Red Blood Cell Lysis Research Articles

A microfluidic paper analytical device using capture aptamers for the detection of PfLDH in blood matrices

BackgroundThe prevalence and death rate arising from malaria infection, and emergence of other diseases showing similar symptoms to malaria require the development of malaria-specific and sensitive devices for its diagnosis. To address this, the design and fabrication of low-cost, rapid, paper-based analytical devices (µPAD) using surface-immobilized aptamers to detect the presence of a recombinant malarial biomarker—Plasmodium falciparum lactate dehydrogenase (rPfLDH)—is reported in this study.MethodsTest zones on paper surfaces were created by covalently immobilizing streptavidin to the paper, subsequently attaching biotinylated aptamers to streptavidin. Aptamers selectively bound rPfLDH. The measurement of captured rPfLDH enzyme activity served as the means of detecting this biomarker. Enzyme activity across three replicate sensors was digitally quantified using the colorimetric Malstat assay.ResultsScreening of several different aptamers reported in the literature showed that aptamers rLDH7 and 2008s immobilized in this manner specifically recognised and captured PfLDH. Using rLDH7, the sensitivity of the µPAD sensor was evaluated and the µPAD sensor was applied for preferential detection of rPfLDH, both in buffered solutions of the protein and in spiked serum and red blood cell lysate samples. In buffered solutions, the test zone of the µPAD sensor exhibited a KD of 24 ± 11 nM and an empirical limit of detection of 17 nM, respectively, a limit similar to commercial antibody-based sensors exposed to rPfLDH. The specific recognition of 133 nM rPfLDH in undiluted serum and blood samples was demonstrated by the µPAD.ConclusionThe reported µPAD demonstrates the potential of integrating aptamers into paper-based malarial rapid diagnostic tests.Graphical The assembly of µPAD sensors using APTEC assay principles for the detection the malarial biomarker, lactate dehydrogenase enzymes from Plasmodium falciparum (PfLDH). The aptamers immobilized at the test zones capture the PfLDH in samples. After washing the unbound sample components from the zones, Malstat assay reagents are added for colour development, proportional to the amount of captured PfLDH.

A Rare Cause of Fever in a Term Neonate.

A Rare Cause of Fever in a Term Neonate.

Use of silver biosynthesized nanoparticles from bacteria pseudomonas aeruginosa in the treatment of induced bacterial injury infection

This research, we discuss how biosynthesis silver nanoparticles and used to treat wounds in the skin and compare them with antibiotics and find out which are more effective for healing. at first, nanosilver is synthesis from Pseudomonas aeruginosa it is characterized by confirmation test to proved this material is silver nanoparticles , as well as measuring the susceptibility of the nanomaterial to lysis of red blood cells after that, tests are done for this material in vitro and vivo and that was done by measuring the ability of silver nanoparticles to inhibit the growth of bacteria and comparing it with the inhibition of antibiotics also, the ability of silver nanoparticles to heal was measured on rats at different times and at different doses and compared with the antibiotic.

Accurate profiling of blood components in microliter with position-insensitive coplanar electrodes-based cytometry

Differentiating and counting blood cell subtypes are essential for medical diagnosis. Microfluidic impedance-based cytometry has been discussed in the past decade as cost-effective and label-free cell classification using multi-cellular electrical properties. The coplanar electrode configuration is particularly desirable since it can dramatically reduce the device fabrication complexity and cost. However, very few research demonstrated accurate leukocyte classification with coplanar electrodes due to the in-channel height-dependent sensitivity variation in cell profiling caused by nonuniform electric field. Herein we propose a microfluidic high-throughput (up to 1000 cells/second) impedance cytometry with a unique coplanar electrode configuration to eliminate height-dependent sensitivity variation for accurate electrical cell profiling. The proposed position-insensitive cytometry enables an accurate 3-part leukocyte classification and quantification comparable with fluorescence-based cytometry. In addition, unlike conventional leukocyte classification approaches that require red blood cell lysis to remove noises caused by erythrocytes, the position-insensitive cytometry can distinguish erythrocytes simply based on cell sizes and characterize the mean size and distribution width of erythrocytes with leukocyte classification simultaneously, offering red blood cell indices for diagnosis with reduced test time. The position-insensitive impedance cytometry has the great potential to provide a point-of-care blood analysis for clinical utility in many diseases (i.e., infections, cancers and HIV).

Neuregulin-1/ErbB4 signaling modulates Plasmodium falciparum HRP2-induced damage to brain cortical organoids

SummaryHuman cerebral malaria (HCM) is a severe complication of Plasmodium falciparum (P.f.) infection that is characterized by capillary occlusions, rupture of the blood-brain barrier (BBB), perivascular cellular injury, and brain swelling. P.f.histidine-rich protein 2 (HRP2), a byproduct of parasitized red blood cell (pRBC) lysis, crosses the BBB when compromised to cause brain injury. We hypothesized that HRP2-induced neuronal damage can be attenuated by Neuregulin-1 (NRG1), an anti-inflammatory neuroprotective factor. Using brain cortical organoids, we determined that HRP2 upregulated cell death and inflammatory markers and disorganized brain organoid tissue. We identified toll-like receptors (TLR1 and 2) as potential mediators of HRP2-induced cellular damage and inflammation. Exogenous acute treatment of organoids with NRG1 attenuated HRP2 effects. The results indicate that HRP2 mediates malaria-associated HRP2-induced brain injury and inflammation and that NRG1 may be an effective therapy against HRP2 effects in the brain.

Continuous-Flow Magnetic Fractionation of Red Blood Cells Based on Hemoglobin Content and Oxygen Saturation—Clinical Blood Supply Implications and Sickle Cell Anemia Treatment

Approximately 36,000 units of red blood cells (RBCs) are used every day in the U.S. and there is a great challenge for hospitals to maintain a reliable supply, given the 42-day expiration period from the blood donation date. For many years, research has been conducted to develop ex vivo storage solutions that limit RBC lysis and maintain a high survival rate of the transfused cells. However, little attention is directed towards potential fractionation methods to remove unwanted cell debris or aged blood cells from stored RBC units prior to transfusion, which could not only expand the ex vivo shelf life of RBC units but also avoid adverse events in transfused patients. Such fractionation methods could also limit the number of transfusions required for treating certain pathologies, such as sickle cell disease (SCD). In this work, magnetic fractionation is studied as a potential technology to fractionate functional and healthy RBCs from aged or sickle cells. It has been reported that during ex vivo RBC storage, RBCs lose hemoglobin (Hb) and lipid content via formation of Hb-containing exosomes. Given the magnetic character of deoxygenated- or met-Hb, in this work, we propose the use of a quadrupole magnetic sorter (QMS) to fractionate RBCs based on their Hb content from both healthy stored blood and SCD blood. In our QMS, a cylindrical microchannel placed inside the center of the quadrupolar magnets is subjected to high magnetic fields and constant field gradients (286 T/m), which causes the deflection of the paramagnetic, Hb-enriched, and functional RBCs from their original path and their collection into a different outlet. Our results demonstrated that although we could obtain a significant difference in the magnetic mobility of the sorted fractions (corresponding to a difference in more than 1 pg of Hb per cell), there exists a tradeoff between throughput and purity. Therefore, this technology when optimized could be used to expand the ex vivo shelf life of RBC units and avoid adverse events in transfused individuals or SCD patients requiring blood exchange therapy.

Optimized S-nitrosohemoglobin Synthesis in Red Blood Cells to Preserve Hypoxic Vasodilation Via βCys93.

Classic physiology links tissue hypoxia to oxygen delivery through control of microvascular blood flow (autoregulation of blood flow). Hemoglobin (Hb) serves both as the source of oxygen and the mediator of microvascular blood flow through its ability to release vasodilatory S-nitrosothiol (SNO) in proportion to degree of hypoxia. β-globin Cys93Ala (βCys93Ala) mutant mice deficient in S-nitrosohemoglobin (SNO-Hb) show profound deficits in microvascular blood flow and tissue oxygenation that recapitulate microcirculatory dysfunction in multiple clinical conditions. However, the means to replete SNO in mouse red blood cells (RBCs) to restore RBC function is not known. In particular, although methods have been developed to selectively S-nitrosylate βCys93 in human Hb and intact human RBCs, conditions have not been optimized for mouse RBCs that are used experimentally. Here we show that loading SNO onto Hb in mouse RBC lysates can be achieved with high stoichiometry and β-globin selectivity. However, S-nitrosylation of Hb within intact mouse RBCs is ineffective under conditions that work well with human RBCs, and levels of metHb are prohibitively high. We developed an optimized method that loads SNO in mouse RBCs to maintain vasodilation under hypoxia and shows that loss of SNO loading in βCys93Ala mutant RBCs results in reduced vasodilation. We also demonstrate that differences in SNO/met/nitrosyl Hb stoichiometry can account for differences in RBC function among studies. RBCs loaded with quasi-physiologic amounts of SNO-Hb will produce vasodilation proportionate to hypoxia, whereas RBCs loaded with higher amounts lose allosteric regulation, thus inducing vasodilation at both high and low oxygen level. SIGNIFICANCE STATEMENT: Red blood cells from mice exhibit poor hemoglobin S-nitrosylation under conditions used for human RBCs, frustrating tests of vasodilatory activity. Using an optimized S-nitrosylation protocol, mouse RBCs exhibit hypoxic vasodilation that is significantly reduced in hemoglobin βCys93Ala mutant RBCs that cannot carry S-nitrosothiol allosterically, providing genetic validation for the role of βCys93 in oxygen delivery.

On-line Coupling of Aptamer Affinity Solid-Phase Extraction and Immobilized Enzyme Microreactor Capillary Electrophoresis-Mass Spectrometry for the Sensitive Targeted Bottom-Up Analysis of Protein Biomarkers.

In this paper, we present a fully integrated valve-free method for the sensitive targeted bottom-up analysis of proteins through on-line aptamer affinity solid-phase extraction and immobilized enzyme microreactor capillary electrophoresis-mass spectrometry (AA-SPE-IMER-CE-MS). The method was developed analyzing α-synuclein (α-syn), which is a protein biomarker related to different neurodegenerative disorders, including Parkinson’s disease. Under optimized conditions, on-line purification and preconcentration of α-syn, enzymatic digestion, electrophoretic separation, and identification of the tryptic peptides by mass spectrometry was achieved in less than 35 min. The limit of detection was 0.02 μg mL–1 of digested protein (66.7% of coverage, i.e., 8 out of 12 expected tryptic peptides were detected). This value was 125 and 10 times lower than for independent on-line digestion by IMER-CE-MS (2.5 μg mL–1) and on-line preconcentration by AA-SPE-CE-MS (0.2 μg mL–1). The repeatability of AA-SPE-IMER-CE-MS was adequate (at 0.5 μg mL–1,% RSD ranged from 3.7 to 16.9% for peak areas and 3.5 to 7.7% for migration times of the tryptic peptides), and the modified capillary could be reused up to 10 analyses with optimum performance, similarly to IMER-CE-MS. The method was subsequently applied to the analysis of endogenous α-syn from red blood cell lysates. Ten α-syn tryptic peptides were detected (83.3% of coverage), enabling the characterization and localization of post-translational modifications of blood α-syn (i.e., N-terminal acetylation).

Inflammatory phenotype and T‐cell mitochondrial reactive oxygen species in young adults with major depressive disorder

BackgroundMajor depressive disorder (MDD) is prevalent in young adults and projected to rise further with the COVID pandemic. Notably, MDD is associated with an increased risk of cardiovascular disease (CVD) and mortality. Although the mechanisms are unclear, evidence supports chronic systemic inflammation as a major contributor to CVD risk in older adults with MDD. However, whether an inflammatory phenotype is present in young adults with MDD prior to the onset of CVD and other comorbidities is equivocal. Recent work has implicated the sympathetic nervous system and specifically norepinephrine as a potent driver of inflammation via stimulating an increase in T‐cell mitochondrial reactive oxygen species (T‐cell mitoROS). Given previous findings of elevated plasma norepinephrine and inflammation in MDD, the purpose of this study was to examine the potential for a T‐cell mitoROS pro‐inflammatory pathway in young otherwise healthy adults with MDD. We hypothesized that young adults with MDD but otherwise free of clinical disease would have augmented circulating pro‐inflammatory cytokines and T‐cell mitoROS compared to young healthy adults without MDD (HA).MethodsAll participants underwent a structured diagnostic neuropsychiatric interview to determine the presence or absence of MDD based on the DSM‐5 diagnostic criteria for MDD. Systemic pro and anti‐inflammatory cytokines and T‐cell mitoROS were measured in 14 healthy young adults with MDD of mild‐to‐moderate severity (non‐medicated; age: 23±2 years) and 11 HA (age: 22±1 years). Serum cytokines were detected using LEGENDplex Human Essential Immune Response panel and assessed by flow cytometry. Seven serum cytokines related to the norepinephrine‐T‐cell mitoROS‐inflammation pathway were measured. Immune cells were isolated from whole blood by red blood cell lysis. T‐cell mitoROS was measured by labeling with antibodies against CD3+ (total T cells) and mitoROS targeted fluorogenic dye (MitoSoxRED) and assessed by flow cytometry. All data are expressed as mean ± SE.ResultsFor pro‐inflammatory cytokines, there were no differences between MDD and HA for IL‐6 (41±18 pg/mL; 92± 66 pg/mL, P=0.66), IL‐17A (7±3 pg/mL; 17±8 pg/mL, P=0.13), TNF‐α (19±9 pg/mL; 36±18 pg/mL, P=0.20), IFNγ (120±70 pg/mL; 201±137 pg/mL, P=0.34) or IL‐2 (5±2 pg/mL; 13±6 pg/mL, P=0.25), respectively. Likewise, no differences were found in anti‐inflammatory cytokines between MDD and HA for IL‐10 (18±9 pg/mL; 78±57 pg/mL, P=0.19) or IL‐4 (33±15 pg/mL; 95±46 pg/mL, P=0.13), respectively. In contrast, T‐cell mitoROS was significantly elevated in MDD (34402±13353 a.u) compared to HA (6258±2942 a.u, P<0.05).ConclusionThese preliminary findings suggest T‐cell mitoROS but not circulating cytokine profiles are different between young adults with and without MDD. Thus, an elevated T‐cell mitoROS may represent an early marker of immune system alterations and inflammation in MDD.

Green synthesis of iron (Fe) nanoparticles using Plumeria obtusa extract as a reducing and stabilizing agent: Antimicrobial, antioxidant and biocompatibility studies

In the current study, a green and facile route for the synthesis of iron nanoparticles (FeNPs) was adopted. The FeNPs were fabricated via a single step green route using aqueous leaves extract of Plumeria obtusa ( P . obtusa ) as a capping/reducing and stabilizing agents. The FeNPs were characterized by UV/Vis (Ultraviolet/Visible), FTIR (Fourier Transform Infra-Red spectroscopy), TEM (Transmission Electron Microscopy), SEM (Scanning Electron Microscopy) and XRD (X-Ray Diffraction) techniques. The FeNPs were of spheroidal shape with average size of 50 nm. The biosynthesized FeNPs were further evaluated for their biological activities like antimicrobial, antioxidant and biocompatibility. The FeNPs displayed auspicious antimicrobial activity against bacterial ( E. coli , B. subtilis ) and fungal strains ( A. niger ) and S. commune . The test performed against red blood cells (RBCs) lysis (1.22 ± 0.02%) and macrophage (31 ± 0.09%) showed biocompatible nature of FeNPs. In vitro cytotoxicity against AU565 (82.03 ± 0.08–23.65 ± 0.065%) and HeLa (88.61 ± 0.06–33.34 ± 0.06%) cell lines showed cell viability loss in dose dependent manner (FeNPs 25–100 μg/mL). The antioxidant activities values were determined through DPPH, TRPA, NO and H 2 O 2 assays with values 70.23 ± 0.02%, 76.65 ± 0.02 μg AAE/mg, 74.43 ± 0.04% and 67.34 ± 0.03%, respectively. Based on the bioactivities, the green synthesized FeNPs have potential for therapeutic applications.

Iron Deposition in the Spleen in a Murine Model of Acute Radiation Syndrome

Total body radiation (TBI) can result in death associated with hematopoietic insufficiency. Although radiation causes apoptosis of white blood cells, red blood cells (RBC) undergo hemolysis due to hemoglobin denaturation. RBC lysis post‐irradiation results in the release of iron into the plasma, producing a secondary toxic event following TBI. We investigated the impact of radiation‐induced iron release on the spleen following TBI and the effects of the radiation mitigator captopril. RBC and HCT were reduced within 7 days, with the nadir at ~14 days post‐TBI. Prussian blue staining revealed ~20‐60‐fold increased Fe3+ in the spleen 7‐14 days post‐irradiation. Increased iron was associated with altered expression of iron binding and transport proteins, determined by qPCR, western blotting, and immunohistochemistry. Captopril did not prevent iron deposition in the spleen, and did not significantly modulate most iron‐binding proteins. Spleen volumes were markedly decreased 7‐14 days post‐irradiation, correlating high Fe3+ levels. At this time point, caspase‐3 was activated and we identified four additional markers of ferroptosis, an iron‐dependent programmed cell death. Interestingly, we did not observe increased expression of p21/Waf1, a marker of accelerated senescence. We investigated the effects of radiation and Fe3+ on the J774.A1 murine macrophage cell line in culture. Radiation induced p21/Waf1 and ferritin within 24 h. Radiation alone and with iron upregulated several markers of M1; radiation + iron also modulated a marker of M2 polarization. Our data indicate that following TBI, iron is deposited in the spleen where it modulates the expression of iron binding proteins and triggers ferroptosis.

In vitro evaluation of the cytotoxicity of Dissotis rotundifolia (Sm.) Triana (Melastomataceae) and Emilia sagittata (Vahl) DC. (Asteraceae) plants used in intimate hygiene among women in Mbandaka, Democratic Republic of the Congo

Plants used by Pygmy women in Mbandaka, Equateur Province, Democratic Republic of Congo include Dissotis rotundifolia (Sm.) Triana (Melastomataceae) and Emilia sagittata (Vahl) DC. (Asteraceae) known as the scarlet gland flower, and are geographically native to parts of Africa, particularly West and East Africa, whose leaves are used for birth spacing, sexual appetite and vaginal shrinkage respectively. The use of these plants as nutraceuticals for several diseases for decades, without any scientific data on their safety is at risk, because although they are natural, they are not completely free of toxic effects or other undesirable effects, hence the evaluation of the toxic effects of these phytomedicines on red blood cells to ensure its safety. For this purpose, the hydro-methanolic extracts of the leaves were prepared by maceration and human red blood cells were used. The results obtained showed a significant hemolytic effect of these extracts, depending on the concentration and the incubation time (15 and 60 minutes). However, the concentrations of 25 and 100 mg/mL of D. rotundifolia induced 34.2 and 51.2% of red blood cell lysis while those of E. sagittata induced only 20.8 and 38.7% of hemolysis. This led to the conclusion that the hydro-methanol extracts of D. rotundifolia are more hemolytic than those of E. sagittata. In view of this finding, the application of D. rotundifolia to the mucosa would cause more damage than that of E. sagittata. However, no significant difference was observed (p>0.05), after Student's test.

Induction and suppression of cell lysis in an electrokinetic microfluidic system.

The ability to strategically induce or suppress cell lysis is critical for many cellular-level diagnostic and therapeutic applications conducted within electrokinetic microfluidic platforms. The chemical and structural integrity of sub-cellular components is important when inducing cell lysis. However, metal electrodes and electrolytes participate in undesirable electrochemical reactions that alter solution composition and potentially damage protein, RNA, and DNA integrity within device microenvironments. For many biomedical applications, cell viability must be maintained even when device-imposed cell-stressing stimuli (e.g., electrochemical reaction byproducts) are present. In this work, we explored a novel and tunable method to accurately induce or suppress device-imposed artifacts on human red blood cell (RBC) lysis in non-uniform AC electric fields. For precise tunability, a dielectric hafnium oxide (HfO2 ) layer was used to prevent electron transfer between the electrodes and the electric double layer and thus reduce harmful electrochemical reactions. Additionally, a low concentration of Triton X-100 surfactant was explored as a tool to stabilize cell membrane integrity. The extent of hemolysis was studied as a function of time, electrode configuration (T-shaped and star-shaped), cell position, applied non-uniform AC electric field, with uncoated and HfO2 coated electrodes (50nm), and absence and presence of Triton X-100 (70µM). Tangible outcomes include a parametric analysis relying upon literature and this work to design, tune, and operate electrokinetic microdevices to intentionally induce or suppress cellular lysis without altering intracellular components. Implications are that devices can be engineered to leverage or minimize device-imposed biological artefacts extending the versatility and utility of electrokinetic diagnostics.

Phenotypic and genotypic changes of Staphylococcus aureus in the presence of the inappropriate concentration of chlorhexidine gluconate

BackgroundChlorhexidine gluconate (CHG) is a disinfectant agent with different applications in health care. Improper use of CHG causes antimicrobial resistance in bacteria as a public health threat. Since Staphylococcus aureus is a common bacteria, it is expected usually exposed to CHG in the hospital and community. The present study aimed to correlate the phenotypic and genotypic changes in a S. aureus strain upon serial adaptation with supra-inhibitory CHG concentration for 50 days.ResultsAfter in vitro serial culture of 5 × 105 CFU/ml of a clinical vancomycin-susceptible S. aureus strain (VAN-S) into brain heart infusion (BHI) broth containing CHG 1/4, 1/2, 1, and 2 × minimal inhibitory concentration (MIC) values of VAN-S in 37 °C during 50 days, we isolated a S. aureus strain (CHGVan-I) with a ≥ twofold decrease in susceptibility to CHG and vancomycin. CHG-induced CHGVan-I strain was considered as a vancomycin-intermediate S. aureus (VISA) strain with a VAN MIC of 4 μg/ml using the broth macro dilution method. However, reduced resistance was observed to tetracycline family antibiotics (doxycycline and tetracycline) using a modified Kirby-Bauer disk diffusion test. Moreover, a remarkable reduction was detected in growth rate, hemolysis activity (the lysis of human red blood cells by alpha-hemolysin), and colony pigmentation (on BHI agar plates). Biofilm formation (using the Microtiter plate method and crystal violet staining) was significantly increased upon CHG treatment. Adaptive changes in the expression of a set of common genes related to the development of VISA phenotype (graTSR, vraTSR, walKR, agr RNAIII, sceD, pbpB, and fmtA) were analyzed by Reverse Transcription quantitative PCR (RT-qPCR) experiment. Significant changes in vraTSR, agr RNAIII, sceD, and pbpB expression were observed. However, gene sequencing of the two-component system vraTSR using the Sanger sequencing method did not detect any non-synonymous substitution in CHGVan-I compared to wild-type. The clonality of VAN-S and CHGVan-I strains was verified using the pulsed-field gel electrophoresis (PFGE) method.ConclusionsThe importance of the present study should be stated in new detected mechanisms underlying VISA development. We found a link between the improper CHX use and the development of phenotypic and genotypic features, typical of VISA clinical isolates, in a CHG-induced strain.Since disruption of the cell wall biosynthesis occurs in VISA isolates, our CHG-induced VISA strain proved new insights into the role of CHG in the stimulation of the S. aureus cell wall.

Alternatingly Amphiphilic Antimicrobial Oligoguanidines: Structure–Property Relationship and Usage as the Coating Material with Unprecedented Hemocompatibility

With the emergence of antibiotic resistance in recent years, the combat against bacteria is becoming more difficult and critical in human healthcare. In addition to the development of new chemical entities and derivatives of antibiotics and disinfectants, the antimicrobial surface is recognized as a potential way to prevent healthcare-associated infections, particularly those caused by medical devices such as implants and various catheters. However, traditional ways of engineering antimicrobial surfaces using coated metal nanostructures or blended-in antibiotics inevitably face the problem of metal ion or antibiotic leakage, leading to surface deactivation, toxicity, and antibiotic resistance. A new strategy developed in recent years involves the use of coated cationic antimicrobial polymers, which serve as resistance-resistant, surface-anchored antibacterial agents, but they also usually come with hemolytic activity resulting from their membrane-disrupting-type mechanism of action. In this report, we show that the combinatorial approach can be highly useful for the selection of alternatingly amphiphilic oligomers, which are essentially molecular nanorods as revealed by molecular dynamic simulations. The combinatorial process allowed the structure–property relationship of these molecular nanorods to be revealed and yielded oligoguanidine hit with high antimicrobial activity, fast killing kinetics, and extremely low hemolytic activity through assay screening. We demonstrate that the covalent conjugation of such a prescreened oligoguanidine nanorod to glass slides or polycaprolactone films could produce antimicrobial surfaces with excellent performance. A catheter prototype made using such a modified polycaprolactone film could transport bacteria-contaminated liquid, efficiently removing all bacteria without causing lysis of red blood cells.

Rapid potency assessment of autologous peripheral blood stem cells by intracellular flow cytometry: the PBSC-IL-3-pSTAT5 assay

Background aimsThe current gold standard for stem cell product potency assessment, the colony-forming unit (CFU) assay, delivers results that are difficult to standardize and requires a substantial amount of time (up to 14 days) for cellular growth. Recently, the authors developed a rapid (<24 h) flow cytometry assay based on the measurement of intracellular phosphorylated STAT5 (pSTAT5) in CD34+ cord blood stem and progenitor cells in response to IL-3 stimulation. The present work presents a novel adaptation of the protocol for use with autologous peripheral blood stem cells (PBSCs) and a performance comparison with the CFU assay. MethodsThe flow cytometry intracellular staining assay was optimized for PBSCs, and patient samples were analyzed using the PBSC-IL-3-pSTAT5 and CFU assays. Warming events were also simulated to emulate impaired potency products. ResultsOptimization led to minor protocol adjustments, such as removal of the red blood cell lysis step, the addition of a formaldehyde fixation step and an increase in anticoagulant concentration. The PBSC-IL-3-pSTAT5 assay discriminated between normal and impaired samples and identified 100% (18 of 18) of the impaired samples, thus showing better specificity than the CFU assay. ConclusionsThe updated IL-3-pSTAT5 potency assay has several important advantages, such as accelerating the release of autologous stem cell products and enabling the detection of potentially impaired products. The assay could also be used to rapidly assess the potency of any cryopreserved allogeneic stem cell product, such as those processed during the coronavirus disease 2019 pandemic.

Antimalarial Effect of Doxorubicin on Plasmodium Falciparum: An in Vitro Study in FCR-3 Strain

Plasmodium falciparum is the most common species of Plasmodium that causes malaria in Southeast Asia. Artemisinin, a drug with the mechanism of action by inducing oxidative stress in infected red blood cells (RBC) is currently used as the main therapy for malaria, after resistance to chloroquine has been found. However, evidence of artemisinin resistance was discovered in several regions in Southeast Asia. Therefore, a research is required to prove the existence of other drugs that have anti-malaria effects. A drug candidate, doxorubicin also can induce the formation of oxidative stress inside the cells. This study aims to determine the activity of doxorubicin to inhibit the development of P. falciparum in vitro. Red blood cell (RBC) infected with P. falciparum were treated with various concentrations of doxorubicin. Giemsa technique was applied to detect P. falciparum inside RBC. After 48 hours of incubation, the culture was observed to measure the number and the confluence of RBC and P. falciparum in the medium. This study revealed that doxorubicin reduced the number of RBC infected with P. falciparum lysis. The effective dose of doxorubicin-inhibit RBC cell lysis is 0.4 μM, which only reduces 81% RBC cell lysis compared to the control group that reduces 95% RBC cell lysis. At this concentration also found a decrease in the number of P. falciparum cells in the medium. The results proved that doxorubicin has an inhibitory effect on the development of P. falciparum and can decrease the lysis of RBC due to P. falciparum infection. This findings provide an insight that doxorubicin is a potential candidate for antimalarial drugs.

Quantification of histone H2AX phosphorylation in white blood cells induced by ex vivo gamma irradiation of whole blood by both flow cytometry and foci counting as a dose estimation in rapid triage.

A quick, reliable, and reproducible biological assay to distinguish individuals with possible life-threatening risk following radiological or nuclear incidents remains a quest in biodosimetry. In this paper, we examined the use of a γ-H2AX assay as an early dose estimation for rapid triage based on both flow cytometry and image analyses. In the experiment, whole blood from 11 donors was irradiated ex vivo inside a water phantom by gamma rays from Co-60 at 0.51 Gy/min. After the lysis of red blood cells, the white blood cells were collected for immunofluorescence labeling of γ-H2AX, CD45, and nuclear stained for signal collection and visualization. Analysis by flow cytometry showed that the relative γ-H2AX intensities of lymphocytes and granulocytes increased linearly with absorbed doses from 0 to 6 Gy with a large variation among individuals observed above 2 Gy. The relative γ-H2AX intensities of lymphocytes assessed by two different laboratories were highly correlated (ICC = 0.979). Using confocal microscopic images, γ-H2AX foci were observed to be discretely distributed inside the nuclei and to increase proportionally with doses from 0 to 2 Gy, whereas large plagues of merged foci appeared at 4 and 6 Gy, resulting in the saturation of foci counts above 4 Gy. The number of total foci per cell as well as the number of foci per plane were significantly different at 0 vs 1 and 2 vs 4 Gy doses (p < 0.01). Blind tests at 0.5 Gy and 1 Gy doses showed that dose estimation by flow cytometry had a mean absolute difference of less than 0.5 Gy from the actual value. In conclusion, while flow cytometry can provide a dose estimation with an uncertainty of 0.5 Gy at doses ≤ 1 Gy, foci counting can identify merged foci that are prominent at doses ≥ 4 Gy.

Targeting the Pseudomonas aeruginosa Virulence Factor Phospholipase C With Engineered Liposomes.

Engineered liposomes composed of the naturally occurring lipids sphingomyelin (Sm) and cholesterol (Ch) have been demonstrated to efficiently neutralize toxins secreted by Gram-positive bacteria such as Streptococcus pneumoniae and Staphylococcus aureus. Here, we hypothesized that liposomes are capable of neutralizing cytolytic virulence factors secreted by the Gram-negative pathogen Pseudomonas aeruginosa. We used the highly virulent cystic fibrosis P. aeruginosa Liverpool Epidemic Strain LESB58 and showed that sphingomyelin (Sm) and a combination of sphingomyelin with cholesterol (Ch:Sm; 66 mol/% Ch and 34 mol/% Sm) liposomes reduced lysis of human bronchial and red blood cells upon challenge with the Pseudomonas secretome. Mass spectrometry of liposome-sequestered Pseudomonas proteins identified the virulence-promoting hemolytic phospholipase C (PlcH) as having been neutralized. Pseudomonas aeruginosa supernatants incubated with liposomes demonstrated reduced PlcH activity as assessed by the p-nitrophenylphosphorylcholine (NPPC) assay. Testing the in vivo efficacy of the liposomes in a murine cutaneous abscess model revealed that Sm and Ch:Sm, as single dose treatments, attenuated abscesses by >30%, demonstrating a similar effect to that of a mutant lacking plcH in this infection model. Thus, sphingomyelin-containing liposome therapy offers an interesting approach to treat and reduce virulence of complex infections caused by P. aeruginosa and potentially other Gram-negative pathogens expressing PlcH.

Carrier-Free Deferoxamine Nanoparticles against Iron Overload in Brain

Carrier-Free Deferoxamine Nanoparticles against Iron Overload in Brain