Dense Albumin Research Articles

A preliminary electrochemical study of crosslinked albumin and collagen membranes as diffusion barriers for potentially degradable chronic wound biosensors

AbstractThe biochemical monitoring of chronic wounds may help to tailor therapy with better clinical outcomes. Wound surface sensors would be appropriate for this, but their adhesion to the wound is likely to lead to tissue damage on retrieval. Degradable sensors offer a practical solution to this, but this would necessitate external degradable diffusion control membranes. In this study, we evaluated glutaraldehyde crosslinked albumen and collagen as potentially biodegradable barrier membranes. The diffusional transport of H2O2, ascorbate, and glucose was studied electrochemically. Albumin proved more selective for H2O2 against ascorbate, possibly due to its net negative charge. The thick (500–700 μm) membranes produced led to long response times (half‐life [t0.5], 500–700 s) but had the advantage of stabilizing the response in unstirred solution. This was possibly because the thicker membrane on the electrode surface moved the developing diffusion layer in solution further away from the electrode surface. A dense albumin layer was produced using sebacoyl chloride as a crosslinker which was impermeable to solute and could be used in future as a degradable sensor base. Also, non‐toxic poly(2,3‐dihydrothieno‐1,4‐dioxin)‐poly(styrenesulfonate) dispersed in crosslinked albumin furnished a conducting membrane able to function as a working electrode. Thus, crosslinked proteins offer scope for diverse membrane properties that might be useful for future, degradable wound monitoring sensors.

Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation

The most potent antigen presenting cell present in peripheral blood, lymphoid and non-lymphoid tissue is the dendritic cell (DC). The study of human DC has been restricted by their low frequency in the tissues and the lack of a truly DC specific surface marker to assist in identification and isolation. Standard techniques for the isolation of blood DC generally employ a period of in vitro culture followed by flotation on dense albumin gradients, or more recently, discontinuous gradients of metrizamide. Dense albumin gradients are time consuming to prepare, giving low and variable yields of DC. Metrizamide is more convenient, although exposure of monocytes to metrizamide can decrease the expression of CD14 and alter the accessory cell properties of antigen presenting cells. Here we demonstrate that Nycodenz gradient centrifugation of 16 h cultured, T lymphocyte depleted, peripheral blood mononuclear cells (PBMC) reliably yields a population of low density cells that is highly enriched for DC. Most B and residual T lymphocytes are depleted and NK cell numbers are reduced two-fold from the interface cell population. The high density pellet fraction exhibits very little allostimulatory activity, indicating that few DC pass into the pellet. The low density fraction contains a significant population (20 ± 5 (SD)%, n = 8) of cells which fail to stain for the lineage markers CD3, CD11b, CD14, CD16, CD19 and CD57. Nycodenz exhibits low toxicity, does not alter the allostimulatory activity of antigen presenting cells, and is therefore ideal for the isolation of cultured DC.

A technique for the isolation of highly viable pancreatic B-cells from ob/ob mice.

A method has been developed to prepare free islet cells in suspension from adult ob/ob-mice. About 200 collagenase-isolated pancreatic islets were pooled in 4 ml of calcium-free Krebs-Ringer-HEPES buffer supplemented with 1 mM EGTA and 10 micrograms/ml DNAase. The islets were gently shaken in a water-bath for 10 min at 30 degrees C. Then, the cell suspension was filtered through a nylon screen and centrifuged through ice-cold, dense albumin. The isolated cells, of which more than 99% were B-cells, appeared well preserved both in light- and electron-microscopy. Out of the isolated cells, 7.1 +/- 0.5% took up Evans Blue and were thus considered non-viable.

A Comparison of Murine Epidermal Langerhans Cells with Spleen Dendritic Cells

To establish if epidermal Langerhans cells (LC) are related to spleen dendritic cells, we have considered the morphology, phenotype, and function of the 2 cell types in culture. Cultured LC could be partially enriched (up to 50%) on the basis of 2 simple physical properties: nonadherence to plastic, and low buoyant density in dense albumin columns. The morphology of cultured LC and spleen dendritic cells were similar. In particular both cell types had many cell processes and/or veils, and cultured LC lost their distinguishing Birbeck granules. Freshly isolated LC exhibited nonspecific esterase and ATPase, as well as the F4/80 (alpha-macrophage) and 2.4G2 (alpha-Fc receptor) antigens. However all these traits were lost in culture, while Ia and Mac-1 antigens persisted. As a result, the cytochemical and antigenic phenotype of LC became similar to spleen dendritic cells. The one exception was that LC lacked the 33D1 dendritic cell antigen. The function of LC at first differed from spleen dendritic cells in that fresh LC were weak stimulators of T cell proliferation in the mixed leukocyte reaction and in sodium periodate-induced mitogenesis. However, stimulatory activity per cell increased at least 30 fold in culture so that by 2-3 days, LC were 3-10 times more potent than dendritic cells. Maturation of LC function was radioresistant and was accompanied by a small increase in cell surface Ia antigens. Although LC have been likened both to lymphoid dendritic cells and to macrophages, our data suggest a different conclusion. LC seem to be dendritic cell precursors and are immunologically immature. Possibly, lymphoid dendritic cells are in general derived from substantial pools of precursors in nonlymphoid tissues, such as epidermal LC.

Human dendritic cells. Enrichment and characterization from peripheral blood.

Previous studies demonstrated that lymphoid tissues of mice and rats contain small numbers (less than 1 percent of nucleated cells) of dendritic cells (DC) with special cytologic, surface, and functional properties. We show here that similar DC represent 0.1-0.5 percent of human peripheral blood mononuclear cells. DC can be enriched to 20-60 percent purity by a multistep procedure analogous to that used in mice. Adherent peripheral blood mononuclear cells are cultured overnight, and the released cells are depleted of monocytes and B cells by readherence to plastic, rosetting with erythrocytes coated with anti-human IgG, and centrifugation in dense albumin columns. Enriched DC have similar cytologic features to rodent DC by light and electron microscopy. DC express HLA, and HLA-DR and the leukocyte-common antigens. They lack phagocytic capacity, receptors for antibody-coated and neuraminidase-treated erythrocytes, surface and intracellular Ig, esterase, peroxidase, and azurophilic granules. DC do not react with several monoclonal antibodies directed to phagocytes (OKM 1, "mac-1," 63D3, and 61D3) and T cells (OKT 3, 6, 8). Unlike the mouse, human DC express complement receptors. When maintained in culture for 4 d, human DC did not give rise to either B cells or monocytes. Therefore, DC identified by cytologic criteria are distinct from other leukocytes. Enriched populations of DC have been compared to fractions enriched in monocytes, B cells, and T cells in three functional assays: stimulation of the primary allogeneic mixed leukocyte reaction, stimulation of the primary syngeneic MLR, and accessory function for the proliferation of periodate- modified T cells. In each case, the DC fraction was 10-fold or more active than other cell fractions. We conclude that DC circulate in man, and represent the principal cell type required for the initiation of several immune responses.

Hydrolytic enzymes of human blood monocytes and neutrophils with special reference to lysozyme (muramidase).

In order to elucidate the cause of markedly increased serum and urine lysozyme (muramidase) in patients with monocytic leukemia on the cellular basis, monocytes and neutrophils were compared on their hydrolytic enzyme contents and their characteristics. Both types of leukocytes were separated from normal human blood or from patients with monocytic leukemia by means of floatation in dense albumin, utilizing the difference in the specific gravity of both cell types. Equivalent lysozyme and acid phosphatase activities were found to be present in monocytes and neutrophils and very low or negligible activities of alkaline phosphatase in monocytes. The subcellular distributions of these enzymes were comparable in the pattern and specific activity in both cell populations and found to be associated with granules. The phagocytic rate and the readiness of release of lysozyme into the extracellular medium during phagocytosis were similar in both cell populations. Leukemic leukocytes, neutrophils and monocytes, showed lower hydrolase activities than normal counterparts, especially alkaline phosphatase being markedly suppressed in leukemic cells. The possibility was discussed that the mechanism for elevated lysozyme in serum and urine of patients with monocytic leukemia may be accounted for by the developmental potential of monocytes in extravascular sites into macrophages with marked increase in lysosomal enzymes, the relatively ready release of lysozyme from cells, and a stable nature of lysozyme.

The isolation and selected properties of blood monocytes.

A technique is described for the quantitative recovery of monocytes from horse blood by means of flotation on dense albumin solutions. Monocytes are concentrated in a surface pellicle along with a few lymphocytes which are then removed when the monocytes adhere to a glass surface. The in vitro cultivation of homogeneous populations of monocytes results in an increase in (a) cell size, (b) number of mitochondria, and (c) phase-dense granules of the centrosphere. The phase-dense granules are osmiophilic and acid phosphatase positive. Quantitative biochemical analysis during cultivation have revealed increased levels of cytochrome oxidase, acid phosphatase, arylsulfatase, and BPN hydrolase. In addition, glucose utilization and lactic acid production are stimulated under the same conditions. The uptake of both bacteria and colloidal gold is stimulated during in vitro cultivation. The phagocytic activity of cultured monocytes may be enhanced by a purified bacterial lipopolysaccharide. These data are consistant with the in vitro maturation of monocytes to macrophages, a cell with greater metabolic and functional potentional.