Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration

The Ca2+-binding protein calretinin is currently used as a positive marker for identifying epithelioid malignant mesothelioma (MM) and reactive mesothelium, but calretinin’s likely role in mesotheliomagenesis remains unclear. Calretinin protects immortalized mesothelial cells in vitro from asbestos-induced cytotoxicity and thus might be implicated in mesothelioma formation. To further investigate calretinin’s putative role in the early steps of MM generation, primary mesothelial cells from calretinin knockout (CR−/−) and wildtype (WT) mice were compared. Primary mouse mesothelial cells from WT and CR−/− mice were investigated with respect to morphology, marker proteins, proliferation, cell cycle parameters and mobility in vitro. Overexpression of calretinin or a nuclear-targeted variant was achieved by a lentiviral expression system. CR−/− mice have a normal mesothelium and no striking morphological abnormalities compared to WT animals were noted. Primary mouse mesothelial cells from both genotypes show a typical “cobblestone-like” morphology and express mesothelial markers including mesothelin. In cells from CR−/− mice in vitro, we observed more giant cells and a significantly decreased proliferation rate. Up-regulation of calretinin in mesothelial cells of both genotypes increases the proliferation rate and induces a cobblestone-like epithelial morphology. The length of the S/G2/M phase is unchanged, however the G1 phase is clearly prolonged in CR−/− cells. They are also much slower to close a scratch in a confluent cell layer (2D-wound assay). In addition to a change in cell morphology, an increase in proliferation and mobility is observed, if calretinin overexpression is targeted to the nucleus. Thus, both calretinin and nuclear-targeted calretinin increase mesothelial cell proliferation and consequently, speed up the scratch-closure time. The increased rate of scratch closure in WT cells is the result of two processes: an increased proliferation rate and augmented cell mobility of the border cells migrating towards the empty space. We hypothesize that the differences in proliferation and mobility between WT and CR−/− mesothelial cells are the likely result from differences in their developmental trajectories. The mechanistic understanding of the function of calretinin and its putative implication in signaling pathways in normal mesothelial cells may help understanding its role during the processes that lead to mesothelioma formation and could possibly open new avenues for mesothelioma therapy, either by directly targeting calretinin expression or indirectly by targeting calretinin-mediated downstream signaling

CT enteroclysis: technique and clinical applications

CT enteroclysis (CTE) has been gradually evolving with technical developments of spiral and multidetector row CT technology. It has nowadays become a well-defined imaging modality for the evaluation of various small bowel disorders. Volume challenge of 2L of enteral contrast agent administrated to the small bowel via a nasojejunal catheter ensures luminal distension, the prerequisite for the detection of mural abnormalities, also facilitating the accurate visualization of intraluminal lesions. CT acquisition is centered on small bowel loops, reconstructed in thin axial slices and completed by multiplanar views. Image analysis is essentially done in cine-mode on work-stations. CTE is of particular diagnostic value in intermediate or advanced stages of Cohn's disease, including the depiction of extraintestinal complications. It has become the imaging modality of choice for the localization and characterization of small bowel tumors. The cause and degree of low-grade small bowel obstruction is more readily analyzed with the technique of CTE than conventional CT. Limitations of CTE concern the assessment of pure intestinal motility disorders, superficial mucosal lesions and arteriovenous malformations of the small bowel, which are not consistently visualized. CTE should be selectively used to answer specific questions of the small bowel. It essentially contributes to the diagnostic quality of modern small bowel imaging, and therefore deserves an established, well-defined place among the other available technique

Decreased activity of inducible nitric oxide synthase type 2 and modulation of the expression of glutathione S-transferase alpha, bcl-2, and metallothioneins during the differentiation of CaCo-2 cells.

Reactive oxygen species modulate the cell growth of a wide variety of mammalian cells. To determine whether oxidative metabolism is altered during the differentiation process, we studied the expression of pro- and antioxidant proteins in proliferating and differentiated CaCo-2 cells, a human colon adenocarcinoma cell line. Nitric oxide synthase type 2 (iNOS) produces nitric oxide (NO). Depending on its rate of synthesis, NO may either promote cellular and DNA damage or reduce the ability of other free radicals to induce cell injury. Using Western and Northern blot analysis and arginine conversion assay, we demonstrate that the expression of iNOS decreases when cells undergo differentiation. This biological event entails a diminished production of NO metabolites and correlates with the loss of activation of soluble guanylate cyclase activity. In differentiated cells, a 2-fold down-regulation of the nuclear factor kappa B activity was observed, suggesting that nuclear factor kappa B could be one of the iNOS gene regulatory factors in the CaCo-2 model. In parallel, we studied the expression of other antioxidant proteins including glutathione S-transferase alpha (GST alpha), bcl-2, and the metallothioneins (MTs). We show that the protein levels of GST alpha and MT increase during the differentiation of CaCo-2 cells, whereas bcl-2 levels decrease. Our investigation indicates that the expression of iNOS, GST alpha, bcl-2, and MT is associated with the enterocytic differentiation. The shift in the expression of specific antioxidant genes during CaCo-2 cell differentiation may occur to avoid alterations in the cell redox potential

Stem cell factor-based identification and functional properties of in vitro-selected subpopulations of malignant mesothelioma cells

Malignant mesothelioma (MM) is an aggressive neoplasm characterized by a poor patient survival rate, because of rapid tumor recurrence following first-line therapy. Cancer stem cells (CSCs) are assumed to be responsible for initiating tumorigenesis and driving relapse after therapeutic interventions. CSC-enriched MM cell subpopulations were identified by an OCT4/SOX2 reporter approach and were characterized by (1) increased resistance to cisplatin, (2) increased sensitivity toward the FAK inhibitor VS-6063 in vitro, and (3) a higher tumor-initiating capacity in vivo in orthotopic xenograft and allograft mouse models. Overexpression of NF2 (neurofibromatosis 2, merlin), a tumor suppressor often mutated or lost in MM, did not affect proliferation and viability of CSC-enriched MM populations but robustly decreased the viability of reporter-negative cells. In contrast, downregulation of calretinin strongly decreased proliferation and viability of both populations. In summary, we have enriched and characterized a small MM cell subpopulation that bears the expected CSC characteristics

Diagnostic performance of MRI for detection of intestinal fistulas in patients with complicated inflammatory bowel conditions

The diagnostic performance of magnetic resonance imaging (MRI) for detection of intestinal fistulas, other than perianal, in patients with known complicated inflammatory bowel conditions (CIBC) was investigated. Our study group consisted of 20 patients (12 women, mean age 43years) with CIBC, including Crohn's disease (n=13), colonic diverticulitis (n=3), colitis after radiotherapy (n=3) and of postoperative origin (n=1). Eleven surgically proven enteral fistulas were known in ten (50%) of these patients, being of enterovesical (n=3), enterocolic (n=2), enteroenteral (n=2), rectovaginal (n=2), rectovaginovesical (n=1) and of entercutaneous (n=1) localisation. The other ten patients (50%), used as the control group, showed MR features of CIBC, although without any fistulous tract. Multiplanar T1- and T2-weighted sequences had been performed, including gadolinium-enhanced acquisition with fat saturation (1.5T). MR findings were independently blindly and retrospectively reviewed by three radiologists for the presence and etiology of any fistula, as well as visualization and characterization of the fistulous tract. Results were compared with surgical findings (n=16) and clinical evolution (n=4). Interobserver agreement was calculated. Interobserver agreement kappa for fistula detection was 0.71. Overall sensitivity, specificity and accuracy for fistula detection were 78.6%, 75% and 77.2%, respectively. Sensitivity for fistula characterization was 80.6%, with visualization of the fistulous tract in all cases, whereby T1-weighted gadolinium-enhanced fat-saturated images were considered the most useful sequences. Gadolinium-enhanced MRI is a reliable and reproducible tool for detection of enteral fistulas secondary to inflammatory condition

Stem Cell Factor-Based Identification and Functional Properties of In Vitro-Selected Subpopulations of Malignant Mesothelioma Cells

Malignant mesothelioma (MM) is an aggressive neoplasm characterized by a poor patient survival rate, because of rapid tumor recurrence following first-line therapy. Cancer stem cells (CSCs) are assumed to be responsible for initiating tumorigenesis and driving relapse after therapeutic interventions. CSC-enriched MM cell subpopulations were identified by an OCT4/SOX2 reporter approach and were characterized by (1) increased resistance to cisplatin, (2) increased sensitivity toward the FAK inhibitor VS-6063 in vitro, and (3) a higher tumor-initiating capacity in vivo in orthotopic xenograft and allograft mouse models. Overexpression of NF2 (neurofibromatosis 2, merlin), a tumor suppressor often mutated or lost in MM, did not affect proliferation and viability of CSC-enriched MM populations but robustly decreased the viability of reporter-negative cells. In contrast, downregulation of calretinin strongly decreased proliferation and viability of both populations. In summary, we have enriched and characterized a small MM cell subpopulation that bears the expected CSC characteristics

Development of a matrix-based technology platform for the high throughput analysis of 3D cell cultures

The screening of large cell libraries is an important process in pharmaceutical discovery and R&D, e.g. to define drug targets or develop effective medicines. The goal of this project is the implementation of a screening platform based on 3D cultivation of primary human mesothelioma cells encapsulated in alginate hydrogels. To this end, new hydrogel compositions will be designed, tested and finally utilized in the Nanoliter Reactor (NLR) cultivation system that enables high throughput analysis of 3D cell cultures

EH-myomesin splice isoform is a novel marker for dilated cardiomyopathy

The M-band is the prominent cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. To investigate M-band alterations in heart disease, we analyzed the expression of its main components, proteins of the myomesin family, in mouse and human cardiomyopathy. Cardiac function was assessed by echocardiography and compared to the expression pattern of myomesins evaluated with RT-PCR, Western blot, and immunofluorescent analysis. Disease progression in transgenic mouse models for dilated cardiomyopathy (DCM) was accompanied by specific M-band alterations. The dominant splice isoform in the embryonic heart, EH-myomesin, was strongly up-regulated in the failing heart and correlated with a decrease in cardiac function (R=−0.86). In addition, we have analyzed the expressions of myomesins in human myocardial biopsies (N=40) obtained from DCM patients, DCM patients supported by a left ventricular assist device (LVAD), hypertrophic cardiomyopathy (HCM) patients and controls. Quantitative RT-PCR revealed that the EH-myomesin isoform was up-regulated 41-fold (P<0.001) in the DCM patients compared to control patients. In DCM hearts supported by a LVAD and HCM hearts, the EH-myomesin expression was comparable to controls. Immunofluorescent analyses indicate that EH-myomesin was enhanced in a cell-specific manner, leading to a higher heterogeneity of the myocytes' cytoskeleton through the myocardial wall. We suggest that the up-regulation of EH-myomesin denotes an adaptive remodeling of the sarcomere cytoskeleton in the dilated heart and might serve as a marker for DCM in mouse and human myocardiu

Habitat Selection by Demersal Nekton: Analysis of Videotape Data

In the past, analysis of habitat choice by marine nekton has been hampered by limited access to its environment. We suggest a method to facilitate study of habitat choice, using data gathered from videotapes. The aims of this study were (a) to identify members of a particular nekton assemblage, and (b) to identify environmental variables important to the assemblage as a whole. Data on species and environmental variables came from videotapes of a sandy-bottom shelf area (60 m depth) in the Gulf of Mexico off Pensacola, Florida, taken by a remote-controlled submersible travelling along defined transects. We analyzed these videotapes to derive information on habitat use of several species of fishes and large invertebrates. We divided the transects into sections which were homogeneous for bottom type and algal coverage, and in each section measured habitat variables and abundances of the most common and reliably identifiable species of demersal nekton. Factor analysis of species\u27 means for environmental variables identified patterns of habitat use among these species. The analysis identified these patterns by generating axes that represented environmental gradients. The patterns of habitat use by these species related to their preferences for different amounts of three-dimensional structure, algae, and infaunal and epifaunal organisms. We compared species distributions and habitat distributions on these axes to find which environmental gradients were of most importance in habitat selection by these species. We found that more species selected habitats on the basis of particular amounts of structure, fewer selected on the basis of algal coverage and infaunal organisms. Only one species seemed to select habitats on the basis of types or abundances of epifaunal organisms. Thus, amount of three-dimensional structure seemed an important variable to the sandy-bottom assemblage overall, followed by amount of algal coverage and types of infaunal organisms