Flow Chamber Research Articles

Endothelial Dysfunction in the Context of Blood-Brain Barrier Modeling.

Here, we discuss pathophysiological approaches to the defining of endothelial dysfunction criteria (i.e., endothelial activation, impaired endothelial mechanotransduction, endothelial-to-mesenchymal transition, reduced nitric oxide release, compromised endothelial integrity, and loss of anti-thrombogenic properties) in different in vitro and in vivo models. The canonical definition of endothelial dysfunction includes insufficient production of vasodilators, pro-thrombotic and pro-inflammatory activation of endothelial cells, and pathologically increased endothelial permeability. Among the clinical consequences of endothelial dysfunction are arterial hypertension, macro- and microangiopathy, and microalbuminuria. We propose to extend the definition of endothelial dysfunction by adding altered endothelial mechanotransduction and endothelial-to-mesenchymal transition to its criteria. Albeit interleukin-6, interleukin-8, and MCP-1/CCL2 dictate the pathogenic paracrine effects of dysfunctional endothelial cells and are therefore reliable endothelial dysfunction biomarkers in vitro, they are non-specific for endothelial cells and cannot be used for the diagnostics of endothelial dysfunction in vivo. Conceptual improvements in the existing methods to model endothelial dysfunction, specifically, in relation to the blood–brain barrier, include endothelial cell culturing under pulsatile flow, collagen IV coating of flow chambers, and endothelial lysate collection from the blood vessels of laboratory animals in situ for the subsequent gene and protein expression profiling. Combined with the simulation of paracrine effects by using conditioned medium from dysfunctional endothelial cells, these flow-sensitive models have a high physiological relevance, bringing the experimental conditions to the physiological scenario.

Abstract 132: VLX-1005 Effectively Treats Heparin-induced Thrombocytopenia (HIT) Without Increasing The Risk For Bleeding

Heparin-induced thrombocytopenia (HIT) is a rare, but deadly disease that occurs in a small percentage of patients following administration of heparin. In these patients, heparin interacts with platelet factor 4 (PF4) resulting in an immune response to the heparin/PF4 complex. This immune complex can then bind to the immune receptor on the platelet, FcγRIIa, to induce platelet activation, clot formation, thrombosis, consumption, and bleeding. This potentially deadly disease results in both a thrombotic event as well as bleeding. Currently, there is only one FDA-approved intervention for HIT, the direct thrombin inhibitor argatroban. However, many patients on argatroban remain at risk for thrombosis, bleeding, and in some cases death. Therefore, we sought to develop a new drug to intervene in patients with HIT by targeting the 12-lipoxygenase enzyme in the platelet which regulates FcγRIIa activity in the platelet. Using mouse models of HIT as well as ex vivo administration of the 12-lipoxygenase inhibitor VLX-1005 (previously known as ML355). In mice expressing the human immune receptor on their platelets, we were able to show that administration of VLX-1005 following induction of HIT in these mice resulted in a blunted thrombocytopenia as well as reduced platelet activation and thrombus formation in the blood. We further demonstrated that coagulation was not impacted by VLX-1005 using thromboelestography while argatroban significantly delayed onset of coagulation and clot formation. Furthermore, bleeding time in these mice was not altered with VLX-1005, while mice on argatroban required cauterization of their tails to stop the bleeding. Finally, Human whole blood was shown in whole blood aggregometry as well as high shear arterial flow chamber experiments to be protected from platelet activation and clot formation in the presence of VLX-1005. The studies presented here demonstrate the potential effectiveness of VLX-1005 in intervention of platelet activation, clot formation, and thrombosis in both mouse models and human blood and support VLX-1005 as a new class of drug for the treatment of HIT in patients without the risk of bleeding.

Obesity Differentially Effects Visceral vs. Subcutaneous Adipose Arteries: Role of Shear Activated Kir2.1 and Alterations to the Glycocalyx in Mediating Endothelial Dysfunction

Obesity imposes well‐established deficits to endothelial function; however, obesity‐induced endothelial dysfunction is not observed in all vascular beds. Visceral adipose arteries (VAA), but not subcutaneous adipose arteries (SAA), exhibit endothelial dysfunction. Here we aimed to determine if differences in SAA vs. VAA endothelial function observed in obesity are attributed to differential impairment of Kir channels and alterations to the glycocalyx. We used a mouse model of diet‐induced obesity and recruited obese human subjects undergoing planned bariatric surgery to this study. The effects of obesity on flow‐induced vasodilation (FIV) in VAA and SAA were assessed ex vivo. Functional downregulation of endothelial Kir2.1 was accomplished by transducing adipose arteries from mice and obese humans with adenovirus containing a dominant‐negative Kir2.1 construct. Kir function was tested in freshly isolated endothelial cells seeded in a flow chamber for electrophysiological recordings under fluid shear. Atomic force microscopy was used to assess biophysical properties of the glycocalyx. Endothelial dysfunction was observed in VAA of obese mice and humans. Downregulating Kir2.1 blunted FIV in SAA, but had no effect on VAA, from obese mice and humans. Kir shear‐sensitivity was observed in SAA endothelial cells from obese mice and effects on SAA glycocalyx were mild. In contrast, Kir shear‐sensitivity was abolished in VAA endothelial cells and the biophysical properties of the VAA glycocalyx was significantly exacerbated in VAA of obese mice. Our findings show differences in Kir function and alterations to the glycocalyx that we propose contribute to the dichotomy in SAA vs. VAA endothelial function with obesity.

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment.

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges’ role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia’s unconventional actin cytoskeleton has an important role in supporting parasite attachment.

Finite element analysis on mechanical state on the osteoclasts under gradient fluid shear stress.

Mechanical loading, such as fluid shear stress (FSS), is regarded as the main factor that regulates the biological responses of bone cells. Our previous studies have demonstrated that the RAW264.7 osteoclast precursors migrate toward the low-FSS region under the gradient FSS field by a cone-and-plate flow chamber, in which the FSS in the outer region is larger than that in the inner region along the radial direction. Whether the FSS distribution on a cell depends on the gradient direction of FSS field should be clarified to explain this experimental observation. In this study, the finite element models of the discretely distributed or closely packed cells adherent on the bottom plate in a cone-and-plate flow chamber were constructed, and cells were regarded as compressible isotropic Hookean solid. Results showed that the average FSS of each discretely distributed cell at the quarter sector far from the center (SFC) was about 0.1% greater than that at the quarter sector near the center (SNC). In the bands with different orientations for a cell, the relative difference between the average FSS in the SFC and the SNC becomes smaller with increased band height. For the hexagonal closely packed cells, the relative value of SFC and SNC increases with increasing cell spacing. The difference between the local wall FSS in the SFC and the SNC may activate mechanosensitive ion channels and further regulate the migration of osteoclast precursors toward the low-FSS region under the gradient FSS field.

Down-regulation of CLEC3B facilitates epithelial-mesenchymal transition, migration and invasion of lung adenocarcinoma cells

BackgroundLung adenocarcinoma (LUAD) belongs to non-small cell lung carcinoma, and the metastasis is the main cause of death in LUAD patients. It is generally accepted that cell adhesion is closely associated with tumor metastasis. Accordingly, the purpose of this study is to investigate the functions of CLEC3B to the invasion, adhesion, and migration of LUAD cells. MethodsThrough bioinformatic analysis, CLEC3B level was found markedly down-regulated in LUAD tissue. Parallel-plate flow chamber, wound-healing and Transwell assays were taken to detect the cell adhesion, invasion, and migration. qRT-PCR and western blot analyzed expression of CLEC3B, p53 and epithelial-mesenchymal transition (EMT) marker. ResultsWhen CLEC3B was lowly-expressed in LUAD cell lines, the cell adhesion capability was also lowered, with the EMT, migration and invasion of the cells progressed. Abnormal expression of CLEC3B was related to p53 signaling pathway. ConclusionAbove all, a further investigation of the function of CLEC3B in LUAD helps us further understand the molecular mechanism of the tumor metastasis.

Tenascin-X Mediates Flow-Induced Suppression of EndMT and Atherosclerosis.

Endothelial-to-mesenchymal transition (EndMT) has been identified as a critical driver of vascular inflammation and atherosclerosis, and TGF-β (transforming growth factor β) is a key mediator of EndMT. Both EndMT and atherosclerosis are promoted by disturbed flow, whereas unidirectional laminar flow limits EndMT and is atheroprotective. How EndMT and endothelial TGF-β signaling are regulated by different flow patterns is, however, still poorly understood. Flow chamber experiments in vitro and endothelium-specific knockout mice were used to study the role of tenascin-X in the regulation of EndMT and atherosclerosis as well as the underlying mechanisms. In human endothelial cells as well as in human and mouse aortae, unidirectional laminar flow but not disturbed flow strongly increased endothelial expression of the extracellular matrix protein TN-X (tenascin-X) in a KLF4 (Krüppel-like factor 4) dependent manner. Mice with endothelium-specific loss of TN-X (EC-Tnxb-KO) showed increased endothelial TGF-β signaling as well as increased endothelial expression of EndMT and inflammatory marker genes. When EC-Tnxb-KO mice were subjected to partial carotid artery ligation, we observed increased vascular remodeling. EC-Tnxb-KO mice crossed to low-density lipoprotein receptor-deficient mice showed advanced atherosclerotic lesions after being fed a high-fat diet. Treatment of EC-Tnxb-KO mice with an anti-TGF-beta antibody or additional endothelial loss of TGF-beta receptors 1 and 2 normalized endothelial TGF-beta signaling and prevented EndMT. In in vitro studies, we found that TN-X through its fibrinogen-like domain directly interacts with TGF-β and thereby interferes with its binding to the TGF-β receptor. In summary, we show that TN-X is a central mediator of flow-induced inhibition of EndMT, endothelial inflammation and atherogenesis, which functions by binding to and by blocking the activity of TGF-β. Our data identify a novel mechanism of flow-dependent regulation of vascular TGF-β, which holds promise for generating new strategies to prevent vascular inflammation and atherosclerosis.

Effects of groundwater fluctuation on migration characteristics and representative elementary volume of entrapped LNAPL

Understanding migration characteristics and the representative elementary volume (REV) variation of the entrapped light non-aqueous phase fluids (LNAPL) under dynamic groundwater conditions is significant in quantifying the effective parameters and formulating pollution treatment strategies. This paper investigates the effect of the initial water table height, the fluctuation intensity, and the fluctuation sequence on redistribution characteristics and REVs of LNAPL in a two-dimensional (2D) flow chamber. The migration behaviors of pollution plumes and associated REVs were quantified by spatial moments and the criterion χi. The results indicated that the redistribution location of residual pollutants was mainly determined by the fluctuation sequence. The REV gradually decreases as the rising water table, while the minimum So-REV frequency distribution becomes closer to a regular Gaussian distribution. These results shed light upon the effect of dynamic water tables on the morphology features and REVs of LNAPL, which can offer valuable guidance for designing remediation schemes.

An Improved One-Dimensional Flow Model for Side Chambers of Centrifugal Pumps Considering the Blade Slip Factor

Abstract The blade slip factor significantly influences the prediction accuracy of the self-closure one-dimensional flow model for side chambers of centrifugal pumps. Wiesner's and Stodola's slip factors, which are used to formulate the blade outlet pressure and served as the boundary condition for the model, are examined, which is an improvement of the previous study (Gu et al., 2020, “A Pressure Model for Open Rotor–Stator Cavities: An Application to an Adjustable-Speed Centrifugal Pump With Experimental Validation,” ASME J. Fluids Eng., 142(10), p. 101301). Both computational fluid dynamics (CFD) simulations and experiments for the centrifugal pump are conducted to support the improvement. A good agreement exists between the performance at the best efficiency points (BEPs) of different rotating speeds obtained by simulations and experiments. Through the CFD analysis, the flow in the impeller remarkably deviates from the blade-congruent flow, especially in the quasi-triangular regions downstream of the throats. Meanwhile, the reason for pressure over-predictions of the side chamber one-dimensional flow model that embeds Wiesner's slip factor (FMW) is that Wiesner's expression underestimates the impeller flow deflection and overestimates pressure boundary. By contrast, the side chamber one-dimensional flow model with Stodola's slip factor (FMS) is closer to CFD in terms of relative flow angle and chamber inlet pressure. Compared with the side chamber pressure measurements, the accuracy of FMS is upgraded approximately by 3.5% than FMW. At the BEPs of different rotating speeds, FMS generates lower shroud thrust coefficients but slightly greater volumetric efficiencies than FMW. This work provides a simple approach to better calculate flow characteristics in the side chambers.

Characterization of microbubble-enhanced molecular delivery to cells in acoustofluidic channels

Cell therapies are rapidly emerging as a promising approach for treatment of many diseases, such as cancer and cardiovascular diseases. Non-viral methods for molecular delivery are currently in development to increase safety and reduce varability during manufacturing of cell therapies. Ultrasound-mediated microbubble cavitation has been utilized as an effective approach for non-viral molecular delivery via sonoporation or other mechanisms. However, prior ultrasound studies have primarily utilized static sample chambers, which have limited throughput and are generally not practical for cell therapy manufacturing processes. To address these limitations, we are developing novel acoustofluidic platforms to enable intracellular delivery of biomolecules as cells continuously pass through an ultrasound field in a flow chamber. We have investigated a range of fundamental parameters that influence acoustofluidic-mediated molecular delivery to human cells, including microbubble properties, channel geometry, and ultrasound parameters. In addition, we investigated biological factors that influence acoustofluidic-mediated molecular delivery to cells, including cellular properties and extracellular metabolite levels. The results of these studies will be presented to provide new insights into the key parameters and mechanisms that affect the efficiency of molecular delivery to human cells in acoustofluidic channels. Further development of acoustofluidic platforms may enable improved processes for manufacturing of cell therapies.

Ultrasound-assisted membrane permeabilization of endothelial cells under flow conditions

Ultrasound-stimulated microbubbles have been shown a feasible approach for localized therapeutic delivery. As applications of this technique span many anatomical sites, so too do the local fluid dynamics experienced by the circulating microbubbles and the adjacent endothelial cells. Our objective was to assess the relative effectiveness of endothelial cell sonoporation as a function of flow conditions. Human umbilical vein (HUVECs) or human brain endothelial cells (HBECs) were cultured as a monolayer in flow chamber slides connected to a fluidic system and placedupon an acoustically-coupled microscope. A suspension of diluted lipid-encapsulated microbubbles and propidium iodide (PI), used as a sonoporation marker, was constantly perfused over the monolayer at either 5 or 30 ml/min. Cells were treated with 1 MHz ultrasound (PRI= 1 ms, 20 cycles, duration = 2 s), and the video-microscopy data were quantified offline to assess the number of PI-positive cells. Our results demonstrate a marked increase in sonoporation efficiency at 30 ml/min as compared to 5 ml/min in both endothelial cell lines under identical acoustic conditions (9.7-fold increase and 2.3-fold increase for HUVECs and HBECs respectively, p < 0.001). Our results suggest the local fluid flow environment plays a role in US-mediated endothelial perforation efficiency and can modulate treatment strategies.

Does biochar affect soil wettability and flow pattern?

Despite the known benefits of biochar application to soils, little is known about its effect on soil wettability and the consequences on soil water movement across the soil profile. Focusing on the latter, three types of commercial biochar derived from wheat, corn, and rice straw produced under low temperature (350–450 ℃) were mixed with a wettable sandy soil at rates of 0%, 2%, 5%, and 10% w/w. Soil water repellency (SWR) of the biochars and biochar–soil mixtures was determined by water drop penetration time (WDPT) test and sessile-drop contact angle (CA) measurement. The WDPT results (< 5 s) showed complete wettability for the biochar–soil mixtures, whereas CA results indicated some water repellency. The mean initial CA of the three biochars varied between 105.3° and 113.9°, with ∼ 30° for the pure sand, and between 56.8° and 75.7° for the biochar–soil mixtures. SWR increased with biochar-application rate, whereas biochar particle size (< 1 mm and > 1 mm) had no significant effects on SWR. A 2-D flow chamber experiment with point source water application at the surface was used to continuously monitor the flow pattern and soil water content (SWC) distribution across the soil profile during infiltration and redistribution periods. Biochar–soil mixtures (2% and 5% w/w) with the three biochar types were studied. Flow chamber results showed substantial differences in plumes shape and internal SWC distribution in the biochar–soil mixtures compared to the untreated sand during the wetting and drainage. The plumes' shape and nonmonotonic spatial SWC distribution suggested unstable flow in the biochar–soil mixtures. The internal fingers developed in the biochar treated sand indicate that SWR induced by biochar addition seemed to be physically induced via the blending of the biochar and soil particles, rather than chemically induced, via coating of the soil particles with amphiphilic molecules. Under field conditions, the primary and inner finger-like plumes may eventually form preferential flow paths that will affect the spatial distribution of water and fertilizer in soil profiles and their availability to plant roots.

Specific inhibition of the transporter MRP4/ABCC4 affects multiple signaling pathways and thrombus formation in human platelets.

The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentration-dependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)-labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.

A human bone infection organ model for biomaterial research

The aim of this work was to establish an organ model for staphylococcal infection of human bone samples and to investigate the influence and efficacy of a microporous β-tricalcium phosphate ceramic (β-TCP, RMS Foundation) loaded with hydrogels (alginate, alginate-di-aldehyde (ADA)-gelatin) and clindamycin on infected human bone tissue over a period of 28 days. For this purpose, human tibia plateaus, collected during total knee replacement surgery, were used as a source of bone material. Samples were infected with S. aureus ATCC29213 and treated with differently loaded β-TCP composites (alginate +/- clindamycin, ADA-gelatin +/- clindamycin, unloaded). The loading of the composites was carried out by means of a flow chamber. The infection was observed for 28 days, quantifying bacteria in the medium and the osseus material on day 1, 7, 14, 21 and 28. All samples were histologically processed for bone vitality evaluation. Bone infection could be consistently performed within the organ model. In addition, a strong reduction in bacterial counts was recorded in the groups treated with ADA-gelatin + clindamycin and alginate + clindamycin, while the bacterial count in the control groups remained constant. No significant differences between groups could be observed in the number of lacunae filled with osteocytes suggesting no differences in bone vitality among groups. In an ex-vivo human bone infection model, over a period of 28 days bacterial growth could be reduced by treatment with ADA-Gel + CLI and ALG + CLI -releasing β-TCP composites. This could be relevant for its clinical use. Further work will be necessary to improve the loading of β-TCP and the bone infection organ model itself. Statement of SignificanceThe common treatment of bone infections is debridement and systemic administration of antibiotics. In some cases, antibiotic-containing carriers are already used, but these must be removed again. Our work is intended to show another treatment option. The scaffold we have developed, made of a calcium phosphate ceramic and a hydrogel as the active substance carrier, can, in addition to releasing the active substance, also assume a load-bearing function of the bone and is biodegradable. In addition, the model we developed can also be used for the analysis and treatment of bone infections other than those of the musculoskeletal system. More importantly, it can also serve as a substitute for previously used animal experiments.

Adsorption-desorption kinetics and phosphorus loss standard curve in erosive weathered granite soil: Stirred flow chamber experiments

Weathered granite has a high soil adsorption rate and it is difficult to apply phosphate fertilizers. In order to effectively use phosphorus, it is very important to study the intensity and speed of soil retention and desorption of phosphorus. The purpose of this study is to evaluate the kinetics of adsorption and desorption in soils with different erodibility. We selected three weathered granite soils with hugely different erosions: topsoil layer (TL-Tillage layer), laterite layer (LL-Laterite layer) and sand layer (SL-Sand layer). Phosphorus adsorption and desorption experiments were successively carried out in the stirred flow reaction chamber, and the stirred flow experiment under unsaturated adsorption conditions was carried out for the first time. The maximum absorption capacity (qamax) and desorption capacity (qdmax) of phosphorus in TL soil was both greater than that in LL and SL soil. The adsorption process mainly occurs within the first 250 min of the reaction. The desorption ratio (Dr) in the TL soil is smaller than the LL and SL soil. The order of the fast reaction sites proportion (F) is SL > LL > TL in adsorption process and LL > SL > TL in desorption process. Seven key soil property indicators can fit qamax and qdmax values well. In the determination coefficient (R2) of the standard curve of phosphorus loss, SL soil is the larger than TL and LL. The qamax and Dr values measured in the stirred flow experiment are both larger than those in the batch experiment. Among the three soils, LL has the lowest adsorption capacity for P and the highest accessibility. The slow adsorption sites in TL account for the main part, and the adsorption in LL and SL mainly occurs at fast adsorption sites. Through the establishment of the phosphorus loss standard curve, the maximum phosphorus loss in the entire region can be predicted and calculated. This study can provide theoretical basis and reference for phosphorus management in similar soil types or physical geographical areas with severely eroded soils.

Characterization of cultivable intestinal microbiota in Rhynchophorus palmarum Linnaeus (Coleoptera: Curculionidae) and determination of its cellulolytic activity.

Rhynchophorus palmarum Linnaeus is an agricultural pest that affects various palm crops, including coconut (Cocos nucifera) plantations which are prominent in the economy of Northeastern Brazil. Characterization of the intestinal microbiota of R. palmarum, as well as elucidation of aspects related to the biochemistry and physiology of the insect's digestion, isessential for intervention in specific metabolic processes as a form of pest control. Thus, this study aimed to characterize the intestinal microbiota of R. palmarum and investigate its ability to degrade cellulosic substrates, to explore new biological control measures. Intestinal dissection of eight adult R. palmarum insects was performed in a laminar flow chamber, and the intestines were homogenized in sterile phosphate-buffered saline solution. Subsequently, serial dilution aliquots of these solutions were spread on nutritive agar plates for the isolation of bacteria and fungi. The microorganisms were identified by matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometry and evaluated for their ability to degrade cellulose. Fourteen bacterial genera (Acinetobacter, Alcaligenes, Arthrobacter, Bacillus, Citrobacter, Enterococcus, Kerstersia, Lactococcus, Micrococcus, Proteus, Providencia, Pseudomonas, Serratia, and Staphylococcus) and two fungal genera (Candida and Saccharomyces)-assigned to the Firmicutes, Actinobacteria, Proteobacteria, and Ascomycota phyla-were identified. The cellulolytic activity was exhibited by six bacterial and one fungal species; of these, Bacillus cereus demonstrated the highest enzyme synthesis (enzymatic index = 4.6). This is the first study characterizing the R. palmarum intestinal microbiota, opening new perspectives for the development of strategies for the biological control of this insect.

Increased platelet thrombus formation under flow conditions in whole blood from polycythaemia vera patients.

Polycythaemia vera is a myeloproliferative neoplasm characterised by a high incidence of thrombosis. The contribution of platelets, key players in haemostasis, in this setting is still unclear. So far, the majority of studies have been focussed on specific platelet abnormalities but not on their actual capacity to form thrombi. The aim of this study was to characterise, ex vivo under flow conditions, the capacity of platelets from patients with polycythaemia vera to adhere to collagen and induce thrombus formation. Thirty-nine patients and 30 healthy controls were studied. Thrombus formation was induced by perfusing whole blood over a collagen-coated surface, in a parallel-plate flow chamber coupled to a fluorescent microscope. This dynamic system enables platelet adhesion and thrombus formation to be followed in real time and also allows measurements of the extent of the thrombus and platelet surface antigen expression. Laboratory data were analysed in the light of the patients' main haematological parameters and therapies. Platelet adhesion was significantly greater in patients than in control subjects. Patient thrombi were usually larger and more complex than those formed by control platelets. A significant positive correlation was found between platelet adhesion and both the haematocrit and red blood cell count. These parameters remained significantly correlated with platelet adhesion also after multivariable analysis adjusted for gender, age, therapy and JAK2V617F allele burden. Furthermore, subjects with a haematocrit >45% had significantly greater platelet adhesion than subjects with a haematocrit <45%. Our data indicate that increased platelet adhesion participates in the thrombotic diathesis of patients with polycythaemia vera, and that the haematocrit level can affect the adhesive and thrombus forming capacities of platelets.

Study on pressure distribution of pump chamber and axial force in particle-laden flow of centrifugal pump

To explore the pressure distribution characteristics of pump chambers, and the law governing the axial force in a particle-laden flow centrifugal pump, a semi-open impeller centrifugal pump was selected as the research object. The particle model and heterogeneous model were used to develop the numerical calculations of the particle-laden flow field within the centrifugal pump. For the liquid phase we adopted the shear–stress–transport (SST) turbulence model, while for the solid phase we adopted the discrete phase zero equation model. The external characteristics, pressure distribution and axial forces were compared and analyzed (1) for a constant solid particle diameter, d, of 0.1 mm and particle volume fractions, Cv, of 0, 1, 5, and 10%, and (2) for a constant particle volume fraction, Cv, of 10% and solid particle diameters, d, of 0, 0.01, 0.1, and 0.5 mm. The correlations between the particle volume fractions and solid particle diameters with the pump chamber pressure distributions and axial forces were obtained. The results show that when Cv increased from 1–10%, or d from 0.01–0.5 mm, the pump head and efficiency decreased continuously under the same flow rate. At the same time, larger particle volume fractions resulted in larger pressures at the same radius, while for larger solid particle diameters, smaller pressures were obtained at the same radius. Under the same flow rate, when Cv increased from 1–10%, the total axial force of the centrifugal pump increased, but it was smaller than what was achieved under the clean-water condition. When d increased from 0.01 to 0.5 mm, the total axial force of the centrifugal pump decreased. This study provides a reference for the calculation and balance of the total axial force in a particle-laden flow centrifugal pump.

Polyvalent Immunoglobulin Preparations Inhibit Pneumolysin-Induced Platelet Destruction.

Platelets play an important role in the development and progression of respiratory distress. Functional platelets are known to seal inflammatory endothelial gaps and loss of platelet function has been shown to result in loss of integrity of pulmonary vessels. This leads to fluid accumulation in the pulmonary interstitium, eventually resulting in respiratory distress. Streptococcus pneumoniae is one of the major pathogens causing community-acquired pneumonia. Previously, we have shown that its major toxin pneumolysin forms pores in platelet membranes and renders them nonfunctional. In vitro, this process was inhibited by polyvalent intravenous immunoglobulins (IVIGs). In this study, we compared the efficacy of a standard IVIG preparation (IVIG, 98% immunoglobulin G [IgG]; Privigen, CSL Behring, United States) and an IgM/IgA-enriched immunoglobulin preparation (21% IgA, 23% IgM, 56% IgG; trimodulin, Biotest AG, Germany) to inhibit pneumolysin-induced platelet destruction. Platelet destruction and functionality were assessed by flow cytometry, intracellular calcium release, aggregometry, platelet viability, transwell, and flow chamber assays. Overall, both immunoglobulin preparations efficiently inhibited pneumolysin-induced platelet destruction. The capacity to antagonize pneumolysin mainly depended on the final IgG content. As both polyvalent immunoglobulin preparations efficiently prevent pneumolysin-induced platelet destruction and maintain platelet function in vitro, they represent promising candidates for clinical studies on supportive treatment of pneumococcal pneumonia to reduce progression of respiratory distress.

Ex vivo observation of granulocyte activity during thrombus formation

BackgroundThe process of thrombus formation is thought to involve interactions between platelets and leukocytes. Leukocyte incorporation into growing thrombi has been well established in vivo, and a number of properties of platelet-leukocyte interactions critical for thrombus formation have been characterized in vitro in thromboinflammatory settings and have clinical relevance. Leukocyte activity can be impaired in distinct hereditary and acquired disorders of immunological nature, among which is Wiskott-Aldrich Syndrome (WAS). However, a more quantitative characterization of leukocyte behavior in thromboinflammatory conditions has been hampered by lack of approaches for its study ex vivo. Here, we aimed to develop an ex vivo model of thromboinflammation, and compared granulocyte behavior of WAS patients and healthy donors.ResultsThrombus formation in anticoagulated whole blood from healthy volunteers and patients was visualized by fluorescent microscopy in parallel-plate flow chambers with fibrillar collagen type I coverslips. Moving granulocytes were observed in hirudinated or sodium citrate-recalcified blood under low wall shear rate conditions (100 s−1). These cells crawled around thrombi in a step-wise manner with an average velocity of 90–120 nm/s. Pre-incubation of blood with granulocyte priming agents lead to a significant decrease in mean-velocity of the cells and increase in the number of adherent cells. The leukocytes from patients with WAS demonstrated a 1.5-fold lower mean velocity, in line with their impaired actin polymerization. It is noteworthy that in an experimental setting where patients’ platelets were replaced with healthy donor’s platelets the granulocytes’ crawling velocity did not change, thus proving that WASP (WAS protein) deficiency causes disruption of granulocytes’ behavior. Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. As most of the crawling granulocytes carried procoagulant platelets teared from thrombi, we propose that the role of granulocytes in thrombus formation is that of platelet scavengers.ConclusionsWe have developed an ex vivo experimental model applicable for observation of granulocyte activity in thrombus formation. Using the proposed setting, we observed a reduction of motility of granulocytes of patients with WAS. We suggest that our ex vivo approach should be useful both for basic and for clinical research.