Biophysical Properties and Motility of Human Mature Dendritic Cells Deteriorated by Vascular Endothelial Growth Factor through Cytoskeleton Remodeling.

Dendritic cells (DCs), the most potent antigen-presenting cells, play a central role in the initiation, regulation, and maintenance of the immune responses. Vascular endothelial growth factor (VEGF) is one of the important cytokines in the tumor microenvironment (TME) and can inhibit the differentiation and functional maturation of DCs. To elucidate the potential mechanisms of DC dysfunction induced by VEGF, the effects of VEGF on the biophysical characteristics and motility of human mature DCs (mDCs) were investigated. The results showed that VEGF had a negative influence on the biophysical properties, including electrophoretic mobility, osmotic fragility, viscoelasticity, and transmigration. Further cytoskeleton structure analysis by confocal microscope and gene expression profile analyses by gene microarray and real-time PCR indicated that the abnormal remodeling of F-actin cytoskeleton may be the main reason for the deterioration of biophysical properties, motility, and stimulatory capability of VEGF-treated mDCs. This is significant for understanding the biological behavior of DCs and the immune escape mechanism of tumors. Simultaneously, the therapeutic efficacies may be improved by blocking the signaling pathway of VEGF in an appropriate manner before the deployment of DC-based vaccinations against tumors.

Investigations of the functional states of dendritic cells under different conditioned microenvironments by Fourier transformed infrared spectroscopy.

BACKGROUND: Dendritic cells are potent and specialized antigen presenting cells, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses. The dendritic cell-based vaccination against cancer has been clinically achieved promising successes. But there are still many challenges in its clinical application, especially for how to identify the functional states. METHODS: The CD14+ monocytes were isolated from human peripheral blood after plastic adherence and purified to approximately 98% with cocktail immunomagnetic beads. The immature dendritic cells and mature dendritic cells were induced by traditional protocols. The resulting dendritic cells were cocultured with normal cells and cancer cells. The functional state of dendritic cells including immature dendritic cells (imDCs) and mature dendritic cells (mDCs) under different conditioned microenvironments were investigated by Fourier transformed infrared spectroscopy (FTIR) and molecular biological methods. RESULTS: The results of Fourier transformed infrared spectroscopy showed that the gene transcription activity and energy states of dendritic cells were specifically suppressed by tumor cells (P < 0.05 or 0.01). The expression levels of NF-kappa B (NF-κB) in dendritic cells were also specifically inhibited by tumor-derived factors (P < 0.05 or 0.01). Moreover, the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were closely correlated with the expression levels of NF-κB (R2:0.69 and R2:0.81, respectively). CONCLUSION: Our results confirmed that the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were positively correlated with the expression levels of NF-κB, suggesting that Fourier transformed infrared spectroscopy technology could be clinically applied to identify the functional states of dendritic cell when performing dendritic cell-based vaccination. It's significant for the simplification and standardization of dendritic cell-based vaccination clinical preparation protocols.

[The capping protein on the slow-growing end of actin filament: erythrocyte tropomodulin].

Erythrocyte tropomodulin (E-Tmod) is first isolated from human erythrocyte membrane as a TM-binding protein. Its N-terminus contains two TM-binding sites and one TM-dependent actin capping domain and C-terminus contains 5 leucine-rich repeats and a TM-independent actin capping domain. As the unique capping protein at the slow-growing end of F-actin, E-Tmod binds to N-terminus of TM and actin and decreases the TM-coated F-actin depolymerization. E-Tmod encoding gene is highly conserved and E-Tmod is distributed widely in erythrocytes and cardiomyocytes, etc. E-Tmod plays a pivotal role in organizing F-actin and cytoskeleton and maintaining the mechanical properties of the cells.

Improving abnormal hemorheological parameters in ApoE-/- mice by Ilex kudingcha total saponins.

The aim of this study was to test the effects of Ilex kudingcha total saponins on hemorheology of ApoE-/- mice suffering from hypercholesterolemia induced by high-cholesterol diet. The mice were randomly divided into six groups: the control group, the high-cholesterol diet group, 50 mg/kg atorvastatin treatment group, 75, 150 and 300 mg/kg Ilex kudingcha saponins treatment groups, and all the drug treatment groups were fed with a high-cholesterol diet. After administration with saponins (150 mg/kg or more) and atorvastatin (50 mg/kg) for six weeks, the plasma total cholesterol (TC), whole blood viscosity (WBV), plasma viscosity (PV), and erythrocyte aggregation index (EAI) had a remarkable decrease compared with that of the high-cholesterol diet group, but the hematocrit (Hct) and erythrocyte deformation index (DI) had no significant changes. In addition, it is found that the improving effects of saponins on reducing plasma fibrinogen (Fg) levels and prolonging the blood coagulation times including activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT). In conclusion, the Ilex kudingcha total saponins may have a significant therapy application of hypercholesterolemia and atherosclerosis by considering its actions on hemorheological characteristics.

Effects of myakuryu on hemorheological characteristics and mesenteric microcirculation of rats fed with a high-fat diet.

There is evidence that hyperlipidemia can induce hemorheological and microcirculatory disturbances. Myakuryu, a Chinese traditional medicine is efficacious in promoting lipid metabolism and protecting oxidative stress, but whether this drug can ameliorate rheologic disturbances caused by hyperlipidemia is still unknown. The present study was conducted to investigate the effects of myakuryu on hemorheological and microcirculatory disturbances induced by hyperlipidemia. Wistar rats were divided into a group on control diet (n=8) and a group on high-fat diet (HFD, n=44). Eight weeks later, plasma triglyceride (TG) and total cholesterol (TC) were determined. Sixteen animals with the highest levels of hyperlipidemia from the HFD group were randomly divided into two sub-groups: the untreated hyperlipidemia group (n=8) and the group treated with myakuryu (n=8). At the end of the sixteenth week, rheological and microcirculatory parameters were measured. Chemical analysis showed that myakuryu treatment caused significant reductions of plasma TG and TC levels (P<0.01), and the cholesterol/phospholipid ratio in the erythrocyte membrane (P<0.05). Rheological and microcirculatory measurements showed that myakuryu treatment led to a significant decrease in the erythrocyte aggregation index, plasma viscosity and blood viscosity at shear rates of 50, 100 and 150 s(-1) and in adherent leukocytes in mesenteric venules. There was a significant increase in erythrocyte deformation, electrophoretic mobility, membrane fluidity and F-actin content in the erythrocyte membrane as well as in red cell velocity in mesenteric venules. Our findings suggest that myakuryu treatment can improve blood flow and reduce adherent leukocytes in the venules of rats fed with HFD by ameliorating blood viscosity, erythrocyte deformability and aggregation, and other hemorheological characteristics.

Effects of cardiotonic pill on RBC rheologic abnormalities in HFD-induced mice and LPL deficient mice.

The lipoprotein lipase deficient (LPL-/-) mice and high fat-diet (HFD) induced hypertriglyceridemic mice were used to investigate the effects of cardiotonic pill (CP) on RBC rheologic abnormalities. Mice were randomly divided into the following five groups: the control group; the untreated HFD group; the untreated LPL-/- group; the treated HFD group; and the treated LPL-/- group, and the treated HFD and LPL-/- mice were administered with CP twice a day (400 mg/kg/day) orally for four weeks. Then, plasma triglyceride (TG), RBC deformation index (DI), orientation index (DI)or and RBC electrophoretic time (EPT) were measured. Compared with the untreated HFD mice, TG level and EPT reduced and DI and (DI)or increased markedly in the treated HFD mice (P<0.05). However, compared with the untreated LPL-/- mice, these parameters in the treated LPL-/- mice had no statistically significant changes (P>0.05). Our data show that CP can lower plasma TG level and ameliorate RBC rheologic abnormalities in the HFD-induced hypertriglyceridemic mice, but it losses its capacity in the LPL deficient animals. The results indicate that LPL may be one of the important targets for CP regulating lipometabolism and rheologic abnormalities.

The measurement of shear modulus and membrane surface viscosity of RBC membrane with Ektacytometry: a new technique.

A new technique is proposed to estimate the shear modulus (mu) and membrane surface viscosity (eta(m)) of red blood cell (RBC). Theoretical formulae for finding these two parameters are first derived based on the force balance on a RBC in a flow field of low viscosity. Different types of Ektacytometry are then used to measure relevant quantities. The obtained values (mu=6.1 x 10(-6)N/m, eta(m)=8.8 x10 (-7)Ns/m for normal RBC) are consistent with those previously found by micropipette technique and in AC electric field. The present technique is, however, much easier to operate and more advantageous in reflecting the average properties of a large quantity of RBCs, and it is much cheaper to be applied in clinical practice than any other method of measuring the two parameters. The sensitivity of the technique is demonstrated by testing RBCs treated with glutaraldehyde of different concentrations. This technique was demonstrated by the flow chamber.

Synergistic effect of cytokines EPO, IL-3 and SCF on the proliferation, differentiation and apoptosis of erythroid progenitor cells.

Erythroblasts were obtained from murine spleen. After cultured for 12 hr, the cells were divided into four groups with the use of the following cytokines in culture: EPO, EPO+SCF, EPO+IL-3, and EPO+IL-3+SCF. Cell proliferation assay was done. Apoptosis rates were obtained by using a flow cytometer. Mitochondrial membrane potential (MMP) was assessed in flow cytometry (FCM) by labeling with rhodamine 123. Mitochondrial enzyme activity (MEA) was evaluated with MTT colorimetric assay. The cells were labeled with Fluo-3/Am Ester and Ca(2+) concentration was measured. The expression of Bax mRNA and Bcl-2 mRNA was analyzed by RT-PCR. At same time, the expression of Bax and Bcl-2 was analyzed by western blotting. Our results showed that IL-3 and SCF have synergistic effects with EPO on the proliferation, differentiation and apoptosis of erythroid progenitors.

Effects of TFAR19 gene on the in vivo biorheological properties and pathogenicity of mouse erythroleukemia cell line MEL.

After injecting VP16, MEL cells and MEL-TF19 cells into the body of mice, with those injected with the same dose of saline as the control group, we observed the mice for their blood pictures, histological changes of the liver and spleen, and the hemorheological indexes within 4 weeks. The results indicated that after injecting MEL cells, the mice entered into a pathological status similar to erythroleukemia, which had the following exhibitions: the tissue structures of the liver and spleen were damaged, a mass of proerythroblasts, basophil erythroblasts and polychromatophilic erythroblasts could be observed on the smears of the bone marrow and spleen, and the deformability and orientation ability of erythrocytes were both depressed. The pathogenicity of MEL-TF19 cells carrying TFAR19 gene was obviously lower than that of MEL cells, and the MEL-TF19 cells even lost their faintish pathogenicity under the apoptosis-inducing effect of the chemotherapeutic reagent. The outcome from the animal experiments suggests that the TFAR19 gene suppresses the pathogenicity of MEL cells to the mice, and the effect may be better exerted with the synergy of the chemotherapeutic reagent.

Biochemical and biophysical studies on the precursor cells of mouse erythrocytes at different stages.

The aim of this research is to study the biochemical and biophysical properties of the precursor cells of mouse erythrocytes at different stages and the molecular mechanisms of their regulation. We investigated the degree of terminal differentiation of splenic erythroblasts obtained from mice during the acute phase of disease caused by the anemia-inducing FVAstrain of Friend virus. We analyzed the transcription and protein levels of alpha-globin, beta-globin (erythroid special protein) and GATA-1 (a special erythroid transcription factor). We also have examined the Ca2+ concentration, the distribution and amount of F-actin, important cellular components such as nucleic acids, lipids, and proteins, and the adhesion of precursor cells of RBC at different stages to vascular endothelium. Our results indicated that Ca2+ concentration and the distribution and structure of F-actin changed with the development of proerythroblasts, and that the adhesion rate between the precursor cells and endothelial cells can be correlated with the expression levels of ICAM-1 and P-selectin. These alternations caused changes in biophysical properties of the cell, such as membrane fluidity and deformability.

Biophysical studies on the differentiation of human CD14+ monocytes into dendritic cells.

Dendritic cells (DCs), which are the most efficient antigen-presenting cells (APCs) currently known, can be derived from CD14+ monocytes (DC predecessor cells) in vitro. Immature DCs actively take up antigens and pathogens, generate major histocompatability complex-peptide complexes, and migrate from the sites of antigen acquisition to secondary lymphoid organs to become mature dendritic cells that interact with and stimulate T-lymphocytes. During this process, the cells must undergo deformation to translocate through several barriers, including the basement membrane and interstitial connective tissue in the blood vessel wall. To further understand the mechanisms of the activation of immunological responses and the migration from peripheral tissue to secondary lymphoid organs, we have applied biophysical and microrheological methods to study the development processes of DCs in vitro. The results showed that membrane fluidity, osmotic fragility, membrane viscoelastic properties, infrared spectroscopy, and cytoskeleton organization of DCs exhibit significant differences in different developmental stages.

Studies on the biomechanical properties of maturing reticulocytes.

We investigated the biomechanical properties of reticulocytes obtained from an animal model of hemolytic anemia induced by antibody injection. The hemorheological indices, membrane viscoelasticity, membrane fluidity, and the secondary structure of membrane proteins of the reticulocytes were monitored continuously during the course of their maturation into erythrocytes. The results indicate that reticulocytes had lower deformability, lower membrane fluidity, greater viscoelastic modulus and lesser proportions of alpha-helices and beta-sheets in protein secondary structures than mature erythrocytes. All these indices approached to the level of normal erythrocytes when reticulocytes transformed during maturation. The results help to enhance our understanding of the biomechanical properties of the reticulocytes in their maturing process with clinical diagnosis significances.

[On the biophysics characteristics of reticulocytes].

This paper reports an in vivo study on the biophysics characteristics of reticulocytes. Anemia was induced by injection of phenylhydrazine in rabbits. The measurements, including electrophoresis rate, hematolytic rate, fluorescent polarization and the changing anisotropic value, were performed in vivo for 72 hours in the process of reticulocytes growing into erythrocytes. It was shown that there were obvious changes in the biophysics characteristics of reticulocytes in this course. Therefore, the findings are of significance to basic, theoretical and clinical studies.

TFAR19 gene changes the biophysical properties of murine erythroleukemia cells.

TFAR19 is a novel apoptosis-related gene and can accelerate cell apoptosis in the presence of apoptosis inducements. Here, we studied the effects of TFAR19 on some biophysical properties of mouse erythroleukemia (MEL) cells and their molecular and structural basis. After transfected with TFAR19 and apoptosis inducement, MEL revealed a high cell membrane fluidity, a decrease in resynthesis of phospholipids, an increase in the proteins/nucleic acids ratio, a relatively orderly cytoskeleton network, an impaired deformability, a low integrin aM expression, and a decrease in adhesion to endothelial cells. These findings suggest the potential of TFAR19 for antitumor cell migration, and thus for antitumor gene therapy.

Adhesion of monocyte-derived dendritic cells to human umbilical vein endothelial cells in flow field decreases upon maturation.

Dendritic cells (DC) are sentinels of the immune system. They and their precursors undergo complex migration to perform their function in vivo. Binding of DC to vascular endothelial cells in a flow field has not been investigated. We therefore determined adhesion of DC and their precursors, MO, to human umbilical vein endothelial cells (HUVECs) under various shear stresses by using a flow chamber system. The results showed that the binding was reduced with developmental stages of DC, which partially depended on CD11a and cell surface charges. The data had potential relevance for anti-cancer immunotherapy strategies favoring the application of mDC.

Alterations in hemorheological properties of erythrocytes in nude mice with erythroleukemia and the treatment effects of etoposide.

The purpose of this study was to examine the changes of hemorheological properties of erythrocytes in the nude mice with erythroleukemia and the treatment effects of etoposide (VP16). Thirty mice were randomly divided into three groups: the control group (C group), injected with 1 ml saline solution, the MEL group (M group) injected with 1 ml MEL (murine erythroleukemia cell line) and the MEL + VP16 group (V group) injected with 1 ml MEL and from the 8th day after injection, 20 microl VP16 (1 microg/microl) was injected intraperitoneally every five days. One week after MEL injection, erythroblastic cells increased in the bone marrow and proerythroblasts were found in the peripheral blood, suggesting that erythroleukemia was induced. Abnormalities were also found in spleens and livers later. At around twenty days after injection, the mice in M group died and about four weeks after injection, the mice in V group also died. Compared with C group, the hemorheological indexes [the deformation index DI, orientation index (DI(or)), and the small deformation index (DI(d))], electrophoretic mobility, membrane fluidity as well as osmotic fragility of red blood cells (RBC) in M and V groups changed significantly. But after VP16 administration, the changes of above parameters in V group were less significant than those of M group. The results above suggested that intraperitoneal injection of MEL cells could cause erythroleukemia in nude mice, VP16 could alleviate the erythroleukemia symptom and improve the hemorheological properties, and could prolong V group nude mice survival.

Effects of Compound Dan-shen Root Dropping Pill on hemorheology in high-fat diet induced hyperlipidemia in dogs.

The purpose of this study was to examine the effects of Compound Dan-shen Root Dropping Pill (CDRDP) (Tasly Group, Tianjing, China) on hemorheology and biorheology of dogs suffering from hyperlipidemia induced by high-fat diet. Eighteen dogs were randomly divided into two groups: the high-fat diet group (H group); the control group (C group), fed with a standard laboratory diet. Six month later, six dogs in the H group were chosen as the drug-taking group (D group), to which CDRDP was administered, fed with the same diet as H group. In the 4th month, blood was taken from the veins of the dogs, and blood triglyceride (TG), total cholesterol (TC), RBC hemorheological indexes as well as malondialdehyde (MDA), glutathione transferase (GSH-ST) and superoxide dismutase (SOD) activities in plasma and erythrocytes were measured. Compared with H group, TC, TG, plasma MDA levels, the whole blood viscosity, RBC osmotic fragility and the value of CHOL (cholesterol)/PL (phospholipid) of the membrane of D group decreased, however, erythrocyte GSH-ST, histopathological changes in liver, deformation index (DI), orientation index (DI)or, small deformation index (DI)d, electrophoresis ratio and microfluidity of the membrane lipid bilayer of RBCs, increased distinctly. CDRDP can improve micro-hemorheological characteristics, therefore has a significant therapy application of hyperlipidemia.

The biomechanical alterations in the CD14+ monocytes of patients with living donor renal transplantation.

Living-donor renal transplantation is an ideal treatment for patients with end stage renal disease because it affords earlier transplantation and better graft for long term survival. CD14+ monocytes are the predominant inflammatory cells in renal allograft intimal arteritis. The biomechanical alterations in CD14+ monocytes would affect the function of graft. The aim of the present study was to explore the changes in the biorheological properties of CD14+ monocytes before and after the living donor renal transplantation. We found that the viscoelastic properties of CD14+ monocytes were greatly decreased after renal transplantation. Confocal microscopy showed that the F-actin content was increased when the oral immunosuppressive agents started. We also found that two cytoskeletal regulatory proteins, cofilin1 and profilin1, changed. Our results suggest that the immunosuppressive agents could significantly change the biorheological characteristics of the CD14+ mononuclear cells and the biomechanical changes may greatly affects their function, which would play a critical role to gain longer immune-tolerance stage.

Influence of TRAIL gene on biomechanical properties of the human leukemic cell line Jurkat.

We cloned the cDNA fragment of human TNF-related apoptosis inducing ligand (TRAIL) into RevTet-On, a Tet-regulated and high-level gene expression system. Making use of the TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the biomechanics properties of Jurkat through measuring changes of cellular biomechanics properties before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the TRAIL gene expression led to significant changes in cellular biomechanics properties. The osmotic fragility increased and the cell stiffness increased after the expression of TRAIL gene. Thus, the apoptosis-inducing TRAIL gene caused significant changes in the biomechanics properties of Jurkat cells.

Rheological studies on precursor cells at different stages in mice.

The deformability and membrane fluidity of the precursor cells for mice at different growth stages were studied; and it was found that the deformability and membrane fluidity were increased with the growth of the precursor cells. It is demonstrated that the normoblast was just placed in a turning point in the growth stage of the precursor cells. The changes are associated with the smaller the nucleus, the smaller the ratio of nucleus and cytoplasm, and the expression of membrane skeleton protein, and so on. It is also found that the order parameter (S) obtained by ESR is negatively correlated with the small deformation index (DI)(d,max) obtained by LBY-BX2 ektacytometer, while the value of R2 (square of correlation coefficient) is 0.9816. Such conclusion shows that the order parameter (S) is basically in accordance with the small deformation index (DI)(d,max).