Function Of Blood Cells Research Articles

L-cysteine improves blood fluidity impaired by acetaldehyde: In vitro evaluation.

Blood fluidity is reportedly influenced by the volume and function of blood cells and plasma and is a predictor of primary cardiovascular events in patients with traditional cardiovascular risk factors. Heavy alcohol consumption was shown to be associated with a higher risk for cardiovascular diseases. Acetaldehyde (ACD), an oxidizing substance formed from ethanol, reportedly stimulates monocyte adhesion, causes abnormalities in the red blood cell (RBC) membrane, and decreases RBC deformability. In addition, it was reported that blood ACD levels are reduced in mice pretreated with L-cysteine. However, there are no studies on the effect of ACD and/or L-cysteine on blood fluidity. In the present study, we evaluated whether ACD impairs blood fluidity. In addition, the effect of L-cysteine on blood fluidity impaired by ACD was examined. Blood samples were obtained from 10 healthy, non-smoking, male volunteers (age: 23.4 ± 1.2 years, body mass index: 21.8 ± 2.6 kg/m2). ACD or ACD and L-cysteine were added to the blood samples before each experiment. We measured the passage time of 100 μL blood and RBC suspension using Kikuchi’s microchannel method. Percentage of microchannel obstruction and the number of adherent white blood cells (WBCs) on microchannel terrace were counted. The blood passage time, percentage of microchannel obstruction, and numbers of adherent WBCs on the microchannel terrace increased after adding ACD in a concentration-dependent manner, whereas they decreased after adding ACD and L-cysteine in a L-cysteine concentration-dependent manner. No significant effects were observed in passage time for 100 μL RBC suspension after adding ACD and L-cysteine. This study suggested that blood fluidity impaired by ACD might improve after adding L-cysteine.

Inhibition of Tropomyosin Receptor Kinase A Signaling Negatively Regulates Megakaryopoiesis and induces Thrombopoiesis

Neurotrophin signaling modulates the differentiation and function of mature blood cells. The expression of neurotrophin receptors and ligands by hematopoietic and stromal cells of the bone marrow indicates that neurotrophins have the potential to regulate hematopoietic cell fate decisions. This study investigates the role of neurotrophins and Tropomyosin receptor kinases (Trk) in the development of megakaryocytes (MKs) and their progeny cells, platelets. Results indicate that primary human MKs and MK cells lines, DAMI, Meg-01 and MO7e express TrkA, the primary receptor for Nerve Growth Factor (NGF) signaling. Activation of TrkA by NGF enhances the expansion of human MK progenitors (MKPs) and, to some extent, MKs. Whereas, inhibition of TrkA receptor by K252a leads to a 50% reduction in the number of both MKPs and MKs and is associated with a 3-fold increase in the production of platelets. In order to further confirm the role of TrkA signaling in platelet production, TrkA deficient DAMI cells were generated using CRISPR-Cas9 technology. Comparative analysis of wild-type and TrkA-deficient Dami cells revealed that loss of TrkA signaling induced apoptosis of MKs and increased platelet production. Overall, these findings support a novel role for TrkA signaling in platelet production and highlight its potential as therapeutic target for Thrombocytopenia.

Antiviral Responses and Biological Concequences of Piscine orthoreovirus Infection in Salmonid Erythrocytes.

Salmonid red blood cells are the main target cells for Piscine orthoreovirus (PRV). Three genotypes of PRV (PRV-1,2,3) infect Atlantic salmon (Salmo salar), Chinook salmon (Onchorhynchus tshawytscha), Coho salmon (Oncorhynchus kisutch), rainbow trout (Onchorhynchus mykiss) and brown trout (Salmo trutta), and can cause diseases like heart and skeletal muscle inflammation (HSMI), jaundice syndrome, erythrocyte inclusion body syndrome (EIBS) and proliferative darkening syndrome (PDS). Purified PRV administrated to fish has proven the causality for HSMI and EIBS. During the early peak phase of infection, salmonid erythrocytes are the main virus-replicating cells. In this initial phase, cytoplasmic inclusions called “virus factories” can be observed in the erythrocytes, and are the primary sites for the formation of new virus particles. The PRV-infected erythrocytes in Atlantic salmon mount a strong long-lasting innate antiviral response lasting for many weeks after the onset of infection. The antiviral response of Atlantic salmon erythrocytes involves upregulation of potential inhibitors of translation. In accordance with this, PRV-1 protein production in erythrocytes halts while virus RNA can persist for months. Furthermore, PRV infection in Coho salmon and rainbow trout are associated with anemia, and in Atlantic salmon lower hemoglobin levels are observed. Here we summarize and discuss the recently published findings on PRV infection, replication and effects on salmonid erythrocytes, and discuss how PRV can be a useful tool for the study of innate immune responses in erythrocytes, and help reveal novel immune functions of the red blood cells in fish.

Myeloid-Specific Deletion of Mcl-1 Yields Severely Neutropenic Mice That Survive and Breed in Homozygous Form.

Mouse strains with specific deficiency of given hematopoietic lineages provide invaluable tools for understanding blood cell function in health and disease. Whereas neutrophils are dominant leukocytes in humans and mice, there are no widely useful genetic models of neutrophil deficiency in mice. In this study, we show that myeloid-specific deletion of the Mcl-1 antiapoptotic protein in Lyz2Cre/CreMcl1flox/flox (Mcl1ΔMyelo) mice leads to dramatic reduction of circulating and tissue neutrophil counts without affecting circulating lymphocyte, monocyte, or eosinophil numbers. Surprisingly, Mcl1ΔMyelo mice appeared normally, and their survival was mostly normal both under specific pathogen-free and conventional housing conditions. Mcl1ΔMyelo mice were also able to breed in homozygous form, making them highly useful for in vivo experimental studies. The functional relevance of neutropenia was confirmed by the complete protection of Mcl1ΔMyelo mice from arthritis development in the K/B×N serum-transfer model and from skin inflammation in an autoantibody-induced mouse model of epidermolysis bullosa acquisita. Mcl1ΔMyelo mice were also highly susceptible to systemic Staphylococcus aureus or Candida albicans infection, due to defective clearance of the invading pathogens. Although neutrophil-specific deletion of Mcl-1 in MRP8-CreMcl1flox/flox (Mcl1ΔPMN) mice also led to severe neutropenia, those mice showed an overt wasting phenotype and strongly reduced survival and breeding, limiting their use as an experimental model of neutrophil deficiency. Taken together, our results with the Mcl1ΔMyelo mice indicate that severe neutropenia does not abrogate the viability and fertility of mice, and they provide a useful genetic mouse model for the analysis of the role of neutrophils in health and disease.

Neutrophil Extracellular Traps Are an Intrinsic Feature in Sickle Cell Disease

Polymerisation of deoxygenated-HbS in sickle cell disease (SCD) leads to alteration of the shape and function of the red blood cells (RBC), resulting in hemolytic anemia and recurrent episodes of micro-vascular occlusion, triggering a cascade of downstream events and inflammation that underlies the progressive multi-systemic organ damage. Neutrophils have a critical role in promoting sickle pathophysiology; patients with higher leukocyte count, particularly neutrophils, tend to have a higher risk for more severe manifestations as well as earlier mortality. SCD neutrophils display a specific overreactive phenotype, including increased adhesive properties and production of reactive oxygen species. Activated neutrophils can also produce extracellular traps (NETs) in response to pathogens or other inflammatory stimuli. Studies have shown that prolonged exposure of healthy neutrophils to SCD plasma leads to formation of mature string-like NETs. These strings of decondensed chromatin covered with elastase (NE) or myeloperoxidase (MPO) might contribute to SCD pathophysiology. Here, we investigated if SCD neutrophils had enhanced intrinsic potential for NETs formation (i.e. could form NETs in the absence of a stimulus).We evaluated NETosis both indirectly, in which neutrophils isolated from healthy subjects were incubated with plasma from HbSS patients and directly, in which neutrophils isolated from HbSS patients were compared with those isolated from healthy donors. 17 HbSS patients in steady-state (all on hydroxyurea) and 11 race and sex matched healthy donors were included in the direct assays; the NETosis-related events were observed over a period of 7 minutes to 4 hours. For the indirect assay, we used plasma from 19 HbSS patients (14 on hydroxyurea, 5 off hydroxyurea and/or transfused), all of whom provided samples in steady-state and acute pain crisis (paired samples); the healthy neutrophils were treated with SCD plasma for up to 7 hours. A pain crisis was defined as an episode of acute pain that has no evident cause other than SCD, resulting in hospitalization and treatment with parenteral opioids. Steady state was defined as the period from at any time 8 weeks prior to or after a crisis. Patients were excluded when they were <18 or >80 years of age, pregnant or had a history of blood transfusion in the previous 8 weeks. The IRB approved the studies under NIH protocol numbers NCT00081523 (SCD subjects) and 03-H-0015 (healthy controls and SCD subjects). The NETosis features (histone citrullination H4cit3, nuclear decondensation, DNA-NE-MPO strings) were assessed by imaging flow cytometry and fluorescence microscopy.In the indirect assays, healthy neutrophils made more string NETs when treated with the SCD plasma (from both steady state and crisis) compared to plasma from healthy donors (Figure 1A), and NETs response was further increased with plasma from acute compared to their paired steady state sample (N=19, p=0.0114) (Figure 1B). Plasma from patients on or off hydroxyurea induced comparable NETosis responses. This confirmed a higher ability for the crisis milieu to modulate NETosis.Directly, neutrophils isolated from SCD patients as compared to those from healthy controls, showed increased number of decondensed H4cit3 positive nuclei, after only 7 min of incubation in RPMI (N=7, p=0.0688). By comparison the response to high concentration (20 uM) of Hemin - a regular product of hemolysis, was similar in the SCD and the healthy neutrophils (p=0.6871) (Figure 1C). This data suggested that the SCD environment might activate neutrophils to produce NETs in the absence of a specific stimulus. Fluorescence microscopy confirmed that neutrophils isolated from the patients did produce significantly more DNA-NE strings without any stimulation compared to healthy neutrophils (N=9, p=0.0079). After Hemin treatment, NETs response in SCD neutrophils was also higher than that of the healthy controls, but not statistically significant (Figure 1D).Our data provide the first direct in vitro evidence that neutrophils in SCD are innately prone to NETosis but whether and how these might contribute to the initiation or progress of SCD vaso-occlusive crises is yet to be determined. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Highly Efficient Editing of the Beta-Globin Gene in Patient Derived Hematopoietic Stem and Progenitor Cells to Treat Sickle Cell Disease

Sickle cell disease (SCD) is an inherited blood disorder associated with a debilitating chronic illness. SCD is caused by a point mutation in the β-globin gene (HBB). A single nucleotide substitution converts glutamic acid to a valine that leads to the production of sickle hemoglobin (HbS), which impairs the function of red blood cells. Here we show that delivery of Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex (RNP) together with a short single-stranded DNA donor (ssODN) template into CD34+ hematopoietic stem and progenitor cells (HSPCs) from SCD patients' bone marrow (BM) was able to correct the sickling HBB mutation, with up to 33% homology directed repair (HDR) without selection. Further, CRISPR/Cas9 cutting of HBB in SCD HSPCs induced gene conversion between the HBB sequences in the vicinity of the target locus and the homologous region in δ-globin gene (HBD), with up to 4.4% additional gene correction mediated by the HBD conversion in cells with Cas9 cutting only. The erythrocytes derived from gene-edited cells showed a marked reduction of the HbS level, increased expression of normal adult hemoglobin (HbA), and a complete loss of cell sickling, demonstrating the potential in curing SCD.We performed extensive off-target analysis of gene-edited SCD HSPCs using the in-silico prediction tool COSMID and unbiased, genome-wide assay Guide-Seq, revealing a gross intrachromosomal rearrangement event between the on- and off-target Cas9 cutting sites. We used a droplet digital PCR assay to quantify deletion and inversion events from Day 2 to Day 12 after RNP delivery, and found that large chromosomal deletion decreased from 1.8% to 0.2%, while chromosomal inversion maintained at 3.3%. We demonstrated that the use of high-fidelity SpCas9 (HiFi Cas9 by IDT) significantly reduced off-target effects and completely eliminated the intrachromosome rearrangement events, while maintaining the same level of on-target gene editing, leading to high-efficiency gene correction with increased specificity.In order to determine if gene-corrected SCD HSPCs retain the ability to engraft, CD34+ cells from the BM of SCD patients were treated with Cas9/gRNA RNP and ssODN donor for HBB gene correction, cryopreserved at Day 2 post genome editing, then intravenously transplanted into NSG mice shortly after thawing. These mice were euthanized at Week 16 after transplantation, and the BM was harvested to determine the engraftment potential. An average of 7.5 ±5.4% of cells were double positive for HLA and hCD45 in mice injected with gene-edited CD34+ cells, compared to 16.8 ±9.3% with control CD34+ cells, indicating a good level of engraftment of gene-corrected SCD HSPCs. A higher fraction of human cells were positive for CD19 (66 ±28%), demonstrating lymphoid lineage bias. DNA was extracted from unsorted cells, CD19 or CD33 sorted cells for gene-editing analysis; the HBB editing rates were respectively 29.8% HDR, 2.4% HBD conversion, and 42.8% non-homologous end joining (NHEJ) pretransplantation, and editing rates at Week 16 posttransplantation were respectively 8.8 ±12% HDR, 1.8 ±1.7% HBD conversion, and 24.5 ±12% NHEJ. The highly variable editing rate and indel diversity in gene-edited cells at Week 16 in all four transplanted mice suggest clonal dominance of a limited number of HSPCs after transplantation.Taken together, our results demonstrate highly efficient gene and phenotype correction of the sickling mutation in BM HSPCs from SCD patients mediated by HDR and HBD conversion, and the ability of gene-edited SCD HSPCs to engraft in vivo. We also demonstrate the importance of genome-wide analysis for off-target analysis and the use of HiFi Cas9. Our results provide further evidence for the potential of moving genome editing-based SCD treatment into clinical practice.Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G. B.), and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) DisclosuresPorteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

PO-067 Effects of oral Lycium barbarum juice in red blood cell antioxidant biomarkers and physical function during 8 days of aerobic exercise

Objective Lycium barbarum polysaccharide (LBP) is the main active components of Lycium barbarum, its benefits to anti-aging, vision, kidney, and liver functions. Nevertheless, there is still a scarcity of experimental evidence to support the effect of Lycium barbarum on aerobic exercise.This a randomized controlled trial was observed the effects of oral Lycium barbarum juice in red blood cell antioxidant biomarkers and physical function during 8 days of aerobic exercise.&#x0D; Methods 28 healthy male college students were divided into control group（16）and experimental group（12），and underwent interval running once every other day，total of 8 days. Exercise program: An exercise session includes two 30-minute aerobic exercises at 60%VO2max and a five-minute break. For the duration of the 8 days period, participants exercised one time every other day and the experimental group drank 100ml Lycium barbarum juice (each LBP content 360-440mg%) at bedtime every night. In ninth days, all the experimenters did exhaustive exercise with 80%VO2max on a treadmill with 8°.simultaneous recording of motion duration. The levels of red blood cell SOD, MDA, GSH-PX, serum CAT, serum TAC and other oxygenation stress markers and BLA, Glu, Urea, CK, Urine eight items and other physical function indexes of the subjects were determined before the experiment and after the completion of each intensity exercise. Differences between before and after intervention values were tested using a paired t test.And to compare the mean of outcomes in quantitative variables between the 2 groups, a independent t-test was used. The SPSS software (version 17, SPSS Inc, Chicago, IL, USA) was applied for data analysis and statistical significance was accepted at P &lt; 0.05.&#x0D; Results (1)After 8 days of oral Lycium barbarum juice, the exhaustion time of exercise force in the experimental group was 30.76 ±12.19min, while the control group was 23.64±8.56min. Compared with the control group, the average exercise exhaustion time of the experimental group was prolonged 7.12min. (2)The red blood cell SOD in the two groups after 8 days of aerobic exercise had significant and significant improvement (P &lt; 0.05, P &lt; 0.01), and moreover, the increase of the experimental group was significantly higher than that of the control group (P &lt; 0.05).As well as, the blood erythrocyte GSH-PX and serum TAC were significantly enhanced after the experiment (P &lt; 0.01).It is suggested that increasing the levels of SOD and GSH-PX in vivo is beneficial in scavenging the free radicals produced by body movement. (3)After the 8 days oral Lycium barbarum juice, the decrease of MDA in blood red blood cells in the experimental group was greater than that of the control group (P &lt; 0.01), indicating that the juice of Lycium barbarum could reduce the production of lipid peroxide products. (4) Exhaustion exercise after 8 days of oral Lycium barbarum juice, the physical function indexes of the experimental group, such as BLA, Urea, and CK were reduced. The positive rate of eight urine items was less than that in the control group, 8 in the control group, 2 for bilirubin positive, 3 in the urinary occult blood and 5 in the urine protein, while only 1 in the experimental group were positive for urine protein.&#x0D; &#x0D; Conclusions Oral Lycium barbarum juice can improve the activity of antioxidant enzymes during aerobic exercise, reduce the formation of lipid peroxides in the body, protect the biological function of red blood cells, improve the physical function and postpone the production of sports fatigue.

Expression of CD markers in JAK2V617F positive myeloproliferative neoplasms: Prognostic significance.

Myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by the presence of JAK2V617F mutation. Thrombohemorrhagic as well as autoimmune or inflammatory phenomena are common clinical outcomes of these disorders. Recent studies have shown that abnormality in frequency and function of blood cells manifested by an alteration in CD markers’ expression patterns play a key role in these complications. So, there may be a relationship between CD markers’ expressions and prognosis of JAK2V617F positive MPNs. Therefore, in this review, we have focused on these abnormalities from the perspective of changing expressions of CD markers and assessment of the relationship between these changes with prognosis of JAK2V617F positive MPNs. It can be stated that the abnormal expression of a large number of CD markers can be used as a prognostic biomarker for clinical outcomes including thrombohememorrhagic events, as well as autoimmune and leukemic transformation in JAK2V617F positive MPNs. Considering the possible role of CD markers’ expressions in JAK2V617F MPNs prognosis, further studies are needed to confirm the relationship between the expression of CD markers with prognosis to be able to find an appropriate therapeutic approach via targeting CD markers.

The effect of canceroembryonic antigen on cytokine production by immunocompetent blood cells in patients with breast cancer

Aim. Investigation of the influence of canceroembryonic antigen (CEA) on the immune cells in patients with breast tumors.Materials and methods. Peripheral blood of 67 patients with invasive ductal carcinoma (n = 44), fibroadenoma (n = 12) and breast pre-cancer (n = 11). In the supernatants of the blood cells cytokine production induced by CEA was determined by ELISA.Results.It was found that CEA stimulated production of IL-6, IL-8, IL-10, IL-1β, IL-1Ra, TNFα, G-CSF, GM-CSF and the protein MCP-1 by blood immune cells in patients with fibroadenoma compared with groups of patients with invasive ductal carcinoma and precancerous lesions. Stimulation index of CEA on cytokine production by immunocompetent cells in patients in these groups was lower than in groups of patients with fibroadenoma due to initially high levels of spontaneous cytokine production.Results. In patients with fibroadenoma CEA was found to stimulate the production of IL-6, IL-8, IL-10, IL-1β, IL-1Ra, TNFa, G-CSF, GM-CSF and MCP-1 compared with groups of patients with invasive ductal carcinoma and precancerous conditions. Indexes of the influence of CEA on cytokine production by immunocompetent cells in these groups was lower than in the fibroadenoma due to initially high spontaneous production of cytokines.Conclusion. Evident spontaneous cytokine-producing function of immunocompetent blood cells was revealed in patients with invasive ductal carcinoma and precancerous conditions compared to patients with fibroadenoma. The differences between the indices of CEA influence on cytokine production in patients with malignant, benign and precancerous conditions can serve as a basis for the development of methods of differential diagnosis of breast tumors.

Abnormal Microcirculation and Red Blood Cell Function as a Cardiovascular Risk Factor in Metabolic Syndrome

The metabolic syndrome is a highly prevalent condition associated with increased cardiovascular risk in the population. Microvasculature is the terminal part of the cardiovascular system which primarily reacts to the increased secretion of the pro inflammatory adipokines typical for the metabolic syndrome. Microcirculation and blood cell abnormalities are the leading mechanisms of cardiovascular events development in this condition. Prevalence of microcirculation abnormalities and red blood cell dysfunction in metabolic syndrome and their role in the increased blood viscosity and cardiovascular events development are covered by the paper. The microcirculation abnormalities with a special focus on red blood cell dysfunction (impaired aggregation, stiffness) seen in metabolic syndrome and associated conditions are discussed in detail. The impact of abnormal red blood cell deformability (secondary to cholesterol accumulation in the cellular membranes) on the hemorheological abnormalities is revealed. Abnormal red blood cell surface charge due to proinflammatory changes associated with insulin resistance in diabetes mellitus is highlighted. These abnormalities lead to increased red blood cell aggregation and plasma viscosity that are the essential components of cardiovascular events pathogenesis. Their timely diagnosis is crucial for effective cardiovascular prevention.

Analysis of mitochondrial respiratory function in tissue biopsies and blood cells.

The review provides an overview on latest methodological strategies to assess mitochondrial respiratory function in tissue biopsies or blood cells. In addition, it summarizes the recent literature related to this topic. Today, the study of mitochondrial function in key metabolic active tissues has been become more relevant, with increasing focus in clinical applications. In addition, assessment of mitochondrial function in blood cells by respirometry might be a sensitive biomarker of disease progression. High-Resolution Respirometry provides a modern tool to study mitochondrial respiratory physiology which allows direct measurement of cellular metabolic function during health and disease. Moreover, standard operating procedures are required regarding instrumental settings, sample collection and preparation, protocol design and respirometric data analysis of mitochondrial respiratory function in tissue biopsies (such as skeletal muscle, liver and adipose tissue), as well as isolated blood cells. Mitochondrial function is a key factor in many metabolic diseases. Although various analytical approaches are available, certain well-established protocols for isolated mitochondria are limited for the analysis of mitochondrial function in tissue biopsies or blood cells. Thus, cautious considerations in selecting appropriate protocols and analytical endpoints are crucial for the interpretation of the gained data and to draw robust conclusions.

Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study

SummaryBackgroundHuman genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms—many related to the structure or function of red blood cells—and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress.MethodsWe did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria.FindingsBetween June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11–0·20; p=2·61 × 10−58), blood group O (0·74, 0·66–0·82; p=6·26 × 10−8), and –α3·7-thalassaemia (0·83, 0·76–0·90; p=2·06 × 10−6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63–0·92; p=0·001) and FREM3 (0·64, 0·53–0·79; p=3·18 × 10−14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49–0·68; p=3·22 × 10−11), as was homozygosity (0·26, 0·11–0·62; p=0·002).InterpretationBoth ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health.FundingWellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative.

Disturbed Red Blood Cell Structure and Function: An Exploration of the Role of Red Blood Cells in Neurodegeneration

The structure of red blood cells is affected by many inborn and acquired factors, but in most cases this does not seem to affect their function or survival in physiological conditions. Often, functional deficits become apparent only when they are subjected to biochemical or mechanical stress in vitro, or to pathological conditions in vivo. Our data on the misshapen red blood cells of patients with neuroacanthocytosis illustrate this general mechanism: an abnormal morphology is associated with an increase in the susceptibility of red blood cells to osmotic and mechanical stress, and alters their rheological properties. The underlying mutations may not only affect red cell function, but also render neurons in specific brain areas more susceptible to a concomitant reduction in oxygen supply. Through this mechanism, an increased susceptibility of already compromised red blood cells to physiological stress conditions may constitute an additional risk factor in vulnerable individuals. Also, susceptibility may be induced or enhanced by systemic pathological conditions such as inflammation. An exploration of the literature suggests that disturbed red blood cell function may play a role in the pathophysiology of various neurodegenerative diseases. Therefore, interventions that reduce the susceptibility of red blood cells to physiological and pathological stress may reduce the extent or progress of neurodegeneration.

Multiple faces of succinate beyond metabolism in blood.

Succinate is an essential intermediate of the tricarboxylic acid cycle that exerts pleiotropic roles beyond metabolism in both physiological and pathological conditions. Recent evidence obtained in mouse models shows its essential role regulating blood cell function through various mechanisms that include pseudohypoxia responses by hypoxia-inducible factor-1α activation, post-translational modifications like succinylation, and communication mediated by succinate receptor 1. Hence, succinate links metabolism to processes like gene expression and intercellular communication. Interestingly, succinate plays key dual roles during inflammatory responses, leading to net inflammation or anti-inflammation depending on factors like the cellular context. Here, we further discuss current suggestions of the possible contribution of succinate to blood stem cell function and blood formation. Further study will be required in the future to better understand succinate biology in blood cells. This promising field may open new avenues to modulate inflammatory responses and to preserve blood cell homeostasis in the clinical setting.

Mitochondria-Targeted Artificial "Nano-RBCs" for Amplified Synergistic Cancer Phototherapy by a Single NIR Irradiation.

Phototherapy has emerged as a novel therapeutic modality for cancer treatment, but its low therapeutic efficacy severely hinders further extensive clinical translation and application. This study reports amplifying the phototherapeutic efficacy by constructing a near‐infrared (NIR)‐responsive multifunctional nanoplatform for synergistic cancer phototherapy by a single NIR irradiation, which can concurrently achieve mitochondria‐targeting phototherapy, synergistic photothermal therapy (PTT)/photodynamic therapy (PDT), self‐sufficient oxygen‐augmented PDT, and multiple‐imaging guidance/monitoring. Perfluorooctyl bromide based nanoliposomes are constructed for oxygen delivery into tumors, performing the functions of red blood cells (RBCs) for oxygen delivery (“Nano‐RBC” nanosystem), which can alleviate the tumor hypoxia and enhance the PDT efficacy. The mitochondria‐targeting performance for enhanced and synergistic PDT/PTT is demonstrated as assisted by nanoliposomes. In particular, these “Nano‐RBCs” can also act as the contrast agents for concurrent computed tomography, photoacoustic, and fluorescence multiple imaging, providing the potential imaging capability for phototherapeutic guidance and monitoring. This provides a novel strategy to achieve high therapeutic efficacy of phototherapy by the rational design of multifunctional nanoplatforms with the unique performances of mitochondria targeting, synergistic PDT/PTT by a single NIR irradiation (808 nm), self‐sufficient oxygen‐augmented PDT, and multiple‐imaging guidance/monitoring.

On the Effects of Reactive Oxygen Species and Nitric Oxide on Red Blood Cell Deformability.

The main function of red blood cells (RBCs) is the transport of respiratory gases along the vascular tree. To fulfill their task, RBCs are able to elastically deform in response to mechanical forces and, pass through the narrow vessels of the microcirculation. Decreased RBC deformability was observed in pathological conditions linked to increased oxidative stress or decreased nitric oxide (NO) bioavailability, like hypertension. Treatments with oxidants and with NO were shown to affect RBC deformability ex vivo, but the mechanisms underpinning these effects are unknown. In this study we investigate whether changes in intracellular redox status/oxidative stress or nitrosation reactions induced by reactive oxygen species (ROS) or NO may affect RBC deformability. In a case-control study comparing RBCs from healthy and hypertensive participants, we found that RBC deformability was decreased, and levels of ROS were increased in RBCs from hypertensive patients as compared to RBCs from aged-matched healthy controls, while NO levels in RBCs were not significantly different. To study the effects of oxidants on RBC redox state and deformability, RBCs from healthy volunteers were treated with increasing concentrations of tert-butylhydroperoxide (t-BuOOH). We found that high concentrations of t-BuOOH (≥ 1 mM) significantly decreased the GSH/GSSG ratio in RBCs, decreased RBC deformability and increased blood bulk viscosity. Moreover, RBCs from Nrf2 knockout (KO) mice, a strain genetically deficient in a number of antioxidant/reducing enzymes, were more susceptible to t-BuOOH-induced impairment in RBC deformability as compared to wild type (WT) mice. To study the role of NO in RBC deformability we treated RBC suspensions from human volunteers with NO donors and nitrosothiols and analyzed deformability of RBCs from mice lacking the endothelial NO synthase (eNOS). We found that NO donors induced S-nitrosation of the cytoskeletal protein spectrin, but did not affect human RBC deformability or blood bulk viscosity; moreover, under unstressed conditions RBCs from eNOS KO mice showed fully preserved RBC deformability as compared to WT mice. Pre-treatment of human RBCs with nitrosothiols rescued t-BuOOH-mediated loss of RBC deformability. Taken together, these findings suggest that NO does not affect RBC deformability per se, but preserves RBC deformability in conditions of oxidative stress.

The effects of gold nanoparticles on the human blood functions

The gold nanoparticles (AuNPs) have been widely used as drug delivery systems at several biomedical fields. However, the effect of AuNPs on the components of human blood is not well characterized. AuNPs firstly interacted with blood when using AuNPs as the drug delivery carries. Therefore, it is urgent to investigate the effect of AuNPs (including the ligands of AuNPs) on human blood, especially its components. In this study, we investigated the possible effects of polyethylene glycol-coated AuNPs (PEG@AuNPs) and citric acid-coated AuNPs (CT-AuNPs) on the blood cell function and distribution of those nanoparticles in blood components including erythrocytes, leukocytes, platelets (PLTs) cells and plasma. We found that the amount of CT-AuNPs engulfed by leukocytes was four folds more compared to PEG@AuNPs, indicating that PEGylation might have the ability to escape the immune system. We found that each individual leukocyte uptaked more AuNPs particles than individual platelet. However, there are more PLTs than leukocytes in the blood. The total amount of AuNPs including PEG@AuNPs and CT-AuNPs accumulation in PLTs were more than in leukocytes. Moreover, we found that both CT-AuNPs and PEG@AuNPs-induced PLTs activation at high concentration. We therefore concluded that the interactions between nanodrug delivery systems (AuNPs) and human blood components, especially the PLTs should be careful evaluated prior to their clinical applications.

A simple and rapid method for measuring human natural killer cell function in whole blood by anti-NKp30-conjugated nanoparticles

Abstract Natural killer (NK) cells play a central role as innate immune effector cells to virus infected cells and tumor cells. The standard assay to evaluate NK activity is 51Cr release assay using radio-labeled K562 cells, however, new methods using fluorescence-labeled targets or flow cytometry to examine degranulation markers have been developed recently. In addition to cytotoxicity, NK cells enable to produce various cytokines to regulate the adaptive immune responses in infection, inflammation and cancer. Here we generated nanoparticles conjugated with various antibody for NK receptors and examined to stimulate human whole blood with these nanoparticles instead of K562 target cells. We found that various cytokines such as IFN-γ, IL-6, IL-1β and CCL2 were produced by NK cells in whole blood by anti-NKp30-conjugated nanoparticles in dose-dependent manner more than by anti-NKp46 or anti-NKG2D nanoparticles. Production of IFN-γ was detected within 4 hours and reached maximum level at 24 hours after stimulation. IFN-γ production was relatively high at incubation temperature of 37°C, but that was significantly decreased over 39°C. Importantly, we could evaluate the reproducible cytokine production in same donors at another blood collection date because of the stable stimulus of nanoparticles and less variation data by human manipulation for lymphocyte separation. Additionally, cytokine secretion did not correlate with NK cell-mediated lysis of K562 target cells and NK cell numbers in whole blood. These data indicated that this method using NKp30 nanoparticles might be useful for measurement of individual differences of NK function especially correlated in adaptive immune response and inflammation.

Construction of anti-thrombotic and anti-oxidative surfaces with elastomer/Pluronic F127 assembled microfibers

The thrombus formation and oxidation of blood components caused by blood-contacting biomaterials were main reasons for poor hemocompatibility of biomaterials. To construct the anti-thrombotic and anti-oxidative surface, we electrospun styrene-b-(ethylene-co-butylene)-b-styrene elastomer (SEBS)/acylated Pluronic F127 (A-F127)/L-ascorbic acid (AA) microfibers based on the SEBS substrate in one step. Our strategy was based on that the phase-separation between SEBS and A-F127 during electrospinning resulted in hydrophilic surface of microfibers, the crosslinking of electrospun meshes rendered the surface stable, and the AA release from the microfibers interacted with blood cells actively to prevent blood cells from oxidative damage. We demonstrated that the self-assembly of SEBS/A-F127 blends resulted in the similar structure as core-shell microfibers with the A-F127 richness in the sheath; the presence of A-F127 on the out layer made the microfibers hydrophilic and rendered the microfibers anti-thrombotic; the release of AA from the microfibers in the PBS solution was temperature-dependent; AA acted as the antioxidant to decrease the oxidative damage of blood cells after long-term contact between biomaterials and blood. Our work paves a new way to develop long-term hemocompatible biomaterials, which may be helpful to prevent blood cells from oxidative damage to maintain the normal function of blood cells in vivo.

Translational Application of Measuring Mitochondrial Functions in Blood Cells Obtained from Patients with Acute Poisoning.

It is conservatively estimated that 5,000 deaths per year and 20,000 injuries in the USA are due to poisonings caused by chemical exposures (e.g., carbon monoxide, cyanide, hydrogen sulfide, phosphides) that are cellular inhibitors. These chemical agents result in mitochondrial inhibition resulting in cardiac arrest and/or shock. These cellular inhibitors have multi-organ effects, but cardiovascular collapse is the primary cause of death marked by hypotension, lactic acidosis, and cardiac arrest. The mitochondria play a central role in cellular metabolism where oxygen consumption through the electron transport system is tightly coupled to ATP production and regulated by metabolic demands. There has been increasing use of human blood cells such as peripheral blood mononuclear cells and platelets, as surrogate markers of mitochondrial function in organs due to acute care illnesses. We demonstrate the clinical applicability of measuring mitochondrial bioenergetic and dynamic function in blood cells obtained from patients with acute poisoning using carbon monoxide poisoning as an illustration of our technique. Our methods have potential application to guide therapy and gauge severity of disease in poisoning related to cellular inhibitors of public health concern.