James Hoffman

The Protective Effect of β1–3D‐Glucan‐Derivatized Plastic Beads against Escherichia coli Infection in Mice

Pretreatment with β‐1, 3‐D‐glucan‐derivated plastic beads conferred strong protection against Escherichia coli infection in mice. The protective effect showed a dose‐response relationship to the amount of beads injected and was dependent on the time point of the injection relative to the infection with E. coli. A similar protection could be obtained in nude mice. Experiments with radioactively labelled bacteria as well as beads indicated a systemic effect of the beads. macrophages extracted from animals treated with glucan plastic beads appeared highly stimulated. This was also true of cells that did not contain beads and presumably therefore not glucan, which seems to indicate a soluble stimulatory factor.

Coupling of polysaccharides activated by means of chloroacetaldehyde dimethyl acetal to amines or proteins by reductive amination

Abstract A novel method has been developed for the coupling of modified polysaccharides to proteins or other amines. Chloroacetaldehyde dimethyl acetal has been used for the introduction of O -(2,2-dimethoxyethyl) groups into amylose, dextran, and a linear (1→3)-β- d -glucan. In amylose and the (1→3)-β- d -glucan, these groups were attached preponderantly at O-6 and in dextran at O-2. Mild treatment with acid then gave polysaccharide derivatives substituted with aldehyde groups which were coupled in good yields to proteins and other amines by reductive amination with sodium cyanoborohydride in aqueous solution at pH 7. An aminated (1→3)-β- d -glucan derivative that induced antitumor activity in mouse macrophages in vitro is reported.

Stimulatory Effect of Immobilized Glycans on Macrophages in Vitro

Mouse macrophages were cultured on chemically modified plastic dishes. On dishes covered with immobilized glycans, the macrophages were stimulated as judged by increased 14C‐glucosamine incorporation, increased cytostatic and cytolytic capacities and by morphology as seen by scanning electron microscopy. The corresponding soluble glycans did not have the capacity to stimulate macrophages as measured by these criteria. Plastic surfaces covered with polyethylenimine showed stimulation of the macrophages with regard to some of the parameters measured. These results may indicate that the stimulation is a multistep process and that, contrary to earlier findings, it is not a prerequisite for stimulation that the glycan be intracellular. The results support the idea that a fixed steric arrangement of glycans is necessary for the stimulation of macrophages in vitro.

In Vivo Activation of Mouse Macrophages with β‐l,3‐D‐Glucan‐Derivatized Plastic Beads

Macrophages obtained from animals treated withβ‐1.3‐D‐glucan‐derivatized plastic beads were greatly stimulated, as judged by morphology, esterase release, and cytostatic effect on L‐929 tumour cells in vitro. The pretreatment of mice with such heads conferred an apparent absolute local resistance to an otherwise lethal pneumococcal infection but had no effect on the growth of intrapcritoneal AA ascites sarcoma. Moreover, peritoneal cells from animals pretreated with glucan beads did nut protect the animals in a Winn assay.

Studies on the blood-anticoagulant activity of sulphated polysaccharides with different uronic acid content

The anticoagulant activities of sulphated alginic acids, amylose, cellulose, chitosan, curdlan, dextran, guaran, laminaran and locust bean gum have been studied. The alginic acids have been partially reduced and some of the neutral polysaccharides have been partially oxidised at C-6 of the glycopyranosyl residues. The activities of sulphated polymers containing different proportions of uronic acid and neutral sugar residues have been compared. n nThe results suggest that polysaccharides with an average of at least one sulphate group on each monomeric unit, a molecular weight higher than 10 000 and a high proportion of sulphated primary hydroxyl functions will display activity in the activated, partial thromboplastin time assay (APTT). Sulphated guaran and locust bean gum had the highest activities of the polymers investigated (70 IU/mg, heparin has 130-50 IU/mg). In the concentration range investigated, none of the sulphated polymers showed any significant activity in an anti-factor Xa assay.

The Effect of Splenectomy on Escherichia coli Sepsis and Its Treatment with Semisoluble Aminated Glucan

Rats were subjected to sham laparotomy or splenectomy and were challenged with either 0.2 X 10(9) Escherichia coli intravenously or 1 X 10(9) E. coli intraperitoneally. By means of quantitative blood culturing asplenic animals were shown to have a significantly impaired ability to clear the bacteria in both forms of challenge. Treatment with intraperitoneally injected semisoluble aminated glucan (SAG), known to have strong macrophage-stimulatory properties, compensated completely for the asplenic state. The substance protected against postsplenectomy sepsis both when given before and when given after removal of the spleen. This protective effect of SAG seemed to last at least 3 weeks.

Protection by Aminated Glucan in Experimental Endogenous Peritonitis

The effect of prophylactic intraperitoneal aminated glucan on the survival rate and formation of adhesions and abscesses was investigated in rats with acute and subacute peritonitis, respectively induced by cecal perforation and ileal ligation. A significantly reduced mortality was found in both forms of peritonitis. Pretreatment by aminated glucan also significantly reduced the number of abscesses and peritoneal adhesions. An about threefold increase in peritoneal macrophages in aminated glucan-treated rats compared to controls was noted. In vitro studies, using 32P-labelled Escherichia coli, demonstrated that peritoneal macrophages from aminated glucan-treated rats had an enhanced ability to engulf and degrade bacteria. Scanning electron microscopy showed that macrophages from aminated glucan-treated animals were highly spread with prominent ruffling and bacteria located intracellularly, as opposed to macrophages from controls which were rounded with bacteria on the cell surface.

Mechanisms of anticoagulant effects of some sulphated polysaccharides.

The anticoagulant activity of a partially reduced sulphated alginic acid, a partially reduced aminated and sulphated alginic acid and sulphated guaran have been studied. The anticoagulant activities in the APTT assay were 28, 39 and 70 IU/mg respectively. None showed any activity in anti-factor Xa assay. Studies on binding to Antithrombin III - Sepharose showed that sulphated guaran and a fraction of the aminated and sulphated alginic acid was bound, whereas no binding occurred with sulphated alginic acid. The inhibition of thrombin activity by these polysaccharides was studied in purified systems with or without added Antithrombin III, using both fibrinogen clotting and chromogenic peptide substrate assays. The two alginic acid preparations showed Antithrombin III-dependent inhibition of thrombin, whereas the sulphated guaran inhibits both by Antithrombin III-dependent and independent mechanisms.