White Blood Cell Membrane Research Articles

Ligand fishing via a monolithic column coated with white blood cell membranes: A useful technique for screening active compounds in Astractylodes lancea

The cell membrane-coated monolithic column (CMMC) ligand fishing assay is an interesting approach set up for the study of natural products (NPs). NPs such as Atractylodes lancea contain many compounds. Traditional methods used to separate compounds and determine active compounds by pharmacological tests are time-consuming and inefficient. Therefore, an alternative method is required to determine active compounds in NPs. Here, white blood cells were broken, and the white blood cell membranes (WBCMs) were immobilized on the surface of a monolithic column to form a CMMC. The column was characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and confocal laser scanning microscopy. Combined with gas chromatography/mass spectrometry (GC/MS), the CMMC was used to screen active compounds in Atractylodes lancea. Three potential active compounds including hinesol, β-eudesmol, and 4-phenylbenzaldehyde were discovered. A molecular docking assay demonstrated that these compounds could bind to MD-2 laid on WBCMs. In addition, antiinflammatory effects by the discovered compound in vitro were confirmed, and β-eudesmol showed a concentration-dependent inhibitory effect on the tumor necrosis factor (TNF)-α of a RAW264.7 cell (P < 0.05). The CMMC ligand fishing assay exhibits good selectivity, great speed effects and is a potentially reliable tool for drug discovery in NPs.

Use of Inert Gases for the Preservation of Nuclear Blood Cells

Abstract The subject of the study was the stability of human white blood cell membranes subject to noble gases (xenon ad krypton, 0.6 mPa) clathrate cryoanabiosis (‒80°C). A unique portable stainless steel low pressure container with a compartment for flexible plastic container was designed to ensure that the cells are saturated with gases. The samples were warmed after 1 and 30 days in a water bath (+38°C) for 35-50 sec, while the container was being tilted (2-3 times per second), until the temperature of the biological object reached +3±1°C. It was demonstrated that after 30 days of clathrate anabiosis (-80°C) over 95% (of the original number) of leukocytes remain viable, and cell membranes of 54.5±3.4% of them is resistant to trypan blue; granulocyte survival rate is 73.5±2.7%, original lipid peroxidation rate and antioxidant activity are retained. Biological object cryopreservation in noble gases environment is a promising trend in biology and medicine.

The Cryoprotectant Effect of Polysaccharides from Plants and Microalgae on Human White Blood Cells.

The use of carbohydrates as cryoprotectants is increasing. In this study the effects of incorporating polysaccharides extracted from plants and microalgae originating in northern Russia, into cryoprotectant solutions used to preserve human white blood cells were investigated. Cells in the presence of the polysaccharides were cooled to either -40°C or -80°C, using a two-step cooling process. The morphological and functional indicators of the cryopreserved leukocytes were assessed by light microscopy. When combined with glycerol, the pectin-polysaccharides Lemnan from common duckweed (Lemna minor L.) and Comaruman from marsh cinquefoil (Comarum palustre L), were capable of lowering the freezing point of the cryoprotectant solution and helped to preserve the integrity of the human white blood cell membranes at temperatures below zero. In addition, the increase in phagocytic activity of neutrophils was confirmed. In the context of the contemporary search for effective cell cryoprotectants, the results of this research demonstrate that the cryopreservation of biospecimens in a polysaccharide environment is a promising trend in applied medicine, which can be considered an alternative to traditional cryogenic nitrogen techniques.

White Blood Cell Signaling and Defense Mechanisms in Patients with Diabetes Mellitus Type 2 and Periodontitis

White blood cell membrane and surface structures are affected by the metabolic disorders and complications found in diabetes mellitus. Therefore, cellular activation, signal propagation, intracellular signaling as well as bactericidal effector functions are altered. Most likely diabetic symptoms can be corrected by the systemic intervention and treatment of the patients (Antidiabetic Therapy/ADT, i.e. anti-diabetic medication, diet and dietetic supervision, physiotherapy and physical exercises). We hypothesize that simultaneously blood cell functions will improve. Gum diseases like periodontitis have long been associated with and termed complications of uncontrolled diabetes mellitus. Vice versa, after diabetic conditions are corrected, periodontitis treatment will be proven effective, when oral hygiene regimen, full mouth decontamination (FD, i.e. the oral use of topical antiseptics prior and after professional mechanical tooth cleaning, tooth as well as root surface planing, polishing as well as gum and soft tissue decontamination in combination with systemic antibiotics) are performed. To reinforce gum healing, reinfection prevention (RP) as well as supportive periodontal therapy (SPT) will be administered by dental professionals on an individual basis and a detailed schedule. If periodontal pockets critical for participant's self care are not eliminated by FD including RP and SPT, and niches > 5mm after 6 month persist, patients are informed and offered surgical intervention as indicated for gum disease elimination.

Identification of squalamine in the plasma membrane of white blood cells in the sea lamprey, Petromyzon marinus

It is well established that innate mechanisms play an important role in the immunity of fish. Antimicrobial peptides have been isolated and characterized from several species of teleosts. Here, we report the isolation of an antimicrobial compound from the blood of bacterially challenged sea lamprey, Petromyzon marinus. An acetic acid extract from the blood cells of challenged fish was subjected to solid-phase extraction, cation-exchange chromatography, gel-filtration chromatography, and reverse-phase high-performance liquid chromatography, with the purified fractions assayed for antimicrobial activity. Surprisingly, antimicrobial activity in these fractions originated from squalamine, an aminosterol previously identified in the dogfish shark, Squalus acanthias. Further chromatographic and mass spectrometric analyses confirmed the identity of squalamine, an antimicrobial and antiangiogenic agent, in the active fraction from the sea lamprey blood cells. Immunocytochemical analysis localized squalamine to the plasma membrane of white blood cells. Therefore, we postulate that squalamine has an important role in the innate immunity that defends the lamprey against microbial invasion. The full biochemical and immunological roles of squalamine in the white blood cell membrane remain to be investigated.

Screening for the anti-inflammatory activity of fractions and compounds from Atractylodes macrocephala koidz

The aim of this study was to screen for the anti-inflammatory activity of fractions and compounds from Atractylodes macrocephala Koidz. The rhizomes of Atractylodes macrocephala were treated with supercritical CO 2 fluid and the extract was separated by normal-phase and reverse-phase column chromatography. The separated samples were screened with white blood cell membrane (WBCM) chromatography (WBCM-C). The anti-inflammatory effects of these fractions and components were tested pharmacologically in vivo. The results indicated that the retention characteristics of the petrol-ether (1:1, v/v) fraction (BZC-2) of the supercritical CO 2 extract, the atractylenolide I and 14-acetoxy-12-senecioyloxytetradeca-2 E,8 E,10 E-trien-4, 6-diyn-1-ol isolated from BZC-2 as active fractions and components were similar to that of dexamethasone in WBCM-C. Therefore, they may act on WBCM and its receptors. BZC-2 has shown anti-inflammatory effects in acute and chronic inflammation models in rats and mice. Oral administration of atractylenolide I and 14-acetoxy-12-senecioyloxytetradeca-2 E,8 E,10 E-trien-4,6-diyn-1-ol produced significant anti-inflammatory effects in acute and chronic inflammation models in mice. The screening results with WBCM-C were correlated significantly with pharmacological effects in vivo. Atractylenolide I and 14-acetoxy-12-senecioyloxytetradeca-2 E,8 E,10 E-trien-4,6-diyn-1-ol were the main components of Atractylodes macrocephala that were effective as anti-inflammatory agents.

Effect of Preoperative Immunonutrition on Body Composition in Patients Undergoing Abdominal Cancer Surgery

Preoperative immunonutrition may induce changes that modulate stress responses and improve the outcome of patients undergoing abdominal cancer surgery. We evaluated the effectiveness of preoperative immunonutrition using an immune-enhancing diet product called Impact. Forty patients aged 20-75 years, who were scheduled to undergo abdominal cancer surgery, were given Impact for 5 days preoperatively, at 1000 ml/day, in addition to a regular diet. We took various measurements before and after Impact administration. All but two patients tolerated a daily intake >900 ml (mean: 924 ml). The serum retinol-binding protein level increased from 3.21 to 3.76 mg/dl and the arginine level increased from 91.9 to 112.0 mmol/ml after Impact intake. The urinary excretion of uracil increased significantly, from 57.6 to 88.9 mmol/g creatinine, as did the content of n-3 fatty acids and the n-3/n-6 ratio in membrane phospholipids from the white blood cells. These changes were not observed in the two patients who did not tolerate Impact. There was no significant improvement in clinical outcome. Preoperative immunonutrition was well tolerated by cancer patients. It induced structural changes in the white blood cell membranes and increased the body store of arginine and nucleotides. These effects may modulate the response to surgical stress.

Establishment of the model of white blood cell membrane chromatography and screening of antagonizing TLR4 receptor component from Atractylodes macrocephala Koidz

A model of white blood cell membrane chromatography (WB-CMC) was established to screen active component from Atractylodes macrocephala Koidz. The component can antagonize Toll-like receptor 4 (TLR4) and inhibit inflammatory reaction. In the model of WB-CMC, cell membrane stationary phase (CMSP) was prepared by immobilizing the rabbit white blood cell membrane (WBCM) onto the surface of silica carrier and taxinol was used as a model molecule. The active component which can act on WBCM and its receptor (such as TLR4) as an effective target in A. macrocephala was determined by using a replacement experiment. The anti-inflammatory effects of the active component were tested by using pharmacological methods in vivo. The results indicated that the retention characteristics of atractylenolide I as active component was similar to that of taxinol in the model of WB-CMC. And so, atractylenolide I acted on the WBCM and TLR4 and its anti-inflammatory activity was related with antagonizing TLR4. Therefore, the interaction between the active component and WBCM and its receptor can be simulated by the model of WB-CMC in vitro. This model can be used to screen active components and to study effective characteristics for acting on definite targets.

Mechanics of leukocyte deformation and adhesion to endothelium in shear flow.

The mechanics of leukocyte [white blood cell (WBC)] deformation and adhesion to endothelial cells (EC) in shear flow has been investigated. Experimental data on transient WBC-EC adhesion were obtained from in vivo measurements. Microscopic images of WBC-EC contact during incipient WBC rolling revealed that for a given wall shear stress, the contact area increases with time as new bonds are formed at the leading edge, and then decreases with time as the trailing edge of the WBC membrane peels away from the EC. A two-dimensional model (2D) was developed consisting of an elastic ring adhered to a surface under fluid stresses. This ring represents an actin-rich WBC cortical layer and contains an incompressible fluid as the cell interior. All molecular bonds are modeled as elastic springs distributed in the WBC-EC contact region. Variations of the proportionality between wall shear stress (tau(w)) in the vicinity of the WBC and the resulting drag force (F(s)), i.e., F(s)/tau(w), reveal its decrease with WBC deformation and increasing vessel channel height (2D). The computations also find that the peeling zone between adherent WBC and EC may account for less than 5% of the total contact interface. Computational studies describe the WBC-EC adhesion and the extent of WBC deformation during the adhesive process.

Image enhancement of the in vivo leukocyte-endothelium contact zone using optical sectioning microscopy.

A major determinant of the strength of leukocyte [white blood cell (WBC)] to endothelium [endothelial cell (EC)] adhesion is the contact area formed between the two cells, which is often obscured by out-of-focus information inherent to intravital microscopy. To improve visualization of the WBC-EC contact zone, techniques of optical sectioning microscopy were developed to enhance brightfield images of WBC-EC adhesion in postcapillary venules of the mesentery of the rat. A 50x/1.0 NA objective was held in a piezoelectric mount that was computer-driven, and video images were obtained by digitizing images from a CCD camera while focusing through the vertical direction in 1 micron steps over a depth of 16 microns. Using measurements of the microscope's optical transfer function, deconvolution of the central image was performed in the Fourier domain using the technique of singular value decomposition with Tikhonov-Miller regulation to remove out-of-focus information. Measurement of the length of the WBC-EC contact zone (LC) in the original images yielded values on the order of 4.32 +/- 1.08 microns (mean +/- SD). The enhanced images showed a significantly 35% smaller LC equal to 2.78 +/- 0.70 micron. Topical application of the chemoattractant f-met-leu-phe resulted in a 26% increase in LC to 3.49 +/- 0.72 micron, thus suggesting that upregulation of adhesion molecules on the WBC membrane results in the recruitment of additional membrane area from surface ruffles into the zone of adhesion. Other advantages of the deconvolution were to visualize structural characteristics of the microvascular wall and parenchymal tissue in greater detail. Thus, brightfield optical sectioning microscopy may provide a valuable tool for in vivo studies of the microvasculature, and serves as a useful alternative to fluorescence microscopy without the undesirable effects of exogenous fluorophores and exposure to ultraviolet radiation.

Effects of cytokines on the expression of cell adhesion molecules by cultured human omental mesothelial cells.

Cultured mesothelial cells (HOMES) are very responsive to the proinflammatory cytokines, interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-alpha). E-selectin, ICAM-1 and VCAM-1 are known to play an important role, because they are presented by diverse cell types, for example endothelial cells (ECs), and interact with co-responding ligands on white blood cell membranes. In this study, the expression of ICAM-1, VCAM-1, E-selectin as well as PECAM-1 on cultured HOMES was studied over 5, 24, 48 and 72 h exposure to IL-1 beta, interferon-gamma and TNF-alpha. In previous studies we have shown that IL-1 beta and TNF-alpha increase the expression of ICAM-1, E-selectin and VCAM-1 on the cytoplasmatic membranes of HUVECs, HSVECs and HAFECs (ECs from human umbilical vein, saphenous vein and femoral artery, respectively). Using a comparative quantitative cell enzyme immunoassay, we found that expression of the adhesion molecules ICAM-1 and VCAM-1 was significantly increased on HOMES in a dose- and time-dependent manner, compared to nonstimulated cells. Thus, ICAM-1 increased dramatically after 5 h incubation with TNF-alpha. Values of about 450% of the control level were measured. VCAM-1 was similarly stimulated after 24 h incubation with the same cytokine, although its level of expression was significantly lower than that of ICAM-1. In contrast to findings in the literature, VCAM-1 was not found to be expressed constitutively. E-selectin was neither constitutively expressed nor markedly inducible on HOMES. Only weak expression was found after 24 h incubation with high-dose IL-1 beta. PECAM-1 was expressed constitutively, as became evident in antibody dilution studies. These data indicate that HOMES respond to inflammatory stimuli, in some ways in a similar fashion to vascular endothelial cells, but also show a specific pattern of antigen presentation. The results are important for a better understanding of inflammatory processes in serous cavities. The data are also relevant for the improvement of antithrombogenous surfaces of the lumina of vascular prostheses by cell seeding.

Comparative Studies on Vascular Endothelium in vitro

Endothelial cells (EC) are very responsive to proinflammatory cytokines, e.g. interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha), as well as to bacterial lipopolysaccharide. EC are stimulated by these substances to secrete chemotactic factors and to increase expression of cell adhesion molecules (CAM), leading to dramatically altered interactions with leukocytes, e.g. granulocytes and monocytes. In these interactions E-selectin, ICAM-1 and VCAM-1 are known to play an important role, as they are presented by the EC and interact with corresponding ligands on the white blood cell membranes. These adhesion molecules have been studied worldwide in a variety of in vitro experiments using cultured EC. Different passages and mixtures of passages have been used in these experiments, often without any regard to the comparability of the results. In this study the expression of E-selectin, ICAM-1 and VCAM-1 on cultured human umbilical vein EC (HUVEC) obtained from different passages (passages 1-6) was studied after 4, 8 and 24 h of exposure to IL-1 beta and TNF alpha. In previous studies, we have shown that IL-1 beta and TNF alpha increase the expression of E-selectin and ICAM-1 on the cytoplasmatic membranes of HUVEC and human adult EC from the saphenous vein and femoral artery in a similar fashion. Using a comparative quantitative cell enzyme immunoassay, we found that the expression of the adhesion molecules was significantly reduced with increasing passages. There was also a decreased persistence of CAM comparing different periods of stimulation between 6 and 24 h in the different passages. These data indicate that the number of passages plays an important role in the expression of adhesion molecules on EC. The results are relevant for the meaningful planning of comparative in vitro studies on EC presentation of CAM.

Spontaneous allergic autocytotoxicity in bronchial asthma associated with food allergy

Spontaneous allergic autocytotoxicity of white blood cells was assessed in six patients with bronchial asthma and 18 normal control volunteers. The observed alterations in non-primed white blood cell membrane were revealed as an increased uptake of trypan blue exclusion dye, an indicator of cell death. The phenomenon of spontaneous allergic autocytotoxicity might be associated with increased permeability of the white blood cell membrane leading to enhanced releasability of chemical mediators of anaphylaxis, which probably bypasses immunoglobulin E mechanisms and T suppressor cell intervention. Of six patients with bronchial asthma, three were sensitive to wheat, two had cows milk sensitivity, and one had corn sensitivity. When white blood cells of these patients were studied in the direct allergic autocytotoxicity assay, augmentation of spontaneous allergic autocytotoxicity by specific food antigens was observed. Two of the patients were professional sports coaches. It is plausible to suggest that increased membrane permeability, presumably indicating increased releasability (i.e., for histamine), may be associated with a low membrane threshold for physical trauma expressed in exercise-induced anaphylaxis syndromes.