In vitro hematological and in vivo vasoactivity assessment of dextran functionalized graphene.

The intravenous, intramuscular or intraperitoneal administration of water solubilized graphene nanoparticles for biomedical applications will result in their interaction with the hematological components and vasculature. Herein, we have investigated the effects of dextran functionalized graphene nanoplatelets (GNP-Dex) on histamine release, platelet activation, immune activation, blood cell hemolysis in vitro, and vasoactivity in vivo. The results indicate that GNP-Dex formulations prevented histamine release from activated RBL-2H3 rat mast cells, and at concentrations ≥ 7 mg/ml, showed a 12-20% increase in levels of complement proteins. Cytokine (TNF-Alpha and IL-10) levels remained within normal range. GNP-Dex formulations did not cause platelet activation or blood cell hemolysis. Using the hamster cheek pouch in vivo model, the initial vasoactivity of GNP-Dex at concentrations (1-50 mg/ml) equivalent to the first pass of a bolus injection was a brief concentration-dependent dilation in arcade and terminal arterioles. However, they did not induce a pro-inflammatory endothelial dysfunction effect.

Techniques for time-efficient isolation of human skin dendritic cell subsets and assessment of their antigen uptake capacity.

Dendritic cells (DCs) residing in skin are important sentinels for foreign antigens. Methods to facilitate studies of subsets of skin DCs are important to increase the understanding of various pathogens, allergens, topical treatments or vaccine components targeting the skin. In this study, we developed a new DC purification method using a skin graft mesher, clinically used for expansion of skin grafts, to accelerate processing of skin into nets that allowed efficient enzymatic disruption and single cell isolation. The reduction in processing time using the skin graft mesher enabled processing of larger skin samples and also limited the ex vivo handling of the specimens which is associated with maturation of DCs. In addition, a skin explant model to functionally monitor early events of antigen uptake by DC subsets in situ was developed. DCs isolated from epidermis represented a uniform CD1a(+) HLA-DR(+) CD11c(+) Langerin(+) DC-SIGN(-) DC-LAMP(int) DEC-205(int) Langerhans cell (LC) population whereas three subtypes of HLA-DR(+) CD11c(+) DCs were isolated from dermis based on their varying expression of CD1a. Epidermal LCs showed a significantly higher antigen uptake capacity of fluorescently-labelled ovalbumin (OVA) and dextran as compared to any of the dermal DC (dDC) subsets. In contrast, injection of antigen directly into skin explants followed by in situ imaging revealed that the majority of DCs with internalized antigen were localized in the dermis, likely as a consequence of the anatomical site for antigen delivery. These methods offer potency for various applications addressing antigen uptake, microbial DC interactions or other antigenic stimulation targeting the skin and can enhance our knowledge of basic DC biology in human skin.

Expanding to fill the gap: a possible role for inert biopolymers in regulating the extent of the 'macromolecular crowding' effect.

We discuss the potential for inert biopolymers existing in cells to play a role in regulating the macromolecular crowding effect via their ability to undergo shape changing structural transitions. We have explored this possibility by the use of theory and experiment. The theoretical component utilized Monte-Carlo based simulations to examine the folding of a hypothetical protein in a concentrated environment of hard spheres which are themselves capable of reversible expansion and contraction. The experimental component of the study involved examination of the effect of different sized crowding agents on the thermally induced denaturation of cytochrome c [in phosphate buffered saline solution containing 1.0M guanidinium hydrochloride at pH 7.0]. On the basis of our findings we suggest that in a crowded solution environment the presence of a non-reactive polymer capable of reversible expansion/contraction via folding and unfolding may alter the excluded volume component of the solution. This ability would confer on the non-reactive polymer a novel role in influencing other processes in solution affected by macromolecular crowding.

[Comparative stability assessment of laccases from the basidiomycetes Coriolus hirsutus and Coriolus zonatus in the presence of effectors]. / Sravnitel'noe izuchenie stabil'nosti lakkaz iz bazidiomitsetov Coriolus hirsutus i Coriolus zonatus v prisutstvii éffectorov.

Stability characteristics of the laccases of the basidiomycetes Coriolus hirsutus and Coriolus zonatus were measured comparatively at temperatures 25 and 40 degrees C in the presence of various effectors (proteins, salts, polyalcohols, polyacids, and polyelectrolytes). Stabilization effects of cations on the laccases from C. hirsutus and C. zonatus decreased in the descending series Cu2+ > Mg2+ > Ca2+ and Ca2+ > Mg2+ > Mn2+, respectively. Tween 20 caused insignificant stabilization of the two enzymes. The C. zonatus laccase was also insignificantly stabilized as a result of treatment with bovine serum albumin. The enzymatic activity of the laccase preparations from C. hirsutus and C. zonatus was conserved virtually completely after vacuum drying (84 and 93%, respectively). The most effective stabilizer of the C. hirsutus laccase was found to be dextran (17 kD). Dry preparations treated with this agent conserved up to 95% of the enzymatic activity. The most effective stabilizer of the C. zonatus was polyacrylic acid (102% of the initial activity).

Pharmacological assessment of the nitric-oxide synthase isoform involved in eosinophilic inflammation in a rat model of sephadex-induced airway inflammation.

Excessive local production of nitric oxide (NO) has been suggested to play a role in rodent models of airway inflammation and in pulmonary diseases such as asthma. However, even given the plethora of data available including gene expression data, pharmacological data, and gene deletion studies in animal models, it is still not clear which nitric-oxide synthase (NOS) isoform is involved in eosinophilic airway inflammation. In this rat study, the nonselective NOS inhibitor L-NAME (N(G)-nitro-L-arginine methyl ester), but not a selective inducible NOS (iNOS) inhibitor 1400W (N-3-(aminomethyl)benzyl)acetamidine), impacted on Sephadex-induced inflammation by significantly inhibiting lung edema, eosinophil infiltration, tumor necrosis factor alpha, interleukin-13, and eotaxin levels in the lung tissue. Furthermore, iNOS gene expression was not induced following Sephadex administration, which confirms that iNOS does not play a role in this model. To demonstrate that this phenomenon was not restricted to this model of asthma, L-NAME, but not 1400W, was shown to reduce eosinophilia in an antigen-induced model. However, in contrast to the Sephadex model, there was an induction of iNOS gene expression after antigen challenge. In a model of aerosolized lipopolysaccharide-induced inflammation, where iNOS gene expression is increased, 1400W inhibited the increased neutrophilia. These data suggest that the compound has been administered using an appropriate dosing regimen for iNOS inhibition in the rat lung. In conclusion, it appears that constitutive, not inducible, NOS isoforms are important in NO production in models of allergic inflammation, which questions whether there is a role for iNOS inhibitors as therapy for the treatment of asthma.

Assessment of dextran 10-benzene-tetracarboxylate-hemoglobin, an oxygen carrier, using guinea pig isolated bowel model.

With the aim of assessing of dextran-benzene-tetracarboxylate hemoglobin as an oxygen carrier, we studied histological changes in the intestinal loop in anesthetized guinea pig. The intestinal tissue being very sensitive to hypoxia, an innervated loop was vascularly perfused with open-flow during one hour at zero hematocrit. To estimate the capacity of hemoglobin solution to oxygenate this tissue, we observed the mechanical and histological changes in the organ and the arterio-venous difference in PO2, oxyhemoglobin, deoxyhemoglobin and we compared them with human albumin, Tyrode and non-modified hemoglobin. The PO2 arteriovenous differences were 51.9 +/- 7.1 torr (m +/- SEM) for Tyrode, 40.2 +/- 6.4 torr for albumin solution, 113.7 +/- 6.5 torr for non-modified hemoglobin and 132.7 +/- 6.8 torr for dex-BTC-Hb. Compared to albumin and Tyrode solutions, hemoglobin solutions transferred more oxygen to tissues. The desaturation of dex-BTC-Hb was significantly superior (p < 0.05) to the one non-modified hemoglobin. With Hb solutions, this desaturation increased with time and it depended on the perfusion flow. The structure of jejunal villi when perfused with a hemoglobin solution, remained almost normal and the loop was still active. Nevertheless, non-modified hemoglobin leaked from the vessels to the lumen and caused edema and a rupture of overlapping epithelium at the tip of the villi. With dex-BTC-Hb, such histological modifications were less significant. With albumin and Tyrode, all villi were totally necrosed and the loop was completely inert. We have demonstrated that dextran-benzene-tetracarboxylate hemoglobin had the ability to maintain the tissue alive thanks to its good capacity to release oxygen and its satisfactory vascular persistence. Dex-BTC-Hb solution can answer to needs of tissue.

Assessment of red blood cell aggregation with dextran by ultrasonic interferometry.

Aggregation of human red blood cells (RBCs) induced by dextrans of various molecular weight has been studied by using a new ultrasonic interferometry method. This method, based on A-mode echography, allowed for the measurement of the accumulation rate of particles on a solid plate which is related to their sedimentation rate (i.e., to their mean size). The initial aggregation process, the mean and the maximum sedimentation rate of aggregates and the packing of the sedimented RBCs have been investigated. Effects of hematocrit, molecular weight of dextrans and inhibition by dextran 40 on the RBC aggregation induced by dextran of higher molecular weight have been determined by analysing variations of the aggregate size. Results obtained confirm the aggregation effect of dextrans of molecular weights equal or higher than 70,000 dalton and disaggregation effect of dextran 40,000 dalton on aggregation by dextrans of higher molecular weight.

Assessment of procollagen processing defects by fibroblasts cultured in the presence of dextran sulphate.

The culture of skin fibroblasts in the presence of 0.01% (w/v) dextran sulphate results in complete proteolytic processing of procollagen to collagen. Processing occurs predominantly via a pN-collagen intermediate, suggesting that C-propeptide cleavage occurs early during the processing pathway. The processed collagen is associated with the cell-layer fraction. This method of inducing procollagen processing was evaluated for use in detecting procollagen processing abnormalities in heritable connective-tissue diseases. Abnormal type I procollagen processing was clearly demonstrated in two cases with known defects of pN-propeptide cleavage. In one, the cleavage deficiency was due to diminished N-proteinase activity (dermatosparaxis) and in the other case (Ehler's-Danlos syndrome type VIIA) the cleavage site was deleted. In a case of osteogenesis imperfecta (type II) the slow electrophoretic migration of type I collagen alpha-chains due to over-modification of lysine was readily demonstrated. Inefficient procollagen processing was also evident in this patient, as had been previously reported [de Wet, Pihlanjaniemi, Myers, Kelly & Prockop (1983) J. Biol. Chem. 258, 7721-7728]. Thus this method of culture in the presence of dextran sulphate provides a simple and rapid procedure for the detection of procollagen processing defects and electrophoretic abnormalities.

Assessment of haemodynamic changes and acid-base equilibrium during hypovolaemia and after infusion of plasma substitutes in dogs.

The following haemodynamic values were determined in anaesthetized mongrel dogs: heart rate, systolic blood pressure in the ascending aorta, left ventricular pressure at the peak dp/dt, left ventricular end-diastolic pressure, time interval from Q in ECG to the onset of the systolic wave of dp/dt, time interval from Q in ECG to peak dp/dt, maximum rate of left ventricular pressure rise, femoral arterial flow, and certain indices of left ventricular contractility. It was concluded from the results of these experiments that infusion of a modified gelatin solution Fluigel prevented haemodynamic and metabolic changes produced by experimental hypovolaemia more effectively than infusion of Plasmagel.

Rapid slide technique with dextranomer beads for bacteriologic assessment of wounds in the elderly: comparison with quantitative biopsy method.

A rapid slide technique to provide quantitative bacteriologic assessment of wounds in elderly and debilitated patients is described. It involves the use of material from dextranomer-bead (Debrisan) wound dressings to replace tissue biopsy for deciding when a wound is ready for closure or when a specific therapy is no longer efficacious. In 27 patients an 81 percent correlation was demonstrated between the bacterial count as determined by the new method and that determined by the more complicated tissue biopsy.

Assessment of pharmacological effects on cerebral blood flow.

The effect of various drugs on hemispheric and regional cerebral blood flow (CBF) was investigated in a total of 410 patients. While a few drugs (midodrin, proxazole, vincamine, hexobendine, extract of ginkgo biloba, dextran and ouabain) were able to improve hemispheric CBF, only ephedrine combined with xanthines decreased CBF. For vincamine the dependency of the effect on certain plasma levels was established. Only ouabain of the tested cardiac glycosides effected CBF; their similar hemodynamic actions suggest here an influence of ouabain on cerebral vessels. For the evaluation of drug effects on rCBF the detection of heterogeneous responses is important. Such responses may be quantified by regression analysis. While intracerebral steal effects were observed only under certain circumstances, inverse cerebral steal phenomena may be caused by diverging actions of several drugs. If treatment is aimed at improvement of cerebral hemodynamics, only drugs with a demonstrated effect, at least on perfusion of ischemic regions, should be employed.