The effect of phospholipase C on human blood platelets.

The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied. Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis. Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C. A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment. Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine. Sphingomyelin was not a substrate for the enzyme under the conditions used. The platelets contained no detectable endogenous phospholipase C activity. The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin. Total platelet factor 3 releasable by freezing and thawing was reduced. Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed. When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred. Sphingomyelinase alone gave no aggregation. The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens. The distribution of phospholipids in the platelet membrane is discussed.

The effect of phospholipase C on DNA synthesis, morphology and phospholipid content of isolated HeLa cell nuclei.

Isolated HeLa cell nuclei have been treated with purified phospholipase C (Bacillus cereus) and sphingomyelinase (Staphylococcus aureus). The phospholipids of untreated nuclei consisted of about 67% phosphatidylcholine, 23% phosphatidylethanolamine, 7% sphingomyelin, 2% phosphatidylserine and 1% phosphatidylinositol. Phospholipase C degraded 80-90% of the total phospholipids of the nuclei. Such nuclei seemed ultrastructurally intact, and had an average diameter and a protein loss during incubation which were not significantly different from those of controls. Their rate of DNA synthesis was only slightly reduced after treatment with phospholipase C alone and slightly more reduced when phospholipase C was used in combination with sphingomyelinase. This suggests that the polar head-groups of the nuclear phospholipids are of very limited importance in DNA synthesis. Since it has been reported that phospholipase C treatment releases nascent DNA from a membrane complex, the absence of a concommitant reduction in DNA synthesis may suggest that this complex is not necessary for the replication of DNA. Phospholipase C did not significantly influence the stability of the DNA product and gave only a slight inhibition of cytosol and nuclear DNA polymerases when tested with exogenous template.

The effect of phospholipase C on platelet factor 3 in human blood platelets.

Intact human platelets isolated by gel filtration have been treated with purified phospholipase C. The effect of the enzyme on available and total platelet factor 3 has been tested. The available procoagulant platelet factor 3 was very low. A further small reduction was observed after incubation with phospholipase C when the enzyme was washed away before testing. External attack on platelets by phospholipase C led to a marked inactivation of total platelet factor 3.

Trace amounts of ganglioside GM1 in human milk inhibit enterotoxins from Vibrio cholerae and Escherichia coli.

Gangliosides were isolated from human milk fat and purified by silica gel column chromatography and high performance liquid chromatography (HPLC). Low amounts of the ganglioside GM1, detected by high performance thin layer chromatography (HPTLC)-immunoassay, were found in all fractions with enterotoxin-inhibitory activity, while fractions without GM1 were inactive. It is concluded that GM1 is responsible for enterotoxin-inhibitory activity in the ganglioside fraction from human milk.

Non-antibody components in human milk inhibit Escherichia coli heat labile enterotoxin measured by an enzyme-linked immunosorbent assay.

Milk from 11 Norwegian women was fractionated by ammonium sulphate precipitation and column chromatography. The milk samples inhibited the binding of heat labile E. coli enterotoxin to antibodies coated on microtiter plates in an enzyme-linked immunosorbent assay. Inhibiting activity was not detected when the toxin was measured in an adrenal cell assay. The inhibiting activity was of a non-immunoglobulin nature with an apparent molecular weight of greater than 400 000 in gel filtration experiments.

Identification of low levels of heat labile enterotoxin in Escherichia coli from children with diarrhoea.

Out of 40 children with gastroenteritis and massive growth of E. coli in faeces, 7 yielded growth of E. coli strains producing heat-labile enterotoxin (LT), as identified by enzyme-linked immunosorbent assay (ELISA) using anticholera toxin coated plates or ganglioside coated plates. The toxin production in vitro was low, and decreased upon subculturing for 3 months in the laboratory. Only two of the strains were identified as LT-positive by the YI adrenal cell test. In addition, an LT-producing strain was isolated from an adult who had recently returned from Jordan. Sonication of the strains after subculturing released cell-bound LT. The clinical importance of such low toxin producing E. coli strains is not known.

Effect of fractions of Ethiopian And Norwegian colostrum on rotavirus and Escherichia coli heat-labile enterotoxin.

Samples of colostrum from both Ethiopian and Norwegian women contained antirotavirus activities of immunoglobulin and non-immunoglobulin nature. No significant differences in rotavirus immunoglobulin A or in rotavirus-inhibiting activity were found between samples from the two countries. The non-immunoglobulin inhibitory activity was trypsin sensitive and heat stable (100 degrees C for 10 min). Escherichia coli heat-labile enterotoxin antibodies were measured in the colostrum samples by enzyme-linked immunosorbent assay. No E. coli enterotoxin-specific immunoglobulin A was detected, possibly due to the high background caused by the nonspecific adsorption of immunoglobulin A to the enzyme-linked immunosorbent assay plates in the absence of toxin. A total of 5 of 15 Ethiopian colostrum samples and 0 of 11 Norwegian colostrum samples neutralized the effect of E. coli heat-labile enterotoxin on YI adrenal cells. Both the Ethiopian and the Norwegian colostrum samples contained a non-immunoglobulin enterotoxin-inhibitory activity when the toxin was measured by enzyme-linked immunosorbent assay. This inhibitory activity was not trypsin sensitive, and extraction by chloroform-methanol indicated that the inhibitor was of a lipid nature.

Non-immunoglobulin fraction of human milk protects rabbits against enterotoxin-induced intestinal fluid secretion.

Human milk was fractionated by ammonium sulphate precipitation and column chromatography. A milk fraction depleted of secretory immunoglobulin A and with an apparent molecular weight of greater than 400,000 inhibited fluid secretion induced by cholera toxin and Escherichia coli heat-labile toxin in rabbit ileal loops.

The effect of human milk fractions on rotavirus in relation to the secretory IgA content.

Human milk from healthy Norwegian women was fractionated by ammonium sulphate precipitation and gel filtration. The protein content, lactoferrin and secretory IgA were measured. Specific antirotavirus IgA, detected by indirect immunofluorescence was found in one out of five milk samples before fractionation, while a more concentrated immunoglobulin fraction from the other four milk samples contained such IgA. Before fractionation, 3 of 5 milk samples neutralized human rota-virus infection of LLC-MK2 cells, whereas concentrated, IgA-rich fractions of all 5 milk samples neutralized human rotavirus. Some fractions without detectable IgA also neutralized human rotavirus. This suggests that human milk contains rotavirus specific IgA as well as rotavirus neutralizing activity of non-immunoglobulin nature.

Chlamydial secretory IgA antibodies in human milk.

Colostrum from 10 of 30 randomly chosen women contained IgA antibodies to Chlamydia trachomatis as shown by an enzyme-linked immunosorbent assay and a single-antigen immunofluorescence test. Specific colostral IgA was present only in seropositive women. In addition, Chlamydial-specific IgA was also detected in milk from 5 of 6 women who were shown to harbour C. trachomatis in the lower genital tract during delivery. There was a close correlation between chlamydial-specific IgA and the chlamydial secretory immunoglobulin titres in colostrum and milk samples but not between chlamydial IgA titres and the total secretory IgA content. No agreement was observed between the specific IgA antibodies in milk and corresponding serum samples. It is suggested that chlamydial-specific IgA in milk is induced by genital infections.

Inhibition of enterotoxin from Escherichia coli and Vibrio cholerae by gangliosides from human milk.

Inhibitory activity of enterotoxin from Escherichia coli and Vibrio cholerae was associated with the ganglioside fraction of human milk. Both the milk fat and skim milk contained gangliosides that inhibited the toxins. The most purified milk fraction contained three glycolipid components, of which two migrated close to ganglioside GM1 on thin-layer chromatography plates. A component with a slightly different mobility from GM1 appeared to be associated with the inhibitory activity. Milk ganglioside fraction, derived from 2 ml of human milk, contained 1 to 4 micrograms of lipid-bound sialic acid and completely inhibited 0.1 micrograms of cholera toxin in rabbit intestinal loop experiments. It is suggested that human milk gangliosides, although present only in trace amounts, may be important in protecting infants against enterotoxin-induced diarrhea.

Human and bovine milk: comparison of ganglioside composition and enterotoxin-inhibitory activity.

Milk gangliosides inhibit Vibrio cholerae enterotoxin and Escherichia coli heat-labile enterotoxin. Human milk gangliosides showed considerably higher enterotoxin-inhibitory activity compared to bovine and formula milk gangliosides as measured in vitro by enzyme-linked immunosorbent assay and in vivo in rabbit small bowel loops. While gangliosides from less than 1 ml human milk inhibited 0.1 microgram choleratoxin in vitro and in vivo, five to 10 times higher amounts of bovine milk gangliosides were necessary to achieve similar results. Analysis of the ganglioside composition in human, bovine, and bovine milk-based formula milk showed that the ganglioside patterns in human and bovine milk differed markedly. The ganglioside patterns of bovine milk and formula milk appeared identical. In human or bovine milk, the total amount of gangliosides was 11 mg/liter compared to 6 mg/liter in formula milk. The predominating ganglioside in human milk, monosialoganglioside 3 (74% of total gangliosides), was only a minor component (3%) of bovine milk gangliosides. Disialoganglioside 3 represented 80% of bovine milk gangliosides compared to 25% of the human milk gangliosides. Trace amounts of monosialoganglioside 1 were detected in human, as well as in bovine, milk by a sensitive high performance thin-layer chromatography immunoassay. The monosialoganglioside 1 content in human milk was 10 times higher than in bovine milk. We conclude that the higher nonimmunoglobulin enterotoxin-inhibitory activity in human milk compared to bovine milk is associated with the differences in the ganglioside fraction.

Enrichment of human milk lymphocytes by discontinuous density gradient sedimentation.

A simple method is described for the enrichment of lymphocytes from human colostrum and early milk. Washed leucocytes from colostrum or milk were suspended in balanced salt solution containing 25% human serum and separated by discontinuous density gradient centrifugation over Lymphoprep (1.077 g/ml) and Nycodenz (1.070 g/ml). The cell fraction harvested from the interface between these two media contained a mean of 56% lymphocytes (9-92%), as compared to 5.6% (0-17%) in the unseparated samples, representing an average enrichment of 12.6 fold. The percentage yield of lymphocytes ranged from 70 to 100%. Enriched preparations of lymphocytes from human colostrum and early milk should prove valuable in studies of the distribution of lymphocyte subpopulations in breast milk and could lead to more definitive studies of their functions in vitro.

Plasma lactoferrin measured by an enzyme-linked immunosorbent assay (ELISA). Measurements on adult and infant plasma.

The lactoferrin content of human plasma has been measured by an enzyme-linked immunosorbent assay. In cord blood the level was 0.02-0.3 mg/l, corresponding to 3-44 X 10-(10) mol/l lactoferrin; in plasma 5 d post partum the level had not changed. In adults the level was 0.02-0.2 in 29 out of 30 plasma samples and above 1 mg/l in 1 sample. Similar results were obtained with EDTA, citrate or heparin as anti-coagulant.

Purification of human milk gangliosides by silica gel chromatography and analysis of trifluoroacetate derivatives by gas chromatography.

Two of the main gangliosides in human milk were purified by silica gel (230-400 mesh) column chromatography. The gangliosides were identified as GD3 and GM3 by methanolysis (2 M hydrochloric acid; 60 or 85 degrees C) and gas chromatography of trifluoroacetate derivatives on a fused-silica capillary column. The molar ratios of galactose, glucose and sialic acid were 1:1:2 and 1:1:1, respectively, and the sequence in both gangliosides comprised sialic acid--galactose--glucose--ceramide, as indicated by the time course of cleavage of individual components during methanolysis at 60 degrees C.