Halldor Thormar

Inactivation of enveloped viruses and killing of cells by fatty acids and monoglycerides.

Lipids in fresh human milk do not inactivate viruses but become antiviral after storage of the milk for a few days at 4 or 23 degrees C. The appearance of antiviral activity depends on active milk lipases and correlates with the release of free fatty acids in the milk. A number of fatty acids which are normal components of milk lipids were tested against enveloped viruses, i.e., vesicular stomatitis virus, herpes simplex virus, and visna virus, and against a nonenveloped virus, poliovirus. Short-chain and long-chain saturated fatty acids had no or a very small antiviral effect at the highest concentrations tested. Medium-chain saturated and long-chain unsaturated fatty acids, on the other hand, were all highly active against the enveloped viruses, although the fatty acid concentration required for maximum viral inactivation varied by as much as 20-fold. Monoglycerides of these fatty acids were also highly antiviral, in some instances at a concentration 10 times lower than that of the free fatty acids. None of the fatty acids inactivated poliovirus. Antiviral fatty acids were found to affect the viral envelope, causing leakage and at higher concentrations, a complete disintegration of the envelope and the viral particles. They also caused disintegration of the plasma membranes of tissue culture cells resulting in cell lysis and death. The same phenomenon occurred in cell cultures incubated with stored antiviral human milk. The antimicrobial effect of human milk lipids in vitro is therefore most likely caused by disintegration of cellular and viral membranes by fatty acids. Studies are needed to establish whether human milk lipids have an antimicrobial effect in the stomach and intestines of infants and to determine what role, if any, they play in protecting infants against gastrointestinal infections. Images

In Vitro Killing of Candida albicans by Fatty Acids and Monoglycerides

ABSTRACT The susceptibility of Candida albicans to several fatty acids and their 1-monoglycerides was tested with a short inactivation time, and ultrathin sections were studied by transmission electron microscopy (TEM) after treatment with capric acid. The results show that capric acid, a 10-carbon saturated fatty acid, causes the fastest and most effective killing of all three strains of C. albicans tested, leaving the cytoplasm disorganized and shrunken because of a disrupted or disintegrated plasma membrane. Lauric acid, a 12-carbon saturated fatty acid, was the most active at lower concentrations and after a longer incubation time.

Cultivation of visna virus in tissue culture

The virus of Visna, a slow, demyelinating leucoencephalitis of sheep, has been cultivated in tissue culture. The cells employed are derived from the chorioid plexus of sheep. The virus causes characteristic cytopathic changes in the culture, so that the method may be used to detect virus activity and measure the activity of virus containing material. Virus which had undergone 3, 11, and 12 passages in TC was injected intracerebrally into sheep and found to produce typical Visna lesions. Neutralizing antibody has been detected in sera from a certain proportion of sheep affected with Visna. The rate of virus multiplication in tissue culture after inocula of varying size has been studied. Small inocula tend to give rise to a mild infection which persists in the culture for long periods of time without destroying more than a certain proportion of the cells. The possible relationship between this relatively stable balance between virus and cells and the extraordinarily slow progress of Visna in the CNS of sheep is discussed.

Antimicrobial activity of lipids added to human milk, infant formula, and bovine milk.

Lipids previously shown to have antiviral and antibacterial activity in buffers were added to human milk, bovine milk, and infant formulas to determine whether increased protection from infection could be provided to infants as part of their diet. Fatty acids and monoglycerides with chain lengths varying from 8 to 12 carbons were found to be more strongly antiviral and antibacterial when added to milk and formula than long chain monoglycerides. Lipids added to milk and formula inactivated a number of pathogens including respiratory syncytial virus (RSV), herpes simplex virus type 1 (HSV-1), Haemophilus influenzae, and Group B streptococcus. The results presented in this study suggest that increased protection from infection may be provided to infants at mucosal surfaces, prior to the digestion of milk and formula triglycerides, by the addition of antimicrobial medium chain monoglycerides to an infants diet.

Killing of Gram-positive cocci by fatty acids and monoglycerides.

The susceptibilities of three Gram‐positive cocci to medium‐chain saturated and long‐chain unsaturated fatty acids and their one‐monoglycerides were studied. The bacteria were incubated with equal volumes of lipid solutions for 10 min. Lauric acid, palmitoleic acid and monocaprin reduced the number of CFU by 6.0 log10 or greater at 5 mM concentration for streptococci of group A (GAS) and group B (GBS). When further compared at lower concentrations and after longer incubation time monocaprin proved to be the most active. Capric acid showed the highest activity against Staphylococcus aureus at 10 mM. However, at lower concentrations monocaprin was the only lipid that showed significant activity against S. aureus. The mode of action of monocaprin against GBS was studied by a novel two‐color fluorescent assay of bacterial viability and by electron microscopy. The results indicate that the bacteria are killed by disintegration of the cell membrane by the lipid, leaving the bacterial cell wall intact. The highly lethal effect of monocaprin indicates that this lipid might be useful as a microbicidal agent for prevention and treatment of infections caused by these bacteria.

Bactericidal effects of fatty acids and monoglycerides on Helicobacter pylori

The susceptibility of Salmonella spp., Escherichia coli and Helicobacter pylori to fatty acids and monoglycerides was studied. None of the lipids showed significant antibacterial activity against Salmonella spp. and E. coli but eight of 12 lipids tested showed high activity against H. pylori, monocaprin and monolaurin being the most active. The high activity of monoglycerides against H. pylori suggests that they may be useful as active ingredients in pharmaceutical formulations.

Antiviral and antibacterial lipids in human milk and infant formula feeds.

Human milk and two infant formula feeds were tested for antiviral and antibacterial activity before being given to 21 low birthweight (LBW) infants; neither was present. When samples were aspirated from the stomachs of the infants within one to three hours of feeding, however, they reduced titres of enveloped virus and also killed both Staphylococcus epidermidis and Escherichia coli. The lipid fraction of the gastric aspirate from an infant who had been given human milk as well as those from four infants who had been given a conventional LBW infant formula feed, showed antiviral and antibacterial activities at least equal to the activities of the unfractionated aspirates. There was no consistent difference in antiviral or antibacterial activity of either the stomach aspirates or the lipid fractions of these aspirates between infants given human milk and those given formula feeds. The antiviral and antibacterial activities of the gastric aspirates seem to result from intragastric production of monoglycerides and fatty acids from the triglyceride content of the ingested feeds.

Stable Concentrated Emulsions of the 1-Monoglyceride of Capric Acid (Monocaprin) with Microbicidal Activities against the Food-Borne Bacteria Campylobacter jejuni, Salmonella spp., and Escherichia coli

ABSTRACT Of 11 fatty acids and monoglycerides tested against Campylobacter jejuni, the 1-monoglyceride of capric acid (monocaprin) was the most active in killing the bacterium. Various monocaprin-in-water emulsions were prepared which were stable after storage at room temperature for many months and which retained their microbicidal activity. A procedure was developed to manufacture up to 500 ml of 200 mM preconcentrated emulsions of monocaprin in tap water. The concentrates were clear and remained stable for at least 12 months. They were active against C. jejuni upon 160- to 200-fold dilution in tap water and caused a >6- to 7-log10 reduction in viable bacterial count in 1 min at room temperature. The addition of 0.8% Tween 40 to the concentrates as an emulsifying agent did not change the microbicidal activity. Emulsions of monocaprin killed a variety of Campylobacter isolates from humans and poultry and also killed strains of Campylobacter coli and Campylobacter lari, indicating a broad anticampylobacter activity. Emulsions of 1.25 mM monocaprin in citrate-lactate buffer at pH 4 to 5 caused a >6- to 7-log10 reduction in viable bacterial counts of Salmonella spp. and Escherichia coli in 10 min. C. jejuni was also more susceptible to monocaprin emulsions at low pH. The addition of 5 and 10 mM monocaprin emulsions to Campylobacter-spiked chicken feed significantly reduced the bacterial contamination. These results are discussed in view of the possible utilization of monocaprin emulsions in controlling the spread of food-borne bacteria from poultry to humans.

The Role of Milk-Derived Antimicrobial Lipids as Antiviral and Antibacterial Agents

Milk lipids are not only nutrients but also non-immunoglobulin protective factors1–3. The lipids in human milk do not initially have antimicrobial activity, but become antiviral2, antibacteria14,5 and antiprotozoal6 in vitro following storage and in vivo following digestion in the gastrointestinal tract of the milk-fed infant2,5,7. Microbial killing by milk lipids is due primarily to free fatty acids (FFAs) and monoglycerides (MGs) released from milk triglycerides by lipases and can be duplicated using purified FFAs and MGs8,9.

The Emergence of Antigenic Variants is a Rare Event in Long-term Visna Virus Infection in vivo

Six sheep persistently infected with visna virus were studied for 4 1/2 to 5 3/4 years until they became ill. Virus was isolated at intervals from peripheral blood leukocytes and cerebrospinal fluid (CSF), and at the time of sacrifice from various parts of the brain and the lungs. Both brain and lungs showed lesions typical of advanced visna/maedi. All the sheep formed antibodies in sera and CSF. Virus isolates from each sheep were tested in neutralization tests against sera and CSF collected from the same animal. In one sheep all isolates were found to be identical to the inoculated virus by this test. In each of the other sheep an antigenic variant emerged from 1 to 3 years after inoculation and remained in circulation even after the formation of autologous antibodies. In one case a variant was isolated from the lungs, whereas in all cases the virus isolated from the brain was identical to the inoculated virus. The results show that antigenic variants are rare in visna and do not seem to have a role in the pathogenesis of the disease.