Red Blood Cell Lysis Research Articles

PSIV-28 Evaluation of Possible Interactions Between Platelets and Neutrophil in Cow Blood

Abstract Neutrophils have a central role in the inflammatory response and fight infection. Platelets are essential for hemostasis and thrombosis and fighting vascular damage. Studies in man show that the interaction between these cells can modulate their function. We aimed to assess the effect of possible relationships between the platelet count in whole blood on neutrophil activation. Blood samples (50 mL) from five clinically healthy Holstein Friesian cows at the NC A&T Dairy research unit were collected. Differential white blood cell counts were conducted using a VetScan (Zoetis). Neutrophils were isolated through differential centrifugation and hypotonic lysis of red blood cells. Duplicate samples of neutrophils were maintained in phosphate-buffered saline for 30 minutes at 37 °C. The total and viable neutrophil count was evaluated using TC20 (Bio-Rad). Samples were centrifuged. The supernatant was used to determine the concentration of total secreted protein using the Bicinchoninic acid assay (Pierce). Enumeration of bovine blood platelets and white blood cells was accomplished using the Vetscan study found that there was no significant difference observed in the total cell count or percent viability in neutrophils between the five cows (P > 0.05). However, protein concentration showed a significant change between animals. Correlations between total platelet count, neutrophil count, viability, and protein secretion were evaluated. No correlation was found between neutrophil count, protein secretion in isolated neutrophils and concentration of platelets in whole cow blood. The number of circulating platelets was not related to neutrophil viability or protein secretion. This approach can be used in further investigation using imaging and is needed to evaluate the effect and functional consequences of interactions between platelets and neutrophils in cow blood.

The Pyruvate Kinase (PK) Activator AG-946 Improves PK Properties and Red Blood Cell (RBC) Characteristics upon Ex Vivo treatment of RBCs from Patients with Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) consist of a heterogenous group of clonal myeloid malignancies, characterized by dysplasia, ineffective hematopoiesis and cytopenias. MDS patients frequently suffer from anemia, directly affecting quality of life. The limited therapeutic options often do not result in the desired clinical improvement. Interestingly, in MDS the activity of the red blood cell (RBC) enzyme pyruvate kinase (PK), a key regulatory enzyme of glycolysis, may be decreased. The activation of PK via small molecules is hypothesized to be beneficial in a wide range of anemias. Currently, a phase 2a, open-label, proof of concept trial is running on the use of the PK activator AG-946 in low-risk MDS patients (NCT05490446). In light of these developments, we investigated the effect of ex vivo treatment of MDS RBCs with AG-946. Objectives: To evaluate RBC PK and cellular properties of patients with MDS, and to determine the effect of ex vivo treatment with the PK activator AG-946. Methods: Eleven low-risk non-transfusion dependent MDS patients and six healthy controls (HCs) were studied. Baseline PK activity and PK thermostability were measured in RBC lysates of purified RBCs, under V max conditions (phosphoenolpyruvate (PEP) 5mM final concentration). Hexokinase (HK) activity was included to relate PK activity to mean RBC age. Ex vivo treatment was performed by incubating MDS RBCs for 16 hours at 37 °C in presence or absence of the PK activator AG-946 (10μM or 5μM) using dimethylsulfoxide (DMSO) as a blank. Effect was assessed by measuring PK activity (using a final concentration of PEP 0.5mM) and levels of adenosine triphosphate (ATP) (LC-MS/MS). Functional RBC analysis was performed by osmotic gradient ektacytometry (Lorrca MaxSis). To investigate the effect on PK thermostability, RBC lysates were incubated with AG-946 (2μM or 1μM) or DMSO, after which residual PK activity was measured (PEP 0.5mM). The effect of AG-946 on erythroid development was studied using peripheral blood mononuclear cells, cultured in MethoCult TM H4434 medium for 14 days in the presence or absence of AG-946 (10μM or 625nM, DMSO as blank). Differences between conditions were determined using Unpaired T-test, ANOVA with Dunnett's test or Kruskal-Wallis with Dunn's test via GraphPad Prism. Results: Mean PK/HK ratio was significantly lower in RBCs from MDS patients compared to HCs (6.1 (SD 1.8) versus 10.5 (SD 1.5), p<0.001). Upon ex vivo treatment, PK activity significantly increased (independent of dosage) when compared to DMSO (10μM AG-946, mean increase 30% ( p<0.001); 5μM, 28% ( p<0.0001)) (Figure 1A). MDS RBCs displayed decreased PK thermostability at baseline under V max conditions (HCs 79% residual activity versus 68% in MDS, p<0.01). This was restored by ex vivo treatment with AG-946 (DMSO, 33% residual activity, 2μM AG-946, 85% ( p<0.0001); 1μM, 87% ( p<0.0001)). The increase in PK activity was accompanied by significantly increased ATP levels (N=10, DMSO, mean ATP 14.9 μg/mL RBC; 10μM AG-946, 21.0 μg/mL RBC ( p<0.01); 5μM, 21.1 μg/mL RBC ( p<0.01)) (Figure 1B). Functionally, RBC properties improved upon ex vivo treatment with AG-946 as reflected by the modest yet significant increase in O hyper, indicating improved hydration status (10μM AG-946, mean increase 2.3% ( p<0.001); 5μM, 2.9% ( p<0.0001)). To date, culture assays were performed in 8 patients; 3/8 showed an increase in the number of burst forming units-erythroid (BFU-Es) upon treatment with AG-946 when compared to DMSO. The percentual increases differed per patient: increase of 25% with 10μM AG946 and 150% with 625nM, increase of 40% (10μM) and 39% (625nM), and for the third an increase of 22% (10μM) and 39% (625nM). Conclusion: Our findings show that RBCs from MDS patients have decreased PK activity and thermostability. We demonstrate that ex vivo treatment with AG-946 increases PK activity and ATP levels and restores PK thermostability. Moreover, ex vivo treatment with AG-946 improves RBC hydration, suggesting that improved energy status directly improves RBC functional properties. The preliminary results of the culture assay could indicate that in certain patients, dyserythropoiesis may be ameliorated upon PK activation. To better understand the effects on erythropoiesis additional experiments are required. In conclusion, our data might support a rationale for the use of PK activators as a novel therapeutic option for MDS.

Microglial FoxO3a deficiency ameliorates ferroptosis-induced brain injury of intracerebral haemorrhage via regulating autophagy and heme oxygenase-1.

Microglial HO-1 regulates iron metabolism in the brain. Intracerebral haemorrhage (ICH) shares features of ferroptosis and necroptosis; hemin is an oxidized product of haemoglobin from lysed red blood cells, leading to secondary injury. However, little is known about the underlying molecular mechanisms attributable to secondary injury by hemin or ICH. In this study, we first show that FoxO3a was highly co-located with neurons and microglia but not astrocytes area of ICH model mice. Hemin activated FoxO3a/ATG-mediated autophagy and HO-1 signalling resulting in ferroptosis invitro and in a mice model of brain haemorrhage. Accordingly, autophagy inhibitor Baf-A1 or HO-1 inhibitor ZnPP protected against hemin-induced ferroptosis. Hemin promoted ferroptosis of neuronal cells via FoxO3a/ATG-mediated autophagy and HO-1 signalling pathway. Knock-down of FoxO3a inhibited autophagy and prevented hemin-induced ferroptosis dependent of HO-1 signalling. We first showed that hemin stimulated microglial FoxO3a/HO-1 expression and enhanced the microglial polarisation towards the M1 phenotype, while knockdown of microglial FoxO3a inhibited pro-inflammatory cytokine production in microglia. Furthermore, the microglia activation in the striatum showed significant along with a high expression level of FoxO3a in the ICH mice. We found that conditional knockout of FoxO3a in microglia in mice alleviated neurological deficits and microglia activation as well as ferroptosis-induced striatum injury in the autologous blood-induced ICH model. We demonstrate, for the first time, that FoxO3a/ATG-mediated autophagy and HO-1 play an important role in microglial activation and ferroptosis-induced striatum injury of ICH, identifying a new therapeutic avenue for the treatment of ICH.

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells.

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

Construction of “Coral” SERS sensor for ultrasensitive and rapid detection of harmful component macrophage migration inhibitory factor in Platelet-rich Plasma

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory factor produced by residual red blood cell lysis, which can significantly influence the curative effect of Platelet-rich plasma (PRP) therapy used for osteoarthritis (OA) treatment. In this study, we proposed a novel approach for detecting the concentration of MIF in PRP using a dopamine-coated antibody-Au (core)-Ag (shell)-SERS sensor, which enables ultrasensitive and rapid detection of MIF. The best experimental conditions have a detection limit of only 90.05 pg/mL and a good linear relationship between 1-5000 ng/mL. In 40 PRP samples collected from actual clinical patients, we detected MIF concentrations ranging from 2.0-3.6 ng/mL. This indicated that the Coral SERS sensor not only allows for results highly consistent with the traditional ELISA method, but also costs less ($0.40-$0.70), needs shorter testing time (integration time is only 10s), and consumes less PRP that can greatly improve the sample quality and maximize the curative effect in clinical applications for OA treatment with PRP.

“Lysis and Concentration Technique” improves the parasitological diagnosis of Trypanosoma vivax

The “Lysis and Concentration Technique” (LCTe) involves lysing red blood cells and concentrating parasites to increase the chances of visualization in low parasitemia and in scenarios of evaluators with less knowledge. The lysis of red blood cells reduced the time of diagnosis by 21 s, showing that the effect produced by the treatment is comparable to the effect of the experience in parasitological examination. In addition, the concentration of parasites was 39.18% higher for slides with high parasitemia and 131.03% for slides with low parasitemia in relation to the standard slide. LCTe proved to be inexpensive, with a total cost of approximately US$0.07 per slide made, which allows it to be easily implemented in most laboratories.

A-075 Development and Validation of Image-based Cell Counter Microscanner C3 for CD34-positive Hematopoietic Stem Cell Enumeration

Abstract Background Accurate measurement of CD34-positive hematopoietic stem cells (HSCs) is critical for successful peripheral blood stem cell transplantation (PBSCT). Flow cytometry is by far the gold standard for measuring CD34-positive stem cells. However, its complicated procedure requires time, cost and highly-trained operators, thus the necessity of simpler and cheaper alternative method is emerging. We have developed a new image-based cell counter, Microscanner C3 (MSC3, Biozentech, South Korea) as an alternative method for enumeration of CD34-positive HSCs and evaluated its performance. Methods 100 µl of apheresis product was stained with CD45-BB515 and CD34-PE antibodies (Becton Dickinson, USA) at room temperature in the dark. Red blood cell lysis with 500 µl of OptiLyze C solution (Beckman Coulter, USA) was followed by centrifugation. After the removal of supernatants, the sample was resuspended with 500 µL PBS, which then infused to filter paper imbedded BZ-1 microchip (Biozentech, South Korea). Thirty-fields of the loaded microchip was auto-scanned by MSC3 and its dedicated software analyzed the images and yielded CD34-positive cell fraction out of CD45-positive cells as percentage. Results of thirty-six samples were compared to that of FacsLyric (Beckton Dickinson, USA) flow cytometry and accuracy and imprecision (pentaplicate measurements for three samples), repeatability (two experimenters performed triplicate measurements for three samples each), linearity (triplicate measurements for five levels), limit of detection (hexaplicate measurements for five levels) and specificity (using whole blood specimen from three donors with monoclonal gammopathy, thrombocytosis or monocytosis) was evaluated. Results CV% values of inaccuracy, imprecision and repeatability did not exceed 15%. The method was found to be linear across the measuring range. LOD was evaluated to be 7 cells/µL and no background interference was detected. The difference between the measured fraction of MSC3 and FacsLyric was within allowable limit, with an R-value greater than 0.98. Conclusion We developed a reliable method for quantifying CD34-positive HSCs based on image analysis. The limitation involved in MSC3 applications in CD34-positive HSCs is that, like dual-platform flow cytometry, the results should be combined with the results of conventional hematology analyzer in order to define absolute HSC numbers. However, with its simpler and faster procedures, MSC3 exhibited a great potential to be used as an alternative to conventional flow cytometry in CD34-positive HSCs enumeration and other applications as well.

Rapid acoustofluidic mixing by ultrasonic surface acoustic wave-induced acoustic streaming flow

Ultrasonic surface acoustic wave (SAW)-induced acoustic streaming flow (ASF) has been utilized for microfluidic flow control, patterning, and mixing. Most previous research employed cross-type SAW acousto-microfluidic mixers, in which the SAWs propagated perpendicular to the flow direction. In this configuration, the flow mixing was induced predominantly by the horizontal component of the acoustic force, which was usually much smaller than the vertical component, leading to energy inefficiency and limited controllability. Here, we propose a vertical-type ultrasonic SAW acousto-microfluidic mixer to achieve rapid flow mixing with improved efficiency and controllability. We conducted in-depth numerical and experimental investigations of the vertical-type SAW-induced ASF to elucidate the acousto-hydrodynamic phenomenon under varying conditions of total flow rate, acoustic wave amplitude, and fluid viscosity conditions. We conducted computational fluid dynamics simulations for numerical flow visualization and utilized micro-prism-embedded microchannels for experimental flow visualization for the vertical SAW-induced ASF. We found that the SAW-induced vortices served as a hydrodynamic barrier for the co-flow streams for controlled flow mixing in the proposed device. For proof-of-concept application, we performed chemical additive-free rapid red blood cell lysis and achieved rapid cell lysis with high lysis efficiency based on the physical interactions of the suspended cells with the SAW-induced acoustic vortical flows. We believe that the proposed vertical-type ultrasonic SAW-based mixer can be broadly utilized for various microfluidic applications that require rapid, controlled flow mixing.

Fragment-based and structure-guided discovery of perforin inhibitors

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands.

The anti-infective crotalicidin peptide analog RhoB-Ctn[1–9] is harmless to bovine oocytes and able to induce parthenogenesis in vitro

Crotalicidin is a cathelicidin-related anti-infective (antimicrobial) peptide expressed in the venom glands of the South American rattlesnake Crotalus durissus terrificus. Congener peptides of crotalicidin, named vipericidins, are found in other pit vipers inhabiting South America. Crotalicidin is active against bacteria and pathogenic yeasts and has anti-proliferative activity for some cancer cells. The structural dissection of crotalicidin produced fragments (e.g., Ctn [15–34]) with multiple biological functionalities that mimic the native peptide. Another structural characteristic of crotalidicin and congeners is a unique repetitive stretch of amino acid sequences in tandem embedded in their primary structures. One of the encrypted vipericidn peptides (Ctn [1–9]) was synthesized, and the analog covalently conjugated with rhodamine B (RhoB-Ctn [1–9]) displayed considerable antimicrobial activity and selective cytotoxicity. Methods to evaluate antimicrobial peptides' toxicity include lysis of red blood cells (hemolysis) in vitro and cytotoxicity of healthy cultured cells (e.g., fibroblasts). Here, as a non-conventional model of toxicity, the bovine oocytes were exposed to two standardized concentrations of RhoB-Ctn [1–9], and embryo viability and development at its first stage of cleavage (division of cells) and blastocyst formation were evaluated. Oocytes treated with peptide at 10 and 40 μM induced cleavage rates of 44.94% and 51.53%, resulting in the formation of blastocysts of 7.07% and 11.73%, respectively. Light sheet microscopy and in silico prediction analysis indicated that RhoB-Ctn [1–9] peptide interacts with zona pellucida and internalizes into bovine oocytes and developing embryos. The ADMET prediction estimated good bioavailability of RhoB-Ctn [1–9]. In conclusion, the peptide appeared harmless to bovine oocytes and, remarkably, activated the parthenogenesis in vitro.

Snapshot of resistome, virulome and mobilome in aquaculture

Aquaculture environments can be hotspots for resistance genes through the surrounding environment. Our objective was to study the resistome, virulome and mobilome of Gram-negative bacteria isolated in seabream and bivalve molluscs, using a WGS approach. Sixty-six Gram-negative strains (Aeromonadaceae, Enterobacteriaceae, Hafniaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Vibrionaceae, and Yersiniaceae families) were selected for genomic characterization. The species and MLST were determined, and antibiotic/disinfectants/heavy metals resistance genes, virulence determinants, MGE, and pathogenicity to humans were investigated. Our study revealed new sequence-types (e.g. Aeromonas spp. ST879, ST880, ST881, ST882, ST883, ST887, ST888; Shewanella spp. ST40, ST57, ST58, ST60, ST61, ST62; Vibrio spp. ST206, ST205). >140 different genes were identified in the resistome of seabream and bivalve molluscs, encompassing genes associated with β-lactams, tetracyclines, aminoglycosides, quinolones, sulfonamides, trimethoprim, phenicols, macrolides and fosfomycin resistance. Disinfectant resistance genes qacE-type, sitABCD-type and formA-type were found. Heavy metals resistance genes mdt, acr and sil stood out as the most frequent. Most resistance genes were associated with antibiotics/disinfectants/heavy metals commonly used in aquaculture settings. We also identified 25 different genes related with increased virulence, namely associated with adherence, colonization, toxins production, red blood cell lysis, iron metabolism, escape from the immune system of the host. Furthermore, 74.2 % of the strains analysed were considered pathogenic to humans. We investigated the genetic environment of several antibiotic resistance genes, including blaTEM-1B, blaFOX-18, aph(3″)-Ib, dfrA-type, aadA1, catA1-type, tet(A)/(E), qnrB19 and sul1/2. Our analysis also focused on identifying MGE in proximity to these genes (e.g. IntI1, plasmids and TnAs), which could potentially facilitate the spread of resistance among bacteria across different environments. This study provides a comprehensive examination of the diversity of resistance genes that can be transferred to both humans and the environment, with the recognition that aquaculture and the broader environment play crucial roles as intermediaries within this complex transmission network.

A rapid and high-throughput T cell immunophenotyping assay for cellular therapy bioprocess using the Cellaca® PLX image cytometer

In cellular therapies chimeric antigen receptor (CAR) T or NK cells undergo phenotypic analysis at multiple stages during discovery and development of novel therapies. Patient samples are routinely analyzed via flow cytometry for population identification and distribution of CD3, CD4, and CD8 positive T cells. As an alternative or orthogonal method, image cytometry systems have been used to perform simple cell-based assays in lieu of flow cytometry. Recently, a new image cytometry system, the Cellaca® PLX (Revvity Health Sciences, Inc., Lawrence, MA), was developed for high-throughput cell counting and viability, immunophenotyping, transfection/transduction efficiency, and cell health assays. This novel instrument allows investigators to quickly assess several critical quality attributes (CQAs) such as cell identity, viability, and other relevant biological functions recommended by the International Organization for Standardization using the ISO Cell Characterization documents focused on cellular therapeutic products. In this work, we demonstrate a rapid and high-throughput image cytometry detection method for cellular immunophenotyping and viability using the Cellaca PLX system for samples throughout the cellular therapy workflow. Freshly isolated peripheral blood mononuclear cells (PBMCs) underwent red blood cell (RBC) lysis and CD3 enrichment. Samples were then subsequently stained with Hoechst/CD3/CD4/CD8 or Hoechst/CD3/CD8/RubyDead Dye surface marker kits and measured on the Cellaca PLX and three different flow cytometers for side-by-side comparison and assay validation. Acquisition and analysis of cell viability and cell populations was shown to be faster and more efficient process compared to flow while achieving highly comparable results between the two technology platforms. This data shows that the Cellaca PLX Image Cytometer may provide a rapid alternative or orthogonal method for PBMC immunophenotyping experiments, as well as potentially streamline the workflow to quickly move precious patient samples downstream within the development processes.

Antioxidant and anti-inflammatory potentials of aerial and floral parts of Neurada procumbens extracts: In vitro and in vivo studies

Neurada procumbens (Neuradaceae) is an importent plant of the Cholistan desert and is traditionally been used for the treatment and control of diabetes, fever, inflammations and jaundice. The aim of the current project is to investigate the bioactive compounds, free radical scavenging capacity and the anti-inflammatory potential of Neurada procumbens separately for its aerial and floral parts in six different extracts. The phytochemical profile (total bioactive contents, HPLC polyphenolic quantification), anti-oxidant (DPPH and FRAP assays), anti-inflammatory (HRBC stabilization) activities of the Aqu, MetOH, n-But, EtAc, n-Hex, and DCM extracts from the aerial and floral parts of Neurada procumbens were quantified. Based on the current results, the aerial and floral parts of N. procumbens extracts have found to contain a significant amount of active metabolites including polyphenolic compounds such as 2,3 di MeO benzoic acid, p-coumaric acid, chlorogenic acid, quercetin dihydrate and t-ferrulic acid. The total phenolic and total flavonoid contents of the plant were found to range from 28.13 to 78.9 GAE mg/g of plant and 17.23 to 68.23 RE mg/g of plant, respectively. DCM and n-But extracts of floral part exhibited comparatively higher antioxidant potential in DPPH (IC50 < 100 μg/ml) and FRAP (IC50 < 55 μg/ml) assays compared to aerial part. The dichloromethane floral extract demonstrated impressive anti-inflammatory activity in the hemolytic red blood cell lysis assay, with 83 % protection of HRBC lysis at an IC50 of 469.6 µg/ml (p < 0.01). In vivo, a 300 mg/kg body weight of DCM floral extract reduced carrageenan-induced paw oedema by 8.51 ± 0.35 mm to 7.65 ± 0.38 mm, a highly significant difference (p < 0.001).The toxicity studies revealed that the floral-DCM dose extract was found to be safe up to 2000 mg/kg BW, while its lethal dose (LD50) was found to be 4472.13 mg/kg BW in rats. Due to quite low toxicity effects, the floral part of the plant could be recommended as a safe pharmacological agent for various therapeutic applications.

Insight into Increased Recovery and Simplification of Genomic DNA Extraction Methods from Dried Blood Spots.

There is no consensus on how to perform the manual extraction of nucleic acids from dried blood spots (DBSs). Current methods typically involve agitation of the DBSs in a solution for varying amounts of time with or without heat, and then purification of the eluted nucleic acids with a purification protocol. We explored several characteristics of genomic DNA (gDNA) DBS extraction such as extraction efficiency, the role of red blood cells (RBCs) in extraction and critical kinetic factors to understand if these protocols can be simplified while maintaining sufficient gDNA recovery. We found that agitation in a RBC lysis buffer before performing a DBS gDNA extraction protocol increases yield 1.5 to 5-fold, depending upon the anticoagulant used. The use of an alkaline lysing agent along with either heat or agitation was sufficient to elute quantitative polymerase chain reaction (qPCR) amplifiable gDNA in 5 minutes. This work adds insight into the extraction of gDNA from DBSs with the intention of informing a simple, standardized manual protocol for extraction.

Red blood cell exposure increases chondrocyte susceptibility to oxidative stress following hemarthrosis

The detrimental effects of blood exposure on articular tissues are well characterized, but the individual contributions of specific whole blood components are yet to be fully elucidated. Better understanding of mechanisms that drive cell and tissue damage in hemophilic arthropathy will inform novel therapeutic strategies. The studies here aimed to identify the specific contributions of intact and lysed red blood cells (RBCs) on cartilage and the therapeutic potential of Ferrostatin-1 in the context of lipid changes, oxidative stress, and ferroptosis. Changes to biochemical and mechanical properties following intact RBC treatment were assessed in human chondrocyte-based tissue-engineered cartilage constructs and validated against human cartilage explants. Chondrocyte monolayers were assayed for changes to intracellular lipid profiles and the presence of oxidative and ferroptotic mechanisms. Markers of tissue breakdown were observed in cartilage constructs without parallel losses in DNA (control: 786.3 (102.2) ng/mg; RBCINT: 751 (126.4) ng/mg; P=0.6279), implicating nonlethal chondrocyte responses to intact RBCs. Dose-dependent loss of viability in response to intact and lysed RBCs was observed in chondrocyte monolayers, with greater toxicity observed with lysates. Intact RBCs induced changes to chondrocyte lipid profiles, upregulating highly oxidizable fatty acids (e.g., FA 18:2) and matrix disrupting ceramides. RBC lysates induced cell death via oxidative mechanisms that resemble ferroptosis. Intact RBCs induce intracellular phenotypic changes to chondrocytes that increase vulnerability to tissue damage while lysed RBCs have a more direct influence on chondrocyte death by mechanisms that are representative of ferroptosis.

Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies

The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.

Calcium Phosphate Treatment Enhances Osteogenic Differentiation of Porcine Adipose-Derived Stem Cells on Fibrin Scaffolds

This study was designed to develop a fibrin scaffold for optimum in vitro osteogenic differentiation of porcine ASCs. Fibrin scaffolds physically and chemically modified to be stiffer or have higher concentrations of calcium and phosphate ions were hypothesized to enhance osteogenic differentiation compared to control fibrin scaffolds. Treatments during coagulation resulted in six scaffold types for comparison: whole blood controls, red blood cell lysis buffer, calcium chloride, calcium hydrogen phosphate, vacuum, and mechanical compression. Based on the results, fibrin supplemented with granules of calcium hydrogen phosphate (CaHPO4) was determined to be the most suitable formulation of those examined. Using calcium phosphate resulted in a fibrin scaffold that coagulated faster (p = 0.022), had a rougher surface, higher stiffness, and desirable properties for practical use during surgical operations and scaffolds used in bone tissue engineering. Further, osteogenic differentiation was enhanced on scaffolds treated with calcium phosphate. In addition, fibrin scaffolds treated with RBC lysis buffer were also stiffer than untreated controls. These results are partly explained by ASC attachment and fibrin polymerization during coagulation.

Engineering Therapeutics to Detoxify Hemoglobin, Heme, and Iron.

Hemolysis (i.e., red blood cell lysis) can increase circulatory levels of cell-free hemoglobin (Hb) and its degradation by-products, namely heme (h) and iron (Fe). Under homeostasis, minor increases in these three hemolytic by-products (Hb/h/Fe) are rapidly scavenged and cleared by natural plasma proteins. Under certain pathophysiological conditions, scavenging systems become overwhelmed, leading to the accumulation of Hb/h/Fe in the circulation. Unfortunately, these species cause various side effects such as vasoconstriction, hypertension, and oxidative organ damage. Therefore, various therapeutics strategies are in development, ranging from supplementation with depleted plasma scavenger proteins to engineered biomimetic protein constructs capable of scavenging multiple hemolytic species. In this review, we briefly describe hemolysis and the characteristics of the major plasma-derived protein scavengers of Hb/h/Fe. Finally, we present novel engineering approaches designed to address the toxicity of these hemolytic by-products.

Extent of Tissue Washing Can Significantly Alter the Composition of Adipose-Derived Stromal Vascular Fraction Cell Preparations: Implications for Clinical Translation.

Stromal vascular fraction (SVF) cell preparations have recently attracted much interest as a form of autologous cell therapy. These heterogenous cell populations typically include some proportion of blood-derived cells (BDCs)-including both red blood cells (RBCs) and leukocytes (WBCs). The objectives of this paper were to evaluate the effects of tissue washing and hypotonic RBC lysis-separately and together-on BDC concentrations within SVF, and further to explore whether BDCs can confer detectable and modifiable effects on adipose-derived cell activity. Using various cell culture assays, flow cytometry and ELISA analysis of human-derived SVF preparations, we show that thorough washing of adipose tissue prior to enzymatic dissociation effectively removes RBCs from SVF preparations as well as standard lysis methods and significantly alters the type and relative quantities of WBCs. In addition, these studies demonstrate that potentially toxic RBC components are detectable for up to 1 week in cultures containing RBC lysate, but not those with intact RBCs, and, that culture-expanded cells proliferate significantly more in the presence of intact RBCs versus RBC lysis products or control media. Broadly, these data exemplify how different seemingly mundane tissue processing steps can significantly influence SVF identity/composition, purity, and potency. Based on the findings of this work, we propose that translational efforts in the field would benefit by a better understanding of the impact of RBCs, WBCs, and non-viable cells on the in vivo therapeutic activity of SVF therapies.

Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood.

PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detect T. pallidum DNA by targeting the polA gene. Simulation media with 106 to 100 treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. The polA assay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of the polA assay reached 1 × 102 treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 104 treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield of T. pallidum DNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs. IMPORTANCE Syphilis is a sexually transmitted disease caused by T. pallidum that can spread into the blood. T. pallidum DNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to blood T. pallidum DNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations of T. pallidum DNA was improved, and the low sensitivity of blood-based T. pallidum PCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring blood T. pallidum DNA.