Nonoxynol-9 causes rapid exfoliation of sheets of rectal epithelium.

Nonoxynol-9 (N-9) containing spermicides and other N-9 containing products are commonly used as lubricants during rectal intercourse. We have previously demonstrated that rectal application of N-9 products in mice can cause exfoliation of epithelial cells, increasing the probability of infection by HSV-2. To determine if N-9-containing products would have a similar effect on the rectal epithelium in humans, the application of K-Y Plus and ForPlay, both over-the-counter (OTC) N-9 products, were compared to the application of two formulations, carrageenan and methyl cellulose, that do not contain N-9. The effects of each formulation were evaluated in 4 human participants. Light and electron microscope examination of rectal lavage specimens collected 15 min post application of N-9 products revealed the presence of sheets of epithelium. Each sheet contained hundreds of epithelial cells that included columnar and goblet cells, varieties of cells typical of rectal epithelial morphology. Sheets of epithelium were not observed in rectal lavage specimens collected 8 to 12 hr post N-9 product use or in either of the timed lavages involving non-N-9 containing formulations. In addition, no sheets of epithelial cells were observed in the baseline lavage specimens. We conclude that the rectal use of N-9-containing products causes a rapid exfoliation of extensive areas of the rectal epithelium. Exfoliation of the epithelium is no longer observed at 8 hr. It is reasonable to assume that the loss of the protective epithelium would render a person more at risk for infection by HIV and other sexually transmitted pathogens. We, therefore, caution against the use of N-9-containing products during rectal intercourse.

Carrageenan-based nonoxynol-9 spermicides for prevention of sexually transmitted infections.

BACKGROUND AND OBJECTIVES: Carrageenan-based nonoxynol-9 (N-9)-containing formulations were developed in response to the concern that over the counter (OTC) spermicides may not protect people from HIV infection and other sexually transmitted pathogens. GOAL OF THIS STUDY: The goal of this study was to compare the efficacy of carrageenan-based formulations to OTC spermicides in protecting mice from infection by herpes simplex virus 2 (HSV-2). STUDY DESIGN: Mice were challenged with vaginal inoculation of HSV-2 after pretreatment with test formulations. RESULTS: Carrageenan-based formulations were significantly more effective than currently marketed spermicides containing the same amount of N-9. Efficacy was demonstrated over a wide pH range. The carrageenan-based formulations could be autoclaved without losing antiviral activity and remained active in the vagina for several hours. CONCLUSIONS: We conclude that carrageenan-based N-9 formulations are likely to provide a more significant degree of protection against HSV-2 and other enveloped viruses than current OTC spermicides while providing comparable spermicidal activity.

Nonoxynol-9 enhances rectal infection by herpes simplex virus in mice.

Pretreating the rectum of mice with nonoxynol-9 (N9) or spermicides containing N9 before infecting via the rectal route with herpes simplex virus 2 (HSV-2) increased the likelihood of infection and shortened the time until infection. Microscopic analysis indicated that N9 rapidly caused exfoliation of the rectal epithelium, exposing the underlying connective tissue. These findings suggest that use of N9-containing products during rectal intercourse may increase the risk of infection with HSV-2 or other sexually transmitted pathogens.

Mechanism of monocyte-macrophage-mediated transmission of HIV.

Observations from our laboratory support the theory that HIV-infected monocyte-macrophages present in genital tract secretions have an important role in sexual transmission of HIV. Light and electron microscopy were used to study the behavior of HIV-infected, primary human monocytes. These cells progress on surfaces, putting forward a leading pseudopod from which they secrete HIV. When added to cultures of CD4-, cervix-derived epithelial cells, monocytes advanced between epithelial cells while secreting virus anteriorly. Epithelial cells subsequently become productively infected. Infection of epithelia could be blocked by sera from HIV-seropositive individuals. These findings support the supposition that transmission of HIV may occur via cell-mediated infection of intact epithelia. The observations also hint at the possibility that HIV-infected monocyte-macrophages in semen or cervical-vaginal secretions could cross intact epithelia by passing between epithelial cells. To test this hypothesis supravital-stained mouse macrophages were inoculated into the vaginas of mice. Four hours later numerous stained cells were observed in the connective tissue beneath the vaginal epithelium and in the iliac lymph nodes. We speculate that direct infection of epithelial cells and/or cell trafficking across epithelia may be involved in sexual transmission of HIV.

Cell-mediated HTLV-I infection of a cervix-derived epithelial cell line.

There is considerable evidence that sexual transmission of human T-cell leukemia virus-I (HTLV-I) is mediated by virus-infected lymphocytes in genital tract secretions. However, it is not clear whether infection occurs through lesions in the genital tract epithelium or takes place via an intact epithelium. We have carried out experiments to test the hypothesis that sexual transmission of HTLV-I is initiated by lymphocyte-mediated infection of intact genital tract epithelia. To examine this question we added either free virus or HTLV-I producing MT-2 cells to cultures of a cervix-derived epithelial cell line, MS751. Although free virus did not infect MS751 cells, MS751 cells which had been coincubated with MT-2 cells became infected. These cultures produced about 50 pg/ml of HTLV-I p24 antigen per 10(6) cells over a 24 h period on the sixth day following exposure to donor T-cells. Proviral DNA could be detected in target MS751 epithelial cells by PCR. Infection of epithelia could be blocked, in a dose-dependent manner, by the sulfated polysaccharides dextran sulfate, heparin, and fucoidan, and by the enzymes fucosidase and mannosidase, but not by a number of other agents that were tested. Since MT-2 cells were observed to attach to the epithelial monolayer, we examined the ability of agents to inhibit adhesion. Adherence was inhibited by the same agents that inhibited infection. Based on these findings, we hypothesize that sexual transmission of HTLV-I may involve lymphocyte-mediated infection of genital tract epithelia and that lymphocyte adhesion to the epithelium is a critical event in transmission of HTLV-I. We speculate that a sugar moiety on the epithelium, possibly mannose or fucose, may be involved in adhesion of T-cells to epithelial cells. As sulfated polysaccharides block both adhesion and productive infection of the epithelium, these compounds might be used as active ingredients in a vaginal formulation to help prevent HTLV-I transmission.

Vaginal formulations of carrageenan protect mice from herpes simplex virus infection.

The observations from the present study indicate that vaginal formulations of the sulfated polysaccharide carrageenan are highly effective in protecting mice from herpes simplex virus type 2 (HSV-2) infection. Test formulations were placed in the vaginas of progestin-treated mice prior to inoculation with HSV-2. Infection was determined by the presence of inflammation in the genital region and death. At a dose of virus that infected half of the control animals, 1% solutions of either lambda, kappa, or iota carrageenan prevented infection of almost all of the animals. Concentrations as low as 0.05% protected a large majority of the mice. At a dose of virus that infected all of the control mice, 1% solutions of carrageenans protected 85% of the inoculated mice. Other sulfated polysaccharides were less effective or showed no efficacy in preventing HSV-2 infection. These findings suggest that a vaginal formulation of carrageenan may be effective in blocking sexual transmission of HSV-2 in women.

A role for cell migration in the sexual transmission of HIV-1?

The issue of how human immunodeficiency virus-1 (HIV-1) enters the body following sexual contact has been the subject of considerable controversy. Several possible routes for the initial infection have been suggested [1-6], including the possibility that the transmission is mediated by HIV-1-infected lymphocytes or macrophages in serum and female genital tract secretions, rather than by free virus. We recently reported that HIV-1-infected, activated primary monocytes can migrate between epithelial cells grown in confluent monolayer cultures in vitro [7]. We report here on experiments carried out in mice to test the hypothesis that mononuclear blood cells are capable of migrating through intact epithelia, and thus of carrying a virus into an animal. We placed double-stained, activated mononuclear blood cells into the vaginas of mice; four hours later, numerous double-stained cells were observed in the connective tissue beneath the vaginal epithelium and the iliac lymph nodes of the experimental mice. We speculate that such migration may be involved in the sexual transmission of HIV-1.

Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles.

The proline-rich tandem repeat domain of human mucin MUC1 forms an extended structure containing large repeating loops that are crested by a turn. We show that the repeating-loop structure of MUC1 can be replaced by an antibody complementarity-determining region loop of a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing antibody to create a chimeric, multivalent, mucin-like, anti-HIV-1 compound. We used 8 residues of an antibody molecule to replace 8 of 20 residues of the MUC1 tandem-repeat sequence. The antiviral peptide discussed here contains three copies of a 20-residue tandem repeat, (IYYDYEEDPAPGSTAPPAHG)3, for a total of 60 residues. We demonstrate that the mucin-antibody chimera retains the binding specificity of the parent antibody (monoclonal antibody F58), GPGR of the HIV-1 gp120 V3 neutralizing epitope, and the ability to neutralize virus particles. In inhibition enzyme-linked immunosorbent assay, the mucin-antibody chimeric peptide could inhibit 71 to 84% of binding to a V3 loop peptide by monoclonal antibodies known to be specific for GPGR in the V3 loop. The mucin-antibody chimeric peptide could also inhibit monoclonal antibody binding to native gp120 captured from virus particles. In addition, the chimeric peptide neutralized the homologous HIV-IIIB virus in a standard neutralization assay. The methods of antiviral peptide design and construction presented here are general and theoretically limited only by the size of the antibody repertoire. This approach could be used to synthesize peptides for a variety of therapeutic applications.

An assay for HIV infection of cultured human cervix-derived cells.

There is a critical need to develop new strategies to prevent sexual transmission of HIV. Condoms have limited acceptance, and a vaccine may not be available for many years. A vaginal formulation could provide an alternative method if a compound that inhibits sexual transmission of HIV can be identified or synthesized, and if this agent can be formulated for vaginal use. In this report we describe an infection assay for testing compounds that may be useful in a vaginal formulation. This assay system utilizes a cell line (ME-180) derived from the human cervix which, on the basis of morphological features, is an appropriate model of female and male genital and urinary tract epithelia. These cells can be productively infected with HIV upon exposure to HIV-infected T-cell lines. Blocking experiments can be readily carried out in this model because in this p24 ELISA assay system the quantity of virus released by the infected epithelium over a 24-h period is 40 times background.

Mechanisms of sexual transmission of HIV: does HIV infect intact epithelia?

The prevailing view of sexual transmission of HIV has been that the virus enters the body through lesions in the epithelium of the genital tract. We propose that transmission of HIV can occur via the infection of intact epithelial cells, and that it is mediated by HIV-infected mononuclear cells in genital-tract secretions.

Association of mycoplasma with HIV-1 and HTLV-I in human T lymphocytes.

Recent findings from a number of investigators suggest associations between mycoplasma and HIV or AIDS. We used a quantitative morphometric technique to analyze electron micrographs of human T lymphocytes that were infected with both mycoplasma and/or HIV-1. We observed that lymphocytes which were associated with HIV-1 were much more likely to be associated with mycoplasma than cells that were not (p < .001). Similarly, cells with associated HTLV-I were more likely to be associated with mycoplasma than cells which were not associated with mycoplasma (p < .0001). In addition, mycoplasma and virus were observed in the same region in 90% of cases. These observations suggest that adherence of mycoplasma to lymphocytes that are chronically infected with human retrovirus may trigger viral release.

Lymphocyte-facilitated infection of epithelia by human T-cell lymphotropic virus type I.

After the addition of human T-cell lymphotropic virus type I (HTLV-I)-infected lymphocytes to enterocyte monolayers, the lymphocytes adhered via microvilli from both cell types and shed virus onto the enterocyte surface. Virus fused with the epithelial membrane and infected these cells as confirmed by electron microscopic immunocytochemistry, in situ hybridization, and amplification by polymerase chain reaction.

Microwave irradiation-accelerated in situ hybridization technique for HIV detection.

High frequency irradiation generated in a common household microwave oven was used to establish an in situ hybridization technique for rapid detection of HIV sequences in infected cells. A biotin-labeled DNA probe was subsequently detected either by an alkaline phosphatase-based colorimetric reaction or by fluorescence. When compared to standard hybridization procedures with radioactive or nonradioactive probes, microwave energy-mediated hybridization results in equal sensitivity and diminished background. The main advantage of this method, however, is the drastic reduction in time, allowing completion of the whole procedure, from sample preparation to hybrid signal visualization, within one hour. In addition to HIV detection, the approach described can be applied for the diagnosis of other viral infections and may stimulate the development of nucleic acid hybridization techniques based on microwave irradiation.

Structure of the cumulus oophorus at the time of fertilization.

Ultrastructural and morphometric techniques were employed to examine the ovulated cumulus oophorus of hamsters and rats. Observations on cumuli prepared in a variety of ways including different chemical fixation techniques and cryofixation freeze substitution were compared. It was concluded that the cumulus mucus is not arranged in lamellae or granules as has previously been suggested but is composed of molecules which form very fine filaments when properly fixed. Morphometric analysis of cumuli fixed either in situ or after being explanted into medium revealed that the distance between neighboring cumulus cells was greater with increasing distance from the oocyte. Morphometry revealed that, when placed into medium, the cumulus expands possibly due to hydration. Thus physiological experiments carried out on cumuli should be performed very shortly after cumuli are isolated. From their ultrastructure cumulus cells appear to be actively involved in protein synthesis and secretion as well as steroid production.

Tubules in semen of infertile patients. Case report.

Numerous groups of small tubules were observed in the semen of two infertile patients. These structures were about 80 nm in diameter and could be observed within larger concentric tubules. They did not resemble typical microtubules, and did not appear similar to unit membrane.