tight barrier across the cell layer are lacking.One model that simulates naturally occurring cell polarization is achieved in vitro when the epithelial cells are cultured on permeable membranes that permit cells to feed basolaterally and allow formation of distinct apical and basolateral surfaces. Such polarized cell cultures more closely resemble the in vivo situation and have proven useful in studies on several aspects of C. trachomatis infection (38). For instance, C. trachomatis entry into epithelial cells (39), the growth and production of infective progeny (35, 39), properties of persistent infection (17, 18), and even antimicrobial drug effects (40) are altered in polarized cell cultures. Although the polarized epithelial cell culture model mimicking the natural epithelial cell barrier appears to be a rational choice for studying the pathogenesis of respiratory tract infections, there are no reports of its use with C. pneumoniae infection. Therefore, in the present study, we set up a polarized cell culture model for C. pneumoniae using two different transformed lung epithelial cell lines. Human lung carcinoma cell line A549 exhibits metabolic and transport properties of type II pulmonary epithelial cells (7). This cell line is widely used in polarized cell studies, although some reports show that these cells do not necessarily form functional tight junctions when grown on permeable supports (15, 37). Calu-3 cells, a lung adenocarcinoma cell line, possess characteristics of serous gland epithelial cells (5, 22). The Calu-3 cell line is reported to form a strong functional barrier in a polarized culture (6, 37). Both cell lines have previously been shown to be susceptible to C. pneumoniae infection when grown on impermeable supports (9, 42). The purpose of the present study was to characterize C. pneumoniae infection in filter-grown/polarized cultures of respiratory epithelial cells. We demonstrate that the epithelial cells cultured on a semipermeable membrane are a suitable model for studying C. pneumoniae infection. Our results show that the characteristics of infection (i.e., kinetics, production of infectious progeny, and sensitivity to antimicrobials) might drastically differ between models that use flat and filter-grown cells.     

Relationships of brain white matter microstructure with clinical and MR measures in relapsing‐remitting multiple sclerosis

Purpose:

To assess the relationships of microstructural damage in the cerebral white matter (WM), as measured by diffusion tensor imaging (DTI), with clinical parameters and magnetic resonance imaging (MRI) measures of focal tissue damage in patients with multiple sclerosis (MS).

Materials and Methods:

Forty‐five relapsing‐remitting (RR) MS patients (12 male, 33 female; median age = 29 years, Expanded Disability Status Scale (EDSS) = 1.5, disease duration = 3 years) were studied. T2‐lesion masks were created and voxelwise DTI analyses performed with Tract‐Based Spatial Statistics (TBSS).

Results:

T2‐lesion volume (T2‐LV) was significantly (P < 0.05, corrected) correlated with fractional anisotropy (FA) in both lesions and normal‐appearing WM (NAWM). Relationships (P = 0.08, corrected) between increasing EDSS score and decreasing FA were found in the splenium of the corpus callosum (sCC) and along the pyramidal tract (PY). All FA associations were driven by changes in the perpendicular (to primary tract direction) diffusivity. No significant global and voxelwise FA changes were found over a 2‐year follow‐up.

Conclusion:

FA changes related to clinical disability in RR‐MS patients with minor clinical disability are localized to specific WM tracts such as the sCC and PY and are driven by changes in perpendicular diffusivity both within lesions and NAWM. Longitudinal DTI measurements do not seem able to chart the early disease course in the WM of MS patients. Imaging 2010; 31:309–316. © 2010 Wiley‐Liss, Inc.     

Potential gonadotoxicity of treatment in relation to quality of life and mental well-being of male survivors of childhood acute lymphoblastic leukemia

Purpose

Results of earlier studies concerning quality of life (QOL) and psychosocial coping of childhood acute lymphoblastic leukemia (ALL) survivors have been inconsistent. Some treatments for ALL affect testicular function and we hypothesized that this may influence the QOL and psychosocial coping of male survivors. Our aims were to assess the QOL and psychosocial coping of male long-term ALL survivors and to evaluate the effect of both testosterone level and the potential gonadotoxicity of various treatment modalities on them.

Methods

Fifty-two male long-term survivors treated for childhood ALL at Helsinki University Hospital between 1970 and 1995, and 56 age- and gender-matched controls were studied. The participants completed a self-report questionnaire including questions on sociodemographics, RAND-36 to assess QOL, General Health Questionnaire and Beck Depression Inventory to assess mental well-being, and CAGE to assess alcohol abuse/dependence. Testosterone levels were measured, and treatment details were reviewed.

Results

ALL survivors in general had QOL close to that of controls or population norms. Decreased QOL was seen in physical health-related subscales, and vitality and emotional well-being were lowered in survivors with more gonadotoxic treatment modalities. No single independent factor in the treatment or the level of testosterone could, however, be found to clearly explain the variation in QOL scores of the survivors. Mental well-being of most of the survivors was good, but a subgroup with previous cyclophosphamide treatment or testicular irradiation showed increased risk of psychiatric morbidity.

Conclusions

The male ALL survivors generally cope well, but increased focus on specific risk groups seems to be necessary. Further studies using patient interviews would probably point out issues concerning the QOL and psychosocial coping of ALL survivors, which may not emerge in these screening studies.

Implications for Cancer Survivors

In general, more attention should be paid for physical functioning of childhood ALL survivors. Increased focus should also be on QOL and mental well-being of survivors with more gonadotoxic treatment modalities and those whose diagnosis was made in their adolescence.     

Strain‐encoded (SENC) magnetic resonance imaging to evaluate regional heterogeneity of myocardial strain in healthy volunteers: Comparison with conventional tagging

Purpose

To evaluate the ability of strain‐encoded (SENC) magnetic resonance imaging (MRI) for regional systolic and diastolic strain analysis of the myocardium in healthy volunteers.

Materials and Methods

Circumferential and longitudinal peak systolic strain values of 75 healthy volunteers (35 women and 40 men, mean age 44 ± 12 years) were measured using SENC at 1.5T. MR tagging was used as the reference standard for measuring regional function. Diastolic function was assessed in the 10 youngest (24 ± 8 years) and 10 oldest (62 ± 5 years) subjects.

Results

Peak strain values assessed with SENC were comparable to those obtained by MR tagging, showing narrow limits of agreement (limits of agreement ?5.6% to 8.1%). Regional heterogeneity was observed between different segments of the left ventricle (LV) by both techniques (P < 0.001). Longitudinal strain obtained by SENC was also heterogenous (P < 0.001). Interestingly, no age‐ or gender‐specific differences in peak systolic strain were observed, whereas the peak rate of relaxation of circumferential strain rate was decreased in the older group.

Conclusion

SENC is a reliable tool for accurate and objective quantification of regional myocardial systolic as well as diastolic function. In agreement with tagged MRI, SENC detected slightly heterogeneous myocardial strain within LV segments. J. Magn. Reson. Imaging 2009;29:99–105. © 2008 Wiley‐Liss, Inc.