Non-alcoholic steatohepatitis patients exhibit reduced CD47, and increased sphingosine, cholesterol and MCP1 levels in the erythrocyte membranes

Non-alcoholic steatohepatitis (NASH) constitutes a significant cause of deaths, liver transplantations and economic costs worldwide. Despite extended research, investigations on the role of erythrocytes are scarce. Red blood cells from experimental animals and human patients with NASH, present phosphatidylserine exposure which is then recognized by Kupffer cells. This event leads to erythrophagocytosis, and amplification of inflammation through iron disposition. In addition, it has been shown that erythrocytes from NASH patients release the chemokine MCP1, leading to increased TNF-α release from macrophages RAW 264.7. However, erythrophagocytosis can also be caused by reduced CD47 levels. In addition, increased MCP1 release could be either signal-induced, or caused by higher MCP1 levels on the erythrocyte membrane. Finally, erythrocyte efferocytosis could provide additional inflammatory metabolites. In this study, we measured the erythrocyte membrane levels of CD47 and MCP1 by ELISA, and cholesterol and sphingosine with thin-layer chromatography. 18 patients (8 men, 10 women aged 56.7+/-11.5 years) and 14 healthy controls (7 men, 7 women aged 39.3+/-15.5 years) participated in our study. The erythrocyte CD47 levels were decreased in the erythrocyte membranes of NASH patients (844+/-409 pg/ml) compared to healthy controls (2969+/-1936 pg/ml) with P(Healthy>NAFLD)=99.1%, while the levels of MCP1 were increased in NASH patients (389+/-255 pg/ml), compared to healthy controls (230+/-117 pg/ml) with P(Healthy<NAFLD)=88.9%. Moreover, in erythrocyte membranes there was a statistically significant accumulation of sphingosine and cholesterol in NASH patients, compared to healthy controls. Our results imply that erythrocytes release chemotactic (find me signals) MCP1, while containing reduced (do not eat me signals) CD47. These molecules can lead to erythrophagocytosis. Next, increased (goodbye signals) sphingosine and cholesterol could augment inflammation by metabolic reprogramming.

Biochemical and biophysical changes in plasma and erythrocyte membranes of alcohol consuming type 2 diabetics: A clinical study

Background and aims: Effects of alcohol consumption on blood glucose levels is unpredictable and more so with a known type 2 diabetic. Since type 2 diabetes is a chronic condition with impairment of glucose metabolism, influence of excess alcohol consumption in such a derailed metabolism is ought to be investigated. Our aim was to understand the interpolating relationship between the metabolisms of glucose and alcohol, by investigating the biochemical and biophysical changes in plasma and erythrocytes respectively. Methods We performed a clinical study with 20 human subjects wherein non-alcoholics, non-diabetics were considered as controls and the test subjects were categorized as alcoholics, diabetics and alcoholic diabetics. Findings were analysed against the control group. Results Increased plasma AST, ALT, ALP, and LDH enzyme activity; higher levels of nitric oxide, thiobarbituric acid reactive species (TBARS) both in plasma and erythrocyte lysate; higher fasting and postprandial glucose, glycated haemoglobin levels (Hb1Ac) levels; elevated levels of erythrocyte membrane total cholesterol / phospholipids (C/P) ratio and altered erythrocyte membrane fluidity in the alcoholic diabetics was noted. Conclusion Alcohol induced oxidative and nitrosative stress during its metabolism and its worsening effects in type II diabetics leading to a failure in the overall metabolic homeostasis is evident from the study.

Evaluation of erythrocyte membrane lipids and proteins in renal disorders

Background: Membrane lipids and proteins play a significant part in imparting membrane its rheological properties. These parameters are altered in diseased states. Exploring the conformational changes in renal disorders can widen our understanding of its impact on the circulatory system. This could lead to a new diagnostic parameter to study the progress of a disease.Methods: 120 blood samples collected from 30 kidney donors, 30 stage 3-4 Chronic kidney disease (CKD) patients (group 1) and 30 stage 5 CKD patients on dialysis (pre and post dialysis) (group 2) were lysed and washed to obtain erythrocyte ghost membranes. The proteins extracted from these membranes were estimated colorimetrically using Micro BCA kit. Phospholipids were separated and quantified using HPTLC. Fatty acids and cholesterol were analysed using GCMS.Results: The erythrocyte membrane protein profile showed lower values in group 2 participants than group 1 participants, but this difference was not significant. Distinct decreases in percentages of palmitic acid, myristic acid, stearic acid, dodecanoic acid, cholesterol, phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine were observed in both groups, with the lowest values in patients undergoing dialysis. Sphingomyelin and linoleic acid did not show any such trend across groups.Conclusions: The data is suggestive of an altered membrane structure in participants undergoing dialysis patients than the control group. This could be because of uremic toxins in the circulatory system affecting the membrane lipids. Decreased levels of essential phospholipids can impact the functions and lifespan of the erythrocytes. This could be a reason behind anaemia seen in most patients with CKD.

Atomic force microscopy in the assessment of erythrocyte membrane mechanical properties with exposure to various physicochemical agents

Background: Mechanical properties of cell membranes and their structural organization are considered among the most important biological parameters affecting the functional state of the cell. Under the influence of various pathogenic factors, erythrocyte membranes lose their elasticity. The resulting changes in their biomechanical characteristics is an important, but poorly studied topic. It is of interest to study the deformation of native erythrocytes to a depth compatible with their deformation in the bloodstream.Aim: To investigate the patterns of deep deformation and the particulars of structural organization of native erythrocyte membranes before and after their exposure to physicochemical agents in vitro.Materials and methods: Cell morphology, nanostructure characteristics, and membrane deformation of native erythrocytes in a solution of hemoconservative CPD/SAGM were studied with atomic force microscope NTEGRA Prima. Hemin, zinc ions (Zn2+), and ultraviolet (UV) radiation were used as modifiers. To characterize the membrane stiffness, we measured the force curves F(h), hHz (the depth to which the probe immersion is described by interaction with a homogeneous medium), and the Young's modulus values of the erythrocyte membrane.Results: Exposure to hemin, Zn2+ and UV radiation led to transformation of the cell shape, appearance of topological defects and changes in mechanical characteristics of erythrocyte membranes. Under exposure to hemin, Young's modulus increased from 10±4 kPa to 27.2±8.6 kPa (p<0.001), exposure to Zn2+, to 21.4±8.7 kPa (p=0.002), and UV, to 18.8±5.6 kPa (p=0.001). The hHz value was 815±210 nm for the control image and decreased under exposure to hemin to 420±80 nm (p<0.001), Zn2+, to 370±90 nm (p<0.001), and UV, to 614±120 nm (p=0.001).Conclusion: The results obtained contribute to a deeper understanding of interaction between membrane surfaces of native erythrocytes and small vessel walls. They can be useful in clinical medicine as additional characteristics for assessment of the quality of packed red blood cells, as well as serve as a basis for biophysical studies into the mechanisms of action of oxidative processes of various origins.

Erythrocyte Membrane-Enveloped Molybdenum Disulfide Nanodots for Biofilm Elimination on Implants Via Toxin Neutralization and Immune Modulation

Backgrounds: Implant-related infections (IRIs) caused by bacterial biofilms remain a prevalent but tricky clinical issue, which are characterized by drug resistance, toxin impairment and immunity suppression. Recently, antimicrobial therapies based on reactive oxygen species (ROS) or hyperthermia have been developed to effectively destroy biofilms. However, all of them have failed to simultaneously focus on the immunosuppressive microenvironment of biofilms and tissue damage caused by bacterial toxins. Results Herein, we proposed a one-arrow-three-hawks strategy to orchestrate hyperthermia/ROS antibiofilm therapy, toxin neutralization and immunomodulatory therapies through engineering a bioinspired erythrocyte membrane-enveloped molybdenum disulfide nanodots (EM@MoS2) nanoplatform. In biofilm microenvironment, pore-forming toxins actively attack the erythrocyte membranes on the nanodots and be detained, thus keeping toxins away from their targets and mitigating tissue damage. Under near-infrared laser irradiation, MoS2 nanodots, with superb photothermal and peroxidase-like properties, exert a powerful synergistic antibiofilm effect. More intriguingly, we initially identified that they possess the ability to reverse the immunosuppressive microenvironment through skewing the macrophages from an anti-inflammatory phenotype to a proinflammatory phenotype, which would promote the elimination of biofilm debris and prevention of infection relapse. Systematic in vitro and in vivo evaluations have demonstrated that EM@MoS2 achieves remarkable antibiofilm effect. Conclusion The current study integrated the functions of hyperthermia/ROS therapy, virulence clearance and immune regulation, which could provide an effective paradigm for IRIs therapy.

Identifying targets of protective antibodies against severe malaria in Papua, Indonesia using locally expressed domains of Plasmodium falciparum Erythrocyte Membrane Protein 1.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multi-domain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective immunity against malaria. Our group recently studied transcription of the var genes encoding PfEMP1 in individuals from Papua, Indonesia with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal components analysis, antibodies to three of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLβ13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults. Importance Severe Plasmodium falciparum malaria kills many African children, and lack of antibody immunity predisposes to severe disease. A critical antibody target is the P. falciparum erythrocyte membrane 1 (PfEMP1) family of multidomain proteins, which are expressed on the infected erythrocyte surface and mediate parasite sequestration in deep organs. We previously identified var genes encoding PfEMP1 that were differentially expressed between severe and uncomplicated malaria in Papua, Indonesia. Here, we have expressed domains from 32 of these PfEMP1s and measured IgG antibody responses to them in Papuan adults and children. Using Principal Component Analysis, IgG antibodies to three domains distinguished between severe and uncomplicated malaria and were higher in uncomplicated malaria. Domains included CIDRα1.6, implicated in severe malaria; a DBLβ13 domain; and a DBLδ domain of unknown function. Immunity to locally relevant PfEMP1 domains may protect from severe malaria. Targets of immunity show important overlap between Asian adults and African children.

Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

Effects of levamisole on haematological and oxidative stress parameters in packed donkeys

Stress can be caused by psychological, physiological, environmental and physical factors. Strenuous exercise like packing in donkeys modifies haematologic parameters. The aim of the study was to investigate the ameliorative effects of levamisole on stress, in packed donkeys. 15 adult male donkeys aged between 4 – 5 years were selected for this study, divided into groups (A, B, C) of five donkeys each: Groups A and B donkeys were the apparently healthy group; while Group C donkeys were naturally infected with Strongyle spp. All the donkeys participated in load carrying (packing) of 40 kg for 10 km. Groups B and C were treated with levamisole at 7.5 mg/kg, while Group A received no treatment prior to packing. Blood was collected from all the groups for haemogram and oxidative stress biomarker analyses. No significant effect (P > 0.05) was observed between groups: A, B and C for: respiratory rate, pulse rate and rectal temperature; haemogram, and activities of malondialdehyde concentration, superoxide dismutase and catalase. Erythrocyte membranes were osmotically stable at 0.5% NaCl in the treated group in comparison to the controls. Packing of donkeys for 10 km did not induce significant changes in vital parameters, haemogram and biomarkers of oxidative stress, but levamisole improved erythrocyte membrane stability. It was concluded that packing for 10 km did not induce any significant changes in blood cellular components and biomarker of oxidative stress, but levamisole improved erythrocyte membrane stability.

Differential expression of erythrocyte membrane protein band 4.2 in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding erythrocyte membrane protein band 4.2, EPB42, when comparing primary tumors of the breast to the tissue of origin, the normal breast. EPB42 mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of EPB42 in primary tumors of the breast was correlated with overall survival in patients with normal-like subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. EPB42 may be of relevance to initiation, maintenance or progression of cancers of the female breast.