Evaluation of WLBU2 peptide and 3-O-octyl-sn-glycerol lipid as active ingredients for a topical microbicide formulation targeting Chlamydia trachomatis.

Topical microbicides for prevention of sexually transmitted diseases (STDs) would be especially useful for women who are not able to persuade their partner(s) to take precautions. Many topical microbicides are in various stages of development, based on a variety of active ingredients. We investigated the in vitro activity of an engineered antimicrobial peptide (WLBU2) and a lipid (3-O-octyl-sn-glycerol [3-OG]) which could potentially be used as active ingredients in such a product. Using commercially available cytotoxicity reagents [Alamar Blue, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH)], we first determined the toxicity of WLBU2 and 3-OG to the host cells in our assay procedure and excluded toxic concentrations from further testing. To determine activity against Chlamydia trachomatis, we used an assay previously developed by our laboratory in which chlamydial elementary bodies (EBs) were exposed to microbicides prior to contact with epithelial cells: the minimum (microbi)cidal concentration (MCC) assay. To further simulate conditions of transmission, we carried out the same assay in the presence of a simulated vaginal fluid, a simulated seminal fluid, human serum albumin, and a range of pH values which might be found in the human vagina at the time of exposure. Last, we tested WLBU2 and 3-OG in combination to determine if adding them together resulted in synergistic activity. We found that WLBU2 and 3-OG both have excellent activity in vitro against C. trachomatis and significantly more activity when added together. The simulated fluids reduced activity, but the synergy seen is good evidence that they would be effective when combined in a microbicide formulation.

Development of a topical vaginal microbicide: lessons learned from human milk.

Vaccines are not presently available to prevent adherence and transmission of many common pathogens at mucosal surfaces. As a result, sexually transmitted diseases were one of the most commonly reported infections in the US in 1999. New methods are needed to reduce the spread of mucosal infections. Providing nonspecific protective factors, such as lipids and retinoids found in human milk to mucosal surfaces could reduce mucosal infection caused by viruses, e.g., herpes simplex virus-1 (HSV-1) and bacteria, e.g., Pseudomonas aeruginosa. Human milk lipids enzymatically modified to produce monoglycerides were antimicrobial and inactivated enveloped viruses, as well as gram-positive and gram-negative bacteria. Enveloped viruses were inactivated in seconds following contact with antimicrobial lipids, and P. aeruginosa infectivity was reduced by 99.9% after 2 hours. Transmission of pathogens at mucosal surfaces can also be prevented using retinoids that inhibit viral replication. In a human embryonic intestinal cell line the retinoic acid (RA) derivatives all-trans-RA and 9-cis-RA (10 microg/mL) decreased the production of HSV-1 and Echo-6 viruses by 1-2 log10 over a 48-hour period. In addition, all-trans-RA inhibited HSV-1 replication in Vero cells as effectively as interferon beta, reducing viral production by 2.5log10. These studies indicate that lipids and retinoids could be part of a topical microbicide to prevent mucosal infections.

The antimicrobial function of milk lipids.

Milk lipids serve not only as nutrients but as antimicrobial agents that constitute a defense system against microbial infections that occur at mucosal surfaces. The lipid fraction of milk develops antimicrobial activity in the gastrointestinal tract of suckling neonates as a result of lipolytic activity which converts milk triglycerides to antimicrobial fatty acids and monoglycerides. Antimicrobial milk lipids may be particularly important in protecting infants with an inadequate secretory immune response from infection. The lipid-dependent antimicrobial activity of milk is due to medium-chain saturated and long-chain unsaturated fatty acids and their respective monoglycerides released by lipases in the gastrointestinal tract. The antimicrobial activity of fatty acids and monoglycerides is additive and consequently it is their combined concentration that determines the lipid-dependent antimicrobial activity of milk. Microbial inactivation occurs rapidly by membrane destabilization. The antimicrobial activity of milk lipids can be duplicated using purified fatty acids and monoglycerides. It should be possible, therefore, to supplement banked human milk to provide lipid-dependent antimicrobial activity from the moment of ingestion (Schanler et al., 1986). This could reduce the risk of viral transmission from mother to infant through milk. Milk lipids also could be adapted for use at mucosal surfaces other than those in the gastrointestinal tract to reduce vertical transmission of pathogens during birth.

Retinoic acid reduces the yield of herpes simplex virus in Vero cells and alters the N-glycosylation of viral envelope proteins.

Treatment of Vero cells with all-trans-retinoic acid (RA) decreased the production of infectious herpes simplex virus (HSV) by 1000-10000-fold when compared with control cultures. Levels of total HSV envelope glycoproteins gB, gC and gD produced following RA treatment, were comparable with those found in control cultures. Following 24 h of RA treatment, lower molecular weight variants of gB, gC and gD were produced in addition to the typical molecular mass of each protein found in control samples. Between 24 and 48 h of RA treatment, the proportion of the lower molecular mass variants increased. When control and RA treated samples were incubated with peptide N-glycosidase F (PNGase F), which removes N-glycosylated sugars, the molecular weights of the respective gB, gC and gD proteins produced were comparable in both the groups, indicating that RA did not alter the primary sequence of viral proteins during protein synthesis or increase viral protein proteolysis. RA treatment increased [3H]mannose incorporation into glycoproteins in HSV infected cells but did not change [3H]glucosamine incorporation. We conclude that RA treatment does not reduce the synthesis of three major viral envelope glycoproteins but alters their N-glycosylation and postulate that the inhibitory effect of RA is related to its action on N-glycosylation.

Killing of Chlamydia trachomatis by novel antimicrobial lipids adapted from compounds in human breast milk.

The development of new methods for prevention of sexually transmitted Chlamydia trachomatis infection is a top public health priority. Topical self-administered vaginal microbicides represent one such approach in which the organism is eradicated at the time of initial exposure. To this end, we examined the activity of five synthetic lipids adapted from naturally occurring compounds found in human breast milk. C. trachomatis serovar D or F elementary bodies were added to serial dilutions of the lipids and incubated for various times. Aliquots were then cultured in monolayers of McCoy cells, and inclusions were counted. A 7.5 mM concentration of 2-O-octyl-sn-glycerol completely prevented growth of C. trachomatis after 120 min of contact with the organism. The remaining lipids, 1-O-octyl-, 1-O-heptyl-, 2-O-hexyl-, and 1-O-hexyl-sn-glycerol, showed less activity. On electron microscopic examination, the lipids were shown to have disrupted the chlamydial inner membrane, allowing leakage of the cytoplasmic contents from the cell. Lipid activity was unaffected by the presence of 10% human blood or alterations in pH from 4.0 to 8.0, conditions reflecting those sometimes found in the vagina. Our results suggest that these lipids, especially 2-O-octyl-sn-glycerol, may be effective as topical microbicides in preventing the transmission of C. trachomatis. Further efficacy and toxicity studies with these lipids and assessment of their activity against other sexually transmitted disease pathogens are in progress.

Inhibition of herpes simplex virus replication by retinoic acid.

The retinoic acid (RA) isomers all-trans-RA, 9-cis-RA and 13-cis-RA as well as other retinoids were tested for their ability to reduce the yield of herpes simplex virus-1 (HSV-1). RA isomers reduced HSV-1 replication whereas the other retinoids, retinol, retinal, beta-carotene and amide derivatives of RA were not inhibitory. All-trans-RA reduced the yield of HSV-1 by 100-fold at 5 micrograms/ml but 9-cis-RA and 13-cis-RA reduced viral replication by 10-fold. At a concentration of 10 micrograms/ml all-trans-RA and 9-cis-RA reduced virus yield by 1000-fold while 13-cis-RA decreased HSV-1 production by 100-fold. RA isomers at a concentration of 10 micrograms/ml were not cytotoxic for the Vero cells used in these studies. Immunofluorescence studies showed that all-trans-RA treated cell cultures exhibited small foci of virus specific immunostaining while untreated cultures displayed intense HSV-1 immunoreactivity in virtually the entire cell population. RA-dependent inhibition of HSV-1 replication required the presence of RA with the virus. HSV-1 replication proceeded when RA was removed from infected cells. Treatment of cell cultures with RA did not induce gene expression for type-1 interferon (IFN) or for the type-1 IFN inducible genes studied suggesting that RA inhibition of HSV-1 replication is not mediated by IFN. These studies have established the ability of RA to reduce the replication of HSV-1 in vitro.

Inactivation of visna virus and other enveloped viruses by free fatty acids and monoglycerides.

Human milk was found to become antiviral during storage at 4 degrees C because of the release of fatty acids by lipolysis. The stored milk caused more than a 10,000-fold inactivation of visna virus during incubation at 37 degrees C for 30 minutes. Medium-chain saturated and long-chain unsaturated fatty acids inactivated visna virus and other enveloped viruses causing more than a 3000-fold to 10,000-fold reduction in virus titer. 1-Monoglycerides and ethers of medium-chain fatty acids were more antiviral than the corresponding free fatty acids. 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. Lipids commonly found in natural products could possibly be used as antiviral agents against enveloped viruses.

Inactivation of enveloped viruses in human bodily fluids by purified lipids.

Antimicrobial lipids are found in mucosal secretions and are one of a number of nonimmunologic and nonspecific protective factors found at mucosal surfaces. Lipids can inactivate enveloped viruses, bacteria, fungi, and protozoa. Lipid-dependent antimicrobial activity at mucosal surfaces is due to certain monoglycerides and fatty acids that are released from triglycerides by lipolytic activity. Medium chain length antiviral lipids can be added to human blood products that contain HIV-1 and HIV-2 and reduce the cell-free virus concentration by as much as 11 log10 TCID50/ml. The presence of lipids does not interfere with most clinical assays performed on human blood samples. Antimicrobial lipids can disrupt cell membranes and therefore lyse leukocytes which potentially carry virus. Genital mucosal epithelial cells should be protected from damage by the mucous layer. Preliminary studies indicate that lipids decrease sperm motility and viability suggesting that lipids may potentially be used as combination spermicidal and virucidal agents.

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.

Distribution of IgG subclasses in human colostrum and milk.

Immunoglobulin G (IgG) subclasses in human colostrum and milk were quantitated using mouse monoclonal antibodies specific for human IgG subclasses and an enzyme-linked immunosorbent assay (ELISA). The percentage of IgG1 was significantly increased and that of IgG2 was decreased in both colostrum and milk relative to the percentage distribution reported in healthy adult and maternal sera. Percentages of IgG3 and IgG4 in colostrum, milk and sera were similar. All IgG subclass concentrations decreased by 90% during the first week of lactation and remained constant thereafter. The possible antiviral role of high levels of IgG1 in colostrum and milk is discussed.

Cerebral glycine content and phosphoserine phosphatase activity in hyperaminoacidemias.

Chronic hyperphenylalaninemia maintained with the aid of a suppressor of phenylalanine hydroxylase, alpha-methylphenylalanine, increases the glycine concentration and the phosphoserine phosphatase activity of the developing rat brain but not that of liver or kidney. Similar increases occur after daily injections with large doses of phenylalanine alone, while tyrosine, isoleucine, alanine, proline, and threonine, were without effect. Treatment with methionine, which increases the phosphoserine phosphatase activity of the brain and lowered that of liver and kidney, left the cerebral glycine level unchanged. When varying the degrees of gestational or early postnatal hyperphenylalaninemia, a significant linear correlation was found between the developing brains' phosphoserine phosphatase and glycine concentration. Observations on the uptake of injected glycine and its decline further indicate that coordinated rises in the brain's phosphoserine phosphatase and glycine content associated with experimental hyperphenylalaninemia denote a direct impact of phenylalanine on the intracellular pathway of glycine synthesis in immature animals.

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.

Membrane-disruptive effect of human milk: inactivation of enveloped viruses.

Fresh human milk treated with antibody to secretory IgA had no effect on viral infectivity but became antiviral after 1 hr in the stomachs of suckling infants. Antiviral activity also appeared in fresh milk stored at 4 C for at least two days. The antiviral activity, which reduced titers of virus by as much as 10,000-fold, only affected enveloped viruses and was localized in the milk lipid fraction. Its appearance in stored milk was apparently due to fatty acids released by the activity of milk lipases, particularly lipoprotein lipase. Antiviral activity in the infants' stomach, however, most likely resulted from the activity of gastric and lingual lipases on milk triglycerides and caused the release of antiviral fatty acids. Milk and stomach contents that were antiviral also lysed cultured cells by disruption of their plasma membrane. Cell lysis was also caused by purified linoleic acid, which is a normal constituent of human milk triglycerides.

Significance of growth modulators in human milk.

Human milk contains growth modulators of potential clinical importance to the neonate. Evidence from animal and cell culture models as well as ancillary evidence about their stability and the way some of them are processed for secretion suggest they are probably physiologically significant. Their presence in human milk does not by itself establish their importance; however, it is useful to explore their potential functional roles. Growth modulators present in human milk include: EGF, NGF, certain enzymes, and taurine.

Sulfhydryl oxidase in human milk: stability of milk enzymes in the gastrointestinal tract.

Sulfhydryl oxidase (SOX) is present in human milk and in milk from all species that have been studied. The pH optimum of human milk SOX is in the neutral range between 7.0 and 7.5. Human milk SOX is stable in an acid environment: 50% of its activity remains after 1 h at pH 2.5. Acid stability is also characteristic of gamma-glutamyltranspeptidase, another membrane-bound enzyme in skim milk. SOX is resistant to pepsin (4000 U/ml), trypsin (50 micrograms/ml), chymotrypsin (200 micrograms/ml), and to trypsin plus chymotrypsin (25 micrograms each/ml). Milk SOX activity has been detected in the stomach and proximal small intestine contents of suckling rats. Human and bovine SOX are relatively heat stable: 75% of the latter remains after treatment at 62.5 degrees C for 30 min and 65% of the former remains after treatment at 60 degrees C for 10 min. Neither remains after 62.5 degrees C for 30 min.