Biomedical subjects

R Pearce-Pratt

Publications and source records attributed to R Pearce-Pratt.

10 recordsLinked to original sources

PubMed1996

Sulfated polysaccharides inhibit lymphocyte-to-epithelial transmission of human immunodeficiency virus-1.

We have previously suggested that sulfated polysaccharides could be used in a vaginal formulation to inhibit infection by human immunodeficiency virus (HIV-1). This supposition was based on studies in which we developed and employed an in vitro model to simulate the mechanism of HIV-1 transmission during coitus. We found that adhesion of mononuclear cells to epithelia was the initial step in infection and speculated that blocking adhesion would prevent HIV-1 transmission. We observed that certain sulfated polysaccharides prevented adhesion of lymphoma cell lines to epithelial cell lines, which were derived from the genital tract, in concentrations of a few milligrams per milliliter; and we theorized that sulfated polysaccharides could thus be used as active ingredients in a topical "microbicide." In the present in vitro study, evidence is presented that a number of sulfated polysaccharides, including carrageenan, dextran sulfate, heparin, fucoidan, and pentosan polysulfate, are capable of blocking infection by mechanisms other than adhesion at concentrations of a thousand times lower than the dosages that are needed to block cell adhesion. One of these compounds, iota carrageenan, is capable not only of blocking infection of epithelia at concentrations of 1-2 micrograms, but of blocking adhesion to a far greater extent than the other sulfated polysaccharides tested. For this reason, as well as for considerations of safety, stability, and gelling properties, we suggest that iota carrageenan may be the best choice of the sulfated polysaccharides tested for use as a vaginal microbicide. The same in vitro model was employed to decipher the cell surface molecules involved in lymphocyte-to-epithelial adhesion. To accomplish this, we screened for the presence of cell adhesion molecules (CAMs), carbohydrates, proteoglycans, and carbohydrate-binding sites. HIV-1-infected lymphocytic cells expressed a CAM profile typical of activated, infected cells (e.g., HLA-DR+, CD4-, LFA-1+, ICAM-1+, LFA-3+, CD2+) whereas epithelia expressed few CAMs (LFA-3, ICAM-1, VLA-5, CD44, CD26, sLEX). Both cell types expressed heparan sulfate and chondroitin sulfate proteoglycans. A variety of sugars (mannose, fucose, galactose, Nac-galactosamine, Nac-glucosamine) were also present, but these cells expressed few carbohydrate-binding sites; lymphocytes bound beta-galactose. We were unable to block the adhesion with anti-CAM antibodies or with exogenous sugars. When enzymes were used against sulfated cell surface molecules, chondroitinase was found to block the adhesion. Our evidence suggests that this CAM-independent adhesion may be a lectin-glycosaminoglycan interaction.

Acquired Immunodeficiency Syndrome↗

PubMed1995

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.

Cell Line↗

PubMed1995

Sulfated polyanions block Chlamydia trachomatis infection of cervix-derived human epithelia.

Using a cell line derived from the human cervix and a rapid fluorescence cytotoxicity assay, we have shown that Chlamydia trachomatis infection can be blocked by certain sulfated polysaccharides (carrageenan, pentosan polysulfate, fucoidan, and dextran sulfate) and glycosaminoglycans (heparin, heparan sulfate, and dermatan sulfate) but not by other glycosaminoglycans (chondroitin sulfate A or C, keratan sulfate, and hyaluronic acid). The most negatively charged molecules are the most effective at blocking infection. Results of infection at 4 degrees C suggest that sulfated polyanions act by preventing the adherence of chlamydiae to target cells. These and additional blocking studies with enzymes suggest that a heparan sulfate-like glycosaminoglycan on the surface of elementary bodies is involved in the adherence of chlamydiae to target cells, probably through a nonspecific charge interaction or possibly a heparin-binding protein. We previously observed that the same sulfated polysaccharides inhibit transmission of human immunodeficiency virus in vitro and suggested that these compounds could be used in a vaginal formulation to inhibit infection by human immunodeficiency virus. The results of the present study suggest that the same type of formulation may inhibit sexual transmission of chlamydia.

Animals↗

PubMed1994

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.

CD4-Positive T-Lymphocytes↗

PubMed1994

Role of the cytoskeleton in cell-to-cell transmission of human immunodeficiency virus.

We previously observed that when human immunodeficiency virus (HIV)-infected T lymphocytes are added to epithelial cells, they adhere, polarize, and secrete virions unidirectionally onto the epithelium. Epithelial cells subsequently take up virus and become productively infected. We report here that colchicine treatment of T-lymphocyte suspensions induced lymphocyte polarization, redistribution of F-actin into a pseudopod, and secretion of HIV from the pseudopod. Immobilization of T lymphocytes on negatively charged plastic also caused redistribution of F-actin and unidirectional secretion of HIV onto the plastic. As neither colchicine nor adhesion caused an increase in HIV secretion, they apparently act by focusing secretion to the tip of the pseudopod. We speculate that adhesion-induced polar secretion of HIV, from activated mononuclear cells onto epithelia, is a cytoskeleton-mediated process which may be involved in HIV transmission in vivo.

Actins↗

PubMed1993

Studies of adhesion of lymphocytic cells: implications for sexual transmission of human immunodeficiency virus.

Considerable evidence suggests that sexual transmission of human immunodeficiency virus (HIV) is mediated via mononuclear cells that can infect epithelia of the genital tract. We describe here an in vitro model that can be used to examine the mechanism of cell-to-cell transmission of this virus. We have employed the system to identify agents that may be effective in a vaginal formulation to prevent HIV transmission via sexual contact. We have previously shown that chronically HIV-infected mononucleocytes can infect CD4-negative epithelial monolayers in the following manner: adhesion, via multiple microvilli, of HIV-infected mononucleocyte-derived cells to epithelial monolayers activates rapid virion secretion. Virions are then shed from the attached surface of the infected lymphocyte into a partially enclosed, microvilli-laden space between the cells. The shedding results in uptake of the virus and epithelial cell infection as demonstrated by ultrastructural examination and in vitro virological techniques. In this report, we present evidence from time-lapse films that HIV-infected lymphocytes adhere to the epithelium for a few minutes and then shift position to another site on the epithelium. As a result, one infected lymphocyte appears to be able to sequentially infected several cells of the epithelial monolayer. Using a fluorescence-based cell-cell adhesion assay to examine the effect of seminal fluid and a variety of chemical compounds on lymphocyte-to-epithelial adherence, we found that seminal fluid significantly increases the number of lymphocytes adhering to epithelia. This suggests that semen can serve as an effective medium for cell-cell transmission of HIV. On the other hand, sulfated polysaccharides and glutathione effectively inhibit cell-cell adhesion. Since the cell-cell adhesion step is critical to epithelial cell infection by HIV, these results suggest that anti-cell adhesion compounds may be effective in a vaginal formulation to reduce the probability of HIV infection.

Cell Adhesion↗

PubMed1993

Productive infection of a cervical epithelial cell line with human immunodeficiency virus: implications for sexual transmission.

The human cervix-derived epithelial cell line (ME180) used in this study displays a characteristics epithelial morphology, including numerous desmosomes, tonofilaments, and epidermal filaments. When T-cell lines infected with human immunodeficiency virus (HIV) are added to epithelial cultures, they rapidly adhere to the epithelial monolayer. Within a few minutes, the T cells shed numerous virions into narrow spaces formed between the epithelial cell and the adherent T cells. Virions subsequently enter the ME180 cells via large vesicles. A few days after infection, cytopathic effects and syncytium formation were observed. Infected clones of ME180 cells have remained infected for 8 months. p24 enzyme-linked immunosorbent assay and infectivity assays show that one subclone of the cell line produces virus titers equivalent to those of high-secreting HIV-infected T-cell lines. Electron microscopy reveals numerous virions budding from both the basal and apical surfaces of the epithelium. These observations suggest that cervical epithelium has the potential to serve as a site of HIV infection.

CD4 Antigens↗

PubMed1992

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.

Cell Line↗

PubMed1991

Simple colorimetric cell-cell adhesion assay using biotinylated lymphocytes.

A new approach for quantitating lymphocyte adhesion based on labeling the lymphocyte plasma membrane with water-soluble biotin was developed. Adherent biotinylated lymphocytes were quantitated by measuring OD values of a colored substrate representing the amount of bound avidin-peroxidase. The lymphocyte adhesion assay based on the high affinity of avidin to biotin was considerably more sensitive when compared to rose bengal or [3H]thymidine labeling methods. The end-point of sensitivity is approximately 1000 lymphocytes which is clearly an improvement over the rose bengal or radiolabeling techniques with a detection limit of respectively 15 x 10(3) and 7.5 x 10(3) lymphocytes added to wells at the beginning of the assay. The method has the advantage of being rapid and simple and offers an alternative to adhesion assays based on cell ELISAs using cell-specific monoclonal antibodies.

Biotin↗

PubMed1990

False-positive signals in enzyme immunoassay (EIA) interactions between rodent IgG subclasses.

Interactions between mouse and rat monoclonal antibodies (MAbs) in EIA have been detected using panels of mouse and rat MAbs of various isotypes. Mouse and rat antibodies of the IgG2a subclass were found to bind to each other most strongly. Immobilised IgG1 antibodies of the two species showed much less interaction. Rat IgG2b and IgG2c were intermediate in binding activity. We were unable to study examples of mouse IgG2b and IgG3 MAbs. By making use of the kappa light chain allotype in the rat we were also able to show intraspecies interactions of rat MAbs with the IgG in rat serum. The molecular basis of these interactions has not been resolved but may involve the extended hinge region and flexible Fab arms of the IgG2a subclass of Ig permitting strong inter domain bonding between the constant region domains of adjacent molecules of this isotype.

Animals↗