Differential effects of biocompatible peritoneal dialysis fluids on human mesothelial and endothelial cells in 2D and 3D phenotypes.

INTRODUCTION: Peritoneal dialysis (PD) is a life maintaining treatment in patients with end-stage renal disease. Its chronic application leads to peritoneal mesothelial layer denudation and fibrotic transformation along with vascular activation of inflammatory pathways. The impact of different PD fluids (PDF) on mesothelial and endothelial cell function and repair mechanisms are not comprehensively described. MATERIALS AND METHODS: Mesothelial (MeT-5A) and endothelial cells (EA.hy926) were cultured in 1:1 ratio with cell medium and different PDF (icodextrin-based, amino acid-based, and glucose-based). Cell adhesion, cell migration, and cell proliferation in 2D and spheroid formation and collagen gel contraction assays in 3D cell cultures were performed. RESULTS: Cell proliferation and cell-mediated gel contraction were both significantly decreased in all conditions. 3D spheroid formation was significantly reduced with icodextrin and amino acid PDF, but unchanged with glucose PDF. Adhesion was significantly increased by amino acid PDF in mesothelial cells and decreased by icodextrin and amino acid PDF in endothelial cells. Migration capacity was significantly decreased in mesothelial cells by all three PDF, while endothelial cells remained unaffected. CONCLUSIONS: In 3D phenotypes the effects of PDF are more uniform in both mesothelial and endothelial cells, mitigating spheroid formation and gel contraction. On the contrary, effects on 2D phenotypes are more uniform in the icodextrin and amino acid PDF as opposed to glucose ones and affect mesothelial cells more variably. 2D and 3D comparative assessments of PDF effects on the main peritoneal membrane cell barriers, the mesothelial and endothelial, could provide useful translational information for PD studies.

Changes in expression of mesothelial BBS genes in 2D and 3D after lithium chloride and ammonium sulphate induction of primary cilium disturbance: a pilot study.

BACKGROUND: Malignant pleural mesothelioma (MPM), a rare and aggressive pleural tumor, has significant histological and molecular heterogeneity. Primary Cilium (PC), an organelle of emerging importance in malignancies, has been scarcely investigated in MPM. A critical molecular complex for the PC function is the BBSome and here we aimed at assessing its expression patterns in ordinary 2D and spheroid 3D cell cultures. METHODS: A human benign mesothelial cell line (MeT-5A), MPM cell lines (M14K, epithelioid MPM; MSTO, biphasic MPM), and primary MPM cells (pMPM) were used. Primers specific for the human BBS1, 2, 4, 5, 7, 9, 18 transcripts were designed, and quantitative real-time PCR (qRT-PCR) was done with ß-actin as the gene of reference. The relative gene expression across 2D and 3D cultures was analyzed by the expression factor (mean of 1/ΔCt values). With the 2-∆∆Ct method the gene expression fold changes were assessed from qRT-PCR data. Molecular changes using the PC-modulating drugs ammonium sulfate (AS) and lithium chloride (LC) were also determined. RESULTS: PC was present in all cells used in the study at approximately 15% of the observed area. BBSome transcripts were differentially expressed in different dimensions of cell culture (2D vs. 3D) in all cell lines and pMPM. Treatment with AS and LC affected the expression of the ciliary BBS2 and BBS18 genes in the benign as well as in the MPM cells. CONCLUSIONS: These data indicate distinct BBSome molecular profiles in human benign and MPM cells cultured in 2D and 3D dimensions and support the notion that PC genes should be investigated as potential MPM therapeutic targets.

The Alonissos Study: Cross-Sectional Study of the Healthcare Access and User Satisfaction in the Community of a Non-Profit-Line Greek Island.

Healthcare access and a high quality of the provided services to healthcare users are fundamental human rights according to the Alma Ata Declaration of 1978. Although 45 years have passed since then, health inequalities still exist, not only among countries but also within populations of the same country. For example, several small Greek islands have only a small Primary Healthcare Center in order to provide healthcare services to the insular population. In the current study, we investigated the level of self-reported overall, dental and mental health status and the level of satisfaction regarding the access to and the quality of the healthcare services provided by the Primary Healthcare center of Alonissos, along with registering the requirements for transportation to the mainland in order to receive such services. In this questionnaire-based cross-sectional study, 235 inhabitants of the remote Greek island of Alonissos that accounts for nearly 9% of the population participated (115 males and 120 females). The self-reported overall health status was reported to be moderate to very poor at a percentage of 31.49%, and the results were similar for dental and self-reported mental health status. Although nearly 60% of the participants reported very good/good quality of the healthcare provision, only 37.45% reported that the access to healthcare was very good/good, while around 94% had at least one visit to the mainland in order to receive proper healthcare services. Strategies for improving access to healthcare services need to be placed in remote Greek islands like Alonissos.

The Alonissos Study: Cross-Sectional Study of the Community Respiratory Health Status in a Greek Healthcare Access Underprivileged Island.

In this study, we investigated the self-reported (questionnaire-based) prevalence of Obstructive Sleep Apnoea Syndrome (OSAS) and the prevalence of Chronic Obstructive Pulmonary Diseases (COPD) in the context of demographics and adherence to the Mediterranean diet in the general population of Alonissos, a non-profit line island in Greece (i.e., with scarce boat transportation to the mainland). In this cross-sectional study, 236 inhabitants of Alonissos participated (circa 10% of the island's population), and 115 males and 121 females were evaluated with appropriate questionnaires for OSAS, COPD, and adherence to the Mediterranean diet and subsequently underwent spirometry testing to establish COPD diagnosis. The self-reported prevalence of OSAS and COPD was 9.44% and 18.8%, respectively. However, only 8.99% of the participants were diagnosed with COPD based on their spirometry testing. Adherence to the Mediterranean Diet was moderate. The high prevalence of COPD and OSAS in this underprivileged island in terms of healthcare access highlights the need for improvements in health promotion and primary healthcare provision in non-profit line Greek islands.

HΜGB1/sRAGE levels differ significantly between transudates and exudates.

High mobility group box 1(HMGB1) protein operates as an alarmin with multiple roles in immunity and cell homeostasis. It is highly expressed in epithelial barrier sites and acts via the binding to the receptor for advanced glycation end products (RAGE). Production of HMGB1 and soluble RAGE (sRAGE), a decoy receptor for HMGB1, has been implicated in several pulmonary diseases, but both have been scarcely investigated in pleural diseases. The aim of this study was to determine the levels of HMGB1 and sRAGE in transudative, malignant and parapneumonic pleural effusions (PEs) and to investigate the effect of low and high HMGB1 pleural fluid levels on MeT-5A cell adhesion, migration and spheroid formation, in each group. HMGB1 and sRAGE levels were significantly lower and higher in transudative PEs compared to malignant and parapneumonic PEs, respectively. Patients above 65 years of age had significantly lower HMGB1 and higher sRAGE levels compared to patients below 65 years old. Furthermore, incubation of MeT-5A cells with malignant or parapneumonic PEs bearing low or high levels of HMGB1 yielded significant differential effects on MeT-5A cell adhesion, migration and spheroid formation. In all types of effusions, high HMGB1 levels correlated with more adherence compared to low HMGB1 levels. In transudative and malignant PEs high HMGB1 levels correlated with decreased migration of MeT-5A cells while in parapneumonic ones the effect was the opposite. Only samples from parapneumonic PEs high in HMGB1 achieved uniform spheroid formation. These results reveal a clinical context-dependent effect of the HMGB1/sRAGE axis in PEs.

Cell and extracellular matrix interaction models in benign mesothelial and malignant pleural mesothelioma cells in 2D and 3D in-vitro.

Malignant pleural mesothelioma (MPM) is an aggressive tumour that grows in the pleural cavity. MPM spheroids released in the pleural fluid can form new tumour foci. Cell-cell, cell-extracellular matrix (ECM) interactions in 2D and 3D impact malignant cell behaviour during cell adhesion, migration, proliferation and epithelial-mesenchymal transition (EMT). In this study, epithelioid, biphasic and sarcomatoid MPM cell types as well as benign mesothelial cells were tested with regards to the above phenotypes. Fibronectin (FN) and homologous cell-derived extracellular matrix (hcd-ECM) treated substratum differentially affected the above phenotypes. 3D MPM spheroid invasion was higher in FN-collagen matrices in the epithelioid and biphasic cells, while 3D cell cultures of epithelioid and sarcomatoid MPM cells in FN-collagen showed a higher contractility compared to hcd-ECM-collagen. Cell aggregates demonstrated invasive behaviour in hcd-ECM matrices alone. Our results suggest that ECM and the dimensionality affect malignant cell behaviour during cell culture studies.

In Vitro Characterization of Cisplatin and Pemetrexed Effects in Malignant Pleural Mesothelioma 3D Culture Phenotypes.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with poor prognosis. The main treatment for MPM is doublet chemotherapy with Cisplatin and Pemetrexed, while ongoing trials test the efficacy of pemetrexed monotherapy. However, there is lack of evidence regarding the effects of Cisplatin and Pemetrexed on MPM cell phenotypes, especially in three-dimensional (3D) cell cultures. In this study, we evaluated the effects Cisplatin and Pemetrexed on cell viability using homologous cell derived extracellular matrix (hECM) as substratum and subsequently in the following 3D cell culture phenotypes: tumor spheroid formation, tumor spheroid invasion, and collagen gel contraction. We used benign mesothelial MeT-5A cells as controls and the MPM cell lines M14K (epithelioid), MSTO (biphasic), and ZL34 (sarcomatoid). Cell viability of all cell lines was significantly decreased with all treatments. Mean tumor spheroid perimeter was reduced after treatment with Pemetrexed or the doublet therapy in all cell lines, while Cisplatin reduced the mean spheroid perimeter of MeT-5A and MSTO cells. Doublet treatment reduced the invasive capacity of spheroids of cell lines into collagenous matrices, while Cisplatin lowered the invasion of the MSTO and ZL34 cell lines, and Pemetrexed lowered the invasion of MeT-5A and ZL34 cell lines. Treatment with Pemetrexed or the combination significantly reduced the collagen gel contraction of all cell lines, while Cisplatin treatment affected only the MeT-5A and M14K cells. The results of the current study can be used as an in vitro 3D platform for testing novel drugs against MPM for ameliorating the effects of first line chemotherapeutics.

Pleural Effusion IL-33/sST2 Levels and Effects of Low and High IL-33/sST2 Levels on Human Mesothelial Cell Adhesion and Migration.

Interleukin 33 (IL-33) is an alarmin with multiple roles in immunity and cell homeostasis, highly expressed in barrier sites, acting via the suppression of tumorigenicity 2 receptor (ST2). Production of IL-33 and soluble ST2 (sST2), a decoy receptor for IL-33, has been implicated in several pulmonary diseases, but both have been scarcely investigated in pleural diseases. The aim of this study was to determine the levels of IL-33 and sST2 in transudative (TrPEs), malignant (MPEs), and parapneumonic (PPEs) pleural effusions (PEs) and investigate the effect of PE fluids from each group with low and high IL-33/sST2 levels on MeT-5A cell adhesion and migration. IL-33 and sST2 pleural fluid levels were similar among TrPEs, MPEs, and PPEs. However, a significant correlation was found between IL-33 and LDH and in sST2 levels with lymphocyte counts in TrPEs. Additionally, in MPEs the levels of IL-33 correlated with the levels of sST2 and with the red blood cell counts. Furthermore, incubation of MeT-5A cells with MPEs and PPEs bearing low or high levels of IL-33/sST2 yielded significant differential effects on MeT-5A cell adhesion and migration. In MPEs, high IL-33/sST2 levels led to increased adhesion and migration of MeT-5A cells, while in PPEs the effect was the opposite, while no effect in both cell phenotypes was determined for TrPEs. These results reveal a clinical context dependent effect of the IL-33/sST2 axis in PEs.