DEAE-Dextran Enhances the Lentiviral Transduction of Primary Human Mesenchymal Stromal Cells from All Major Tissue Sources Without Affecting Their Proliferation and Phenotype.

Genetic engineering of mesenchymal stromal cells (MSCs) is a tool widely used to explore MSC properties in vitro and in vivo. Lentiviral infection with the use of polycations as an adjuvant is a method that is commonly used to generate stably transduced cells. However, it is known that some polycations can negatively affect primary MSCs and to date, no study has explored the effect of different polycations on the transduction efficiency and properties of all main types of MSCs, namely those derived from umbilical cord, bone marrow and adipose tissue. Here we explore a range of polycations, using transduction protocols with and without spinoculation, to produce stably transduced MSCs from these three tissue sources. We identified that an overnight incubation with diethylaminoethyl-dextran (DEAE-Dextran) is the protocol associated with the best transduction efficiency without compromising the viability of the cells, and which worked consistently with lentiviral particles encoding for different transgenes. Transduced and sorted MSC populations revealed no significant changes in proliferation, morphology and expression of MSC markers compared to naïve MSCs. Following this study, we conclude that DEAE-Dextran is a polycation that can be successfully used to enhance the transduction of MSCs from all major tissue sources.

A novel blood-pooling MR contrast agent: Carboxymethyl-diethylaminoethyl dextran magnetite.

Gadofosveset trisodium is available as a prolonged pooling vascular contrast agent for magnetic resonance imaging. As gadolinium (Gd)-based agents may increase the risk for nephrogenic systemic fibrosis in patients with severe renal insufficiency, the present study synthesized carboxymethyl-diethylaminoethyl dextran magnetite (CMEADM) particles as a blood-pooling, non-Gd­based contrast agent. CMEADM particles carry a negative or positive charge due to the binding of amino and carboxyl groups to the hydroxyl group of dextran. The present study evaluated whether the degree of charge alters the blood­pooling time. The evaluation was performed by injecting four groups of three Japanese white rabbits each with CMEADM­, CMEADM2­, CMEADM+ (surface charges: ­10.4, ­41.0 and +9.6 mV, respectively) or with ultrasmall superparamagnetic iron oxide (USPIO; ­11.5 mV). The relative signal intensity (SIrel) of each was calculated using the following formula: SIrel = (SI post­contrast ­ SI pre­contrast / SI pre­contrast) x 100. Following injection with the CMEADMs, but not with USPIO, the in vivo pooling time was prolonged to >300 min. No significant differences were attributable to the electric charge among the CMEADM­, CMEADM2­ or and CMEADM+ particles when analyzed with analysis of variance and Tukey's HSD test. Taken together, all three differently­charged CMEADM2 particles exhibited prolonged vascular enhancing effects, compared with the USPIO. The degree of charge of the contrast agents used in the present study did not result in alteration of the prolonged blood pooling time.

Formation of polyelectrolyte complexes with diethylaminoethyl dextran: charge ratio and molar mass effect.

The formation of polyelectrolyte complexes (PECs) between carboxymethyl pullulan and DEAE Dextran, was investigated, in dilute solution, with emphasis on the effect of charge density (molar ratio or pH) and molar masses. Electrophoretic mobility measurements have evidenced that insoluble PECs (neutral electrophoretic mobility) occurs for charge ratio between 0.6 (excess of polycation) and 1 (stoichiometry usual value) according to the pH. This atypical result is explained by the inaccessibility of some permanent cationic charge when screened by pH dependant cationic ones (due to the Hoffman alkylation). Isothermal titration calorimetry (ITC) indicates an endothermic formation of PEC with a binding constant around 10(5) L mol(-1). Finally asymmetrical flow field flow fractionation coupled on line with static multi angle light scattering (AF4/MALS) evidences soluble PECs with very large average molar masses and size around 100 nm, in agreement with scrambled eggs multi-association between various polyelectrolyte chains.

[Receptor elements for biosensors in two ways of methylotrophic yeast immobilization].

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 In yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05-1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2-1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzochinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.

Inhibition of oxidized LDL aggregation with the calcium channel blocker amlodipine: role of electrostatic interactions.

Atherogenic low-density lipoproteins (LDL) are characterized by elevations in cholesterol content and increased electronegativity, factors that contribute to aggregation and foam cell formation. This study was designed to test the effect of the positively charged calcium channel blocker (CCB) amlodipine on the aggregation properties of oxidized LDL lipids. Large unilamellar vesicles (LUVs) (100 nm diameter) labeled with a non-exchangeable marker [3H]cholesteryl hexadecyl ether were prepared with lipids extracted from human LDL following oxidation. The LUVs were shown to bind, in a reversible fashion, to charged diethylaminoethyl Sephadex columns. The addition of amlodipine inhibited binding of the oxidized LDL lipids in a dose-dependent fashion with an IC(50) in the nanomolar range as a result of its high lipophilicity and positively charged amino group (pK(a) of 9.02). The activity of amlodipine was reproduced in model membranes that contained fixed amounts of charged phospholipid (glycerophospholipid) in a concentration-dependent manner. By contrast, drugs lacking a formal positive charge, including CCBs (felodipine, nifedipine, diltiazem, verapamil) and an angiotensin-converting enzyme-inhibitor (ramiprilate) had no effect on the column binding of the modified, electronegative lipids. These effects of amlodipine on LDL lipid aggregation and electrostatic properties may represent a novel antiatherosclerotic mechanism of action.

Multiplication of the V4 strain of Newcastle disease virus in Madin Derby bovine kidney cells.

This study describes a reproducible cell culture system that permits the growth and secondary multiplication of the V4 strain of Newcastle disease virus. Allantoic fluid, magnesium chloride and diethylaminoethyl dextran were incorporated in Dulbecco's modified Eagle's medium to encourage secondary viral multiplication without adversely affecting healthy Madin Derby bovine kidney cell growth.

Factors affecting the resolution of dl-menthol by immobilized lipase-catalyzed esterification in organic solvent.

Among 10 lipases tested, Candida rugosa lipase exhibited the best ability to catalyze the resolution of dl-menthol in organic solvent. The lipase was immobilized on different carriers, and the experiment was carried out with different acyl donors. The high yield and optical purity of the product were achieved in cyclohexane with valeric acid as acyl donor using C. rugosa lipase immobilized on DEAE-Sephadex A-25. The conversion of dl-menthol depended on the water content of immobilized lipase and on the pH of the aqueous solution from which lipase was immobilized. The operational stability of the DEAE-Sephadex A-25 immobilized lipase in catalysis of the esterification reaction showed that >85% activity remained after 34 days of repeated use. The resolution of racemic menthol in organic medium catalyzed by immobilized C. rugosa lipase-catalyzed esterification is very convenient, and it represents a significant improvement in the use of enzyme for the preparative production of optically active menthol. This process is readily applicable to large-scale preparation.

Efficient, rapid and reliable establishment of human trophoblast cell lines using poly-L-ornithine.

OBJECTIVE: Human trophoblast cells in primary culture are difficult to use for the rigorous study of trophoblast function because of contamination with other cell types, paucity of numbers, poor viability and inter-experiment variation engendered by the need to prepare fresh cells for each experiment. DNA-transfection to produce immortalized cells, or cells with extended life-span, has been the obvious approach to solve this problem. Although there have been a few reports in the literature describing trophoblast cell lines generated in this way, it is clear that to date the methods are difficult and few lines have been generated. The basic problem is that transfection efficiencies of different methods are cell type-specific. The objectives of this study were therefore to compare the transfection efficiencies of three commonly used techniques, to use the best technique to generate trophoblast cell lines, and to conduct preliminary characterization studies. METHODS: We have compared calcium phosphate co-precipitation, DEAE-dextran and poly-L-ornithine (PLO) DNA transfection protocols. We then used the most efficient to transfect human extravillous trophoblast with pSV3neo. RESULTS: Our modification of the PLO method has a transfection efficiency greater than 30 times that of the next best method. Several cell lines were established which had an extended life span and displayed an invasive phenotype, including the expression of MHC Class I framework antigens, human placental lactogen and human chorionic gonadotrophin, and thus have characteristics of extravillous trophoblast. In addition these cells express the integrin subunits b1, a1, and a3, all of which are known to be expressed in human trophoblast, and respond to IL-1alpha by increased secretion of GM-CSF. CONCLUSIONS: PLO is a highly efficient, rapid, reliable, simple and low-cost technique for the procurement of human trophoblast cell lines which retain most, if not all, the phenotype of the parental cell. These lines are potentially powerful tools in the rigorous study of trophoblast function.

Monitoring DNA dynamics using spin-labels with different independent mobilities.

The electron paramagnetic resonance (EPR) spectra of spin-labeled DNA duplexes, both bound to DEAE-Sephadex and free in solution, have been analyzed. The nitroxide spin-labels are covalently linked to a deoxyuridine residue using either a monoacetylene or diacetylene tether. This difference in tether length produces a dramatic difference in the independent mobility of the nitroxide relative to the DNA. In the case of the monoacetylene tether, the motion of the nitroxide has previously been shown to be tightly coupled to that of the DNA duplex. With the diacetylene tether, there is considerable independent motion of the probe. The diacetylene tether is intended to minimize the possibility of the nitroxide producing a perturbation of the dynamics of DNA. It is demonstrated here that, when coupled via the diacetylene tether, the nitroxide undergoes a rapid uniaxial rotation about the tether. A detailed analysis of the EPR spectrum of duplex DNA in solution, spin-labeled using the diacetylene tether, demonstrates that the motion of the nitroxide can be modeled in terms of this independent uniaxial rotation together with motion of the DNA which is consistent with the global tumbling of the duplex. As was previously found using the monoacetylene tether, there is no evidence of rapid, large-amplitude motions of the base pair in the EPR spectrum of a nitroxide coupled to duplex DNA via the diacetylene tether. This result confirms the small amplitudes of internal motion, local and collective, previously observed in duplex DNA with the monoacetylene-tethered nitroxide.

Disposition characteristics of model macromolecules in the perfused rat kidney.

The disposition characteristics of model macromolecules such as dextran (70 kDa), bovine serum albumin (BSA), and their charged derivatives were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous and urinary recovery patterns and tissue accumulation of radiolabeled compounds were evaluated under filtering or nonfiltering conditions. In the filtering kidney, cationic macromolecules such as diethylaminoethyl-dextran (DEAE-dex) and cationized BSA (cBSA) accumulated in the kidney to a great extent whereas anionic and neutral macromolecules such as BSA, carboxymethyl-dextran (CM-dex), and dextran showed only small uptake. DEAE-dex and cBSA were distributed to both the medulla and cortex regions of the kidney and their recoveries in the kidney decreased as the injected dose increased. Similar tissue uptake was observed in the nonfiltering kidney perfusion system suggesting that they were mainly taken up by the kidney from the renal capillary side based on electrostatic interaction. In addition, the steady-state distribution volumes of cationic macromolecules calculated from venous outflow patterns were larger than those of the intravascular volume estimated from the distribution volumes of neutral and anionic macromolecules, suggesting their reversible interaction with the vascular wall. On the other hand, dextran derivatives with molecular weight distribution were excreted into urine based on glomerular permselectivity; i.e., cationic DEAE-dex and anionic CM-dex showed enhanced and restricted urinary excretion, respectively, compared with neutral dextran. In contrast, no significant excretion was observed for BSA and cBSA. The utility of the isolated rat kidney perfusion experiment for studying the renal disposition of macromolecular drugs was thus demonstrated.

Investigation on the fate of orally administered DEAE-dextran in rats.

The breakdown of the carbohydrates by the colonic bacterial flora can cause intestinal symptoms, such as meteorism, abdominal pain and diarrhoea. The ability of digestive enzymes and colonic bacterial flora to break down the DEAE-dextran, a new lipid lowering resin, was investigated in rats. DEAE-dextran appeared to be unaffected by either enzyme activity in the small intestine or bacterial flora in the large intestine. This may be important when dealing with the pharmacological activity of DEAE-dextran and estimating its side effects. Small intestinal transit rate appeared to be accelerated by oral DEAE-dextran in rats.

Binding of polycation DEAE-dextran to Chinese hamster ovary cells induces reversible Ca2+ influx and inhibits capping of concanavalin A acceptor proteins.

Binding of the polycation DEAE-dextran to the cell surface of HA-1 CHO cells caused a marked increase in 45Ca2+ exchange influx. The effect was fairly selective for Ca2+, undirectional (efflux was not increased) and was rapidly reversed by treatment with polyanion dextran sulfate. 45Ca2+ influx could not be stimulated by treatment with multivalent lectins or fibronectin. In addition to stimulating 45Ca2+ flux, DEAE-dextran inhibited the capping of concanavalin-A acceptor proteins. Inhibition of capping occurred over the same DEAE-dextran concentration range (20-200 micrograms/ml) which stimulated 45Ca2+ uptake, possibly implicating increased cellular [Ca2+] in the inhibition of concanavalin A acceptor protein capping in this cell type. The profound effect of DEAE-dextran on cellular Ca2+ uptake and the rapid reversal of the effect by dextran sulfate might make the polycation a useful agent for the induction of transient increases in cellular [Ca2+].

Influence of charge on filtration across renal basement membrane films in vitro.

The filtration of differently charged species of myoglobin and dextran across films of isolated basement membrane in vitro showed that the filtration behavior of both polymers was influenced by charge. Rejection increased with increasing negative charge. Titration of the isolated basement membrane revealed an isoionic point of pH 5.5 and an isoelectric point of pH 5.7. The net negative charge at pH 7.4 was 0.15 mEq/g protein; this charge was attributed to carboxylate anion. Glycosaminoglycan sulphate did not contribute significantly to the net charge. Filtration of narrow range dextran fractions across films of basement membrane at the isoelectric point markedly reduced differences in filtration due to charge confirming that the differences in behavior found at pH 7.4 were due to charge interactions between the solutes and the membranes. Physical characterization of the charged and uncharged dextran fractions revealed no substantial differences in size or shape for the differently charged species.

Binding of bile acids to anion-exchanging drugs in vitro.

Equal amounts of anion exchanger of the drugs, Secholex, Colestipol, Cuemid, and Questran, were incubated at 37 degrees C with human duodenal fluid containing about 7 mM total bile acid. Binding of bile acid to Qestran, which contains about 45% cholestyramine, was fastest: concentration of unbound bile acid after 2 hours was less than 3 mM compared to about 5 mM in the solutions incubated with the other drugs, including Cuemid, which contains about 83% cholestyramine. After 24 hours, differences were less marked, but binding to Qestran was still greatest. Glycocholic acid was least efficiently bound, especially to Secholex and Cuemid. The differences in rates of binding were unaffected by preincubation of the drugs with 1 N HCl to simulate stomach conditions. Although differences between the cholestyramine components of Cuemid and Questran are not ruled out, it is possible that one or more of the other components of Questran significantly affect the in vitro binding of bile acids. Cholestryramine in the form of Questran may be the drug of choice in the treatment of hypercholesterolemia, in which reduction of the bile acid pool is desirable. In cholegenic diarrhoea, however, one of the drugs with lower affinity for glycocholic acid may be preferable.

Permselectivity of the glomerular capillary wall. Facilitated filtration of circulating polycations.

To examine the electrostatic effects of fixed negative charges on the glomerular capillary wall, polydisperse [(3)H]DEAE dextran, a polycationic form of dextran, was infused into 10 Munich-Wistar rats. Fractional clearances of DEAE ranging in radius from 18 to 44A were determined in these rats, together with direct measurements of the forces and flows governing the glomerular filtration rate of water. These results were compared with data previously obtained in Munich-Wistar rats receiving tritiated neutral dextran (D) and polyanionic dextran sulfate (DS). Measured values for the determinants of the glomerular filtration rate of water in rats given DEAE were found to be essentially identical to those in rats given either D or DS. In addition, DEAE was shown to be neither secreted nor reabsorbed. Fractional clearances of polycationic DEAE were increased relative to both D and DS, the increase relative to D being significant for effective molecular radii ranging from 24 to 44A. Fractional DEAE clearances were also measured in a separate group of six Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN). Fractional DEAE clearances in NSN rats were reduced significantly, relative to values measured in normal rats, for effective DEAE radii ranging from 18 to 42A. Moreover, in NSN rats, fixed negative charges on the glomerular capillary wall were greatly reduced, relative to non-NSN rats, as evidenced by a reduction in intensity of colloidal iron staining. Thus, in NSN rats, DEAE clearances were essentially indistinguishable from values obtained with both neutral D and polyanionic DS.

The fate of the orally administered bile acid sequestrant, polidexide, in humans.

1. The metabolic fate of the insoluble bile acid sequestrant polidexide, (poly-[2-(diethylamino)ethyl] polyglycerylenedextran hydrochloride), was studied in four adult humans following the oral administration of the 14C-labelled substance. 2. The mean cumulative recovery of 14C in faeces was 95-3% (s.e.m. = 1-1) of the administered dose, while mean cumulative recovery in urine was 0-37% (s.e.m. = 0-13) of the oral dose. 3. Only background levels of radioactivity were detectable in plasma samples taken 1-3 days after administration of tracer. 4. The findings suggested that polidexide was not absorbed from the gastrointestinal in man to any significant degree.