Photodynamic sterilization of red cells and its effect on contaminating white cells: viability and mechanism of cell death

BACKGROUND: Phthalocyanines are useful sensitizers for photodynamic sterilization of red cell concentrates. Various lipid-enveloped viruses can be inactivated with only limited red cell damage. Because white cells are involved in the immunomodulatory effects of blood transfusions, the study of the effect of photodynamic treatment on these cells is imperative. STUDY DESIGN AND METHODS: White cell-enriched red cell suspensions were photodynamically treated with either the hydrophobic Pc4 (HOSiPcOSi-(CH3)2(CH2)3N(CH3)2) or water-soluble aluminum phthalocyanine tetrasulfonate (AIPCS4) under virucidal conditions. Viability of white cell subpopulations on Days 0, 1, and 4 after treatment was determined by fluorescence-activated cell sorting by flow cytometric analysis of propidium iodide uptake. Apoptosis induction was studied by DNA ladder formation and staining for an early marker of apoptosis (annexin V). RESULTS: Treatment with Pc4 causes a significant decrease in cell viability of all white cells, as shown by prodidium iodide uptake. Monocytes and granulocytes are the most sensitive, and lymphocytes are relatively more resistant. Some of the cells die by apoptosis, which is induced within 30 minutes after treatment. Treatment with AIPCS4 damages only monocytes; other cell populations are not affected. CONCLUSIONS: Physicochemical properties of the photosensitizers partly determine their effect on white cells. Differences in intracellular localization are likely to be responsible for the effects observed.     

Serum supplement, inoculum cell density, and accessory cell effects are dependent on the cytokine combination selected to expand human HPCs ex vivo

BACKGROUND: The prolonged periods of pancytopenia associated with cord blood transplants suggest that in some cases cell numbers may be limiting. The possibility that limiting cell numbers may be overcome and prolonged periods of pancytopenia abrogated by the transplantation of human umbilical cord blood cells expanded ex vivo has led to efforts to define optimal culture conditions for these cells. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured with three cytokine combinations: SCF+G-CSF+GM-CSF+MGDF (SGGM); IL-6+ SCF+MGDF+Flt3-ligand (6SMF); and IL-1+IL-3+IL-6+G-CSF+GM-CSF+SCF+Epo (GFmix). Serum effects, inoculum concentration (cells/mL) seeding density (cell/cm(2)) and accessory cell effects on the expansion of CD34+ cells were determined. RESULTS: Cellular outputs were significantly higher with fetal calf serum (FCS) than with cord blood serum (CBS) or adult group AB serum (ABS) in the presence of 6SMF, however, CBS was as effective as FCS. The best seeding concentrations varied for each of the cytokine combinations, and inoculum densities exceeding 1000 cells per cm(2) proved detrimental for cultures containing GFmix and SGGM. Accessory cell studies indicated that populations expressing the CD33 antigen inhibited the expansion of purified CD34+ cells in the presence of GFmix or SGGM, but not in the presence of 6SMF. CONCLUSION: Serum supplement, inoculum cell concentration, seeding densities, and accessory cell effects are dependent upon the cytokine combination selected to expand cord blood HPCs ex vivo. Thus, each of these measures should be assessed to establish reproducible and reliable conditions for the selection of different cytokine combinations to culture cord blood HPCs.     

Influence of carboxylic acid functionalization on the cytotoxic effects induced by single wall carbon nanotubes on human endothelial cells (HUVEC)

A vast variety of nanomaterials have been developed in the recent years, being carbon nanotubes (CNTs) the ones that have attracted more attention, due to its unique properties which make them suitable for numerous applications. Consequently, it is predicted that tons of CNTs will be produced worldwide every year, being its exposure of toxicological concern. Nanomaterials, once into the body, can translocate from the uptake sites to the blood circulation or the lymphatic system, resulting in distribution throughout the body. Thus, the vascular endothelium can be in contact with them and can suffer from their toxic effects. In this regard, the aim of this work was to investigate the cytotoxicity of single-walled carbon nanotubes (SWCNTs) on human endothelial cells evaluating the influence of acid carboxylic functionalization and also the exposure time (24 and 48 h). Biomarkers assessed were neutral red uptake, protein content, a tetrazolium salt metabolization and cell viability by means of the Trypan blue exclusion test. Cells were exposed to concentrations between 0 and 800 μg/mL SWCNTs for 24 and 48 h. Results have shown that both SWCNTs and carboxylic acid functionalized single-walled carbon nanotubes (COOH-SWCNTs) induce toxic effects in HUVEC cells in a concentration- and time-dependent way. Moreover, the carboxylic acid functionalization results in a higher toxicity compared to the SWCNTs.     

Lack of cytotoxic and genotoxic effects of Minthostachys verticillata essential oil: Studies in vitro and in vivo

Minthostachys verticillata (peperina) is an aromatic and medicinal plant with several uses and ethnobotanical properties. Numerous studies have demonstrated that its essential oil (Mv-EO) presents antimicrobial capacity and shows immunomodulating and anti-allergic properties in human cell lines. Thus, the goal of this study was to investigate the main chemical composition, analyzed by GC–FID, and the cyto-genotoxic effects of Mv-EO, using Vero cells, human PBMCs and mice bone marrow cells. The Mv-EO was rich in pulegone 60.5% and menthone 18.2%. Our results clearly show that Mv-EO is not cyto-genotoxic in vitro nor in vivo. It not induced cytotoxic effects, as indicated by trypan blue dye exclusion and NRU assays both in Vero cells and human PBMCs. In addition, Mv-EO (100–1000 μg/mL) not induced apoptotic effects on human PBMCs, as indicated by Hoechst staining and DNA fragmentation analysis by agarose gel electrophoresis. The in vivo assay showed that Mv-EO (25–500 mg/kg) not increased the frequency of micronucleus in bone marrow cells of mice. Further, the ratio of polychromatic/normochromatic erythrocytes was not modified. These findings suggest that Mv-EO appears to be safe as a therapeutic agent.     

Cytokine production by co-cultures exposed to monodisperse amorphous silica nanoparticles: the role of size and surface area

The aim of this study was to test the influence of nanoparticle size and surface area (SA) on cytokine secretion by co-cultures of pulmonary epithelial cells (A549), macrophages (differentiated THP-1 cells) and endothelium cells (EA.hy926) in a two-compartment system. We used monodisperse amorphous silica nanoparticles (2, 16, 60 and 104 nm) at concentrations of 5 μg/cm2 cell culture SA or 10 cm2 particle SA/cm2. A549 and THP-1 cells were exposed to nanoparticles for 24h, in the presence of EA.hy926 cells cultured in an insert introduced above the bi-culture after 12h. Supernatants from both compartments were recovered and TNF-α, IL-6, IL-8 and MIP-1α were measured. Significant secretion of all cytokines was observed for the 2 nm particles at both concentrations and in both compartments. Larger particles of 60 nm induced significant cytokine secretion at the dose of 10 cm2 particle SA/cm2. The use of multiple cellular types showed that cytokine secretion in single cell cultures is amplified or mitigated in co-cultures. The release of pro-inflammatory mediators by endothelial cells not directly exposed to nanoparticles indicates a possible endothelium activation after inhalation of silica particles. This work shows the role of size and SA in cellular response to amorphous nanosilica.     

CD39/NTPDase-1 activity and expression in normal leukocytes

INTRODUCTION: CD39/NTPDase-1 is a cell surface enzyme expressed on leukocytes and endothelial cells that metabolizes ATP to ADP and AMP. CD39 is expressed on numerous different types of normal leukocytes, but details of its expression have not been determined previously. METHODS: We examined CD39 expression and activity in leukocytes isolated from healthy volunteers. Expression of CD39 on leukocytes was measured by FACS and activity of CD39 in lymphocytes and neutrophils was determined by an enzymatic radio-TLC assay. RESULTS: We established that CD39 is expressed on neutrophils, lymphocytes, and monocytes. The enzyme is found on >90% of monocytes, neutrophils, and B-lymphocytes, and 6% of T-lymphocytes and natural killer cells. Per cell density of expression varied, with the highest expression on monocytes and B-lymphocytes. ATPase and ADPase activities were highest on B-lymphocytes, lower on neutrophils, lowest on T-lymphocytes. The ratio of ADPase:ATPase activity was 1.8 for neutrophils and B-lymphocytes and 1.4 for T-lymphocytes. Hypertensive volunteers had lower levels of CD39 on their T-lymphocytes and NK cells. No correlation between age, gender, ethnic background, or cholesterol level and CD39 expression was observed. CONCLUSIONS: We conclude that CD39 activity and expression are present to varying degrees on all leukocytes types examined. Differences between leukocyte types should be considered when examining CD39 in disease states.     

An agarose spot chemotaxis assay for chemokine receptor antagonists

Introduction

Chemokines are important players in directing the migration of cancer cells as part of the metastatic process. The aim of this study is to develop an easy-to-perform, reliable, and inexpensive assay for rapid analysis of anti-chemotactic activity of chemokine antagonists under a number of experimental conditions.

Methods

An agarose spot containing the chemokine chemoattractant is applied to a glass petri dish. Live cells in a media, both with and without a chemokine antagonist, are added to the dish and, following cell adhesion, the migration under the agarose spot is observed and analysed by microscopy.

Results

In the absence of CXCL12 in the agarose, no migration under the agarose spot is detected. In the presence of CXCL12, significant migration under the agarose spot is observed which can be retarded if a neutralising monoclonal antibody or a small molecule antagonist is added to the media.

Discussion

This experimental configuration is a reliable, inexpensive and easy-to-perform chemotaxis assay, which enables assessment of the activity of CXCR4 antagonists.     

Role of caspases and Bax protein in saffron-induced apoptosis in MCF-7 cells

Saffron (Crocus sativus), widely used as a spice in Middle Eastern cuisine and is known for anti-cancer properties. The mechanism of saffron-induced cytotoxicity, in tumor cells has not been adequately explored. Therefore, we investigated the role of caspases and Bax protein in saffron-induced apoptosis in MCF-7 cells, a commonly used cell culture system for in vitro studies on breast cancer.     

Interspecies difference in liver-specific functions and biotransformation of testosterone of primary rat,porcine and human hepatocyte in an organotypical sandwich culture

Interspecies difference is an important issue in toxicology research. We compared the potential in vitro metabolism of human, porcine and rat hepatocytes over 2 weeks in culture in an organotypical culture model which reflects the in vivo situation. All three species show similar LDH-rates. Albumin measurements showed that rat cells are about twice as active as human and porcine hepatocytes. The ethoxyresorufin-O-deethylase (EROD) activity of the rat hepatocytes is with about 14 μU/10⁶ cells distinctly higher than those of porcine and human cells (1.8 and 0.5 μU/10⁶ cells respectively), furthermore, the activity of the rat EROD increases slightly during the prolonged time in culture, whereas those of porcine and human enzymes slightly decrease. Concerning ethoxycoumarin-O-deethylase (ECOD), the enzyme activities are found to be in three different ranges where rat cells show the highest activity with 66 μU/10⁶ cells, porcine hepatocytes exhibit an activity of about 23 μU/10⁶ cells, and human activity is lowest with 0.7 μU/10⁶ cells. All three species show a similar decreasing trend of ECOD during the period of study. Regarding the biotransformation of testosterone, human and porcine liver cells form three major metabolites whereas rat cells form a mixture of all measured metabolites. Hence, in vitro metabolism using porcine hepatocytes would be much more scientific sense than one using rat hepatocytes since the metabolic pathways are much closer to human metabolism.