Static Adhesion Research Articles

Basal anti-inflammatory responses in cultured endothelial cells exposed to two conjugated linoleic acids

AbstractConjugated linoleic acid (CLA) isomers have been shown to possess anti-atherosclerotic properties, which may be related to the downregulation of inflammatory pathways. Whether low concentrations of CLAs are able to affect basal, unstimulated endothelial cell (EC) responses is not clear. The aim of this study was to evaluate the effects of two CLAs (cis-9, trans-11 and trans-10, cis-12) on basal inflammatory responses by ECs.EA.hy926 cells (HUVEC lineage) were cultured under standard conditions and exposed to CLAs (1 and 10 μM) for 48 hours. MTT assay was performed to determine cell viability; incorporation of FA was confirmed by gas chromatography; inflammatory mediators were assessed by multiplex immunoassay; the relative expression of genes encoding transcription factors and inflammatory cytokines was assessed through real-time PCR and static adhesion assay was used to evaluate monocyte attachment to the EC monolayer.CLAs were incorporated into ECs in a dose-dependent manner. Pre-treatment with CLA9,11 (1 uM) significantly reduced unstimulated, basal concentrations of MCP-1 (p < 0.05), and CLA10,12 at 10 uM had the same effect (p < 0.05). Both CLAs at 10 uM increased the relative expression of NFκβ (p < 0.01 and p < 0.05, respectively), while decreasing the relative expression of PPARα (p < 0.0001), COX-2 (p < 0.0001) and IL-6 (p < 0.0001). In contrast, no effect was observed in the adhesion assay for either CLA.These results suggest that both CLAs at a low concentration have a neutral or modest anti-inflammatory effect in basal conditions, which may influence endothelial function and risk of vascular disease. Interestingly, at these low CLA concentrations some pro-inflammatory genes are upregulated while others are down regulated. This suggests complex effects of CLAs on inflammatory pathways.

Online Concentration of Bacteria from Tens of Microliter Sample Volumes in Roughened Fused Silica Capillary with Subsequent Analysis by Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

This study presents a timely, reliable, and sensitive method for identification of pathogenic bacteria in clinical samples based on a combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In this respect, a part of a single-piece fused silica capillary was etched with supercritical water with the aim of using it for static or dynamic cell-surface adhesion from tens of microliter sample volumes. The conditions for this procedure were optimized. Adhered cells of Staphylococcus aureus (methicillin-susceptible or methicillin-resistant) and of Pseudomonas aeruginosa were desorbed and preconcentrated from the rough part of the capillary surface using transient isotachophoretic stacking from a high conductivity model matrix. The charged cells were swep and separated again in micellar electrokinetic chromatography using a nonionogenic surfactant. Static adhesion of the cells onto the roughened part of the capillary is certainly volumetric limited. Dynamic adhesion allows the concentration of bacteria from 100 μL volumes of physiological saline solution, bovine serum, or human blood with the limits of detection at 1.8 × 102, 1.7 × 103, and 1.0 × 103 cells mL-1, respectively. The limits of detection were the same for all three examined bacterial strains. The recovery of the method was about 83% and it was independent of the sample matrix. A combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry required at least 4 × 103 cells mL-1 to obtain reliable results. The calibration plots were linear (R2 = 0.99) and the relative standard deviations of the peak area were at most 2.2%. The adhered bacteria, either individual or in a mixture, were online analyzed by micellar electrokinetic chromatography and then collected from the capillary and off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without interfering matrix components.

Neuraminidase-3 Is a Negative Regulator of LFA-1 Adhesion.

Within the plasma membrane environment, glycoconjugate-receptor interactions play an important role in the regulation of cell-cell interactions. We have investigated the mechanism and activity of the human neuraminidase (NEU) isoenzyme, NEU3, on T cell adhesion receptors. The enzyme is known to prefer glycolipid substrates, and we confirmed that exogenous enzyme altered the glycolipid composition of cells. NEU3 was able to modify the sialic acid content of purified LFA-1 in vitro. Enzymatic activity of NEU3 resulted in re-organization of LFA-1 into large clusters on the membrane. This change was facilitated by an increase in the lateral mobility of LFA-1 upon NEU3 treatment. Changes to the lateral mobility of LFA-1 were specific for NEU3 activity, and we observed no significant change in diffusion when cells were treated with a bacterial NEU (NanI). Furthermore, we found that NEU3 treatment of cells increased surface expression levels of LFA-1. We observed that NEU3-treated cells had suppressed LFA-1 adhesion to an ICAM-1 coated surface using an in vitro static adhesion assay. These results establish that NEU3 can modulate glycoconjugate composition and contribute to the regulation of integrin activity. We propose that NEU3 should be investigated to determine its role on LFA-1 within the inflammatory cascade.

Reveal the Neutrophil: Elucidating the Role of a Neutrophil-Specific JAK2-V617F Mutation

Introduction: Myeloproliferative neoplasms (MPN) are characterized by monoclonal proliferation of myeloid cells. The clinical presentation of classic BCR-ABL negative MPN includes blood count changes, splenomegaly, chronic inflammation and, prognostically relevant, thrombosis. Within polycythemia vera, essential thrombocythemia and primary myelofibrosis, the most frequent genetic alteration is the point mutation V617F (VF) in the Jak2 gene. Due to an activation of leucocyte β1 and β2 integrins, in particular on neutrophils, JAK2-VF is driving thrombus formation (Edelmann et al., JCI, 2018). Thus, understanding the role of neutrophils within JAK2-VF-positive MPN is of special interest. We developed a murine JAK2-VF Catchup model of MPN that is characterized by the neutrophil-specific expression of JAK2-VF in combination with the fluorescent protein tdTomato allowing in-vivo two-photon microscopy. Neutrophil-specificity was achieved through a knock-in allele of Cre recombinase and tdTomato replacing exon 1 of Ly6G (Hasenberg et al., Nat Methods, 2015). Methods: The murine JAK2-VF Catchup model was developed by crossing JAK2+/loxP-VF-loxP mice and Ly6G+/Cre-tdTomato CatchupIVM-red mice. Experiments were performed in 12 weeks old JAK2+/loxP-VF-loxP Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/VF Catchup) mice (n=5) and JAK2+/+ Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/+ Catchup) mice (n=5) as wildtype (WT) controls. Whole blood cell counts and spleen weight were determined. Bone marrow (BM) cells were harvested from hind limbs followed by isolation of granulocytes using negative selection. In order to determine hematopoietic stem and progenitor cells (HSPCs), BM and spleen cell suspensions were analyzed by flow cytometry (FC). Using ICAM-1, VCAM-1, E-selectin and P-selectin, each with and without Fc-tag, adhesion properties of the isolated granulocytes were examined by either FC or microplate reader analysis. The expression of β1 integrin was further analyzed by FC. Results and Discussion: JAK2+/VF Catchup mice showed a slight but significant increase in platelet counts [1544±58.02 Gpt/l; WT: 1190±90.50 Gpt/l; p=0.011], with the mean value still remaining within the normal range. Hematocrit, red blood cell, white blood cell and neutrophil count as well as spleen weight were also within the normal range showing no differences from their WT controls. Interestingly, neutrophil and platelet counts of JAK2+/VF Catchup mice significantly correlated with one another [p=0.0251; R2=0.8529]. MPN-typical changes in number and composition of c-Kit+ HSPCs in either BM or spleen could not be found in JAK2+/VF Catchup mice. Directly compared to reliably MPN-developing Vav-1-Cre JAK2+/VF mice, JAK2+/VF Catchup mice showed significantly less megakaryocyte-erythroid progenitor (MEP), Lin- Sca1+ c-Kit+ (LSK) and multipotent progenitor cells (MPP) in the BM. In spleen of JAK2+/VF Catchup mice, the fractions of Lin- c-Kit+ (LK) and granulocyte-macrophage progenitor cells (GMP) were significantly lower compared to Vav-1-Cre JAK2+/VF mice. Thus, the neutrophil-specific JAK2-VF mutation does not lead to apparent numerical or compositional changes of HSPCs. These results show that a neutrophil-specific JAK2-VF mutation is not sufficient to induce a characteristic phenotype of JAK2-VF-positive MPN. The expression of β1 integrin on granulocytes was significantly increased in JAK2+/VF Catchup mice [fold change vs. control; 1.126±0.041; WT: 1.0±0.011; p=0.0194]. Unexpectedly, despite increased β1 integrin expression, the binding of granulocytes from JAK2+/VF Catchup mice to either ICAM-1, VCAM-1, E-selectin or P-selectin corresponded to that of their WT controls. Static adhesion and soluble ligand binding assays showed similar results. Since adhesion of granulocytes appeared unchanged in-vitro, we hypothesize that expression of JAK2-VF in neutrophils is not sufficient to overactivate integrins but secondary extrinsic signals (e.g. cytokines) may play a dominant role. Further experimentation is under way to elucidate this molecular mechanism. Conclusions: In order to dissect the properties of JAK2-VF-positive neutrophils, the JAK2-VF Catchup model represents a promising murine model of MPN. Herewith, the initiation of thrombosis and the development of other granulocyte-driven disease manifestations of JAK2-VF induced MPN can be thoroughly examined. Disclosures No relevant conflicts of interest to declare.

RESEARCH OF ANTIBACTERIAL PROPERTIES OF THE SURFACE OF THE TITANIUM ALLOYS VT5-1 AND VT6 CONVERTED TO ANATASE TO PREVENT BACTERIAL ADHESION

Aim: to evaluate the effectiveness of preventing the formation of microbial biofilms on the surfaces of implants made of titanium alloys VT5-1, VT6 with a transformed surface into anatase. Materials and methods. The study was carried out in the bacteriological laboratory of the Severodonetsk multidisciplinary city hospital from January 2019 to September 2019. The ability to prevent the formation of microbial biofilms of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa was evaluated. The static cell adhesion method and the disco diffuse method were used. The surface of the plates was examined on a XS 6220 UCMJS05100KPA microscope. The intensity of the formation of microbial biofilms on the plates was evaluated visually by the four-cross system. Results. On all coatings with anatase, in comparison with unmodified plates, the intensity of initial adhesion and the number of bacteria fixed on the surface of the colonies markedly decreased. On surfaces with silica crystals, biofilm formation was minimal. The altered surface of titanium alloys VT5-1 and VT6 anatase and anatase with silicon dioxide crystals does not inhibit microflora growth. Conclusions. The transformed surface of titanium implants into anatase and anatase with silicon dioxide crystals prevents the fixation of biofilms of microbial associations.

Influence of Loading Conditions and Temperature on Static Friction and Contact Aging of Hydrogels with Modulated Microstructures.

Biological tribosystems enable diverse functions of the human body by maintaining extremely low coefficients of friction via hydrogel-like surface layers and a water-based lubricant. Although stiction has been proposed as a precursor to damage, there is still a lack of knowledge about its origin and its relation to the hydrogel's microstructure, which impairs the design of soft matter as replacement biomaterials. In this work, the static friction of poly(acrylamide) hydrogels with modulated composition was investigated by colloidal probe lateral force microscopy as a function of load, temperature, and loading time. Temperature-dependent studies enable to build a phase diagram for hydrogel's static friction, which explains stiction via (polymer) viscoelastic and poroelastic relaxation, and a subtle transition from solid- to liquid-like interfacial behavior. At room temperature, the static friction increases with loading time, a phenomenon called contact aging, which stems from the adhesion of the polymer to the colloid and from the drainage-induced increase in contact area. Contact aging is shown to gradually vanish with increase in temperature, but this behavior strongly depends on the hydrogel's composition. This work scrutinizes the relation between the microstructure of hydrogel-like soft matter and interfacial behavior, with implications for diverse areas of inquiry, not only in biolubrication and biomedical applications but also in soft robotics and microelectromechanical devices, where the processes occurring at the migrating hydrogel interface are of relevance. The results support that modulating both the hydrogel's mesh size and the structure of the near-surface region is a means to control static friction and adhesion. This conceptual framework for static friction will foster further understanding of the wear of hydrogel-like materials.

Myxozoan Adhesion and Virulence: Ceratonova shasta on the Move.

Motility factors are fundamental for parasite invasion, migration, proliferation and immune evasion and thus can influence parasitic disease pathogenesis and virulence. Salmonid enteronecrosis is caused by a myxozoan (Phylum Cnidarian) parasite, Ceratonova shasta. Three parasite genotypes (0, I, II) occur, with varying degrees of virulence in its host, making it a good model for examining the role of motility in virulence. We compare C. shasta cell motility between genotypes and describe how the cellular protrusions interact with the host. We support these observations with motility gene expression analyses. C. shasta stages can move by single or combined used of filopodia, lamellipodia and blebs, with different behaviors such as static adhesion, crawling or blebbing, some previously unobserved in myxozoans. C. shasta stages showed high flexibility of switching between different morphotypes, suggesting a high capacity to adapt to their microenvironment. Exposure to fibronectin showed that C. shasta stages have extraordinary adhesive affinities to glycoprotein components of the extracellular matrix (ECM). When comparing C. shasta genotypes 0 (low virulence, no mortality) and IIR (high virulence, high mortality) infections in rainbow trout, major differences were observed with regard to their migration to the target organ, gene expression patterns and proliferation rate in the host. IIR is characterized by rapid multiplication and fast amoeboid bleb-based migration to the gut, where adhesion (mediated by integrin-β and talin), ECM disruption and virulent systemic dispersion of the parasite causes massive pathology. Genotype 0 is characterized by low proliferation rates, slow directional and early adhesive migration and localized, non-destructive development in the gut. We conclude that parasite adhesion drives virulence in C. shasta and that effectors, such as integrins, reveal themselves as attractive therapeutic targets in a group of parasites for which no effective treatments are known.

Aronia berry extract inhibits TNF-α-induced vascular endothelial inflammation through the regulation of STAT3.

BackgroundInflammation in endothelial cells induces production of inflammatory cytokines and monocytes adhesion, which are crucial events in the initiation of atherosclerosis. Aronia berry (Aronia meranocalpa), also called black chokeberry, contains abundant anthocyanins that have received considerable interest for their possible relations to vascular health.ObjectiveThe aim of this study was to investigate whether an anthocyanin-rich extract obtained from aronia berry can attenuate inflammatory responses in vascular endothelial cells.MethodsAs a model of vascular endothelial inflammation, human umbilical vein endothelial cells (HUVECs) pretreated with aronia berry extract were stimulated with tumor necrosis factor-alpha (TNF-α). The expression levels of cytokines and adhesion molecules were analyzed. To investigate the effects of aronia berry extract on the adhesion of THP-1 monocytic cell, the static adhesion assay was carried out. The possible molecular mechanisms by which aronia berry extract regulated vascular inflammatory responses were explored.ResultsThe mRNA expressions of interleukins (IL-1β, IL-6, and IL-8) and monocyte chemoattractant protein-1 (MCP-1) upregulated by TNF-α were significantly suppressed by pretreatment with aronia berry extract. Aronia berry extract decreased TNF-α-induced monocyte/endothelial adhesion and suppressed vascular cell adhesion molecule-1 (VCAM-1) expression, but did not affect intercellular adhesion molecule-1 (ICAM-1) expression. Moreover, aronia berry extract decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the nuclear levels of STAT3 and interferon regulatory transcription factor-1 (IRF1). The nuclear translocation of nuclear factor-kappa B (NF-κB) was not inhibited by aronia berry extract.ConclusionAronia berry extract could exert anti-atherosclerotic effects on TNF-α-induced inflammation through inhibition of STAT3/IRF1 pathway in vascular endothelial cells.

Mechanisms utilized by feline adipose-derived mesenchymal stem cells to inhibit T lymphocyte proliferation

BackgroundFeline adipose-derived mesenchymal stem cells (ASCs) have been successfully used in clinical trials for the treatment of immune-mediated diseases with T cell dysregulation. However, the immunomodulatory pathways utilized by feline ASCs to suppress T cell activation have not been fully determined. We investigated the mechanisms used by feline ASCs to inhibit T cell proliferation, including the soluble factors and the cell-cell contact ligands responsible for ASC-T cell interaction.MethodsThe immunomodulatory activity of feline ASCs was evaluated via cell cycle analysis and in vitro mixed leukocyte reaction using specific immunomodulatory inhibitors. Cell-cell interactions were assessed with static adhesion assays, also with inhibitors.ResultsFeline ASCs decrease T cell proliferation by causing cell cycle arrest in G0–G1. Blocking prostaglandin (PGE2), but not IDO, partially restored lymphocyte proliferation. Although PDL-1 and CD137L are both expressed on activated feline ASCs, only the interaction of intercellular adhesion molecule 1 (ICAM-1, CD54) with its ligand, lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), was responsible for ASC-T cell adhesion. Blocking this interaction reduced cell-cell adhesion and mediator (IFN-γ) secretion and signaling.ConclusionsFeline ASCs utilize PGE2 and ICAM-1/LFA-1 ligand interaction to inhibit T cell proliferation with a resultant cell cycle arrest in G0–G1. These data further elucidate the mechanisms by which feline ASCs interact with T cells, help define appropriate T cell-mediated disease targets in cats that may be amenable to ASC therapy, and may also inform potential translational models for human diseases.

Depletion interaction forces contribute to erythrocyte-endothelial adhesion in diabetes

Abnormal adhesion of red blood cells (RBC) to the endothelium has been linked to the pathophysiology of several diseases associated with vascular disorders. Various biochemical changes on the outer membrane of RBC, as well as plasma protein levels, have been identified as possibly playing key roles, but the detailed interplay between plasma factors and cellular factors often remains unclear. In this work, we identified an alternative pathway by demonstrating that non-adsorbing macromolecules can also have a marked impact on the adhesion efficiency of RBC from patients with type 2 Diabetes (T2DM) to endothelial cells (EC). RBC isolated from blood samples of T2DM patients were suspended in isotonic solutions of dextran in order to mimic the impact of non-adsorbing macromolecules. Static and continuous flow adhesion assays were used to determine the adhesion behavior of T2DM RBC with EC and the results compared with those of normal controls. We found that the presence of non-adsorbing molecules promotes an increase in T2DM RBC - EC adhesion and that these RBC exhibit much greater adhesion than normal red cells. Our results thus suggest that the depletion mechanism might be an alternative phenomenon through which plasma proteins could cause enhanced RBC-EC adhesion in diabetes mellitus. These findings contribute towards the comprehensive understanding of pathophysiological mechanisms of vascular complications in diabetes and other diseases with similar vascular sequelae.

Effect of polymer structure modified on RO membrane surfaces via surface-initiated ATRP on dynamic biofouling behavior

Biofouling is a major drawback for most polyamide reverse osmosis (RO) membranes, which results in water flux decline and higher energy demand. To prevent biofouling, it is important to minimize the interaction between bacterial and membrane surfaces. In this research, we investigated the influence of surface modification with various hydrophilic polymers on biofouling behavior. First, polyamide RO membranes were modified with well-structural controlled hydrophilic polymers, poly(2-hydroxyethyl methacrylate) (pHEMA), poly[poly(ethylene glycol)methacrylate] (pPEG), and poly[(2-methacryloyloxy)ethyl]dimethyl[3-sulfopropyl]ammonium hydroxide (pMEDSAH) via the surface-initiated atomic transfer radical polymerization (SI-ATRP). The biofouling properties were assessed by a static bacterial adhesion and a dynamic biofouling filtration. The membranes modified with a longer main chain of pMEDSAH and a long side chain of pPEG had good resistance against both the bacterial adhesion and dynamic biofouling. Experimental results between the static and dynamic biofouling behavior did not correlate with each other. Molecular dynamics (MD) simulation clarified that pPEG had a longer side chain than pHEMA and pMEDSAH, and that pMEDSAH possessed stronger hydration than pPEG and pHEMA. Experimental results and MD simulation indicated that the main chain length, side chain length, and hydration of the modifying polymer should be taken into account for developing anti-biofouling membranes.

Physicochemical characterization of Sargassum fusiforme fucoidan fractions and their antagonistic effect against P-selectin-mediated cell adhesion.

P-selectin, mediated adhesion between endothelium and neutrophils, is a promising target for the therapeutics of acute inflammatory-related diseases. It is reported that brown algal fucoidans can antagonize P-selectin function. However, the fractionation and physicochemical characterization of Sargassum fusiforme fucoidan, and the screening of fucoidan fractions with P-selectin antagonistic capability have not been investigated. In this study, we isolated and fractionated systematically the S. fusiforme fucoidan by ion-exchange chromatography and size exclusion chromatography to obtain eight fucoidan fractions. Their physicochemical characterization was determined by chemical methods, HPLC and FT-IR. The inhibitory capacity of the fucoidan fractions in P-selectin-mediated leukocyte adhesion was evaluated by static adhesion assay and parallel-plate flow chamber. Results showed that fucoidan fractions possessed distinct physicochemical properties, including total carbohydrate, uronic acid and sulfate contents, molecular weight, and monosaccharide compositions. Among all the fucoidan fractions, SFF-32 and SFF-42 showed better blocking ability against P-selectin-mediated cell adhesion.

Amyloid Peptide β1-42 Induces Integrin αIIbβ3 Activation, Platelet Adhesion, and Thrombus Formation in a NADPH Oxidase-Dependent Manner.

The progression of Alzheimer's dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects of β amyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptide β1-42 (Aβ1-42), Aβ1-40, and Aβ25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to Aβ1-42 compared to other Aβ peptides. In addition, significant platelet spreading was observed over Aβ1-42, while Aβ1-40, Aβ25-35, and the scAβ1-42 control did not seem to induce any platelet spreading, which suggested that only Aβ1-42 activates platelet signalling in our experimental conditions. Aβ1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other Aβ peptides did not. The molecular mechanism of Aβ1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrin αIIbβ3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, Aβ1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished Aβ1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrin αIIbβ3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptide β1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer's patients.

Effect of interfacial voltage on tribological characteristics of polysilicon micro-contacts tested in ambient air under dynamic impact conditions

A polycrystalline silicon microtribometer was used to examine the evolution of tribological properties under dynamic mechanical impact and hot switching conditions. Changes in surface properties were tracked via periodic static adhesion tests. Surfaces experienced two distinct periods in the course of the dynamic lifetime—a run-in phase with little measurable surface modification and a degradation phase with significant surface modification and corresponding increases in the measured adhesion force. No statistically significant differences were found in either the run-in period length or the degradation rate between hot switching conditions and purely mechanical impacting. However, when combining these two parameters, it was discovered that longer run-in periods correspond to slower degradation rates for the mechanical switching but correspond to faster degradation rates for the hot switching. This can be attributed to the action of the surface hydrocarbons physisorbed from the ambient air. These hydrocarbons have high surface mobility because they are bound by weak van der Waals forces. Under hot switching conditions, mobility is enhanced by increasing surface temperature caused by electrical current flow through nano-asperities. Mobile hydrocarbons diffuse across the surface (driven by concentration gradients) to damaged areas resulting in a healing effect that is dominant in the hot switching tests. Results are interpreted in light of the principle tribological degradation and healing mechanisms and the experimental sources of these mechanisms.

Combined Administration of Recombinant TGF-β1 and DMSO Decreases the in Vitro Inflammatory Properties of Neutrophils from Sickle Cell Anemia Individuals

Sickle cell anemia (SCA) is a chronic inflammatory disease, in which activated neutrophils play a role in initiating vaso-occlusion. Two populations of circulating neutrophils have been described, denominated as low-(LDN) and high-(HDN) density neutrophils. Circulating numbers of LDN (a less inflammatory subset) are normally minimal, but this population augments under inflammatory stress, such as that seen in cancer. Transforming growth factor beta-1 (TGF-β1) is a cytokine with anti-inflammatory properties that is elevated in SCA. In conditions such as Chron's disease, TGF-β1 has protective effects, mediated by its immuno-suppressive functions. In macrophages, it is thought to trigger the polarization from pro- (M1) to anti-inflammatory phenotypes (M2), which hypothetically occur in neutrophils too (N1 and N2). Moreover, dimethyl sulfoxide (DMSO) reportedly increases TGF-β receptors expression on epithelial cells. We aimed to characterize the subsets of circulating neutrophils in SCA patients and investigate the effects of TGF-β1 and DMSO on these cells. Neutrophils from healthy (CON) and SCA individuals, in steady state and without blood transfusion for 90 days, were isolated from peripheral blood by Ficoll-Paque density gradient centrifugation. HDN and LDN were obtained from the granulocyte and mononuclear layers, respectively, and stained with CD66b for neutrophil identification by flow cytometry. As no significant effect of hydroxyurea (HU) therapy on the data obtained was observed, patients' data were grouped together irrespective of HU use. Percentages of LDN were calculated based on the total of gated CD66b+ cells. SCA patients had higher levels of LDN than CON (3.2±0.9%, N=7 vs 1.3±0.3%, N=13; p=0.02). We next investigated the presence of CD66b+/CD206- and CD66b+/CD206+ cells, to infer the presence of N1 and N2 phenotypes, respectively. N2 were more frequent in the LDN than in the HDN subset (CON: 68.1±3.3% vs 52.0±4.4%, N=9, p=0.01; SCA: 77.6±8.9% vs 44.1±5.0%, N=3, p=0.03). To determine whether TGF-β1 and DMSO could shift HDN to a LDN profile, cells were treated (2h) with TGF-β1 (50pM) and/or DMSO (1.5%). Treatments with DMSO alone or combined with TGF-β1 increased the percentage of CD206+ cells in CON (45.7±2.1% vs 61.9±7.6 and 53.6±2.6% respectively, N=6, p=0.04), as well as CD206+ expression on each cell (mean fluorescence intensity, MFI) (137.5±16.9 MFI vs 293.6±71.2 MFI and 210.1±23.9 MFI, respectively, p=0.03). In SCA, only the combined TGF-β1/DMSO treatment increased the MFI of CD206 in HDN (115.7±10.2 vs 255.8±29.7 MFI, N=4, p=0.03). We next investigated whether TGF-β1/DMSO could reduce the adhesion of HDN to fibronectin ligand (FN, 20μg/mL) using static adhesion assays (30 min, 37ºC). HDN from CON and SCA were treated with TGF-β1 and/or DMSO (90min) and stimulated with TNF-α (200ng/mL, 30min). Although TGF-β1 alone did not reduce the adhesion of HDN to FN (p>0.05), the addition of DMSO decreased TNF-α-induced adhesion in CON (16.5±1.8% to 11.3±1.5%, p=0.03, N=10) and SCA HDN (38.9±23.9% to 13.9±1.5%, p=0.04, N=3). Subsequently, HDN were stimulated (4h) with LPS (100ng/mL) and INF-γ (20ng/mL), to induce N2 polarization, in the presence/absence of TGF-β1 and DMSO. The combined treatment again reduced adhesion in both groups (CON: 11.1±1.5% to 4.2±1.2%, p<0.01, N=4; SCA: 47.4±12.3% vs 21.9±4.5%, p=0.04, N=4). To assess whether TGF-β1 and DMSO could affect the production of proinflammatory cytokines by HDN after stimulation with LPS/INF-γ, TNF-α and IL-1β levels in cell supernatants were measured by ELISA. TGF-β1 and DMSO, in combination, decreased both TNF-α and IL-1β release from CON (TNF-α: 39.7±8.7pg/mL to 8.7±0.9pg/mL, p<0.01, N=6; IL-1β: 66.9±10.5pg/mL to 16.5±5.1pg/mL, p=0.02, N=4) and SCA HDN (TNF-α: 174.0±55.5pg/mL to 21.8±6.6pg/mL, p=0.01, N=8; IL-1β: 103.6±25.6pg/mL to 43.1±15.5pg/mL, p=0.01, N=8). Our results demonstrate for the first time the presence of elevated numbers of LDN in SCA patients, indicating an increased basal response to inflammatory stress. However, this shift in the anti-inflammatory subset does not appear enough to control inflammatory responses in the disease, and the use of agents capable of inducing this polarization may be a promising approach. Moreover, the anti-inflammatory effects of TGF-β1 on HDN seem to be enhanced by DMSO and suggest this combination as an effective modulator of the inflammatory profile of neutrophils. DisclosuresNo relevant conflicts of interest to declare.

Heme Induces Significant Neutrophil Adhesion in Vitro Via an Nfκb-Dependent Pathway

Background: Intravascular hemolysis, a major complication of sickle cell anemia and malaria among other diseases, incurs the release of excessive quantities of hemoglobin and heme from red blood cells. If not adequately sequestered by hemoglobin- and heme-binding proteins, these molecules may incur oxidative stress, thrombosis and cellular activation. Heme is now recognized as a red cell DAMP (damage-associated molecular pattern) that stimulates inflammasome formation in macrophages and can induce neutrophil extracellular trap release under certain circumstances. Aim: This study evaluated the in vitro effects of heme on the adhesive properties of human neutrophils. Methods: Neutrophils were separated from the peripheral blood of healthy individuals and their adhesion in the presence/absence of heme was compared by static adhesion assays using myeloperoxidase, for quantification of cell adhesion (30 min, 37oC, 5% CO2). Results: Heme (50 µM) significantly increased the adhesion of neutrophils to fibronectin (FN) and to recombinant ICAM-1 (an endothelial ligand), when compared to non-treated neutrophils (43.7±4.6%; 10.9±21.4 %, respectively, n=5, P<0.001 for FN and 38.1±3.6%; 6.9±0.8%, respectively, n=6, P<0.001 for ICAM-1). Interestingly, heme induced neutrophil adhesion even more efficiently than the potent pro-inflammatory cytokine, TNF-α (200 ng/ml) (35.5±5.2% adhesion to FN, n=6, P<0.05). Furthermore, inhibition of the NFκB transcription factor with the pharmacological inhibitor, BAY 11-7082 (20 μM), abolished heme-stimulated neutrophils adhesion to FN (reduced from 41.8±6.7% to 7.4±0.5%. n=6; P<0.001). Flow cytometry demonstrated that while TNF-α significantly increases the expression of the Mac-1 integrin subunit, CD11b (data not shown, P<0.05), but not the LFA-1 integrin subunit CD11a (data not shown, P>0.05), on the surface of neutrophils, heme did not augment CD11b or CD11a expression (P>0.05). In contrast, heme significantly augmented the active conformations of these two β2 integrin subunits, as demonstrated by epitope-specific antibodies (CD11b; heme: 520.5±51.8; basal 123.7±16.7 MFI, n=10, P<0.001 and CD11a; heme: 69.8±3.9; basal: 43.8±2.0 MFI, n=6, P>0.001). Inhibition of NFκB translocation with BAY 11-7082 (20 μM) significantly decreased the activity of the LFA-1 integrin on the surface of neutrophils after heme stimulation (reduced to 47.6±4.2 MIF, n=6; P<0.05). To assess whether heme-induced neutrophil adhesive properties are mediated by cytoskeletal rearrangements, we evaluated the effects of cytochalasin D (0.5 μg/ml), an inhibitor of actin polymerization. While cytochalasin D inhibited TNF-α-induced neutrophils adhesion (data not shown, P<0.05), this compound did not significantly alter the adhesive properties of heme-stimulated neutrophils to FN (heme: 22.8±2.3%, p<0.001; cytochalasin D: 22.5±2.0%, p=0.4; n=6). Pre-incubation of heme-stimulated neutrophils with the antioxidants, ascorbic acid (120 µM; 3.5 hours) and α-tocopherol (1mM; 15 min), reduced their adhesion to FN by 14.4±9.2% and 46.5±5.5% respect. n=6, p<0.05). Conclusion: We therefore demonstrate, herein, that heme is a potent activator of neutrophil adhesive properties, increasing the ligand affinity of the β2 integrins via a mechanism that is apparently mediated by an NFkB-dependent pathway. The mechanism of neutrophil activation appears to differ from that stimulated by TNF-α and may involve, in part, the generation of reactive oxygen species. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in the vaso-occlusive process in sickle cell disease and other vascular inflammatory processes, our findings further support the idea that cell-free heme represents a major therapeutic target in the hemolytic diseases. DisclosuresNo relevant conflicts of interest to declare.

Unfractionated and Low Molecular Weight Heparin Reduce Platelet Induced Epithelial-Mesenchymal Transition in Pancreatic and Prostate Cancer Cells.

The interaction with platelets is of crucial importance for tumor cells passing through hematogenous metastasis. Platelets protect cancer cells from immune surveillance and exhibit many other prometastatic effects. Notably, platelets can change the epithelial tumor phenotype, a process termed epithelial-mesenchymal transition (EMT), which confers stem cell-like properties onto tumor cells associated with an increased motility and drug resistance. The aim of the study is to investigate the impact of heparin on the platelet induced EMT program in pancreatic and prostate tumor cells. Platelet activation and interaction with cancer cells were determined by static adhesion assays. Applying ELISAs, the platelet release of EMT inducing mediators was quantified. EMT marker protein expression by tumor cells was explored by western blot and qPCR. Our data show that different tumor cell entities have different platelet binding capacities and also that a weak interaction is sufficient to change tumor cell phenotype. Additionally, unfractionated heparin (UFH) as well as low molecular weight heparin (LMWH) reduced tumor cell platelet interaction. Subsequently, attenuated platelet-derived mediator release resulted in reduced EMT marker protein and transcription factor expression by the cancer cells and decreased cell migration. These data suggest that heparin reduces platelet induced EMT program and prevents the formation of cancer cells with stem cell-like properties. This additional mechanism argues for the use of heparin in oncological applications.

Dual-functionalization of polymeric membranes via cyclodextrin-based host-guest assembly for biofouling control

We demonstrated a dual-functionalization strategy of polymeric membrane with both “defensive” (adhesion resistance) and “offensive”(bacterial inactivation) efficacies via combining external-hydrophilic β-cyclodextrins (β-CDs) and bactericidal enrofloxacin (ENR) for combating biofouling. β-CDs were immobilized onto polydopamine (PDA) pre-modified membrane surface, followed by host-guest assembly of ENR. Both static adhesion tests and dynamic fouling experiments implied that the CD-modified membranes showed significantly reduced adsorption and lower water flux decline against organic foulant. The enhanced “defensive” (fouling resistance) capacity of CD-modified membrane was mainly attributed to hydrophilicity improvement. Next, antimicrobial tests demonstrated that the integration of ENR in the functional layer could improve the “offensive” (biocidal) activity of modified membrane. Conspicuous reduction in biomass and ATP content further illustrated that the PVDF/CD-ENR membranes could effectively suppress biofilm formation during dynamic cross-flow biofouling tests. Finally, flow cytometry (FCM) assessment was first combined with heterotrophic plate counts (HPC) method in the present study, implying that the antimicrobial mechanism of the dual-functional membrane lies in inhibition of bacterial breed without disrupting the cell membrane.

Wet-adhesion properties of microstructured surfaces inspired by newt footpads

To understand wet adhesion of newt foot pads, we firstly examined and evaluated the attachment and climbing abilities of newts under three different wetting conditions. Then we characterized the micro and nanostructures of newts’ foot pads by using the scanning electron microscopy. Followed by observing the micro and nanoscale structural features of newts’ foot pads, four different micropatterns, including round pillars, hexagonal pillars, and two hybrid patterns with closed and semi-closed hexagonal ridges, were designed and fabricated on PDMS. The static friction and adhesion of microstructured surfaces were measured by using a multi-functional surface meter and an adhesion testing equipment, respectively. Effects of micropattern on static friction, and effects of retraction speed, amount of liquid, approach-retraction cycle on adhesion were investigated experimentally. Results suggested that a larger amount of liquid decreases the adhesion and friction, but a little liquid increases the adhesion and friction. It was found that there exists an optimum amount of liquid (about 0.1 μl) that can enhance the adhesion. Our results can give insights into the repeated shear movements of a newt’s foot pads in the stream condition as well as the possible functions of dense nanopillar arrays on newt foot pads.

β2ARs stimulation in osteoblasts promotes breast cancer cell adhesion to bone marrow endothelial cells in an IL-1β and selectin-dependent manner

Progression and recurrence of breast cancer, as well as reduced survival of patients with breast cancer, are associated with chronic stress, a condition known to impact the hypothalamic-pituitary axis and the autonomic nervous system. Preclinical and clinical evidence support the involvement of the sympathetic nervous system in the control of bone remodeling and in pathologies of the skeleton, including bone metastasis. In experimental mouse models of skeletal metastasis, administration of the βAR agonist isoproterenol (ISO), used as a surrogate of norepinephrine, the main neurotransmitter of sympathetic neurons, was shown to favor bone colonization of metastatic breast cancer cells via an increase bone marrow vascularity. However, successful extravasation of cancer cells into a distant organ is known to be favored by an activated endothelium, itself stimulated by inflammatory signals. Based on the known association between high sympathetic outflow, the expression of inflammatory cytokines and bone metastasis, we thus asked whether βAR stimulation in osteoblasts may alter the vascular endothelium to favor cancer cell engraftment within the skeleton. To address this question, we used conditioned medium (CM) from PBS or ISO-treated bone marrow stromal cells (BMSCs) in adhesion assays with bone marrow endothelial cells (BMECs) or the endothelial cell line C166. We found that ISO treatment in differentiated BMSCs led to a robust induction of the pro-inflammatory cytokines interleukin-1 beta (IL-1β) and interleukin-6 (IL-6). The CM from ISO-treated BMSCs increased the expression of E- and P-selectin in BMECs and the adhesion of human MDA-MB-231 breast cancer cells to these cells in short-term static and dynamic adhesion assays, and a blocking antibody against IL-1β, but not IL-6, reduced this effect. Direct IL-1β treatment of BMECs had a similar effect, whereas the impact of IL-6 treatment on the expression of adhesion molecules by BMECs and on the adhesion of cancer cells to BMECs was negligible. Collectively, these in vitro results suggest that in the context of the multicellular and dynamic bone marrow environment, sympathetic activation and subsequent βAR stimulation in osteoblasts may profoundly remodel the density but also the activation status of bone marrow vessels to favor the skeletal engraftment of circulating breast cancer cells.