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The COVID-19 pandemic continues to impose a major impact on global health and economy since its identification in early 2020, causing significant morbidity and mortality worldwide. Caused by the SARS-CoV-2 virus, along with a growing number of variants, COVID-19 has led to 651,918,402 confirmed cases and 6,656,601 deaths worldwide (as of December 27, 2022; https://covid19.who.int/ ). Despite advances in our understanding of COVID-19 pathogenesis, the precise mechanism by which SARS-CoV2 causes epithelial injury is incompletely understood. In this current study, robust application of global-discovery proteomics identified highly significant induced changes by the Spike S1 protein of SARS-CoV-2 in the proteome of alveolar type II (ATII)-like rat L2 cells that lack ACE2 receptors. Systems biology analysis revealed that the S1-induced proteomics changes were associated with three significant network hubs: E2F1, CREB1/RelA, and ROCK2/RhoA. We also found that pretreatment of L2 cells with high molecular weight hyaluronan (HMW-HA) greatly attenuated the S1 effects on the proteome. Western blotting analysis and cell cycle measurements confirmed the S1 upregulation of E2F1 and ROCK2/RhoA in L2 cells and the protective effects of HMW-HA. Taken as a whole, our studies revealed profound and novel biological changes that contribute to our current understanding of both S1 and hyaluronan biology. These data show that the S1 protein may contribute to epithelial injury induced by SARS-CoV-2. In addition, our work supports the potential benefit of HMW-HA in ameliorating SARS CoV-2-induced cell injury.

Secondary human lymphoid tissue immune reactions take place in a highly coordinated environment with compartmentalization representing a fundamental feature of this organization.In situprofiling methodologies are indispensable for the understanding of this compartmentalization. Here, we propose a complementary experimental approach aiming to reveal different aspects of this process. The analysis of human tonsils, using a combination of single cell phenotypic analysis based on flow cytometry and multiplex imaging and mass spectrometry-based methodologies, revealed a compartmentalized organization at cellular and molecular level. More specifically, the skewed distribution of highly specialized immune cell subsets and relevant soluble mediators was accompanied by a compartmentalized localization of several lipids across different anatomical areas of the tonsillar tissue. The performance of such combinatorial experimental approaches could lead to the identification of novelin situinteractions and molecular targets for thein vivomanipulation of lymphoid organ, particularly the germinal center, immune reactions.

Ranolazine (RAN) has previously been shown to lower the onset of cholinergic atrial fibrillation in intact animals; however, its efficacy in the setting of atrial tachycardia (AT) is unknown. The purpose of this study was to investigate the effects of RAN alone or in combination with amiodarone (AMIO) on rapid pacing-evoked right AT in rabbit hearts. Right atrial monophasic action potentials (MAPs) were recorded in 11 anesthetized rabbits, using combination MAP pacing catheters. Vulnerability to AT was tested by employing consecutive trains of rapid burst pacing prior to and after 2.4 mg/kg of RAN alone delivered intravenously and then in combination with 3 mg/kg of AMIO as a 15-minute infusion. Primary endpoints were postdrug AT reproducibility as well as cycle length (CL) and tachycardia duration. MAP duration at 75% repolarization and the effective refractory period (ERP) were assessed during programmed pacing to calculate the atrial postrepolarization refractoriness (aPRR = ERP - MAPD75%). AT was elicited in eight out of 11 rabbits; only these animals were included for further investigation. RAN did not abolish the inducibility of AT in any experiment; however, it prolonged its CL (baseline vs. RAN: 120 ± 16 ms vs. 138 ± 18 ms; p = 0.053). Supplemental AMIO further increased the AT CL (baseline vs. RAN + AMIO: 120 ± 16 ms vs. 152 ± 23 ms; p = 0.006), without affecting arrhythmia reinducibility. Slowing of the tachycardia after RAN or RAN + AMIO was associated with spontaneous termination of the arrhythmia. RAN prolonged the aPRR significantly, while AMIO in addition to RAN potentiated this effect. Neither RAN alone nor its combination with AMIO abolished the elicitation of AT in this model. However, both agents synergistically prolonged the aPRR, resulting in the slowing of AT and promoting spontaneous termination of the arrhythmia.

Owing to a high-volume industrial usage of the halogens chlorine (Cl(2)) and bromine (Br(2)), they are stored and transported in abundance, creating a risk for accidental or malicious release to human populations. Despite extensive efforts to understand the mechanisms of toxicity upon halogen exposure and to develop specific treatments that could be used to treat exposed individuals or large populations, until recently, there has been little to no effort to determine whether there are specific features and or the mechanisms of halogen exposure injury in newborns or children. We established a model of neonatal halogen exposure and published our initial findings. In this review, we aim to contrast and compare the findings in neonatal mice exposed to Br(2) with the findings published on adult mice exposed to Br(2) and the neonatal murine models of bronchopulmonary dysplasia. Despite remarkable similarities across these models in overall alveolar architecture, there are distinct functional and apparent mechanistic differences that are characteristic of each model. Understanding the mechanistic and functional features that are characteristic of the injury process in neonatal mice exposed to halogens will allow us to develop countermeasures that are appropriate for, and effective in, this unique population.

The COVID-19 pandemic continues to impose a major impact on global health and economy since its identification in early 2020, causing significant morbidity and mortality worldwide. Caused by the SARS-CoV-2 virus, along with a growing number of variants that have been characterized to date, COVID-19 has led to 571,198,904 confirmed cases, and 6,387,863 deaths worldwide (as of July 15th, 2022). Despite tremendous advances in our understanding of COVID19 pathogenesis, the precise mechanism by which SARS-CoV2 causes epithelial injury is incompletely understood. In this current study, robust application of global-discovery proteomics applications combined with systems biology analysis identified highly significant induced changes by the Spike S1 protein of SARS-CoV-2 in an ATII-like Rat L2 cells that include three significant network hubs: E2F1, CREB1/ RelA, and ROCK2/ RhoA. Separately, we found that pre-treatment with High Molecular Weight Hyaluronan (HMW-HA), greatly attenuated the S1 effects. Immuno-targeted studies carried out on E2F1 and Rock2/ RhoA induction and kinase-mediated activation, in addition to cell cycle measurements, validated these observations. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with standard immuno-targeted and cell cycle measurements revealed profound and novel biological changes that contribute to our current understanding of both Spike S1 and Hyaluronan biology. This data shows that the Spike S1 protein may contribute to epithelial injury induced by SARS-CoV-2. In addition, our work supports the potential benefit of HMW-HA in ameliorating SARS CoV2 induced cell injury.

Background and aims Chronic viral hepatitis is a prevalent disease with major health implications. Its underlying pathophysiological mechanisms are not fully understood. IL-1β and the NLRP3 inflammasome involvement has been suggested in recent years, from in vitro data and data from peripheral blood samples. Therefore, we investigated IL-1β and the NLRP3 inflammasome in liver tissues in an effort to clarify their role in the pathophysiology of chronic viral hepatitis. Methods We studied liver biopsies from patients with a new diagnosis of either chronic hepatitis B (CHB) and chronic hepatitis C (CHC) or patients with chronic hepatitis B in remission (CHB-rem). The biopsies were separated in two parts. The first part was sent to histology to determine the grade of inflammation and fibrosis. From the second part, RNA was extracted and converted to cDNA used in semi-quantitative Real-Time PCR to measure the levels of IL1B, CASP1, NLRP3, ASC and IL1RA. The cell lines used in the in vitro experiments were Huh7.5, LX2 and THP-1 in variety of combinations of monocultures, co-cultures and triple cultures with one of the cell lines infected with the JFH-1 HCV clone. From the cell cultures RNA was extracted and converted to cDNA. For cell lines, we focused in the expression of IL1B and NLRP3. Results The expression of IL1B, CASP1 and NLRP3 were found significantly different between our groups (p = 0.001, p = 0.001 and p = 0.038, respectively). CHB patients displayed significantly higher IL1B and CASP1 mRNA levels compared to both CHB-rem and CHC patients. IL1B expression significantly correlates with liver biochemical data in CHB patients (AST: p = 0.006, r = 0.457; ALT p = 0.002, r = 0.497). Finally, mRNA levels of IL1B in CHB patients significantly correlate with the degree of inflammation (p = 0.016) but not the stage of fibrosis (p = 0.362). Interestingly, the relative expression of IL1B in triple culture experiments in vitro was below of 1.5-fold, suggesting no activation of IL1B. Moreover, no activation of NLRP3 was demonstrated in all investigated in vitro conditions. Conclusion IL-1β might play an important role in the pathogenesis of chronic hepatic inflammation from HBV, but not from HCV.

Triggering factors of Acute Chest Syndrome (ACS) is a leading cause of death in patients with Sickle Cell Disease (SCD) and targeted therapies are limited. Chlorine (Cl2) inhalation happens frequently, but its role as a potential trigger of ACS has not been determined. In this study, we hypothesized that Cl2 exposure resembling that in the vicinity of industrial accidents induces acute hemolysis with acute lung injury, reminiscent of ACS in humanized SCD mice. When exposed to Cl2 (500 ppm for 30 min), 64% of SCD mice succumbed within 6 h while none of the control mice expressing normal human hemoglobin died (p, Graphical abstract Image 1, Highlights • Chlorine inhalation induces exaggerated hemolysis in sickle mice. • Free heme results in fatal acute chest syndrome in sickle versus control mice. • Chlorinated lipids mediate the effects of chlorine on the red blood cells. • Hemopexin alleviates chlorine induced hypoxia, and acute lung injury. • Therefore, postexposure hemopexin injection improves the survival of sickle mice.

AimWe investigated the mechanisms by which N1-(β-d-ribofuranosyl)-5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase (AMPK), decreases lung injury and mortality when administered to mice post exposure to bromine gas (Br2).MethodsWe exposed male C57BL/6 mice and heme oxygenase-1 (HO-1)-deficient (HO-1−/−) and corresponding wild-type (WT) littermate mice to Br2 (600 ppm for 45 or 30 min, respectively) in environmental chambers and returned them to room air. AICAR was administered 6 h post exposure (10 mg·kg−1, intraperitoneal). We assessed survival, indices of lung injury, high mobility group box 1 (HMGB1) in the plasma, HO-1 levels in lung tissues and phosphorylation of AMPK and its upstream liver kinase B1 (LKB1). Rat alveolar type II epithelial (L2) cells and human club-like epithelial (H441) cells were also exposed to Br2 (100 ppm for 10 min). After 24 h we measured apoptosis and necrosis, AMPK and LKB1 phosphorylation, and HO-1 expression.ResultsThere was a marked downregulation of phosphorylated AMPK and LKB1 in lung tissues and in L2 and H441 cells post exposure. AICAR increased survival in C57BL/6 but not in HO-1−/− mice. In WT mice, AICAR decreased lung injury and restored phosphorylated AMPK and phosphorylated LKB1 to control levels and increased HO-1 levels in both lung tissues and cells exposed to Br2. Treatment of L2 and H441 cells with small interfering RNAs against nuclear factor erythroid 2-related factor 2 or HO-1 abrogated the protective effects of AICAR.ConclusionsOur data indicate that the primary mechanism for the protective action of AICAR in toxic gas injury is the upregulation of lung HO-1 levels.

Coronaviruses have been known to cause respiratory infections in humans and intestinal infections in other mammals. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, is the 7th virus of the Coronaviridae family that is known to infect humans. Until 2002, four Coronaviruses infecting humans were described (HCoV- NL63, HCoV-229E, HCoV- OC43 and HKU1). These viruses caused only mild respiratory diseases in immunocompetent hosts. Since 2002, three highly pathogenic viruses from this family have been identified. SARS-CoV (also referred to as SARS-CoV-1) is an enveloped, positive-sense, single-stranded RNA virus which infects the epithelial cells within the lungs. The virus enters the host cell by binding to angiotensin-converting enzyme 2 [1]. It infects humans, bats, and palm civets [1]. It emerged in 2002–2003 with an approximate 10% mortality but limited transmissibility (approximately 8100 cases, R0, “Cyclosporine inhibits the replication of coronaviruses and could potentially suppress the cytokine storm associated with coronavirus infections”

Display Omitted CLIC3 and CLIC4 gene expression is induced in malignant pleural mesothelioma.CLIC3 and CLIC4 gene expressions are significantly positively associated.Functional Enrichment for GO reveals a role in EMT pathways and water transport. BackgroundChloride Intracellular Channels (CLICs) are contributing to the regulation of multiple cellular functions. CLICs have been found over-expressed in several malignancies, and therefore they are currently considered as potential drug targets. The goal of our study was to assess the gene expression levels of the CLIC's 1-6 in malignant pleural mesothelioma (MPM) as compared to controls. MethodsWe used gene expression data from a publicly available microarray dataset comparing MPM versus healthy tissue in order to investigate the differential expression profile of CLIC 1-6. False discovery rates were calculated and the interactome of the significantly differentially expressed CLICs was constructed and Functional Enrichment Analysis for Gene Ontologies (FEAGO) was performed. ResultsIn MPM, the gene expressions of CLIC3 and CLIC4 were significantly increased compared to controls (p=0.001 and p

The aim of our study was to assess the differential gene expression of Parkinson protein 7 (PARK7) interactome in malignant pleural mesothelioma (MPM) using data mining techniques to identify novel candidate genes that may play a role in the pathogenicity of MPM. We constructed the PARK7 interactome using the ConsensusPathDB database. We then interrogated the Oncomine Cancer Microarray database using the Gordon Mesothelioma Study, for differential gene expression of the PARK7 interactome. In ConsensusPathDB, 38 protein interactors of PARK7 were identified. In the Gordon Mesothelioma Study, 34 of them were assessed out of which SUMO1, UBC3, KIAA0101, HDAC2, DAXX, RBBP4, BBS1, NONO, RBBP7, HTRA2, and STUB1 were significantly overexpressed whereas TRAF6 and MTA2 were significantly underexpressed in MPM patients ( network 2). Furthermore, Kaplan-Meier analysis revealed that MPM patients with high BBS1 expression had a median overall survival of 16.5 vs. 8.7 mo of those that had low expression. For validation purposes, we performed a meta-analysis in Oncomine database in five sarcoma datasets. Eight network 2 genes (KIAA0101, HDAC2, SUMO1, RBBP4, NONO, RBBP7, HTRA2, and MTA2) were significantly differentially expressed in an array of 18 different sarcoma types. Finally, Gene Ontology annotation enrichment analysis revealed significant roles of the PARK7 interactome in NuRD, CHD, and SWI/SNF protein complexes. In conclusion, we identified 13 novel genes differentially expressed in MPM, never reported before. Among them, BBS1 emerged as a novel predictor of overall survival in MPM. Finally, we identified that PARK7 interactome is involved in novel pathways pertinent in MPM disease.

Background Human exposure to engineered nanoparticles has been linked to pleural effusion, inflammation and fibrosis. Silver nanoparticles (AgNPs) are widely used in medical and domestic products, increasing the risk of occupational and domestic exposure. We assessed the influence of AgNPs on adhesion and proliferation of sheep primary pleural mesothelial cells. Materials and methods Cells were used for cell adhesion (90 min) and proliferation experiments (3 days) while exposed to 20 nm and 60 nm AgNPs (0.2 μg/ml and 2 μg/ml) using colorimetric assays. Results Exposure to 0.2 μg/ml of 20 nm and 60 nm AgNPs significantly increased cell adhesion, while at 2 μg/ml this effect was not elicited. Cell proliferation was significantly increased by both 20 nm and 60 nm AgNPs at 0.2 μg/ml, while at 2 μg/ml this effect was only elicited by the 60 nm AgNPs. Conclusion AgNPs alter the adhesive and proliferative properties of primary pleural mesothelial cells.

Introduction: The role of ENaC in pleural fluid turnover is important in pleural effusions, a clinical hallmark of MPM. Biochemical and functional expression of ENaC is reported both in normal and in MPM cells. ENaC activity also influences cell cycle progression thus it may influence cell proliferation and thus tumor growth. TnT was recently described as a structure favoring direct distant cell-cell communication, however ENaC contribution in TnT development has not been investigated. Aim: To assess the role of ENaC in cell proliferation and TnT formation in the context of MPM pathobiology. Methods: MeT-5A (benign mesothelial cells), M14K (epithelioid MPM cells), MSTO (biphasic MPM cells) and ZL34 (sarcomatoid MPM cells) were used in the study. Cell proliferation over 3 days and TnT formation in 1 day was tested with 10% FBS RPMI (Controls) or by ENaC inhibition with Amiloride (AM, 10-5 M in 10% FBS RPMI). Crystal violet staining was used to assess changes in cell proliferation and for visualizing TnTs. Results: Cell proliferation post AM treatments were lower in MSTO and ZL34 cell lines at day 3, (MSTO Control 1888.3±106%, AM 1610.5±115.5%, p Conclusions: MeT-5A and M14K cell proliferation was unaffected by ENaC inhibition. AM significantly lowered cell proliferation on MSTO and ZL34 cell lines. TnT formation was unaffected in all cell lines regardless of ENaC activity.

Vascular endothelial growth factor (VEGF), a cytokine that increases vascular permeability to water and proteins and induces angiogenesis, has been implicated in the development of pleural effusions. Inflammatory and malignant pleural effusions are rich in VEGF content while mesothelial cells produce and excrete VEGF. In this report we aimed at investigating by means of electrophysiology the direct effects of VEGF on the parietal and visceral sheep pleura as well as the type of receptors that mediate this effect. Our findings show that VEGF has a direct effect on the pleural mesothelium rendering it more permeable and this effect is mediated through the stimulation of VEGF receptor 2. Our findings shed more light to the role of VEGF in the pathogenesis of pleural effusions and provide functional evidence for a role of VEGFR2 on the pleural mesothelium that has never been studied before.

Airway smooth muscle cells (ASMCs) participate in tissue remodeling characteristic of airway inflammatory diseases like asthma. Inflammation and hypoxia pathways are often interconnected and the regulatory subunit of the hypoxia inducible factor, HIF-1α, has been recently shown to be induced by cytokines. Here we investigate the effect of individual or combined treatment of ASMCs with the inflammatory mediator TNFα and/or hypoxia on the expression of HIF-1α, HIF-1 targets and inflammation markers. TNFα enhances HIF-1α protein and mRNA levels, under both normoxia and hypoxia. TNFα-mediated induction of HIF-1α gene transcription is repressed by inhibition of the NF-κB pathway. Despite the up-regulation of HIF-1α protein, the transcription of HIF-1 target genes remains low in the presence of TNFα at normoxia and is even reduced at hypoxia. We show that the reduction in HIF-1 transcriptional activity by TNFα is due to inhibition of the interaction of HIF-1α with ARNT and subsequent blocking of its binding to HREs. Comparison between hypoxia and TNFα for their effects on the expression of inflammatory markers shows significant differences: hypoxia up-regulates the expression of IL-6, but not RANTES or ICAM, and reduces the induction of VCAM by TNFα. Finally, ex vivo treatment of rabbit trachea strips with TNFα increases HIF-1α protein levels, but reduces the expression of HIF-1 targets under hypoxia. Overall, TNFα induces HIF-1α mRNA synthesis via an NF-κB dependent pathway but inhibits binding of HIF-1α to ARNT and DNA, while hypoxia and TNFα have distinct effects on ASMC inflammatory gene expression.

Ranolazine is a relatively novel antiischemic/antianginal compound with antiarrhythmic properties. We investigated its ability to shorten the time to conversion of postoperative atrial fibrillation (POAF) when added to amiodarone after coronary artery bypass graft (CABG) surgery. In this prospective, randomized, allocation-concealed, single-blind, single-site clinical trial, we enrolled consecutive eligible patients who developed POAF after elective on-pump CABG surgery. Participants were randomized to receive either ranolazine 375 mg twice daily orally plus intravenous amiodarone (active group) or intravenous amiodarone alone (control group). We enrolled 41 patients; 20 in the active and 21 in the control group. There were no significant differences between the groups in terms of age, procedural duration, extracorporeal circulation time, and aortic cross-clamp time. Mean time of conversion was significantly shorter in the active group (19.9 ± 3.2 vs 37.2 ± 3.9 hours, P < .001), suggesting that compared to amiodarone alone, the ranolazine–amiodarone combination had a superior antiarrhythmic effect against POAF.

Mesothelium is an important part of the peritoneal barrier for water and ion transport, essential for effective peritoneal dialysis (PD). Peritoneal fibrosis has been associated with PD treatment failure. Endothelin-1 (ET-1) is a potent vasoactive peptide, involved in pathologic fibrotic processes. Its action is mediated mainly by endothelin type A (ETA ) and type B (ETB ) receptors. The aim of this study was to investigate, by Ussing chamber experiments, the effect of ET-1 on the transmesothelial electrical resistance (RTM ) of the isolated visceral sheep peritoneum. Intact sheets of visceral peritoneum were obtained from 40 adult sheep and mounted in Ussing-type chambers. ET-1 (10(-7) M), BQ-123 (ETA receptor antagonist; 10(-6) M), BQ-788 (ETB receptor antagonist; 10(-6) M), and their combinations were added on the apical and the basolateral side of the peritoneum. RTM was measured before and serially after addition of the substances, and changes were registered as percentage (ΔRTM %). RTM increased within 1 min after addition of ET-1 apically (ΔRTM 65.03 ± 15.87%; P

Nanoparticles have been implicated in the development of pleural effusions in exposed factory workers while in experimental animal studies it has been shown that they induce inflammation, fibrosis and carcinogenesis in the pleura. The scope of this study was to investigate the direct effects of silver nanoparticles exposure on the membrane permeability of sheep parietal pleura, of primary sheep pleural cell monolayers and on a human mesothelial cell line. Our findings suggest that acute (30min) exposure increases the pleural permeability ex vivo, while longer (24h) exposure in vivo leads to late decrease of the pleural cell monolayers permeability.

Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy. In MPM cells the amiloride sensitive sodium channel (ENaC) is expressed, however its role in cell migration is unknown. Aim: Our aim was to investigate the role of ENaC inhibition on MPM cell migration during wound healing. Materials and methods: The human cell lines MeT-5A (benign mesothelial cells), M14K (epithelioid MPM), MSTO (biphasic MPM) and ZL34 (sarcomatoid MPM) were used in this study. Cell migration was tested at 4 hrs on fibronectin (FN) and at 8 hrs on homologous cell derived extracellular matrix (ECM) coated plates. ENaC inhibition was done by amiloride (AM, 10 -5 M). Migration index (MI) was used as a measure of cell migration. Results: The migration of MeT-5A (MI=0.06±0.01; p Conclusion: ENaC inhibition differentially decreases the cell migration of normal mesothelial and MPM cells in a histological and substartum dependent manner.

The purpose of this paper is to study the ionic permeability of the leptomeninges related to the effect of ouabain (sodium-potassium-ATPase inhibitor) and amiloride (epithelial sodium channel (ENaC) inhibitor) on the tissue, as well as identify the presence of ion channels.Cranial leptomeningeal samples from 26 adult sheep were isolated. Electrophysiological measurements were performed with Ussing system and transmembrane resistance values (R(TM) in Ω*cm(2)) obtained over time. Experiments were conducted with the application of ouabain 10(-3) M or amiloride 10(-5) M at the arachnoidal and pial sides. Immunohistochemical studies of leptomeningeal tissue were prepared with alpha-1 sodium-potassium-ATPase (ATP1A1), beta-ENaC, and delta-ENaC subunit antibodies.The application of ouabain at the arachnoidal side raised the transmembrane resistance statistically significantly and thus decreased its ionic permeability. The addition of ouabain at the pial side led also to a significant but less profound increment in transmembrane resistance. The addition of amiloride at the arachnoidal or pial side did not produce any statistical significant change in the R(TM) from controls (p0.05). Immunohistochemistry confirmed the presence of the ATP1A1 and beta- and delta-ENaC subunits at the leptomeninges.In summary, leptomeningeal tissue possesses sodium-potassium-ATPase and ENaC ion channels. The application of ouabain alters the ionic permeability of the leptomeninges thus reflecting the role of sodium-potassium-ATPase. Amiloride application did not alter the ionic permeability of leptomeninges possibly due to localization of ENaC channels towards the subarachnoid space, away from the experimental application sites. The above properties of the tissue could potentially be related to cerebrospinal fluid turnover at this interface.

The vascular surface distribution of the subarachnoid space has not been studied extensively. The aim of our study was to develop a method of computer-assisted estimation of the distribution of the vascular network in the cortical leptomeninges and subarachnoid space and model it to aid the study of the physiology of brain surface dialysis. Nine sheets of leptomeningeal tissue were obtained from adult sheep. Fourteen image sample areas of 4 cm² each were acquired and processed with ImageJ. The vascular and nonvascular areas of the cortical subarachnoid space were identified using a "projected surface" approach. The modeling equations were used to predict the behavior of brain surface dialysis processes. The mean surface area of identified subarachnoid vessels was 0.354 ± 0.02 cm² per 1 cm² of tissue. The mean meningeal area with unidentified vessels was 0.646 ± 0.02 cm²/1 cm², and the difference between these surfaces was significant (p < 0.0001). The modeling equations used predict that modifying the vessel diameter of the subarachnoid space could alter the efficiency of brain surface dialysis. The computer-assisted modeling of the vascular surface of the cortical subarachnoid space may be a useful tool in depicting its morphology and assessing the physiology during brain surface dialysis.

AIM: Insulin promotes ion transportation across epithelia, mainly kidneys, leading to water and electrolyte abnormalities, possibly causing ‘insulin oedema syndrome’, which rarely presents as pleural effusion. Direct stimulation of sheep pleura by insulin and the possible electrophysiology mechanisms involved were investigated. MATERIAL AND METHODS: Sheep visceral and parietal pleural specimens were mounted between Ussing chambers. Insulin solutions (10 −9 to 10 −5 M), L-NAME, Nitroprussid sodium, amiloride and ouabain were used. Trans-mesothelial Resistance was determined. Immunohistochemistry for presence of Insulin Receptors was performed. RESULTS: Insulin increased Trans-mesothelial Resistance within 1st minute when added mesothelially of visceral (p=0.008) and parietal pleura (p=0.046) for concentrations higher than 10 −7 M. L-NAME or Nitroprussid sodium didn’t but amiloride and ouabain inhibited insulin's effect. Immunohistochemistry revealed the presence of Insulin Receptors. CONCLUSION: Insulin changes the permeability of sheep pleura by altering its electrophysiology and may interfere in pleural effusion formation. Involvement of Insulin Receptors may be suggested.

Potassium channel openers are known to act on potassium ATP-dependent channels in cardiac tissue. Such agents may exacerbate acceleration of acute ischemia-induced ventricular repolarization and aggravate arrhythmias. To test whether activation of K ATP channels during the healing period of myocardial infarction (MI) can still influence the electrophysiologic properties and the type of inducible arrhythmias, we investigated the effects of bimakalim (BIM) on sustained ventricular tachycardia (VT) 4 days after ligation of the left anterior descending (LAD) coronary artery in pigs. Programmed stimulation was performed to elicit VT prior to and after intravenous (IV) BIM. Combination monophasic action potential (MAP)/PACING catheters were used to enable simultaneous ventricular MAP recording and pacing. Ventricular effective refractory period (ERP) and MAP duration determined at 50% and 90% repolarization were measured prior to and after BIM. After completion of baseline measurements, BIM was consecutively given at 0.5, 1, and 3 mg/kg bolus followed by 0.025, 0.05, and 0.1 mg/kg per minute maintenance infusion, respectively. From a total of 23 pigs subjected to LAD ligation, 4 animals succumbed to infarction and the remaining 19 animals were studied by programmed stimulation. Only animals that exhibited reproducible and hemodynamically stable monomorphic VTs during control stimulation were selected for evaluation (n = 14). After the first, second, and third dose of BIM, the mean VT rate was increased by 6%, 14% (P

Quercetin, a flavonoid with anti-oxidant, metal chelating, kinase modulating and anti-proliferative properties, can induce hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia, but its mechanism of action has not been determined. In this study we characterized the induction of HIF-1alpha and the inhibition of cell proliferation caused by quercetin in HeLa and ASM (airway smooth muscle) cells and examined the effect of iron on these processes. Furthermore, we investigated the relevance of the intracellular levels of quercetin to HIF-1alpha expression and cell proliferation. Our data demonstrate that quercetin depletes intracellular calcein-chelatable iron and that supplying additional iron from extracellular or intracellular pools abrogates the induction of HIF-1alpha by quercetin. Moreover, addition of iron reverses the quercetin-induced inhibition of DNA synthesis, cell proliferation and cycle progression, but to different extents, depending on cell type. We propose that quercetin stabilises HIF-1alpha and inhibits cell proliferation predominantly by decreasing the concentration of intracellular iron through chelation.

It is well-known that parapneumonic effusions lead to the formation of inflammatory exudates which contain an increasing amount of inflammatory cells, especially polymorphonuclear. At these pathological conditions characterized by oxidative stress, ascorbic acid (AA) plays an important role in quenching free radicals, so that it could protect neutrophils and mesothelial cells from oxidative damage. Besides that ascorbic acid and its metabolite dehydroascorbic acid (DHA) alters the sheep visceral and parietal pleura permeability. More specific ascorbic acid as well as dehydroascorbic acid decreases the permeability of pleura after addition on apical and basolateral side in both visceral and parietal pleurae. It seems that, AA and DHA have an opposite action upon pleura from that of the inflammatory mediators, like VEGF, which increases the permeability of pleura and causes mesothelial barrier dysfunction. The decrease of pleura permeability induced by AA and DHA suggest the hypothesis that AA and/or its metabolite DHA during inflammatory reactions not only protects mesothelial cells from oxidative damage, but also contributes to maintaining the mesothelial barrier function. Consequently, the inflammatory pleural fluid may be trapped in pleural space and the inflammation may be restricted, and have extension avoided.

Malignant pleural mesothelioma (MPM) is an aggressive cancer. MPM cells express aquaporin-1 (AQP1) that in other cancers has been shown to participate in the tumor metastasis processes. However, in MPM patients AQP1 overexpression is an independent prognostic factor favoring survival. In this study we aimed at evaluating the role of AQP1 in cell adhesion, migration, and tumor sphere formation in nonmalignant mesothelial cells (MeT-5A) and in epithelioid (M14K) and sarcomatoid (ZL34) MPM cell lines. We used fibronectin (FN) or homologous cell-derived extracellular martrix (ECM) substratum to investigate the role of AQP1 in these experimental phenotypes, inhibiting AQP1 by 10−5 M mercury chloride (MC). Deposited ECM during cell culture exhibited significant concentration differences among cell types. ZL34 cell adhesion was significantly higher than MeT-5A or M14K cells on FN and ECM. MeT-5A and M14K cell adhesion on FN was sensitive to AQP1 inhibition, whereas AQP1 inhibition on ECM was limited to M14K cells. Wound healing in ZL34 cells was significantly higher than MeT-5A and M14K cells on FN and ECM. AQP1 inhibition significantly lowered cell migration in ZL34 cells on FN and ECM. Sphere formation was not dependent on FN or ECM in the media. AQP1 inhibition in FN media reduced sphere formation in M14K cells, whereas, in ECM, all three cell types were sensitive to AQP1 inhibition.

Aim: Malignant pleural mesothelioma (MPM) is an aggresive malignancy. Recently the role of water channels in MPM has started being investigated and patients with aquaporin-1 (AQP1) have favorable survival. In MPM the amiloride sensitive sodium channel (EnaC) is also expressed. Our aim was to investigate the role of AQP1 and ENaC in cell adhesion in mesothelial cells and in MPM cells. Methods: The human cell lines Met5A (benign mesothelial cells), M14K (epithelioid mesothelioma), ZL35 (sarcomatoid mesothelioma) were used. Cell adhesion was tested in fibronectin coated plates and in homologous cell derived extracellular matrix coated plates. In some experiments AQP1 inhibition or ENaC inhibition was performed with HgCl 2 and amiloride respectively. Results: Inhibition of AQP1 resulted in significant decrease of Met5A and M14K cell adhesion irrispective of plate coating (p Conclusion: In cell adhesion of mesothelioma cells, AQP1 inhibition is cell-type dependent while EnaC inhibition is cell-type and extracellular matrix-type dependent. These results render AQP1 and/or EnaC drug targeting worth investigating in MPM pathobiology.

Objective: Exposure to engineered nanoparticles (NPs) has been associated with lung and pleura pathology such as inflammation, fibrosis and cancer. Silver (Ag) NPs are used in a large number of consumer products and medical devices increasing therefore the possibility of user exposure. The aim of this study was to investigate the possible effects of engineered AgNPs on the function and expression of ENaC on parietal sheep pleura. Materials and methods: In the present study the short circuit current (I SC ) and the transmesothelial resistance (R TM ) of sheep parietal pleura was monitored in Ussing chambers after the preincubation of the tissue with spherical AgNPs of 20nm in size for 30 min. The effect of the AgNPs on the function of the epithelial sodium channel (ENaC) was investigated by the addition of the inhibitor amiloride (10 -5 M) apically. Moreover, the expression of αENaC subunit was analyzed in these samples by Western Blotting in order to assess effects on the protein level. Results: AgNPs (20 nm; 2μg/ml) increased the amiloride sensitive I SC indicating that ENaC function is enhanced by this treatment. The R TM of the sheep parietal pleura was not altered, suggestive of no effects on the tight junctions and the tissue integrity. Finally, the levels of αENaC expression were similar in all groups. Conclusions: AgNPs of 20 nm affect sodium permeability of the sheep parietal pleura by increasing ENaC activity. This effect is not due to increased αENaC expression. These data suggest that AgNPs induce changes in the function of pleural ENaC.

Cells adjust to osmotic stress regulating their volume by absorbing or secreting osmolytes and water. Even though pleural mesothelial cells face osmolality changes in pleural effusions, their cell volume regulation properties have been poorly studied. In this study we aim to investigate the effects of osmotic stress on the adhesion and migration rate of benign and malignant pleural mesothelial cells. The effect of osmotic gradients in cell adhesion was studied by crystal violet assay and in cell migration by wound healing assay in Met5-A, M14K and ZL-34 cell lines. The adhesion of M14K and ZL-34 cells was partially inhibited by hyposmotic stress (∼255mOsm) in comparison to isomotic condition (∼295mOsm). Hyperosmotic stress (∼350mOsm) significantly promoted adhesion of Met5-A cells. The migration rate was partially inhibited by hyperosmotic conditions in Met5-A cells and by hyposmotic conditions in ZL-34 cells. M14K cells migration rate was decreased under both hyposmotic and hyperosmotic conditions. Our results indicate that osmolality gradient differentially affects the migratory and adhesive properties of pleural benign and malignant mesothelial cells.

Summary Background Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative disorder. Cytokines contribute an important but yet undefined role to its pathogenesis. Objectives The present study aims to compare serum levels of cytokines involved in Th-1 and Th-2 immunity, such as interleukins (IL) IL-2, IL-4, IL-8, IL-10, interferon- γ (IFN- γ ) and IL-12 (p40) in patients with IPF and healthy volunteers. Twenty patients with IPF and 40 healthy controls (HC) participated. Methods Cytokines were assessed by enzyme-linked immunoabsorbent assay (ELISA). Results Median values of serum IL-2, IL-8, IL-10, IL-12 (p40) were higher in the IPF than the control group: IPF group: 1.05U/ml, 12.55, 10.13, 44.17pg/ml; control group: 0.05U/ml, 6.91, 0.75, 4.51pg/ml, respectively \( P 0.05 \) . IFN- γ serum levels were lower in the IPF (0.19pg/ml) than in the control group (0.49pg/ml). IL-4 values did not differ in a statistically significant way among the groups: 8.40pg/ml in the IPF group, and 7.46pg/ml in the control group \( P > 0.05 \) . IL-4 positively correlated to fast expiratory volume in 1s (FEV 1 %) and forced vital capacity (FVC%), while IL-8 negatively correlated to the respective values \( P 0.005 \) . Conclusions IL-2, IL-8, IL-10 and IL-12 (p40) were found to be elevated in the sera of patients with IPF. IFN- γ was found to be decreased in the sera of patients with IPF.

The peritoneal mesothelium constitutes an ion transport barrier that is taken advantage of in peritoneal dialysis. The aim of this study was to investigate the effects of epinephrine on the electrical transmesothelial resistance (R(TM)) of the isolated parietal sheep peritoneum by means of Ussing-type chamber experiments. Intact parietal (diaphragmatic) peritoneal samples were obtained from adult sheep immediately after sacrifice and transferred within 0.5 h to the laboratory in a cooled Krebs-Ringer bicarbonate solution (4 degrees C, pH 7.5), bubbled with 95% O2-5% CO2. A parietal peritoneal planar sheet was mounted in a Ussing-type chamber. Epinephrine (10(-7) M) was added to the apical and the basolateral side. The R(TM) was measured before and serially after the addition of epinephrine for 30 min. As active ion transport is temperature-dependent, all measurements were performed at 37 degrees C. The results were calculated as means with standard errors (x +/- SE) of six independent experiments. The control R(TM) was 20.05 +/- 0.61 ohm x cm2. The addition of epinephrine to the basolateral side within 1 min induced an increase of R(TM) to 21.8 +/- 0.45 ohm x cm2, which decreased thereafter progressively to reach control values again after 15 min. A similar effect of epinephrine on the apical side was apparent with a rapid rise of R(TM) to 22.5 +/- 0.66 ohm x cm2 and a subsequent decrease (P < 0.05). A clear association between the R(TM) and active ion transport was established from previous studies. The results of our study indicate a rapid action of epinephrine on the parietal peritoneum permeability.

Objectives Assess whether age influences standard biochemical parameters used in the differential diagnosis of transudative and exudative pleural effusions. Design and methods We retrospectively analyzed data from the database of our clinic from 225 patients with pleural effusions categorized based on their final diagnosis in 5 groups: transudates 41 (18%), uncomplicated parapneumonic 26 (12%), complicated parapneumonic 20 (9%), tuberculosis 35 (15%) and lung cancer 103 (46%). We tested whether age correlated with pleural fluid protein or lactate dehydrogenase. Results There was a statistically significant inverse correlation only between the age and the pleural fluid protein content in patients with uncomplicated parapneumonic effusions with correlation coefficient r = − 0.6 [(95% CI = − 0.8 to − 0.28); p = 0.001]. Linear regression analysis showed that this association is given by the equation: age = 101.998 − 10.03 protein. In the same group of patients age was not correlated with serum protein content. Conclusions Our study shows that age may be a confounding factor in the differential diagnosis of transudative and exudative pleural effusions. Clinicians should be aware of this finding especially when dealing with elders.

In order to test the serum levels of ICAM-1, ICAM-2 and ICAM-3 in patients with idiopathic pulmonary fibrosis (IPF), twenty patients with IPF and eleven with secondary interstitial fibrosis (SIF), as well as forty healthy volunteers (HV) were studied. Serum intracellular adhesion molecules (ICAM) 1, 2 and 3 were assessed by ELISA. Functional respiratory tests, which included spirometry and lung diffusing capacity were simultaneously performed. Median values of serum ICAM-1 and ICAM-2 were higher in the patients' than in the healthy volunteers' (HV) group: IPF group: 946.60 ng/ml and 400.14 ng/ml; SIF group: 901.58 ng/ml and 378.27 ng/ml; HV group: 308.40 ng/ml and 217.55 ng/ml, respectively (p

The goal of the present investigation was to describe the prevalence of and clinical factors associated with sleep-disordered breathing in children and adolescents. Children and adolescents (3,680 in all, 1–18 years old) attending schools in central Greece were surveyed by questionnaires distributed to parents. We found a similar prevalence of habitual snoring (present every night) among three different age groups (5.3%, 4%, and 3.8% in 1–6-, 7–12-, and 13–18-year-old subjects, P = NS). Several children with an adenoidectomy and/or tonsillectomy were snoring every night (6.1%), whereas sleepiness at school was more common in habitual snorers than in nonhabitual snorers (4.6 vs. 2%, P = 0.03). Seventy randomly selected subjects among 307 snorers without adenoidectomy and/or tonsillectomy underwent polysomnography. The estimated frequency of obstructive sleep apnea-hypopnea among children without adenoidectomy and/or tonsillectomy was 4.3%. Factors associated with snoring were: male gender (odds ratio 1.5 (confidence interval, 1.2–1.9)); chronic rhinitis (2.1 (1.6–2.7)); snoring in father (1.5 (1.2–1.9)), mother (1.5 (1.1–2.0)), or siblings (1.7 (1.2–2.4)); adenoidectomy in mother (1.5 (1.0–2.2)); and passive smoking (1.4 (1.1–1.8)). In conclusion, snoring every night was equally prevalent in younger and older ages, more frequent in males, and present even in some children with a history of adenoidectomy and/or tonsillectomy. Chronic rhinitis, family history of snoring, and exposure to cigarette smoke were associated with an increased frequency of habitual snoring. Pediatr Pulmonol. 2004; 37:499–509. © 2004 Wiley-Liss, Inc.

Left-handedness has been associated with asthma and allergic disorders. The Geschwind-Behan-Galaburda (GBG) hypothesis could explain this association. In view of previous findings, we investigated the distribution of laterality scores among asthmatic children and controls aged 4-8 years old. Seventy families with asthmatic children were administered the International Study of Asthma and Allergy in Childhood (ISAAC) questionnaire and the Edinburgh Left-handedness Inventory. A sample of 70 families with non-asthmatic, healthy children was used as controls. The majority of children had mild asthma. Ambidexterity was the main feature in the asthmatic children. A statistically significant difference in the laterality quotient (LQ) distribution was found in the group of asthmatic children with allergic rhinitis (LQ mean value in the asthmatic children with allergic rhinitis: 42.85 vs. 79.50 in the rest of the asthmatic children). These results suggest that there is a tendency towards left-handedness in asthmatic children and lend support to the GBG hypothesis.

The fluid and solute transport properties of human parietal pleura were studied and compared with sheep parietal pleura in vitro. The pleura was mounted as a planar sheet between Ussing-type chambers. The results presented are the mean values of nine different experiments. The transepithelial electrical resistance (R(TE)) of both pleurae species was measured before and after the addition of amiloride in both sides of pleura. The R(TE) for human was 25.74 +/- 1.23 Ohm x cm(2), while for the sheep it was 38.18 +/- 0.83 Ohm x cm(2). The addition of amiloride to the serosal bathing solution increased the R(TE) of human pleura to 30.48 +/- 1.01 Ohm x cm(2) and sheep pleura to 40.32 +/- 0.82 Ohm x cm(2), while amiloride had no effect on the basolateral side. From the above, it is strongly suggested that the human pleura seems to be more leaky than sheep pleura. Although the R(TE) was increased in both pleurae, the elevation in human pleura was significantly higher, thus results from experiments in sheep pleura could only partly be extrapolated in human pleura.

Left-handedness has often been associated with asthma and allergic disorders. In view of previous findings, we investigated the distribution of laterality scores, using the Edinburgh Handedness Inventory, among 172 children. These children were asthmatic and allergic children of pre-school age and adolescence, who visited the Lung Function Laboratory of the Physiology Department of the University of Thessaly, and their controls. We failed to find an association of left-handedness and asthma at pre-school age. We found an association of left-handedness and other allergies at pre-school age and an association of left-handedness and asthma in adolescence, due to hereditary reasons. Our data suggest that both asthma or other allergies and handedness are inherited, especially through the maternal line.

Purpose: The aim of this study was to correlate athletic performance with both lung function and diet. Design: Open, uncontrolled trial. Materials and Methods: Sixty-eight swimmers (28 females and 40 males), 9-15 years old, were registered for the study. The average age was 11.75 years, the average height was 151.65 cm and the average weight was 42.88 kg. Every swimmer completed a questionnaire which included details about eating habits and performances in the 100 m freestyle and backstroke. They were submitted to spirometry tests and diffusion capacity measurements (with a simple inspiration of carbon monoxide). All swimmers had three spirometry tests. The best one was recorded and compared to the expected values. Results: The average diffusing capacity was 29.07% higher than expected and the average forced expiratory volume in the first second was 4.21% higher than expected. Swimmers who consumed 5.7 meals per week based on meat and 4.43 meals per week based on vegetables performed better than swimmers ...

This article reports on the development of a simple and cost-effective bioassay for the detection of biotin in urine and serum, based on the very selective binding of avidin and biotin. Avidin was allowed to react without isolating it from egg white. Egg white was treated with the dye HABA, which binds to avidin. Upon subsequent treatment with biotin, HABA is released due to the high affinity of biotin to avidin. The amount of HABA released is proportional to the amount of biotin used.