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ALCOHOL MAY INCREASE RISK OF HIV FROM ORAL SEX
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Study indicates alcohol facilitates transmission of virus in mouth cells
"...Our results indicate that in cell culture conditions, the ranges of concentrations of alcohol that are commercially available are able to stimulate the infection efficiency of HIV in primary oral epithelial cells..."
By Dyana Bagby
Southern Voice
January 14, 2005
Drinking beer may cause the cells that line your mouth to become more vulnerable to HIV, according to lab experiments conducted by a team of researchers in California.
The study, titled "Ethanol Stimulation of HIV Infection of Oral Epithelial Cells" and conducted by researchers at the University of California at Los Angeles, reports that exposure to four percent alcohol - a common amount for most beers - can weaken mouth cells against HIV. The report was published in the Dec. 1 2004 issue of the Journal of Acquired Immune Deficiency Syndromes.
But scientists stress that the findings were determined in a lab setting and not with actual people.
"In people, you have mucous, saliva and other things that could inactivate the stimulation or at least interfere with it," said Nabila Wassef, program officer with the National Institute of Allergy & Infectious Diseases Division of AIDS at the National Institutes of Health in Maryland. The NIH partially funded the study.
"We have to stress the fact these are in vitro findings [and did not include mucous or saliva]," Wassef added.
According to the report, scientists took mouth cells and placed them in a petri dish, soaking them in between zero and four percent ethanol for 10 minutes. The cells exposed to the four percent alcohol showed they were up to six times as likely to contract the HIV strain used in the experiment.
The findings startled AIDS educators who often teach oral sex is safer than unprotected anal sex when attempting to avoid HIV.
"It's almost like a double whammy," said Cara Emery, treatment education program manager at AIDS Survival Project in Atlanta.
"Oral sex does have the reputation of being safer and obviously alcohol use can increase your chances of contracting HIV due to riskier behaviors. This is telling someone they may be at more increased risk than they already were," Emery said.
But Dr. Tri D. Do, president of the Gay & Lesbian Medical Association, cautioned against taking the findings too seriously.
"It's really hard to take what you learn in a lab setting and put it in real-world situations," he said.
"I don't think the results of this study should change what people are doing. If people want to play it safe, they should avoid drinking before having sex ... because it impairs judgment," Do said. "My bottom line is oral sex is safe sex."
The study
Scientists with UCLA reported they took oral epithelial cells - cells that line the interior of the mouth - and exposed them to alcohol for up to 10 minutes before inserting a fluorescent HIV strain to make the transmissions visible.
Dr. Shen Pang with the UCLA Dental Institute, one of the researchers involved with the study, said the cells were obtained from leftover cell samples taken between 1994-1996 from two or three HIV-negative people. The cells used were taken from healthy mouths and did not take into account such things as mouth sores, Pang said.
The study was conducted about two years ago, he added.
The alcohol used was a 200 percent lab-based ethanol that was diluted to four percent for use in the study. Pang said alcohol higher than four percent used in the study quickly killed the HIV strain.
"Most beer is between three and five percent, depending on which country you live in, and that's why we put in four percent," Pang said.
The report also summarizes that beer is "the most widely consumed alcoholic beverage of Americans."
According to Wassef, alcohol at, for example, 12 percent - roughly equivalent to a 24-proof wine - would inactivate the virus.
"What this study states is that at these medium concentrations [of four percent] that you get after one or two drinks, this is what happens in a culture setting," Wassef said. "The findings are definitely there. But we don't know what would happen if we did this with people."
Wassef said the oral epithelial cells were used because they are CD-4 negative, which is a protein present in T-cells. HIV strains often bind themselves to the CD-4 proteins to get into T-cells, she explained. So how exactly the HIV entered into the mouth cells is uncertain at this time other than the presence of alcohol facilitated the transmission, she said.
"This adds to our body of knowledge about AIDS, but whether it really applies to people, that I cannot say," Wassef said.
Pang said he and the other scientists, led by Dr. Jun-ying Zheng of UCLA, believe the findings indicate people must be more careful during oral sex.
The study results would suggest that alcohol would stimulate the transmission of other STDs during oral sex, but none of those were studied, so no definitive conclusions can be drawn, he said.
Pang said this was a very specific, scientific study that only looked at the relationship between oral epithelial cells and HIV.
"Many papers have shown that saliva can kill HIV, but we can't study all this in the lab. We have to go one step by one step," he said.
Oral sex and HIV
The chance of contracting HIV through oral sex is about 1 in 10,000 chance per act, according to Do of the GLMA.
"Various studies have showed this to be the average number, but the science is not very good in this area," he said. "I think it's probably closer to 1 in a million - you probably have a better chance of getting struck by lightning than getting HIV through oral sex."
Risk factors such as mouth sores and cuts must also be considered, Do said. AIDS educators often teach that people should not floss or brush before performing oral sex as it risks opening a sore.
Other estimates of the risk of contracting HIV through oral sex vary widely. A study by the Centers for Disease Control & Prevention in 2000 indicated that eight of 102 cases of HIV infection among gay and bisexual men were "likely" due to receptive oral sex without a condom.
The Gay Men's Health Crisis in New York posts on its Web site a summary of a report that states an eight-year study of nearly 1,000 men found HIV transmission through oral sex too rare to be measured, but not completely without risk.
Those who engage in unprotected receptive anal sex with an HIV-positive partner have a 1 in 100 chance per act of contracting the virus, Do said. Receptive vaginal sex with an HIV-positive person poses a 1 in 1,000 per act chance of contracting HIV, he said.
Dr. Pat Hawkins with the Whitman-Walker Clinic in Washington, D.C., said determining the numbers in risk factors is difficult. The best defense, she said, is a good offense, including the use of condoms.
"Some things are more risky than others, but if you want to be safe, you can," she said.
Ethanol Stimulation of HIV Infection of Oral Epithelial Cells
[Basic Science]
JAIDS Journal of Acquired Immune Deficiency Syndromes: Volume 37(4) 1 December 2004 pp 1445-1453
Zheng, Jun MD, PHD*; Yang, Otto O MD; Xie, Yiming MS; Campbell, Richard BS*; Chen, Irvin S. Y PHD; Pang, Shen PHD*
From the *Division of Oral Biology and Medicine and University of California at Los Angeles (UCLA) Dental Institute, UCLA School of Dentistry, Los Angeles, CA; Division of Infectious Diseases, Department of Medicine, UCLA School of Medicine, Los Angeles, CA; and Departments of Medicine and Microbiology and Immunology and UCLA AIDS Institute, UCLA School of Medicine, Los Angeles, CA.
Summary:
Oral mucosal cells can be infected by exogenous HIV during receptive oral sex or breast-feeding. The risk of oral mucosal infection depends on the infection efficiency of the HIV strains present in the oral cavity, the viral titers, and the defense mechanisms in the oral cavity environment. It is expected that alcohol can weaken the host defense mechanism against HIV infection in the oral cavity. We modified an HIV strain, NL4-3, by inserting the enhanced green fluorescent protein gene and used this virus to infect oral epithelial cells obtained from patients. Various concentrations of ethanol (0%-4%) were added to the infected cells. HIV-infected cells were detected by fluorescent microscopy or fluorescence-activated cell sorting. We found that ethanol significantly increases HIV infection of primary oral epithelial cells (POEs). POEs pretreated with 4% ethanol for less than 10 minutes demonstrated 3- to 6-fold higher susceptibility to infection by the CXCR-4 HIV strain NL4-3. Our studies also demonstrated that HIV infects POEs through a gp120-independent mechanism. We tested an HIV CCR5 strain, JRCSF, and also found its infection efficiency to be stimulated by alcohol. Our results indicate that in cell culture conditions, the ranges of concentrations of alcohol that are commercially available are able to stimulate the infection efficiency of HIV in POEs.
HIV infects not only CD4-positive T lymphocytes but some types of CD4-negative cells as well.1-10 Infection of oral epithelial cells and female vaginal epithelial cells may be important for HIV-1 transmission.4,5 Studies by Qureshi et al4 indicate that the virus can be detected in oral epithelial cells isolated from 69% of HIV-1-positive patients. Of these patients, 33% showed high levels of infection, with 1% to 4% of the cells testing positive. CD4-negative epithelial cells and stromal cells derived from female reproductive tract tissue also showed susceptibility to HIV-1 infection. Howell et al5 tested epithelial cells derived from the female reproductive tract and found that all the epithelial cells tested could be infected by HIV-1 strains JR-FL and Harwi. These results suggest that infection of epithelial cells in either the female reproductive tract or in oral cells may be the earliest event of HIV-1 transmission.
In this report, we focus on the HIV infection of primary oral epithelial cells. Receptive oral sex (ROS) plays an important role in spreading the AIDS epidemic.11-13 HIV oral transmission may also occur through breast-feeding by infected mothers.14 Therefore, an understanding of the mechanisms involved in oral HIV transmission is important.
Oral transmission of HIV uses an infection pathway similar to that of other transmucosal infections, by which more than 80% of HIV infections occur. Normally, the oral cavity is shielded by epithelial cell layers, which block access of the virus to its target cells such as CD4-positive T lymphocytes, monocytes/macrophages, and dendritic cells. Because these epithelial cells do not express the CD4 molecule, which is known to act as the primary receptor for HIV, these cells are not infected via the typical pathway involving the interaction of gp120 and CD4. There are 2 hypotheses proposed to explain how HIV crosses the barrier formed by epithelial cell layers. One means is transcytosis, whereby HIV uses endocytosis to enter epithelial cells on the apical surface, is transported to the basolateral surface, and is released.15-17 The other hypothesis is that HIV infects some types of CD4-negative epithelial cells, including oral epithelial cells, although its infection efficiency is lower for these cells than for CD4-positive cells.1,4,18-21 The virus replicates in these cells and is subsequently released from the basolateral side of an epithelial layer to encounter CD4-positive target cells. Reported evidence supports the likelihood that HIV possesses the ability to enter these cells and undergo the full replicative cycle.19-22 It has been demonstrated that either chemokine receptors (CXCR4 or CCR5) or galactosylceramide (GalCer) may be required for transcytosis or infection to occur.17,19-23 HIV may use both mechanisms to cross the CD4-negative epithelial cell layers.
The study of HIV infection of oral epithelial cells represents an important aspect in understanding HIV oral transmission. Unlike other mucosal sites, the integrity of the mucosal membrane of the oral cavity can be directly affected by food and beverages. Previous reports have demonstrated that clinically relevant ethanol concentrations significantly increase the expression of tumor necrosis factor-α (TNFα)-inducible nuclear factor-κB (NFκB) in CD4-positive T cells, suggesting that ethanol may upregulate HIV gene expression.24 Alcohol has been shown to increase CCR5 expression and thus to increase the infection of blood monocyte-derived macrophages.25 There is no report demonstrating that ethanol has a direct effect on oral epithelial cell infection, however. In some reports, ethanol was shown to have a negative role in HIV infection in that it inhibited TNFα expression in vitro26 and neutralized the gp120 glycoprotein.27
Compared with HIV infection of CD4-positive cells, such as activated CD4-positive T lymphocytes or macrophages, HIV infects most types of CD4-negative cells with a lower infection efficiency. This feature causes difficulties in quantifying HIV infection of these cells.
To confirm HIV infection and replication in CD4-negative cells, the p24 assay and coculturing with peripheral blood lymphocytes (PBLs) have been commonly used. Infection of these types of cells by HIV-1 can be monitored by measuring the amount of an HIV-1 Gag protein, p24, is released by the infected cells following a time course or by adding PBLs to the infected culture to rescue the virus that was generated by the infected CD4-negative cells. There are some disadvantages to using these 2 methods, however. Because the infection efficiency of CD4-negative cells by HIV is much lower compared with the infection efficiency of CD4-positive cells, the p24 protein level generated by the infected CD4-negative cell culture is low. Detection of the low count of p24 is difficult because of the background of residual p24 from the input virus. The background p24 is derived from the HIV input for infection. In a general testing procedure, a certain amount of HIV with a p24 titer from 1 to 1000 ng is required to infect 104 CD4-negative cells. After the infection, no matter how many washes are used, a significant amount of p24 protein remains because of the fact that some p24 protein molecules stick to the cells or the plates. These p24 molecules can be slowly released into the culture medium. This p24 background can be ignored if the HIV infection level is high. If only a low percentage of cells are infected, however, the background p24 level causes problems. We found that it is quite common for an infected CD4-negative cell culture with only a few infected cells to demonstrate almost the same level of p24 compared with another infected CD4-negative cell culture with 10-fold more infected cells. Therefore, the p24 assay is not a sensitive method to quantify the infection of CD4-negative cells. Although coculturing with PBLs to detect HIV infection of CD4-negative cells is a more sensitive method, it is not quantitative.
Because the p24 assay and PBL coculturing do not offer both sensitive and quantitative results, an alternative method must be developed. Our laboratory, in addition to several others, modified HIV strains by inserting the enhanced green fluorescent protein (EGFP) gene.28-34 Insertion of the EGFP gene into the location of the HIV-1 nef gene does not affect the efficiency of viral replication, packaging, or infection.32 The infected cells are directly detectable by fluorescent microscopy or fluorescence-activated cell sorting (FACS) analysis. In other words, these modified viruses offer a direct, sensitive, and quantitative approach to investigate viral infection and viral gene expression in infected cells.
We made additional modifications by deleting a 581-base pair (bp) fragment of the env sequence from the viral genome, which not only introduces a deletion into Env gp160 (the precursor of gp120 and gp41) but causes a frame shift. This makes it possible to generate an HIV-1 Env-negative virus. Because the deleted 581-bp sequence does not overlap the open reading frames (ORFs) of other viral genes, the deletion only abrogates env gene expression.33 By cotransfecting or coinfecting this virus with vectors carrying the HIV-1 env gene from various strains, virions pseudotyped by Env from various strains of HIV-1 can be generated. Such a strategy offers an approach to generate an HIV-1 strain with no Env protein (neither gp41 nor gp120) on its envelope.
In this study, we used the EGFP-modified HIV strains CXCR4-tropic NL4-3 and CCR5-tropic JRCSF to infect primary cells and cell lines of oral epithelial cells in cell culture conditions. Cells treated with various concentrations of ethanol were infected with these viral strains. By measuring the expression of EGFP, the percentages of infected cells were assessed. We also compared the infection of oral epithelial cells with gp120-negative HIV. Through these studies, the effects of ethanol on oral epithelial cells in cell culture conditions were quantitatively characterized.
DISCUSSION
Ethanol is widely consumed by adults. Although some reports demonstrated that alcoholism may be related to HIV transmission, most related studies have focused on alcohol-related sexual behavior; thus, the direct effect of ethanol in HIV infection has been overlooked. In this study, we found that 3% to 4% ethanol treatment significantly stimulated HIV infection of POEs, suggesting that ethanol may play a direct role in HIV oral transmission. The concentration of 3% to 4% ethanol is similar to that contained in various beers (with beer being the most widely consumed alcoholic beverage of Americans), so the study of the effect of ethanol in HIV infection of oral cells may provide useful knowledge to prevent oral transmission.
Our results demonstrate that HIV strains do not express gp120-infected POEs with an efficiency identical to that of strains containing gp120 on the viral envelope, suggesting that the infection mechanism is largely gp120 independent. Studies of the HIV life cycle have indicated that HIV uses a patch of host membrane as its envelope. It is undeniable that the HIV envelope contains many cellular membrane proteins. For infection of CD4-negative cells, HIV may not use gp120 to infect the target cells; instead, it may use certain cellular membrane proteins to bind and fuse to the target cells. Our previous studies demonstrated that some types of CD4-negative cells are much more susceptible to HIV infection than others. For example, HIV NL4-3 had high infection efficiency in the prostate cancer cell line LNCaP but poor infection efficiency for HeLa-CD4 cells.33,34 In the current study, HIV showed higher infection efficiency for POEs than for the SCC4 oral cell line. These results suggest that gp120-independent infection likely involves certain membrane proteins specifically present on some types of cells but not on others.
Our results indicate that HIV can infect POEs and oral cancer cell lines, suggesting that there are specific receptors on the oral cell membrane responsible for gp120-independent infection. Ethanol can only stimulate HIV infection of POE cells, however. One explanation is that there are 2 types of receptors for gp120-independent HIV infection. One type may be present on the surface of POEs and cell lines, and the other type may be exclusively present on the surface of POEs. The addition of ethanol may either change the conformation of these types of proteins so that these cells become more susceptible to HIV infection or may upregulate the expression of these proteins.
Many protein receptors are responsive to ethanol, for example, N-methyl-d-aspartate (NMDA) receptors,41-44 gamma-aminobutyric acid (GABA) receptors,41,45 and anandamide and cannabinoid receptors.46 Ethanol may also induce receptor-specific endocytosis.47 Some of these receptors are expressed in oral cells.48 It is difficult to predict whether these receptors expressed in oral cells are involved in gp120-independent HIV infection. Because HIV can also infect nerve cells that are CD4-negative and have high expression of those ethanol-susceptible receptors, it is possible that some of these receptors are involved in HIV infection of POEs.
Ethanol may also change the properties of lipid rafts in the cell membrane. One component, glycosphingolipid GalCer, of the lipid raft structure has been identified as an essential component for transcytosis.16,17 Both the gp41 and gp120 viral envelope glycoproteins of HIV use the lipid raft as a transport mechanism to get from the apical side to the basolateral side of the cell.49 Because GalCer has been identified as the key component for CD4-negative cell infection by gp120-dependent pathways, it is expected that the lipid raft plays some role in HIV infection of some CD4-negative cells. Because the ethanol-related POE infection is largely gp120 independent, it is unlikely that the molecule GalCer in lipid rafts plays a key role. It may still be possible that some unidentified components in the lipid raft would play an important role, however. It is possible that a specific receptor that is responsible for POE infection is highly enriched in the lipid raft structure.
Our results also demonstrated that ethanol treatment has a relatively long-term effect on HIV infection of POEs. Cell cultures pretreated with ethanol 2 hours before infection have an infection profile identical to that of cultures with 4% ethanol maintained in the culture medium. Two models can be proposed to explain such long-term effects of ethanol treatment: the receptors with changed conformations induced by ethanol have a long life, so these oral cells maintain their susceptibility to HIV, or ethanol rapidly triggers the expression of HIV infection-related proteins, and once the expression is triggered, expression can stay high for at least 2 hours even when ethanol is removed. More study is required to distinguish between these 2 mechanisms.
It is also of interest to quantify the infection of POEs by other HIV strains. Our results demonstrated that the 2 CCR5-tropic HIV strains NFN-SX and JRCSF also infected POEs (see Fig. 1). Currently, we are modifying the genomes of these 2 strains by addition of the EGFP gene. We expect that the use of EGFP-modified HIV clones will enable us to compare the infection efficiency of these two strains quantitatively with the well-characterized HIV strain NL4-3. It is also of interest to quantify the infection of other HIV subtypes or recombined HIV strains. The knowledge gained should be useful in future studies of HIV infection of oral epithelial cells.
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