High performance gel filtration using monodisperse highly cross-linked agarose as a one-step system for mucin purification.

High performance gel filtration on monodisperse cross-linked agarose (Superose 6) has been assessed as a system for purification of mucus glycoproteins. Comparison with the conventional two-step purification of mucus glycoprotein by Sepharose CL4B gel filtration followed by caesium chloride density gradient centrifugation shows that purification by high performance gel filtration is at least as thorough, yielding mucin that is free from non-mucin glycoproteins as defined by buoyant density, mobility on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and absence of Concanavalin A binding (mannose-containing) material. This technique allows mucus glycoprotein to be purified from lyophilized crude mucin in 120 min compared with approximately 72 h using the conventional techniques. This makes the comparative study of mucus glycoprotein changes in disease states much more feasible.

Ion exchange chromatography of purified colonic mucus glycoproteins in inflammatory bowel disease: absence of a selective subclass defect.

Previous reports of a selective mucin subclass defect in ulcerative colitis have been reassessed using high performance chromatography (Superose 6 and Mono Q) for mucin purification and fractionation coupled with analysis of the fractions obtained using a combination of enzyme linked lectin and mucin antibody assays. Mucin samples purified from snap frozen rectal biopsy specimens obtained from patients with ulcerative colitis (n = 12), Crohn's disease (n = 5), and non-inflammatory bowel disease control subjects (n = 9) were subject to ion exchange chromatography using a continuous 0-0.35 mol/l NaCl salt gradient with a final 2.5 mol/l NaCl step. In all samples the major proportion (mean (SD) 86.7 (8.9)%) of the mucin detectable by wheat germ agglutinin binding eluted between 0.15 and 0.35 mol/l NaCl with no significant difference in elution profile between ulcerative colitis and control subjects. Significant elution of glycoprotein at less than 0.15 mol/l NaCl did occur, however, when a lower molecular weight mucin containing fraction which contained concanavalin A positive (glucose or mannose containing) material was analysed similarly. Similar ion exchange profiles were obtained when (3H)N-acetylglucosamine labelled mucins were studied after tissue culture of rectal biopsy specimens. No significant alteration in the ion exchange profile of purified mucins in ulcerative colitis has been shown in these studies. It is possible that the previously reported relative depletion of mucin subclass IV (eluting with 0.20 mol/l NaCl) may simply have reflected mucin depletion.

Characterisation of a monoclonal antibody capable of neutralising the haemorrhagic activity of West African Echis carinatus (carpet viper) venom.

A murine monoclonal antibody (SV-1/F10) was highly specific immunologically for West African E.carinatus venom both by ELISA and immunoblotting. In cross-protection tests in vivo, it possessed strong antihaemorrhagic activity. This IgGl monoclonal antibody recognised an epitope present in a protein band of 124,000 mol. wt using immunoblotting of non-reduced Nigerian and Ghanaian E.carinatus venoms, as well as in a second protein band of 105,000 mol. wt in Ghanaian E.carinatus venom. The SV-1/F10 monoclonal antibody is of potential use for the isolation of West African E.carinatus venom haemorrhagin from whole venom, allowing the possibility of elucidation of the mechanism of its actions as well as its interactions with antibody.

A study of the pathogenesis of local skin necrosis induced by Naja nigricollis (spitting cobra) venom using simple histological staining techniques.

The course of degeneration and regeneration of mouse skin following intradermal injection of spitting cobra (N. nigricollis) venom was investigated using simple histological staining techniques. Early changes observed were vascular congestion, oedema and degeneration of the skeletal muscle cell layer (panniculus carnosus) in the area local to the injection site. Fuchsin staining of degenerative muscle cells in haematoxylin-basic fuchsin-picric acid (HBFP) stained sections appeared long before any detectable change was obvious in serial sections stained with haematoxylin and eosin (H and E). Positive reactions were apparent as early as 5 min after venom injection. Infiltration by a mixed population of cells was observed 1 hr after injection. Twenty four hours after injection large numbers of neutrophils, eosinophils, macrophages and mast cells were observed in and around the necrotic tissue and there was evidence of fibrin deposition in the blood vessels. A newly formed epithelium and muscle cell layer was visible after 18 days. The destroyed tissue tended to slough, leaving behind granular scar tissue. Dense granular scar tissue had generally replaced the damaged tissue after 28 days. It appears likely that myonecrosis was due to the direct myolytic action of one or more venom components, since signs of damage were apparent as early as 5 min after injection. However, the venom also caused fibrin deposition, suggesting possible thrombus formation later, and so it is also probable that some contribution to the degenerative state may be attributable to ischaemia brought about by a diminished blood supply.

Biological properties of the venom of the red-necked keel-back snake (Rhabdophis subminiatus).

Studies in mice on the biological activity of the venom of the red-necked keel-back snake, Rhabdophis subminiatus, showed that the venom contained a potent Factor X activator and had intense defibrinogenating activity; the overall proteolytic activity of the venom was low, and this correlated well with its negligible fibrinogenolytic and fibrinolytic activities. Only one antivenom tested was shown to have weak neutralizing activity against the venom in mice. This species of snake has recently been added to the schedule of the Dangerous Wild Animals Act, 1976.

An investigation of venom secretion by the venom gland cells of the carpet viper (Echis carinatus).

The indirect immunofluorescent antibody technique was applied to the study of Echis carinatus pyramidum venom antigens in venom gland tissue using semi-thin frozen sections. A total of four rabbit antisera, two monoclonal antibodies active against E. carinatus venom, two monoclonal antibodies active against the rodent malaria parasite, Plasmodium chabaudi, and two monoclonal antibodies active against the human malaria parasite, Plasmodium falciparum, were investigated. The results of this study suggest that each secretory cell within the main part of the gland produces all the venom constituents. The resultant venom is therefore considered to be produced as a single package by each individual secretory cell. The different constituents of the venom studied are not produced at the same time or at the same rate throughout the secretory cycle, some being produced at the beginning and others at a later stage.

Liposomal immunisation against snake venoms.

A method is described which produces a high, permanent antibody response following a single injection of venom. Animals (mice, rabbits, sheep) were given intravenous, subcutaneous or orally administered Nigerian Echis carinatus (carpet viper) venom which had been incorporated into sphingomyelin-cholesterol liposomes whose membranes had been stabilized by cross-linking adjacent molecules of sphingomyelin using osmium tetroxide. Before use the preparations were thoroughly dialysed to remove any unbound osmium tetroxide. Antibody levels were estimated using enzyme immunoassay. Venom treated in this way and administered i.v. or s.c. produced a powerful, sustained and protective antibody response lasting for the lifetime of a mouse. We also report the development of significant antibody responses after oral administration of liposome-entrapped but not free venom.

Protective effect of snake venom antibodies in sera of previous snake bite victims.

Three out of seven serum samples from Ecuadorian Indians had very high antibody levels against Bothrops nasutus venom, and IgG concentrates of these sera effectively neutralized this venom when subsequently injected into mice. It is concluded that the high mortality rate among these Indians would be even higher if there were not such natural protection. Further research into active immunization of humans should be encouraged.