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Circulating RNAs have been investigated systematically for over 20 years, both as constituents of circulating extracellular vesicles (EVs) or, more recently, non-EV RNA carriers, such as exomeres and supermeres. The high level of variability and low reproducibility rate of EV/extracellular RNA (exRNA) results generated even on the same biofluids promoted several efforts to limit pre-analytical variability by standardizing sample collection and sample preparation, along with instrument validation, setup and calibration. Anticoagulants (ACs) are often chosen based on the initial goal of the study and not necessarily for the later EV and/or exRNA analyses. We show the effects of blood collection on EV size, abundance, and antigenic composition, as well on the miRNAs. Our focus of this work was on the effect of ACs on the number and antigenic composition of circulating EVs and on a set of circulating miRNA species, which were shown to be relevant as disease markers in several cancers and Alzheimer's disease. Results show that while the number of plasma EVs, their relative size, and post-fluorescence labeling profile varied with each AC, their overall antigenic composition, with few exceptions, did not change significantly. However, the number of EVs expressing platelet and platelet-activation markers increased in serum samples. For overall miRNA expression levels, EDTA was a better AC, although this may have been associated with stimulation of cells in the blood collection tube. Citrate and serum rendered better results for a set of miRNAs that were described as circulating markers for Alzheimer's disease, colon, and papillary thyroid cancers., Competing Interests: John Tigges is consulting for Acoerela., (© 2024 The Author(s). Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

BACKGROUNDCardiorenal syndrome (CRS) - renal injury during heart failure (HF) - is linked to high morbidity. Whether circulating extracellular vesicles (EVs) and their RNA cargo directly impact its pathogenesis remains unclear.METHODSWe investigated the role of circulating EVs from patients with CRS on renal epithelial/endothelial cells using a microfluidic kidney-on-chip (KOC) model. The small RNA cargo of circulating EVs was regressed against serum creatinine to prioritize subsets of functionally relevant EV-miRNAs and their mRNA targets investigated using in silico pathway analysis, human genetics, and interrogation of expression in the KOC model and in renal tissue. The functional effects of EV-RNAs on kidney epithelial cells were experimentally validated.RESULTSRenal epithelial and endothelial cells in the KOC model exhibited uptake of EVs from patients with HF. HF-CRS EVs led to higher expression of renal injury markers (IL18, LCN2, HAVCR1) relative to non-CRS EVs. A total of 15 EV-miRNAs were associated with creatinine, targeting 1,143 gene targets specifying pathways relevant to renal injury, including TGF-β and AMPK signaling. We observed directionally consistent changes in the expression of TGF-β pathway members (BMP6, FST, TIMP3) in the KOC model exposed to CRS EVs, which were validated in epithelial cells treated with corresponding inhibitors and mimics of miRNAs. A similar trend was observed in renal tissue with kidney injury. Mendelian randomization suggested a role for FST in renal function.CONCLUSIONPlasma EVs in patients with CRS elicit adverse transcriptional and phenotypic responses in a KOC model by regulating biologically relevant pathways, suggesting a role for EVs in CRS.TRIAL REGISTRATIONClinicalTrials.gov NCT03345446.FUNDINGAmerican Heart Association (AHA) (SFRN16SFRN31280008); National Heart, Lung, and Blood Institute (1R35HL150807-01); National Center for Advancing Translational Sciences (UH3 TR002878); and AHA (23CDA1045944).

Extracellular vesicles (EVs) are membrane-bound structures released by cells and tissues into biofluids, involved in cell-cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell-type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities, and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC-derived microvesicles (RBC-MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis, and vesicular transport, such as of sorcin, stomatin, annexin A7, and RAB proteins. Cryo-electron microscopy identified two separate pathways of RBC-MV-neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC-MVs decrease neutrophil ability to phagocytose E. coli but do not affect their survival at 24 hrs. This work brings new insights regarding the complexity of the RBC-MVs biogenesis, as well as their possible role in circulation., Competing Interests: conflict-of-interest statements CCP is a senior translational data scientist at Sanofi and declares no competing financial interests. The remaining authors declare no competing financial interests that are relevant to the content of this article.

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

MicroRNAs are short, non-coding RNA sequences involved in gene expression regulation. Quantification of miRNAs in biological fluids involves time consuming and laborious methods such as Northern blotting or PCR-based techniques. Molecular beacons (MB) are an attractive means for rapid detection of miRNAs, although the need for sophisticated readout methods limits their use in research and clinical settings. Here, we introduce a novel method based on delayed electrophoretic mobility, as a quantitative means for detection of miRNAs-MB hybridization. Upon hybridization with the target miRNAs, MB form a fluorescent duplex with reduced electrophoretic mobility, thus bypassing the need for additional staining. In addition to emission of light, the location of the fluorescent band on the gel acts as an orthogonal validation of the target identity, further confirming the specificity of binding. The limit of detection of this approach is approximately 100 pM, depending on the MB sequence. The method is sensitive enough to detect specific red blood cell miRNAs molecules in total RNA, with single nucleotide specificity. Altogether, we describe a rapid and affordable method that offers sensitive detection of single-stranded small DNA and RNA sequences., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized in endothelial cells and stored in Weibel-Palade bodies (WPBs). Understanding the mechanisms underlying WPB biogenesis and exocytosis could enable therapeutic modulation of endogenous VWF, yet optimal targets for modulating VWF release have not been established. Because biogenesis of lysosomal related organelle-2 (BLOC-2) functions in the biogenesis of platelet dense granules and melanosomes, which like WPBs are lysosome-related organelles, we hypothesized that BLOC-2-dependent endolysosomal trafficking is essential for WPB biogenesis and sought to identify BLOC-2-interacting proteins. Depletion of BLOC-2 caused misdirection of cargo-carrying transport tubules from endosomes, resulting in immature WPBs that lack endosomal input. Immunoprecipitation of BLOC-2 identified the exocyst complex as a binding partner. Depletion of the exocyst complex phenocopied BLOC-2 depletion, resulting in immature WPBs. Furthermore, releasates of immature WPBs from either BLOC-2 or exocyst-depleted endothelial cells lacked high-molecular weight (HMW) forms of VWF, demonstrating the importance of BLOC-2/exocyst-mediated endosomal input during VWF maturation. However, BLOC-2 and exocyst showed very different effects on VWF release. Although BLOC-2 depletion impaired exocytosis, exocyst depletion augmented WPB exocytosis, indicating that it acts as a clamp. Exposure of endothelial cells to a small molecule inhibitor of exocyst, Endosidin2, reversibly augmented secretion of mature WPBs containing HMW forms of VWF. These studies show that, although BLOC-2 and exocyst cooperate in WPB formation, only exocyst serves to clamp WPB release. Exocyst function in VWF maturation and release are separable, a feature that can be exploited to enhance VWF release., (© 2020 by The American Society of Hematology.)

Extracellular vesicles (EVs), such as exosomes and microvesicles, are nonreplicating lipid bilayer particles shed by most cell types which have the potential to revolutionize the development and efficient delivery of clinical therapeutics. This article provides an introduction to the landscape of EV-based vectors under development for the delivery of protein- and nucleic acid-based therapeutics. We highlight some of the most pressing measurement and standardization challenges that limit the translation of EVs to the clinic. Current challenges limiting development of EVs for drug delivery are the lack of: standardized cell-based platforms for the production of EV-based therapeutics; EV reference materials that allow researchers/manufacturers to validate EV measurements and standardized measurement systems for determining the molecular composition of EVs.

Protein "AND-gate" systems, in which a ligand acts only on cells with two different receptors, direct signaling activity to a particular cell type and avoid action on other cells. In a bifunctional AND-gate protein, the molecular geometry of the protein domains is crucial. Here we constructed a tissue-targeted erythropoietin (EPO) that stimulates red blood cell (RBC) production without triggering thrombosis. The EPO was directed to RBC precursors and mature RBCs by fusion to an anti-glycophorin A antibody V region. Many such constructs activated EPO receptors in vitro and stimulated RBC and not platelet production in mice but nonetheless enhanced thrombosis in mice and caused adhesion between RBCs and EPO-receptor-bearing cells. On the basis of a protein-structural model of the RBC surface, we rationally designed an anti-glycophorin-EPO fusion that does not induce cell adhesion in vitro or enhance thrombosis in vivo . Thus, mesoscale geometry can inform the design of synthetic-biological systems.

Extracellular vesicles (EVs) have recently emerged as intercellular conveyors of biological information and disease biomarkers. Identification and characterization of RNA species in single EVs are currently challenging. Molecular beacons (MBs) represent an attractive means for detecting specific RNA molecules. Coupling the MBs to cell-penetrating peptides (CPPs) provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol. Here, we generated RBCs-derived EVs by complement activation and tested the ability of MBs coupled with CPP to detect miRNAs from RBC-EVs. Our results showed that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nano-flow cytometry. MB-based detection of RNA via nano-flow cytometry creates a powerful new analytical framework in which a simple addition of a reagent allows profiling of specific RNA species present within certain EV subsets., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: We have developed a novel, easily implementable methodology using magnetic levitation to quantify circulating leukocyte size, morphology, and magnetic properties, which may help in rapid, bedside screening for sepsis., Objective: Our objectives were to describe our methodological approach to leukocyte assessment, and to perform a pilot investigation to test the ability of magnetic levitation to identify and quantify changes in leukocyte size, shape, density, and/or paramagnetic properties in healthy controls and septic patients., Methods: This prospective, observational cohort study was performed in a 56,000/y visit emergency department (ED) and affiliated outpatient phlebotomy laboratory. Inclusion criteria were admittance to the hospital with suspected or confirmed infection for the septic group, and we enrolled the controls from ED/outpatient patients without infection or acute illness. The bench-top experiments were performed using magnetic levitation to visualize the leukocytes. We primary sought to compare septic patients with noninfected controls and secondary to assess the association with sepsis severity. Our covariates were area, length, width, roundness, and standard deviation (SD) of levitation height. We used unpaired t test and area under the curve (AUC) for the assessment of accuracy in distinguishing between septic and control patients., Results: We enrolled 39 noninfected controls and 22 septic patients. Our analyses of septic patients compared with controls showed: mean cell area in pixels (px) 562 ± 111 vs. 410 ± 45, P < 0.0001, AUC = 0.89 (0.80-0.98); length (px), 29 ± 2.5 vs. 25 ± 1.9, P < 0.0001, AUC = 0.90 (0.83-0.98); and width (px), 27 ± 2.4 vs. 23 ± 1.5, P < 0.0001, AUC = 0.92 (0.84-0.99). Cell roundness: 2.1 ± 1.0 vs. 2.2 ± 1.2, P = 0.8, AUC = 0.51. SD of the levitation height (px) was 72 ± 25 vs. 47 ± 16, P < 0.001, AUC = 0.80 (0.67-0.93)., Conclusions: Septic patients had circulating leukocytes with especially increased size parameters, which distinguished sepsis from noninfected patients with promising high accuracy. This portal-device compatible technology shows promise as a potential bedside diagnostic.

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks., Competing Interests: The authors declare no conflict of interest.

Magnetic levitation has emerged as a technique that offers the ability to differentiate between cells with different densities. We have developed a magnetic levitation system for this purpose that distinguishes not only different cell types but also density differences in cells of the same type. This small-scale system suspends cells in a paramagnetic medium in a capillary placed between two rare earth magnets, and cells levitate to an equilibrium position determined solely by their density. Uniform reference beads of known density are used in conjunction with the cells as a means to quantify their levitation positions. In one implementation images of the levitating cells are acquired with a microscope, but here we also introduce a cell phone-based device that integrates the magnets, capillary, and a lens into a compact and portable unit that acquires images with the phone's camera. To demonstrate the effectiveness of magnetic levitation in cell density analysis we carried out levitation experiments using red blood cells with artificially altered densities, and also levitated those from donors. We observed that we can distinguish red blood cells of an anemic donor from those that are healthy. Since a plethora of disease states are characterized by changes in cell density magnetic cell levitation promises to be an effective tool in identifying and analyzing pathologic states. Furthermore, the low cost, portability, and ease of use of the cell phone-based system may potentially lead to its deployment in low-resource environments.

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.

Sickle cell disease affects 25% of people living in Central and West Africa and, if left undiagnosed, can cause life threatening "silent" strokes and lifelong damage. However, ubiquitous testing procedures have yet to be implemented in these areas, necessitating a simple, rapid, and accurate testing platform to diagnose sickle cell disease. Here, we present a label-free, sensitive, and specific testing platform using only a small blood sample (<1 μl) based on the higher density of sickle red blood cells under deoxygenated conditions. Testing is performed with a lightweight and compact 3D-printed attachment installed on a commercial smartphone. This attachment includes an LED to illuminate the sample, an optical lens to magnify the image, and two permanent magnets for magnetic levitation of red blood cells. The sample is suspended in a paramagnetic medium with sodium metabisulfite and loaded in a microcapillary tube that is inserted between the magnets. Red blood cells are levitated in the magnetic field based on equilibrium between the magnetic and buoyancy forces acting on the cells. Using this approach, we were able to distinguish between the levitation patterns of sickle versus control red blood cells based on their degree of confinement.

Magnetic levitation, which uses a magnetic field to suspend objects in a fluid, is a powerful and versatile technology. We develop a compact magnetic levitation platform compatible with a smart-phone to separate micro-objects and estimate the density of the sample based on its levitation height. A 3D printed attachment is mechanically installed over the existing camera unit of a smart-phone. Micro-objects, which may be either spherical or irregular in shape, are suspended in a paramagnetic medium and loaded in a microcapillary tube which is then inserted between two permanent magnets. The micro-objects are levitated and confined in the microcapillary at an equilibrium height dependent on their volumetric mass densities (causing a buoyancy force toward the edge of the microcapillary) and magnetic susceptibilities (causing a magnetic force toward the center of the microcapillary) relative to the suspending medium. The smart-phone camera captures magnified images of the levitating micro-objects through an additional lens positioned between the sample and the camera lens cover. A custom-developed Android application then analyzes these images to determine the levitation height and estimate the density. Using this platform, we were able to separate microspheres with varying densities and calibrate their levitation heights to known densities to develop a technique for precise and accurate density estimation. We have also characterized the magnetic field, the optical imaging capabilities, and the thermal state over time of this platform.

A simple, yet powerful magnetic-levitation-based device is reported for real-time, label-free separation, as well as high-resolution monitoring of cell populations based on their unique magnetic and density signatures. This method allows a wide variety of cellular processes to be studied, accompanied by transient or permanent changes in cells' fundamental characteristics as a biological material., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (~25-30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ~60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane., (© 2015 Gokhin et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Objective: Complement system is activated in patients with trauma. Although complement activation is presumed to contribute to organ damage and constitutional symptoms, little is known about the involved mechanisms. Because complement components may deposit on RBCs, we asked whether complement deposits on the surface of RBC in trauma and whether such deposition alters RBC function., Design: A prospective experimental study., Setting: Research laboratory., Subjects: Blood samples collected from 42 trauma patients and 21 healthy donors., Intervention: None., Measurements and Main Results: RBC and sera were collected from trauma patients and control donors. RBCs from trauma patients (n = 40) were found to display significantly higher amounts of C4d on their surface by flow cytometry compared with RBCs from control (n = 17) (p < 0.01). Increased amounts of iC3b were found in trauma sera (n = 27) (vs 12 controls, p < 0.01) by enzyme-linked immunosorbent assay. Incubation of RBC from universal donors (type O, Rh negative) with trauma sera (n = 10) promoted C4d deposition on their surface (vs six controls, p< 0.05). Complement-decorated RBC (n = 6) displayed limited their deformability (vs six controls, p < 0.05) in two-dimensional microchannel arrays. Incubation of RBC with trauma sera (n = 10) promoted the phosphorylation of band 3, a cytoskeletal protein important for the function of the RBC membrane (vs eight controls, p < 0.05), and also accelerated calcium influx (n = 9) and enhanced nitric oxide production (n = 12) (vs four and eight controls respectively, p < 0.05) in flow cytometry., Conclusions: Our study found the presence of extensive complement activation in trauma patients and presents new evidence in support of the hypothesis that complement activation products deposit on the surface of RBC. Such deposition could limit RBC deformability and promote the production of nitric oxide. Our findings suggest that RBC in trauma patients malfunctions, which may explain organ damage and constitutional symptoms that is not accounted for otherwise by previously known pathophysiologic mechanisms.

Humans and other higher primates are unique among mammals in using complement receptor 1 (CR1, CD35) on red blood cells (RBC) to ligate complement-tagged inflammatory particles (immune complexes, apoptotic/necrotic debris, and microbes) in the circulation for quiet transport to the sinusoids of spleen and liver where resident macrophages remove the particles, but allow the RBC to return unharmed to the circulation. This process is called immune-adherence clearance. In this study we found using luminometric- and fluorescence-based methods that ligation of CR1 on human RBC promotes ATP release. Our data show that CR1-mediated ATP release does not depend on Ca(2+) or enzymes previously shown to mediate an increase in membrane deformability promoted by CR1 ligation. Furthermore, ATP release following CR1 ligation increases the mobility of the lipid fraction of RBC membranes, which in turn facilitates CR1 clustering, and thereby enhances the binding avidity of complement-opsonized particles to the RBC CR1. Finally, we have found that RBC-derived ATP has a stimulatory effect on phagocytosis of immune-adherent immune complexes.

Objective: Systemic lupus erythematosus (SLE) is characterized by intravascular activation of the complement system and deposition of complement fragments (C3 and C4) on plasma membranes of circulating cells, including red blood cells (RBCs). The aim of this study was to address whether this process affects the biophysical properties of RBCs., Methods: Serum and RBCs were isolated from patients with SLE and healthy controls. RBCs from healthy universal donors (type O, Rh negative) were incubated with SLE or control serum. We used flow cytometry to assess complement fragment deposition on RBCs. RBC membrane deformability was measured using 2-dimensional microchannel arrays. Protein phosphorylation levels were quantified by Western blotting., Results: Incubation of healthy universal donor RBCs with sera from patients with SLE, but not with control sera, led to deposition of C4d fragments on the RBCs. Complement-decorated RBCs exhibited significant decreases in both membrane deformability and flickering. Sera from SLE patients triggered a transitory Ca(++) influx in RBCs that was associated with decreased phosphorylation of β-spectrin and with increased phosphorylation of band 3, two key proteins of RBC cytoskeleton. Finally, incubation with SLE sera led to the production of nitric oxide by RBCs, whereas this did not occur with control sera., Conclusion: Our data suggest that complement activation in patients with SLE leads to calcium-dependent cytosketeletal changes in RBCs that render them less deformable, probably impairing their flow through capillaries. This phenomenon may negatively affect the delivery of oxygen to the tissues., (Copyright © 2011 by the American College of Rheumatology.)

This chapter is an overview of basic principles of fluorescence microscopy, including a brief history on the invention of this type of microscopy. The chapter highlights important points related to properties of fluorochromes, resolution in fluorescence microscopy, phase contrast and fluorescence, fluorescence filters, construction of a fluorescence microscope, and tips on the correct use of this equipment.

Microbes as well as immune complexes and other continuously generated inflammatory particles are efficiently removed from the human circulation by red blood cells (RBCs) through a process called immune-adherence clearance. During this process, RBCs use complement receptor 1 (CR1, CD35) to bind circulating complement-opsonized particles and transfer them to resident macrophages in the liver and spleen for removal. We here show that ligation of RBC CR1 by antibody and complement-opsonized particles induces a transient Ca(++) influx that is proportional to the RBC CR1 levels and is inhibited by T1E3 pAb, a specific inhibitor of TRPC1 channels. The CR1-elicited RBC Ca(++) influx is accompanied by an increase in RBC membrane deformability that positively correlates with the number of preexisting CR1 molecules on RBC membranes. Biochemically, ligation of RBC CR1 causes a significant increase in phosphorylation levels of β-spectrin that is inhibited by preincubation of RBCs with DMAT, a specific casein kinase II inhibitor. We hypothesize that the CR1-dependent increase in membrane deformability could be relevant for facilitating the transfer of CR1-bound particles from the RBCs to the hepatic and splenic phagocytes.

Present techniques for prenatal diagnosis are invasive and present significant risks of fetal loss. Noninvasive prenatal diagnosis utilizing fetal nucleated red blood cells (fNRBC) circulating in maternal peripheral blood has received attention, since it poses no risk to the fetus. However, because of the failure to find broadly applicable identifiers that can differentiate fetal from adult NRBC, reliable detection of viable fNRBC in amounts sufficient for clinical use remains a challenge. In this Letter we show that fNRBC light-scattering spectroscopic signatures may lead to a clinically useful method of minimally invasive prenatal genetic testing.

This article reports the development of an optical imaging technique, confocal light absorption and scattering spectroscopic (CLASS) microscopy, capable of noninvasively determining the dimensions and other physical properties of single subcellular organelles. CLASS microscopy combines the principles of light-scattering spectroscopy (LSS) with confocal microscopy. LSS is an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index, and shape. The multispectral nature of LSS enables it to measure internal cell structures much smaller than the diffraction limit without damaging the cell or requiring exogenous markers, which could affect cell function. Scanning the confocal volume across the sample creates an image. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to optical microscopy. It provides unique capabilities to study functions of viable cells, which are beyond the capabilities of other techniques.