Genome sequencing of Chlamydia trachomatis serovars E and F reveals substantial genetic variation.

Chlamydia trachomatis (Ctr) is a bacterial pathogen that causes ocular, urogenital and lymph system infections in humans. It is highly abundant and among its serovars, E, F and D are most prevalent in sexually transmitted disease. However, the number of publicly available genome sequences of the serovars E and F, and thereby our knowledge about the molecular architecture of these serovars, is low. Here we sequenced the genomes of six E and F clinical isolates and one E lab strain, in order to study the genetic variance in these serovars. As observed before, the genomic variation inside the Ctr genomes is very low and the phylogenetic placement in comparison to publicly available genomes is as expected by ompA gene serotyping. However, we observed a large InDel carrying four to five open reading frames in one clinical E sample and in the E lab strain. We have also observed substantial variation on nucleotide and amino acid levels, especially in membrane proteins and secreted proteins. Furthermore, these two groups of proteins are also target for recombination events. One clinical F isolate was genetically heterogeneous and revealed the highest differences on nucleotide level in the pmpE gene.

Broad recruitment of mGBP family members to Chlamydia trachomatis inclusions.

Chlamydia, the most common sexually transmitted pathogen, is an exquisitely adapted Gram-negative obligate intracellular bacterium. Intracellular Chlamydia trachomatis replicate in a specialized vacuole, termed inclusion, which shields the bacterium from antimicrobial immunity of the host cells and acts as a signalling interface. Previously it was shown that members of the interferon induced guanylate binding protein (mGBP) family, in particular murine GBP1 and mGBP2, were found to accumulate at the bacterial inclusions, similar to previously published recruitment of GBPs to the parasitophorous vacuole of Toxoplasma gondii. Here, we provide a wide comparison of mGBPs roles within the host cell in the context of Chlamydia and Toxoplasma infection. By confocal microscopy on fixed and living infected cells we show localization of mGBP3, mGBP6, mGBP7, mGBP9, and mGBP10, in addition to mGBP1 and mGBP2, at chlamydia inclusions. In time lapse videos using GFP expressing Chlamydia we show rapid and transient dynamics of mGBP9 accumulation onto chlamydia inclusions. Taken together this study reveals a broad activation of mGBP recruitment towards Chlamydia trachomatis inclusions after infection and provides evidence for time limited action of mGBP9 at the chlamydia inclusion.

Vaccination with the polymorphic membrane protein A reduces Chlamydia muridarum induced genital tract pathology.

Chlamydia trachomatis serovars D-K are one of the most frequent causes of sexually transmitted infections of the female genital tract, with possible complications such as hydrosalpinx, pelvic inflammatory disease, extra-uterine gravidity or infertility. We used the murine genital tract infection model with C. muridarum for vaccination studies and found that more than 70% of the infected mice suffered from uterus dilatations and/or hydrosalpinx. Systemic consequences of the vaginal infection were apparent by splenomegaly ten to fifteen days post infection. While cultivable microorganisms were detectable for the first 23days post infection, the first lesions of the genital tract developed at day 15, however, many lesions occurred later in the absence of cultivable bacteria. Lesions were not accompanied by pro-inflammatory cytokines such as IFNÉ£, TNF and IL-6, since these cytokines were almost undetectable in the genital tract 43days post infection. To prevent genital tract lesions, we vaccinated mice with the polymorphic membrane protein (Pmp) A in combination with CpG-ODN 1826 as adjuvant. The vaccine lowered the chlamydial burden and the differences were significant at day 10 post infection but not later. More importantly the vaccine decreased the rate and severity of genital tract lesions. Interestingly, control vaccination with the protein ovalbumin plus CpG-ODN 1826 enhanced significantly the severity but not the rate of pathologic lesions, which was presumably caused by the activation of innate immune responses by the adjuvant in the absence of a C. muridarum-specific adaptive immune response. In summary, vaccination with recombinant PmpA plus CpG-ODN 1826 significantly reduced C. muridarum-induced tissue damage, however, CpG-ODN 1826 may aggravate C. muridarum-induced tissue injuries in the absence of a protective antigen.

The Chlamydia trachomatis Ctad1 invasin exploits the human integrin ß1 receptor for host cell entry.

Infection of human cells by the obligate intracellular bacterium Chlamydia trachomatis requires adhesion and internalization of the infectious elementary body (EB). This highly complex process is poorly understood. Here, we characterize Ctad1 (CT017) as a new adhesin and invasin from C. trachomatis serovar E. Recombinant Ctad1 (rCtad1) binds to human cells via two bacterial SH3 domains located in its N-terminal half. Pre-incubation of host cells with rCtad1 reduces subsequent adhesion and infectivity of bacteria. Interestingly, protein-coated latex beads revealed Ctad1 being an invasin. rCtad1 interacts with the integrin ß1 subunit on human epithelial cells, and induces clustering of integrins at EB attachment sites. Receptor activation induces ERK1/2 phosphorylation. Accordingly, rCtad1 binding to integrin ß1-negative cells is significantly impaired, as is the chlamydial infection. Thus interaction of C. trachomatis Ctad1 with integrin ß1 mediates EB adhesion and induces signaling processes that promote host-cell invasion.

Multicenter immunohistochemical ALK-testing of non-small-cell lung cancer shows high concordance after harmonization of techniques and interpretation criteria.

INTRODUCTION: Detection of anaplastic lymphoma kinase (ALK)-gene rearrangements in non-small-cell lung cancer (NSCLC) is mainly performed by fluorescence in-situ hybridization (FISH). The question was raised if FISH might be replaced by immunohistochemistry (IHC) in a reliable and reproducible manner across different laboratories. METHODS: After calibration of the staining instruments and training of the observers to binary interpretation (positive versus negative), 15 NSCLC were independently tested for ALK protein expression by IHC only in a multicenter setting (16 institutes). Each laboratory utilized the VENTANA ALK-D5F3 IHC assay. As demonstrated by FISH the samples displayed unequivocal ALK break-positivity (6×) and negativity (7×), as well as ALK positive-"borderline" character (2×), which is challenging for FISH diagnosis and thus was RT-PCR-confirmed. RESULTS: All seven ALK FISH-negative cases were homogenously scored as ALK-IHC negative. All 16 participants scored the two ALK positive-"borderline" samples as unequivocally positive according to their protein expression. Concordant IHC interpretation was also noticed in four of six unequivocal ALK break positive cases. In two of six some observers described a weak/heterogeneous ALK-IHC staining. This would have resulted in a subsequent ALK-testing (FISH/PCR) in a routine diagnostic setting. CONCLUSIONS: This so-called "ALK-Harmonization-Study" shows for the first time that predictive semiquantitative IHC reveals reliable and reproducible results across several labs when methodology and interpretation are strictly defined and the pathologists are uniquely trained. The application of validated ALK IHC assays and its comparison to ALK-FISH is highly needed in future clinical trials. This might answer the question if ALK-IHC cannot only serve as a prescreening tool, but as a stand-alone test at least in cases displaying an unequivocally staining pattern as well as an alternative predictive test in samples with reduced FISH interpretability.

Characterization of the interaction between the chlamydial adhesin OmcB and the human host cell.

In a previous study, we reported that the OmcB protein from Chlamydia pneumoniae mediates adhesion of the infectious elementary body to human HEp-2 cells by interacting with heparin/heparan sulfate-like glycosaminoglycans (GAGs) via basic amino acids located in the first of a pair of XBBXBX heparin-binding motifs (K. Moelleken and J. H. Hegemann, Mol. Microbiol. 67:403-419, 2008). In the present study, we show that the basic amino acid at position 57 (arginine) in the first XBBXBX motif, the basic amino acid at position 61 (arginine) in the second motif, and another amino acid (lysine 69) C terminal to it play key roles in the interaction. In addition, we show that discrimination between heparin-dependent and -independent adhesion by C. trachomatis OmcBs is entirely dependent on three variable amino acids in the so-called variable domain C terminal to the conserved XBBXBX motif. Here, the predicted conformational change in the secondary structure induced by the proline at position 66 seems to be crucial for heparin recognition. Finally, we performed neutralization experiments using different anti-heparan sulfate antibodies to gain insight into the nature of the GAGs recognized by OmcB. The results suggest that C. trachomatis serovar L2 OmcB interacts with 6-O-sulfated domains of heparan sulfate, while C. pneumoniae OmcB apparently interacts with domains of heparan sulfate harboring a diverse subset of O-sulfations.

Factors predicting poor survival after lung-sparing radical pleurectomy of IMIG stage III malignant pleural mesothelioma.

OBJECTIVES: The role of radical pleurectomy (RP) in the management of IMIG stage III in malignant pleural mesothelioma (MPM) remains controversial. The aim of the study was to investigate the feasibility and outcome as well as to determine factors predicting poor survival. METHODS: Patients having IMIG stage III MPM were identified within a prospective multimodality treatment study (RP followed by chemoradiation) between 2002 and 2010 at a single institution. Kaplan-Meier analyses, log-rank test and Cox regression analyses were used to estimate survival and to determine predictors of survival. RESULTS: A total of 78 patients (66.3 ± 2.5 years, 65 males) underwent RP followed by chemoradiation. A total of 42 (54%) had IMIG stage III. Mortality and morbidity were 4.8 and 31%, respectively. Median survival and 5-year survival were 21 months and 28%, respectively, for stage III patients. Progression-free survival was 11 months. The sites of failure were predominantly locoregional (20/42, 47.6%). Pathological detection of tumour spread at the resected thoracoscopy incisions (median survival 12 vs 35 months, P < 0.001), incomplete resections (median survival 13 vs 35 months, P = 0.01) and male gender (median survival 18 vs 68 months, P < 0.039) were associated with inferior survival in the univariate analyses. Histology, lymph node metastases, laterality and age had no significant impact on survival. The tumour spread at the resected previous incisions remained the only significant prognostic factor (hazard ratio (HR) = 4.3; P = 0.027) in the multivariate analysis. Patients having tumour spread had survival comparable to that of patients at stage IV in the complete patient cohort (median survival 12 vs 8 months; P = 0.39). CONCLUSIONS: Lung-sparing RP for IMIG stage III MPM is feasible and offers promising long-term survival. The tumour spread at the resected previous incisions is associated with more incomplete resections and was a negative prognosticator for long-term survival. The tumour spread at the resected previous incisions or chest tube sites should be considered as T4 or stage IV according to the IMIG staging system.

Assessment of postischemic neurogenesis in rats with cerebral ischemia and propofol anesthesia.

BACKGROUND: Postischemic endogenous neurogenesis can be dose-dependently modulated by volatile anesthetics. The intravenous anesthetic propofol is used during operations with a risk of cerebral ischemia, such as neurosurgery, cardiac surgery, and vascular surgery. The effects of propofol on neurogenesis are unknown and, therefore, the object of this study. METHODS: Eighty male Sprague-Dawley rats were randomly assigned to treatment groups with propofol administration for 3 h: 36 mg x kg(-1) x h(-1) propofol with or without cerebral ischemia and 72 mg x kg(-1) x h(-1) propofol with or without cerebral ischemia. In addition, 7 rats with propofol administration for 6 h and 14 treatment-naive rats were investigated. Forebrain ischemia was induced by bilateral carotid artery occlusion and hemorrhagic hypotension. Animals received 5-bromo-2-deoxyuridine for 7 days. 5-Bromo-2-deoxyuridine-positive neurons were counted in the dentate gyrus after 9 and 28 days. Spatial learning in the Barnes maze and histopathologic damage of the hippocampus were analyzed. RESULTS: Propofol revealed no impact on basal neurogenesis. Cerebral ischemia increased the amount of new neurons. After 28 days, neurogenesis significantly increased in animals with low-dose propofol administered during cerebral ischemia compared with naive animals, whereas no significant difference was observed in animals with high-dose propofol during ischemia. Neuronal damage in the CA3 region was increased at 28 days with high-dose propofol. Postischemic deficits in spatial learning were not affected by propofol. CONCLUSIONS: Independent effects of propofol are difficult to ascertain. Peri-ischemic propofol administration may exert secondary effects on neurogenesis by modulating the severity of histopathologic injury and thereby regenerative capacity of the hippocampus.