Less Deformable Erythrocyte Subpopulations Biomechanically Induce Endothelial Inflammation in Sickle Cell Disease.

Sickle cell disease (SCD) is canonically characterized by reduced red blood cell (RBC) deformability leading to microvascular obstruction and inflammation. While the biophysical properties of sickle RBCs are known to influence SCD vasculopathy, the contribution of poor RBC deformability to endothelial dysfunction has yet to be fully explored. Leveraging interrelated in vitro and in silico approaches, we introduce a new paradigm of SCD vasculopathy in which poorly deformable sickle RBCs directly cause endothelial dysfunction via mechanotransduction, where endothelial cells sense and pathophysiologically respond to aberrant physical forces independently of microvascular obstruction, adhesion, or hemolysis. We demonstrate that perfusion of sickle RBCs or pharmacologically-dehydrated healthy RBCs into small venule-sized "endothelialized" microfluidics leads to pathologic physical interactions with endothelial cells that directly induce inflammatory pathways. Using a combination of computational simulations and large venule-sized endothelialized microfluidics, we observed that perfusion of heterogeneous sickle RBC subpopulations of varying deformability, as well as suspensions of dehydrated normal RBCs admixed with normal RBCs leads to aberrant margination of the less-deformable RBC subpopulations towards the vessel walls, causing localized, increased shear stress. Increased wall stress is dependent on the degree of subpopulation heterogeneity and oxygen tension and leads to inflammatory endothelial gene expression via mechanotransductive pathways. Our multifaceted approach demonstrates that the presence of sickle RBCs with reduced deformability leads directly to pathological physical (i.e., direct collisions and/or compressive forces) and shear-mediated interactions with endothelial cells and induces an inflammatory response, thereby elucidating the ubiquity of vascular dysfunction in SCD.

Redefining hyperviscosity in acute leukemia: Potential implications for red cell transfusions in the microvasculature.

Hyperleukocytosis is an emergency of acute leukemia leading to blood hyperviscosity, potentially resulting in life-threatening microvascular obstruction, or leukostasis. Due to the high number of red cells in the circulation, hematocrit/hemoglobin levels (Hct/Hgb) are major drivers of blood viscosity, but how Hct/Hgb mediates hyperviscosity in acute leukemia remains unknown. In vivo hemorheological studies are difficult to conduct and interpret due to issues related to visualizing and manipulating the microvasculature. To that end, a multi-vessel microfluidic device recapitulating the size-scale and geometry of the microvasculature was designed to investigate how Hct/Hgb interacts with acute leukemia to induce "in vitro" leukostasis. Using patient samples and cell lines, the degree of leukostasis was different among leukemia immunophenotypes with respect to white blood cell (WBC) count and Hct/Hgb. Among lymphoid immunophenotypes, severe anemia is protective against in vitro leukostasis and Hct/Hgb thresholds became apparent above which in vitro leukostasis significantly increased, to a greater extent with B-cell acute lymphoblastic leukemia (ALL) versus T-cell ALL. In vitro leukostasis in acute myeloid leukemia was primarily driven by WBC with little interaction with Hct/Hgb. This sets the stage for prospective clinical studies assessing how red cell transfusion may affect leukostasis risk in immunophenotypically different acute leukemia patients.

The Coexistence of TRPV6 Variants With Other Pancreatitis-Associated Genes Affects Pediatric-Onset Pancreatitis.

OBJECTIVES: Recently, a genetic risk for chronic pancreatitis (CP) was found to be conferred by pathogenic variants in the transient receptor potential cation channel, subfamily V, member 6 ( TRPV6 ). Interestingly, 20%-57% of patients with functionally defective TRPV6 variants have other susceptibility genes such as cationic trypsinogen, serine protease inhibitor Kazal type 1, chymotrypsin C, cystic fibrosis transmembrane conductance regulator, and carboxypeptidase A1. In this study, we focused on pediatric patients with acute recurrent pancreatitis or CP with at least 1 variant in these 5 genes and investigated the presence of coexisting TRPV6 mutations. METHODS: Ninety Japanese pediatric patients (median age at first onset, 8.0 years) who had at least 1 variant of these 5 genes were enrolled in this study. DNA samples were extracted for analysis from peripheral blood leukocytes. Coding regions of TRPV6 were screened by Sanger sequencing. RESULTS: Regardless of functional defects or non-defects in TRPV6 variants, 14 of the 90 patients (15.6%) were trans-heterozygous for TRPV6 variants [p.A18S (n = 3), p.C197R (n = 3), p.I223T (n = 3), p.D324N (n = 4), p.M418V (n = 3), p.V540F (n = 1), p.A606T (n = 1), and p.M721T (n = 3)] and the 5 susceptibility genes noted above. Of these variants, p.D324N, p.V540F, and p.A606T are associated with pancreatitis. Three patients had the ancestral haplotype [p.C197R + p.M418V + p.M721T]. CONCLUSIONS: Overall, 4 of 90 patients (4.4%) had the coexistence of clearly pathogenic TRPV6 variants with pancreatitis-associated variants. The cumulative accumulation of these genetic factors may contribute to the development of pancreatitis at a young age.

Hemostasis-on-a-chip / incorporating the endothelium in microfluidic models of bleeding.

Currently, point-of-care assays for human platelet function and coagulation are used to assess bleeding risks and drug testing, but they lack intact endothelium, a critical component of the human vascular system. Within these assays, the assessment of bleeding risk is typically indicated by the lack of or reduced platelet function and coagulation without true evaluation of hemostasis. Hemostasis is defined as the cessation of bleeding. Additionally, animal models of hemostasis also, by definition, lack human endothelium, which may limit their clinical relevance. This review discusses the current state-of-the-art of hemostasis-on-a-chip, specifically, human cell-based microfluidic models that incorporate endothelial cells, which function as physiologically relevant in vitro models of bleeding. These assays recapitulate the entire process of vascular injury, bleeding, and hemostasis, and provide real-time, direct observation, thereby serving as research-enabling tools that enhance our understanding of hemostasis and also as novel drug discovery platforms.

The human body's response to stop bleeding after a vascular injury involves a complex but finely tuned cascade of interactions between the blood, the blood vessel wall, and the physical flow of the blood. Accordingly, in vitro models that incorporate those aspects that occur in vivo are highly needed for research and clinical purposes. Here, we review the state of the art of these technologies, hemostasis-on-a-chip devices that aim to achieve those goals. These physiologically relevant "microchips" mimic the bleeding process as well as the cessation thereof, and can be leveraged as research-enabling tools, platforms for drug discovery, and clinical testing.

Getting a good view: in vitro imaging of platelets under flow.

As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myriad of biophysical and biochemical stimuli within vasculature and heterogeneous blood clots. Highly controlled, reductionist in vitro imaging studies have been instrumental in providing a detailed and quantitative understanding of platelet biology and behavior, and have helped elucidate some surprising functions of platelets. In this review, we highlight the tools and approaches that enable visualization of platelets in conjunction with precise control over the local biofluidic and biochemical microenvironment. We also discuss next generation tools that add further control over microenvironment cell stiffness or enable visualization of the interactions between platelets and endothelial cells. Throughout the review, we include pragmatic knowledge on imaging systems, experimental conditions, and approaches that have proved to be useful to our in vitro imaging studies of platelets under flow.

Extracellular fluid tonicity impacts sickle red blood cell deformability and adhesion.

Abnormal sickle red blood cell (sRBC) biomechanics, including pathological deformability and adhesion, correlate with clinical severity in sickle cell disease (SCD). Clinical intravenous fluids (IVFs) of various tonicities are often used during treatment of vaso-occlusive pain episodes (VOE), the major cause of morbidity in SCD. However, evidence-based guidelines are lacking, and there is no consensus regarding which IVFs to use during VOE. Further, it is unknown how altering extracellular fluid tonicity with IVFs affects sRBC biomechanics in the microcirculation, where vaso-occlusion takes place. Here, we report how altering extracellular fluid tonicity with admixtures of clinical IVFs affects sRBC biomechanical properties by leveraging novel in vitro microfluidic models of the microcirculation, including 1 capable of deoxygenating the sRBC environment to monitor changes in microchannel occlusion risk and an "endothelialized" microvascular model that measures alterations in sRBC/endothelium adhesion under postcapillary venular conditions. Admixtures with higher tonicities (sodium = 141 mEq/L) affected sRBC biomechanics by decreasing sRBC deformability, increasing sRBC occlusion under normoxic and hypoxic conditions, and increasing sRBC adhesion in our microfluidic human microvasculature models. Admixtures with excessive hypotonicity (sodium = 103 mEq/L), in contrast, decreased sRBC adhesion, but overswelling prolonged sRBC transit times in capillary-sized microchannels. Admixtures with intermediate tonicities (sodium = 111-122 mEq/L) resulted in optimal changes in sRBC biomechanics, thereby reducing the risk for vaso-occlusion in our models. These results have significant translational implications for patients with SCD and warrant a large-scale prospective clinical study addressing optimal IVF management during VOE in SCD.

Cellular softening mediates leukocyte demargination and trafficking, thereby increasing clinical blood counts.

Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a well-documented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid- and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids. Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing.

Urinary angiotensinogen in pediatric urinary tract infection.

BACKGROUND: Urinary tract infection (UTI) is one of the most common diseases in children, and urinary angiotensinogen (U-AGT) is a new biomarker gathering attention in many renal diseases. U-AGT reflects intrarenal renin-angiotensin system (RAS) activity. We conducted a study to measure U-AGT in children <4 months old with UTI. METHODS: All children <4 months old who came to Toshima Hospital with fever between January 2015 and December 2015 were included. Patients were divided into a UTI group and a non-UTI group, and U-AGT was measured. RESULTS: Median U-AGT was higher in patients with UTI compared with patients without UTI: (0.56 ng/dL, range, 0.025-2.753 ng/dL vs 0.13 ng/dL, range, 0.008-1.697 ng/dL, respectively; P < 0.05). CONCLUSIONS: U-AGT is elevated in UTI patients, and RAS activation may contribute to renal injury caused by UTI.

Single-platelet nanomechanics measured by high-throughput cytometry.

Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot's capability to stem haemorrhage are its changing mechanical properties, the major drivers of which are the contractile forces exerted by platelets against the fibrin scaffold. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders. Here, we report a high-throughput hydrogel-based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.

Microfluidic Transduction Harnesses Mass Transport Principles to Enhance Gene Transfer Efficiency.

Ex vivo gene therapy using lentiviral vectors (LVs) is a proven approach to treat and potentially cure many hematologic disorders and malignancies but remains stymied by cumbersome, cost-prohibitive, and scale-limited production processes that cannot meet the demands of current clinical protocols for widespread clinical utilization. However, limitations in LV manufacture coupled with inefficient transduction protocols requiring significant excess amounts of vector currently limit widespread implementation. Herein, we describe a microfluidic, mass transport-based approach that overcomes the diffusion limitations of current transduction platforms to enhance LV gene transfer kinetics and efficiency. This novel ex vivo LV transduction platform is flexible in design, easy to use, scalable, and compatible with standard cell transduction reagents and LV preparations. Using hematopoietic cell lines, primary human T cells, primary hematopoietic stem and progenitor cells (HSPCs) of both murine (Sca-1+) and human (CD34+) origin, microfluidic transduction using clinically processed LVs occurs up to 5-fold faster and requires as little as one-twentieth of LV. As an in vivo validation of the microfluidic-based transduction technology, HSPC gene therapy was performed in hemophilia A mice using limiting amounts of LV. Compared to the standard static well-based transduction protocols, only animals transplanted with microfluidic-transduced cells displayed clotting levels restored to normal.

Normal saline is associated with increased sickle red cell stiffness and prolonged transit times in a microfluidic model of the capillary system.

OBJECTIVE: Vaso-occlusive crisis (VOC) is a complex process that occurs in patients with sickle cell disease (SCD) and is often associated with pain and urgent hospitalization. A major instigator of VOC is microvascular obstruction by pathologically stiffened sickle red blood cells (RBCs), and thus, therapy relies heavily on optimizing intravenous fluid (IVF) hydration to increase RBC deformability. However, no evidence-based guidelines regarding the choice of IVF currently exist. We therefore analyzed alterations in biomechanical properties of sickle RBCs isolated from patients with homozygous SCD (hemoglobin SS) after exposure to different osmolarities of clinical IVF formulations. METHODS: Atomic force microscopy (AFM) was used to assess stiffness of RBCs after exposure to different IVFs. A microfluidic model of the human capillary system was used to assess transit time (TT) and propensity to occlusion after exposure to the different IVF formulations. RESULTS: Sickle RBCs exposed to normal saline (NS) had increased stiffness, TTs, and propensity to microchannel occlusion compared to other osmolarities. CONCLUSION: NS, an IVF formulation often used to treat patients with SCD during VOC, may induce localized microvascular obstruction due to alterations of sickle RBC biomechanical properties.

Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach.

The mechanism of action of the widely used in vivo ferric chloride (FeCl3) thrombosis model remains poorly understood; although endothelial cell denudation is historically cited, a recent study refutes this and implicates a role for erythrocytes. Given the complexity of the in vivo environment, an in vitro reductionist approach is required to systematically isolate and analyze the biochemical, mass transfer, and biological phenomena that govern the system. To this end, we designed an "endothelial-ized" microfluidic device to introduce controlled FeCl3 concentrations to the molecular and cellular components of blood and vasculature. FeCl3 induces aggregation of all plasma proteins and blood cells, independent of endothelial cells, by colloidal chemistry principles: initial aggregation is due to binding of negatively charged blood components to positively charged iron, independent of biological receptor/ligand interactions. Full occlusion of the microchannel proceeds by conventional pathways, and can be attenuated by antithrombotic agents and loss-of-function proteins (as in IL4-R/Iba mice). As elevated FeCl3 concentrations overcome protective effects, the overlap between charge-based aggregation and clotting is a function of mass transfer. Our physiologically relevant in vitro system allows us to discern the multifaceted mechanism of FeCl3-induced thrombosis, thereby reconciling literature findings and cautioning researchers in using the FeCl3 model.

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition.

Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to "self-deposit" additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbß3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium.

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation.

As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin αIIbß3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation.

Genetic Analysis of Japanese Children With Acute Recurrent and Chronic Pancreatitis.

OBJECTIVES: Causes of acute recurrent pancreatitis (ARP) or chronic pancreatitis (CP) are sometimes difficult to determine in children. In such patients, genetic analysis may prove helpful. The present study analyzed mutations of cationic trypsinogen (PRSS1), serine protease inhibitor Kazal type 1 (SPINK1), chymotrypsin C (CTRC), and carboxypeptidase A1 (CPA1) and investigated the clinical features of children with these mutations. METHODS: Genetic analyses of mutations in these 4 genes were conducted in 128 patients with ARP or CP. Characteristics of the patients showing mutations were investigated using medical records. RESULTS: Fifty of the 128 (39.1%) subjects had at least 1 mutation (median age at onset, 7.6 years). Abdominal pain was the presenting symptom of pancreatitis in 48 of the 50 patients (96%). Fifteen of those 50 patients (30.0%) had a family history of pancreatitis. Gene mutations were present in PRSS1 in 26 patients, SPINK1 in 23, CTRC in 3, and CPA1 in 5. In the 31 patients with mutations in SPINK1, CTRC, or CPA1, 16 (51.6%) had homozygous or heterozygous mutations with other mutations. Three patients underwent surgery and another 4 patients underwent endoscopy to manage ARP or CP. Although 3 of the 7 patients complained of mild abdominal pain, none of those 7 patients experienced any obvious episode of ARP after treatment. CONCLUSIONS: In pediatric patients with idiopathic ARP and CP, genetic analysis is useful for identifying the cause of pancreatitis. Early endoscopic or surgical treatment prevents ARP by extending the interval between episodes of pancreatitis in this population.

Dual biologics for severe asthma and atopic dermatitis: Synopsis of two cases and literature review.

The efficacy and safety of the combination of biologic therapies remain unclear with an ineffective and insufficient single biologic for managing asthma. Herein, we report two cases using dual biologics for severe asthma and atopic dermatitis. A 52-year-old male patient who received dupilumab and mepolizumab, benralizumab, or tezepelumab, followed by bronchial thermoplasty, and a 41-year-old male patient who received dupilumab and omalizumab, both experienced improved asthma and atopic dermatitis. To date, 38 cases are using dual biologics for severe asthma. The success rate was 84%, with no major adverse effects. We report the first case of severe asthma receiving dual biologics with tezepelumab and furthermore bronchial thermoplasty, and comprehensive literature review on dual biologics. Dual biologics may be an effective treatment method for severe asthma, requiring further investigation.

Biomechanics of haemostasis and thrombosis in health and disease: from the macro- to molecular scale.

Although the processes of haemostasis and thrombosis have been studied extensively in the past several decades, much of the effort has been spent characterizing the biological and biochemical aspects of clotting. More recently, researchers have discovered that the function and physiology of blood cells and plasma proteins relevant in haematologic processes are mechanically, as well as biologically, regulated. This is not entirely surprising considering the extremely dynamic fluidic environment that these blood components exist in. Other cells in the body such as fibroblasts and endothelial cells have been found to biologically respond to their physical and mechanical environments, affecting aspects of cellular physiology as diverse as cytoskeletal architecture to gene expression to alterations of vital signalling pathways. In the circulation, blood cells and plasma proteins are constantly exposed to forces while they, in turn, also exert forces to regulate clot formation. These mechanical factors lead to biochemical and biomechanical changes on the macro- to molecular scale. Likewise, biochemical and biomechanical alterations in the microenvironment can ultimately impact the mechanical regulation of clot formation. The ways in which these factors all balance each other can be the difference between haemostasis and thrombosis. Here, we review how the biomechanics of blood cells intimately interact with the cellular and molecular biology to regulate haemostasis and thrombosis in the context of health and disease from the macro- to molecular scale. We will also show how these biomechanical forces in the context of haemostasis and thrombosis have been replicated or measured in vitro.

Membrane curvature and PS localize coagulation proteins to filopodia and retraction fibers of endothelial cells.

Prior reports indicate that the convex membrane curvature of phosphatidylserine (PS)-containing vesicles enhances formation of binding sites for factor Va and lactadherin. Yet, the relationship of convex curvature to localization of these proteins on cells remains unknown. We developed a membrane topology model, using phospholipid bilayers supported by nano-etched silica substrates, to further explore the relationship between curvature and localization of coagulation proteins. Ridge convexity corresponded to maximal curvature of physiologic membranes (radii of 10 or 30 nm) and the troughs had a variable concave curvature. The benchmark PS probe lactadherin exhibited strong differential binding to the ridges, on membranes with 4% to 15% PS. Factor Va, with a PS-binding motif homologous to lactadherin, also bound selectively to the ridges. Bound factor Va supported coincident binding of factor Xa, localizing prothrombinase complexes to the ridges. Endothelial cells responded to prothrombotic stressors and stimuli (staurosporine, tumor necrosis factor-α [TNF- α]) by retracting cell margins and forming filaments and filopodia. These had a high positive curvature similar to supported membrane ridges and selectively bound lactadherin. Likewise, the retraction filaments and filopodia bound factor Va and supported assembly of prothrombinase, whereas the cell body did not. The perfusion of plasma over TNF-α-stimulated endothelia in culture dishes and engineered 3-dimensional microvessels led to fibrin deposition at cell margins, inhibited by lactadherin, without clotting of bulk plasma. Our results indicate that stressed or stimulated endothelial cells support prothrombinase activity localized to convex topological features at cell margins. These findings may relate to perivascular fibrin deposition in sepsis and inflammation.

Allergy to Omalizumab: Lessons from a Reaction to the Coronavirus 2019 Vaccine.

Omalizumab can cause hypersensitivity reactions. We herein report the first case of an 18-year-old woman with refractory cough-predominant asthma that correlated with allergic reactions caused by omalizumab and the coronavirus disease 2019 (COVID-19) vaccine. The patient developed angioedema after taking omalizumab. She had previously experienced intense coughing immediately after receiving a COVID-19 vaccine. A skin prick test was positive for polysorbate 20, which was probably the cause of the allergic reactions to omalizumab and the COVID-19 vaccine. Clinicians should check for an allergic reaction, irrespective of its intensity, triggered by polysorbate and be careful when prescribing biologics to patients in order to avoid allergic reactions.