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Female Reproductive Tract |
Department of General Physiology and Biochemistry,3 University of Milan, Milan, Italy
The Population Council,4 New York, New York 10021
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cervix, female reproductive tract, ovulatory cycle, toxicology, vagina
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A concept that has been supported by some evidence is that HIV transmission is mediated by HIV-infected lymphocytes and/or monocyte/macrophages. Jay Levy originally proposed the idea in 1988 [15]. A few years later, Anderson [16] used the term Trojan horse to describe the theory that HIV-producing mononuclear cells could somehow pass through the intact epithelial barrier to the connective tissue, where CD4-positive lymphocytes and macrophages reside. Since then, research has shown that HIV-infected mononuclear blood cells are present in both semen and cervical/vaginal secretions [17]. The idea that mononuclear blood cells are capable of exiting the vaginal vault through an intact epithelium is not unreasonable. Many cells of the immune system traffic through the body to perform their normal functions. Mononuclear and polymorphonuclear blood cells migrate from the basolateral to the apical surface of the vaginal, uterine [18, 19], and intestinal epithelia [20]. Transmigration of polymorphonuclear and mononuclear blood cells from the lumenal to the basal surface of endothelia is a basic element of the immune and inflammatory responses [21].
Ten years ago, we observed that mononuclear cells could adhere to epithelial cells derived from the cervical epithelium [7]. We also found that adhesion triggered cytoskeletal events that produced a pseudopod from which HIV was secreted [8] and that adhesion of mononuclear cells to epithelia could be blocked by sulfated polysaccharides [7, 22]. Recently, Carreno et al. [23] presented evidence that adhesion of macrophages to epithelial cells is mediated by binding of LFA-1 to intercellular adhesion molecule (ICAM) 2 and ICAM-3. These workers also showed that this adhesion caused transmigration of macrophages across tight epithelial monolayers in vitro.
Ibata et al. [24] tested the Trojan horse concept directly by placing supravitally stained, activated mononuclear blood cells in the vaginas of mice. When they killed the animals, they observed stained cells in the connective tissue beneath the vaginal epithelium and in the iliac lymph nodes [24]. We carried out similar experiments and reported that macrophages can pass through epithelia of the genital tract of female mice [25]. Recently, Khanna et al. [26] used a somewhat different system to test the Trojan horse model. These workers placed human peripheral blood lymphocytes (HuPBL) from uninfected blood into the peritoneum of severe combined immunodeficiency mice. They subsequently placed HIV-infected HuPBL in the vaginas of these animals, resulting in infection of the uninfected HuPBL. In contrast, placing free virus in the vagina of mice did not cause infection of HuPBL in the peritoneum. Thus, several studies carried out by different laboratory groups support the hypothesis that one mechanism of sexual transmission of HIV may involve trafficking of HIV-infected mononuclear cells across the genital epithelium.
Microbicide administration as a preventative treatment is receiving increasing attention from the biomedical community. These products are designed to prevent infection when topically applied prior to sexual intercourse. The term microbicide is misleading becuase the products work on a variety of mechanisms in addition to inactivating HIV. A carrageenan formulation, Carraguard, is the Population Council's lead candidate microbicide. Its active ingredient is carrageenan, a sulfated polysaccharide that represents most of the dry weight of red algae. Carrageenan has been effective in preventing HIV infection in in vitro assays [22]. The carrageenan formulation also has been used to prevent infection by other sexually transmitted pathogens in animals [27] and has been deemed safe in clinical trials [28].
In this study, we carried out experiments to determine whether mononuclear cells introduced into the vagina could migrate farther than the regional lymph nodes and enter the bloodstream of the recipient. Using the carrageenan formulation and an apparently inert methylcellulose formulation, we also determined whether the system of transmigration of macrophages could be used for assaying microbicide efficacy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Biotinylated rat monoclonal anti-F4/80 antibody and Erythrolyse were purchased from Serotec (Oxford, U.K.), and BSA fraction V, mouse IgG, culture media, fetal bovine serum (FBS), and 3-4,5-dimethylthiazol-2-Yl-2, 5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). Gentamicin was from Gibco BRL (Life Technology Srl, San Giulinao, Italy). The fluorescent supravital dye 4-chloromethylbenzoylaminotetramethyl rhodamine (CellTracker Orange CMTMR), streptavidin-Alexa Fluor 488, and Prolong Antifade were from Molecular Probes Europe B (Leiden, The Netherlands). Medroxyprogesterone acetate (Depo-Provera) was purchased from Upjohn (Kalamazoo, MI), and Brewer thioglycollate broth was obtained from Difco (Detroit, MI). Ketamine hydrochloride and xylazine hydrochloride were purchased from Fort Dodge Laboratories (Fort Dodge, TX). O.C.T compound was from Sakura Finetek Europe B.V. (Zoeterwoude, The Netherlands), and Vectashield was from Vector Laboratories (Burlingame, CA). SDS was purchased from BioRad (Hercules, CA), and N,N-dimethylformamide was from Merck (Dalstad, Germany). All other chemicals were purchased from Sigma.
Animals
Nine- to 11-wk-old female BALB/c mice (Charles River, Calco, Italy) were maintained on a 12L:12D cycle in the departmental animal care facility of the University of Milan in accordance with the European Communities Council Directive 86/609/EEC regarding mammalian research. Five days prior to vaginal inoculation, recipient animals were injected s.c. with 0.1 ml of a 25-mg/ml Depo-Provera solution in PBS.
Macrophages
Mice were inoculated i.p. with Brewer thioglycollate broth [29]. Five days later, animals were killed by CO2 asphyxiation. Inflammatory macrophages were harvested by peritoneal lavage with 5 ml of RPMI 1640 medium containing 5% heat-inactivated FBS and 50 µg/ml gentamicin (RPMI 5) and centrifuged at 400 x g for 5 min at 4°C.
Viability was assessed by propidium iodide (PI) exclusion test, for which 106 cells were incubated in 0.5 ml RPMI 5 containing 1 µg/ml PI for 5 min at room temperature in the dark [30]. The percentage of PI-stained dead or dying cells was evaluated with a DAS Mikroskop epifluorescence microscope (Leica) equipped with standard filter sets for Hoechst/4',6'-diaminidino-2-phenylindole, fluorescein, tetramethylrhodamine, with a band pass filter for fluorescein (527/20) and with phase contrast optics. Cell viability was >90% (93–95 %).
Immunophenotyping of the cell population was carried out by indirect immunofluorescence using monoclonal antibody anti-F4/80, a marker of resident and elicited peritoneal macrophages [31]. One million cells were washed with cold PBS, fixed with 2% paraformaldehyde in PBS for 10 min at room temperature, thoroughly washed in PBS, preincubated for 20 min in 90 µl of 0.2% mouse IgG in PBS containing 1% BSA, and incubated for 30 min at room temperature in the same solution to which 10 µl of biotinylated monoclonal rat anti-F4/80 had been added. After thorough rinsing in PBS/BSA, cells were incubated for 30 min in 5 µg/ml of streptavidin-Alexa Fluor 488 in PBS containing 1 µg/ml Hoechst 33342 to counterstain nuclei. Cells were then washed in PBS and mounted in Prolong Antifade, and F4/80-positive cells were counted with the epifluorescence microscope. F4/80-positive cells accounted for 96–98% of the cells harvested.
Donor cells were present in the iliac and inguinal lymph nodes and in the spleen. When mice received only a vaginal inoculation of macrophages, the recipient animals had an average of 55 labeled donor cells in the draining lymph nodes and an average of 558 labeled cells in the spleen. In mice that received a vaginal preinoculation of the carrageenan formulation, an average of only four labeled cells were counted in the draining lymph nodes, and an average of only 28 labeled cells were observed in the spleen. The difference between untreated animals and those treated with the carrageenan formulation was highly significant (P < 0.01). When the recipients were preinoculated with methylcellulose, the number of donor cells that reached lymph nodes and spleen averaged 26 in the lymph nodes and 153 in the spleen. The difference between the carrageenan formulation-treated mice and methylcellulose-treated mice was significant (P < 0.05), whereas the difference between untreated mice and methylcellulose-preinoculated mice was not significant (P > 0.05). No fluorescent cells were observed in control mice that had been inoculated with frozen-thawed CMTMR-stained macrophages.
T Lymphocytes
The mesenteric, iliac, axillary, and mandibular lymph nodes were rapidly removed from donor mice and minced in RPMI 1640. Cell suspensions were prepared as previously described [25]. The cells were centrifuged at 400 x g for 10 min at 4°C and counted. The cell suspension was enriched for T cells by nonadherence to nylon fibers using a prepacked, sterile nylon fiber column (Polysciences,Warrington, PA) according to the established procedures [32]. Cell viability, as assessed by the PI exclusion test, was >90% (92–96%). Yield of T cells was determined by direct immunofluorescence using a monoclonal anti-CD3 (clone 29B) antibody, a pan T-cell marker (Sigma). One million cells eluted from the nylon column were washed in cold PBS, preincubated for 30 min in 90 µl of 0.2% mouse IgG in PBS containing 1% BSA, and incubated for 45 min in the same solution to which 4 µl of anti-CD3-fluorescein isothiocyanate (FITC) and 1 µl Hoechst 33342 had been added. The cells were kept at 4°C throughout immunolabelling. The cells were then thoroughly washed in PBS, fixed with 2% paraformaldehyde for 10 min at room temperature, washed in PBS, mounted, and examined with epifluorescence microscopy as indicated above. CD3-positive cells represented 95–97% of the cells eluted from the nylon fiber column.
Supravital Fluorochrome Labeling
The CellTracker Orange CMTMR dye was sterilely dissolved in anhydrous dimethyl sulfoxide, separated into aliquots, and stored as a 10 mM stock solution at -30°C. The stock solution was diluted to a final working concentration of 5 mM in RPMI. A total of 1.5 x 107 viable macrophages in RMPI 5 or 2.5 x 107 viable T cells were centrifuged at 400 x g for 5 min at 4°C and resuspended in 1 ml of CMTMR working solution prewarmed at 37°C. The cells were incubated for 30 min at 37°C in humidified 5% CO2. Staining was quenched by adding 10 ml of RPMI 5, and the cells were washed once with RPMI 5 and twice with RPMI without serum and used for inoculation immediately following a final viability test. Cell viability after fluorochrome staining, as assessed with the trypan blue exclusion test [33], was 90–93%.
Vaginal Inoculation
Immediately prior to inoculation, recipient mice were sedated with an i.m. injection of 0.2 ml of a ketamine/xylazine mixture containing 0.12 mg ketamine hydrochloride and 0.13 mg xylazine hydrochloride. Four x 106 viable CMTMR-stained cells were resuspended in a volume of 20 µl of RMPI 1640 and inoculated into the vagina by gently inserting a yellow pipette tip mounted on a P 20 Pipetman (Gilson, Villiers-le-Belle, France) into the vaginal vault. Four hours after inoculation, mice were killed by CO2 asphyxiation, and the vagina, spleen, and iliac and inguinal nodes were removed and processed for microscopic examination. Mice were killed at 4 h postinoculation because in previous experiments [25] discrete numbers of donor cells were already present in the iliac lymph nodes of the recipient at that time. However, there are data suggesting that the life span is very limited for mononucleated cells experimentally inoculated into the vagina or deposited after ejaculation [34]. To be confident that the fluorescence detected in the recipient tissues was not due to leakage of dye from inoculated cells, two control mice were inoculated with repeatedly frozen-thawed CMTMR-labeled cells.
Microscopic Examination
Vaginas were fixed with 2% paraformaldehyde in PBS for 2 h at room temperature, thoroughly washed in PBS, and infiltrated over a 12-h period through a sucrose gradient, from 10% to 30%, in PBS. Vaginas were then coated with O.C.T., quickly frozen in liquid nitrogen-cooled 2-methylbutan, and stored at -80°C until use. Cryostat sections of 10 µm were attached to slides coated with 0.01% polylysine (molecular weight >300 000), air dried, washed three times in PBS, mounted in Vectashield, and examined with an epifluorescence microscope with tetramethylrhodamine filters and phase contrast optics. Serial sections of the vaginas were also cut and examined to determine whether inoculation had produced lesions of the mucosa. No evidence of lesions was detected.
The iliac and inguinal lymph nodes and spleens were rapidly removed from recipient mice and cells suspensions prepared as previously described [25]. Erythrocytes were removed from spleen preparations using Erythrolyse according to the manufacturer's instructions. Cell suspensions were then washed in PBS, fixed in 2% paraformaldehyde for 10 min, rinsed in PBS, and counted. Iliac and inguinal lymph nodes yielded 2.2–5.2 x 106 and 0.9–1.6 x 106 cells, respectively. Spleens yielded 0.9–1.2 x 108 cells. Lymph node cells were resuspended in 20 µl Vectashield for microscopic examination. Cells from spleens were mounted at a final cell density of 1.5 x 106 cells/10 µl. Aliquots of 4 µl were spotted on eight ring slides and examined for the presence of fluorescent CMTMR-labeled cells. Because the spleen contains so many cells, a pilot study aimed at establishing the size of a representative sample was carried out. Volumes representing fractions of increasing sizes, from 5% to 33% of the total cell suspension, were examined, fluorescent cells were counted, and their number was rounded off to the hypothetical total number of fluorescent cells present in the whole spleen preparation. When the sample size was increased from 20% to 25% and to 30% of the volume of the cell suspension, the differences in the evaluation of the total number of CMTMR-labeled cells were lower than ±10%. We concluded that a volume corresponding to 33% of the spleen suspension could be safely regarded as representative of the whole suspension. The cell suspensions were examined with epifluorescence microscopy and phase microscopy as indicated above.
Carrageenan Formulation and Transepithelial Migration
To study the effect of the carrageenan formulation on macrophage migration, mice were vaginally preinoculated with 20 µl of the carrageenan formulation 20 min prior to macrophage inoculation. Control mice were inoculated with 20 µl of a methylcellulose formulation [27, 35] before inoculation with macrophages. The 20-min pretreatment with the carrageenan formulation or with methylcellulose was chosen to allow spreading of the formulations over the vaginal surface. A similar time point had been previously employed when the formulations were tested for efficacy in protecting mice from herpes simplex virus type 2 infection [27, 35]. The effect of the carrageenan formulation and of methylcellulose on macrophage migration to lymphoid organs was evaluated with an unpaired t-test and one-way ANOVA, using the program MultiStat 1.1 (BioSoft, Cambridge, U.K.). A P value of 
0.05 was considered significant.
Localization of the Carrageenan Formulation in the Vagina
Vaginas from mice inoculated with the carrageenan formulation and CMTMR-stained macrophages were fixed with paraformaldehyde, processed, and quick-frozen. Cryostat sections of 10 µm were attached to aminoalkysilane-coated slides (Sigma), air dried, and washed five times in PBS for a total period of 30 min. The sections were preincubated for 15 min in 0.2% BSA in PBS, incubated in 5 µg/ml polylysine-FITC (molecular weight 15 000) in PBS/BSA for 30 min at room temperature in the dark, rinsed with PBS, and mounted in Vectashield for microscopy. Two types of negative controls were performed: 1) sections of vagina from mice inoculated only with CMTMR-stained macrophages were labeled with polylysine-FITC as indicated above, and 2) sections of vaginas from mice inoculated with the carrageenan formulation and macrophages were preincubated in 5 µg/ml of unlabeled polylysine for 30 min and thoroughly washed with PBS before treatment with polylysine-FITC.
Cytotoxicity
The potential cytotoxic effect of the carrageenan formulation and of methylcellulose on macrophages and on epithelial cells was evaluated using the MTT-based colorimetric test according to the procedures of Niks and Otto [36]. Elicited peritoneal macrophages (37 x 104) in 625 µl of phenol red-free RPMI 1640 supplemented with 2 mM glutamine, 10% heat-inactived FBS, and 50 µg/ml gentamicin (RPMI 10) were plated in 24-well flat-bottom microwell plates (Corning, Ithaca, NY) and maintained at 37°C in humidified 5% CO2. After 1 h, the wells were washed three times with warm medium to remove nonadherent cells and were then incubated in 625 µl of a mixture of equal volumes of the carrageenan formulation or methylcellulose and RPMI 10. After 2 or 4 h, the carrageenan formulation and methylcellulose mixtures were gently aspirated and the wells thoroughly washed four times with warm RPMI 10. The wells were then incubated for 1 h at 37°C in 625 µl of a 0.5-mg/ml MTT solution in RPMI 10. At the end of the incubation time, 625 µl of 20% SDS in 50% N, N-dimethylformamide, pH 4.7, was added to each plate to solubilize the formazan product. Solubilization was carried out for 30 min at room temperature, with continuous and vigorous mixing [36]. The optical density of the solubilized formazan from each well was measured immediately at a wavelength of 570 nm, with subtraction of a blank (medium with SDS in N, N-dimethylformamide) and with background subtraction at 630 nm. As controls, 37 x 104 cells were incubated in RPMI 10 for the same time intervals and washed with fresh medium as many times as the experimental wells before undergoing the MTT assay.
Cytotoxicity tests on epithelial cells were carried out using the human cervical epithelial cell line ME-180 (ICLC, Genoa, Italy). Exponentially growing cells were seeded into 24-well plates at a density of 2 x 105 cells/well and incubated in phenol red-free RPMI 10 for 24 h at 37°C. On the day of treatment, culture medium was replaced with the carrageenan formulation and methylcellulose mixtures as indicated above. After 2 and 4 h of incubation, the MTT assay was carried out as described above.
All tests were performed in triplicate. Reproducibility between replicates was high, with SEMs of <10%. Results are presented as mean ± SEM of n independent experiments (indicated in the figure captions). Analysis of the results was performed with a paired t-test and one-way ANOVA. A P value of 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We examined cryostat sections for the presence of fluorescent cells 4 h following insertion of CMTMR-stained macrophages or T cells. We labeled mononuclear cells with CellTracker Orange CMTMR because this relatively new supravital dye is easily loaded into the cells, where it reacts with intracellular proteins via glutathione-S-transferase and forms adducts that are fixable with aldehydes [37]. Long-term in vitro studies have demonstrated excellent cell retention and low cytotoxicity [38, 39]. In our model, this dye was an excellent supravital dye for tracking adoptively transferred mononuclear cells. It stained 100% of cells, although the intensity of staining of macrophages was somewhat heterogeneous. Its fluorescence was only minimally quenched by aldehyde fixation. Therefore, its compatibility with fixation represented a great asset for microscopic analysis of the recipient tissues. Donor cells were observed in the vaginal epithelium and through the connective tissue of the lamina propria and submucosa. Fluorescent cells were observed migrating through the mucosa at the vaginal vault, where the epithelium is made up of one or two layers of cuboidal or columnar cells (Fig. 1A), and at more caudal levels, where the vaginal canal is lined by a columnar multilayered epithelium (Fig. 1B).
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Mononuclear Cells in Secondary Lymphoid Organs
Fluorescent cells of the donor were easily distinguishable in the cell suspensions obtained from the lymph nodes draining the vagina and from the spleens of the experimental animals (Fig. 1C). All the recipients were positive, although there was considerable variation in the number of fluorescent cells among the mice. Donor cells were present in both the iliac and the inguinal lymph nodes. When we inoculated macrophages intravaginally, the recipient animals had an average of 55 labeled macrophages in the draining lymph nodes and an average of 558 labeled macrophages in the spleen (Table 1). When we inoculated T cells intravaginally (n = 4), the number of the donor cells detectable in the draining lymph nodes ranged from 15 to 48, with an average of 31, whereas in the spleen suspensions the number of the donor cells ranged from 291 to 750, with an average of 519. The difference in the number of macrophages and the number of T cells homing to the spleen and lymph nodes was not significant (P > 0.05). No fluorescent cells were observed in mice that had been inoculated with frozen-thawed CMTMR-stained mononuclear cells.
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In mice that received vaginal preinoculation treatment with the carrageenan formulation before macrophage inoculation, an average of only 4 labeled cells were counted in the draining lymph nodes and an average of only 28 labeled cells were observed in the spleen (Table 1). The difference in average number of labeled cells between untreated mice and those pretreated with the carrageenan formulation was highly significant (P < 0.01). When the recipients were preinoculated with methylcellulose, the number of donor macrophages that reached lymph nodes and spleen averaged 26 and 153, respectively (Table 1). These averages were lower than those in animals that were inoculated only with macrophages. However, the difference in the number of migrated cells between untreated mice and methylcellulose-treated mice was not significant (P > 0.05). The number of donor cells that migrated to the local lymph nodes and to the spleen of recipients preinoculated with methylcellulose was significantly higher than that in recipients preinoculated with the carrageenan formulation (P < 0.05).
Cytotoxicity
The MTT assay was carried out using an in vitro system to ascertain whether the inhibition of vaginal transmigration of macrophages was due to a cytotoxic effect of the formulations. We carried out this test on both the elicited peritoneal macrophages and on the human cervical epithelial cell line ME 180, using the same concentrations of compounds as those used in the experimental animals. After 2 and 4 h of incubation, viability of macrophages treated with the carrageenan formulation and with methylcellulose was lower than that in matched controls (P < 0.05). However the difference in viability between the carrageenan formulation-treated and methylcellulose-treated cells was not significant (P > 0.05) (Fig. 2a). Neither of the compounds had a significant effect on the viability of ME180 cells (P > 0.05) (Fig. 2b).
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Localization of Carrageenan
To investigate the possibility that the mechanism of action of the carrageenan formulation was due to binding of carrageenan to macrophages or to vaginal epithelium, we examined cytochemical localization of carrageenan. Because carrageenan is water soluble and is not fixable by aldehydes, the technique detects only firmly bound carrageenan. Carrageenan was observed on both the surface of macrophages and the lumenal surface of the vaginal epithelium (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of the present study extend our previous findings and those of other investigators. We used populations of macrophages and T cells that were almost homogeneous, whereas all the previous studies employed mononuclear cell populations of a mixed or undetermined composition [24–26]. The conditions we applied indicate that inflammatory macrophages and T cells have similar intrinsic capacities for migration. We found that both types of mononuclear cells placed in the vagina can migrate to the regional lymph nodes and can enter the bloodstream of the recipient and home to the spleen. The numerical data suggest that the donor cells, after migrating into the regional lymph nodes, recirculate quickly. En route from the recipient vagina to lymphoid organs and ultimately to the bloodstream, a single infected donor cell is likely to interact with and infect several CD4-positive lymphocytes and cells of the monocytic lineage. Vaginal transmigration of mononuclear cells, therefore, may enhance dissemination of the HIV infection to target cells of the host.
In the second part of this study, we used macrophages rather than T lymphocytes to study a possible effect of the carrageenan formulation on mononuclear cell migration, because a large body of evidence suggests that M-tropic viruses are preferentially transmitted during sexual contact. We found that significantly fewer macrophages reached the lymph nodes and spleen when the carrageenan formulation was placed in the vagina prior to inoculation of macrophages. We used methylcellulose as a control because this compound is not toxic and has a viscosity similar to that of the carrageenan formulation [35].
Several mechanisms may account for blocking of vaginal transmigration by the carrageenan formulation. Toxicity is a possibility, especially because macrophages are exposed to the carrageenan formulation for 4 h. Both the carrageenan formulation and methylcellulose inhibited trafficking compared with the PBS control. However, because both the carrageenan formulation and methylcellulose were equally cytotoxic, cytotoxicity probably was only partly responsible for inhibition of cell trafficking. Neither the carrageenan formulation nor methylcellulose was cytotoxic to human cervical cells. Another possibility is that the viscosity of the vaginal formulations trapped macrophages and thus prevented them from migrating from the vagina. However, this scenario is unlikely because the methylcellulose control formulation and the carrageenan formulation have a similar viscosity, but the inhibitory effects of the two formulations differ significantly.
The carrageenan formulation appears to have a specific effect on cell migration. Because the carrageenan formulation is primarily composed of lambda carrageenan, which has three sulfate groups per disaccharide unit, it may bind to the surface of macrophages and epithelial cells through interactions between anionic/cationic surface charges and prevent the cells from binding to each other. To investigate this possibility, we used cytochemical stains on sections of vagina of recipient mice after inoculation of the carrageenan formulation and macrophages. The carrageenan formulation bound to both the surfaces of the vagina and macrophages. This finding suggests that the carrageenan formulation prevents cell trafficking from the vaginal mucosa by coating the surfaces of cells with a negatively charged coat that prevents adhesion of macrophages to the epithelial surface. The mucus present in the vagina did not interfere with the binding of the carrageenan formulation to the macrophages and epithelium surfaces.
Cell trafficking is only one of several theories of how HIV is sexually transmitted. Thus, the finding that the carrageenan formulation blocks cell trafficking from the vaginal lumen is not an assurance that it will prevent sexual transmission of HIV. However, because clinical trials of microbicides are problematic and costly, it is prudent to enter into efficacy trials with a candidate that has a broad spectrum of activities and that acts on systems that represent the different plausible modes of HIV transmission.
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FOOTNOTES |
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2 Correspondence: David M. Phillips, The Population Council, 1230 York Ave., New York, NY 10021. FAX: 212 327 7678; dphillips{at}popcouncil.org
Received: 13 December 2002.
First decision: 31 December 2002.
Accepted: 6 March 2003.
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  G. N. Milligan, C.-F. Chu, C. G. Young, and L. R. Stanberry Effect of Candidate Vaginally-Applied Microbicide Compounds on Recognition of Antigen by CD4+ and CD8+ T Lymphocytes Biol Reprod, November 1, 2004; 71(5): 1638 - 1645. [Abstract] [Full Text] [PDF]
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