A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells

PubMed Central

Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang

2017-01-01

Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P < 0.05). There was no significant difference in islet viability among the four groups. The islet purity, function, yield, and cost of our method were superior to those of the Ficoll-400 and 1077 methods, but inferior to the handpicking method. However, the handpicking method may cause wrist injury and visual impairment in researchers during large-scale islet isolation (>1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized. PMID:28207765

A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

PubMed

Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang; Baifeng, Li

2017-01-01

Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P < 0.05). There was no significant difference in islet viability among the four groups. The islet purity, function, yield, and cost of our method were superior to those of the Ficoll-400 and 1077 methods, but inferior to the handpicking method. However, the handpicking method may cause wrist injury and visual impairment in researchers during large-scale islet isolation (>1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

Immunopotentiation by SGP and Quil A. II. Identification of responding cell populations.

PubMed

Flebbe, L M; Braley-Mullen, H

1986-04-15

The adjuvants SGP (a starch-acrylamide polymer) and Quil A (purified saponin) were shown to markedly augment antibody responses to T-independent (TI) antigens, suggesting that their adjuvant effects may be at least partially mediated through B cells. The ability of both adjuvants to augment primary responses to trinitrophenyl (TNP)-Ficoll (TI-2 antigen) in athymic nude mice further suggested these adjuvants affect B cells. SGP, however, did not induce a response to the T-dependent (TD) antigen dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) in athymic nude mice, indicating it was unable to replace the requirement for T-helper cells for responses to TD antigens. Responses to TNP-lipopolysaccharide (LPS) were augmented by SGP in CBA/N X Balb/c immune defective (xid) mice. However, SGP was unable to induce a response to TNP-Ficoll in xid mice. The SGP and Quil A augmented responses to TNP-Ficoll were completely inhibited by the mitotic inhibitor, Velban, indicating that SGP and Quil A increased the plaque-forming cell (PFC) response primarily by stimulating cell proliferation, and not by recruitment of antigen-reactive cells. The effects of the adjuvants on secondary responses were investigated using adoptive transfer experiments. SGP and A1(OH)3 both increased the induction of hapten-specific memory B cells in mice primed with DNP-KLH. SGP, Quil A, and A1(OH)3 also increased priming of carrier specific T cells. Priming of memory B cells with DNP-KLH and either A1(OH)3 or SGP was prevented when T cells were depleted with anti-lymphocyte serum (ALS) at the time of antigen priming, indicating that the augmentation of memory B-cell priming by SGP and A1(OH)3 was dependent on the presence of functional T cells. SGP and Quil A were both unable to augment memory cell induction to the TI antigen, TNP-Ficoll, even though both adjuvants markedly augmented primary IgM and IgG responses to this antigen. Based on these results, it is suggested that SGP and Quil A can mediate their adjuvant effects primarily by a direct or indirect effect on B cells although the adjuvants may also affect T cells to some extent.

Technique for obtaining highly enriched, quiescent immature Langerhans cells suitable for ex vivo assays.

PubMed

Tchou, Isabelle; Sabido, Odile; Lambert, Claude; Misery, Laurent; Garraud, Olivier; Genin, Christian

2003-03-03

Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a. These cells can capture a pathogen and then migrate and differentiate to a more mature stage. During this maturation process, dentritic cells express surface markers differentially. In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells. For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery. This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage. After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process. Both systems were equally efficient in terms of purification and yield. By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a(+) LCs, contrary to the repeated Ficoll floatation. This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells. Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.

[Establishment and evaluation of an in vitro method for neutrophil extracellular trap generation and degradation].

PubMed

Li, Jinlong; Zhang, Yidan; Zhou, Xin; Ji, Wenjie; Zhao, Jihong; Wei, Luqing; Li, Yuming

2014-09-01

To evaluate a novel method for in vitro generation and degradation of neutrophil extracellular traps (NETs), which are a newly recognized structure that is involved in the pathogenesis of autoimmune diseases and thrombosis. Neutrophils from peripheral blood of healthy donors were obtained by Ficoll-Histopaque gradient separation. NET release was initiated by phorbol myristate acetate (PMA) and validated by immunofluorescence staining and agarose gel electrophoresis. NETs degraded by DNase I and healthy human plasma were quantified by fluorescence spectrometry after staining with PicoGreen. HE staining showed that the purity of neutrophils was up to 95% after Ficoll-Histopaque gradient separation. NET immunofluorescent staining revealed that the network structure was mainly composed of DNA and histones, with molecular length more than 10 kb as demonstrated by agarose gel electrophoresis. Moreover, both DNase and healthy human plasma could induce the degradation of NETs, in varying degrees. This work established an efficient method for in vitro generation and degradation of human NETs.

Immunoregulatory effects of covalent antigen-antibody complexes. III. Enhancement or suppression depending on the time of administration of complex relative to a T-independent antigen.

PubMed Central

Tite, J P; Morrison, C A; Taylor, R B

1981-01-01

The photosensitive affinity label NAP (4-azido-2-nitrophenyl) was used to make a stable covalent-bonded monomeric immune complex (Ag2Ab) between rabbit anti-NAP antibody and a bihaptenic compound containing NAP linked to fluorescein (NAP-aminocaproyl-lysyl-Fl). This complex injected into mice had marked effects on their subsequent response to fluorescein coupled to a thymus-independent carrier (Fl-ficoll). Depending on the time at which the complex was administered relative to challenge, it was possible to obtain either enhancing or suppressive effects. The enhancing but not the suppressive effect of complex was dependent on immune recognition of the rabbit IgG carrier. While the suppressive effect probably results from complex-mediated inactivation of T-independent B cells, it is suggested that the enhancing effect results from priming of the T-dependent B cells by Fl-Ficoll followed by their triggering into antibody production by rabbit IgG-specific helper cells. PMID:7007223

Isoelectric focusing of red blood cells in a density gradient stabilized column

NASA Technical Reports Server (NTRS)

Smolka, A. J. K.; Miller, T. Y.

1980-01-01

The effects of Ficoll and cell application pH on red blood cell electrophoretic mobility and focusing pH were investigated by focusing cells in a density gradient stabilized column. Sample loading, cell dispersion, column conductivity, resolution of separation, and the effect of Ampholines were examined.

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization.

PubMed

Niu, Hui-Ran; Zi, Xiang-Dong; Xiao, Xiao; Xiong, Xian-Rong; Zhong, Jin-Cheng; Li, Jian; Wang, Li; Wang, Yong

2014-02-01

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6-42.2%) and blastocyst formation (2.9-8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws. Copyright © 2014 Elsevier Inc. All rights reserved.

Translational Diffusion of Macromolecule-sized Solutes in Cytoplasm and Nucleus

PubMed Central

Seksek, Olivier; Biwersi, Joachim; Verkman, A.S.

1997-01-01

Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficolls in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aqueous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 × 10−7 cm2/s with increasing dextran size (4–2,000 kD). D in cytoplasm relative to that in water (D/Do) was 0.26 ± 0.01 (MDCK) and 0.27 ± 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [RG] 40–300 Å). The fraction of mobile FITC-dextran molecules (fmob), determined by the extent of fluorescence recovery after spot photobleaching, was >0.75 for RG < 200 Å, but decreased to <0.5 for RG > 300 Å. The independence of D/Do on FITC-dextran and Ficoll size does not support the concept of solute “sieving” (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different spot diameters (1.5–4 μm) gave similar D/Do, indicating that microcompartments, if present, are of submicron size. Measurements of D/Do and fmob in concentrated dextran solutions, as well as in swollen and shrunken cells, suggested that the low fmob for very large macromolecules might be related to restrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/Do was 0.25 ± 0.02 (MDCK) and 0.27 ± 0.03 (fibroblasts), and independent of solute size (RG 40–300 Å). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus. PMID:9214387

Isolation of Intact Chloroplasts from Euglena gracilis by Zonal Centrifugation 1

PubMed Central

Vasconcelos, Aurea; Pollack, Marilyn; Mendiola, Leticia R.; Hoffmann, H.-P.; Brown, D. H.; Price, C. A.

1971-01-01

Chloroplasts were separated from Euglena gracilis by zonal centrifugation at low speed in density gradients of Ficoll or dextran. The chloroplasts were intact by the criteria of ultrastructure and their content of ribulose diphosphate carboxylase and soluble protein. The chloroplasts also contained ribosomes and ribosomal RNA uncontaminated by the corresponding cytoplasmic particles. Images PMID:16657599

Phase 1 Study of Safety and Immunogenicity of an Escherichia coli-Derived Recombinant Protective Antigen (rPA) Vaccine to Prevent Anthrax in Adults

DTIC Science & Technology

2010-11-05

AVA sera AVR801, kindly provided by Conrad Quinn, CDC, Atlanta, GA), serum samples and controls were diluted in serum diluent (5% skim milk in PBS...previously published assays [17]. Briefly, whole blood was collected in acid citrate dextrose (ACD) citrate tubes and PBMC were separated by ficoll

Development anmd testing of electrophoresis solutions. Task I.1: Development of optimal buffer system

NASA Technical Reports Server (NTRS)

1985-01-01

Two buffers were explored for testing: low ionic strength electrophoresis buffer with and without density gradient material. It was found that the electrophoresis routine was better tolerated when Ficoll was present. The results of a viability study of primary human fetal kidney (HFK-1) cells at the first passage are shown. Cell strain HFK-1 was used in several experiments at the first and second passage. The HFK consisted mainly of fibroblasts, and HFK-1 has a high epithelioid cell content. The chromosomes of HFK were examined and found to be euploid. The stock medium for cell electrophoresis is described. In this solution density gradient solutes such as sucrose and Ficoll are dissolved to bring the osmolarity to 0.30. Its ionic strength is less than 0.01M, and its conductivity is usually 0.0011 mho/cm. Methods for viability determination included direct microscopic counting of the percent cells attached and spread within 24 hr of plating test cultures or electrophoretically separated fractions. The Cytograf viability assay concept was tested, and shown that blue stained cells scatter less light into the 0.8 to 3.3 deg angular interval than do unstained cells.

Rapid and automated processing of bone marrow grafts without Ficoll density gradient for transplantation of cryopreserved autologous or ABO-incompatible allogeneic bone marrow.

PubMed

Schanz, U; Gmür, J

1992-12-01

The growing number of BMTs has increased interest in safe and standardized in vitro bone marrow processing techniques. We describe our experience with a rapid automated method for the isolation of mononuclear cells (MNC) from large volumes of bone marrow using a Fenwal CS-3000 cell separator without employing density gradient materials. Forty bone marrow harvests with a mean volume of 1650 +/- 307 ml were processed. A mean of 75 +/- 34% (50 percentile range 54-94%) of the original MNCs were recovered in a volume of 200 ml with only 4 +/- 2% of the starting red blood cells (RBC). Removal of granulocytes, immature myeloid precursors and platelets proved to be sufficient to permit safe cryopreservation and successful autologous BMT (n = 25). Allogeneic BMT (n = 14, including three major ABO-incompatible) could be performed without additional manipulation. In both groups of patients timely and stable engraftment comparable to historical controls receiving Ficoll gradient processed autologous (n = 17) or unprocessed allogeneic BMT (n = 54) was observed. Moreover, 70 +/- 14% of the RBC could be recovered from the grafts. They were used for autologous RBC support of donors, rendering unnecessary autologous blood pre-donations.

Dynamics of Model Hydraulic Fracturing Liquid Studied by Two-Dimensional Infrared Spectroscopy

NASA Astrophysics Data System (ADS)

Daley, Kim; Kubarych, Kevin J.

2014-06-01

The technique of two-dimensional infrared (2DIR) spectroscopy is used to expose the chemical dynamics of various concentrations of polymers and their monomers in heterogeneous mixtures. An environmentally relevant heterogeneous mixture, which inspires this study, is hydraulic fracturing liquid (HFL). Hydraulic fracking is a technique used to extract natural gas from shale deposits. HFL consists of mostly water, proppant (sand), an emulsifier (guar), and other chemicals specific to the drilling site. Utilizing a metal carbonyl as a probe, we observe the spectral dynamics of the polymer, guar, and its monomer, mannose, and compare the results to see how hydration dynamics change with varying concentration. Another polymer, Ficoll, and its monomer, sucrose, are also compared to see how polymer size affects hydration dynamics. The two results are as follows: (1) Guar experiences collective hydration at high concentrations, where as mannose experiences independent hydration; (2) no collective hydration is observed for Ficoll in the same concentration range as guar, possibly due to polymer shape and size. HFL experiences extremely high pressure during natural gas removal, so future studies will focus on how increased pressure affects the hydration dynamics of polymers and monomers.

Macromolecular crowding gives rise to microviscosity, anomalous diffusion and accelerated actin polymerization

NASA Astrophysics Data System (ADS)

Rashid, Rafi; Chee, Stella Min Ling; Raghunath, Michael; Wohland, Thorsten

2015-05-01

Macromolecular crowding (MMC) has been used in various in vitro experimental systems to mimic in vivo physiology. This is because the crowded cytoplasm of cells contains many different types of solutes dissolved in an aqueous medium. MMC in the extracellular microenvironment is involved in maintaining stem cells in their undifferentiated state (niche) as well as in aiding their differentiation after they have travelled to new locations outside the niche. MMC at physiologically relevant fractional volume occupancies (FVOs) significantly enhances the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells during chemically induced adipogenesis. The mechanism by which MMC produces this enhancement is not entirely known. In the context of extracellular collagen deposition, we have recently reported the importance of optimizing the FVO while minimizing the bulk viscosity. Two opposing properties will determine the net rate of a biochemical reaction: the negative effect of bulk viscosity and the positive effect of the excluded volume, the latter being expressed by the FVO. In this study we have looked more closely at the effect of viscosity on reaction rates. We have used fluorimetry to measure the rate of actin polymerization and fluorescence correlation spectroscopy (FCS) to measure diffusion of various probes in solutions containing the crowder Ficoll at physiological concentrations. Similar to its effect on collagen, Ficoll enhanced the actin polymerization rate despite increasing the bulk viscosity. Our FCS measurements reveal a relatively minor component of anomalous diffusion. In addition, our measurements do suggest that microviscosity becomes relevant in a crowded environment. We ruled out bulk viscosity as a cause of the rate enhancement by performing the actin polymerization assay in glycerol. These opposite effects of Ficoll and glycerol led us to conclude that microviscosity becomes relevant at the length scale of the reacting molecules within a crowded microenvironment. The excluded volume effect (arising from crowding) increases the effective concentration of actin, which increases the reaction rate, while the microviscosity does not increase sufficiently to lower the reaction rate. This study reveals finer details about the mechanism of MMC.

Macromolecular crowding gives rise to microviscosity, anomalous diffusion and accelerated actin polymerization.

PubMed

Rashid, Rafi; Chee, Stella Min Ling; Raghunath, Michael; Wohland, Thorsten

2015-04-30

Macromolecular crowding (MMC) has been used in various in vitro experimental systems to mimic in vivo physiology. This is because the crowded cytoplasm of cells contains many different types of solutes dissolved in an aqueous medium. MMC in the extracellular microenvironment is involved in maintaining stem cells in their undifferentiated state (niche) as well as in aiding their differentiation after they have travelled to new locations outside the niche. MMC at physiologically relevant fractional volume occupancies (FVOs) significantly enhances the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells during chemically induced adipogenesis. The mechanism by which MMC produces this enhancement is not entirely known. In the context of extracellular collagen deposition, we have recently reported the importance of optimizing the FVO while minimizing the bulk viscosity. Two opposing properties will determine the net rate of a biochemical reaction: the negative effect of bulk viscosity and the positive effect of the excluded volume, the latter being expressed by the FVO. In this study we have looked more closely at the effect of viscosity on reaction rates. We have used fluorimetry to measure the rate of actin polymerization and fluorescence correlation spectroscopy (FCS) to measure diffusion of various probes in solutions containing the crowder Ficoll at physiological concentrations. Similar to its effect on collagen, Ficoll enhanced the actin polymerization rate despite increasing the bulk viscosity. Our FCS measurements reveal a relatively minor component of anomalous diffusion. In addition, our measurements do suggest that microviscosity becomes relevant in a crowded environment. We ruled out bulk viscosity as a cause of the rate enhancement by performing the actin polymerization assay in glycerol. These opposite effects of Ficoll and glycerol led us to conclude that microviscosity becomes relevant at the length scale of the reacting molecules within a crowded microenvironment. The excluded volume effect (arising from crowding) increases the effective concentration of actin, which increases the reaction rate, while the microviscosity does not increase sufficiently to lower the reaction rate. This study reveals finer details about the mechanism of MMC.

Effect of DHEA on Bone in Young Adults

DTIC Science & Technology

1999-09-01

material from female subjects undergoing total hip replacement. Low-density mononuclear cells were isolated by centrifugation on Ficoll histopaque...affected genetic males that is similar to that of unaffected female animals (56). Another natural model is the case of a man with an estrogen receptor...transgenic estrogen receptor knockout mouse, has a BMD 20-25% that of normal mice (58). Similarly, male and female patients with aromatase deficiency have

The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens.

PubMed

Lindsten, T; Yaffe, L J; Thompson, C B; Guelde, G; Berning, A; Scher, I; Kenny, J J

1985-05-01

Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.

Efficient long-term cryopreservation of pluripotent stem cells at −80 °C

PubMed Central

Yuan, Ye; Yang, Ying; Tian, Yuchen; Park, Jinkyu; Dai, Aihua; Roberts, R. Michael; Liu, Yang; Han, Xu

2016-01-01

Current long term cryopreservation of cell stocks routinely requires the use of liquid nitrogen (LN2), because commonly used cryopreservation media containing cell membrane permeating cryoprotectants are thermally unstable when frozen at higher storage temperatures, e.g. −80 °C. This instability leads to ice recrystallization, causing progressive loss of cell viability over time under the storage conditions provided by most laboratory deep freezers. The dependency on LN2 for cell storage significantly increases operational expense and raises issues related to impaired working efficiency and safety. Here we demonstrate that addition of Ficoll 70 to cryoprotectant solutions significantly improves system thermal stability at the working temperature (~−80 °C) of laboratory deep freezers. Moreover, a medium comprised of Ficoll 70 and dimethyl sulfoxide (DMSO) in presence or absence of fetal bovine serum (FBS) can provide reliable cryopreservation of various kinds of human and porcine pluripotent stem cells at −80 °C for periods that extend up to at least one year, with the post-thaw viability, plating efficiency, and full retention of pluripotent phenotype comparable to that achieved with LN2 storage. These results illustrate the practicability of a promising long-term cryopreservation method that completely eliminates the need for LN2. PMID:27694817

Investigation of the hydrodynamic response of cells in drop on demand piezoelectric inkjet nozzles.

PubMed

Cheng, Eric; Yu, Haoran; Ahmadi, Ali; Cheung, Karen C

2016-01-29

Cell motion within a liquid suspension inside a piezoelectrically actuated, cylindrical inkjet printhead was studied using high speed imaging and a low depth of field setup. For each ejected droplet, a cell within the inkjet nozzle was observed to exhibit one of three possible behaviors which are termed: cell travel, cell ejection and cell reflection. Cell reflection is an undesirable phenomenon which may adversely affect an inkjet's capability in dispensing cells and a possible reason why it was previously reported that the rate of cells dispensed did not follow the expected Poisson distribution. Through the study of the cells motions, it was hypothesized that the rheological properties of the media in the cell suspension play an important role in influencing the cell behaviors exhibited. This was experimentally studied with the tracking of cells within the inkjet nozzle in a 10% w/v Ficoll PM400 cell suspension. The effect of cell reflection was eliminated using the higher density and viscosity Ficoll PM400 suspension. The presented work is the first in-depth study of the cell behaviors occurring within a piezoelectric inkjet nozzle during the printing process. The understanding of the hydrodynamics during a droplet ejection and its effect on the suspended cells are imperative towards achieving reliable cell dispensing for biofabrication applications.

Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

PubMed Central

Scoville, Steven D.; Keller, Karen A.; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A.; Freud, Aharon G.

2015-01-01

We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method. PMID:26223896

Triptolide Attenuates Endotoxin- and Staphylococcal Exotoxin-Induced T-Cell Proliferation and Production of Cytokines and Chemokines

DTIC Science & Technology

2005-02-01

were from Sigma (St. Louis, MO, USA). Cell Culture Human PBMC were isolated by Ficoll-Hypaque density gradient centri- fugation of heparinized blood...Cytotoxicity was measured by the uptake of trypan blue . T-cell proliferation was assayed with PBMC (105 cells/well) that were plated in triplicate with...concentration range used in these studies (1–30 nM), as confirmed by trypan blue dye exclusion test. However, at 100 nM triptolide, 20% of PBMC took up

Protein-Glycan Quinary Interactions in Crowding Environment Unveiled by NMR Spectroscopy.

PubMed

Diniz, Ana; Dias, Jorge S; Jiménez-Barbero, Jesús; Marcelo, Filipa; Cabrita, Eurico J

2017-09-21

Protein-glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of 1 H 15 N-HSQC signals. The intensity of the Gal-3 1 H 15 N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of 1 H 15 N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

"Prostatic acid phosphatase?" A comparison of acid phosphatase activities in epithelial cells, granulocytes, monocytes, lymphocytes, and platelets purified by velocity sedimentation in isokinetic gradients of Ficoll in tissue culture medium.

PubMed Central

Helms, S. R.; Brattain, M. G.; Pretlow, T. G.; Kreisberg, J. I.

1977-01-01

Numerous investigators have found several substrates and inhibitors to be particularly suited for the demonstration of acid phosphatase of prostatic origin. There has been much controversy over the specificity or lack of specificity of several substrates and inhibitors. We have investigated acid phosphatase activities obtained from several kinds of purified cells. None of the substrates or inhibitors which we studied permitted us to discriminate "prostatic" acid phosphatase from acid phosphatase activities obtained from other kinds of cells. PMID:560800

LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01).

PubMed

Avettand-Fènoël, Véronique; Chaix, Marie-Laure; Blanche, Stéphane; Burgard, Marianne; Floch, Corinne; Toure, Kadidia; Allemon, Marie-Christine; Warszawski, Josiane; Rouzioux, Christine

2009-02-01

HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/10(6) leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r = 0.900, P < 0.0001). A total of 3,002 specimens from 1,135 infants were tested. The specificity of HIV-DNA and HIV-RNA assays was 100%. HIV-1 infection was diagnosed in nine infants before age 60 days. HIV-DNA levels were low, underlining the need for sensitive assays when highly active antiretroviral therapy (HAART) has been given. The performances of this HIV-DNA assay showed that it is adapted to early diagnosis in children. The results were equivalent to those of HIV-RNA assay. HIV-DNA may be used even in masked primary infection in newborns whose mothers have received HAART. (c) 2008 Wiley-Liss, Inc.

Solubilization and other studies on adenylate cyclase of baker's yeast.

PubMed Central

Varimo, K; Londesborough, J

1976-01-01

1. Adenylate cyclase of Saccharomyces cerevisiae was sedimented from mechanically disintegrated preparations of yeast over an unusually wide range of centrifugal forces. 2. The enzyme was readily solubilized by Ficoll and by Lubrol PX. Lubrol caused a 2-fold activation. 3. Both particle-bound and Lubrol-solubilized enzyme had an apparent Km for ATP of 1.6 mM in the presence of 0.4 mM-cyclic AMP and 5 mM-MnCl2 at pH 6.2 and 30 degrees C. 4. The Lubrol-solubilized enzyme behaved on gel filtration as a monodisperse protein with an apparent mol.wt. of about 450000. PMID:793584

In vitro study of bovine oligodendroglia.

PubMed

Fewster, M E; Blackstone, S

1975-12-01

Oligodendroglia were prepared by 'Ficoll' density gradient centrifugation from the centrum ovale of fetal and adult bovine brains. When cultivated in Rose Chambers, and provided an air bubble was included in the chamber during the cultivation, processes developed on cells around the circumference of the bubble. A sizeable air phase seems to be important for process formation in isolated bovine glial preparations. Various culture systems, media and additions to the cultures were examined for their effect on the behavior of the cultures. Fibroblast overgrowth occurred in oligodendroglial cultures from fetal brains in media supplemented with fetal bovine serum (FBS) but not in medium 199 supplemented with 2.5% FBS.

Heterogeneous and monoclonal helper T cells induce similar anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody populations in the primary adoptive response. II. Lambda light chain dominance and idiotope expression.

PubMed

Smith, F I; Tesch, H; Rajewsky, K

1984-02-01

When the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) is presented on different carrier molecules, different anti-NP antibody responses are stimulated. On stimulation with NP-lipopolysaccharide (LPS) [T-independent type 1 (TI-1) antigen] kappa + antibodies are the major population, whereas on stimulation with NP-Ficoll [T-independent type 2 (TI-2) antigen], NP-keyhole limpet hemocyanin (KLH) or NP-chicken gamma globulin (CG) [T-dependent (TD) antigens], lambda 1+ antibodies dominate. The relative contribution of idiotopes Ac38 or Ac146 to the lambda 1+ anti-NP response was also different on comparison of TI-1 with TI-2 or TD anti-NP responses. We investigated whether light chain- or idiotype-specific T cells are responsible for these differences. Analysis of the anti-NP response of nude mice after immunization with NP-Ficoll showed lambda 1 dominance. Likewise primary adoptive transfer experiments using carrier-specific T cell lines to reconstitute the TD anti-NP response to NP-KLH or NP-CG, showed that help from carrier-specific T cells alone is capable of stimulating the characteristic lambda 1 dominant response. No significant difference could be found in the levels of Ac38 and Ac146 idiotope expression between mice reconstituted with splenic T cells and those reconstituted with T cell lines. These results suggest that light chain- or idiotype-specific T cells are required neither for the production of lambda 1 light chain dominance, nor for the appearance of idiotopes characteristic of the primary anti-NP response. The possible reasons for differences seen in both light chain and idiotope expression between primary anti-NP responses to the TI-1 antigen NP-LPS and those to TD or TI-2 antigens are discussed.

Efficiency and Impact of Positive and Negative Magnetic Separation on Monocyte Derived Dendritic Cell Generation.

PubMed

Kowalewicz-Kulbat, Magdalena; Ograczyk, Elżbieta; Włodarczyk, Marcin; Krawczyk, Krzysztof; Fol, Marek

2016-06-01

The immunomagnetic separation technique is the basis of monocyte isolation and further generation of monocyte-derived dendritic cells. To compare the efficiency of monocyte positive and negative separation, concentration of beads, and their impact on generated dendritic cells. Monocytes were obtained using monoclonal antibody-coated magnetic beads followed the Ficoll-Paque gradient separation of mononuclear cell fraction from the peripheral blood of 6 healthy volunteers. CD14 expression was analyzed by flow cytometry. Both types of magnetic separation including recommended and reduced concentrations of beads did not affect the yield and the purity of monocytes and their surface CD14 expression. However, DCs originated from the "positively" separated monocytes had noticeable higher expression of CD80.

Induction of human antigen-specific suppressor factors in vitro.

PubMed Central

Kontiainen, S; Woody, J N; Rees, A; Feldmann, M

1981-01-01

Based on methods used for the in vitro induction of antigen-specific suppressor cells in the mouse, we have cultured Ficoll-Isopaque-separated human blood cells with high dose of antigen (100 microgram/ml) in Marbrook culture vessels for 4 days. The resulting cells, when further recultured for 24 hr with a low dose of antigen (1 microgram/ml), released into the supernatant material, termed 'suppressor factor', which inhibited, in an antigen-specific manner, the antibody response of mouse spleen cells in culture. The suppressor factor was analysed using immunoabsorbents, and was bound to and eluted from specific antigen, concanavalin A and lentil lectin, anti-human Ia antibodies, and anti-mouse suppressor factor antibodies, but was not bound to antibodies against human IgG. PMID:6169475

Identification of primary and secondary UBA footprints on the surface of ubiquitin in cell-mimicking crowded solution.

PubMed

Munari, Francesca; Bortot, Andrea; Zanzoni, Serena; D'Onofrio, Mariapina; Fushman, David; Assfalg, Michael

2017-04-01

Despite significant advancements in our understanding of ubiquitin-mediated signaling, the influence of the intracellular environment on the formation of transient ubiquitin-partner complexes remains poorly explored. In our work, we introduce macromolecular crowding as a first level of complexity toward the imitation of a cellular environment in the study of such interactions. Using NMR spectroscopy, we find that the stereospecific complex of ubiquitin and the ubiquitin-associated domain (UBA) is minimally perturbed by the crowding agent Ficoll. However, in addition to the primary canonical recognition patch on ubiquitin, secondary patches are identified, indicating that in cell-mimicking crowded solution, UBA contacts ubiquitin at multiple sites. © 2017 Federation of European Biochemical Societies.

Rabbit polymorphonuclear leukocytes do not secrete endogenous pyrogens or interleukin 1 when stimulated by endotoxin, polyinosine:polycytosine, or muramyl dipeptide.

PubMed

Windle, B E; Murphy, P A; Cooperman, S

1983-03-01

Rabbit polymorphonuclear leukocytes were purified from rabbit blood by centrifugation on colloidal silica gradients followed by sedimentation in 4% Ficoll. The purified neutrophils had normal random motility, responded to chemotactic stimuli, phagocytosed zymosan particles, made superoxide, and phagocytosed and killed bacteria. However, they did not secret endogenous pyrogens either spontaneously or in response to stimulation with endotoxin, polyinosine:polycytosine, or muramyl dipeptide. Macrophages isolated on the same gradients secreted some pyrogen spontaneously and secreted considerably more in response to the same three stimuli. This evidence reinforces the idea that macrophages are the only source of endogenous pyrogens, and that pyrogens secreted by cell populations that are rich in neutrophils are to be attributed to the monocytes or macrophages that the cell populations contain.

Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats.

PubMed Central

Wiedermann, U; Hanson, L A; Kahu, H; Dahlgren, U I

1993-01-01

We have previously reported that vitamin A deficiency resulted in a reduced IgA antibody response to cholera toxin (CT) after per-oral immunization. In the present investigation we have studied the in vivo and in vitro immune response in vitamin A-deficient rats to two parenterally applied antigens, beta-lactoglobulin (beta-LG) and picrylsulphonic acid (TNP)-Ficoll. The serum IgG and IgM antibody responses to the T-cell dependent antigen beta-LG were significantly lower in the vitamin A-deficient rats than in the pair-fed control rats. No such differences were seen with the IgG and IgM responses to the T-cell independent antigen TNP-Ficoll. However, the biliary IgA and the serum IgE antibodies against both antigens were decreased in the vitamin A-deficient rats. In vitro lymphocyte stimulation with concanavalin A (Con A) or beta-LG gave higher T-cell proliferation rates in the vitamin A-deficient than in the control rats. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) levels in supernatants from Con A-stimulated mesenteric lymph node cells were also higher in the vitamin A-deficient rats, while IL-6 levels were decreased, which is consistent with an up-regulated Th1 activity. Proliferation studies on purified accessory cells and T cells from the deficient and the control rats, mixed in different combinations, showed that the T cells, but not the accessory cells, were disturbed in the vitamin A-deficient rats. Despite the increased T-cell activity in vitro the vitamin A-deficient rats had a lower delayed-type hypersensitivity (DTH) reaction than the pair-fed control rats. In conclusion, the increased IL-2 and IFN-gamma levels may reflect an up-regulation of Th1 cell function, while the decreased IgA, IgE and IL-6 levels indicate a suppression of Th2 cells. The disturbed T-lymphocyte function is manifested in vivo as a decreased DTH reaction and suppressed antibody production, the latter possibly due to a lack of B-cell switching and proliferation factors in vitamin A-deficient rats. PMID:8307607

Intracellular zidovudine (ZDV) and ZDV phosphates as measured by a validated combined high-pressure liquid chromatography-radioimmunoassay procedure.

PubMed Central

Slusher, J T; Kuwahara, S K; Hamzeh, F M; Lewis, L D; Kornhauser, D M; Lietman, P S

1992-01-01

In vitro studies of zidovudine (ZDV) phosphorylation may not accurately reflect the in vivo dose-response relationship, which is crucial to determining the relationship between ZDV exposure, efficacy, and toxicity. However, measurement of ZDV phosphorylated anabolites in peripheral blood mononuclear cells (PBMCs) from ZDV-treated human immunodeficiency virus (HIV)-infected patients would be extremely useful in the more appropriate utilization of ZDV in the treatment of HIV infection. We developed a specific and sensitive combined high-pressure liquid chromatography (HPLC)-radioimmunoassay (RIA) procedure for the determination of ZDV, ZDV-monophosphate, ZDV-diphosphate, and ZDV-triphosphate in PBMCs taken from ZDV-treated HIV-infected patients. ZDV and its anabolites were extracted from washed, Ficoll-Paque-isolated PBMCs and then separated by HPLC using a strong anion-exchange column. The anabolites were then hydrolyzed to ZDV with acid phosphatase. ZDV was then measured by using a modified commercially available RIA protocol. Our method was validated by measuring [3H]ZDV anabolites generated in Molt-4 cells radioisotopically and simultaneously by the combined HPLC-RIA procedure. The ZDV determinations correlated well (r2 = 0.97) over the range of 0.037 to 5.2 pmol (10 to 1,400 pg) per assay tube. Furthermore, we defined the stability of ZDV anabolites during ficoll isolation and the recovery after extraction and cleanup. We then measured intracellular parent ZDV and its phosphorylated anabolites in PBMCs from six ZDV-treated HIV-infected patients (PBMCs were taken 2 h after a 300-mg oral dose). The mean concentrations ( +/- standard deviations) of parent and of mono-, di-, and triphosphates were 0.15 +/- 0.08, 1.4 +/-, 0.082 +/- 0.02, and 0.081 +/- 0.03 pmol/10(6) PBMC, respectively (one pmol/10(6) PBMC represents a concentration of approximately 1 microm). Concurrent serum ZDV concentrations were between 1.3 and 7.1 microm. This method should provide a useful tool for evaluating in vivo pharmacokinetics of ZDV anabolites in PBMCs and possibly other cell types, even at the low doses of ZDV currently administered therapeutically. PMID:1489190

Rabbit polymorphonuclear leukocytes do not secrete endogenous pyrogens or interleukin 1 when stimulated by endotoxin, polyinosine:polycytosine, or muramyl dipeptide.

PubMed Central

Windle, B E; Murphy, P A; Cooperman, S

1983-01-01

Rabbit polymorphonuclear leukocytes were purified from rabbit blood by centrifugation on colloidal silica gradients followed by sedimentation in 4% Ficoll. The purified neutrophils had normal random motility, responded to chemotactic stimuli, phagocytosed zymosan particles, made superoxide, and phagocytosed and killed bacteria. However, they did not secret endogenous pyrogens either spontaneously or in response to stimulation with endotoxin, polyinosine:polycytosine, or muramyl dipeptide. Macrophages isolated on the same gradients secreted some pyrogen spontaneously and secreted considerably more in response to the same three stimuli. This evidence reinforces the idea that macrophages are the only source of endogenous pyrogens, and that pyrogens secreted by cell populations that are rich in neutrophils are to be attributed to the monocytes or macrophages that the cell populations contain. PMID:6601619

Macrophages induce differentiation of plasma cells through CXCL10/IP-10

PubMed Central

Joo, HyeMee; Clayton, Sandra; Dullaers, Melissa; Herve, Marie-Cecile; Blankenship, Derek; De La Morena, Maria Teresa; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Pascual, Virginia; Oh, SangKon; Banchereau, Jacques

2012-01-01

In tonsils, CD138+ plasma cells (PCs) are surrounded by CD163+ resident macrophages (Mϕs). We show here that human Mϕs (isolated from tonsils or generated from monocytes in vitro) drive activated B cells to differentiate into CD138+CD38++ PCs through secreted CXCL10/IP-10 and VCAM-1 contact. IP-10 production by Mϕs is induced by B cell–derived IL-6 and depends on STAT3 phosphorylation. Furthermore, IP-10 amplifies the production of IL-6 by B cells, which sustains the STAT3 signals that lead to PC differentiation. IP-10–deficient mice challenged with NP-Ficoll show a decreased frequency of NP-specific PCs and lower titers of antibodies. Thus, our results reveal a novel dialog between Mϕs and B cells, in which IP-10 acts as a PC differentiation factor. PMID:22987802

Suppression of unprimed T and B cells in antibody responses by irradiation-resistant and plastic-adherent suppressor cells in Toxoplasma gondii-infected mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Y.; Kobayashi, A.

1983-04-01

In the acute phase of Toxoplasma infection, the function of both helper T and B cells was suppressed in primary antibody responses to dinitrophenol (DNP)-conjugated protein antigens. During the course of infection, the suppressive effect on T cells seems to continue longer than that on B cells, since suppression in responses to sheep erythrocytes, a T-dependent antigen, persisted longer than those to DNP-Ficoll, a T-independent antigen. Plastic-adherent cells from the spleens of Toxoplasma-infected and X-irradiated (400 rads) mice had strong suppressor activity in primary anti-sheep erythrocyte antibody responses of normal mouse spleen cells in vitro. These data suggest that themore » activation of irradiation-resistant and plastic-adherent suppressor cells causes the suppression of both T and B cells in Toxoplasma-infected mice.« less

Selective effect of irradiation on responses to thymus-independent antigen.

PubMed

Lee, S K; Woodland, R T

1985-02-01

Low doses of ionizing radiation have a selective immunosuppressive effect on in vivo B cell responses to thymus-independent (TI) antigens. The B cell response, assayed as direct anti-trinitrophenyl (TNP)-specific plaque-forming cells (PFC), induced by type 2, TI antigens (TNP-Ficoll or TNP-Dextran), was reduced, on the average, by 10-fold in animals exposed to 200 rad of ionizing radiation 24 hr before antigen challenge. In contrast, PFC responses to type 1, TI antigens (TNP-lipopolysaccharide or TNP-Brucella abortus) are unaffected in mice exposed to the same dose of radiation. Adoptive transfers showed that this selective immunosuppression is a result of the specific inactivation of the B cell subpopulation responding to type 2, TI antigens. These experiments suggest that physiologic differences exist in the B cell subpopulations of normal mice which respond to type 1, or type 2, TI antigens.

Calcium-activated potassium channels in basolateral membranes of colon epithelial cells; reconstitution and functional properties.

PubMed

Wiener, H; Turnheim, K

1990-10-26

Using differential sedimentation, isopycnic and Ficoll-400 barrier centrifugation, basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon were enriched 34- and 9-fold, respectively. 86Rb(+)-uptake into these vesicles, driven by an electrical potential difference, was stimulated by submicromolar Ca2+ activities and inhibited by Ba2+. These findings indicate the presence of Ca2(+)-activated K+ channels. The K+ channels in surface and crypt cell membranes differed with respect to inhibition by the bee venom apamin, the scorpion venom charybdotoxin and tetraethylammonium and exhibited a different pH dependence. Fusion of basolateral membrane vesicles with planar phospholipid bilayers revealed the presence of high-conductance Ba2(+)-sensitive K+ channels which were activated by micromolar Ca2+ and inhibited by crude scorpion venom and trifluoperazine. These K+ channels may be involved in the coupling of apical and basolateral membrane conductances during Na+ absorption and Cl- secretion, but they may also play a role in cell volume regulation.

Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts.

PubMed

Gao, Xiao-Yu; Liao, Xing-Cheng; Wu, Ruo-Lai; Liu, Ting; Wang, Hai-Xing; Lu, Ling-Li

This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The results revealed that preheating cellulase and macerozyme at 50 °C for 5 min significantly accelerated the cell wall degradation. For the most optimal conditions for mesophyll protoplast isolation, the mixture of fresh leaves and cell lysates was followed by a 2-h-long vibration. The protoplast lysate for vacuole isolation was diluted, and 0.675 mmol/L was identified as the most appropriate 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) level, in which S. alfredii large vacuoles are characterized by a high metal and malic acid content. For the best vacuole purification results, we established that 0.8 mol/L was the most optimal mannitol level in the vacuole buffer in terms of vacuole protection during centrifugation, whereas a Ficoll concentration of 0.10 g/ml was adopted in the density-gradient centrifugation.

Effect of Environmental Density and Buoyancy on Growth and Gravitropic Response in Maize Roots

NASA Astrophysics Data System (ADS)

Robbins, J. L.; Mulkey, T. J.

2008-06-01

The mechanism by which plants sense gravity is not fully understood. The hydrostatic model was proposed as an alternative to the statolith model. These experiments are designed to provide further understanding about the underlying mechanism of the gravitropic sensing. Primary roots of maize with a length of about 1 cm were used. The roots were placed in environments of various density and buoyancy using air, water, sucrose, sucrose/polyethylene glycol 4000 (PEG), PEG 8000, and Ficoll PM 400. The rates of growth and gravitropic curvature were monitored using time-lapse video and digital recordings. Comparison of roots in air to roots in oxygenated water indicate that there is no significant difference in growth rates but the higher density of water and the other test solutions significantly slows the gravitropic response. Altering the environmental density and buoyancy of the solution surrounding the root does not appear to alter sedimentation of statoliths within the root tip.

Investigations on gel forming media use in low gravity bioseparations research

NASA Technical Reports Server (NTRS)

Todd, Paul; Szlag, David C.; Plank, Lindsay D.; Delcourt, Scott G.; Kunze, M. Elaine

1989-01-01

Research on gelling media and conditions suitable for the preservation of the spatial configuration of cell suspensions and macromolecular solutions after separation in free fluid during low gravity experiments is presented. The examples studied included free electrophoresis of cells in a cylindrical column and two-phase aqueous polymer separation. Microgravity electrophoresis experiments were simulated by separating model cell types (animal or human) in a vertical density gradient containing low-conductivity buffer, 1.7-6.5 percent Ficoll, 6.8-5.0 percent sucrose, and 1 percent SeaPrep low-melting temperature agarose. Upon cooling, a gel formed in the column and cells could be captured at the forming locations. Two-phase extraction experiments were simulated using two-polymer solutions in which phase separation occurs in normal saline at temperatures compatible with cell viability and in which one or both phases form a gel upon cooling. Suitable polymers included commercial agaroses (1-2 percent), maltodextrin (5-7 percent), and gelatin (5-20 percent).

Assessment of six different collagenase-based methods to isolate feline pancreatic islets.

PubMed

Zini, Eric; Franchini, Marco; Guscetti, Franco; Osto, Melania; Kaufmann, Karin; Ackermann, Mathias; Lutz, Thomas A; Reusch, Claudia E

2009-12-01

Isolation of pancreatic islets is necessary to study the molecular mechanisms underlying beta-cell demise in diabetic cats. Six collagenase-based methods of isolation were compared in 10 cat pancreata, including single and double course of collagenase, followed or not by Ficoll centrifugation or accutase, and collagenase plus accutase. Morphometric analysis was performed to measure the relative area of islet and exocrine tissue. Islet specific mRNA transcripts were quantified in isolates by real-time PCR. The single and double course of collagenase digestion was successful in each cat and provided similar islet-to-exocrine tissue ratio. Quantities of insulin mRNA did not differ between the two methods. However, on histological examination either method yielded only approximately 2% of pure islets. The other methods provided disrupted islets or insufficient samples in 1-7 cats. Although pancreas digestion with single and double course of collagenase was superior, further studies are needed to improve islet isolation in cats.

Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells.

PubMed

Thayer, S S; Huffaker, R C

1984-05-01

The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.

In vivo traffic of indium-111-oxine labeled human lymphocytes collected by automated apheresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Read, E.J.; Keenan, A.M.; Carter, C.S.

1990-06-01

The in vivo traffic patterns of autologous lymphocytes were studied in five normal human volunteers using lymphocytes obtained by automated apheresis, separated on Ficoll-Hypaque gradients, and labeled ex vivo with {sup 111}In-oxine. Final lymphocyte infusions contained 1.8-3.1 X 10(9) cells and 270-390 microCi (9.99-14.43 MBq) {sup 111}In, or 11-17 microCi (0.41-0.63 MBq) per 10(8) lymphocytes. Gamma imaging showed transient lung uptake and significant retention of radioactivity in the liver and spleen. Progressive uptake of activity in normal, nonpalpable axillary and inguinal lymph nodes was seen from 24 to 96 hr. Accumulation of radioactivity also was demonstrated at the forearm skinmore » test site, as well as in its associated epitrochlear and axillary lymph nodes, in a subject who had been tested for delayed hypersensitivity with tetanus toxoid. Indium-111-oxine labeled human lymphocytes may provide a useful tool for future studies of normal and abnormal lymphocyte traffic.« less

On optimizing the blocking step of indirect enzyme-linked immunosorbent assay for Epstein-Barr virus serology.

PubMed

Lim, Chun Shen; Krishnan, Gopala; Sam, Choon Kook; Ng, Ching Ching

2013-01-16

Because blocking agent occupies most binding surface of a solid phase, its ability to prevent nonspecific binding determines the signal-to-noise ratio (SNR) and reliability of an enzyme-linked immunosorbent assay (ELISA). We demonstrate a stepwise approach to seek a compatible blocking buffer for indirect ELISA, via a case-control study (n=176) of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). Regardless of case-control status, we found that synthetic polymer blocking agents, mainly Ficoll and poly(vinyl alcohol) (PVA) were able to provide homogeneous backgrounds among samples, as opposed to commonly used blocking agents, notably nonfat dry milk (NFDM). The SNRs for NPC samples that correspond to blocking using PVA were approximately 3-fold, on average, higher than those blocking using NFDM. Both intra- and inter-assay precisions of PVA-based assays were <14%. A blocking agent of choice should have tolerable sample backgrounds from both cases and controls to ensure the reliability of an immunoassay. Copyright © 2012 Elsevier B.V. All rights reserved.

Ciliary propulsion of objects in tubes: wall drag on swimming Tetrahymena (Ciliata) in the presence of mucin and other long-chain polymers.

PubMed

Winet, H

1976-04-01

The lubrication effect of three long-chain polymers - mucin, methylcellulose and Ficoll - on ciliary propulsion in tubes is measured by plotting the relative velocities of swimming cilitates as a function of the tube bore diameter. Mucin shows the most unequivocal lubrication, which is found at concentrations between 0% and 9.1% (w/v). This observation, coupled with viscometric measurements which show that ciliary tip shear rates are sufficient to solate mucin, serve as the groundwork for a model of mucin lubrication which explains the optimized lubrication behaviour of thixotropic gelating polymers as an expression of the response to shear by the various stages of polymer clustering during the gelatin process. In addition to the lubricative effect, another wall drag reduction effect by mucin was measured in the clearance region beyond the lubrication layer. This apparent viscosity reduction is optimized in the concentration range between I.7% and 4.I% mucin and may also be explained in terms of the properties of gel clustering.

Multiscale Spectroscopy of Diffusing Molecules in Crowded Environments

NASA Astrophysics Data System (ADS)

Heikal, Ahmed A.

2015-06-01

Living cells are known to be crowded with organelles, biomembranes, and macromolecules such as proteins, DNA, RNA, and actin filaments. It is believed that such macromolecular crowding affect biomolecular diffusion, protein-protein and protein-substrate interaction, and protein folding. In this contribution, I will discuss our recent results on rotational and translational diffusion of small and large molecules in crowded environments using time-resolved anisotropy and fluorescence correlation spectroscopy methods. In these studies, rhodamine green and enhanced green fluorescent protein are used as fluorescent probes diffusing in buffers enriched with biomimetic crowding agents such as Ficoll-70, bovine serum albumin (BSA), and ovalbumin. Controlled experiments on pure and glycerol-rich buffers were carried out as environments with variable, homogeneous viscosity. Our results indicate that the microviscosity differs from the corresponding bulk viscosity, depending on the nature of crowding agents (i.e., proteins versus polymers), the concentration of crowding agents and spatio-temporal scaling of our experimental approach. Our findings provide a foundation for fluorescence-based studies of diffusion and binding of biomolecules in the crowded milieu of living cells.

Combined cytogenotoxic effects of bee venom and bleomycin on rat lymphocytes: an in vitro study.

PubMed

Abd-Elhakim, Yasmina M; Khalil, Samah R; Awad, Ashraf; Al-Ayadhi, Laila Y

2014-01-01

This study was carried out to determine the cytotoxic and genotoxic effects of bee venom (BV) and/or the chemotherapeutic agent bleomycin (BLM) on healthy isolated rat lymphocytes utilizing morphometric and molecular techniques. Using the Ficoll-Histopaque density gradient centrifugation technique, lymphocytes were isolated, divided into groups, and subjected to BV and/or BLM at incubation medium concentrations of 10 or 20 μg/mL respectively for 24 and 72 hrs. An MTT assay and fluorescent microscopy examinations were used to assess the cytotoxic effects. To determine the predominant type of BV and/or BLM-induced cell death, LDH release assay was employed beside quantitative expression analyses of the apoptosis-related genes (Caspase-3 and Bcl-2). The genotoxic effects of the tested compounds were evaluated via DNA fragmentation assay. The results of these assays demonstrated that BV potentiates BLM-induced cytotoxicity through increased LDH release and diminished cell viability. Nevertheless, BV significantly inhibited the BLM-induced DNA damage. The results verify that BV significantly attenuates the genotoxic effects of BLM on noncancerous isolated rat lymphocytes but does not diminish BLM cytotoxicity.

Expression changes of serotonin receptor gene subtype 5HT3a in peripheral blood mononuclear cells from schizophrenic patients treated with haloperidol and Olanzapin.

PubMed

Shariati, Gholam Reza; Ahangari, Ghasem; Hossein-nezhad, Arash; Asadi, Seyed Mohammad; Pooyafard, Farzaneh; Ahmadkhaniha, Hamid Reza

2009-09-01

Serotonin receptors are involved in pathophysiology of schizophrenia and may mediate other neurotransmitter effects. We investigated serotonin receptors gene expression in peripheral blood mononuclear cells (PBMC) of naïve schizophrenic patients, before and after treatment. Also serotonin receptor gene expression was compared in two treatment groups including Haloperidol and Olanzapine. The PBMC was separated from whole blood by Ficoll-hypaque. The total cellular RNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using primer pairs specific for 5HT(3a) serotonin receptor mRNA and beta-actin as internal control. The results showed the presence of subtype of serotonin receptor in lymphocytes. Serotonin gene expression showed significant changes in Olanzapine treatment group which correlated with Clinical Global Impression (CGI) score improvement. In conclusion, the present study has shown that human PBMC express serotonin receptors 5HT(3a). Moreover, clinical symptom improvement of Olanzapin may be demonstrated by a change in serotonin receptor gene expression.

[Microwave stimulated cell marker analysis. Possibilities for more rapid immune diagnosis].

PubMed

Ebener, U; Wehner, S

1993-01-01

We describe a successful rapid APAAP-complex technique using innovative application of microwave irradiation (MIWI) on Ficoll separated peripheral blood mononuclear cell smears of healthy donors. The typing with several monoclonal antibodies (MoAbs) against different cell surface antigens is compared with the conventional APAAP procedure. The commercial domestic microwave oven was operated at 2.45 GHz. Fifteen second irradiation at 350 W during all incubation steps, e.g. primary antibody, bridging antibody and APAAP-complexes produced excellent color reactions with Fast Red TR, Fast Blue BB, New Fuchsin or NBT similar with the conventional immunoenzyme procedure. The routinely usage of a Silicon-Chamber-System developed by us is applicable without limitation under microwave conditions. The results till now have shown that the application of microwave-technique (MIWI) eliminated the need for much longer incubation periods without lost of sensitivity. All immunological markers could be detected in the same degree as observed with the conventional method. We could demonstrate that an immunological diagnosis is possible within 30 minutes using air dried smears in an microwave oven.

Lymphocyte subpopulations of intestinal mucosa in inflammatory bowel disease.

PubMed Central

Eade, O E; Andre-Ukena, S S; Moulton, C; MacPherson, B; Beeken, W L

1980-01-01

Lymphocyte subpopulations in peripheral blood (PBL) and intestinal mucosa (IML) of 10 patients with inflammatory bowel disease (IBD) were compared with those of 11 non-IBD controls. PBL were separated on Ficoll/hypaque gradients, and IML were isolated by incubation in dithiothreitol, EDTA, and collagenase. These methods yielded cells of good viability and with intact HLA A and B-antigens. T-cells, identified by neuraminidase-treated sheep RBC rosettes and non-specific esterase staining, comprised approximately 91% of the IML from normal mucosa of all groups. B-cells, identified by erythrocyte-antibody-complement rosettes and surface immunoglobulins, were only 7% of these IML populations. Cell yields were two-fold or more greater from abnormal IBD mucosa, with T-cells ranging from 55 to 95% and B-cells from 2 to 36%. The percentage of Fc receptor bearing cells was low in all specimens. By these methods, T-lymphocytes predominated in intestinal mucosa of both IBD and non-IBD patients, but there is marked increase in the percentage of B-cells isolated from abnormal mucosa in IBD. Images Fig. 1 Fig. 2 Fig. 3 Fig. 11 PMID:6968706

Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

PubMed Central

Bezek, D M; Baker, J C; Kaneene, J B

1988-01-01

A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood samples collected using ethylenediaminetetraacetic acid as an anticoagulant. Bovine viral diarrhea virus antigen was detected by immunofluorescence between 3 and 11 days postinfection in lymphocyte smears and 3 to 12 days postinfection in buffy coat smears. Isolation of virus from both lymphocytes and buffy coat preparations correlated with detection of immunofluorescence. Serum neutralizing antibody to bovine virus diarrhea virus was detected on day 10 postinfection. Buffy coat smears were as sensitive as lymphocyte smears for the detection of antigen by immunofluorescence. It appeared that immunofluorescent staining of white blood cells was an effective method of detecting bovine virus diarrhea viral antigen. PMID:2836047

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective

PubMed Central

Nombela, Ivan; Carrion, Aurora; Puente-Marin, Sara; Chico, Verónica; Mercado, Luis; Perez, Luis; Coll, Julio; Ortega-Villaizan, Maria del Mar

2017-01-01

Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection  have not been studied yet. Methods: Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs. PMID:29333244

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective.

PubMed

Nombela, Ivan; Carrion, Aurora; Puente-Marin, Sara; Chico, Verónica; Mercado, Luis; Perez, Luis; Coll, Julio; Ortega-Villaizan, Maria Del Mar

2017-01-01

Background : Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection  have not been studied yet. Methods : Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results : IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1 , pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions : Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs.

Utilization of a specific in vitro lymphocyte immunostimulation assay as an aid in detection of brucella-infected cattle not detected by serological tests.

PubMed Central

Kaneene, J M; Johnson, D W; Anderson, R K; Muscoplat, C C

1978-01-01

Studies using the in vitro lymphocyte stimulation test (LST) were conducted with cattle in a dairy herd with a high percentage of reactors to several serological tests for brucellosis. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique. Lymphocytes were cultured using microtitration culture plates. Brucella abortus soluble antigen, at a concentration of 4.4 microgram/culture, was added to the appropriate wells of microtitration culture plates and incubated for 6 days. The lymphocyte stimulation responses were measured by assaying for [3H]thymidine incorporation into DNA. Seroagglutination tests were conducted simultaneously with the LST, and tissues were collected after slaughter of the cattle for bacteriological culture to isolate B. abortus. All 21 animals studied were serologically negative for anti-brucella antibodies. Two of the 21 animals were classified as infected with Brucella by the LST, and B. abortus biotype 1 was isolated from tissues of these same two animals. The LST exhibited significant sensitivity and specificity in this study, and more observations of this nature might strengthen the application of this assay as an aid in the diagnosis of brucellosis. PMID:103888

Immunological responsiveness of frozen-thawed human lymphocytes.

PubMed Central

Strong, D M; Woody, J N; Factor, M A; Ahmed, A; Sell, K W

1975-01-01

Mononuclear cells (10--20 X 10(6)) obtained from human peripheral blood by a standard Ficoll-Hypaque technique were suspended in RPMI 1640 media at 4 degrees C containing 10% foetal calf serum and 7-5% dimethyl sulphoxide (DMSO). Two-millilitre aliquots were cooled at -1 degree C/min in a Cryoson BV-4 programmed freezing system to -30 degrees C, then -5 degrees C/min to -80 degrees C and stored in liquid nitrogen vapor. On the day of testing, cell suspensions were thawed rapidly in a 37 degree C water bath. DMSO was diluted slowly out of the sample and cells resuspended in fresh RPMI 1640. It was found that frozen stored human lymphocytes (FSHL) demonstrated all the characteristics of fresh unfrozen cells. These included their ability to form spontaneous rosettes with sheep erythrocytes ('E' rosettes) and sheep erythrocyte--antibody--complement rosettes ('EAC' rosettes). The presence of surface immunoglobulins and Fc receptors were shown by membrane immunofluorescence to be comparable. In addition, the results show that FSHL respond to mitogens, specific antigens; act as both stimulators and responders in the mixed lymphocyte culture reaction; and exhibit cell-mediated lymphocytotoxicity following in vitro sensitization, or against antibody-coated target cells. PMID:128429

The role of cell size in density gradient electrophoretic separation of mouse leukemia cells according to position in the cell cycle

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Todd, P. W.

1985-01-01

Cultured mouse leukemia cells line L5178Y were subjected to upward electrophoresis in a density gradient and the slower migrating cell populations were enriched in G2 cells. It is indicated that this cell line does not change electrophoretic mobility through the cell cycle. The possibility that increased sedimentation downward on the part of the larger G2 cells caused this separation was explored. Two different cell populations were investigated. The log phase population was found to migrate upward faster than the G2 population, and a similar difference between their velocities and calculated on the basis of a 1 um diameter difference between the two cell populations. The G2 and G1 enriched populations were isolated by Ficoll density gradient sedimentation. The bottom fraction was enriched in G2 cells and the top fraction was enriched with G1 cells, especially when compared with starting materials. The electrophoretic mobilities of these two cell populations did not differ significantly from one another. Cell diameter dependent migration curves were calculated and were found to be different. Families of migration curves that differ when cell size is considered as a parameter are predicted.

Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma.

PubMed

Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Dogan, Askin; Runnebaum, Ingo Bernhard; Dürst, Matthias; Häfner, Norman

2014-02-05

The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*10(5) MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts.

Feasibility studies of oncornavirus production in microcarrier cultures.

PubMed

Manousos, M; Ahmed, M; Torchio, C; Wolff, J; Shibley, G; Stephens, R; Mayyasi, S

1980-06-01

Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis.

Flagellated bacterial motility in polymer solutions

PubMed Central

Martinez, Vincent A.; Schwarz-Linek, Jana; Reufer, Mathias; Wilson, Laurence G.; Morozov, Alexander N.; Poon, Wilson C. K.

2014-01-01

It is widely believed that the swimming speed, v, of many flagellated bacteria is a nonmonotonic function of the concentration, c, of high-molecular-weight linear polymers in aqueous solution, showing peaked v(c) curves. Pores in the polymer solution were suggested as the explanation. Quantifying this picture led to a theory that predicted peaked v(c) curves. Using high-throughput methods for characterizing motility, we measured v and the angular frequency of cell body rotation, Ω, of motile Escherichia coli as a function of polymer concentration in polyvinylpyrrolidone (PVP) and Ficoll solutions of different molecular weights. We find that nonmonotonic v(c) curves are typically due to low-molecular-weight impurities. After purification by dialysis, the measured v(c) and Ω(c) relations for all but the highest-molecular-weight PVP can be described in detail by Newtonian hydrodynamics. There is clear evidence for non-Newtonian effects in the highest-molecular-weight PVP solution. Calculations suggest that this is due to the fast-rotating flagella seeing a lower viscosity than the cell body, so that flagella can be seen as nano-rheometers for probing the non-Newtonian behavior of high polymer solutions on a molecular scale. PMID:25468981

Human cord blood mononuclear cell transplantation for the treatment of premature ovarian failure in nude mice

PubMed Central

Dang, Jianhong; Jin, Zhijun; Liu, Xiaojun; Hu, Dian; Wang, Zhifeng

2015-01-01

Objective: This study explored the potential of human cord blood mononuclear cell (HCMNC) transplantation as a treatment for premature ovarian failure (POF) in a nude mouse model. Methods: Female nude mice were randomly divided into three groups; a normal control group (n = 35), a POF group (POF plus vehicle, n = 35) and a POF plus cell transplantation group (HCMNCs were implanted into the ovaries, n = 35). HCMNCs were isolated by Ficoll density gradient centrifugation and labeled with BrdU. Four weeks after transplantation, the nude mice were sacrificed to determine serum levels of E2, FSH and LH as indicators of ovarian function, and the ovaries were examined both histologically and immunochemically. Results: The transplanted HCMNCs survived in the transplantation group and were detected by BrdU. In the transplantation group, serum levels of E2 significantly increased while serum levels of FSH and LH significantly decreased compared to the POF control group. Additionally, the transplantation group had a recovery in follicle number. Conclusion: HCMNCs can be successfully transplanted into the ovaries of nude mice and can improve ovarian function in POF. PMID:26064319

Induction and identification of rabbit peripheral blood derived dendritic cells

NASA Astrophysics Data System (ADS)

Zhou, Jing; Yang, FuYuan; Chen, WenLi

2012-03-01

Purpose: To study a method of the induction of dendritic cells (DCs) from rabbit peripheral blood. Methods: Peripheral blood cells were removed from rabbit, filtered through nylon mesh. Peripheral blood mononuclear cells (PBMC) were isolated from the blood cells by Ficoll-Hypaque centrifugation (density of 1.077g/cm3).To obtain DCs, PBMC were cultured in RPMI1640 medium containing 10% fetal calf serum, 50U/mL penicillin and streptomycin, referred to subsequently as complete medium, at 37°C in 5% CO2 atmosphere for 4 hours. Nonadherent cells were aspirated, adherent cells were continued incubated in complete medium, supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF, 50ng/ml),and interleukin 4 (IL-4, 50ng/ml) for 9 days. Fluorescein labeled antibodies(anti-CD14, anti-HLA-DR, anti-CD86) were used to sign cells cultured for 3,6,9 days respectively, Then flow cytometry was performed. Results: Ratio of anti-HLA-DR and anti-CD86 labeled cells increased with induction time extension, in contrast with anti-CD14. Conclusion: Dendritic cells can be effectively induced by the method of this experiment, cell maturation status increased with induction time extension.

Combined Cytogenotoxic Effects of Bee Venom and Bleomycin on Rat Lymphocytes: An In Vitro Study

PubMed Central

Abd-Elhakim, Yasmina M.; Khalil, Samah R.; Awad, Ashraf; AL-Ayadhi, Laila Y.

2014-01-01

This study was carried out to determine the cytotoxic and genotoxic effects of bee venom (BV) and/or the chemotherapeutic agent bleomycin (BLM) on healthy isolated rat lymphocytes utilizing morphometric and molecular techniques. Using the Ficoll-Histopaque density gradient centrifugation technique, lymphocytes were isolated, divided into groups, and subjected to BV and/or BLM at incubation medium concentrations of 10 or 20 μg/mL respectively for 24 and 72 hrs. An MTT assay and fluorescent microscopy examinations were used to assess the cytotoxic effects. To determine the predominant type of BV and/or BLM-induced cell death, LDH release assay was employed beside quantitative expression analyses of the apoptosis-related genes (Caspase-3 and Bcl-2). The genotoxic effects of the tested compounds were evaluated via DNA fragmentation assay. The results of these assays demonstrated that BV potentiates BLM-induced cytotoxicity through increased LDH release and diminished cell viability. Nevertheless, BV significantly inhibited the BLM-induced DNA damage. The results verify that BV significantly attenuates the genotoxic effects of BLM on noncancerous isolated rat lymphocytes but does not diminish BLM cytotoxicity. PMID:24822179

Role of macromolecular crowding and salt ions on the structural-fluctuation of a highly compact configuration of carbonmonoxycytochrome c.

PubMed

Kumar, Rajesh; Sharma, Deepak; Jain, Rishu; Kumar, Sandeep; Kumar, Rajesh

2015-12-01

Carbonmonoxycytochrome c refolds to a native-like compact state (NCO-state), where the non-native Fe(2+)-CO interaction persists. Structural and molecular properties extracted from CD, fluorescence and NMR experiments reveal that the NCO-state shows the generic properties of molten globules. Slow thermal-dissociation of CO transforms the NCO-state to native-state (N-state), where the native Fe(2+)-M80 bond recovers. To determine the role of crowding agents and salt ions on the structural-fluctuation of NCO, the kinetic and thermodynamic parameters for CO-dissociation from NCO (NCO→N+CO) were measured at varying concentrations of crowding agents (dextran 70, dextran 40, ficoll 70) and salt ions (anion: ClO4(-), I(-), Br(-), NO3(-), Cl(-); cation: NH4(+), K(+), Na(+)). As [crowding agent] or [ion] is increased, the rate coefficient of CO-dissociation (kdiss) decreases exponentially. Furthermore, the extent of decrease in kdiss is found to be dependent on (i) size, charge density and charge dispersion of the ion, and (ii) size, shape, and viscosity of the crowding agent. Copyright © 2015 Elsevier B.V. All rights reserved.

Multipotent Mesenchymal Stromal Stem Cell Expansion by Plating Whole Bone Marrow at a Low Cellular Density: A More Advantageous Method for Clinical Use

PubMed Central

Mareschi, Katia; Rustichelli, Deborah; Calabrese, Roberto; Gunetti, Monica; Sanavio, Fiorella; Castiglia, Sara; Risso, Alessandra; Ferrero, Ivana; Tarella, Corrado; Fagioli, Franca

2012-01-01

Mesenchymal stem cells (MSCs) are a promising source for cell therapy due to their pluripotency and immunomodulant proprieties. As the identification of “optimal” conditions is important to identify a standard procedure for clinical use. Percoll, Ficoll and whole bone marrow directly plated were tested from the same sample as separation methods. The cells were seeded at the following densities: 100 000, 10 000, 1000, 100, 10 cells/cm2. After reaching confluence, the cells were detached, pooled and re-plated at 1000, 500, 100, and 10 cells/cm2. Statistical analyses were performed. Cumulative Population Doublings (PD) did not show significant differences for the separation methods and seeding densities but only for the plating density. Some small quantity samples plated in T25 flasks at plating densities of 10 and 100 cells/cm2 did not produce any expansion. However, directly plated whole bone marrow resulted in a more advantageous method in terms of CFU-F number, cellular growth and minimal manipulation. No differences were observed in terms of gross morphology, differentiation potential or immunophenotype. These data suggest that plating whole bone marrow at a low cellular density may represent a good procedure for MSC expansion for clinical use. PMID:23715383

Two-dimensional analysis of human lymphocyte proteins. III. Preliminary report on a marker for the early detection and diagnosis of infectious mononucleosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willard, K.E.

1982-04-01

Two-dimensional gel electrophoretic patterns of human peripheral blood leukocytes from 12 patients with infectious mononucleosis were prepared by use of the ISO-DALT system. Before the two-dimensional separation, the leukocytes were purified by Ficoll-Paque gradient centrifugation and labeled overnight with (/sup 35/S) methionine. Quantitative increases in two proteins were detected in the patterns of infected leukocytes from the patients as compared with controls. Fluorescence-activated cell sorting of leukocytes from normal human peripheral blood before subsequent two-dimensional gel analysis revealed that the dramatic increase in one of these proteins (Inmono:2) could be due to shifts in the population ratios of lymphocytes, monocytes,more » and granulocytes. In contrast, the appearance in the infected leukocytes of a second protein, Inmono:1, could not be accounted for by cell-population shifts. Increased amounts of these two proteins have been found in every patient studied who had clinically detectable infectious mononucleosis. In addition, a patient who displayed symptoms of infectious mononucleosis but who did not have a positive result in the MONOSPOT test (Ortho) until three weeks after our analysis also demonstrated increased relative amounts of these proteins in his leukocyte pattern.« less

Vitrification of mouse embryos using the thin plastic strip method

PubMed Central

Hur, Yong Soo; Ann, Ji Young; Maeng, Ja Young; Park, Miji; Park, Jeong Hyun; Yoon, Jung; Yoon, San Hyun; Hur, Chang Young; Lee, Won Don; Lim, Jin Ho

2012-01-01

Objective The aim of this study was to compare vitrification optimization of mouse embryos using electron microscopy (EM) grid, cryotop, and thin plastic strip (TPS) containers by evaluating developmental competence and apoptosis rates. Methods Mouse embryos were obtained from superovulated mice. Mouse cleavage-stage, expanded, hatching-stage, and hatched-stage embryos were cryopreserved in EM grid, cryotop, and TPS containers by vitrification in 15% ethylene glycol, 15% dimethylsulfoxide, 10 µg/mL Ficoll, and 0.65 M sucrose, and 20% serum substitute supplement (SSS) with basal medium, respectively. For the three groups in which the embryos were thawed in the EM grid, cryotop, and TPS containers, the thawing solution consisted of 0.25 M sucrose, 0.125 M sucrose, and 20% SSS with basal medium, respectively. Rates of survival, re-expansion, reaching the hatched stage, and apoptosis after thawing were compared among the three groups. Results Developmental competence after thawing of vitrified expanded and hatching-stage blastocysts using cryotop and TPS methods were significantly higher than survival using the EM grid (p<0.05). Also, apoptosis positive nuclei rates after thawing of vitrified expanded blastocysts using cryotop and TPS were significantly lower than when using the EM grid (p<0.05). Conclusion The TPS vitrification method has the advantages of achieving a high developmental ability and effective preservation. PMID:23346525

Allotype suppression induced in the adoptive transfer system: the variables of the system and an apparent absence of a role for T cells.

PubMed

Lee, S K; Dresser, D W

1981-04-01

A study has been made of the variables concerned in allotype suppression of adult spleen cells in the adoptive transfer system. These are; SRBC (antigen) dose; the dose and timing of injection of anti-allotype serum IgG; the number of spleen cells transferred and whether these cells were taken from primed or unprimed donors. Adoptively transferred primed cells are considerably less susceptible to suppression by concomitantly injected anti-allotype serum IgG than are unprimed spleen cells. Injection of anti-allotype serum during the period after adoptive transfer, has shown that primed cells loose their susceptibility sooner (2 days) than the unprimed cells (4 days). Allotype heterozygous CBA spleen cells are less susceptible heterozygous CBA spleen cells are less susceptible to allotype suppression than either allotypically homozygous or heterozygous non-H-2k cells (H-2b,d, or s). Allotype suppression of the TI IgG response to DNP-Ficoll was measured 7 days after adoptive transfer of allotype-homozygous cells from both normal and nude CBA mice (unprimed). The results indicate that T cells do not play a role in the initiation of short-term allotype suppression in the adoptive transfer system.

Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells.

PubMed

Kazemi, E; Mortazavi, S M J; Ali-Ghanbari, A; Sharifzadeh, S; Ranjbaran, R; Mostafavi-Pour, Z; Zal, F; Haghani, M

2015-09-01

Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated.

Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells

PubMed Central

Kazemi, E.; Mortazavi, S. M. J.; Ali-Ghanbari, A.; Sharifzadeh, S.; Ranjbaran, R.; Mostafavi-pour, Z.; Zal, F.; Haghani, M.

2015-01-01

Background Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. Objective Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. Method 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. Results Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. Conclusion The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated. PMID:26396966

Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma

PubMed Central

Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Dogan, Askin; Runnebaum, Ingo Bernhard; Dürst, Matthias; Häfner, Norman

2014-01-01

The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*105 MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts. Abbreviations: CTC – circulating tumour cells, CxCa – cervical cancer, DD-RT-PCR – Digital-Direct Reverse Transcriptase PCR, HPV – Human Papilloma Virus, MNBC – mononuclear blood cells, ICC – immunocytochemistry. PMID:24496006

CXCR-4 expression by circulating endothelial progenitor cells and SDF-1 serum levels are elevated in septic patients.

PubMed

Patry, Christian; Stamm, Daniela; Betzen, Christian; Tönshoff, Burkhard; Yard, Benito A; Beck, Grietje Ch; Rafat, Neysan

2018-01-01

Endothelial progenitor cell (EPC) numbers are increased in septic patients and correlate with survival. In this study, we investigated, whether surface expression of chemokine receptors and other receptors important for EPC homing is upregulated by EPC from septic patients and if this is associated with clinical outcome. Peripheral blood mononuclear cells from septic patients ( n  = 30), ICU control patients ( n  = 11) and healthy volunteers ( n  = 15) were isolated by Ficoll density gradient centrifugation. FACS-analysis was used to measure the expression of the CXC motif chemokine receptors (CXCR)-2 and - 4, the receptor for advanced glycation endproducts (RAGE) and the stem cell factor receptor c-Kit. Disease severity was assessed via the Simplified Acute Physiology Score (SAPS) II. The serum concentrations of vascular endothelial growth factor (VEGF), stromal cell-derived factor (SDF)-1α and angiopoietin (Ang)-2 were determined with Enzyme linked Immunosorbent Assays. EPC from septic patients expressed significantly more CXCR-4, c-Kit and RAGE compared to controls and were associated with survival-probability. Significantly higher serum concentrations of VEGF, SDF-1α and Ang-2 were found in septic patients. SDF-1α showed a significant association with survival. Our data suggest that SDF-1α and CXCR-4 signaling could play a crucial role in EPC homing in the course of sepsis.

Lymphocyte-platelet crosstalk in Graves' disease.

PubMed

Kuznik, Boris I; Vitkovsky, Yuri A; Gvozdeva, Olga V; Solpov, Alexey V; Magen, Eli

2014-03-01

Platelets can modulate lymphocytes' role in the pathophysiology of thyroid autoimmune diseases. The present study was performed to clarify the status of platelet-lymphocyte subpopulations aggregation in circulating blood in patients with Graves' disease (GD). One hundred and fifty patients with GD (GD group) and 45 hyperthyroid patients with toxic multinodular goiter (TMG group) were recruited in the study. Control group consisted 150 healthy subjects. Immunophenotyping of lymphocytes was performed by flow cytometry. Detection of lymphocyte-platelet aggregates (LPAs) was done using light microscope after Ficoll-gradient centrifugation. The group of GD patients exhibited reduced CD8 lymphocyte and higher CD19 cell counts compared with TMG group and healthy controls. A greater number of activated CD3, HLA-DR+ lymphocytes were observed in GD than in TMG group and control group. GD group was characterized by lower blood platelet count (232 ± 89 × 10 cells/µL) than TMG group (251 ± 97 × 10 cells/µL; P < 0.05) and control group (262 ± 95 × 10 cells/µL; P < 0.05). In GD group, more platelet-bound lymphocytes (332 ± 91 /µL) were found than that in TMG group (116 ± 67/µL, P < 0.005) and control group (104 ± 58 /µL; P < 0.001). GD is associated with higher levels of activated lymphocytes and lymphocyte-platelet aggregates.

Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency

PubMed Central

Walter, Jolan E.; Rucci, Francesca; Patrizi, Laura; Recher, Mike; Regenass, Stephan; Paganini, Tiziana; Keszei, Marton; Pessach, Itai; Lang, Philipp A.; Poliani, Pietro Luigi; Giliani, Silvia; Al-Herz, Waleed; Cowan, Morton J.; Puck, Jennifer M.; Bleesing, Jack; Niehues, Tim; Schuetz, Catharina; Malech, Harry; DeRavin, Suk See; Facchetti, Fabio; Gennery, Andrew R.; Andersson, Emma; Kamani, Naynesh R.; Sekiguchi, JoAnn; Alenezi, Hamid M.; Chinen, Javier; Dbaibo, Ghassan; ElGhazali, Gehad; Fontana, Adriano; Pasic, Srdjan; Detre, Cynthia; Terhorst, Cox

2010-01-01

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro–B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP–keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4+ T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell–activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations. PMID:20547827

[Harringtonine induces apoptosis in NB4 cells through down-regulation of Mcl-1].

PubMed

Wu, Chunxiao; Shen, Hongqiang; Xia, Dajing

2013-07-01

To investigate the growth inhibition effect, cytotoxicity and apoptotic induction of harringtonine (HT) in human acute promyelocytic leukemia (APL) NB4 cells,and the related mechanism. NB4 cells were treated with HT. Total cell numbers were counted by hemocytometer, and cell viabilities were determined by trypan blue exclusion. Apoptotic cells were determined by fluorescence microscopy and FACS after staining with AO and EB or PI, respectively. The cleavage of PARP and the activation of Bax and the expression of anti-apoptotic proteins were determined by Western Blot. siRNA was used to silence the expression of target genes. Primary cells were isolated following Ficoll-Hypaque density gradient centrifugation method. HT inhibited cell growth and induced apoptosis of NB4 cells in a dose- and time-dependent manner. Apoptosis induced by HT was correlated with the down-regulation of Mcl-1 and the cleavage of PARP, while HT did not affect the protein level of Bax and Bak or change the protein level of Bcl-2. The silence of Bcl-XL sensitized HT-induced apoptosis in NB4 cells.Apoptosis induced by HT in primarily cultured APL cells was also correlated with the down-regulation of Mcl-1. HT inhibits cell growth and induces apoptosis in NB4 cells and primarily cultured APL cells, which may be associated with down-regulation of Mcl-1.

A Metabolic Biofuel Cell: Conversion of Human Leukocyte Metabolic Activity to Electrical Currents

PubMed Central

2011-01-01

An investigation of the electrochemical activity of human white blood cells (WBC) for biofuel cell (BFC) applications is described. WBCs isolated from whole human blood were suspended in PBS and introduced into the anode compartment of a proton exchange membrane (PEM) fuel cell. The cathode compartment contained a 50 mM potassium ferricyanide solution. Average current densities between 0.9 and 1.6 μA cm-2 and open circuit potentials (Voc) between 83 and 102 mV were obtained, which were both higher than control values. Cyclic voltammetry was used to investigate the electrochemical activity of the activated WBCs in an attempt to elucidate the mechanism of electron transfer between the cells and electrode. Voltammograms were obtained for the WBCs, including peripheral blood mononuclear cells (PBMCs - a lymphocyte-monocyte mixture isolated on a Ficoll gradient), a B lymphoblastoid cell line (BLCL), and two leukemia cell lines, namely K562 and Jurkat. An oxidation peak at about 363 mV vs. SCE for the PMA (phorbol ester) activated primary cells, with a notable absence of a reduction peak was observed. Oxidation peaks were not observed for the BLCL, K562 or Jurkat cell lines. HPLC confirmed the release of serotonin (5-HT) from the PMA activated primary cells. It is believed that serotonin, among other biochemical species released by the activated cells, contributes to the observed BFC currents. PMID:21569243

In vitro effects of oil's fatty acids on T cell function in gestational diabetic pregnant women and their newborns.

PubMed

Djelti, Farah; Merzouk, Hafida; Merzouk, Sid Ahmed; Narce, Michel

2015-07-01

The aim of this investigation was to determine the in vitro effects of linseed, olive and Nigel oils on T cell proliferation and function in gestational diabetes. Blood samples were collected from 40 control healthy and 32 gestational diabetic mothers and their newborns. Peripheral blood lymphocytes were isolated using a density gradient of Ficoll. T cell proliferation, interleukin-2 and -4 (IL-2, IL-4) secretion, fatty acid composition and intracellular oxidative status were investigated. Mitogen (Concanavalin A) stimulated lymphocyte proliferation, IL-2 secretion, intracellular reduced glutathione levels, superoxide dismutase (SOD) and catalase activities were lower while intracellular malondialdehyde (MDA) and carbonyl proteins were higher in diabetic mothers and in their newborns as compared to their respective controls. Linseed oil induced a reduction in T-lymphocyte proliferation and IL-2 production, and alpha linolenic acid membrane enrichment in both diabetic and control groups. In the presence of Nigel oil, T-lymphocyte proliferation and IL-2 secretion, phospholipid linoleic and oleic acids were enhanced. Olive oil had no effect on lymphocyte proliferation in all groups. Linseed, olive and Nigel oils induced an increase in T cell levels of reduced glutathione levels and in activities of catalase and SOD with a concomitant decrease in MDA and carbonyl protein contents. Linseed, olive and Nigel oils had beneficial effects on T cell functions in gestational diabetes. © 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

An effective purification method using large bottles for human pancreatic islet isolation

PubMed Central

Shimoda, Masayuki; Itoh, Takeshi; Iwahashi, Shuichi; Takita, Morihito; Sugimoto, Koji; Kanak, Mazhar A.; Chujo, Daisuke; Naziruddin, Bashoo; Levy, Marlon F.; Grayburn, Paul A.; Matsumoto, Shinichi

2012-01-01

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification. PMID:23221740

Roles of intracellular ice formation, vitrification of cell water, and recrystallisation of intracellular ice on the survival of mouse embryos and oocytes.

PubMed

Mazur, Peter; Paredes, Estefania

2016-03-01

Mazur and collaborators began examining the validity of initial views regarding mouse oocyte and embryo vitrification and found that most are partially or fully wrong. First, the relative effects of warming and cooling rates on the survival of mouse oocytes subjected to a vitrification procedure were determined. The high sensitivity to warming rate strongly suggests that the lethality of slow warming is a consequence of either the crystallisation of intracellular glassy water during warming or the recrystallisation during slow warming of small intracellular crystals that had formed during cooling. Warming rates of 107°C min-1 were achieved in 0.1-µL drops of ethylene glycol-acetamide-Ficoll-sucrose (EAFS) solution plus a small amount of India ink on Cryotops warmed using an infrared laser pulse. Under these conditions, survival rates of 90% were obtained even when mouse oocytes were suspended in 0.3× EAFS, a concentration that falls in the range that many cells can tolerate. A second important finding was that the survival of oocytes is more dependent on the osmotic withdrawal of much of the intracellular water before vitrification than it is on the penetration of cryoprotective solutes into the cells. Herein we review the roles of internal ice formation, vitrification and recrystallisation. It remains to be seen how widely these findings will be applicable to other types of cells and tissues from other species.

Load- and polysaccharide-dependent activation of the Na+-type MotPS stator in the Bacillus subtilis flagellar motor.

PubMed

Terahara, Naoya; Noguchi, Yukina; Nakamura, Shuichi; Kami-Ike, Nobunori; Ito, Masahiro; Namba, Keiichi; Minamino, Tohru

2017-04-05

The flagellar motor of Bacillus subtilis possesses two distinct H + -type MotAB and Na + -type MotPS stators. In contrast to the MotAB motor, the MotPS motor functions efficiently at elevated viscosity in the presence of 200 mM NaCl. Here, we analyzed the torque-speed relationship of the Bacillus MotAB and MotPS motors over a wide range of external loads. The stall torque of the MotAB and MotPS motors at high load was about 2,200 pN nm and 220 pN nm, respectively. The number of active stators in the MotAB and MotPS motors was estimated to be about ten and one, respectively. However, the number of functional stators in the MotPS motor was increased up to ten with an increase in the concentration of a polysaccharide, Ficoll 400, as well as in the load. The maximum speeds of the MotAB and MotPS motors at low load were about 200 Hz and 50 Hz, respectively, indicating that the rate of the torque-generation cycle of the MotPS motor is 4-fold slower than that of the MotAB motor. Domain exchange experiments showed that the C-terminal periplasmic domain of MotS directly controls the assembly and disassembly dynamics of the MotPS stator in a load- and polysaccharide-dependent manner.

Evaluation of immune functions in captive immature loggerhead sea turtles (Caretta caretta).

PubMed

Rousselet, Estelle; Levin, Milton; Gebhard, Erika; Higgins, Benjamin M; DeGuise, Sylvain; Godard-Codding, Céline A J

2013-11-15

Sea turtles face numerous environmental challenges, such as exposure to chemical pollution and biotoxins, which may contribute to immune system impairment, resulting in increased disease susceptibility. Therefore, a more thorough assessment of the host's immune response and its susceptibility is needed for these threatened and endangered animals. In this study, the innate and acquired immune functions of sixty-five clinically healthy, immature, captive loggerhead sea turtles (Caretta caretta) were assayed using non-lethal blood sample collection. Functional immune assays were developed and/or optimized for this species, including mitogen-induced lymphocyte proliferation, natural killer (NK) cell activity, phagocytosis, and respiratory burst. Peripheral blood mononuclear cells (PBMC) and phagocytes were isolated by density gradient centrifugation on Ficoll-Paque and discontinuous Percoll gradients, respectively. The T lymphocyte mitogens ConA significantly induced lymphocyte proliferation at 1 and 2 μg/mL while PHA significantly induced lymphocyte proliferation at 5 and 10 μg/mL. The B lymphocyte mitogen LPS significantly induced proliferation at 1 μg/mL. Monocytes demonstrated higher phagocytic activity than eosinophils. In addition, monocytes exhibited respiratory burst. Natural killer cell activity was higher against YAC-1 than K-562 target cells. These optimized assays may help to evaluate the integrity of loggerhead sea turtle's immune system upon exposure to environmental contaminants, as well as part of a comprehensive health assessment and monitoring program. Copyright © 2013 Elsevier B.V. All rights reserved.

Methionine enkephalin (MENK) improves lymphocyte subpopulations in human peripheral blood of 50 cancer patients by inhibiting regulatory T cells (Tregs)

PubMed Central

Wang, Qiushi; Gao, Xinghua; Yuan, Zhe; Wang, Zhe; Meng, Yiming; Cao, Yan; Plotnikoff, Nicolas P; Griffin, Noreen; Shan, Fengping

2014-01-01

MENK, a penta-peptide is considered as being involved in the regulatory feedback loop between the immune and neuroendocrine systems, with marked modulation of various functions of human immune cells. The aim of the present work was to investigate change of lymphocyte subpopulations in peripheral blood of 50 cancer patients before and after treatment with MENK. Peripheral blood mononuclear cells (PBMCs) of peripheral blood from 50 cancer patients were isolated by density gradient centrifugation using Ficoll-Paque solution and cultured with MENK. We measured proliferation of total nucleated cells, subpopulations of individual CD4+T cells, CD8+T cells, CD4+CD25+ regulatory T cells (Treg), natural killer cells (NK) before and after treatment with 10-12M MENK in cell culture by flow cytometry (FCM). Our results indicated that MENK showed a strong inhibiting effect on Treg cells while it stimulated marked proliferation of other lymphocyte subpopulations. All data obtained were of significance statistically. It was therefore concluded that MENK could work as a strong immune booster with great potential in restoring damaged human immune system and we could consider MENK as a drug to treat cancer patients, whose immune systems are damaged by chemotherapy or radiotherapy. Furthermore we could consider MENK as a chemotherapy additive, which would sustain immune system of cancer patients during the process of chemotherapy to get maximized efficacy with minimized side effect. PMID:25424790

Load- and polysaccharide-dependent activation of the Na+-type MotPS stator in the Bacillus subtilis flagellar motor

PubMed Central

Terahara, Naoya; Noguchi, Yukina; Nakamura, Shuichi; Kami-ike, Nobunori; Ito, Masahiro; Namba, Keiichi; Minamino, Tohru

2017-01-01

The flagellar motor of Bacillus subtilis possesses two distinct H+-type MotAB and Na+-type MotPS stators. In contrast to the MotAB motor, the MotPS motor functions efficiently at elevated viscosity in the presence of 200 mM NaCl. Here, we analyzed the torque-speed relationship of the Bacillus MotAB and MotPS motors over a wide range of external loads. The stall torque of the MotAB and MotPS motors at high load was about 2,200 pN nm and 220 pN nm, respectively. The number of active stators in the MotAB and MotPS motors was estimated to be about ten and one, respectively. However, the number of functional stators in the MotPS motor was increased up to ten with an increase in the concentration of a polysaccharide, Ficoll 400, as well as in the load. The maximum speeds of the MotAB and MotPS motors at low load were about 200 Hz and 50 Hz, respectively, indicating that the rate of the torque-generation cycle of the MotPS motor is 4-fold slower than that of the MotAB motor. Domain exchange experiments showed that the C-terminal periplasmic domain of MotS directly controls the assembly and disassembly dynamics of the MotPS stator in a load- and polysaccharide-dependent manner. PMID:28378843

Transport of Spherical Particles Through Fibrous Media and a Row of Parallel Cylinders: Applications to Glomerular Filtration.

PubMed

Punyaratabandhu, Numpong; Kongoup, Pimkhwan; Dechadilok, Panadda; Katavetin, Pisut; Triampo, Wannapong

2017-12-01

Viewed in renal physiology as a refined filtration device, the glomerulus filters large volumes of blood plasma while keeping proteins within blood circulation. Effects of macromolecule size and macromolecule hydrodynamic interaction with the nanostructure of the cellular layers of the glomerular capillary wall on the glomerular size selectivity are investigated through a mathematical simulation based on an ultrastructural model. The epithelial slit, a planar arrangement of fibers connecting the epithelial podocytes, is represented as a row of parallel cylinders with nonuniform spacing between adjacent fibers. The mean and standard deviation of gap half-width between its fibers are based on values recently reported from electron microscopy. The glomerular basement membrane (GBM) is represented as a fibrous medium containing fibers of two different sizes: the size of type IV collagens and that of glycosaminoglycans (GAGs). The endothelial cell layer is modeled as a layer full of fenestrae that are much larger than solute size and filled with GAGs. The calculated total sieving coefficient agrees well with the sieving coefficients of ficolls obtained from in vivo urinalysis in humans, whereas the computed glomerular hydraulic permeability also falls within the range estimated from human glomerular filtration rate (GFR). Our result indicates that the endothelial cell layer and GBM significantly contribute to solute and fluid restriction of the glomerular barrier, whereas, based on the structure of the epithelial slit obtained from electron microscopy, the contribution of the epithelial slit could be smaller than previously believed.

Comparative studies of mononuclear phagocyte function in patients with Crohn's disease and colon neoplasms.

PubMed Central

Beeken, W L; St Andre-Ukena, S; Gundel, R M

1983-01-01

Phagocytosis and cellular cytotoxicity by mononuclear phagocytes of blood and intestinal mucosa were studied in patients with Crohn's disease and large bowel neoplasms. Antibody coated sheep erythrocytes were used for phagocytic assays and cellular cytotoxicity in vitro was measured by 24 hour isotope release from 75Selenium methionine-labelled RPMI 4788 human cancer cell cultures in the presence of mononuclear phagocyte-enriched effector populations. The mean percent of mononuclear phagocytes in Ficoll-Hypaque purified mononuclear cell suspensions of blood of healthy controls was 25.9 compared with 44.6 in patients with Crohn's disease, 45.6 in patients with colon neoplasms and 11.6 in intestinal mucosa. Phagocytic indices were similar in all groups, but the phagocytic capacity of mucosal macrophages was twice that of blood monocytes. Mean cytotoxicity of monocytes of patients with Crohn's disease was 12.8% compared with 22.9% for monocytes from normal controls, and 29.4% for patients with colon tumours. Mean cytotoxicity by mucosal macrophages was 18.0% compared with 13.2% by mucosal lymphocyte populations. Exposure of monocytes of Crohn's disease patients to bacterial lipopolysaccharide modestly increased cytotoxicity, but exposure did not alter phagocytosis by monocytes of patients or controls. The results indicate that monocytes of patients with Crohn's disease exhibit subnormal in vitro cytotoxicity. Mucosal macrophages from patients with various diseases show enhanced phagocytosis compared with blood monocytes, and they can mediate cellular cytotoxicity in vitro. PMID:6629113

Evaluation of interleukin 12 and CD56+ lymphocyte cells in pediatric hematopoietic stem cell transplantation for early diagnosis of acute graft versus host disease.

PubMed

Tootoonchian, Raziyeh; Pak, Fatemeh; Ardekani, Ali M; Sehati, Nasrin; Abedi-Valugerdi, Manuchehr; Kokhaei, Parviz

2016-11-01

The present study tried to explain CD56+ lymphocyte cells activities and possible prognostic role of these cells in Graft-Versus-Host-Disease (GVHD). The role of IL-12 activation and function is of interest in this study. Peripheral blood samples of 51 Hematopoietic Stem Cell Transplantation (HSCT) recipients collected at before (day -8) and after (days 7 and 14). PBMC were collected by Ficoll separation and analyzed by Flow Cytometry using triple antibody (CD45-PerCP, CD56-FITC, and CD69-PE staining and control antibody. Levels of the cytokine IL-12 in the patient's serum were evaluated by ELISA. Percentage of CD56+ lymphocytes (CD56+ bright ) cells was significantly increased at day 14 in patients with acute GVHD and percentage of lymphocytes expressing CD69 was significantly increased at days 7 and 14 posts HSCT in patients with acute GVHD in comparison to those in non-GVHD patients. Baseline serum IL-12 levels (pre-HSCT, day -8) were significantly higher in those HSCT recipients who did not develop GVHD. This study showed that post-transplant CD56+ lymphocytes and pre-transplant serum levels of IL-12 play significant roles in the induction of and protection against GVHD, respectively. The increase in the percentage of CD69+ cells indicates the activation of lymphocyte in acute GVHD group. Copyright © 2016 Elsevier B.V. All rights reserved.

[CD34(+)/CD123(+) cell sorting from the patients with leukemia by Midi MACS method].

PubMed

Wang, Guang-Ping; Cao, Xin-Yu; Xin, Hong-Ya; Li, Qun; Qi, Zhen-Hua; Chen, Fang-Ping

2006-10-01

The aim of this study was to sort the CD34(+)/CD123(+) cells from the bone marrow cells of patients with acute myeloid leukemia (AML) by Midi MACS method. Firstly, the bone marrow mononuclear cells (BMMNC) were isolated from the patients with AML with Ficoll Paque, CD34(+) cells were then isolated by Midi MACS method followed by the isolation of CD34(+)/CD123(+) cells from the fraction of CD34(+) cells. The enrichment and recovery of CD34(+) and CD34(+)/CD123(+) cells were assayed by FACS technique. The results showed that the enrichment of CD34(+) cells was up to 98.73%, its average enrichment was 95.6%, and the recovery of CD34(+) was 84.6%, its average recovery was 51% after the first round sorting, by the second round sorting, the enrichment of CD34(+)/CD123(+) cells was up to 99.23%, its average enrichment was 83%. With regard to BMMNCs before sorting, the recovery of CD34(+)/CD123(+) was 34%. But, on the CD34(+) cells obtained by the first round sorting, its recovery was 56%. In conclusion, these results confirmed that the method of Midi MACS sorting can be applied to sort CD34(+)/CD123(+) cells from the bone marrow cells of AML patients, which give rise to the similar enrichment and recovery of the sorted cells with that of literature reported by the method of FACS.

Downregulated Expression of TRPV2 in Peripheral Blood Cells following Acute Myocardial Infarction Is Inversely Correlated with Serum Levels of CRP and Troponin I.

PubMed

Rozenbaum, Zach; Cohen, Lena; Bigelman, Einat; Shacham, Yacov; Keren, Gad; Entin-Meer, Michal

We have recently shown that the transient receptor potential vanilloid 2 (TRPV2) channel is exclusively upregulated in rat/murine peri-infarct monocytes/macrophages following an acute myocardial infarction (AMI), and that this overexpression might be detrimental for cardiac recovery. We aimed to characterize the expression levels of TRPV2 in peripheral blood mononuclear cells (PBMCs) of AMI patients relative to individuals with normal coronaries, and to analyze potential associations with inflammatory and cardiac ischemic markers. Patients who underwent coronary angiography due to AMI or chest pain were prospectively included. PBMCs were isolated from whole blood by Ficoll gradient centrifugation. TRPV2 expression was analyzed by real-time PCR. C-reactive protein (CRP) and troponin I (TpI) levels were determined at the central chemistry laboratory; interleukin 6 and insulin-like growth factor (IGF)-1 levels were tested by ELISA. Following AMI, the number of TRPV2-expressing PBMCs was reduced when compared to in patients with normal coronaries. An inverse correlation was documented between the numbers of circulating macrophages and TRPV2 expression. Additionally, TRPV2 expression was inversely correlated with CRP and TpI and directly correlated with serum IGF-1. We assume that peripheral TRPV2 downregulation occurs concomitantly with the accumulation of TRPV2-white blood cells in the peri-infarct zone. TRPV2 may thus represent a novel target for treatment in the acute phase after MI. © 2018 S. Karger AG, Basel.

Factors affecting storage of Slovak native rabbit semen in the gene bank.

PubMed

Kulíková, Barbora; Oravcová, Marta; Baláži, Andrej; Supuka, Peter; Chrenek, Peter

2017-10-01

In this study, fresh and frozen-thawed semen of Nitra and Zobor rabbit breeds were evaluated for potential inter-breed or inter-male differences in sperm quality traits. Individual male semen from four rabbits of each breed were diluted (v:v; 1:1) in a freezing medium composed of a commercial diluent, 16% of dimethyl sulphoxide (DMSO), 4% of Ficoll 70 and 2% of sucrose and frozen in liquid nitrogen vapours before being plunged into liquid nitrogen. Different motility traits, viability and plasma membrane integrity of fresh and frozen-thawed semen were evaluated in vitro using computer-assisted sperm analysis and flow cytometry. To evaluate the sperm fertilization ability, artificial insemination of fresh and frozen-thawed sperm was performed. Our results showed the effect of breed (P ≤ 0.05) on frozen-thawed sperm viability and plasma membrane integrity. Moreover, individual variability in semen quality among the rabbits was revealed (0.31 to 0.71 among quality traits). Our results thereby confirmed that the cryopreservation procedure could not ensure comparable sperm post-thaw survival for different breeds or males. Nevertheless, correlations between numbers of fresh total motile and progressively moving sperm and several quality parameters measured post thawing were revealed. Therefore, we suggest that the objective assessment of fresh rabbit sperm motility may be an effective indicator of frozen-thawed semen quality. Consequently, regular semen assessment is required in order to preserve good-quality insemination doses from native breeds.

Directly observed reversible shape changes and hemoglobin stratification during centrifugation of human and Amphiuma red blood cells.

PubMed

Hoffman, Joseph F; Inoué, Shinya

2006-02-21

This paper describes changes that occur in human and Amphiuma red blood cells observed during centrifugation with a special microscope. Dilute suspensions of cells were layered, in a centrifuge chamber, above an osmotically matched dense solution, containing Nycodenz, Ficoll, or Percoll (Pharmacia) that formed a density gradient that allowed the cells to slowly settle to an equilibrium position. Biconcave human red blood cells moved downward at low forces with minimum wobble. The cells oriented vertically when the force field was increased and Hb sedimented as the lower part of each cell became bulged and assumed a "bag-like" shape. The upper centripetal portion of the cell became thinner and remained biconcave. These changes occurred rapidly and were completely reversible upon lowering the centrifugal force. Bag-shaped cells, upon touching red cells in rouleau, immediately reverted to biconcave disks as they flipped onto a stack. Amphiuma red cells displayed a different type of reversible stratification and deformation at high force fields. Here the cells became stretched, with the nucleus now moving centrifugally, the Hb moving centripetally, and the bottom of the cells becoming thinner and clear. Nevertheless, the distribution of the marginal bands at the cells' rim was unchanged. We conclude that centrifugation, per se, while changing a red cell's shape and the distribution of its intracellular constituents, does so in a completely reversible manner. Centrifugation of red cells harboring altered or missing structural elements could provide information on shape determinants that are still unexplained.

Introducing a New Experimental Islet Transplantation Model using Biomimetic Hydrogel and a Simple High Yield Islet Isolation Technique.

PubMed

Mohammadi Ayenehdeh, Jamal; Niknam, Bahareh; Hashemi, Seyed Mahmoud; Rahavi, Hossein; Rezaei, Nima; Soleimani, Masoud; Tajik, Nader

2017-07-01

Islet transplantation could be an ideal alternative treatment to insulin therapy for type 1 diabetes Mellitus (T1DM). This clinical and experimental field requires a model that covers problems such as requiring a large number of functional and viable islets, the optimal transplantation site, and the prevention of islet dispersion. Hence, the methods of choice for isolation of functional islets and transplantation are crucial. The present study has introduced an experimental model that overcomes some critical issues in islet transplantation, including in situ pancreas perfusion by digestive enzymes through common bile duct. In comparison with conventional methods, we inflated the pancreas in Petri dishes with only 1 ml collagenase type XI solution, which was followed by hand-picking isolation or Ficoll gradient separation to purify the islets. Then we used a hydrogel composite in which the islets were embedded and transplanted into the peritoneal cavity of the streptozotocin-induced diabetic C57BL/6 mice. As compared to the yield of the classical methods, in our modified technique, the mean yield of isolation was about 130-200 viable islets/mouse pancreas. In vitro glucose-mediated insulin secretion assay indicated an appropriate response in isolated islets. In addition, data from in vivo experiments revealed that the allograft remarkably maintained blood glucose levels under 400 mg/dl and hydrogel composite prevents the passage of immune cells. In the model presented here, the rapid islet isolation technique and the application of biomimetic hydrogel wrapping of islets could facilitate islet transplantation procedures.

Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

PubMed Central

Chico, Verónica; Puente-Marin, Sara; Ciordia, Sergio; Mena, María Carmen; Carracedo, Begoña; Mercado, Luis; Coll, Julio

2018-01-01

Primitive nucleated erythroid cells in the bloodstream have long been suggested to be more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo morphological changes, especially when exposed to stress, and enter a new cell stage that we have coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission electron microscopy (TEM) micrographs, Wright–Giemsa staining, cell marker immunostaining, and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm, hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine (PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic studies resulted in the identification of proteins involved in pathways related to the regulation of cell morphogenesis involved in differentiation, cellular response to stress, and immune system process. In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 β (IL1β), interferon ɣ (IFNɣ), and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in roles related to immune response mediation, homeostasis, and the differentiation and development of blood cells. PMID:29671811

Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

PubMed

Chico, Verónica; Puente-Marin, Sara; Nombela, Iván; Ciordia, Sergio; Mena, María Carmen; Carracedo, Begoña; Villena, Alberto; Mercado, Luis; Coll, Julio; Ortega-Villaizan, María Del Mar

2018-04-19

Primitive nucleated erythroid cells in the bloodstream have long been suggested to be more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo morphological changes, especially when exposed to stress, and enter a new cell stage that we have coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission electron microscopy (TEM) micrographs, Wright⁻Giemsa staining, cell marker immunostaining, and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm, hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine (PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic studies resulted in the identification of proteins involved in pathways related to the regulation of cell morphogenesis involved in differentiation, cellular response to stress, and immune system process. In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 β (IL1β), interferon ɣ (IFNɣ), and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in roles related to immune response mediation, homeostasis, and the differentiation and development of blood cells.

Synthesis of α-L-Rhamnosyl Ceramide and Evaluation of its Binding with Anti-Rhamnose Antibodies

PubMed Central

Long, David E.; Karmakar, Partha; Wall, Katherine A.; Sucheck, Steven J.

2014-01-01

An α-L-rhamnosyl ceramide (1, α-L-RhaCer) has been prepared that was recognized by anti-L-rhamnose (anti-Rha) antibodies. During these studies we explored the use of an α-L-rhamnosyl thioglycoside and a trichloroacetimidate as a glycosyl donors. Subsequently, the acceptors desired for glycosylation, 3-O-benzoylazidosphingosine or 3-O-alloxycarbonylsphingosine, were prepared from D-xylose. The thioglycoside donor, 2,3,4-tri-O-acetyl-1-(4-tolyl)thio-α-L-rhamnopyranoside, and the trichloroacetimidate donor, 2,3,4-tri-O-acety-1-(2,2,2-trichloroethanimidate)-α-L-rhamnopyranoside, were synthesized in 50% and 78% yield overall, respectively. The synthesis of the glycosylation acceptor employed an addition-fragmentation olefination that was successfully carried out in 53% yield. With the successful synthesis of key intermediates, α-L-RhaCer (1) was prepared without any insurmountable obstacles. Anti-Rha antibodies were prepared in BALB/c mice by immunizing them with rhamnose-Ficoll with Sigma Adjuvant System (SAS) and the anti-L-Rha antibodies were isolated from the blood sera. Liposomes and EL4 tumor cells were used as model systems to demonstrate the ability of 1 to insert into a lipid bilayer. The interaction of the liposomes or the EL4 cells with α-L-RhaCer (1) and anti-Rha antibodies were investigated by fluorescence microscopy and flow cytometry, respectively, to confirm the ability of glycolipid 1 to be displayed on the tumor cell surface as well as the ability to be recognized by anti-Rha antibodies. PMID:25172148

Apoptosis of lymphocytes in the presence of Cr(V) complexes: role in Cr(VI)-induced toxicity.

PubMed

Vasant, C; Balamurugan, K; Rajaram, R; Ramasami, T

2001-08-03

Cr(VI) compounds have been declared as a potent occupational carcinogen by IARC (1990) through epidemiological studies among workers in chrome plating, stainless-steel, and pigment industries. Studies relating to the role of intermediate oxidation states such as Cr(V) and Cr(IV) in Cr(VI)-induced carcinogenicity are gaining importance. In this study, issues relating to toxicity elicited by Cr(V) have been addressed and comparisons made with those relating to Cr(VI) employing human peripheral blood lymphocytes. Lymphocytes have been isolated from heparinized blood by Ficoll-Hypaque density gradient centrifugation and exposed to Cr(V) complexes viz. sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[Cr(V)O(ehba)(2)], 1 and sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[Cr(V)O(hmba)(2)], 2 and Cr(VI). The phytohemagglutinin (PHA)-induced proliferation of lymphocytes has been found to be inhibited by the two complexes of Cr(V) and chromate Cr(VI) in a time- and concentration-dependent manner. Viability of cells decreases in the presence of Cr(V). Apoptosis appears to be the mode of cell death in the presence of both Cr(V) and Cr(VI). Pretreatment of cells with antioxidants before exposure to chromium(V) complexes reverse apoptosis partially. Possibility for the formation and implication of reactive oxygen species in Cr(V)-induced apoptosis of human lymphocyte cells has been indicated in this investigation. The intermediates of Cr(V) and radical species in the biotoxic pathways elicited by Cr(VI) seems feasible. Copyright 2001 Academic Press.

Inhibition of human polymorphonuclear leukocyte function by components of human colostrum and mature milk.

PubMed

Pickering, L K; Cleary, T G; Caprioli, R M

1983-04-01

To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 +/- 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-(14)C]glucose utilization, and Fc receptors were significantly (P < 0.05 to P < 0.001) less in PMNL exposed to whole human colostrum or colostral lipid than in non-lipid-exposed cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-(3)H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.

Quality testing of an innovative cascade separation system for multiple cell separation

NASA Astrophysics Data System (ADS)

Pierzchalski, Arkadiusz; Moszczynska, Aleksandra; Albrecht, Bernd; Heinrich, Jan-Michael; Tarnok, Attila

2012-03-01

Isolation of different cell types from mixed samples in one separation step by FACS is feasible but expensive and slow. It is cheaper and faster but still challenging by magnetic separation. An innovative bead-based cascade-system (pluriSelect GmbH, Leipzig, Germany) relies on simultaneous physical separation of different cell types. It is based on antibody-mediated binding of cells to beads of different size and isolation with sieves of different mesh-size. We validated pluriSelect system for single parameter (CD3) and simultaneous separation of CD3 and CD15 cells from EDTA blood-samples. Results were compared with those obtained by MACS (Miltenyi-Biotech) magnetic separation (CD3 separation). pluriSelect separation was done in whole blood, MACS on Ficoll gradient isolated leukocytes, according to the manufacturer's protocols. Isolated and residual cells were immunophenotyped (7-color 8-antibody panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLADR) on a CyFlowML flow cytometer (Partec GmbH). Cell count (Coulter), purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (92-98%), yield (50-60%) and viability (92-98%) of isolated cells. PluriSelect separation was slightly faster than MACS (1.15 h versus 1.5h). Moreover, no preenrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two cell subpopulation directly from whole blood and can provide a simple alternative to FACS. The isolated cells can be used for further research applications.

Tumor necrosis factor-α inhibits effects of aryl hydrocarbon receptor ligands on cell death in human lymphocytes.

PubMed

Ghatrehsamani, Mahdi; Soleimani, Masoud; Esfahani, Behjat A Moayedi; Shirzad, Hedayatollah; Hakemi, Mazdak G; Mossahebimohammadi, Majid; Eskandari, Nahid; Adib, Minoo

2015-01-01

Activation of aryl hydrocarbon receptor (AhR) leads to diverse outcome in various kinds of cells. AhR activation may induce apoptosis or prevent of apoptosis and cell death. Recent studies suggest that apoptosis effects of AhR can be modulated by inflammatory cytokine like tumor necrosis factor alpha (TNF-α). In this study, we try to investigate the possible interaction of TNF-α with the 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand of AhR, on peripheral lymphocytes. Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by discontinuous density gradient centrifugation on ficoll. Isolated PBMCs were divided into four groups: Control group, TNF-α administered group, TCDD administered group, co-administered group with TCDD and TNF-α. Cells were maintained for a week in lymphocyte culture condition. Then, TNF-α was added to group 2 and 4. Finally, apoptosis and necrosis were analyzed in all samples using flowcytometry. In group 4, the mean percent of necrosis and apoptosis in TCDD treatment groups was significantly larger than other groups; (P < 0.05). Furthermore, there was no significant difference between the mean percent of cell death in TNF-α administered group and TCDD administered group (P > 0.05). However, the mean percent of cell death in co-administered group with TCDD and TNF-α was significantly lower than other groups; (P < 0.05). TNF-α could significantly inhibit effects of TCDD on lymphocytes apoptosis. Combination effects of TNF-α and TCDD on lymphocyte increase cell survival.

Effect of two-chambered bicarbonate lactate-buffered peritoneal dialysis fluids on peripheral blood mononuclear cell and polymorphonuclear cell function in vitro.

PubMed

Sundaram, S; Cendoroglo, M; Cooker, L A; Jaber, B L; Faict, D; Holmes, C J; Pereira, B J

1997-11-01

Low pH, high osmolality, increasing glucose concentration, and glucose degradation products (GDP) formed during heat sterilization of conventional peritoneal dialysis (PD) fluids have been shown to have a detrimental effect on cells involved in peritoneal host defense. The two-chambered PD fluid bag in which glucose at pH approximately 3 is separated from a bicarbonate (25 mmol/L)-lactate (15 mmol/L) buffer during heat sterilization permits PD fluids with lower GDP to be delivered to the patient at neutral pH. To establish the possible benefit of two-chambered bag PD fluids on peripheral blood mononuclear cell (PBMC) and polymorphonuclear (PMN) cell function, we compared conventional 1.5% Dianeal (1.5%D) with 1.5% two-chambered bag bicarbonate-lactate (1.5%D-B), and conventional 4.25% Dianeal (4.25%D) with 4.25% two-chambered bag bicarbonate-lactate (4.25%D-B). Furthermore, to study the effect of the sterilization process on PBMC and PMN function, we compared filter-sterilized 4.25%D (4.25%D-F) with 4.25%D and 4.25%D-B. PBMC were harvested by Ficoll-Hypaque separation, and 2.5 x 10(6) cells in RPMI were incubated with an equal volume of the test fluids for 4 hours, pelleted, and resuspended in RPMI containing 10 ng endotoxin for a further 20 hours. Tumor necrosis factor alpha (TNF-alpha) production by endotoxin-stimulated PBMC was not significantly different (P = 0.10) between 1.5%D-B and 1.5%D, but was significantly higher (P = 0.01) with 4.25%D-B compared with 4.25%D. PBMC exposed to filter-sterilized fluid (4.25%D-F) showed significantly higher endotoxin-stimulated TNF-alpha production compared with 4.25%D (P = 0.02), but was not significantly different from 4.25%D-B (P = 0.40). PMN were harvested by Ficoll-Hypaque separation and 10 x 10(6) cells incubated with test fluids for 30 minutes. After incubation, phagocytosis (phagocytosis index) was determined by the uptake of 14C-labeled Staphylococcus aureus, oxidative burst by reduction of ferricytochrome C to ferrocytochrome C on stimulation with PMA, and enzyme release by measurement of endotoxin-stimulated bactericidal/permeability increasing protein (BPI). Bicarbonate-lactate two-chambered fluids of similar osmolality and glucose concentration conferred a significant improvement in phagocytosis (P = 0.02 for 1.5%D-B and P < 0.001 for 4.25%D-B). Oxidative burst and BPI release were significantly higher in 4.25%D-B compared with 4.25%D (P < 0.001). Filter-sterilized 4.25%D-F conferred a significant improvement in phagocytosis and oxidative burst compared with 4.25%D (P < 0.001) or 4.25%D-B (P < 0.001). Furthermore, conventional 4.25%D was associated with significantly lower BPI release compared with 4.25%D-F (P = 0.01). GDP's acetaldehyde and 5-HMF were analyzed in 4.25%D-B, 4.25%D, and 4.25%D-F. Acetaldehyde was below the lower limit (0.79 ppm) of the standard curve in 4.25%D-B and 4.25%D-F fluids but was detected (3.76 to 5.12 ppm) in all of the 4.25%D fluids. Relative levels of 5-HMF in the 4.25%D-B (0.032 to 0.041 Abs @ 284 nm) and 4.25%D (0.031 to 0.036 Abs @ 284 nm) were similar. The lowest levels (0.001 Abs @ 284 nm) were observed in the filter-sterilized 4.25%D-F. The beneficial effects of two-chambered bicarbonate lactate-buffered PD fluids on PBMC and PMN function are probably related to reduction of GDP from heat sterilization of glucose in a separate chamber at a lower pH. This improvement in biocompatibility could have a beneficial affect on peritoneal defenses.

High IL-35 pleural expression in patients with tuberculous pleural effusion.

PubMed

Dong, Xuan; Yang, Jiong

2015-05-03

IL-35 is a novel anti-inflammatory and immunosuppressive cytokine primarily produced by Treg cells, and is involved in inflammatory diseases and autoimmune diseases. However, its roles in tuberculous pleural effusion (TPE) remain unknown. We aimed to investigate the potential involvement of IL-35 in TPE. Thirty TPE patients and 20 lung cancer patients with malignant pleural effusion (MPE) were recruited. Samples of pleural effusion (100 mL) were collected after traditional pleurocentesis. Blood was sampled from TPE patients. Mononuclear cells were isolated by Ficoll-Hypaque gradient. Proportions of Th1, Th17, and IL-35-producing cells were analyzed by flow cytometry. IL-35 was assessed by real-time RT-PCR, ELISA, and immunofluorescence. An ELISPOT assay was used to assess the effect of IL-35 on pleural effusion mononuclear cells (PEMCs). Proportions of IL-35-producing cells were higher in TPE compared with MPE (49.4±6.0 vs. 15.8±5.4%, P<0.001) and blood from TPE patients (49.4±6.0% vs. 16.6±3.1, P<0.001). IL-35, IL-17 and IFN-γ were elevated in TPE compared with MPE (all P<0.01). ELISPOT assay showed that IL-35 reduced the proportion of IFN-γ-producing CD4+ T cells in TPE. IL-35 mRNA expression was higher in TPE compared with MPE (P<0.001). Immunofluorescence showed that IL-35-positive cells were present in pleural tissues from TPE patients. Results suggest that there is an imbalance in IL-35 metabolism in TPE. However, further studies are required to assess the exact relationship with the immune system response to tuberculosis. IL-35 might play a role in TPE and might be targeted as a treatment for TPE.

Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer.

PubMed

Drake, Andrew C; Lee, Youngjoo; Burgess, Emma M; Karlsson, Jens O M; Eroglu, Ali; Higgins, Adam Z

2018-01-01

Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants.

Cytomegalovirus cultured from different major leukocyte subpopulations: association with clinical features in CMV immunoglobulin G-positive renal allograft recipients.

PubMed

Schäfer, P; Tenschert, W; Cremaschi, L; Schröter, M; Gutensohn, K; Laufs, R

2000-08-01

Cytomegalovirus (CMV) cultured from peripheral blood mononuclear cells (PBMCs) was shown to be associated more closely with clinical manifestations than infectious CMV in polymorphonuclear leukocytes (PMNLs) of renal allograft recipients with secondary CMV infection. Shell vial culture was carried out with ficoll-purified PBMCs and PMNLs of 71 CMV IgG-positive patients after kidney transplantation. Thirty-six patients experienced active CMV infections. Of these, 17 developed clinical symptoms. The diagnostic value of PMNLs and PBMCs viremia was determined in comparison to pp65 antigenemia, leukoDNAemia, plasma DNAemia, and detection of cytomegalic endothelial cells. In both PMNLs and PBMCs (with or without detectable endothelial cells), frequencies and levels of viremia were significantly higher among symptomatic patients. Regarding the occurrence of clinical CMV manifestations, the sensitivity of culture from PMNLs and from PBMCs fractions was 100%. Viremia in PBMCs, however, was far more specific (94%) than in PMNLs (74%). Cutoff values established previously for pp65 antigenemia and leukoDNAemia, standard markers in the laboratory, had similar specificity (96% each) to PBMCs viremia, but were less sensitive (88% each). Plasma DNA-emia was both less sensitive (82%) and less specific (69%) than PBMCs viremia. Detection of endothelemia showed maximal specificity (100%), but inferior sensitivity (47%). All patients had PBMCs viremia before the onset of symptoms. In conclusion, infectious CMV present in PBMCs may prove to be a determinant of clinical CMV manifestations in seropositive immunocompromised individuals. Factors involved in PBMCs tropism may help to understand the pathogenetic mechanisms of CMV dissemination in this group of patients. Copyright 2000 Wiley-Liss, Inc.

Function of beta 2-adrenergic receptors in chronic localized myalgia.

PubMed

Maekawa, Kenji; Kuboki, Takuo; Inoue, Eitoku; Inoue-Minakuchi, Mami; Suzuki, Koji; Yatani, Hirofumi; Clark, Glenn T

2003-01-01

To investigate alteration of beta 2-adrenergic receptor (beta 2 AR) function in chronic localized myalgia subjects by evaluating levels of the beta 2 AR second messenger, cyclic adenosine monophosphate (cAMP), in mononuclear cells after beta AR-agonist stimulation. Eleven chronic localized myalgia subjects and 21 matched healthy controls participated in this study. Peripheral blood (30 cc) was drawn from the subjects' anterocubital vein. Mononuclear cells were isolated from the total blood by using the Ficoll-Hypaque gradient technique. Basal and stimulated intracellular cAMP levels were determined by enzyme immunoassay using a commercially available kit. Aliquots of 5 x 10(6) cells were incubated with or without stimulation of the beta AR-agonist isoproterenol for 5 minutes. Five different concentrations of isoproterenol (10(-3) M to 10(-7) M) were utilized. cAMP levels in both groups were tested statistically by a 2-way repeated-measures ANOVA with 2 predictors, group difference and isoproterenol concentration difference. As with isoproterenol stimulation, the cAMP responses to forskolin, which activates adenylyl cyclase directly and produces cAMP, bypassing the cell surface receptors were also measured. The basal cAMP levels in both groups (myalgia: 0.33 +/- 0.02 pmol/5 x 10(6) cells; control: 0.43 +/- 0.10 pmol/5 x 10(6) cells) were almost identical, and isoproterenol-produced cAMP levels increased dose-dependently in both groups. No significant differences in the mean cAMP levels were observed between the groups (P = .909). Significant increases were observed according to the isoproterenol concentration increase (P < .0001). The cAMP responses to forskolin stimulation also showed no significant group difference (P = .971). These results suggest that beta 2 AR function is not different between localized myalgia subjects and healthy individuals.

The Dominance of Warming Rate Over Cooling Rate in the Survival of Mouse Oocytes Subjected to a Vitrification Procedure✰

PubMed Central

Seki, Shinsuke; Mazur, Peter

2009-01-01

The formation of more than trace amounts of ice in cells is lethal. The two contrasting routes to avoiding it are slow equilibrium freezing and vitrification. The cryopreservation of mammalian oocytes by either method continues to be difficult, but there seems a slowly emerging consensus that vitrification procedures are somewhat better for mouse and human oocytes. The approach in these latter procedures is to load cells with high concentrations of glass-inducing solutes and cool them at rates high enough to induce the glassy state. Several devices have been developed to achieve very high cooling rates. Our study has been concerned with the relative influences of warming rate and cooling rate on the survival of mouse oocytes subjected to a vitrification procedure. Oocytes suspended in an ethylene glycol-acetamide-Ficoll-sucrose solution were cooled to −196°C at rates ranging from 37°C/min to 1827°C/min between 20°C and −120°C, and for each cooling rate, warmed at rates ranging from 139°C/min to 2950°C/min between −70°C and −35°C. The results are unambiguous. If the samples were warmed at the highest rate, survivals were >80% over cooling rates of 187°C/min to 1827°C/min. If the samples were warmed at the lowest rate, survivals were near 0% regardless of the cooling rate. We interpret the lethality of slow warming to be a consequence of it allowing time for the growth of small intracellular ice crystals by recrystallization. PMID:19427303

Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus.

PubMed

Nombela, Ivan; Puente-Marin, Sara; Chico, Veronica; Villena, Alberto J; Carracedo, Begoña; Ciordia, Sergio; Mena, Maria Carmen; Mercado, Luis; Perez, Luis; Coll, Julio; Estepa, Amparo; Ortega-Villaizan, Maria Del Mar

2017-01-01

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1 ) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail.

Improvements in the production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture.

PubMed

Cistué, Luis; Romagosa, I; Batlle, F; Echávarri, B

2009-05-01

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C(17) induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars 'Ciccio' and 'Claudio' an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F(1) crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named 'Lanuza', has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.

Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer

PubMed Central

Burgess, Emma M.; Karlsson, Jens O. M.; Eroglu, Ali

2018-01-01

Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants. PMID:29304068

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum.

PubMed

Paschoal, Daniela Martins; Sudano, Mateus José; Guastali, Midyan Daroz; Dias Maziero, Rosiára Rosária; Crocomo, Letícia Ferrari; Oña Magalhães, Luis Carlos; da Silva Rascado, Tatiana; Martins, Alicio; da Cruz Landim-Alvarenga, Fernanda

2014-05-01

The objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

Lutein accumulates in subcellular membranes of brain regions in adult rhesus macaques: Relationship to DHA oxidation products

PubMed Central

Erdman, John W.; Kuchan, Matthew J.; Neuringer, Martha; Johnson, Elizabeth J.

2017-01-01

Objectives Lutein, a carotenoid with anti-oxidant functions, preferentially accumulates in primate brain and is positively related to cognition in humans. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid (PUFA), is also beneficial for cognition, but is susceptible to oxidation. The present study characterized the membrane distribution of lutein in brain regions important for different domains of cognitive function and determined whether membrane lutein was associated with brain PUFA oxidation. Methods Adult rhesus monkeys were fed a stock diet (~2 mg/day lutein or ~0.5 μmol/kg body weight/day) (n = 9) or the stock diet plus a daily supplement of lutein (~4.5 mg/day or~1 μmol/kg body weight/day) and zeaxanthin (~0.5 mg/day or 0.1 μmol/kg body weight/day) for 6–12 months (n = 4). Nuclear, myelin, mitochondrial, and neuronal plasma membranes were isolated using a Ficoll density gradient from prefrontal cortex (PFC), cerebellum (CER), striatum (ST), and hippocampus (HC). Carotenoids, PUFAs, and PUFA oxidation products were measured using HPLC, GC, and LC-GC/MS, respectively. Results All-trans-lutein (ng/mg protein) was detected in all regions and membranes and was highly variable among monkeys. Lutein/zeaxanthin supplementation significantly increased total concentrations of lutein in serum, PFC and CER, as well as lutein in mitochondrial membranes and total DHA concentrations in PFC only (P<0.05). In PFC and ST, mitochondrial lutein was inversely related to DHA oxidation products, but not those from arachidonic acid (P <0.05). Discussion This study provides novel data on subcellular lutein accumulation and its relationship to DHA oxidation in primate brain. These findings support the hypothesis that lutein may be associated with antioxidant functions in the brain. PMID:29049383

Treatment of pressure ulcers with autologous bone marrow nuclear cells in patients with spinal cord injury

PubMed Central

Sarasúa, J González; López, S Pérez; Viejo, M Álvarez; Basterrechea, M Pérez; Rodríguez, A Fernández; Gutiérrez, A Ferrero; Gala, J García; Menéndez, Y Menéndez; Augusto, D Escudero; Arias, A Pérez; Hernández, J Otero

2011-01-01

Context Pressure ulcers are especially difficult to treat in patients with spinal cord injury (SCI) and recurrence rates are high. Prompted by encouraging results obtained using bone marrow stem cells to treat several diseases including chronic wounds, this study examines the use of autologous stem cells from bone marrow to promote the healing of pressure ulcers in patients with SCI. Objective To obtain preliminary data on the use of bone marrow mononuclear cells (BM-MNCs) to treat pressure ulcers in terms of clinical outcome, procedure safety, and treatment time. Participants Twenty-two patients with SCI (19 men, 3 women; mean age 56.41 years) with single type IV pressure ulcers of more than 4 months duration. Interventions By minimally invasive surgery, the ulcers were debrided and treated with BM-MNCs obtained by Ficoll density gradient separation of autologous bone marrow aspirates drawn from the iliac crest. Results In 19 patients (86.36%), the pressure ulcers treated with BM-MNCs had fully healed after a mean time of 21 days. The number of MNCs isolated was patient dependent, although similar clinical outcomes were observed in each case. Compared to conventional surgical treatment, mean intra-hospital stay was reduced from 85.16 to 43.06 days. Following treatment, 5 minutes of daily wound care was required per patient compared to 20 minutes for conventional surgery. During a mean follow-up of 19 months, none of the resolved ulcers recurred. Conclusions Our data indicate that cell therapy using autologous BM-MNCs could be an option to treat type IV pressure ulcers in patients with SCI, avoiding major surgical intervention. PMID:21756569

Tier-2 studies on monocrotaline immunotoxicity in C57BL/6 mice.

PubMed

Deyo, J A; Kerkvliet, N I

1991-01-01

Monocrotaline (MCT) is a member of a class of naturally occurring phytotoxins known as pyrrolizidine alkaloids, and is a toxicological concern to both man and his livestock. The purpose of these studies was to evaluate the effect of a 14-day oral MCT (0-100 mg/kg per day) exposure on the functional integrity of various immunocyte effector systems in C57BL/6 mice, as well as to investigate potential mechanisms for its immunotoxicity. Decreases in lymphoid organ weights and cellularity, and resident peritoneal exudate cell (PEC) number were only observed after exposure to the highest dose of 100 mg/kg MCT. This dose also inhibited NK cell cytotoxicity, while the total number of NK lytic units per spleen was decreased (-53%) after exposure to 50 mg/kg MCT. Following i.p. injection of SRBC, the percentage of PEC macrophages containing engulfed SRBC was significantly increased in MCT-exposed mice, while the percentage of large vacuolated (activated) macrophages was decreased in a dose-dependent manner. Exposure to MCT significantly decreased the total number of Ig+ cells without altering the number of CD4+ and CD8+ cells. The antibody responses (PFC/10(6) spleen cells) to two T cell-independent antigens, TNP-LPS and DNP-Ficoll, were significantly decreased at all MCT doses, and the degree of suppression of both responses was identical at coincident doses. MCT exposure (25 mg/kg) significantly suppressed the blastogenic response to the T cell mitogen concanavalin A (-38%), and to the B cell mitogen lipopolysaccharide (-58%). These results indicate that exposure to MCT can alter the functional integrity of various immune effector responses in a dose-dependent manner, and suggest that the B cell may be a relatively more sensitive target of MCT immunotoxicity compared to T cells, macrophages and NK cells.

[Influence of Cryopreservation on Human Peripheral Blood Mononuclear Cell Immunocompetence].

PubMed

Pan, Xue-Feng; Lu, Chun-Xia; Yang, Li-Li; Shu, Chang; Yao, Na; Zuo, Hong-Bin; Cui, Li-Feng

2016-08-01

To establish a method for isolation, cryopreservation and recovery of the highly viable human peripheral blood monomuclear cells (PBMNCs) so as to achieve the long-term preservation of PBMNCs. A total of 80-100 ml peripheral blood were collected from the healthy volumteers aged over 50 years old. The PBMNCs were isolated by the Ficoll density gradient technique and cryopreserved gradually by program control method in liquid nitrogen freezer of -196 °C. The serum-free medium and autoloqous plasma medium were test for preservation of PBMNCs. The cell viability was assessed at time point of 1, 2, 4, 8, 12 and 24 months after thawing. Finally, the proliferation ability, purity and cytotoxicity were compared between the autologous immune lymphocytes (AIL) induced from cryopreserved PBMNCs and AIL as control from fresh PBMNCs. After separating, the cell viability was 99.6%±0.4%, and the recovery rate of lymphocytes was 58.4%±6.52%. The cell recovery rate of lymphocyte was 89.7%±3.82% at 24 months. The quality assurance program was reliable within 2 years of running. The AIL cells induced with cryopreserved PBMNCs were not significantly different from those induced from fresh PBMNCs in terms of proliferative action, purity and cytotoxicity(CD3(+)CD8(+) ≥45%,CD3(+)CD56(+) NKT≥10%,CD4(+)CD25(+) NKT≤10%). Manual separation of lymphocytes in vitro can get enough high-quality PBMNCs. The long-term cryopreserved PBMNC still maintain their high viability. The reinfusion of the clinical autologous immune cells would be advantageous for early tumor immunotherapy. Human AIL induced from cryopreserved PBMNC maintain their anti-tumor ability. These findings have the important implications for the application of these cells to adoptive cellular therapy.

Comparison of two methodologies for the enrichment of mononuclear cells from thawed cord blood products: The automated Sepax system versus the manual Ficoll method.

PubMed

Kaur, Indreshpal; Zulovich, Jane M; Gonzalez, Marissa; McGee, Kara M; Ponweera, Nirmali; Thandi, Daljit; Alvarez, Enrique F; Annandale, Kathy; Flagge, Frank; Rezvani, Katayoun; Shpall, Elizabeth

2017-03-01

Umbilical cord blood (CB) is being used as a source of hematopoietic stem cells (HSCs) and immune cells to treat many disorders. Because these cells are present in low numbers in CB, investigators have developed strategies to expand HSCs and other immune cells such as natural killer (NK) cells. The initial step in this process is to enrich mononuclear cells (MNCs) while depleting unwanted cells. The manual method of MNC enrichment is routinely used by many centers; however, it is an open system, time-consuming and operator dependent. For clinical manufacturing, it is important to have a closed system to avoid microbial contamination. In this study, we optimized an automated, closed system (Sepax) for enriching MNCs from cryopreserved CB units. Using Sepax, we observed higher recovery of total nucleated cells (TNC), CD34 + cells, NK cells and monocytes when compared to manual enrichment, despite similar TNC and CD34 + viability with the two methods. Even though the depletion of red blood cells, granulocytes and platelets was superior using the manual method, significantly higher CFU-GM were obtained in MNCs enriched using Sepax compared to the manual method. This is likely related to the fact that the automated Sepax significantly shortened the processing time (Sepax: 74 - 175 minutes versus manual method: 180 - 290 minutes). The use of DNAse and MgCl 2 during the Sepax thaw and wash procedure prevents clumping of cells and loss of viability, resulting in improved post-thaw cell recovery. We optimized enrichment of MNCs from cryopreserved CB products in a closed system using the Sepax which is a walk away and automated processing system. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Multiscale diffusion of a molecular probe in a crowded environment: a concept

NASA Astrophysics Data System (ADS)

Currie, Megan; Thao, Chang; Timerman, Randi; Welty, Robb; Berry, Brenden; Sheets, Erin D.; Heikal, Ahmed A.

2015-08-01

Living cells are crowded with macromolecules and organelles. Yet, it is not fully understood how macromolecular crowding affects the myriad of biochemical reactions, transport and the structural stability of biomolecules that are essential to cellular function and survival. These molecular processes, with or without electrostatic interactions, in living cells are therefore expected to be distinct from those carried out in test tube in dilute solutions where excluded volumes are absent. Thus there is an urgent need to understand the macromolecular crowding effects on cellular and molecular biophysics towards quantitative cell biology. In this report, we investigated how biomimetic crowding affects both the rotational and translation diffusion of a small probe (rhodamine green, RhG). For biomimetic crowding agents, we used Ficoll-70 (synthetic polymer), bovine serum albumin and ovalbumin (proteins) at various concentrations in a buffer at room temperature. As a control, we carried out similar measurements on glycerolenriched buffer as an environment with homogeneous viscosity as a function of glycerol concentration. The corresponding bulk viscosity was measured independently to test the validity of the Stokes-Einstein model of a diffusing species undergoing a random walk. For rotational diffusion (ps-ns time scale), we used time-resolved anisotropy measurements to examine potential binding of RhG as a function of the crowding agents (surface structure and size). For translational diffusion (μs-s time scale), we used fluorescence correlation spectroscopy for single-molecule fluctuation analysis. Our results allow us to examine the diffusion model of a molecular probe in crowded environments as a function of concentration, length scale, homogeneous versus heterogeneous viscosity, size and surface structures. These biomimetic crowding studies, using non-invasive fluorescence spectroscopy methods, represent an important step towards understanding cellular biophysics and quantitative cell biology.

Recovery of CD45(-)/Lin(-)/SSEA-4(+) very small embryonic-like stem cells by cord blood bank standard operating procedures.

PubMed

Chang, Yu-Jen; Tien, Kuei-Erh; Wen, Cheng-Hao; Hsieh, Tzu-Bou; Hwang, Shiaw-Min

2014-04-01

Very small embryonic-like (VSEL) stem cells are a rare cell population present in bone marrow, cord blood and other tissues that displays a distinct small cell size and the ability to give rise to cells of the three germ layers. VSEL stem cells were reported to be discarded in the red blood cell fraction by Ficoll-Paque density gradient centrifugation during the processing of bone marrow and cord blood specimens. However, most cord blood banks do not include density gradient centrifugation in their procedures while red blood cells are removed by Hespan sedimentation following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP). To clarify the retention of VSEL stem cells, we investigated the recovery of VSEL stem cells following COBLT SOP guidelines. The recovery of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells of umbilical cord blood was examined by flow cytometry before and after COBLT SOP processing, and relative expression of pluripotent genes was analyzed by quantitative polymerase chain reaction. CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells were mostly recovered in the final products following COBLT SOP guidelines. The expression of pluripotent genes could be maintained at >80% in products after hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) processing. The rare sub-population of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells survived after Hespan sedimentation. This finding suggests that umbilical cord blood units cryopreserved by COBLT SOP in cord blood banks should retain most VSEL stem cells present in the un-processed specimens. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Detection and sequencing of measles virus from peripheral mononuclear cells from patients with inflammatory bowel disease and autism.

PubMed

Kawashima, H; Mori, T; Kashiwagi, Y; Takekuma, K; Hoshika, A; Wakefield, A

2000-04-01

It has been reported that measles virus may be present in the intestine of patients with Crohn's disease. Additionally, a new syndrome has been reported in children with autism who exhibited developmental regression and gastrointestinal symptoms (autistic enterocolitis), in some cases soon after MMR vaccine. It is not known whether the virus, if confirmed to be present in these patients, derives from either wild strains or vaccine strains. In order to characterize the strains that may be present, we have carried out the detection of measles genomic RNA in peripheral mononuclear cells (PBMC) in eight patients with Crohn's disease, three patients with ulcerative colitis, and nine children with autistic enterocolitis. As controls, we examined healthy children and patients with SSPE, SLE, HIV-1 (a total of eight cases). RNA was purified from PBMC by Ficoll-paque, followed by reverse transcription using AMV; cDNAs were subjected to nested PCR for detection of specific regions of the hemagglutinin (H) and fusion (F) gene regions. Positive samples were sequenced directly, in nucleotides 8393-8676 (H region) or 5325-5465 (from noncoding F to coding F region). One of eight patients with Crohn disease, one of three patients with ulcerative colitis, and three of nine children with autism, were positive. Controls were all negative. The sequences obtained from the patients with Crohn's disease shared the characteristics with wild-strain virus. The sequences obtained from the patients with ulcerative colitis and children with autism were consistent with being vaccine strains. The results were concordant with the exposure history of the patients. Persistence of measles virus was confirmed in PBMC in some patients with chronic intestinal inflammation.

Methodological considerations for implementation of lymphocyte subset analysis in a clinical reference laboratory.

PubMed

Muirhead, K A; Wallace, P K; Schmitt, T C; Frescatore, R L; Franco, J A; Horan, P K

1986-01-01

As the diagnostic utility of lymphocyte subset analysis has been recognized in the clinical research laboratory, a wide variety of reagents and cell preparation, staining and analysis methods have also been described. Methods that are perfectly suitable for analysis of smaller sample numbers in the biological or clinical research setting are not always appropriate and/or applicable in the setting of a high volume clinical reference laboratory. We describe here some of the specific considerations involved in choosing a method for flow cytometric analysis which minimizes sample preparation and data analysis time while maximizing sample stability, viability, and reproducibility. Monoclonal T- and B-cell reagents from three manufacturers were found to give equivalent results for a reference population of healthy individuals. This was true whether direct or indirect immunofluorescence staining was used and whether cells were prepared by Ficoll-Hypaque fractionation (FH) or by lysis of whole blood. When B cells were enumerated using a polyclonal anti-immunoglobulin reagent, less cytophilic immunoglobulin staining was present after lysis than after FH preparation. However, both preparation methods required additional incubation at 37 degrees C to obtain results concordant with monoclonal B-cell reagents. Standard reagents were chosen on the basis of maximum positive/negative separation and the availability of appropriate negative controls. The effects of collection medium and storage conditions on sample stability and reproducibility of subset analysis were also assessed. Specimens collected in heparin and stored at room temperature in buffered medium gave reproducible results for 3 days after specimen collection, using either FH or lysis as the preparation method. General strategies for instrument optimization, quality control, and biohazard containment are also discussed.

Mesenchymal stem cells from sternum: the type of heart disease, ischemic or valvular, does not influence the cell culture establishment and growth kinetics.

PubMed

Dias, Lucinara Dadda; Casali, Karina Rabello; Ghem, Carine; da Silva, Melissa Kristocheck; Sausen, Grasiele; Palma, Patrícia Bonini; Covas, Dimas Tadeu; Kalil, Renato A K; Schaan, Beatriz D; Nardi, Nance Beyer; Markoski, Melissa Medeiros

2017-07-25

In an attempt to increase the therapeutic potential for myocardial regeneration, there is a quest for new cell sources and types for cell therapy protocols. The pathophysiology of heart diseases may affect cellular characteristics and therapeutic results. To study the proliferative and differentiation potential of mesenchymal stem cells (MSC), isolated from bone marrow (BM) of sternum, we made a comparative analysis between samples of patients with ischemic (IHD) or non-ischemic valvular (VHD) heart diseases. We included patients with IHD (n = 42) or VHD (n = 20), with average age of 60 years and no differences in cardiovascular risk factors. BM samples were collected (16.4 ± 6 mL) and submitted to centrifugation with Ficoll-Paque, yielding 4.5 ± 1.5 × 10 7  cells/mL. Morphology, immunophenotype and differentiation ability had proven that the cultivated sternal BM cells had MSC features. The colony forming unit-fibroblast (CFU-F) frequency was similar between groups (p = 0.510), but VHD samples showed positive correlation to plated cells vs. CFU-F number (r = 0.499, p = 0.049). The MSC culture was established in 29% of collected samples, achieved passage 9, without significant difference in expansion kinetics between groups (p > 0.05). Dyslipidemia and the use of statins was associated with culture establishment for IHD patients (p = 0.049 and p = 0.006, respectively). Together, these results show that the sternum bone can be used as a source for MSC isolation, and that ischemic or valvular diseases do not influence the cellular yield, culture establishment or in vitro growth kinetics.

Analysis of peripheral blood lymphocytes using flow cytometry in polymyalgia rheumatica, RS3PE and early rheumatoid arthritis.

PubMed

Shimojima, Y; Matsuda, M; Ishii, W; Gono, T; Ikeda, S

2008-01-01

Clinical pictures of poly-myalgia rheumatica (PMR) and remitting seronegative symmetrical synovitis with pitting edema (RS3PE) are often indistinguishable from those of early rheumatoid arthritis (RA). To investigate whether there is a difference in immunological aspects among these 3 disorders, we performed a phenotypic analysis of peripheral blood lymphocytes. Eleven patients with early RA, 14 with PMR and 11 with RS3PE were enrolled in this study. After separation of mononuclear cells from peripheral blood using the Ficoll-Hypaque method, surface markers and intracellular cytokines of lymphocytes were analyzed by 2- or 3-color flow cytometry. Both PMR and RS3PE showed a significant decrease in CD8⁺CD25⁺ cells (p<0.05), and significant increases in CD4⁺IFN-gamma⁺IL-4- (p<0.05), CD8⁺IFN-gamma⁺IL-4- (p<0.05 and p<0.01, respectively) and CD4⁺TNF-alpha⁺ cells (p<0.05) compared with early RA. CD3⁺CD4⁺ cells were higher in PMR than in RS3PE (p<0.01), but there were no significant differences in any other phenotypes between these disorders. A decrease in activated cytotoxic/suppressor T cells and increases in circulating Th1 and Tc1 cells may be common characteristics of PMR and RS3PE in comparison with early RA. Both disorders are clearly different from early RA, and probably belong to the same disease entity with regard to phenotypes of peripheral blood lymphocytes.

Prevalence and Quantitation of Species C Adenovirus DNA in Human Mucosal Lymphocytes

PubMed Central

Garnett, C. T.; Erdman, D.; Xu, W.; Gooding, Linda R.

2002-01-01

The common species C adenoviruses (serotypes Ad1, Ad2, Ad5, and Ad6) infect more than 80% of the human population early in life. Following primary infection, the virus can establish an asymptomatic persistent infection in which infectious virions are shed in feces for several years. The probable source of persistent virus is mucosa-associated lymphoid tissue, although the molecular details of persistence or latency of adenovirus are currently unknown. In this study, a sensitive real-time PCR assay was developed to quantitate species C adenovirus DNA in human tissues removed for routine tonsillectomy or adenoidectomy. Using this assay, species C DNA was detected in Ficoll-purified lymphocytes from 33 of 42 tissue specimens tested (79%). The levels varied from fewer than 10 to greater than 2 × 106 copies of the adenovirus genome/107 cells, depending on the donor. DNA from serotypes Ad1, Ad2, and Ad5 was detected, while the rarer serotype Ad6 was not. When analyzed as a function of donor age, the highest levels of adenovirus genomes were found among the youngest donors. Antibody-coated magnetic beads were used to purify lymphocytes into subpopulations and determine whether viral DNA could be enriched within any purified subpopulations. Separation of T cells (CD4/8- expressing and/or CD3-expressing cells) enriched viral DNA in each of nine donors tested. In contrast, B-cell purification (CD19-expressing cells) invariably depleted or eliminated viral DNA. Despite the frequent finding of significant quantities of adenovirus DNA in tonsil and adenoid tissues, infectious virus was rarely present, as measured by coculture with permissive cells. These findings suggest that human mucosal T lymphocytes may harbor species C adenoviruses in a quiescent, perhaps latent form. PMID:12368303

Andrographolide as an anti-H1N1 drug and the mechanism related to retinoic acid-inducible gene-I-like receptors signaling pathway.

PubMed

Yu, Bin; Dai, Cong-qi; Jiang, Zhen-you; Li, En-qing; Chen, Chen; Wu, Xian-lin; Chen, Jia; Liu, Qian; Zhao, Chang-lin; He, Jin-xiong; Ju, Da-hong; Chen, Xiao-yin

2014-07-01

To observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1. Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR). The optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-β promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05). The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Tumour-cytolytic human monocyte-derived macrophages: a simple and efficient method for the generation and long-term cultivation as non-adherent cells in a serum-free medium.

PubMed

Streck, R J; Hurley, E L; Epstein, D A; Pauly, J L

1992-01-01

We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the results of ongoing studies in which scanning electron microscopy and confocal laser scanning microscopy are being used to define MAC function in different immunological reactions, and examples of these observations are presented herein.

ANTI-INFLAMMATORY ACTIVITY OF EUCALYPTUS SPP. AND PISTASCIA LENTISCUS LEAF EXTRACTS.

PubMed

Qabaha, Khaled; Ras, Sari Abu; Abbadi, Jehad; Al-Rimawi, Fuad

2016-01-01

Eucalyptus spp. and Pistascia lentiscus are among the Palestinian trees that are traditionally used in folkloric medicine in treating many diseases; leaves of which are thought to have anti-inflammatory, antibacterial and antioxidant effects. The goal of this study is to evaluate the in vitro inhibitory effect of Eucalyptus spp . and Pistascia lentiscus extracts on Lipopolysacaride (LPS)-induced Interlukin-6 (Il-6) and Tumor Necrosis Factor-α (TNF-α) by polymorphonuclear Cells (PMNCs). Polymorphonuclear cells were isolated from the whole blood using Histopaque (Ficol-1077) method and then cultured in an enriched Roswell Park Memorial Institute (RBMI) medium. Supernatants' Interlukin-6 (IL-6) and Tumor Necrosis Factor (TNF-α) levels were determined 24 hour after LPS stimulation. HPLC was employed to determine the concentration of phenolic compounds in the extracts. The concentrations of TNF-α and IL-6 were compared using paired-samples t test. Eucalyptus spp . and Pistascia lentiscus leaves extracts have shown significant reduction in the levels of both Il-6 and TNF-α Gallic acid; a strong anti-inflammatory agent was found to be the major phenolic compound in both leaf extracts. However, other anti-inflammatory phenolic compounds were detected in Pitascia lentiscus extract including syringic acid and p-coumaric acid, while chlorogenic acid was detected in Eucalyptus spp . leaf extract. Reduction in the levels of Il-6 and TNF-α upon the effect of both Eucalyptus spp . and Pistascia lentiscus extract is an indication of their anti-inflammatory effects. Our results may also indicate that the observed anti-inflammatory effect of the above extracts may be due to the presence of gallic acid and other phenolic compounds. List of Abbreviations and Nomenclature: LPS: Lipopolysacaride, Il-6: Interlukin-6, TNF-α: Tumor Necrosis Factor-α, PMNCs: Polymorphonuclear Cells, HPLC: High Performance Liquid Chromatography, ELISA: Enzyme Linked Immune Sorbent Assay, EDTA: Ethylene Diamine Tetra Acetic acid, PBS: phosphate buffered saline, RPMI: Roswell Park Memorial Institute medium FBS: Fetal Bovine Serum.

VCAM-1 expression is upregulated by CD34+/CD133+-stem cells derived from septic patients

PubMed Central

Remmé, Christoph; Betzen, Christian; Tönshoff, Burkhard; Yard, Benito A.; Beck, Grietje; Rafat, Neysan

2018-01-01

CD34+/CD133+- cells are a bone marrow derived stem cell population, which presumably contain vascular progenitor cells and are associated with improved vascular repair. In this study, we investigated whether the adhesion molecules ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular adhesion molecule-1), E-selectin und L-selectin, which are involved in homing of vascular stem cells, are upregulated by CD34+/CD133+-stem cells from septic patients and would be associated with improved clinical outcome. Peripheral blood mononuclear cells from intensive care unit (ICU) patients with (n = 30) and without sepsis (n = 10), and healthy volunteers (n = 15) were isolated using Ficoll density gradient centrifugation. The expression of VCAM-1, ICAM-1, E-selectin and L-selectin was detected on CD34+/CD133+-stem cells by flow cytometry. The severity of disease was assessed by the Simplified Acute Physiology Score (SAPS) II. Serum concentrations of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 were determined by Enzyme-linked immunosorbent assay. The expression of VCAM-1, ICAM-1, E-selectin and L-selectin by CD34+/CD133+-stem cells was significantly upregulated in septic patients, and correlated with sepsis severity. Furthermore, high expression of VCAM-1 by CD34+/CD133+-stem cells revealed a positive association with mortalitiy (p<0.05). Furthermore, significantly higher serum concentrations of VEGF and Ang-2 were found in septic patients, however none showed a strong association with survival. Our data suggest, that VCAM-1 upregulation on CD34+/CD133+-stem cells could play a crucial role in their homing in the course of sepsis. An increase in sepsis severity resulted in both and increase in CD34+/CD133+-stem cells and VCAM-1-expression by those cells, which might reflect an increase in need for vascular repair. PMID:29601599

TIM-1 signaling in B cells regulates antibody production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Juan; Usui, Yoshihiko; Department of Ophthalmology, Tokyo Medical University, 6-7-1 Nishi-shinjuku-ku, Tokyo 160-0023

Highlights: {yields} TIM-1 is highly expressed on anti-IgM + anti-CD40-stimulated B cells. {yields} Anti-TIM-1 mAb enhanced proliferation and Ig production on activated B cell in vitro. {yields} TIM-1 signaling regulates Ab production by response to TI-2 and TD antigens in vivo. -- Abstract: Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressedmore » on B cells and inducible on anti-CD3{sup +} anti-CD28-stimulated CD4{sup +} T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.« less

Effect of curcumin on accumulation in mononuclear cells and secretion in incubation medium of Аβ(40) and cytokines under local excess of Аβ(42)-homoaggregates.

PubMed

Sokolik, V V; Shulga, S M

2016-01-01

The aim of the work was to investigate accumulation of endogenous Aβ40 and cytokines (IL-1β, TNFα, IL-6, IL-10) in mononuclear cells and their secretion into incubation medium under Aβ42-aggregates’ toxicity and anti-inflammatory effects of curcumin. Mononuclear cells were isolated in Ficoll-Urografin density gradient from venous blood of healthy donors, resuspended and used for testing of homoaggregates of Aβ42 (15 nM), curcumin (54 pM) and their combinations on various timescales (0, 1, 2, 3, 6 and 24 hours). Endogenous Aβ40 and cytokines were detected in mononuclear cells and (separately) in incubation medium by ELISA. We demonstrated for the first time that homoaggregates of Aβ42 cause rapid accumulation of endogenous Aβ40 in mononuclear cells and accelerate its secretion into incubation medium. We found increased concentration of TNFα after 3 hours of incubation, and no changes in IL-1β concentration due to secretion of these pro-inflammatory factors into incubation medium. The concentrations of IL-6 in mononuclear cells were increased under effects of Aβ42 homoaggregates, and it was being secreted profoundly into incubation medium. Aβ42 did not affect IL-10 secretion, yet caused an increase in its intracellular concentration after 1 hour of incubation, which was subsequently suppressed. Curcumin prevented the increase in Aβ40 concentration in mononuclear cells and significantly decreased its secretion resulting from Aβ42 toxicity. Curcumin negated the activating effect of Aβ42 on pro-inflammatory cytokines, starting immediately for IL-1β and on 3-6 hours for TNFα, which resulted in decreased extracellular concentrations of these cytokines. The polyphenol also potentiated repleni­shing of intracellular IL-6 and IL-10 concentrations and their secretion into incubation medium.

Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus

PubMed Central

Nombela, Ivan; Puente-Marin, Sara; Chico, Veronica; Villena, Alberto J.; Carracedo, Begoña; Ciordia, Sergio; Mena, Maria Carmen; Mercado, Luis; Perez, Luis; Coll, Julio; Ortega-Villaizan, Maria del Mar

2018-01-01

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. PMID:29527292

Diagnostic Applications and Methods to Isolate Circulating Tumor Cells (CTCs) from Blood

NASA Astrophysics Data System (ADS)

Tang, Cha-Mei

2013-03-01

Each year a million new cancer cases are diagnosed in the United States. Ninety percent of the deaths will be the result of metastasis, not from the primary tumor. Tissue biopsy is a universally accepted tool for cancer diagnosis and determination of treatment. The procedure varies, but is invasive, costly, and can be fatal, and for these reasons is seldom repeated after initial diagnosis. Monitoring of treatment response and for possible relapse is usually done by CT or MRI scan, both of which are expensive and require the tumor to change size perceptibly. Further, cancer can mutate or develop resistance to therapeutics and require modification of the treatment regimen. The initial tissue biopsy often cannot reflect the disease as it progresses, requiring new biopsy samples to determine a change of treatment. All carcinomas, about 80% of all cancer, shed tumor cells into the circulation, most often at the later stages when treatment is more critical. These circulating tumor cells (CTCs) are the cause of metastasis, and can be isolated from patient blood to serve as ``liquid biopsy''. These CTCs contain a valuable trove of information that help both patient and clinician understand disease status. In addition to counting the number of CTCs (known to be a prognostic indicator of survival), CTCs can provide biomarker information such as protein expressions and gene mutations, amplifications, and translocations. This information can be used to determine treatment. During treatment, the number of intact and apoptotic CTCs can be measured on a repeated basis to measure the patient's response to treatment and disease progression. Following treatment, liquid biopsy can be repeated at regular intervals to watch for relapse. Methods to isolate CTCs can be grouped into three categories: i) immunocapture based on surface markers of CTCs, ii) size exclusion based on CTC size, typically larger than blood cells, and iii) negative selection utilizing red blood cell lysis, white blood cell depletion or FICOLL. Various implementations of the CTC isolation methods will be presented.

Direct Measurement of Helical Cell Motion of the Spirochete Leptospira

PubMed Central

Nakamura, Shuichi; Leshansky, Alexander; Magariyama, Yukio; Namba, Keiichi; Kudo, Seishi

2014-01-01

Leptospira are spirochete bacteria distinguished by a short-pitch coiled body and intracellular flagella. Leptospira cells swim in liquid with an asymmetric morphology of the cell body; the anterior end has a long-pitch spiral shape (S-end) and the posterior end is hook-shaped (H-end). Although the S-end and the coiled cell body called the protoplasmic cylinder are thought to be responsible for propulsion together, most observations on the motion mechanism have remained qualitative. In this study, we analyzed the swimming speed and rotation rate of the S-end, protoplasmic cylinder, and H-end of individual Leptospira cells by one-sided dark-field microscopy. At various viscosities of media containing different concentrations of Ficoll, the rotation rate of the S-end and protoplasmic cylinder showed a clear correlation with the swimming speed, suggesting that these two helical parts play a central role in the motion of Leptospira. In contrast, the H-end rotation rate was unstable and showed much less correlation with the swimming speed. Forces produced by the rotation of the S-end and protoplasmic cylinder showed that these two helical parts contribute to propulsion at nearly equal magnitude. Torque generated by each part, also obtained from experimental motion parameters, indicated that the flagellar motor can generate torque >4000 pN nm, twice as large as that of Escherichia coli. Furthermore, the S-end torque was found to show a markedly larger fluctuation than the protoplasmic cylinder torque, suggesting that the unstable H-end rotation might be mechanically related to changes in the S-end rotation rate for torque balance of the entire cell. Variations in torque at the anterior and posterior ends of the Leptospira cell body could be transmitted from one end to the other through the cell body to coordinate the morphological transformations of the two ends for a rapid change in the swimming direction. PMID:24411236

Direct measurement of helical cell motion of the spirochete leptospira.

PubMed

Nakamura, Shuichi; Leshansky, Alexander; Magariyama, Yukio; Namba, Keiichi; Kudo, Seishi

2014-01-07

Leptospira are spirochete bacteria distinguished by a short-pitch coiled body and intracellular flagella. Leptospira cells swim in liquid with an asymmetric morphology of the cell body; the anterior end has a long-pitch spiral shape (S-end) and the posterior end is hook-shaped (H-end). Although the S-end and the coiled cell body called the protoplasmic cylinder are thought to be responsible for propulsion together, most observations on the motion mechanism have remained qualitative. In this study, we analyzed the swimming speed and rotation rate of the S-end, protoplasmic cylinder, and H-end of individual Leptospira cells by one-sided dark-field microscopy. At various viscosities of media containing different concentrations of Ficoll, the rotation rate of the S-end and protoplasmic cylinder showed a clear correlation with the swimming speed, suggesting that these two helical parts play a central role in the motion of Leptospira. In contrast, the H-end rotation rate was unstable and showed much less correlation with the swimming speed. Forces produced by the rotation of the S-end and protoplasmic cylinder showed that these two helical parts contribute to propulsion at nearly equal magnitude. Torque generated by each part, also obtained from experimental motion parameters, indicated that the flagellar motor can generate torque >4000 pN nm, twice as large as that of Escherichia coli. Furthermore, the S-end torque was found to show a markedly larger fluctuation than the protoplasmic cylinder torque, suggesting that the unstable H-end rotation might be mechanically related to changes in the S-end rotation rate for torque balance of the entire cell. Variations in torque at the anterior and posterior ends of the Leptospira cell body could be transmitted from one end to the other through the cell body to coordinate the morphological transformations of the two ends for a rapid change in the swimming direction. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A novel tool for high-throughput screening of granulocyte-specific antibodies using the automated flow cytometric granulocyte immunofluorescence test (Flow-GIFT).

PubMed

Nguyen, Xuan Duc; Dengler, Thomas; Schulz-Linkholt, Monika; Klüter, Harald

2011-02-03

Transfusion-related acute lung injury (TRALI) is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT) has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti-HNA 3a, n = 3; anti-HNA-1b, n = 1) and GAT (anti-HNA-2a, n = 1). The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of blood products.

Bone marrow uptake of 99mTc-MIBI in patients with multiple myeloma.

PubMed

Fonti, R; Del Vecchio, S; Zannetti, A; De Renzo, A; Di Gennaro, F; Catalano, L; Califano, C; Pace, L; Rotoli, B; Salvatore, M

2001-02-01

In a previous study, we showed the ability of technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) scan to identify active disease in patients with multiple myeloma (Eur J Nucl Med 1998; 25: 714-720). In particular, a semiquantitative score of the extension and intensity of bone marrow uptake was derived and correlated with both the clinical status of the disease and plasma cell bone marrow infiltration. In order to estimate quantitatively 99mTc-MIBI bone marrow uptake and to verify the intracellular localization of the tracer, bone marrow samples obtained from 24 multiple myeloma patients, three patients with monoclonal gammopathy of undetermined significance (MGUS) and two healthy donors were studied for in vitro uptake. After centrifugation over Ficoll-Hypaque gradient, cell suspensions were incubated with 99mTc-MIBI and the uptake was expressed as the percentage of radioactivity specifically retained within the cells. The cellular localization of the tracer was assessed by micro-autoradiography. Twenty-two out of 27 patients underwent 99mTc-MIBI scan within a week of bone marrow sampling. Whole-body images were obtained 10 min after intravenous injection of 555 MBq of the tracer; the extension and intensity of 99mTc-MIBI uptake were graded using the semiquantitative score. A statistically significant correlation was found between in vitro uptake of 99mTc-MIBI and both plasma cell infiltration (Pearson's coefficient of correlation r=0.69, P<0.0001) and in vivo score (Spearman rank correlation coefficient r=0.60, P<0.01). No specific tracer uptake was found in bone marrow samples obtained from the two healthy donors. Micro-autoradiography showed localization of 99mTc-MIBI inside the plasma cells infiltrating the bone marrow. Therefore, our findings show that the degree of tracer uptake both in vitro and in vivo is related to the percentage of infiltrating plasma cells which accumulate the tracer in their inner compartments.

Chemically Defined and Xeno-Free Cryopreservation of Human Adipose-Derived Stem Cells

PubMed Central

López, Melany; Bollag, Roni J.; Yu, Jack C.; Isales, Carlos M.; Eroglu, Ali

2016-01-01

The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs). These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Therefore, ASCs offer exceptional opportunities for tissue engineering and regenerative medicine. However, current practices involving ASCs typically use fetal bovine serum (FBS)-based cryopreservation solutions that are associated with risks of immunological reactions and of transmitting infectious diseases and prions. To realize clinical applications of ASCs, serum- and xeno-free defined cryopreservation methods are needed. To this end, an animal product-free chemically defined cryopreservation medium was formulated by adding two antioxidants (reduced glutathione and ascorbic acid 2-phosphate), two polymers (PVA and ficoll), two permeating cryoprotectants (ethylene glycol and dimethylsulfoxide), a disaccharide (trehalose), and a calcium chelator (EGTA) to HEPES-buffered DMEM/F12. To limit the number of experimental groups, the concentration of trehalose, both polymers, and EGTA was fixed while the presence of the permeating CPAs and antioxidants was varied. ASCs suspended either in different versions of the defined medium or in the conventional undefined cryopreservation medium (10% dimethylsulfoxide+10% DMEM/F12+80% serum) were cooled to -70°C at 1°C/min before being plunged into liquid nitrogen. Samples were thawed either in air or in a water bath at 37°C. The presence of antioxidants along with 3.5% concentration of each penetrating cryoprotectant improved the freezing outcome to the level of the undefined cryopreservation medium, but the plating efficiency was still lower than that of unfrozen controls. Subsequently, increasing the concentration of both permeating cryoprotectants to 5% further improved the plating efficiency to the level of unfrozen controls. Moreover, ASCs cryopreserved in this defined medium retained their multipotency and chromosomal normality. These results are of significance for tissue engineering and clinical applications of stem cells. PMID:27010403

Good manufacturing practice-compliant expansion of marrow-derived stem and progenitor cells for cell therapy.

PubMed

Gastens, Martin H; Goltry, Kristin; Prohaska, Wolfgang; Tschöpe, Diethelm; Stratmann, Bernd; Lammers, Dirk; Kirana, Stanley; Götting, Christian; Kleesiek, Knut

2007-01-01

Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14-CD45- and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 x 10(6) total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.

Probiotic Yogurt Culture Bifidobacterium Animalis Subsp. Lactis BB-12 and Lactobacillus Acidophilus LA-5 Modulate the Cytokine Secretion by Peripheral Blood Mononuclear Cells from Patients with Ulcerative Colitis.

PubMed

Sheikhi, A; Shakerian, M; Giti, H; Baghaeifar, M; Jafarzadeh, A; Ghaed, V; Heibor, M R; Baharifar, N; Dadafarin, Z; Bashirpour, G

2016-06-01

There are some evidences for the immunomodulation disorders in the response to intestinal microbiota in inflammatory bowel disease. Yogurt is a fermented milk product made with a starter culture consisting of different probiotics which could be colonized in intestine. However, the role of probiotics in the aetiopathogenesis of ulcerative colitis (UC) has not been clarified. To determine how the immune system responds to these bacteria this study was planned. Bifidobacterium lactis BB-12 (B. lactis) and Lactobacillus acidophilus LA-5 (L. acidophilus) were cultivated on MRS broth. PBMCs of 36 UC patients were separated by Ficoll-Hypaque centrifugation and co-cultured with different concentrations of UV killed bacteria in RPMI-1 640 plus 10% FCS for 48/72 h. IL-10, TGF-β, IFN-γ and TNF-α were measured in supernatant of PBMCs by ELISA. Both bacteria significantly augmented IL-10, TGF-β, IFN-γ and TNF-α compared to control (p<0.001). The secretion levels of IL-10 and TGF-β by B. lactis- compared to L. acidophilus-stimulated PBMCs were significantly higher (p<0.05, p<0.01 respectively). The secretion levels of TNF-α and IFN-γ by PBMCs after 72 h were significantly lower compared to 48 h stimulation by B. lactis (p<0.001, p<0.035 respectively). These data show that both probiotics may trigger the pro- and anti-inflammatory immune response of UC patients. It seems that IL-10/TGF-β uprising by B. lactis could be the reason of TNF-α/IFN-γ reduction. Therefore albeit B. lactis still stimulates the effector Th cells but because of more stimulatory effect on Tregs, it could be a good potential therapeutic candidate for further investigation. © Georg Thieme Verlag KG Stuttgart · New York.

Pulmonary vascular responsiveness in rats following neonatal exposure to high altitude or carbon monoxide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tucker, A.; Penney, D.G.

1993-01-01

Exposure of adult and neonatal rats to high altitude increases pulmonary vascular responsiveness during the exposure. A study was undertaken to determine if a short exposure of neonatal rats to either high-altitude or carbon monoxide (CO) hypoxia would cause persistent alterations in pulmonary vascular responsiveness postexposure. One-day-old male Sprague-Dawley rats were obtained as 16 litters of 10-12 pups each. At 2 days of age, 4 litters were exposed to CO (500 ppm) for 32 days, and 4 litters were exposed to ambient air (AIR) in Detroit (200 m). Another 4 litters were exposed to 3500 m altitude (ALT) in amore » chamber for 32 days, and 3 litters were exposed to ambient conditions in Fort Collins (CON, 1524 m). After the exposures, all rats were maintained at 1524 m. At 2, 40, 76 and 112 days postexposure, lungs were isolated and perfused with Earle's salt solution (+Ficoll, 4 g%). Pulmonary vascular responsiveness was assessed by dose responses to angiotensin II (AII, 0.025-0.40 [mu]g) and acute hypoxia (3% O[sub 2] for 3 min). AII responses were higher in ALT vs CON rats at 2 and 40 days postexposure, but no differences were noted between CO and AIR rats. Baseline pulmonary vascular resistance and pulmonary arterial pressure (in isolated lungs) were higher in ALT rats at all four ages compared to the other three groups. Both the ALT and CO rats displayed hypertrophy of the right ventricle (RV) and the left ventricle (LV) at the termination of treatment and elevated hematocrit. LV hypertrophy and polycythemia regressed with time, but RV hypertrophy remained significant in the ALT rats through 112 days postexposure. The results indicate that neonatal exposure to ALT, but no CO, causes a persistent increase in pulmonary vascular responsiveness and RV hypertrophy for at least 112 days after termination of the exposure. 40 refs., 3 figs., 2 tabs.« less

Quality Assurance of Samples and Processes in the Spanish Renal Research Network (REDinREN) Biobank.

PubMed

Calleros-Basilio, Laura; Cortés, María Alicia; García-Jerez, Andrea; Luengo-Rodríguez, Alicia; Orozco-Agudo, Ana; Valdivielso, José Manuel; Rodríguez-Puyol, Diego; Rodríguez-Puyol, Manuel

2016-12-01

Biobanks are useful platforms to build bridges between basic, translational, and clinical research and clinical care. They are repositories of high-quality human biological samples ideal for evaluating their histological characteristics and also their genome, transcriptome, and proteome. The Spanish Renal Research Network Biobank contains more than 76,500 well-preserved frozen samples of a wide variety of kidney diseases, collected from 5450 patients seen by over 70 nephrology services throughout the Spanish territory. To determine and to report the results of the quality control of samples and processes conducted in our biobank, implemented in accordance with the requirements of the ISO 9001:2008 international standard. Two types of quality controls were performed: (1) systematic, that is, measurement of viable peripheral blood mononuclear cells (PBMCs) obtained and purity of nucleic acids and (2) ad-hoc, that is, viability of thawed PBMC, DNA extraction process reproducibility, and the integrity and functionality of nucleic acids, implemented on a routine basis. PBMC isolation by Ficoll yielded reproducible results and its cryopreserved viability was >90%. Acceptable A260/A280 ratios were obtained for the vast majority of the DNA (n = 2328) and RNA (n = 78) samples analyzed. DNA integrity was demonstrated by agarose gels and by β-globulin gene polymerase chain reaction (PCR) amplification of 1327 and 989 bp fragments. DNA of acceptable quality had at least three bands of β-globulin amplified obtained (n = 26/30). RNA integrity number (RIN) determinations obtained RIN numbers ≥7 (n = 87/96). The amplifiability of nucleic acids was confirmed by qPCR and RT-qPCR of β-actin and GAPDH genes. Long storage or delayed processing time did not affect the quality of the samples analyzed. The processes of DNA extraction also yielded reproducible results. These results clearly indicate that our PBMC, DNA, and RNA stored samples meet the required quality standards to be used for biomedical research, ensuring their long-term preservation.

Circulating tumor cells in patients with testicular germ cell tumors.

PubMed

Nastały, Paulina; Ruf, Christian; Becker, Pascal; Bednarz-Knoll, Natalia; Stoupiec, Małgorzata; Kavsur, Refik; Isbarn, Hendrik; Matthies, Cord; Wagner, Walter; Höppner, Dirk; Fisch, Margit; Bokemeyer, Carsten; Ahyai, Sascha; Honecker, Friedemann; Riethdorf, Sabine; Pantel, Klaus

2014-07-15

Germ cell tumors (GCTs) represent the most frequent malignancies among young men, but little is known about circulating tumor cells (CTCs) in these tumors. Considering their heterogeneity, CTCs were investigated using two independent assays targeting germ cell tumor and epithelial cell-specific markers, and results were correlated with disease stage, histology, and serum tumor markers. CTCs were enriched from peripheral blood (n = 143 patients) and testicular vein blood (TVB, n = 19 patients) using Ficoll density gradient centrifugation. For CTC detection, a combination of germ cell tumor (anti-SALL4, anti-OCT3/4) and epithelial cell-specific (anti-keratin, anti-EpCAM) antibodies was used. In parallel, 122 corresponding peripheral blood samples were analyzed using the CellSearch system. In total, CTCs were detected in 25 of 143 (17.5%) peripheral blood samples, whereas only 11.5% of patients were CTC-positive when considering exclusively the CellSearch assay. The presence of CTCs in peripheral blood correlated with clinical stage (P < 0.001) with 41% of CTC positivity in patients with metastasized tumors and 100% in patients with relapsed and chemotherapy-refractory disease. Histologically, CTC-positive patients suffered more frequently from nonseminomatous primary tumors (P < 0.001), with higher percentage of yolk sac (P < 0.001) and teratoma (P = 0.004) components. Furthermore, CTC detection was associated with elevated serum levels of α-fetoprotein (AFP; P = 0.025), β-human chorionic gonadotropin (βHCG; P = 0.002), and lactate dehydrogenase (LDH; P = 0.002). Incidence and numbers of CTCs in TVB were much higher than in peripheral blood. The inclusion of germ cell tumor-specific markers improves CTC detection in GCTs. CTCs occur frequently in patients with more aggressive disease, and there is a gradient of CTCs with decreasing numbers from the tumor-draining vein to the periphery. ©2014 American Association for Cancer Research.

CD22 Promotes B-1b Cell Responses to T Cell-Independent Type 2 Antigens.

PubMed

Haas, Karen M; Johnson, Kristen L; Phipps, James P; Do, Cardinal

2018-03-01

CD22 (Siglec-2) is a critical regulator of B cell activation and survival. CD22 -/- mice generate significantly impaired Ab responses to T cell-independent type 2 (TI-2) Ags, including haptenated Ficoll and pneumococcal polysaccharides, Ags that elicit poor T cell help and activate BCR signaling via multivalent epitope crosslinking. This has been proposed to be due to impaired marginal zone (MZ) B cell development/maintenance in CD22 -/- mice. However, mice expressing a mutant form of CD22 unable to bind sialic acid ligands generated normal TI-2 Ab responses, despite significantly reduced MZ B cells. Moreover, mice treated with CD22 ligand-binding blocking mAbs, which deplete MZ B cells, had little effect on TI-2 Ab responses. We therefore investigated the effects of CD22 deficiency on B-1b cells, an innate-like B cell population that plays a key role in TI-2 Ab responses. B-1b cells from CD22 -/- mice had impaired BCR-induced proliferation and significantly increased intracellular Ca 2+ concentration responses following BCR crosslinking. Ag-specific B-1b cell expansion and plasmablast differentiation following TI-2 Ag immunization was significantly impaired in CD22 -/- mice, consistent with reduced TI-2 Ab responses. We generated CD22 -/- mice with reduced CD19 levels (CD22 -/- CD19 +/- ) to test the hypothesis that augmented B-1b cell BCR signaling in CD22 -/- mice contributes to impaired TI-2 Ab responses. BCR-induced proliferation and intracellular Ca 2+ concentration responses were normalized in CD22 -/- CD19 +/- B-1b cells. Consistent with this, TI-2 Ag-specific B-1b cell expansion, plasmablast differentiation, survival, and Ab responses were rescued in CD22 -/- CD19 +/- mice. Thus, CD22 plays a critical role in regulating TI-2 Ab responses through regulating B-1b cell signaling thresholds. Copyright © 2018 by The American Association of Immunologists, Inc.

Cryopreservation of feline oocytes by vitrification using commercial kits and slush nitrogen technique.

PubMed

Fernandez-Gonzalez, L; Jewgenow, K

2017-04-01

Assisted reproductive techniques are a valuable tool for conservation breeding of endangered species. Cryopreservation methods are the basis of gamete banks, supporting genetic diversity preservation. Unfortunately, cryopreservation of feline oocytes is still considered an experimental technique. The aim of this study was to compare two commercial kits, with our protocol for vitrification of cat oocytes (IZW), which comprises a three-step method with ethylene glycol, DMSO, fetal calf serum, trehalose and Ficoll PM-70. Furthermore, we applied slush nitrogen (SN 2 ) for ultra-rapid freezing to improve survival rates. Cumulus-oocyte complexes were collected from domestic cat ovaries by slicing and vitrified at immature stage using Cryotop as storage device. Vit Kit ® Freeze/Thaw (n = 89) showed the lowest maturation percentage obtained after warming (10.1%). A significant difference in maturation percentage of oocytes was found between Kitazato ® kit (38.7%, n = 137) and IZW protocol (24.5%, n = 143). The cleavage after ICSI of warmed and matured oocytes (20.7% and 28.6%, respectively) and the morula percentage (18. 2% and 22.5%, respectively), however, did not reveal any significant difference between the two methods. Application of SN 2 did not result in any improvement of oocytes' cryopreservation. Maturation percentage of the oocytes vitrified by IZW method with SN 2 (n = 144) decreased until 6.1%, without any cleavage after fertilization. For Kitazato ® (n = 62), only 17.7% were able to undergo maturation and cleavage percentage dropped to 18.2%, not reaching morula stage. These data demonstrate that feline oocytes can be vitrified either by our IZW method or by commercial Kitazato ® kit, but the use of SN 2 is improving neither maturation nor cleavage percentages when combined with these procedures. © 2016 Blackwell Verlag GmbH.

[Effect of G-CSF in vitro Stimulation on Distribution of Peripheral Lymphocyte Subsets in the Healthy Persons].

PubMed

Zhao, Sha-Sha; Fang, Shu; Zhu, Cheng-Ying; Wang, Li-Li; Gao, Chun-Ji

2018-02-01

To investigate the effect of granulocyte-colony stimulating factor (G-CSF) in vitro stimulation on the distribution of lymphocyte subset in healthy human. Peripheral blood mononuclear cells (PBMNCs) were collected from 8 healthy volunteers by density gradient centrifugation on Ficoll-Paque TM . In vitro 200 ng/ml G-CSF or 200 ng/ml G-CSF plus 10 µg/ml ConA directly act on PBMNCs, then the colleted cells were cultivated for 3 days. Lymphocyte subsets were stained with the corresponding fluoresce labeled antibodies and detected by flow cytometry. The levels of T cells in G-CSF group and G-CSF+ConA group were both higher than that in the control group (P<0.001, P<0.05). However, there were not significantly different in B cells and NK cells levels among the 3 groups. Furthermore, analysis of the effect of G-CSF on T cell subsets indicated that the levels of CD4 + T cells and CD8 + T cells in G-CSF group were both significantly higher than those in control group (P<0.01, P<0.05), Treg cells was not different between G-CSF and control group. Compared with the control group, the level of CD4 + T cells, CD8 + T cells and Treg cells in G-CSF+ConA group significantly increased (P<0.05, P<0.01, P<0.01). Analysis of G-CSF receptor (G-CSFR) expression showed that G-CSFR expression on T cells in G-CSF+ConA group dramatically increased, as compared with control group (P<0.01). The levels of CD4 + T cells and CD8 + T cells in healthy human peripheral blood can be increased by G-CSF stimulation. ConA can enhance the level of T cells and induce G-CSFR expression on T cells.

Single-cell antibody nanowells: a novel technology in detecting anti-SSA/Ro60- and anti-SSB/La autoantibody-producing cells in peripheral blood of rheumatic disease patients.

PubMed

Esfandiary, Lida; Gupta, Nirupama; Voigt, Alexandria; Wanchoo, Arun; Chan, Edward K L; Sukumaran, Sukesh; Nguyen, Cuong Q

2016-05-17

Anti-SSA/Ro60 and anti-SSB/La are essential serological biomarkers for rheumatic diseases, specifically Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Currently, laboratory detection technology and platforms are designed with an emphasis on high-throughput methodology; therefore, the relationship of sensitivity with specificity remains a significant area for improvement. In this study, we used single-cell antibody nanowells (SCAN) technology to directly profile individual B cells producing antibodies against specific autoantigens such as SSA/Ro60 and SSB/La. Peripheral blood mononuclear cells were isolated using Ficoll gradient. Fluorescently labeled cells were added to fabricated nanowells and imaged using a high-speed epifluorescence microscope. The microengraving process was conducted using printed slides coated with immunoglobulins. Printed slides were hybridized with fluorescence-conjugated immunoglobulin G (IgG), SSA/Ro60, and SSB/La antigens. Microarray spots were analyzed for nanowells with single live B cells that produced antigen-specific autoantibodies. Our results indicate that SCAN can simultaneously detect high frequencies of anti-SSA/Ro60 and anti-SSB/La with a specific IgG isotype in peripheral blood mononuclear cells of patients, as well as measure their individual secretion levels. The data showed that patients with SS and SLE exhibited higher frequency and greater concentration of anti-SSA/Ro60- and anti-SSB/La-producing B cells in the IgG isotype. Furthermore, individual B cells of patients produced higher levels of IgG-specific anti-SSA/Ro60 autoantibody, but not IgG-specific anti-SSB/La autoantibody, compared with healthy control subjects. These results support the application of SCAN as a robust multiparametric analytical bioassay that can directly measure secretion of autoantibody and accurately report antigen-specific, autoantibody-producing cells.

AB033. Preimplantation genetic diagnosis of spinal muscular atrophy in Vietnam

PubMed Central

Khoa, Tran Van; Nga, Nguyen Thi Thanh; Tao, Nguyen Dinh; Sang, Trieu Tien; Giang, Ngo Truong; Dung, Vu Chi

2015-01-01

Objective Spinal muscular atrophy (SMA) is a severe neurodegenerative autosomal recessive disorder. Most of patients are caused by the homozygous absence of exon 7 of the telomeric copy of the SMN gene (SMNt) on chromosome 5. Setting up a molecular diagnostic protocol for detecting exon 7 gen SMNT homozygous deletion in single cell is basic to preimplantation genetic diagnosis of spinal muscular atrophy. Methods This study was carried out on 17 patients and their parents. Firstly, lymphocytes of patients and their parents were isolated from fresh blood by ficoll. Taking a lymphocyte on stereoscopic microscope, lysing the cell, amplifying whole genome, then amplifying exon 7 of SMNT gene by using a polymerase chain reaction, followed by HinfI restriction digest enzyme of the PCR enabling the important SMNT gene to be distinguished from the centromic SMN gene (SMNc) which has no clinical phenotype to detect mutation. Electrophoresis PCR products after digesting by restriction enzyme and analysis. Besides, the minisequencing technique has also been used to detect the absence of exon 7 of SMNT gene based on the difference of one nucleotide at 214-position in exon 7 (C-SMNT, T-SMNc). Secondly, the application of the protocol was set up on one lymphocyte to preimplantation genetic diagnosis of spinal muscular atrophy on biopsied blastomeres. Results Two different protocols which were PCR-RFLP and minisequencing, were set up on 200 lymphocytes from 17 patients and their parents to screen the homozygous deletion in exon 7 SMNT gene with the PCR efficiency in 96%. The results were similar with the gene diagnosed from fresh blood. The methods were also efficient, providing interpretable result in 96.55% (28/29) of the blastomeres tested. Three couples were treated using this method. Three normal embryos were transfer which resulted in one clinical pregnancy. Conclusions We have successfully applied the technique of PCR-RFLP and minisequencing for the preimplantation genetic diagnosis of spinal muscular atrophy.

Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

PubMed Central

Boos, Anja M; Loew, Johanna S; Deschler, Gloria; Arkudas, Andreas; Bleiziffer, Oliver; Gulle, Heinz; Dragu, Adrian; Kneser, Ulrich; Horch, Raymund E; Beier, Justus P

2011-01-01

Abstract Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto-transplanted versus in vitro expanded MSC with or without bone morphogenetic protein-2 (BMP-2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto-transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT-PCR analysis before subcutaneous implantation in combination with BMP-2 and β-tricalcium phosphate/hydroxyapatite (β-TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT-PCR evaluation. Sheep MSC were CD29+, CD44+ and CD166+ after selection by Ficoll gradient centrifugation, while directly auto-transplanted MSC-populations expressed CD29 and CD166 at lower levels. Both, directly auto-transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto-transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β-TCP/HA matrix comparable to the application of 60 μg/ml BMP-2 only or implantation of expanded MSC. Bone matrix proteins were up-regulated in constructs following direct auto-transplantation and in expanded MSC as well as in BMP-2 constructs. Up-regulation was detected using immunohistology methods and RT-PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto-transplanted or expanded MSC with β-TCP/HA granules alone. Hence BMP-2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering. PMID:20636333

Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo

PubMed Central

Jusof, Wan-Hafizah Wan; Khan, Nor-Ashikin Mohamed Noor; Rajikin, Mohd Hamim; Satar, Nuraliza Abdul; Mustafa, Mohd-Fazirul; Jusoh, Norhazlin; Dasiman, Razif

2015-01-01

Background Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos. Materials and Methods In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test. Results A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001). Conclusion Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification. PMID:26246881

Increase in peripheral blood mononuclear cell Toll-like receptor 2/3 expression and reactivity to their ligands in a cohort of patients with wet age-related macular degeneration

PubMed Central

Zhu, Yi; Liang, Liang; Qian, Dan; Yu, Hongsong; Yang, Peizeng; Lei, Bo

2013-01-01

Purpose To investigate Toll-like receptor (TLR) expression and reactivity in patients with the wet form age-related macular degeneration (AMD). Methods Blood samples were collected from 25 patients with wet AMD and 25 age-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Hypaque density gradient centrifugation. Expression of TLR1 to TLR10 mRNAs in PBMCs from 15 patients with wet AMD and 15 controls was assessed with real-time PCR. TLR2 and TLR3 protein levels in PBMCs from six patients with wet AMD and six controls were measured with flow cytometry. After PBMCs were stimulated with peptidoglycan (PGN) and poly(I:C), the specific ligands of TLR2 and TLR3, cytokines interleukin-6 (IL-6), IL-8, VEGF, and monocyte chemoattractant protein-1 (MCP-1) production in 11 patients with wet AMD and 11 controls were assessed. Results TLR2 and TLR3 mRNA and protein expression in the PBMCs of the patients with wet AMD was significantly higher than that in the controls. However, the difference in TLR1 and TLR4–10 mRNA expression between the two groups was not significant. The PBMCs of the patients with wet AMD produced more IL-6 and IL-8 proteins than the controls in response to PGN, a ligand for TLR2, and more IL-6 protein than the controls in response to poly(I:C), the ligand for TLR3. However, there was no significant difference in vascular endothelial growth factor and monocyte chemoattractant protein-1 production between the wet AMD group and the control group when the PBMCs were stimulated with PGN or poly(I:C). Conclusions Our data suggested that upregulation of TLR2 and TLR3 may be associated with the pathogenesis of wet AMD. PMID:23946637

Identification and characterization of B cell precursors in rat lymphoid tissues. I. Adoptive transfer assays for precursors of TI-1, TI-2, and TD antigen-reactive B cells.

PubMed

Whalen, B J; Goldschneider, I

1993-10-01

Quantitative adoptive transfer assays were developed to detect the precursors of TI-1, TI-2, and TD antigen-reactive B cells in rat lymphoid tissues. Studies on the immune responses in normal and athymic nude rats validate the use of TNP-lipopolysaccharide as a TI-1 antigen, TNP-Ficoll as a TI-2 antigen, and SRBC as a TD antigen in rats. The precursors to these immunologically competent B cells are detected, following transfer into irradiated histocompatible recipients, by their ability to generate expanded populations of antigen-reactive B cells capable of mounting antibody responses (splenic IgM plaque-forming cells) to these antigens. Maximal numbers of antigen-reactive B cells emerge in antigenically naive rats after an interval of 7-12 days following transfer of donor lymphoid cells and decline rapidly thereafter. The delayed responses in adoptive recipients reconstituted with spleen cells are proportional to the numbers of spleen cells transferred and are shown to be primarily donor derived using histocompatible Ig kappa chain alloantigen disparate rat strain combinations. The precursors of TI-1, TI-2, and TD antigen-reactive B cells are present in both donor spleen and bone marrow. However, precursor cells to TI-1 and TD antigens are largely absent from donor lymph node cells, whereas precursors to the TI-2 antigen are as prevalent in donor lymph node as in donor spleen. These results support the hypothesis that newly formed virginal B cells represent transient populations of precursor cells that undergo further proliferation and differentiation in the spleen before acquiring immunological competence. The results also suggest that the precursors of TI-2 antigen-reactive B cells differ developmentally from those of TI-1 and TD antigen-reactive B cells, and that the antigen-reactive progeny of these precursors require additional stimulation in order to join the pool of long-lived peripheral B cells.

ANTI-INFLAMMATORY ACTIVITY OF EUCALYPTUS SPP. AND PISTASCIA LENTISCUS LEAF EXTRACTS

PubMed Central

Qabaha, Khaled; Ras, Sari Abu; Abbadi, Jehad; Al-Rimawi, Fuad

2016-01-01

Background: Eucalyptus spp. and Pistascia lentiscus are among the Palestinian trees that are traditionally used in folkloric medicine in treating many diseases; leaves of which are thought to have anti-inflammatory, antibacterial and antioxidant effects. The goal of this study is to evaluate the in vitro inhibitory effect of Eucalyptus spp. and Pistascia lentiscus extracts on Lipopolysacaride (LPS)-induced Interlukin-6 (Il-6) and Tumor Necrosis Factor-α (TNF-α) by polymorphonuclear Cells (PMNCs). Materials and Methods: Polymorphonuclear cells were isolated from the whole blood using Histopaque (Ficol-1077) method and then cultured in an enriched Roswell Park Memorial Institute (RBMI) medium. Supernatants’ Interlukin-6 (IL-6) and Tumor Necrosis Factor (TNF-α) levels were determined 24 hour after LPS stimulation. HPLC was employed to determine the concentration of phenolic compounds in the extracts. The concentrations of TNF-α and IL-6 were compared using paired-samples t test. Results: Eucalyptus spp. and Pistascia lentiscus leaves extracts have shown significant reduction in the levels of both Il-6 and TNF-α Gallic acid; a strong anti-inflammatory agent was found to be the major phenolic compound in both leaf extracts. However, other anti-inflammatory phenolic compounds were detected in Pitascia lentiscus extract including syringic acid and p-coumaric acid, while chlorogenic acid was detected in Eucalyptus spp. leaf extract. Conclusion: Reduction in the levels of Il-6 and TNF-α upon the effect of both Eucalyptus spp. and Pistascia lentiscus extract is an indication of their anti-inflammatory effects. Our results may also indicate that the observed anti-inflammatory effect of the above extracts may be due to the presence of gallic acid and other phenolic compounds. List of Abbreviations and Nomenclature: LPS: Lipopolysacaride, Il-6: Interlukin-6, TNF-α: Tumor Necrosis Factor-α, PMNCs: Polymorphonuclear Cells, HPLC: High Performance Liquid Chromatography, ELISA: Enzyme Linked Immune Sorbent Assay, EDTA: Ethylene Diamine Tetra Acetic acid, PBS: phosphate buffered saline, RPMI: Roswell Park Memorial Institute medium FBS: Fetal Bovine Serum. PMID:28487887

Evaluation of Ginger (Zingiber officinale Roscoe) Bioactive Compounds in Increasing the Ratio of T-cell Surface Molecules of CD3+CD4+:CD3+CD8+ In-Vitro.

PubMed

Tejasari, Dr

2007-09-01

The potential ability of ginger bioactive compounds in increasing the ratio of T-cell surface molecules of CD3+CD4+:CD3+CD8+ was investigated using dual tagging FITC and PE of monoclonal antibody anti-human with its fluorescence measured by flow cytometer. Oleoresin was extracted using sinkhole distillation technique. Its components namely, gingerol in fraction-1, shogaol in fraction 2 and zingeron in fraction-3 were separated by column vacuum chromatography method. The doses of oleoresin, gingerol, shogaol, and zingeron tested were 50, 100,150, 200, and 250 μg/ml. Lymphocytes (2x106 cell/ml) from human peripheral blood were isolated using ficoll density gradient technique, and cultured in the presence of the compounds in RPMI-1640 medium and phytohemaglutinin (PHA) mitogen for 96 h under normal conditions. Percentages of T-cell surface molecules (CD4+ and CD8+) were determined using dual-tagging FITC and PE fluorescents labeled on monoclonal antibody anti human. The fluorescence-labeled bands on the T-cell surface molecules were counted using flow cytometer. The experiment revealed that oleoresin and its three fractions increased the percentage of CD3+CD4+. The compound in fraction 3 of oleoresin at 200 μg/ml increased by the highest percentage of CD3+CD4+ of 9%, but slightly decreased the percentage of CD3+CD8+. These ginger bioactive compounds increased the ratio of CD3+CD4:CD3+CD8+ T-cells with the highest increment of 30% from effects of 200 μg/ml fraction 3 of oleoresin. This in vitro finding revealed that ginger bioactive compounds potentially increased cellular and humoral immune response. Further clinical studies are needed to confirm the benefits of these ginger bioactive compounds as a potential functional food for testing on HIV infected patients.

Comparative study on anti-oxidant and anti-inflammatory activities of Caesalpinia crista and Centella asiatica leaf extracts

PubMed Central

Ramesh, B. N.; Girish, T. K.; Raghavendra, R. H.; Naidu, K. Akhilender; Rao, U. J. S. Prasada; Rao, K. S.

2014-01-01

Background: Amyloidosis, oxidative stress and inflammation have been strongly implicated in neurodegenerative disorders like Alzheimer's disease. Traditionally, Caesalpinia crista and Centella asiatica leaf extracts are used to treat brain related diseases in India. C. crista is used as a mental relaxant drink as well as to treat inflammatory diseases, whereas C. asiatica is reported to be used to enhance memory and to treat dementia. Objective: The present study is aimed to understand the anti-oxidant and anti-inflammatory potential of C. asiatica and C. crista leaf extracts. Materials and Methods: Phenolic acid composition of the aqueous extracts of C. crista and C. asiatica were separated on a reverse phase C18 column (4.6 x 250 mm) using HPLC system. Antioxidant properties of the leaf extracts were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and the reducing potential assay. The anti-inflammatory activities of aqueous extracts of C. crista and C. asiatica were studied using 5-lipoxygenase assay. Polymorphonuclear leukocytes (PMNLs) were isolated from blood by Ficoll-Histopaque density gradient followed by hypotonic lysis of erythrocytes. Results: Gallic, protocatechuic, gentisic, chlorogenic, caffeic, p-coumaric and ferulic acids were the phenolic acids identified in C. crista and C. asiatica leaf aqueous extracts. However, gallic acid and ferulic acid contents were much higher in C. crista compared to C. asiatica. Leaf extracts of C. asiatica and C. crista exhibited antioxidant properties and inhibited 5-lipoxygenase (anti-inflammatory) in a dose dependent manner. However, leaf extracts of C. crista had better antioxidant and anti-inflammatory activity compared to that of C. asiatica. The better activity of C. crista is attributed to high gallic acid and ferulic acid compared to C. asiatica. Conclusions: Thus, the leaf extract of C. crista can be a potential therapeutic role for Alzheimer's disease. PMID:24741275

Ex vivo expansion of human umbilical cord blood-derived T-lymphocytes with homologous cord blood plasma.

PubMed

Kim, Yong-Man; Jung, Min-Hyung; Song, Ha-Young; Yang, Hyun Ok; Lee, Sung-Tae; Kim, Jong-Hyeok; Kim, Young-Tak; Nam, Joo-Hyun; Mok, Jung-Eun

2005-02-01

This study was designed to establish a more effective and safe culture system for adoptive immunotherapy by investigating the use of homologous cord blood plasma (HCBP) instead of fetal bovine serum (FBS), which has various limitations including ethical problems for the ex vivo expansion of human umbilical T lymphocytes. Fresh human umbilical mononuclear cell fractions were isolated by Ficoll-Hypaque density centrifugation. Nonadherent mononuclear cell fractions were cultured with anti-CD3 antibody (5 microg/ml), IL-2 (175 U/ml), and either 10% FBS or 10% HCBP. On day 8, the cellular proliferation rate and cell surface markers were assessed. There was no significant difference in proliferation when human umbilical cord blood T lymphocytes were grown in medium supplemented with FBS or HCBP (p > 0.05). In medium containing FBS, the proportion of CD3(+)CD4(+) (markers for helper T cell), CD3(+)CD8(+) (cytotoxic T cell), CD3(+)CD25(+) (activated T cell), CD3(+)CD38(+) (immature T cell), and CD3(+)CD45RO(+) (memory T cell) cells was significantly increased (p < 0.05), whereas proportion of CD3(+)CD45RA(+) (naive T cell) and CD16(+)CD56(+) (NK cell) cells was significantly decreased (p < 0.05). In HCBP supplemented medium, the proportion of CD3(+)CD8(+), CD3(+)CD25(+), CD3(+)CD45RA(+), and CD3(+)CD45RO(+) cells was significantly increased (p < 0.05). The proportion of CD3(+)CD4(+), CD3(+)CD45RO(+) and CD3(+)CD38(+) cells was significantly higher, but proportion of CD3(+)CD45RA(+) and CD3(+)CD8(+) cells was significantly lower in FBS compared with HCBP supplemented medium (p < 0.05). Our results support the feasibility of ex vivo expansion of human umbilical cord blood T lymphocytes in medium supplemented with HCBP for future adoptive cellular immunotherapy.

Role of shear stress in nitric oxide-dependent modulation of renal angiotensin II vasoconstriction.

PubMed

Endlich, K; Muller, C; Barthelmebs, M; Helwig, J J

1999-08-01

1. Renal vasoconstriction in response to angiotensin II (ANGII) is known to be modulated by nitric oxide (NO). Since shear stress stimulates the release of a variety of vasoactive compounds from endothelial cells, we studied the impact of shear stress on the haemodynamic effect of ANGII in isolated perfused kidneys of rats under control conditions and during NO synthase inhibition with L-NAME (100 microM). 2. Kidneys were perfused in the presence of cyclo-oxygenase inhibitor (10 microM indomethacin) with Tyrode's solution of relative viscosity zeta=1 (low viscosity perfusate, LVP) or, in order to augment shear stress, with Tyrode's solution containing 7% Ficoll 70 of relative viscosity zeta=2 (high viscosity perfusate, HVP). 3. Vascular conductance was 3.5+/-0.4 fold larger in HVP as compared with LVP kidneys, associated with an augmentation of overall wall shear stress by 37+/-5%. During NO inhibition, vascular conductance was only 2.5+/-0.2 fold elevated in HVP vs LVP kidneys, demonstrating shear stress-induced vasodilatation by NO and non-NO/non-prostanoid compound(s). 4. ANGII (10 - 100 pM) constricted the vasculature in LVP kidneys, but was without effect in HVP kidneys. During NO inhibition, in contrast, ANGII vasoconstriction was potentiated in HVP as compared with LVP kidneys. 5. The potentiation of ANGII vasoconstriction during NO inhibition has been shown to be mediated by endothelium-derived P450 metabolites and to be sensitive to AT2 receptor blockade in our earlier studies. Accordingly, in HVP kidneys, increasing concentrations of the AT2 receptor antagonist PD123319 (5 and 500 nM) gradually abolished the potentiation of ANGII vasoconstriction during NO inhibition, but did not affect vasoconstriction in response to ANGII in LVP kidneys. 6. Our results demonstrate, that augmentation of shear stress by increasing perfusate viscosity induces vasodilatation in the rat kidney, which is partially mediated by NO. Elevated levels of shear stress attenuate renal ANGII vasoconstriction through enhanced NO production and are required for AT2 sensitive potentiation during NO inhibition.

Role of shear stress in nitric oxide-dependent modulation of renal angiotensin II vasoconstriction

PubMed Central

Endlich, Karlhans; Muller, Catherine; Barthelmebs, Mariette; Helwig, Jean-Jacques

1999-01-01

Renal vasoconstriction in response to angiotensin II (ANGII) is known to be modulated by nitric oxide (NO). Since shear stress stimulates the release of a variety of vasoactive compounds from endothelial cells, we studied the impact of shear stress on the haemodynamic effect of ANGII in isolated perfused kidneys of rats under control conditions and during NO synthase inhibition with L-NAME (100 μM).Kidneys were perfused in the presence of cyclo-oxygenase inhibitor (10 μM indomethacin) with Tyrode's solution of relative viscosity ζ=1 (low viscosity perfusate, LVP) or, in order to augment shear stress, with Tyrode's solution containing 7% Ficoll 70 of relative viscosity ζ=2 (high viscosity perfusate, HVP).Vascular conductance was 3.5±0.4 fold larger in HVP as compared with LVP kidneys, associated with an augmentation of overall wall shear stress by 37±5%. During NO inhibition, vascular conductance was only 2.5±0.2 fold elevated in HVP vs LVP kidneys, demonstrating shear stress-induced vasodilatation by NO and non-NO/non-prostanoid compound(s).ANGII (10–100 pM) constricted the vasculature in LVP kidneys, but was without effect in HVP kidneys. During NO inhibition, in contrast, ANGII vasoconstriction was potentiated in HVP as compared with LVP kidneys.The potentiation of ANGII vasoconstriction during NO inhibition has been shown to be mediated by endothelium-derived P450 metabolites and to be sensitive to AT2 receptor blockade in our earlier studies. Accordingly, in HVP kidneys, increasing concentrations of the AT2 receptor antagonist PD123319 (5 and 500 nM) gradually abolished the potentiation of ANGII vasoconstriction during NO inhibition, but did not affect vasoconstriction in response to ANGII in LVP kidneys.Our results demonstrate, that augmentation of shear stress by increasing perfusate viscosity induces vasodilatation in the rat kidney, which is partially mediated by NO. Elevated levels of shear stress attenuate renal ANGII vasoconstriction through enhanced NO production and are required for AT2 sensitive potentiation during NO inhibition. PMID:10482926

Ketamine, as adjuvant analgesics for patients with refractory cancer pain, does affect IL-2/IFN-γ expression of T cells in vitro?: A prospective, randomized, double-blind study.

PubMed

Zhou, Naibao; Fu, Zhijian; Li, Hao; Wang, Kaiguo

2017-04-01

Ketamine has been used as an analgesic adjuvant with morphine in the treatment of refractory cancer pain recently. But both morphine and ketamine have been reported to produce a number of immunomodulatory effects. The current study was performed to assess whether the concentration of ketamine, as adjuvant analgesics for patient with refractory cancer pain, was related to its effect on T cells interleukin-2 (IL-2)/interferon-γ (IFN-γ) expression in vitro. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood of patients with refractory cancer pain over a Ficoll-Hypaque density gradient. T cells were isolated from by positive selection using anti-CD3 beads. T cells were then treated with vehicle (C group), morphine (200 ng/mL, M group), morphine (200 ng/mL), and different dose of ketamine (100, 200, 1000 ng/mL; MK1, MK5, MK10 group) for 24 hours before stimulation with anti-CD3 and anti-CD28. Then supernatant IL-2 and IFN-γ protein analysis, quantitative reverse transcription polymerase chain reaction (RT-PCR) for IL-2 and IFN-γ were done. There were no significant difference of supernatant IL-2 and IFN-γ among C group, M group, and MK1 group, but the mRNA of M group and MK1 group were decreased compared with C group (P < .05). Compared with C group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all significantly decreased (P < .01). Compared with M group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all decreased (P < .05), while supernatant IL-2 and the mRNA of MK10 group were significantly decreased (P < .01). In conclusion, we confirmed that just as morphine, ketamine dose-dependently suppressed IL-2 and IFN-γ of activated T lymphocyte of patients with refractory cancer pain in vitro, but the inhibitory action of low dose ketamine could be neglected.

Ketamine, as adjuvant analgesics for patients with refractory cancer pain, does affect IL-2/IFN-γ expression of T cells in vitro?

PubMed Central

Zhou, Naibao; Fu, Zhijian; Li, Hao; Wang, Kaiguo

2017-01-01

Abstract Background: Ketamine has been used as an analgesic adjuvant with morphine in the treatment of refractory cancer pain recently. But both morphine and ketamine have been reported to produce a number of immunomodulatory effects. The current study was performed to assess whether the concentration of ketamine, as adjuvant analgesics for patient with refractory cancer pain, was related to its effect on T cells interleukin-2 (IL-2)/interferon-γ (IFN-γ) expression in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood of patients with refractory cancer pain over a Ficoll-Hypaque density gradient. T cells were isolated from by positive selection using anti-CD3 beads. T cells were then treated with vehicle (C group), morphine (200 ng/mL, M group), morphine (200 ng/mL), and different dose of ketamine (100, 200, 1000 ng/mL; MK1, MK5, MK10 group) for 24 hours before stimulation with anti-CD3 and anti-CD28. Then supernatant IL-2 and IFN-γ protein analysis, quantitative reverse transcription polymerase chain reaction (RT-PCR) for IL-2 and IFN-γ were done. Results: There were no significant difference of supernatant IL-2 and IFN-γ among C group, M group, and MK1 group, but the mRNA of M group and MK1 group were decreased compared with C group (P < .05). Compared with C group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all significantly decreased (P < .01). Compared with M group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all decreased (P < .05), while supernatant IL-2 and the mRNA of MK10 group were significantly decreased (P < .01). Conclusion: In conclusion, we confirmed that just as morphine, ketamine dose-dependently suppressed IL-2 and IFN-γ of activated T lymphocyte of patients with refractory cancer pain in vitro, but the inhibitory action of low dose ketamine could be neglected. PMID:28422864

[The induction and cryopreservation of erythroid progenitor cells derived from umbilical cord blood mononuclear cells].

PubMed

Chen, Lin; Xie, Xiaoyan; Xi, Jiafei; Lyu, Yang; Tian, Yu; Liu, Daqing; Yue, Wen; Li, Yanhua; Nan, Xue; Li, Siting; Fan, Zeng; Pei, Xuetao

2016-01-01

To discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs). UCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing. With the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective. This non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.

A cell transportation solution that preserves live circulating tumor cells in patient blood samples.

PubMed

Stefansson, Steingrimur; Adams, Daniel L; Ershler, William B; Le, Huyen; Ho, David H

2016-05-06

Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90% viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs after days of storage. Therefore, we suggest an effective and economical transportation of cancer patient blood samples containing live CTCs can be achieved.

The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs.

PubMed

Krickhahn, Mareike; Bühler, Christoph; Meyer, Thomas; Thiede, Arnulf; Ulrichs, Karin

2002-01-01

Clinical islet allotransplantation has become an increasingly efficient "routine" therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 +/- 720 islet equivalents per gram organ (IEQ/g); n = 25; 2-3 years old; RB] and also from young hybrid pigs [2868 +/- 260 IEQ/g; n = 65; 4-6 months old; HY] using LiberasePI and a modified version of Ricordi's digestion-filtration technique. As expected, isolations from RB showed significantly better results (p < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of organs from RB (80%) contained mainly large islets (diameter > 200 microm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RB(Large Islets): 5680 +/- 3,318 IEQ/g (n= 20); RB(Small Islets): 1353 +/- 427 IEQ/g (n = 5); p < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrep density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrep was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the isolation process. Under these conditions highly successful isolations can reliably be performed even from young market pigs.

A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture

PubMed Central

Jaquinod, Michel; Villiers, Florent; Kieffer-Jaquinod, Sylvie; Hugouvieux, Véronique; Bruley, Christophe; Garin, Jérôme; Bourguignon, Jacques

2007-01-01

To better understand the mechanisms governing cellular traffic, storage of various metabolites and their ultimate degradation, Arabidopsis thaliana vacuoles proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker α-mannosidase, the enrichment factor of the vacuoles was estimated at approximately 42 fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomic study. Therefore, a proteomic approach was developed in order to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, (iii) a pre-fractionation of proteins by short migration on SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, 2/3 of which copurify with the membrane hydrophobic fraction and 1/3 with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were previously known to be associated with vacuolar activities. The proteins identified are involved in: ion and metabolite transport (26%), stress response (9%), signal transduction (7%), metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein- and sugar-hydrolysis. The sub-cellular localization of several putative vacuolar proteins was confirmed by transient expression of GFP-fusion constructs. PMID:17151019

Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model.

PubMed

Boos, Anja M; Loew, Johanna S; Deschler, Gloria; Arkudas, Andreas; Bleiziffer, Oliver; Gulle, Heinz; Dragu, Adrian; Kneser, Ulrich; Horch, Raymund E; Beier, Justus P

2011-06-01

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto-transplanted versus in vitro expanded MSC with or without bone morphogenetic protein-2 (BMP-2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto-transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT-PCR analysis before subcutaneous implantation in combination with BMP-2 and β-tricalcium phosphate/hydroxyapatite (β-TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT-PCR evaluation. Sheep MSC were CD29(+), CD44(+) and CD166(+) after selection by Ficoll gradient centrifugation, while directly auto-transplanted MSC-populations expressed CD29 and CD166 at lower levels. Both, directly auto-transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto-transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β-TCP/HA matrix comparable to the application of 60 μg/ml BMP-2 only or implantation of expanded MSC. Bone matrix proteins were up-regulated in constructs following direct auto-transplantation and in expanded MSC as well as in BMP-2 constructs. Up-regulation was detected using immunohistology methods and RT-PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto-transplanted or expanded MSC with β-TCP/HA granules alone. Hence BMP-2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

HIV-1-based defective lentiviral vectors efficiently transduce human monocytes-derived macrophages and suppress replication of wild-type HIV-1

PubMed Central

Zeng, Lingbing; Planelles, Vicente; Sui, Ziye; Gartner, Suzanne; Maggirwar, Sanjay B.; Dewhurst, Stephen; Ye, Linbai; Nerurkar, Vivek R.; Yanagihara, Richard; Lu, Yuanan

2010-01-01

Background Human monocytes play an important role in mediating human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS), and monocytes-derived macrophages (MDM) represent a major viral reservoir within the brain and other target organs. Current gene transduction of MDM is hindered by a limited efficiency. In this study we established a lentiviral vector-based technique for improved gene transfer into human MDM cultures in vitro and demonstrated significant protection of transduced MDM from super-infection with wild-type HIV-1. Methods HIV-1-based lentiviral vector stocks were prepared in 293T cells by the established calcium phosphate transfection method. Human monocytes were isolated from donors' blood by Ficoll-Paque separation and cultured in vitro. To establish an effective technique for vector-mediated gene transfer, primary cultures of human MDM were transduced at varying multiplicities of infection (MOI) and at a range of time points following initial isolation of cells (time-in-culture). Transduced cells were then examined for transgene (green fluorescent protein (GFP)) expression by fluorescent microscopy and reverse transcription polymerase chain reaction (RT-PCR). These cultures were then exposed to wild-type HIV-1, and viral replication was quantitated by p24 assay; production of neurotoxic effector molecules by the transduced MDM was also examined, using indicator neurons. Results We have demonstrated that primary human MDM could be efficiently transduced (>50%) with concentrated HIV-1-based defective lentiviral vectors (DLV). Furthermore, DLV-mediated gene transduction was stable, and the transduced cells exhibited no apparent difference from normal MDM in terms of their morphology, viability and neurotoxin secretion. Challenge of DLV-transduced MDM cultures with HIV-1Ba-L revealed a 4- to 5-fold reduction in viral replication, as measured by p24 antigen production. This effect was associated with the mobilization of the GFP-expressing DLV construct by the wild-type virus. Conclusions These data demonstrate the inhibition of HIV-1 replication in primary MDM, by a DLV vector that lacks any anti-HIV-1 transgene. These findings lay the initial groundwork for future studies on the ability of DLV-modified monocytes to introduce anti-HIV-1 genes into the CNS. Lentiviral vector-mediated gene delivery to the CNS by monocytes/macrophages is a promising, emerging strategy for treating neuro-AIDS. PMID:16142830

Time and force dependence of the rupture of glycoprotein IIb-IIIa-fibrinogen bonds between latex spheres.

PubMed Central

Goldsmith, H L; McIntosh, F A; Shahin, J; Frojmovic, M M

2000-01-01

We studied the shear-induced breakup of doublets of aldehyde/sulfate (A/S) latex spheres covalently linked with purified platelet GPIIb-IIIa receptor, and cross-linked by fibrinogen. Flow cytometry with fluorescein isothiocyanate-fibrinogen showed than an average of 22,500 molecules of active GPIIb-IIIa were captured per sphere, with a mean K(d) = 56 nM for fibrinogen binding. The spheres, suspended in buffered 19% Ficoll 400 containing 120 or 240 pM fibrinogen, were subjected to Couette flow in a counter-rotating cone-plate rheoscope. Doublets, formed by two-body collisions at low shear rate (G = 8 s(-1)) for < or =15 min, were subjected to shear stress from 0.6 to 2.9 Nm(-2), their rotations recorded until they broke up or were lost to view. Although breakup was time dependent, occurring mostly in the first 2 rotations after the onset of shear, the percentage of doublets broken up after 10 rotations were almost independent of normal hydrodynamic force, F(n): at 240 pN, 15.6, 16.0, and 17.0% broke up in the force range 70-150 pN, 150-230 pN, and 230-310 pN. Unexpectedly, at both [fibrinogen], the initial rate of breakup was highest in the lowest force range, and computer simulation using a stochastic model of breakup was unable to simulate the time course of breakup. When pre-sheared at low G for >15 min, no doublets broke up within 10 rotations at 70 < F(n) < 310 pN; it required >3 min shear (>1110 rotations) at F(n) = 210 pN for significant breakup to occur. Other published work has shown that binding of fibrinogen to GPIIb-IIIa immobilized on plane surfaces exhibits an initial fast reversible process with relative low affinity succeeded by transformation of GPIIb-IIIa to a stable high-affinity complex. We postulate that most doublet breakups observed within 10 rotations were from a population of young doublets having low numbers of bonds, by dissociation of the initial receptor complex relatively unresponsive to force. The remaining, older doublets with GPIIb-IIIa in the high-affinity complex were not broken up in the time or range of forces studied. PMID:10692309

The Action of Red Cell Calcium Ions on Human Erythrophagocytosis in Vitro

PubMed Central

Romero, Pedro J.; Hernández-Chinea, Concepción

2017-01-01

In the present work we have studied in vitro the effect of increasing red cell Ca2+ ions on human erythrophagocytosis by peripheral monocyte-derived autologous macrophages. In addition, the relative contribution to phagocytosis of phosphatidylserine exposure, autologous IgG binding, complement deposition and Gárdos channel activity was also investigated. Monocytes were obtained after ficoll-hypaque fractionation and induced to transform by adherence to glass coverslips, for 24 h at 37°C in a RPMI medium, containing 10% fetal calf serum. Red blood cells (RBC) were loaded with Ca2+ using 10 μM A23187 and 1 mM Ca-EGTA buffers, in the absence of Mg2+. Ca2+-loaded cells were transferred to above coverslips and incubated for 2 h at 37°C under various experimental conditions, after which phagocytosis was assessed by light microscopy. Confirming earlier findings, phagocytosis depended on internal Ca2+. Accordingly; it was linearly raised from about 2–15% by increasing the free Ca2+ content of the loading solution from 0.5 to 20 μM, respectively. Such a linear increase was virtually doubled by the presence of 40% autologous serum. At 7 μM Ca2+, the phagocytosis degree attained with serum was practically equal to that obtained with either 2 mg/ml affinity-purified IgG or 40% IgG-depleted serum. However, phagocytosis was reduced to levels found with Ca2+ alone when IgG-depleted serum was inactivated by heat, implying an involvement of complement. On the other hand, phagocytosis in the absence of serum was markedly reduced by preincubating macrophages with phosphatidylserine-containing liposomes. In contrast, a similar incubation in the presence of serum affected it partially whereas employing liposomes made only of phosphatidylcholine essentially had no effect. Significantly, the Gárdos channel inhibitors clotrimazole (2 μM) and TRAM-34 (100 nM) fully blocked serum-dependent phagocytosis. These findings show that a raised internal Ca2+ promotes erythrophagocytosis by independently triggering phosphatidylserine externalization, complement deposition and IgG binding. Serum appeared to stimulate phagocytosis in a way dependent on Gárdos activity. It seems likely that Ca2+ promoted IgG-binding to erythrocytes via Gárdos channel activation. This can be an important signal for clearance of senescent human erythrocytes under physiological conditions. PMID:29255426

Fluorescence Dynamics of a FRET Probe Designed for Crowding Studies.

PubMed

Currie, Megan; Leopold, Hannah; Schwarz, Jacob; Boersma, Arnold J; Sheets, Erin D; Heikal, Ahmed A

2017-06-15

Living cells are crowded with macromolecules and organelles. As a result, there is an urgent need for molecular sensors for quantitative, site-specific assessment of the macromolecular crowding effects on a myriad of biochemical processes toward quantitative cell biology and biophysics. Here we investigate the excited-state dynamics and translational diffusion of a novel FRET sensor (mCerulean-linker-mCitrine) in a buffer (PBS, pH 7.4) at room temperature. Complementary experiments were carried out on free CFP, YFP, and the cleaved FRET probe as controls. The wavelength-dependent fluorescence lifetime measurements of the donor and acceptor in the FRET probe, using the time-correlated single-photon counting technique, indicate an energy transfer efficiency of 6.8 ± 0.9% in PBS, with distinct excited-state dynamics from the recombinant CFP and YFP. The estimated mCerulean-mCitrine distance in this FRET probe is 7.7 ± 0.2 nm. The energy transfer efficiency increases (11.5 ± 0.9%) as the concentration of Ficoll-70 increases over the range of 0-300 g/L with an estimated mCerulean-mCitrine distance of 6.1 ± 0.2 nm. Complementary time-resolved anisotropy measurements suggest that the rotational diffusion of hetero-FRET in PBS is sensitive to the energy transfer from the donor to the acceptor. The results also suggest that the linker, -(GSG) 6 A(EAAAK) 6 A(GSG) 6 A(EAAAK) 6 A(GSG) 6 -, is rather flexible, and the observed rotational dynamics is likely to be due to a segmental mobility of the FRET pairs rather than an overall tumbling motion of a rigid probe. Comparative studies on a new construct of a FRET probe with a shorter, more flexible linker, mCerulean-(GSG) 18 -mCitrine, reveal enhanced energy transfer efficiency. On the millisecond time scale, fluorescence fluctuation analyses of the acceptor (excited at 488 nm) provide a means to examine the translational diffusion coefficient of the FRET probe. The results also suggest that the linker is flexible in this FRET probe, and the observed diffusion coefficient is faster than predicted as compared to the cleaved FRET probe. Our results serve as a point of reference for this FRET probe in a buffer toward its full potential as a sensor for macromolecular crowding in living cells and tissues.

The influence of thyroxine on intensity of energy metabolism in bone marrow myeloid cells and neutrophilic polymorphonuclear leukocytes of neonatal pig.

PubMed

Babych, H; Antonyak, H; Sklyarov, A Y

2000-06-01

To investigate the participation of thyroxine in the regulation of energy metabolism in neutrophilic polymorphonuclear leukocytes and their bone marrow precursors. The influence of L-thyroxine (T4; 4 mg/kg every 12 hr from day 2 to 10 of age) was estimated on the activity of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH), NADP-dependent isocitrate dehydrogenase (ICDH) and cytochrome C-oxidase in bone marrow myeloid cells and circulating neutrophils of 3, 5 and 10 day (d) old piglets. Serum T4 and 3,5, 3'-triiodothyronine (T3) concentrations were estimated at every stage of experiment by radioimmunoassay. Bone marrow cells of myeloid lineage and blood neutrophilic polymorphonuclear leukocytes were separated by differential centrifugation of haematopoietic cell suspension using Ficoll-Hypaque gradients. The hyperthyroid status resulted in significant increase in PFK and LDH activity in myelokaryocytes of 3 and 3-5 d piglets, while the activity of HK and PK in the cells of 3-10 d animals remained unchanged. Moreover, ICDH activity in myelokaryocytes increased on day 10 and that of cytochrome C oxidase in bone marrow cells at all intervals. Marked increase in HK and LDH activity on day 3-5 was found also in blood polymorphonuclear granulocytes, while PFK and PK activity was increased during the whole period. At the same time even the increase in ICDH and cytochrome C-oxidase activity was observed, respectively, in 3 and 5-10 d old piglet neutrophils. Besides that, T4 inhibited G-6-PDH activity in myeloid cells on day 3 to 10 and did not influence the enzyme activity in circulating leukocytes. The administration of T4 resulted in preferential stimulation of oxidative stages of carbohydrate catabolism in myelocaryocytes, while the activity of glycolytic enzymes in these cells was less affected. On the contrary, the enzymes of glycolysis in blood neutrophils showed higher sensitivity to T4 action as compared to catalysts of oxidative reactions. The intensity of pentose phosphate pathway seems to be inhibited in bone marrow myelocaryocytes of T4 treated animals, while that in blood leukocytes remained unaffected.

Septic Shock Alters Mitochondrial Respiration of Lymphoid Cell-Lines and Human Peripheral Blood Mononuclear Cells: The Role of Plasma.

PubMed

Clere-Jehl, Raphael; Helms, Julie; Kassem, Mohamad; Le Borgne, Pierrick; Delabranche, Xavier; Charles, Anne-Laure; Geny, Bernard; Meziani, Ferhat; Bilbault, Pascal

2018-02-14

In septic shock patients, post-septic immunosuppression state following the systemic inflammatory response syndrome is responsible for nosocomial infections, with subsequent increased mortality. The aim of the present study was to assess the underlying cellular mechanisms of the post-septic immunosuppression state, by investigating mitochondrial functions of peripheral blood mononuclear cells (PBMCs) from septic shock patients over 7 days. Eighteen patients admitted to a French intensive care unit for septic shock were included. At days 1 and 7, PBMCs were isolated by Ficoll density gradient centrifugation. Mitochondrial respiration of intact septic PBMCs was assessed versus control group PBMCs, by measuring O2 consumption in plasma, using high-resolution respirometry. Mitochondrial respiration was then compared between septic plasmas and control plasmas for control PBMCs, septic PBMCs and lymphoid cell-line (CEM). To investigate the role of plasma, we measured several plasma cytokines, among them HMGB1, by ELISA. Basal O2 consumption of septic shock PBMCs was of 8.27 ± 3.39 and 10.48 ± 3.99 pmol/s/10 cells at days 1 and 7 respectively, significantly higher than in control PBMCs (5.37 ± 1.46 pmol/s/10 cells, p < 0.05). Septic patient PBMCs showed a lower response to oligomycin, suggesting a reduced ATP-synthase activity, as well as an increased response to FCCP suggesting an increased mitochondrial respiratory capacity. At 6 hours, septic plasmas showed a decreased O2 consumption of CEM (4.73 ± 1.46 vs. 6.58 ± 1.53, p < 0.05) as well as in control group PBMCs (1.76 ± 0.36 vs. 2.70 ± 0.42, p < 0.05), and triggered a decreased ATP-synthase activity but an increased response to FCCP. These differences are not explained by different cell survival. High HMGB1 levels were significantly associated with reduced PBMCs mitochondrial respiration. Septic plasma impairs mitochondrial respiration in immune cells, with a possible role of the proinflammatory protein HMGB1, leading to a subsequent compensation, probably by enzymatic activation. This compensation result is an improvement of global mitochondrial respiratory capacity, but without restoring ATP-synthase activity.

The Action of Red Cell Calcium Ions on Human Erythrophagocytosis in Vitro.

PubMed

Romero, Pedro J; Hernández-Chinea, Concepción

2017-01-01

In the present work we have studied in vitro the effect of increasing red cell Ca 2+ ions on human erythrophagocytosis by peripheral monocyte-derived autologous macrophages. In addition, the relative contribution to phagocytosis of phosphatidylserine exposure, autologous IgG binding, complement deposition and Gárdos channel activity was also investigated. Monocytes were obtained after ficoll-hypaque fractionation and induced to transform by adherence to glass coverslips, for 24 h at 37°C in a RPMI medium, containing 10% fetal calf serum. Red blood cells (RBC) were loaded with Ca 2+ using 10 μM A23187 and 1 mM Ca-EGTA buffers, in the absence of Mg 2+ . Ca 2+ -loaded cells were transferred to above coverslips and incubated for 2 h at 37°C under various experimental conditions, after which phagocytosis was assessed by light microscopy. Confirming earlier findings, phagocytosis depended on internal Ca 2+ . Accordingly; it was linearly raised from about 2-15% by increasing the free Ca 2+ content of the loading solution from 0.5 to 20 μM, respectively. Such a linear increase was virtually doubled by the presence of 40% autologous serum. At 7 μM Ca 2+ , the phagocytosis degree attained with serum was practically equal to that obtained with either 2 mg/ml affinity-purified IgG or 40% IgG-depleted serum. However, phagocytosis was reduced to levels found with Ca 2+ alone when IgG-depleted serum was inactivated by heat, implying an involvement of complement. On the other hand, phagocytosis in the absence of serum was markedly reduced by preincubating macrophages with phosphatidylserine-containing liposomes. In contrast, a similar incubation in the presence of serum affected it partially whereas employing liposomes made only of phosphatidylcholine essentially had no effect. Significantly, the Gárdos channel inhibitors clotrimazole (2 μM) and TRAM-34 (100 nM) fully blocked serum-dependent phagocytosis. These findings show that a raised internal Ca 2+ promotes erythrophagocytosis by independently triggering phosphatidylserine externalization, complement deposition and IgG binding. Serum appeared to stimulate phagocytosis in a way dependent on Gárdos activity. It seems likely that Ca 2+ promoted IgG-binding to erythrocytes via Gárdos channel activation. This can be an important signal for clearance of senescent human erythrocytes under physiological conditions.

Influence of source and quantity of protein on the development of immunity and resistance to African trypanosomiasis.

PubMed Central

Norton, J D; Yang, S P; Diffley, P

1986-01-01

Although it is well documented that severe protein deprivation inhibits the development of the immune response and exacerbates certain infections, little has been done to study the effects of native diets on endemic diseases or immunity. Therefore, protein-restricted diets were formulated for mice to mimic the sources and amounts measured in human diets of the Batouri region of Cameroon, endemic for African trypanosomiasis. Weanling C57BL/6 female mice were fed a diet that contained 73% of the recommended daily allowance (RDA) of protein. The sources of protein were all plant (cornmeal), all animal (casein), or a ratio that reflected the native diet (2.2 parts plant to 1 part animal protein). Diets were isocaloric on a weight basis, equal in lipids, and adequate in vitamins and minerals. Control mice were fed laboratory chow or two times the RDA of animal protein (casein). Mice fed only cornmeal or the native diets consumed as much food but did not gain as much weight as mice fed only animal protein, indicating the poorer quality of protein in their diets. Upon infection with Trypanosoma brucei gambiense, however, significantly higher numbers of these mice controlled the first peak of parasitemia and survived the infection as compared with mice fed the other three diets. Since all mice developed patent infections and the parasite growth rate was unaffected by diet, innate immune factors were ruled out as the cause for the higher level of resistance to the parasite. To determine whether diet affected the development of the immune system, weanling mice were maintained on diets for 30 days before immunization with sheep erythrocytes or trinitrophenylated Ficoll. Mice fed only plant protein or native diets elicited higher direct plaque-forming-cell responses to both the T-cell-dependent and T-cell-independent antigens. Since variant-specific immunity which controls levels of African trypanosomes in the blood is a T-cell-independent humoral immunoglobulin M response, this suggests that cornmeal, a protein of poor quality, was adequate for the development of humoral immunity and resistance to African trypanosomiasis while casein, an animal protein of high quality, was not. This provides more evidence that diet plays an important role in infection and immunity. PMID:3484725

Synergistic effects of the xid gene in X chromosome congenic mice. I. Inability of C3.CBA/N mice to respond to thymus-dependent antigens in adoptive transfer assays.

PubMed

Kenny, J J; Guelde, G; Hansen, C; Mond, J J

1987-03-01

The xid gene, which causes a B lymphocyte immune defect in CBA/N mice, has been bred onto the C3H/HeN background. The resulting X chromosome congenic mice (C3.CBA/N) exhibit immunologic defects that are much more profound than the defect exhibited by CBA/N mice; thus, the B cells from C3.CBA/N mice not only fail to respond to thymus-independent (TI) type 2 antigens such as TNP-Ficoll, but they fail to respond in vitro to TI-type 1 antigens such as TNP-Brucella abortus (BA) and B cell mitogens such as LPS and Nocardia water-soluble mitogen. In this paper we show that the synergistic defect seen in C3.CBA/N B cells is also elicited in adoptive transfer assays to thymus-dependent (TD) antigens such as TNP-KLH and PC-KLH, antigens to which both parental strains respond. Thus, the secondary adoptive transfer response of C3.CBA/N spleen cells is generally less than 5% of the immune response produced by CBA/N or C3H/HeN spleen cells. This synergistic defect is restricted to the C3.CBA/N B cells, since C3.CBA/N T cells can provide help to CBA/N B cells that is equivalent to the help obtained with CBA/N T cells. The low responsiveness of C3.CBA/N spleen cells to TD antigens, which is elicited in adoptive transfer assays, is not seen when the intact animal is immunized with antigen in CFA; this, intact C3.CBA/N mice produce anti-PC-KLH and anti-TNP-KLH responses only slightly lower than the responses of CBA/N mice to these same antigens. In contrast, when these mice are immunized with phenol-extracted LPS, a TI-type 1 antigen, their antibody responses are severely depressed. These data suggest that under conditions in which T cell help may be limiting or in which the intact physiology of the T and B cells has been disrupted, C3.CBA/N B cells demonstrate profound immunologic impairment; however, when adequate T cell help is available and the splenic architecture is not disrupted, their immune responses appear to progress in a normal fashion.

Morphology and function of lacrimal gland acinar cells in primary culture.

PubMed

Hann, L E; Tatro, J B; Sullivan, D A

1989-01-01

The objectives of the current investigation were fourfold: (1) to establish an effective procedure for the isolation of acinar cells from the rat lacrimal gland; (2) to evaluate the functional capacity of freshly isolated cells; (3) to determine defined culture conditions which permit maintenance of viable, differentiated cells, as well as secretory component (SC) production, during long-term culture; and (4) to characterize the morphological features of cultured cells. Acinar cells were isolated by serial incubation of gland fragments in chelating and enzymatic solutions, followed by centrifugation through a Ficoll gradient. The yield of viable cells/gland appeared to be age-dependent: cell recovery was inversely proportional to the age of the animals. Immunofluorescence analysis of freshly isolated cells showed the presence of SC, the IgA antibody receptor, within isolated cells. In addition, experiments with a labeled analog (Nle4-D-Phe7-alpha MSH) of alpha-melanocyte-stimulating hormone (alpha-MSH) demonstrated specific binding sites on freshly isolated cells; alpha-MSH is a known modulator of acinar protein secretion. Maximum binding of the alpha-MSH analog occurred within 30 min, was dependent upon cell density and was reduced by coincubation with unlabeled alpha-MSH. To determine the culture requirements of acinar cells, cells were cultured on a variety of substrates (plastic or modified plastic [Primaria], coated with or without extracellular matrix [Matrigel]) in the presence or absence of various supplements and/or fetal calf serum (FCS) for 0.7 to 3.5 weeks. Cell attachment, function and long-term viability required an extracellular matrix. Moreover, in long term cultures (25 days), acinar cell attachment was enhanced by the inclusion of supplements to media containing 10% FCS. Replacement of serum with fibroblast growth factor, high-density lipoprotein and an increased concentration of epidermal growth factor resulted in a distinct "cobblestone" morphology characteristic of epithelial cell cultures. Electron microscopic analysis of cells cultured in supplemented serum-free media demonstrated extensive rough endoplasmic reticulum and Golgi, intermediate filaments and numerous secretory granules, as well as tight junctions and desmosomes. In addition to cell maintenance and attachment, acinar cell synthesis and/or secretion of SC was positively influenced by inclusion of supplements in the media. In summary, we have isolated lacrimal gland acinar cells, which express receptors for IgA antibodies and alpha-MSH. In addition, we have defined culture conditions which permit the long-term maintenance of SC-secreting acinar cells.

The effects of an acute exercise bout on GH and IGF-1 in prediabetic and healthy African Americans: A pilot study investigating gene expression.

PubMed

Berry, Nathaniel T; Hubal, Monica; Wideman, Laurie

2018-01-01

The incidence of pre-diabetes (PD) and Type-2 Diabetes Mellitus (T2D) is a worldwide epidemic. African American (AA) individuals are disproportionately more likely to become diabetic than other ethnic groups. Over the long-term, metabolic complications related to diabetes result in significant alterations in growth hormone (GH) and insulin-like growth factor-1 (IGF-1). Considering the limited exercise-related studies in the area of gene expression changes with disease progression, the objective of this study was to examine differences in exercise-induced gene expression related to the GH and IGF-1 pathways in peripheral blood mononuclear cells (PBMCs) of healthy (CON) and PD AA individuals. Ten subjects [5 PD (age = 35±9.3 yr, BMI = 32.1±4.0, FBG = 101.8±1.3 mg/dl) and 5 CON (age = 31±9.4 yr, BMI = 29.4±5.2, FBG = 82.8±9.7 mg/dl)] had blood drawn for RNA isolation prior to exercise (Pre), immediately following acute moderate intensity exercise on a treadmill (Post-1), 6-hours post (Post-6), and 24-hours post (Post-24). Isolation of mRNA from PBMCs was performed using ficoll separation, while the profiling of mRNA expression was performed using Illumina beadchip arrays with standard protocols. Scan results were statistically analyzed for a specific list of genes related to GH and IGF-1. GH and IGF-1 protein levels were also assessed in each sample. To address issues of normality, all GH and IGF-1 data were log-transformed prior to analysis. Statistical significance was set at p<0.05. Group differences for GH2 variant 2 (p = 0.070) and GH2 variant 3 (p = 0.059) were coupled with significant alterations in IGF-1 mRNA over time (p = 0.024). A significant interaction between group and time was observed for GHRH mRNA (p = 0.008). No group differences were observed in GH AUC (p = 0.649), ΔGH (p = 0.331), GHrec (p = 0.294), or IGF-1 AUC (p = 0.865), representing a similar exercise-induced GH and IGF-1 response for both groups. Analysis of GH and IGF-1 related-gene expression indicates that mild elevations in fasting blood glucose and exercise-induced alterations in gene expression are impacted by the prediabetic state.

Sitagliptin inhibit human lymphocytes proliferation and Th1/Th17 differentiation in vitro.

PubMed

Pinheiro, Marcelo Maia; Stoppa, Caroline Lais; Valduga, Claudete Justina; Okuyama, Cristina Eunice; Gorjão, Renata; Pereira, Regina Mara Silva; Diniz, Susana Nogueira

2017-03-30

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new class of anti-diabetic agents that are widely used in clinical practice to improve glycemic control in patients with type 2 diabetes. DPP-4 is also known as lymphocyte cell surface protein, CD26, and plays an important role in T-cell immunity. Recent studies suggest that DPP-4 inhibitors improve beta-cell function and attenuate autoimmunity in type 1 diabetic mouse models. To investigate the direct effect of DPP4 in immune response, human peripheral blood mononuclear cells (PBMC) from healthy volunteers were obtained by Ficoll gradient and cultivated in the absence (control) or presence of phytohemagglutinin (PHA), or stimulated with PHA and treated with sitagliptin. The immune modulation mechanisms analyzed were: cell proliferation, by MTT assay; cytokine quantification by ELISA or cytometric bead array (CBA), Th1/Th2/Th17 phenotyping by flow cytometric analysis and CD26 gene expression by real time PCR. The results showed that sitagliptin treatment inhibited the proliferation of PBMC-PHA stimulated cells in a dose dependent manner and decreased CD26 expression by these cells, suggesting that sitagliptin may interfere in CD26 expression, dimerization and cell signaling. Sitagliptin treatment not only inhibited IL-10 (p<0.05) and IFN-gamma (p=0.07) cytokines, but also completely abolish IL-6 expression by PBMCs (p<0.001). On the other hand, IL-4 were secreted in culture supernatants from sitagliptin treated cells. A statistically significant increase (p<0.05) in the ratio of TGF-beta/proliferation index after sitagliptin treatment (2627.97±1351.65), when comparing to untreated cells (646.28±376.94), was also demonstrated, indicating higher TGF-beta1 production by viable cells in cultures. Sitagliptin treatment induced a significantly (p<0.05) decrease in IL-17 and IFN-gamma intracellular expression compared with PHA alone. Also, the percentage of T CD4 + IL-17 + , T CD4 + IFNgamma + and T CD4 + IL-4 + cells were significantly reduced (p<0.05) by sitagliptin. Our data demonstrated an immunosuppressive effect of sitagliptin on Th1, Th17 and Th2 lymphocytes differentiation that leads to the generation of regulatory TGF-beta1 secreting cells with low CD26 gene expression that may influence the state of pancreatic beta-cells and controlling DM1 patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion.

PubMed

Lakey, J R; Warnock, G L; Shapiro, A M; Korbutt, G S; Ao, Z; Kneteman, N M; Rajotte, R V

1999-01-01

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4 degrees C) Liberase-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35 degrees C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32 degrees C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 +/- 2.6 g vs. 4.6 +/- 2.1 g, mean +/- SEM, p < 0.05). Postdigestion recovery of islets was 471 +/- 83 x 10(3) IE in the perfusion group compared with 391 +/- 57 x 10(3) IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 +/- 45 vs. 251 +/- 28 x 10(3) IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 +/- 0.6 for the perfusion group and 4.2 +/- 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.

Effects of polyphenols including curcuminoids, resveratrol, quercetin, pterostilbene, and hydroxypterostilbene on lymphocyte pro-inflammatory cytokine production of senior horses in vitro.

PubMed

Siard, Melissa H; McMurry, Kellie E; Adams, Amanda A

2016-05-01

Senior horses (aged ≥ 20 years) exhibit increased chronic, low-grade inflammation systemically, termed inflamm-aging. Inflammation is associated with many afflictions common to the horse, including laminitis and osteoarthritis, which are commonly treated with the non-steroidal anti-inflammatory drugs (NSAIDs) flunixin meglumine and phenylbutazone. Although these NSAIDs are effective in treating acute inflammatory problems, long-term treatment with NSAIDs can result in negative side effects. Thus, bioactive polyphenols including curcuminoids, resveratrol, quercetin, pterostilbene, and hydroxypterostilbene were investigated to determine their effectiveness as anti-inflammatory agents in vitro. Heparinized blood was collected via jugular venipuncture from senior horses (n = 6; mean age = 26 ± 2 years), and peripheral blood mononuclear cells (PBMC) were isolated using a Ficoll density gradient. PBMC were then incubated 22 h at 37°C, 5% CO2 with multiple concentrations (320, 160, 80, 40, 20, 10 μM) of all five polyphenols (curcuminoids, resveratrol, quercetin, pterostilbene, and hydroxypterostilbene), dissolved in DMSO to achieve the aforementioned concentrations. PBMC were stimulated the last 4h of the incubation period with phorbol 12-myristate 13-acetate (PMA)/ionomycin and Brefeldin A (BFA). A Vicell-XR counter evaluated cell viability following incubation. PBMC were stained intracellularly for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) and analyzed via flow cytometry. Data was analyzed by one-way analysis of variance (ANOVA). Viability of PBMC incubated with various compound concentrations were compared with PBMC incubated with DMSO alone (positive control) to determine at what concentration each compound caused cytotoxicity. The highest concentration at which cell viability did not significantly differ from the positive control was: 20 μM for curcuminoids, 40 μM for hydroxypterostilbene, 80 μM for pterostilbene, and 160 μM for quercetin and resveratrol. Flunixin meglumine and phenylbutazone were then evaluated within this range of optimal concentrations for the polyphenol compounds (160, 80, 40, 20 μM) to compare the polyphenols to NSAIDs at equivalent concentrations. The highest concentration at which viability did not significantly differ from the positive control was: 40 μM for flunixin meglumine and 160 μM for phenylbutazone. All five polyphenols and flunixin meglumine significantly decreased lymphocyte production of IFN-γ, while only hydroxypterostilbene, pterostilbene, quercetin, and resveratrol significantly reduced lymphocyte production of TNF-α compared to the positive control (p < 0.05). Polyphenols performed similarly to or more effectively than common NSAIDs in reducing lymphocyte production of inflammatory cytokines of the senior horse in vitro. This study therefore supports the further investigation of polyphenols to determine whether they may be effective anti-inflammatory treatments for chronic inflammation in the horse. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification and Properties of Cytidine Deaminase from Normal and Leukemic Granulocytes

PubMed Central

Chabner, Bruce A.; Johns, David G.; Coleman, C. Norman; Drake, James C.; Evans, Warren H.

1974-01-01

Cytidine deaminase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), has been partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70°C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G-150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol. Cytidine deaminase from normal human granulocytes has a greater affinity for its physiologic substrate cytidine (Km = 1.1 × 10−5 M) than for ara-C (8.8 × 10−5 M) or 5-azaC (4.3 × 10−4 M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2′-deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a Ki of 5.4 × 10−8 M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, molecular weight, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52±1.86 × 103/mg protein) than chronic myelocytic leukemia (CML) cells (1.40±0.70 × 103 U/mg protein) or acute myelocytic leukemia (AML) cells (0.19±0.17 × 103 U/mg protein). To explain these differences in enzyme levels in leukemic versus normal cells, the changes in cytidine deaminase levels associated with maturation of normal granulocytes were studied in normal human bone marrow. Myeloid precursors obtained from bone marrow aspirates were separated into mature and immature fractions by Ficoll density centrifugation. Deaminase activity in lysates of mature granulocytes was 3.55-14.2 times greater than the activity found in the lysates of immature cells. Decreased enzyme activity was also found in immature myeloid cells from a patient with CML as compared to mature granulocytes from the same patient. These observations support the conclusion that the greater specific activity of cytidine deaminase in normal mature granulocytes as compared to leukemic cells is related to the process of granulocyte maturation rather than a specific enzymatic defect in leukemic cells. PMID:4521417

An optimized method for extraction and detection of Coconut cadang-cadang viroid(CCCVd) from oil palm.

PubMed

Mohammadi, M R; Vadamalai, G; Joseph, H

2010-01-01

Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.

[Bifidobacterium DNA upregulates Th1 type response of umbilical cord blood mononuclear cell].

PubMed

Zhao, Hui; Wang, Xiao-chuan; Wang, Jing-yi; Yu, Ye-heng; Wang, Chuan-qing; Yang, Yi

2006-06-01

To study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell (CBMC), and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction. Bifidobacterium genomic DNA (bDNA) and human DNA (hDNA) were extracted with phenol/chloroform/isoamyl alcohol and stored at -20 degrees C for later use. Parts of bDNA were completely digested with DNaseI (d-bDNA) at 37 degrees C. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37 degrees C in a 5% CO2 humidified incubator. These cells were divided into four groups, control group: without any stimulant; bDNA group: stimulated with 25 microg/ml bDNA; d-bDNA group: stimulated with 25 microg/ml d-bDNA; hDNA group: stimulated with 25 microg/ml hDNA. The cells were stimulated with different stimulants in vitro, at the end of incubation culture supernatant and cells were collected. IL-12 and IL-10 levels in the culture supernatant were measured by enzyme linked immuno sorbent assay (ELISA); cells secreting IL-4 and IFN-gamma were counted by enzyme linked immunospot (ELISPOT) assay; and total RNA was isolated from the cells to assay T-bet and GATA3 mRNA expression levels by reverse transcription polymerase chain reaction (RT-PCR). Six hours after stimulation there was no significant difference in IL-12 level in supernatant among the four groups; 12 hours after stimulation, IL-12 level in supernatant of bDNA treated group was significantly higher than that of each of the other groups, so were the results obtained at 24 hours and 48 hours after stimulation (P < 0.05). No significant difference could be detected in IL-12 level in supernatant among the other 3 groups. On the other hand, 6 hours after stimulation there was no significant difference in IL-10 level in supernatant among the four groups. But 12 and 24 hours after stimulation IL-10 level in supernatant of bDNA treated group was lower than that of each of the other groups, but the difference was not statistically significant. The count of IFN-gamma secreting cells of bDNA treated group was higher than that of the other groups, while IL-4 secteting cells of bDNA treated group were lower than that of the other groups. After bDNA stimulation, nuclear factor T-box expressed in T cells (T-bet) mRNA expression was conspicuously enhanced as compared to the other three groups (P < 0.05). GATA3 mRNA transcription in CBMC had no significant change after bDNA stimulation. bDNA could promote secretion of Th1 type cytokine IL-12, while Th2 type cytokine IL-10 level of cell supernatant was decreased. bDNA could stimulate secretion of IFN-gamma by CBMC and inhibit secretion of IL-4. T-bet mRNA expression was highly enhanced after bDNA stimulation. bDNA could upregulate Th1 type response, which may be one of important mechanisms by which bifidobacterium inhibit allergic response.

B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma

PubMed Central

Hua, Dong; Sun, Jing; Mao, Yong; Chen, Lu-Jun; Wu, Yu-Yu; Zhang, Xue-Guang

2012-01-01

AIM: To investigate the expression of B7-H1 in human colorectal carcinoma (CRC) to define its regulating effects on T cells in tumor microenvironment. METHODS: One hundred and two paraffin blocks and 33 fresh samples of CRC tissues were subject to this study. Immunohistochemistry was performed for B7-H1 and CD3 staining in CRC tissues. Ficoll-Hypaque density gradient centrifugation was used to isolate peripheral blood mononuclear cells of fresh CRC tissues; flow cytometry and immunofluorescence staining were used for detection of regulatory T cells. Data was analyzed with statistical software. RESULTS: Costimulatory molecule B7-H1 was found strongly expressed in CRC tissues, localized in tumor cell membrane and cytoplasm, while weak or none expression of B7-H1 was detected in pared normal colorectal tissues. Meanwhile, CD3 positive T cells were found congregated in CRC tumor nest and stroma. Statistic analysis showed that B7-H1 expression level was negatively correlated to the total T cell density in tumor nest (P < 0.0001) and tumor stroma (P = 0.0200) of 102 cases of CRC tissues. Among the total T cells, a variable amount of regulatory T cells with a clear Foxp3+ (forkhead box P3) staining could be detected in CRC tissues and patients’ blood. Interestingly, in the 33 samples (15 cases of B7-H1high CRC tissues and 18 cases of B7-H1low CRC tissues) of freshly isolated mononuclear cells from CRC tissues, the percentages of CD4+Foxp3+ and CD8+Foxp3+ regulatory T cells were found remarkably higher in B7-H1high CRC tissues than in B7-H1low CRC tissues (P = 0.0024, P = 0.0182), indicating that B7-H1 expression was involved in proliferation of regulatory T cell. No significant difference was found in CRC peripheral blood (P = 0.0863, P = 0.0678). PD-1 is the specific ligand for B7-H1 pathway transferring inhibitory signal to T cell, which is expressed by activated T cell. Our further analysis of PD-1 expression on T cells in CRC tissues showed that conventional T cells (CD4+Foxp3-/CD8+Foxp3-), which was thought to contribute to the anti-tumor immune response, highly expressed PD-1; while regulatory T cells (CD4+Foxp3+/CD8+Foxp3-) almost failed to express PD-1. The average percentage of PD-1 expression on regulatory T cells was significantly higher than the percentage of PD-1 on conventional T cells (CD4+Foxp3- T cell, P < 0.0001; CD8+Foxp3- T cell, P < 0.0001). The diverse expression of PD-1 might lead to different fate of T cell subsets in B7-H1 over-expression CRC tumor microenvironment. CONCLUSION: B7-H1 expression in tumor cells can inhibit the conventional T cell proliferation in tumor microenvironment through the PD-1 expression on conventional T cells. PMID:22408358

Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified-warmed ovarian tissue survival.

PubMed

Youm, Hye Won; Lee, Jung Ryeol; Lee, Jaewang; Jee, Byung Chul; Suh, Chang Suk; Kim, Seok Hyun

2014-04-01

What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified-warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of α-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified-warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4-58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6-92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma.

PubMed

Hua, Dong; Sun, Jing; Mao, Yong; Chen, Lu-Jun; Wu, Yu-Yu; Zhang, Xue-Guang

2012-03-07

To investigate the expression of B7-H1 in human colorectal carcinoma (CRC) to define its regulating effects on T cells in tumor microenvironment. One hundred and two paraffin blocks and 33 fresh samples of CRC tissues were subject to this study. Immunohistochemistry was performed for B7-H1 and CD3 staining in CRC tissues. Ficoll-Hypaque density gradient centrifugation was used to isolate peripheral blood mononuclear cells of fresh CRC tissues; flow cytometry and immunofluorescence staining were used for detection of regulatory T cells. Data was analyzed with statistical software. Costimulatory molecule B7-H1 was found strongly expressed in CRC tissues, localized in tumor cell membrane and cytoplasm, while weak or none expression of B7-H1 was detected in pared normal colorectal tissues. Meanwhile, CD3 positive T cells were found congregated in CRC tumor nest and stroma. Statistic analysis showed that B7-H1 expression level was negatively correlated to the total T cell density in tumor nest (P < 0.0001) and tumor stroma (P = 0.0200) of 102 cases of CRC tissues. Among the total T cells, a variable amount of regulatory T cells with a clear Foxp3⁺ (forkhead box P3) staining could be detected in CRC tissues and patients' blood. Interestingly, in the 33 samples (15 cases of B7-H1(high) CRC tissues and 18 cases of B7-H1(low) CRC tissues) of freshly isolated mononuclear cells from CRC tissues, the percentages of CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ regulatory T cells were found remarkably higher in B7-H1(high) CRC tissues than in B7-H1(low) CRC tissues (P = 0.0024, P = 0.0182), indicating that B7-H1 expression was involved in proliferation of regulatory T cell. No significant difference was found in CRC peripheral blood (P = 0.0863, P = 0.0678). PD-1 is the specific ligand for B7-H1 pathway transferring inhibitory signal to T cell, which is expressed by activated T cell. Our further analysis of PD-1 expression on T cells in CRC tissues showed that conventional T cells (CD4⁺Foxp3⁻/CD8⁺Foxp3⁻), which was thought to contribute to the anti-tumor immune response, highly expressed PD-1; while regulatory T cells (CD4⁺Foxp3⁺/CD8⁺Foxp3⁻) almost failed to express PD-1. The average percentage of PD-1 expression on regulatory T cells was significantly higher than the percentage of PD-1 on conventional T cells (CD4⁺Foxp3⁻ T cell, P < 0.0001; CD8⁺Foxp3⁻ T cell, P < 0.0001). The diverse expression of PD-1 might lead to different fate of T cell subsets in B7-H1 over-expression CRC tumor microenvironment. B7-H1 expression in tumor cells can inhibit the conventional T cell proliferation in tumor microenvironment through the PD-1 expression on conventional T cells.

Macromolecular crowding: chemistry and physics meet biology (Ascona, Switzerland, 10-14 June 2012).

PubMed

Foffi, G; Pastore, A; Piazza, F; Temussi, P A

2013-08-02

More than 60 years of biochemical and biophysical studies have accustomed us to think of proteins as highly purified entities that act in isolation, more or less freely diffusing until they find their cognate partner to bind to. While in vitro experiments that reproduce these conditions largely remain the only way to investigate the intrinsic properties of molecules, this approach ignores an important factor: in their natural milieu , proteins are surrounded by several other molecules of different chemical nature, and this crowded environment can considerably modify their behaviour. About 40% of the cellular volume on average is occupied by all sorts of molecules. Furthermore, biological macromolecules live and operate in an extremely structured and complex environment within the cell (endoplasmic reticulum, Golgi apparatus, cytoskeletal structures, etc). Hence, to further complicate the picture, the interior of the cell is by no means a simply crowded medium, rather, a most crowded and confining one. In recent times, several approaches have been developed in the attempt to take into account important factors such as the ones mentioned above, at both theoretical and experimental levels, so that this field of research is now emerging as one of the most thriving in molecular and cell biology (see figure 1). [Formula: see text] Figure 1. Left: number of articles containing the word 'crowding' as a keyword limited to the biological and chemical science domains (source: ISI Web of Science). The arrow flags the 2003 'EMBO Workshop on Biological Implications of Macromolecular Crowding' (Embo, 2012). Right: number of citations to articles containing the word 'crowding' limited to the same domains (bars) and an exponential regression curve (source: Elsevier Scopus). To promote the importance of molecular crowding and confinement and provide researchers active in this field an interdisciplinary forum for meeting and exchanging ideas, we recently organized an international conference held in Ascona from 10 to 14 June 2012. In the unique scenario of the Maggiore lake and absorbed in the magic atmosphere of the Centro Stefano Franscini (CSF) at Monte Verità, we enjoyed three-and-a-half days of intense and inspiring activity, where not only many of the most prominent scientists working on macromolecular crowding, but also experts in closely related fields such as colloids and soft matter presented their work. The meeting was intended and has been organized to bring theoreticians and experimentalists together in the attempt to promote an active dialogue. Moreover, we wanted different disciplines to be represented, notably physics and chemistry, besides biology, as cross-fertilization is proving an increasingly fundamental source of inspiration and advancement. This issue of Physical Biology (PB) features a selection of the oral contributions presented at the conference, expanded in the form of research or review articles. PB, one of the scientific journals of the Institute of Physics (IOP), is one of the most dynamic and lively forums active at the interface between biology on one side, and physics and mathematics on the other. As its mission is stated by IOP, PB 'focuses on research in which physics-based approaches lead to new insights into biological systems at all scales of space and time, and all levels of complexity'. For these reasons, and also in view of its high reputation and broad readership, PB appears to be the ideal place for disseminating the thriving pieces of research presented at the conference. We are extremely grateful to PB and its kind and efficient editorial staff who helped make this issue a great scientific follow-up to the conference. The opening lecture of the conference, the first of four day-opening keynote lectures, was given by Allen P Minton from NIH (USA), possibly the most influential among the pioneers in the field. He provided a lucid and well-thought-out overview of the concept of macromolecular crowding through an exhaustive chronological account of the major milestones. It is clear that the concept of excluded volume as a key factor remains central to the concept of molecular crowding. As a consequence, simple descriptive paradigms borrowed essentially from colloid physics may still provide useful tools to understand the subtle effects of crowding and confinement in living matter. The contiguity between crowding, colloids and soft matter further emerged as an important concept in the course of the conference in several theoretical lectures and a few experimental ones. Dave Thirumalai, from the University of Maryland (USA), one of the most active theoreticians in the field of theoretical biophysics, outlined scaling theories, concepts from colloid literature and different simulation techniques to describe scenarios for crowding-induced changes in the structure and dynamics of proteins and RNA. In particular, he showed the importance of the shape of crowding particles in affecting folding oligomerization of amyloidogenic peptides. Johannes Schöneberg, from IMPRS, Mathematics Institute (Germany), illustrated ReaDDy , a newly developed particle-based simulation software tool for reaction-diffusion dynamics, developed in the group of Frank Noe at EMPRS. He showed that ReaDDy makes it possible to bridge the gap between soft matter and molecular dynamics (MD) simulations on the one hand and particle-based stochastic reaction-diffusion simulations on the other. We asked Johannes to organize a tutorial session to lead interested participants into the package and 'get their hands wet' under the guidance of the developers. The tutorial session was indeed successful and the broad possibilities offered by the simulation toolkit appeared to be clear to the participants. Paolo De Los Rios, from the Ecole Polytechnique Fédérale de Lausanne (EPFL, Switzerland), examined the complexity of the effects caused by crowding conditions from the point of view of statistical physics. Starting from a modification of the well-known Smoluchowski approach to calculate the encounter rate of diffusion-limited reactions, he showed how more realistic situations accounting for crowding effects could be treated equally well on the same theoretical grounds. This talk marked an important point in the conference as it reinforced the idea that simple models of theoretical physics still have the power to provide inspiring results in spite of the intrinsic simplifications of such theoretical approaches. Along the same lines, Nicolas Dorsaz, from the University of Cambridge (UK), proposed an extension of the Smoluchowski framework that incorporates repulsive and attracting interactions between the reactants. This approach was illustrated by reaction rates obtained from event-driven Brownian dynamics and dynamical Monte Carlo simulations. Another striking example of the physical subtleties associated with modelling crowding effects was provided by Jeffrey Skolnick, from the Georgia Institute of Technology (USA). He examined the role of hydrodynamic interactions in the self-organization of biological assemblies in the presence of crowding. His results strongly suggest that hydrodynamic interactions greatly affect the kinetics of self-assembly reactions, so that including them in the picture appears crucial for understanding the dynamics of biological systems in vivo . Margareth Cheung, from the University of Houston (USA), emphasized that how the crowded environment inside a cell affects the structural conformation of a protein with a spherical shape is a vital question because the geometry of proteins and protein-protein complexes are far from globules in vivo . Her work demonstrates the malleability of 'native' proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo . Huan-Xiang Zhou, from the Florida State University (USA), focused on atomistic simulations of protein folding and binding under crowding conditions. His lab has developed a post-processing method that allows the atomistic representation of proteins in folding and binding processes under crowding. A comparison with experimental results was also presented. Other lecturers pointed out that there are still aspects not entirely explored in the effects of both crowding and confinement. As suggested in the talk by Gary Pielak, from the University of North Carolina (USA), the currently used synthetic crowding agents are far from being satisfactory in replicating naturally occurring effects associated with crowded environments. For example, non-specific binding seems to play a subtle role in the cell, as natural macromolecules can induce both stabilization and destabilization when used as crowders. It is indeed possible to fine-tune the effect of proteins, as crowders, on the stability of other proteins. Another aspect that became clear is that new, more powerful methods need to be developed to study the effect of crowding, but even more to compare crowding and confinement. Indeed, it appeared clear from the lecture by Pierandrea Temussi, from the University of Naples (Italy), that a reliable comparison of the effects of crowding and confinement on the stability of proteins can only be based on the measurement of the whole stability curve of the same protein. Controversial aspects do not pertain only to the influence of crowding on protein stability, but also to aggregation phenomena in natural fluids. Domenico Sanfelice, from NIMR (London, UK), reported an interesting case of the apparent influence of crowding on aggregation. Hen egg white, a possible natural medium to study macromolecules in crowded conditions can dramatically increase the aggregation kinetics of proteins with an inbuilt tendency to associate. By carefully dissecting the phenomenology, it was shown that only part of this effect is due to crowding, while another factor playing an important role is the interaction with proteins from the milieu . In other words, high-molecular-weight glycoproteins can act as efficient molecular seeds for aggregation. A special topic of great relevance in the conference appeared to be the direct study of crowding in living systems. Alan Verkman, from the University of California, San Francisco (USA), one of the world's leading scientific personalities in the field of experimental investigation of crowding and confinement, was invited to give the second plenary lecture devoted to the experimental study of crowding effects in vivo . In his keynote lecture, Dr Verkman led us on a wide and compelling tour, exploring the main experimental approaches to study molecular crowding in and around cells. After a thorough examination of methods such as fluorescence recovery after photo-bleaching, fluorescence correlation spectroscopy, photo-activation localization microscopy and stochastic reconstruction microscopy, he concluded that the general consensus emerging from experimental studies is that the notion of universally anomalous diffusion in and around cells as a consequence of molecular crowding may not be correct, and that the slowing of diffusion in cells is less marked than has been widely assumed and can be simply described through a five- to sixfold reduction of the normal diffusion coefficient. A Soranno, from the University of Zürich (Switzerland), described how, by employing FRET measurements, it is possible to quantify the effect of molecular crowding on the dimensions of the highly charged, intrinsically disordered protein human prothymosin alpha. For a large variety of polymeric crowders (PEG, PVP, Ficoll, Dextran, PVA, PAA), a collapse of the polypeptide chain is observed with increasing polymer size and polymer concentration. The largest extent of collapse is observed for polymer radii comparable to the dimensions of the protein, in agreement with theoretical considerations. For his contribution, A Soranno was awarded the CSF Award for the best contributed talk. In his most inspiring talk, Clifford Brangwynne, from Princeton University (USA), drew attention to very important objects, namely Ribonucleoprotein (RNP) bodies. These are non-membrane-bound macromolecular assemblies that form from the dynamic interactions of RNA and proteins. The assembly of RNP bodies may sensitively depend on the biophysical features of the surrounding cytoplasm, including the degree of crowding, transport coefficients and mechanical properties. This dependency may have important implications for the RNA processing reactions involved in fundamental biological processes such as developmental cell growth. Remarkably, Brangwynne showed how RNPs behave in the cell as liquid droplets, pointing to a possible entirely new means that the cell could use to control and fine-tune its internal processes, in fact, more than that, a completely unexplored, new state of organization of living matter, and a functional one. Giuseppe Zaccai, from Institut Laue Langevin, Grenoble (France), showed that protein dynamics is more sensitive than structure to environmental factors such as crowding, solvent, temperature or pressure. Furthermore, he convincingly explained how neutron scattering provides unique experimental data to underpin MD calculations in this context. Following up on environment-induced modulations of protein functional dynamics, Ruth Nussinov, from Tel Aviv University (Israel), addressed the important problem of whether cellular signals can travel long distances in a crowded environment. She proposed a model based on the evolution of at least three properties: a modular functional organization of the cellular network, sequences in some key regions of proteins, such as linkers or loops, and compact interactions between proteins, possibly favoured by a crowded environment. The workshop ended on a keynote lecture by Jean-Marie Lehn, from the Université de Strasbourg (France). Lehn, 1987 Nobel Laureate in chemistry, offered a 'supramolecular view' of the field of molecular interactions. Supramolecular chemistry explores the design of systems undergoing self-organization , i.e. systems capable of generating well-defined functional supramolecular architectures by self-assembling from their components, thus behaving as programmed chemical systems . Chemistry may therefore be considered an information science , the science of informed matter. Supramolecular chemistry is intrinsically a dynamic chemistry in view of the ability of the interactions connecting the molecular components of a supramolecular entity and the resulting ability of supramolecular species to exchange their constituents. The same holds for molecular chemistry when the molecular entity contains covalent bonds that may form and break reversibly, so as to allow a continuous change in constitution by the reorganization and exchange of building blocks. These features define a constitutional dynamic chemistry (CDC) on both the molecular and supramolecular levels. CDC takes advantage of dynamic constitutional diversity to allow variation and selection in response to either internal or external factors to achieve adaptation . The merging of the features-information and programmability, dynamics and reversibility, constitution and structural diversity-points towards the emergence of adaptive and evolutive chemistry . The whole workshop could have not taken place without the help of the Centro Stefano Franscini. The CSF is the congress centre of the Swiss Federal Institute of Technology of Zurich (ETH Zurich) and has been situated at Monte Verità since 1989. It is an ideal meeting point for all members of the international scientific community who wish to discuss the state-of-the-art and new challenges of any field of research. The CSF supports 20-25 international conferences every year and, since 2010, up to ten winter doctoral schools 1 . The competence and professionalism of the staff were at the same level of beauty and inspiring character as that of Monte Verità. A meeting of this sort, if successful, leaves the audience with more open questions than settled answers, and this was definitely the case for Crowding 2012. Excluded volume is clearly a fundamental concept that has allowed crowding, a very familiar concept in soft matter, to enter into the domain of biological sciences. However, the complexity of the biological milieu calls for more refined descriptions. What is the role of electrostatic and electrodynamic interactions? What is the role of hydrodynamics interactions? To what extent does the strong spatial inhomogeneity (clustering of molecules, cellular compartmentalization, etc) have to be taken into account? Or, more generally, what are the minimal elements that prove crucial to describe reactions within a cell? How does the diffusion proceed (diffusion, slow diffusion, sub-diffusion) given that the experimental evidences are still controversial? In conclusion, we knew that allowing scientists with very different backgrounds and ideas to mingle was a hazardous attempt. Despite that, the workshop turned out to be a very successful experiment, which was highly enjoyed both by the participants and the organizers. Discussions sparked regularly among ever-changing groups, comprising senior scientists and students, despite the rather tight schedule, adding to the sense of fulfilment ignited by the outstanding level of the presentations. Given the success of the meeting Crowding 2012, a new event has been organized and will take place on the same themes during fall 2013, this time in the beautiful scenery of the Loire valley in France. The workshop 'Macromolecular crowding effects in cell biology: models and experiments' will be held on the CNRS campus in Orléans, France, on 24-25 October 2013. More information can be found on the workshop website: http://dirac.cnrs-orleans.fr/∼piazza/. 1 Source: www.csf.ethz.ch/

Macromolecular crowding: chemistry and physics meet biology (Ascona, Switzerland, 10-14 June 2012)

NASA Astrophysics Data System (ADS)

Foffi, G.; Pastore, A.; Piazza, F.; Temussi, P. A.

2013-08-01

More than 60 years of biochemical and biophysical studies have accustomed us to think of proteins as highly purified entities that act in isolation, more or less freely diffusing until they find their cognate partner to bind to. While in vitro experiments that reproduce these conditions largely remain the only way to investigate the intrinsic properties of molecules, this approach ignores an important factor: in their natural milieu , proteins are surrounded by several other molecules of different chemical nature, and this crowded environment can considerably modify their behaviour. About 40% of the cellular volume on average is occupied by all sorts of molecules. Furthermore, biological macromolecules live and operate in an extremely structured and complex environment within the cell (endoplasmic reticulum, Golgi apparatus, cytoskeletal structures, etc). Hence, to further complicate the picture, the interior of the cell is by no means a simply crowded medium, rather, a most crowded and confining one. In recent times, several approaches have been developed in the attempt to take into account important factors such as the ones mentioned above, at both theoretical and experimental levels, so that this field of research is now emerging as one of the most thriving in molecular and cell biology (see figure 1). Figure 1. Figure 1. Left: number of articles containing the word 'crowding' as a keyword limited to the biological and chemical science domains (source: ISI Web of Science). The arrow flags the 2003 'EMBO Workshop on Biological Implications of Macromolecular Crowding' (Embo, 2012). Right: number of citations to articles containing the word 'crowding' limited to the same domains (bars) and an exponential regression curve (source: Elsevier Scopus). To promote the importance of molecular crowding and confinement and provide researchers active in this field an interdisciplinary forum for meeting and exchanging ideas, we recently organized an international conference held in Ascona from 10 to 14 June 2012. In the unique scenario of the Maggiore lake and absorbed in the magic atmosphere of the Centro Stefano Franscini (CSF) at Monte Verità, we enjoyed three-and-a-half days of intense and inspiring activity, where not only many of the most prominent scientists working on macromolecular crowding, but also experts in closely related fields such as colloids and soft matter presented their work. The meeting was intended and has been organized to bring theoreticians and experimentalists together in the attempt to promote an active dialogue. Moreover, we wanted different disciplines to be represented, notably physics and chemistry, besides biology, as cross-fertilization is proving an increasingly fundamental source of inspiration and advancement. This issue of Physical Biology (PB) features a selection of the oral contributions presented at the conference, expanded in the form of research or review articles. PB, one of the scientific journals of the Institute of Physics (IOP), is one of the most dynamic and lively forums active at the interface between biology on one side, and physics and mathematics on the other. As its mission is stated by IOP, PB 'focuses on research in which physics-based approaches lead to new insights into biological systems at all scales of space and time, and all levels of complexity'. For these reasons, and also in view of its high reputation and broad readership, PB appears to be the ideal place for disseminating the thriving pieces of research presented at the conference. We are extremely grateful to PB and its kind and efficient editorial staff who helped make this issue a great scientific follow-up to the conference. The opening lecture of the conference, the first of four day-opening keynote lectures, was given by Allen P Minton from NIH (USA), possibly the most influential among the pioneers in the field. He provided a lucid and well-thought-out overview of the concept of macromolecular crowding through an exhaustive chronological account of the major milestones. It is clear that the concept of excluded volume as a key factor remains central to the concept of molecular crowding. As a consequence, simple descriptive paradigms borrowed essentially from colloid physics may still provide useful tools to understand the subtle effects of crowding and confinement in living matter. The contiguity between crowding, colloids and soft matter further emerged as an important concept in the course of the conference in several theoretical lectures and a few experimental ones. Dave Thirumalai, from the University of Maryland (USA), one of the most active theoreticians in the field of theoretical biophysics, outlined scaling theories, concepts from colloid literature and different simulation techniques to describe scenarios for crowding-induced changes in the structure and dynamics of proteins and RNA. In particular, he showed the importance of the shape of crowding particles in affecting folding oligomerization of amyloidogenic peptides. Johannes Schöneberg, from IMPRS, Mathematics Institute (Germany), illustrated ReaDDy , a newly developed particle-based simulation software tool for reaction-diffusion dynamics, developed in the group of Frank Noe at EMPRS. He showed that ReaDDy makes it possible to bridge the gap between soft matter and molecular dynamics (MD) simulations on the one hand and particle-based stochastic reaction-diffusion simulations on the other. We asked Johannes to organize a tutorial session to lead interested participants into the package and 'get their hands wet' under the guidance of the developers. The tutorial session was indeed successful and the broad possibilities offered by the simulation toolkit appeared to be clear to the participants. Paolo De Los Rios, from the Ecole Polytechnique Fédérale de Lausanne (EPFL, Switzerland), examined the complexity of the effects caused by crowding conditions from the point of view of statistical physics. Starting from a modification of the well-known Smoluchowski approach to calculate the encounter rate of diffusion-limited reactions, he showed how more realistic situations accounting for crowding effects could be treated equally well on the same theoretical grounds. This talk marked an important point in the conference as it reinforced the idea that simple models of theoretical physics still have the power to provide inspiring results in spite of the intrinsic simplifications of such theoretical approaches. Along the same lines, Nicolas Dorsaz, from the University of Cambridge (UK), proposed an extension of the Smoluchowski framework that incorporates repulsive and attracting interactions between the reactants. This approach was illustrated by reaction rates obtained from event-driven Brownian dynamics and dynamical Monte Carlo simulations. Another striking example of the physical subtleties associated with modelling crowding effects was provided by Jeffrey Skolnick, from the Georgia Institute of Technology (USA). He examined the role of hydrodynamic interactions in the self-organization of biological assemblies in the presence of crowding. His results strongly suggest that hydrodynamic interactions greatly affect the kinetics of self-assembly reactions, so that including them in the picture appears crucial for understanding the dynamics of biological systems in vivo . Margareth Cheung, from the University of Houston (USA), emphasized that how the crowded environment inside a cell affects the structural conformation of a protein with a spherical shape is a vital question because the geometry of proteins and protein-protein complexes are far from globules in vivo . Her work demonstrates the malleability of 'native' proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo . Huan-Xiang Zhou, from the Florida State University (USA), focused on atomistic simulations of protein folding and binding under crowding conditions. His lab has developed a post-processing method that allows the atomistic representation of proteins in folding and binding processes under crowding. A comparison with experimental results was also presented. Other lecturers pointed out that there are still aspects not entirely explored in the effects of both crowding and confinement. As suggested in the talk by Gary Pielak, from the University of North Carolina (USA), the currently used synthetic crowding agents are far from being satisfactory in replicating naturally occurring effects associated with crowded environments. For example, non-specific binding seems to play a subtle role in the cell, as natural macromolecules can induce both stabilization and destabilization when used as crowders. It is indeed possible to fine-tune the effect of proteins, as crowders, on the stability of other proteins. Another aspect that became clear is that new, more powerful methods need to be developed to study the effect of crowding, but even more to compare crowding and confinement. Indeed, it appeared clear from the lecture by Pierandrea Temussi, from the University of Naples (Italy), that a reliable comparison of the effects of crowding and confinement on the stability of proteins can only be based on the measurement of the whole stability curve of the same protein. Controversial aspects do not pertain only to the influence of crowding on protein stability, but also to aggregation phenomena in natural fluids. Domenico Sanfelice, from NIMR (London, UK), reported an interesting case of the apparent influence of crowding on aggregation. Hen egg white, a possible natural medium to study macromolecules in crowded conditions can dramatically increase the aggregation kinetics of proteins with an inbuilt tendency to associate. By carefully dissecting the phenomenology, it was shown that only part of this effect is due to crowding, while another factor playing an important role is the interaction with proteins from the milieu . In other words, high-molecular-weight glycoproteins can act as efficient molecular seeds for aggregation. A special topic of great relevance in the conference appeared to be the direct study of crowding in living systems. Alan Verkman, from the University of California, San Francisco (USA), one of the world's leading scientific personalities in the field of experimental investigation of crowding and confinement, was invited to give the second plenary lecture devoted to the experimental study of crowding effects in vivo . In his keynote lecture, Dr Verkman led us on a wide and compelling tour, exploring the main experimental approaches to study molecular crowding in and around cells. After a thorough examination of methods such as fluorescence recovery after photo-bleaching, fluorescence correlation spectroscopy, photo-activation localization microscopy and stochastic reconstruction microscopy, he concluded that the general consensus emerging from experimental studies is that the notion of universally anomalous diffusion in and around cells as a consequence of molecular crowding may not be correct, and that the slowing of diffusion in cells is less marked than has been widely assumed and can be simply described through a five- to sixfold reduction of the normal diffusion coefficient. A Soranno, from the University of Zürich (Switzerland), described how, by employing FRET measurements, it is possible to quantify the effect of molecular crowding on the dimensions of the highly charged, intrinsically disordered protein human prothymosin alpha. For a large variety of polymeric crowders (PEG, PVP, Ficoll, Dextran, PVA, PAA), a collapse of the polypeptide chain is observed with increasing polymer size and polymer concentration. The largest extent of collapse is observed for polymer radii comparable to the dimensions of the protein, in agreement with theoretical considerations. For his contribution, A Soranno was awarded the CSF Award for the best contributed talk. In his most inspiring talk, Clifford Brangwynne, from Princeton University (USA), drew attention to very important objects, namely Ribonucleoprotein (RNP) bodies. These are non-membrane-bound macromolecular assemblies that form from the dynamic interactions of RNA and proteins. The assembly of RNP bodies may sensitively depend on the biophysical features of the surrounding cytoplasm, including the degree of crowding, transport coefficients and mechanical properties. This dependency may have important implications for the RNA processing reactions involved in fundamental biological processes such as developmental cell growth. Remarkably, Brangwynne showed how RNPs behave in the cell as liquid droplets, pointing to a possible entirely new means that the cell could use to control and fine-tune its internal processes, in fact, more than that, a completely unexplored, new state of organization of living matter, and a functional one. Giuseppe Zaccai, from Institut Laue Langevin, Grenoble (France), showed that protein dynamics is more sensitive than structure to environmental factors such as crowding, solvent, temperature or pressure. Furthermore, he convincingly explained how neutron scattering provides unique experimental data to underpin MD calculations in this context. Following up on environment-induced modulations of protein functional dynamics, Ruth Nussinov, from Tel Aviv University (Israel), addressed the important problem of whether cellular signals can travel long distances in a crowded environment. She proposed a model based on the evolution of at least three properties: a modular functional organization of the cellular network, sequences in some key regions of proteins, such as linkers or loops, and compact interactions between proteins, possibly favoured by a crowded environment. The workshop ended on a keynote lecture by Jean-Marie Lehn, from the Université de Strasbourg (France). Lehn, 1987 Nobel Laureate in chemistry, offered a 'supramolecular view' of the field of molecular interactions. Supramolecular chemistry explores the design of systems undergoing self-organization , i.e. systems capable of generating well-defined functional supramolecular architectures by self-assembling from their components, thus behaving as programmed chemical systems . Chemistry may therefore be considered an information science , the science of informed matter. Supramolecular chemistry is intrinsically a dynamic chemistry in view of the ability of the interactions connecting the molecular components of a supramolecular entity and the resulting ability of supramolecular species to exchange their constituents. The same holds for molecular chemistry when the molecular entity contains covalent bonds that may form and break reversibly, so as to allow a continuous change in constitution by the reorganization and exchange of building blocks. These features define a constitutional dynamic chemistry (CDC) on both the molecular and supramolecular levels. CDC takes advantage of dynamic constitutional diversity to allow variation and selection in response to either internal or external factors to achieve adaptation . The merging of the features—information and programmability, dynamics and reversibility, constitution and structural diversity—points towards the emergence of adaptive and evolutive chemistry . The whole workshop could have not taken place without the help of the Centro Stefano Franscini. The CSF is the congress centre of the Swiss Federal Institute of Technology of Zurich (ETH Zurich) and has been situated at Monte Verità since 1989. It is an ideal meeting point for all members of the international scientific community who wish to discuss the state-of-the-art and new challenges of any field of research. The CSF supports 20-25 international conferences every year and, since 2010, up to ten winter doctoral schools1. The competence and professionalism of the staff were at the same level of beauty and inspiring character as that of Monte Verità. A meeting of this sort, if successful, leaves the audience with more open questions than settled answers, and this was definitely the case for Crowding 2012. Excluded volume is clearly a fundamental concept that has allowed crowding, a very familiar concept in soft matter, to enter into the domain of biological sciences. However, the complexity of the biological milieu calls for more refined descriptions. What is the role of electrostatic and electrodynamic interactions? What is the role of hydrodynamics interactions? To what extent does the strong spatial inhomogeneity (clustering of molecules, cellular compartmentalization, etc) have to be taken into account? Or, more generally, what are the minimal elements that prove crucial to describe reactions within a cell? How does the diffusion proceed (diffusion, slow diffusion, sub-diffusion) given that the experimental evidences are still controversial? In conclusion, we knew that allowing scientists with very different backgrounds and ideas to mingle was a hazardous attempt. Despite that, the workshop turned out to be a very successful experiment, which was highly enjoyed both by the participants and the organizers. Discussions sparked regularly among ever-changing groups, comprising senior scientists and students, despite the rather tight schedule, adding to the sense of fulfilment ignited by the outstanding level of the presentations. Given the success of the meeting Crowding 2012, a new event has been organized and will take place on the same themes during fall 2013, this time in the beautiful scenery of the Loire valley in France. The workshop 'Macromolecular crowding effects in cell biology: models and experiments' will be held on the CNRS campus in Orléans, France, on 24-25 October 2013. More information can be found on the workshop website: http://dirac.cnrs-orleans.fr/~piazza/. 1Source: www.csf.ethz.ch/