Concentrations Of Protamine Sulfate Research Articles

Limiting Carryover Events Resulting from Cell Surface Retention of VSV-G Pseudotyped HIV-Lentivector Particles on Hematopoietic Targets

Preclinical evidence and clinical trials speak to the therapeutic potential of retrovirus vectors for the heritable genetic modification of cells. Careful evaluation of the antecedent risks is critical to move these applications forward. Others previously demonstrated the persistence of intact vector particles on the surface of target cells. Inadvertent particle transfer after in vivo applications could lead to the transduction of bystander tissues, or provoke immunological responses. We recently demonstrated prolonged adherence of VSV-G pseudotyped, HIV-1 derived lentivirus particles after ex vivo transduction culture of murine hematopoietic target cells (1°) with subsequent transduction of secondary (2°) targets in vitro and in vivo. Extended particle adherence is independent of Env pseudotype and routine wash procedures (Pan et al., J Virol. Jan 2007). We hypothesized that unwanted carryover could be minimized by disrupting the vector particle attachment to 2° cells while maintaining uptake to 1° targets. Initial studies indicated that the transduction of 1° targets at 4°C (to prevent uptake) for up to 6 hours followed by serial PBS washes and subsequent direct co-culture with fibroblasts resulted in undiminished 2° gene transfer compared to transduction at 37°C. Conversely, post-transduction exposure to escalating concentrations of citric acid resulted in a systematic decrease in both 1° and 2° gene transfer rates. This is consistent with separable mechanisms for pH sensitive VSV-G mediated uptake of particles in 1° targets and the receptor independent attachment responsible for carryover and 2° transduction, respectively. Glycosaminoglycans, including heparin, quantitatively bind to pseudotyped vector particles. We found that exposure of particles to heparin effectively abrogated subsequent transduction of cells by disrupting attachment. Remarkably, serial heparin washes at the conclusion of transduction had only minimal effects on gene transfer to 1° targets, but resulted in a two-log reduction in 2° gene transfer. Increases in the concentration of protamine sulfate (a polycation) during transduction partly reversed the effect of heparin (a polyanion), demonstrating the residual impact of electrostatic interactions on attachment of retrovirus particles from the 1° cell. In further studies we showed that trypsin washes following vector exposure incompletely cleaved 1° cell surface bound particles while pronase effectively degraded cell surface bound particles in a dose dependent manner, abrogating carryover. Because pronase at high concentrations also compromised cell surface epitope integrity we studied the expression of chemokine receptor (CXCR) 4, both a critical mediator of progenitor cell homing to the bone marrow and a representative protease-sensitive surface molecule. These experiments revealed a dose dependent degradation of CXCR4 on the cell surface of 1° target cells and rapid regeneration within three hours, critical for applications involving the injection of ex vivo modified hematopoietic cells. In conclusion, our results demonstrate that select wash procedures can disrupt the ability of virus particles to bind secondary targets, degrade residual surface bound particles and reduce gene transfer to inadvertent 2° targets in vitro by up to 99%. These studies are important first steps in understanding and limiting inadvertent carryover in the context of gene therapy while maximizing target cell transduction.

Effect of ovotransferrin, protamine sulfate and EDTA combination on biofilm formation by catheter-associated bacteria

To determine the effect of a composition comprising ovotransferrin (OT), protamine sulfate (PS) and ethylenediaminetetraacetic acid (EDTA) on biofilm formation by catheter-associated bacteria. The in vitro activity of OT, PS and EDTA alone and in combinations against biofilm formation by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis and Staphylococcus epidermidis was investigated. All the three compounds either alone or in combinations failed to inhibit the growth completely at the concentrations tested. However, the subinhibitory concentrations of three compounds in a composition showed synergistic inhibitory effect on biofilm formation by K. pneumoniae, Ps. aeruginosa and S. epidermidis. Furthermore, 79-95% reduction in Ps. aeruginosa and S. epidermidis biofilm formation was observed in a clear vinyl urinary catheter treated with the composition. The subinhibitory concentrations of OT, PS and EDTA in a composition were effective in reducing biofilm formation by catheter-associated bacteria. This study shows that a synergistic composition-comprising non-antibiotic generally regarded as safe (GRAS) compounds such as OT, PS and EDTA may be used in the prevention of catheter-related infections.

Intravesical protamine sulfate and potassium chloride as a model for bladder hyperactivity

Objectives An acute animal model for hyperactive bladder in rats was developed using intravesical infusion of protamine sulfate (PS), an agent thought to break down urothelial barrier function, and physiologic concentrations of potassium chloride (KCl). Methods Continuous cystometrograms (CMGs) were performed in urethane-anesthetized female rats by filling the bladder (0.04 mL/min) with normal saline followed by intravesical infusion of a test solution consisting of either KCl (100 or 500 mM) or PS (10 or 30 mg/mL) for 60 minutes. Subsequently, the 10 mg/mL PS-treated animals were infused intravesically with 100, 300, or 500 mM KCl. Some animals were pretreated with capsaicin (125 mg/kg subcutaneously) 4 days before the experiments. Results Unlike KCl (100 or 500 mM) or a low concentration of PS (10 mg/mL) alone, the intravesical administration of a high concentration of PS (30 mg/mL) produced irritative effects with a decreased intercontraction interval (by 80.6%). After infusion of a low concentration of PS, infusion of 300 or 500 mM KCl produced irritative effects (intercontraction interval decreased by 76.9% or 82.9%, respectively). The onset of irritation occurred more rapidly after 500 mM KCl (10 to 15 minutes) than after 300 mM KCl (20 to 30 minutes). Capsaicin pretreatment delayed the onset (approximately 60 minutes) and reduced the magnitude (intercontraction interval decreased by 35.5%) of irritative effects. Conclusions Intravesical administration of KCl after PS treatment activates capsaicin-sensitive afferents and detrusor muscle and presumably capsaicin-resistant afferents. Modest, noncytotoxic affronts to urothelial barrier function can result in dramatic irritative responses. This model may be useful in the study of bladder irritation and hyperactivity.

Protamine sulfate test for fibrin monomers.

A procedure utilizing protamine sulfate to demonstrate the presence of fibrin monomers is described. The procedure is unaffected by heparin and by fibrinolytic split products. Interference by high levels of plasmin, however, was suggested. The possibility of precipitating protein fractions other than fibrin monomers has been lessened by utilizing a low concentration of protamine sulfate (0.9%) and a temperature of 37 C. This method was more sensitive and less subject to interference than two other procedures used to demonstrate fibrin monomers. Its application in differentiating disseminated intravascular coagulation from primary fibrinolysis is discussed.

Studies on the effect of protamine sulfate on cells of Vicia faba roots

Experiments have shown that a 2 h treatment with various concentrations of protamine sulfate will severely reduce the mitotic index in primary roots of Vicia faba. Since one result of PS treatment is the induction of an abscission layer within or directly behind the region of cell enlargement within 6–15 h, the effect on MI after this time could be due to general death of the root tip. This general death is characterized by loss of DNA from the nuclei. Of more immediate importance is that directly following treatment with PS there is a reduced nuclear incorporation of 3H-lysine and total inhibition of 3H-thymidine incorporation, indicating an effect on protein and DNA synthesis. Studies on uptake vs incorporation of 3H-lysine following PS treatment indicate that PS is affecting incorporation of the radioisotope into protein but not its uptake into the cell. Staining of nuclei with eosin Y and fast green following deamination indicates that the PS is entering the nucleus. It is postulated that the protamine could be behaving as a repressor molecule.