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Simulation with lifelike mannequins is used in schools of nursing and hospital-based education as a method of teaching clinical content, enhancing clinical skills, applying theory to practice, and validating competency. It provides a safe learning environment to enhance nurses' clinical judgment and critical thinking skills in an increasingly complex care environment. Simulation can be used in the practice setting with experienced nurses to teach or reinforce complex information and allow the learner to practice without devastating consequences. Medical-surgical units in some institutions have dedicated beds for patients with cancer but may not be a full oncology unit. Evaluating chemotherapy and biotherapy competency is difficult when extensive time periods exist between chemotherapy administrations. One method for assessing annual chemotherapy competency is to use simulation.

The use of high-fidelity simulation as an accepted substitute for traditional clinical learning experiences in nursing education has gained acceptance over the past decade, as evidenced by the California Board of Registered Nursing now allowing up to 25% of student clinical learning to occur in simulation laboratories. However, little research evidence has documented the efficacy of these simulated learning experiences, particularly on objective outcomes such as examination performance. Therefore, this study examined the effects of high-fidelity simulation participation on knowledge acquisition in 104 undergraduate nursing students. Students who participated in high-fidelity simulation scenarios scored significantly higher on examinations than students who did not. These findings provide beginning evidence that high-fidelity simulation can be an effective substitute for traditional clinical experience. More importantly, the findings may help boards of nursing more effectively regulate the use of high-fidelity simulation in the future., (Copyright 2012, SLACK Incorporated.)

Human patient simulation provides students with experiences and skills they might not otherwise encounter in a clinical rotation. It also offers an experience during which the time is suspended, thus affording students time to think critically, make decisions, and act, as opposed to the fast-paced hospital environment where students may have neither a clear picture of the situation nor adequate time to act. This article presents the design of a simulation center within a school of nursing along with several areas of considerations for successful implementation of the laboratory. A simulation scenario focusing on the acute coronary syndrome used during a final semester critical care nursing course is described and student evaluation of the experience analyzed. The evaluation includes student assessment of the simulation process for the development of necessary patient care skills and the ability to test decision-making and critical thinking skills. The experience with the initial integration of simulation into the nursing curriculum is discussed, inclusive of opportunities for improvement.

An adult mouse model for studies of latency and recurrence after vaginal HSV-2 infection is not available at present, largely because the infection kills most mice within 14 days. We describe here an antiviral therapy that rescues most vaginally infected mice from death. Vaginally infected mice were nearly all rescued by combined treatment with one dose of monoclonal anti-HSV glycoprotein D 3 days after infection plus valacyclovir in the drinking water on days 3, 4, 5, 7, 9, 11, 13, and 15 after infection. At 60 days after infection, PCR measurements revealed that most rescued mice had viral DNA in their lumbosacral dorsal root ganglia, lumbosacral spinal cords, and paracervical autonomic ganglia, consistent with the possibility that latent infections were established. At this time, immunolabeling revealed CD45+ lymphoid cells in these neural tissues in rescued mice but not in normal control mice. In vivo depletion of T lymphocytes with monoclonal antibodies caused a recurrence of herpes illness symptoms earlier and in a larger proportion of rescued mice than was observed in non-depleted rescued mice. Interestingly, many rescued mice (46/114) spontaneously developed a syndrome of typical herpes illness symptoms that began with ruffled fur on a mouse that previously had sleek fur and progressed to arched backs, feeble gait, hindlimb paralysis, and death or euthanasia, or in some cases to recovery to health. This high incidence of apparent spontaneous recurrence of HSV-2 infection in rescued mice suggests that it may be possible, with some refinement of the procedure, to obtain an effective adult mouse model for studies of therapeutic vaccination to inhibit or prevent HSV-2 recurrence after genital tract infection.

Female mice have been used extensively to study mucosal immunity against herpes simplex virus type 2 (HSV-2) infection of the vagina, but comparatively little is known about the spread of this virus to other tissues. Here the authors have used immunolabeling to demonstrate that HSV-2 infected the vaginal epithelium; the epithelium covering the vulva, perineum, and anal canal; and perineal hair follicles and sebaceous glands. The kinetics and basal localization of the immunolabeling indicated that the virus spread horizontally within the epithelial layer, starting in the vagina and then proceeding to the distal epithelial sites. HSV-2 also spread from the vagina to multiple neuronal sites including the paracervical ganglia (PCG), which are the major autonomic ganglia of the pelvis. The authors demonstrated both sympathetic and parasympathetic neurons in the PCG by labeling of acetylcholinesterase and tryosine hydroxlyase, and noted that infection was limited mainly or entirely to parasympathetic neurons. Infection of the PCG was correlated with the presence of virus in the autonomic ganglia in the walls of the rectum and urinary bladder, which in turn correlated with distention of these organs and retention of urine and feces. HSV-2 infection was also detected in cell bodies and axons in the lumbosacral sympathetic chain, in lumbosacral dorsal root ganglia, and in the dorsal portions of the lumbar spinal cord. Collectively, the data show that vaginal HSV-2 infection in mice leads to subsequent infection of multiple neural and epithelial sites. This information should be useful for development of a mouse model that can be used to study HSV-2 latency and for development of therapeutic vaccines to prevent recurrent infections.

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that infects the genital tract. Efforts to develop vaccines to protect women against this and other sexually transmitted pathogens would be facilitated by a better understanding of the immune mechanisms that protect the female reproductive tract against such infections. Such information would be invaluable in developing vaccine strategies to promote the type and magnitude of immune responses in the genital tract that would effectively protect against infection. This review focuses on recent studies using a progestin-treated adult mouse model to explore mucosal immunity to HSV-2 in the vagina. Evidence indicating a major role for both humoral and T cell immunity is presented.

We investigated the involvement of antibody in protection against vaginal herpes simplex virus type-2 (HSV-2) infection by comparing intact and B-cell knockout (KO) mice. Vaginal immunization of intact mice with attenuated HSV-2 markedly reduced an HSV-2 challenge infection in the vagina. In contrast, immunization of B-cell KO mice produced less immunity against the challenge infection and that immunity occurred in a different pattern. At 20 hr after challenge, immunostaining of virus proteins in the vaginal epithelium and shed virus protein titres in the vaginal secretions were not significantly different between immunized and non-immunized B-cell KO mice and were much greater than in immunized intact mice. At 48 hr after challenge, the vaginal infection in immunized B-cell KO mice was markedly less than at 20 hr but remained approximately sevenfold higher than in intact mice. This pattern of challenge infection in the vagina indicates that B cells, and probably the antibody derived from them, provided significant protection against reinfection in intact mice, especially during the first 20 hr after challenge, while other effector mechanisms became important between 20 and 48 hr after challenge. To determine whether T-cell immunity in immunized B-cell KO mice was equal to that in intact mice, we assessed interferon-gamma (IFN-gamma) secretion by memory T cells in vivo in the vagina at 20 hr after challenge. We found no significant differences in the up-regulation of major histocompatibility complex (MHC) class II antigens in the epithelium, up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in vascular endothelium, or recruitment of T cells to the mucosa, indicating that the memory T-cell response to virus challenge was the same in intact and B-cell KO mice.

Lymphocyte recruitment into tissues involves interactions between adhesion molecules on vascular endothelial cells and corresponding ligands on the lymphocyte surface. In the present study we investigated the expression of four endothelial addressins in the vagina and their possible up-regulation by interferon-gamma (IFN-gamma) in immune mice after vaginal challenge with herpes simplex virus type 2. The adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were minimally expressed in the vagina of non-immune mice with or without vaginal challenge and in immune mice before challenge, but both were up-regulated by IFN-gamma, directly or indirectly, in immune mice after challenge. Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was detected in most vaginas but was not up-regulated by IFN-gamma in immune mice after virus challenge. E-selectin was not detected in any vaginas. The results suggest that ICAM-1 and VCAM-1 may be involved in rapid, IFN-gamma-mediated recruitment of lymphocytes to the vaginal mucosal of immune mice after local virus challenge.

We compared nasal and vaginal immunizations using attenuated herpes simplex virus type-2 (HSV-2) for protection against vaginal infection with wild-type HSV-2. Mice were immunized once intranasally, intravaginally after progestin (DP) treatment, or intravaginally with scarification after oestradiol treatment. Compared with vaginal immunizations, nasal immunization did not increase immunoglobulin A (IgA) plasma cell numbers in the vagina or elicit a higher antiviral IgA titre in vaginal secretions. Both types of vaginal immunizations increased the number of immunoglobulin G (IgG) plasma cells in the vagina and the secretion/serum titre ratio of IgG antiviral antibody, indicating local production of virus-specific IgG in these groups. Cell-mediated immunity in the vagina, as indicated by memory T-cell secretion of interferon-gamma (IFN-gamma) in situ 20 hr after HSV-2 challenge, was essentially equivalent in the vaginally immunized groups but significantly lower in the nasal group, while lymphocyte recruitment to the vagina was similar in all three groups. All three immunizations protected all mice from neurological disease after challenge, but vaginal DP immunization induced the greatest immunity against reinfection of the vaginal epithelium.

We investigated the role of interferon gamma (IFN-gamma) in a mouse model of immunity to vaginal infection by herpes simplex virus type 2 (HSV-2). Within 8 h after immune mice were challenged intravaginally with HSV-2, IFN-gamma concentrations in vaginal secretions reached levels that can be antiviral in vitro. This rapid synthesis of IFN-gamma occurred in immune-challenged mice but not in nonimmune-challenged mice, indicating that it required memory T cells. Immunostaining and in situ hybridization revealed that the IFN-gamma was synthesized by cells whose morphological appearance suggested that they were lymphocytes and macrophage-like cells in the mucosa. The presence of IFN-gamma in vaginal secretions was correlated with upregulation of MHC class II antigens in the epithelium and with vigorous (30-fold) recruitment of T and B lymphocytes into the vagina. In vivo administration of anti-IFN-gamma to immune mice 17 h before virus challenge blocked the subsequent appearance of IFN-gamma in vaginal secretions, blocked upregulation of class II antigens, blocked adherence of T cells to endothelium and their recruitment into the vagina, and markedly reduced immunity against reinfection of the vaginal epithelium., (Copyright 1999 Academic Press.)

An immunoglobulin A (IgA) knockout (KO) mouse was used to study the role of IgA in protective immunity against vaginal infection with herpes simplex virus-type 2 (HSV-2). Intact and KO mice were immunized intravaginally (IVAG) with attenuated HSV-2, challenged IVAG with wild-type virus 6 weeks later and evaluated for vaginal infection and neurological disease. Non-immunized/challenged intact and KO mice showed vaginal infection and succumbed to neurological disease, while immunized/challenged mice exhibited reduced or no vaginal infection and no neurological disease. Log 2.5 enzyme-linked immunoassay (ELISA) titres of viral IgA, immunoglobulin G (IgG) and immunoglobulin M (IgM) in vaginal secretions collected from intact immune mice before challenge were 0.6+/-0.3, 6.4+/-0.32 and 0.0, while those in KO immune mice were 0.0, 6.7+/-0.19 and 3.0+/-0.29, respectively. Twenty-four hours after challenge, the percentage of vaginal epithelium that was infected in non-immune intact and KO mice was 2.0+/-0.6 and 2.4+/-0.6, which was reduced to 0.2+/-0.1 and 0.1+/-0.06 in immune intact and KO mice, respectively. No shed virus protein was detected in vaginal secretions 3 days after challenge in any immune mouse, whereas titres were 1400 and 1700 in the two groups of non-immune mice. Thus, immune protection against vaginal HSV-2 infection was similar in both KO and intact mice, indicating that this mucosal immunity does not depend mainly on IgA.

This investigation evaluated immunity to vaginal herpes simplex virus type 2 (HSV-2) infection after local or parenteral immunization with attenuated HSV-2. Vaginal immunization induced sterilizing immunity against challenge with a high dose of wild-type virus, whereas parenteral immunizations protected against neurologic disease but did not entirely prevent infection of the vagina. Vaginal immunization caused 86- and 31-fold increases in the numbers of immunoglobulin G (IgG) plasma cells in the vagina at 6 weeks and 10 months after immunization, whereas parenteral immunizations did not increase plasma cell numbers in the vagina. Vaginal secretion/serum titer ratios and specific antibody activities in vaginal secretions and serum indicated that IgG viral antibody was produced in the vagina and released into vaginal secretions at 6 weeks and 10 months after vaginal immunization but not after parenteral immunizations. In contrast to the case for plasma cells, the numbers of T and B lymphocytes in the vagina were similar in vaginally and parenterally immunized mice. Also, lymphocyte numbers in the vagina were markedly but similarly increased by vaginal challenge with HSV-2 in both vaginally and parenterally immunized mice. Lymphocyte recruitment to the vagina after virus challenge appeared to involve memory lymphocytes, because it was not observed in nonimmunized mice. Thus, local vaginal immunization with attenuated HSV-2 increased the number of IgG plasma cells in the vagina and increased vaginal secretion/serum titer ratios to 3.0- to 4.7-fold higher than in parenterally immunized groups but caused little if any selective homing of T and B lymphocytes to the vagina.

Intravaginal (IVAG) inoculation of wild-type herpes simplex virus type 2 (HSV-2) in mice causes epithelial infection followed by lethal neurological illness, while IVAG inoculation of attenuated HSV-2 causes epithelial infection followed by development of protective immunity against subsequent IVAG challenge with wild-type virus. The role of T cells in this immunity was studied by in vivo depletion of these cells with monoclonal antibodies. Three groups of mice were used for each experiment: nonimmune/challenged mice, immune/challenged mice, and immune depleted mice [immune mice depleted of a T-cell subset(s) shortly before challenge with HSV-2]. Mice were assessed for epithelial infection 24 h after challenge, virus protein in the vaginal lumen 3 days after challenge, and neurological illness 8 to 14 days after challenge. Monoclonal antibodies to CD4, CD8, or Thy-1 markedly reduced T cells in blood, spleen, and vagina, but major histocompatibility complex class II antigens were still partially upregulated in the vaginal epithelium after virus challenge, indicating that virus-specific memory T-cell function was not entirely eliminated from the vagina. Nevertheless, immune mice depleted of CD4+ and CD8+ T cells, Thy-1+ T cells, or CD8+ T cells alone had greater viral infection in the vaginal epithelium than nondepleted immune mice, indicating that T cells contribute to immunity against vaginal HSV-2 infection. All immune depleted mice retained substantial immunity to epithelial infection and were immune to neurological illness, suggesting that other immune mechanisms such as virus-specific antibody may also contribute to immunity.

Progestin-treated female mice are susceptible to vaginal infection by two sexually transmitted disease organisms: herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis. Vaccination of mice with HSV-2 or chlamydial antigens elicits immunity to vaginal infection that may be due in part to secreted antibodies in the vaginal lumen. Analysis of the role of these antibodies in immunity would be aided by information about the vaginal secretion in progestin-treated mice and the antibodies it contains. Gross and histologic observations of progestin-treated mice that were immune to vaginal HSV-2 infection indicated that the vaginal lumen was filled with mucus. A procedure for extraction of immunoglobulin from the mucus was developed and shown to recover at least 98% of the secretory IgA (S-IgA) that was free to diffuse from the mucus. Immunoblotting revealed that the predominant molecular form of IgA in vaginal mucus was dimeric S-IgA. Immunoglobulin concentrations in vaginal secretions were higher in immune mice than in non-immune mice and S-IgA concentrations were higher than those of IgG. The IgG concentration in vaginal secretions of immune mice was 4.5-fold higher than in non-immune mice, while serum IgG increased only 1.5-fold, suggesting local production of IgG or increased transudation in immune mice. Specific IgG antibody to HSV-2 was demonstrated in vaginal secretions of immune mice at a mean ELISA titer of 6200, whereas the titer of specific S-IgA in the same secretions was only 1.9. Thus, while the predominant immunoglobulin by weight in the vaginal mucus of immune mice was S-IgA, the ELISA titers suggested that the virus-specific antibody was almost entirely IgG.

To determine whether lymphocytes and Langerhans cells in vaginal epithelium are migratory, we stained mouse vaginal epithelium, including its lymphoid cells, by intraluminal administration of H33342, a fluorescent, vital dye. Stromal staining was superficial, and no free dye reached the iliac lymph nodes. The numbers and phenotypes of H33342-stained cells that migrated from the vagina to the iliac lymph nodes during the next 48 h were determined in four groups: normal mice, mice infected intravaginally with wild-type herpes simplex virus type 2 (HSV-2), mice that were immune to vaginal HSV-2 infection, and immune mice that received vaginal challenge with HSV-2. H33342-stained cells migrated from the vaginal epithelium to the iliac lymph nodes in all groups and were mainly Thy-1.2+ cells and B220+ cells. The number of migrating Thy-1.2+ cells was similar to the sum of CD4+ and CD8+ cells in all groups and was not significantly different from the number of CD44+ cells, suggesting that most of the migrating T cells were memory cells. B lymphocytes comprised 31, 32, 43, and 68% of the migrating cells in the four groups, respectively. We found no evidence that Langerhans cells or macrophages were migrating. Thus, most MHC class II+ cells in all groups were accounted for by B cells, and migrating cells did not express B7.1 or F4/80 or exhibit indented nuclei or dendritic processes. We suggest that the migrating T cells and B cells probably belonged to a pool of lymphocytes that recirculates from blood to tissues and back to the lymph nodes via their afferent lymphatics.

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that infects the genital tract. The high prevalence of HSV-2 in humans underscores the need to develop an effective vaccine. Efforts to develop vaccines to protect women against this and other sexually transmitted pathogens would be facilitated by a better understanding of the immune mechanisms that protect the female reproductive tract against infections in animal models. Such information would be invaluable in developing vaccine strategies to promote the type and magnitude of immune responses in the genital tract that would effectively protect against infection. This review focuses on recent studies using a progestin-treated adult mouse model to explore mucosal immunity to HSV-2 in the vagina. Evidence indicating a major role for both humoral and T cell immunity is presented.

We investigated the protective role of antibodies in vaginal secretions of mice that were immune to vaginal challenge with herpes simplex virus type 2 (HSV-2). Unfractionated vaginal immunoglobulins from immune and nonimmune mice and affinity-purified immunoglobulin G (IgG) and secretory IgA (S-IgA) from immune secretions were adjusted to their concentrations in vivo. Wild-type HSV-2 was incubated in the immunoglobulin preparations for 15 min in vitro, followed by inoculation into vaginae of nonimmune mice. HSV-2 was neutralized by unfractionated antibody and purified IgG from immune secretions but not by unfractionated nonimmune antibody or by purified immune S-IgA. The protective effect of IgG in vivo was investigated by passively transferring purified serum IgG from immune and nonimmune donors to nonimmune recipients before vaginal challenge infection. Immune IgG significantly reduced the percentage of vaginal epithelium infected, concentrations of shed virus protein in the vaginal lumen, and illness scores, even though the viral antibody titers in serum and vaginal secretions of recipient mice at the time of challenge were only 29 and 8%, respectively, of those in actively immunized mice. Additionally, removal of vaginal secretions from immune mice 10 min before vaginal challenge with HSV-2 significantly increased the concentration of shed virus protein in the vaginal lumen after challenge. Collectively, the data indicate that IgG antibody in vaginal secretions of immune mice provides early protection against vaginal challenge infection, probably by neutralizing virus in the vaginal lumen. In contrast, S-IgA antibody contributed relatively little to immune protection of the vagina.

The mode of heterosexual transmission of human immunodeficiency virus (HIV) is not yet understood. The semen of HIV-infected men contains free virus and infected cells, and it is not known which of these is more important for sexual transmission of the virus to women. Some investigators have presented in vitro studies supporting a cellular mode of transmission of HIV and have suggested that infected lymphoid cells may act as the primary source of infection. This has become known as the "Trojan Horse" hypothesis. In vivo demonstrations of such events are lacking and are not likely to be forthcoming using human subjects. To investigate the ability of normal lymphoid cells to invade the cervicovaginal mucosa in an experimental animal, we stained C3H/He (H-2Kk) mouse peritoneal lymphoid cells with bisbenzimide, a vital fluorescent DNA-binding dye, and inoculated the cells atraumatically into the vaginas of progestin-treated, BALB/c (H-2Kd) recipient mice. Donor cells were identified in recipient tissues by their bisbenzimide-fluorescent nuclei and by fluorescein staining of the membrane antigen, H-2Kk. Donor lymphoid cells were observed in histological sections of recipient cervicovaginal mucosa and also in the iliac lymph nodes of 34 of 36 recipient mice 24 h after inoculation into the vagina. The number of donor cells in the iliac lymph nodes was 8.6 +/- 1.4 (mean +/- SEM) cells per mouse with a range of 0-35 cells per mouse. Approximately 28% of the donor lymphoid cells in recipient lymph nodes expressed CD4, which in humans is the receptor for HIV. We did not detect F4/80, a marker of mature mouse macrophages in the donor cell population, on any of the migrating cells in recipient lymph nodes. However, this negative result is equivocal, because the marker might be down-regulated after transfer or the migrating macrophages might be difficult to dissociate from the recipient lymph node tissue. These observations in mice support the suggestion that HIV-containing lymphoid cells in the semen of infected men may invade the cervicovaginal mucosa after sexual intercourse and deliver the virus to a woman's internal environment. However, both the donor cells and the recipient reproductive tract of the mice in the present study differed in significant respects from their counterparts in humans that might be involved in heterosexual HIV transmission. Further studies are needed to determine whether this possible mode of virus transmission is mainly responsible for heterosexual transmission of HIV in humans.

Advanced practice nursing in a managed care environment--is this an oxymoron? Can these two phrases exist together? Do advanced practice nurses have a role in a managed care environment? As health-care systems continue to merge and managed care impacts the bottom line, advanced practice nurses are challenged to show their worth. In this article, the author reviews the impact managed care has had on the roles of two master's-prepared traditional clinical nurse specialists. Included are examples of how the roles were adapted to fit in the managed care setting. The current role of the advanced practitioners is reviewed, using the common subroles as a basis for discussion. The impact of managed care on each component of the clinical nurse specialist role is identified. The author also demonstrates that advanced practice nurses can have a valuable role in the managed care environment.

An important factor limiting better understanding of the protective role of sIgA at mucosal surfaces is the limited availability of the purified immunoglobulin. Among other things, purified sIgA is needed for use as a standard in measurements of the concentration of this immunoglobulin in mucosal secretions, particularly in mice, where several models of mucosal infections are available. We describe here a simple method by which one can obtain a mean of 3.5 ml of milk per mouse without a breast pump. Immunoblotting studies after native PAGE demonstrated that the milk contained mainly 420 kDa dimeric sIgA and higher polymeric forms of sIgA; only a trace of monomeric IgA was present. Similar immunoblotting studies after SDS-PAGE revealed that a portion of the sIgA was dissociated by this treatment. The 420 kDa sIgA was purified by salt fractionation, gel filtration, and affinity chromatography, and the purity of the final product was demonstrated by immunoblot analysis of biotinylated polypeptides after reduction of biotinylated protein. The concentration of 420 kDa sIgA in whey was measured by densitometry of immunoblot bands, using the purified 420 kDa sIgA as a standard, and found to be 1.0 +/- 0.3 mg/ml.

The synthesis and cellular localization of tumor necrosis factor-alpha (TNF-alpha) were studied in mouse GMG cells, which are activated NK cells in uterine decidual tissue during pregnancy. Synthesis of the protein was demonstrated in GMG cells on days 10 and 14 of pregnancy by in situ hybridization of TNF-alpha message. Immunostaining demonstrated that TNF-alpha protein was localized in the cytoplasmic granules of GMG cells at these times. The results suggest that the cytolytic activity of uterine NK cells may be due in part to TNF-alpha, and that this cytokine may be delivered to target cells intracellularly via transmembrane pores formed by perforin, which is also localized in uterine NK cell granules.

The present study describes the effects of mating on urethral gland acinar cells in male mice. Histological and morphometric analysis demonstrated that there was a depletion of secretory granules in the urethral glands during mating. However, no change occurred in the rough endoplasmic reticulum containing tubular elements. The results indicate that the urethral glands are functional during mating. The timing of their granule depletion suggests that urethral gland secretions may contribute to the formation of semen or the copulation plug.

Background: The role of mucosal immunity in defense of the female genital tract against pathogens such as herpes simplex virus-2 (HSV-2) is poorly understood. Here we explored the use of a new mouse model to determine whether local immune events in the vagina of immune animals may protect them against genital herpes., Experimental Design: The effect of the estrous cycle, pregnancy, and sex hormones on vaginal infection of adult mice by HSV-2 was determined by immunolabeling of virus proteins. The immune response to infection was studied by immunolabeling of T lymphocytes, B lymphocytes, and plasma cells in the vagina of infected mice., Results: Inoculation of attenuated virus (TK-HSV-2) or wild-type virus (TK+HSV-2) into the vagina on day 6 of pregnancy or after treatment with Depo-Provera (DP) caused infection of the vaginal epithelium. In contrast, these viruses did not cause infection after vaginal inoculation at estrus, metestrus, or after treatment with Depo-Estradiol. Infected mice showed immunolabeling of virus in the vaginal epithelium from 24 hrs to 5 days after virus inoculation. The immune response to infection included upregulation of class II MHC antigen in vaginal epithelium, CD8+ T cells in epithelium and stroma, and plasma cells and lymphoid nodules in the stroma. Mice that were infected with TK-HSV-2 did not exhibit infection of vaginal epithelium when challenged 6 weeks later with TK+HSV-2., Conclusions: Progesterone-dominated adult mice become infected after intravaginal inoculation with HSV-2, but estradiol-dominated mice are refractory. Vaginal infection with attenuated HSV-2 produces immunity that protects mice against later infection by wild-type virus. This immunity either prevents infection of vaginal epithelium or severely inhibits viral replication in the epithelium. The observations suggest that the E/DP-treated adult mouse should be a useful model for studies of mucosal immunity to vaginal infection by HSV-2.

Recent studies of the urethral glands in the male mouse and rat have suggested that they are testosterone-dependent glands that may be potential sites for secretory immunity in the male genital tract. In the present study we describe the ultrastructural features of these glands in normal mice and provide quantitative data on the sizes of the acinar cells and their organelles in sham-, oil-, and testosterone-treated castrated mice. Acinar cells in urethral glands from normal mice contain numerous secretory granules, prominent Golgi complexes, elongated mitochondria, and an abundance of rough endoplasmic reticulum (RER) with large and dilated cisternae, all of which are features characteristic of secretory cells. In some acinar cells the cisternae of the RER were filled with closely packed, unbranched, straight, tubular structures that were oriented parallel to one another, that radiated from aggregates of dense material, or that were randomly arranged. In other acinar cells the cisternae of the RER showed a network of branching and anastomosing vesicular-like structures whose limiting membranes were occasionally seen in continuity with the membranes of the RER. Secretory acini showed large, unbranched tubules in the acinar lumen. When cut at right angles the large tubules exhibited a distinct fuzzy outer coat with fine projections radiating outwards. The ultrastructure of the acinar cells and the presence of tubules in the lumen suggests that they are engaged in secretion of a tubular protein. Morphometric analysis of acinar cells in the urethral glands showed that the mean volumes of nuclei, cytoplasm, secretory granules, vacuoles, and mitochondria were significantly reduced in castrated mice in comparison to either normal or testosterone-treated castrated mice. This confirms earlier observations that the urethral glands are targets of testosterone.

The occurrence and possible functions of mucosal immunity in the male urogenital tract have not been extensively investigated. In this study we used immunolabeling to localize secretory component (SC) and immunoglobulin (Ig) A in the urogenital tract of the male mouse. SC was located in the ventral prostate, while SC and IgA plasma cells were both detected in the urethral glands in the pelvic and bulbous portions of the urethra. SC and IgA were not observed elsewhere in the urogenital tract. We also examined the ventral prostate and urethral glands of sham-castrated, oil-treated castrated, and testosterone-treated castrated mice. There was a striking reduction in the size of the ventral prostate and urethral glands in oil-treated castrates compared to the other two groups, based on gross and histological morphology. Morphometric analysis showed that the cell and nuclear sizes of the urethral gland acinar cells were reduced after castration and restored to normal size by testosterone treatment. Androgen receptors (AR) were localized in the nuclei of urethral gland cells by immunocytochemistry using anti-AR antibodies. Labeling of SC and IgA plasma cells was similar in the urethral glands and ventral prostates of sham- and testosterone-treated castrates, but was reduced or absent at these sites in oil-treated castrates. These studies show that the ventral prostate and urethral glands may be sites for secretory immunity in the male murine urogenital tract, and that the urethral glands are targets for testosterone.

It has previously been shown that granulated metrial gland (GMG) cells of the pregnant uterus express abundant quantities of the lymphocyte pore-forming protein, perforin. No perforin was present before implantation of the embryo, but large numbers of perforin-producing GMG cells were observed after implantation, which coincides with decidualization of the uterus. The possible source of the activation factors responsible for perforin gene induction in GMG cells was studied here with the pseudopregnancy model, in which cervical stimulation of mice during estrus leads to a series of hormonal changes resembling those seen in pregnancy, but in the absence of an embryo. Subsequent stimulation of the uterus of pseudopregnant mice with oil causes the stimulated portion of the endometrium to differentiate into decidual tissue. Perforin-containing GMG cells were in fact present in the deciduomata, but not in adjacent nondecidualized tissues of the same mice. These results suggest that maternal factors associated with decidual tissue are responsible for the local expression of perforin in GMG cells.

Previous studies have shown that dendritic cells in the murine vaginal epithelium at diestrus and metestrus can endocytose intravaginally administered soluble protein tracers, but the identity of the dendritic cells was not established. In the investigation reported here, we used a combination of histochemistry and transmission electron microscopy to study the endocytosis of exogenous horseradish peroxidase by vaginal dendritic cells and to identify these cells as Langerhans' cells on the basis of their cellular associations, ultrastructural morphology, and the presence of Langerhans' cell granules.

Langerhans' cells (LCs) have been studied extensively in the epidermis, where they function as antigen-presenting cells. LCs are also present in the stratified epithelia of the murine vagina and cervix, but their function at these sites is not known. Recent reports noted the association of LCs with vaginal epithelial cells undergoing apoptosis and suggested that LCs might be involved in phagocytosis of dead cells. The present study describes the ultrastructural details of this process. The results demonstrate that LCs in murine vaginal epithelium during late metestrus and early diestrus phagocytose apoptotic epithelial cells and may thereby contribute to the normal turnover of the vaginal epithelium during the estrous cycle.

Natural killer (NK) lymphocytes were identified in the mouse uterus by immunostaining their surface membrane marker, LGL-1. The cells were present in large numbers from before mating through Day 14 of pregnancy. Double immunostaining indicated that uterine NK cells began to contain the pore-forming protein, perforin, on Day 6 of pregnancy in mesometrial decidua. Perforin is a probable mediator of cellular cytotoxicity found in lymphokine-activated NK and cytotoxic T lymphocytes. Activation of NK cells to produce perforin continued in mesometrial decidua on Days 8 and 10 of pregnancy and in the peripheral portion of metrial glands (MGs) on Days 12 and 14 of pregnancy, where cells at 3 stages of activation were simultaneously present: small cells with bright surface membrane staining of LGL-1 but no perforin (nonactivated), larger cells with intermediate staining of both markers (partially activated), and large cells with bright staining of perforin but no LGL-1 (fully activated). These observations indicate that activation of uterine NK cells involves loss of membrane LGL-1 as perforin accumulates in the cytoplasm, that the zone of activation shifts from mesometrial decidua to the MG on about Day 11 of pregnancy, and that nonactivated NK cells probably enter activation zones continuously during this period. Resting NK cells may enter activation zones by proliferation and/or migration from other regions of the uterus, rather than from blood, because depletion of circulating NK cells during pregnancy by treatment with NK-1.1 or asialo GM1 antibodies had no effect or only a small effect on the numbers of LGL-1-or perforin-positive cells seen in the uterus later in pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunization in the vagina can lead to the production of specific antibodies in the luminal fluid of this organ. To help understand the immune mechanisms involved in this process, we have studied the occurrence of Langerhans cells (LCs), macrophages, natural killer cells, and T and B lymphocytes in the murine vagina and cervix during the estrous cycle. LCs in the epithelia expressed Ia, F4/80, NLDC-145, and CD45, but not Mac-1, Moma-1, and Moma-2; double-labeling demonstrated phenotypic heterogeneity in this population Ia+, NLDC-145+; Ia+, NLDC-145-; Ia+, F4/80+; Ia+, F4/80-; Ia- F4/80+. T lymphocytes of both helper and cytotoxic/suppressor types were also present in the epithelia, sometimes in close association with LCs, but natural killer cells were not observed. The stroma of the vagina and cervix contained LCs (or interdigitating cells) and macrophages but few T lymphocytes and no B lymphocytes, natural killer cells, or lymphoid nodules. These observations confirm and extend previous reports that the murine vagina and cervix contain epithelial LCs and T lymphocytes and support the suggestion that antigens in the vagina and cervix, as in the epidermis, may be recognized and presented to the immune system by epithelial LCs. However, the paucity of T cells and the absence of B cells and lymphoid nodules from the stroma suggest that antigen presentation may not occur locally but at another site such as in the draining lymph nodes.

Immunostimulating complexes (ISCOMs) are subunit vaccines that are particularly effective in producing immunity against systemic viral infections, but their effectiveness against mucosal infections has received little attention. To study their ability to produce mucosal immune responses in the female reproductive tract, a model ISCOM was prepared containing sheep erythrocyte membrane proteins, and anti-erythrocyte IgA and IgG titres in mouse vaginal washings were measured after immunization at parenteral or local mucosal sites. The ISCOM was prepared by a modified procedure that resulted in incorporation of 10-15% of initial membrane protein compared with 1-5% previously reported. Electrophoretic analysis demonstrated that four out of five erythrocyte membrane proteins were incorporated into the ISCOM, and electron microscopic observations indicated that the ISCOM had a cage-like structure with a diameter of 40 nm, similar to previous ISCOMs. Immunization in the pelvic presacral space (p.s.-p.s.) stimulated significantly higher anti-erythrocyte IgA titres in vaginal fluid than were produced by intraperitoneal (i.p.-i.p.), subcutaneous (s.c.-s.c.), intravaginal (i.vag.-i.vag.), or i.p.-i.vag. immunizations with the same vaccine. Specific IgG titres were less dependent on the route of immunization, with p.s.-p.s., i.p.-i.p. and s.c.-s.c. administration all giving similar high titres while i.p.-i.vag. treatment induced lower titres. These observations using a model ISCOM indicate that mucosal immune responses against membrane proteins were elicited in the female reproductive tract, and that non-mucosal immunization in the pelvis was a more effective route of administration than local application of the ISCOM to the vaginal mucosa.

The mouse uterus during pregnancy contains a large population of lymphoid cells termed granulated metrial gland (GMG) cells. Our observations suggest that these cells are highly activated cytolytic lymphocytes related to NK or lymphokine-activated killer cells. Immunostaining demonstrated asialo GM1 and Thy-1 on GMG cells, both of which are expressed by NK cells. Decidua basalis tissue and isolated GMG cells contained three proteins that are characteristic of activated cytolytic lymphocyte granules: perforin, serine esterase 1, and serine esterase 2. These mediators were demonstrated in GMG cells by Western blot analysis using polyclonal antisera and by Northern blot analysis using specific cDNA probes for their mRNA. The proteins were not detected in normal spleen or liver or in asialo GM1+ cells isolated from those organs, consistent with the absence of these mediators from resting cytolytic cells. The amount of perforin in GMG cells was similar to that present in cloned, IL-2-stimulated, CTL shown previously to contain a large amount of this protein. A large population of NK cells bearing the surface marker LGL-1 was demonstrated at the implantation site by labeling with monoclonal antibody 4D11, but T cells were not detected. Many LGL-1+ cells at the implantation site expressed the GMG cell markers asialo GM1, Thy-1, and perforin. Staining intensities were inversely correlated, with LGL-1-bright cells showing little or no staining of GMG cell markers and LGL-1-faint cells showing more obvious staining of GMG cell markers. This suggests that LGL-1+ NK cells may differentiate in situ to GMG cells, losing LGL-1 and gaining a high concentration of GMG cell markers in the process. Activated cytolytic cells related to NK or lymphokine-activated killer cells may function in the pregnant rodent uterus to intercept and kill aberrant placental or embryonic cells that might otherwise enter the female and proliferate.

Measurements of specific antibody titres in uterine fluid of mice immunized by different routes indicated that two immunizations in the pelvic presacral space using aluminium hydroxide as adjuvant was a simple and effective way to elicit a significant IgA and IgG response. Higher IgA and IgG titres were produced in uterine fluid by subcutaneous immunization with antigen in Freund's complete adjuvant followed by intravaginal boosting without adjuvant, but this immunization involved both a toxic adjuvant and repeated applications of large doses of antigen in the vagina. Intragastric immunization produced an IgA response in the uterus but no IgG. Local intravaginal priming and boosting with large doses of antigen without adjuvant produced an IgA response in uterine fluid, but was less effective for IgG and was inefficient in terms of time and the amount of antigen used. Hysterectomy reduced the concentration of specific IgA in vaginal fluid of immunized mice to no more than 5% of normal, indicating that most of the IgA in vaginal fluid originates in the uterus. In contrast, IgG titres were not significantly different in hysterectomized and intact mice. IgA titres in vaginal fluid were at least partly restored to normal levels in sham-hysterectomized mice.

Intravaginal (ivag) immunization elicits secretory immune responses in the female reproductive tract, but little is known about the safety and effectiveness of adjuvants for such immunization. Mice were immunized intravaginally once daily for 5 days with large doses of horse ferritin combined with aluminum hydroxide (AH), muramyl dipeptide (MDP), monophosphoryl lipid A (MPL), dimethyl dioctadecyl ammonium bromide (DDA) or cholera toxin (CT). Titers of anti-ferritin IgA and IgG were measured in vaginal fluid by ELISA. The most effective adjuvant for ivag primary immunization was AH, while MPL was most effective for ivag boosting. None of the adjuvants caused a detectable tissue reaction in vaginal mucosa. Primary ivag immunization for 5 days with ferritin and AH followed by ivag boosting for 5 days with ferritin and MPL elicited higher IgA titers in vaginal fluid than systemic priming and boosting with ferritin and AH or systemic priming and ivag boosting with ferritin and MPL. Systemically immunized animals exhibited the highest IgG titers in vaginal fluid. The data indicate that adjuvants, particularly AH, can increase local immune responses to intravaginal immunization, but it should be noted that multiple applications of large doses of antigen were used and that this route of sensitization may be relatively inefficient.

Intravaginal immunization causes IgA responses in vaginal fluid, but so far lymphoid nodules in mouse vaginal mucosa have not been detected. The present study was therefore designed to test the hypothesis that IgA responses in the female reproductive tract may be generated in the regional iliac lymph nodes. Two, non-mucosal sites were identified in the female mouse pelvis, the subserous and presacral spaces, from which lymph drains mainly to the iliac nodes. Immunization at these pelvic sites with horse ferritin adsorbed to aluminum hydroxide (AH) caused much higher IgA and IgG titres in vaginal fluid than intravaginal immunization; moreover, the pelvic immunizations caused significantly higher and better sustained IgA titres in vaginal fluid than subcutaneous immunization near the scapulae or in the perineum, while IgG titres in vaginal fluid were similar in these groups. Additional mice were immunized with ferritin subcutaneously near the scapulae or in the presacral pelvic space using dimethyl dioctadecyl ammonium bromide (DDA), AH plus muramyl dipeptide, or the Ribi adjuvant system as adjuvants. Pelvic immunization caused higher IgA titres in vaginal fluid than subcutaneous immunization in each case. The IgA response stimulated by DDA was similar to that produced by AH but higher than the responses caused by the other two adjuvants, while IgG titres were similar with all four adjuvants in both sites. The results suggest that non-mucosal, pelvic immunization is particularly effective in stimulating IgA responses in the female reproductive tract. The observation is consistent with the possibility that the iliac lymph nodes may play a role in the development of IgA responses in the reproductive tract.

Local immunization in the vagina of several species elicits immune responses, but little is known about the uptake, processing and recognition of antigens at this site. We investigated the uptake of intravaginally administered tracers using FITC-bovine albumin, FITC-horse ferritin and FITC-horseradish peroxidase in non-pregnant and pregnant mice. Tracers were detected in cells in the vaginal epithelium and stroma at diestrus, proestrus and metestrus, but not at estrus. During pregnancy, racers were present in vaginal cells on Day 6 but not on Day 13. The distribution of tracers in the vagina was the same in all mice. They were present in vaginal epithelium in cells similar to Langerhans' cells and in the stroma in cells that resembled dendritic cells, fibroblasts or macrophages. In some non-pregnant mice, tracers were present in cells adjacent to lymphatic nodules located in the adventitia between the vagina and urethra. Tracers were seen in phagocytic cells lining the marginal and medullary sinuses of the draining lymph nodes (iliac nodes) in some non-pregnant mice at 4 h after intravaginal administration, or in small, dendritic cells in the paracortex at 17 h. To test the possibility that transfer of proteins into the vagina was due to toxic effects of the tracers, FITC-conjugated proteins were also administered into the lumen of uterine horns, and their distribution in horns, cervix and vagina was studied. In uterine horns, tracers were either absent or were located only in apical vesicles in the luminal epithelium. Tracers were present in the cervix and vagina as described above for intravaginal tracers. This result suggests that uptake of tracers in the vagina was not due to toxic effects, and that the vagina and cervix are major sites of protein uptake into the reproductive tract.

Cells of the metrial glands of mice were isolated by enzymic or mechanical dissociation procedures. Morphological observations indicated that up to half of the enzymically dissociated cells and nearly all of the mechanically dissociated cells were granulated metrial gland cells, but the presence of some fibroblast-like stromal cells among the latter population was not ruled out. Moreover, the granulated metrial gland cells had lost a substantial part of their granule content during isolation. Both cell preparations had little or no natural killer (NK) activity, indicating either that granulated metrial gland cells are not NK-like or that their NK activity was impaired by loss of granule-associated lytic substances or by other factors. Enzymically dissociated metrial gland cells did not bind significantly to the NK target cell YAC-1, nor did they develop granules, NK activity, or the ability to bind YAC-1 cells during culture in vitro, either in normal medium or with the addition of indomethacin or lymphokines. Mechanically dissociated metrial gland cells bound avidly to YAC-1 cells but not to P815 cells or adult thymus cells, which are not NK target cells. Since many if not most of the mechanically dissociated metrial gland cells appeared morphologically to be granulated metrial gland cells, their selective binding to an NK target cell suggests that granulated metrial gland cells may be related in some way to NK cells.

Prostaglandins (PGs) appear to have a role in the appearance of the increased uterine vascular permeability and subsequent decidualization observed at implantation in many species. However, the sites of production of these PGs have not been clearly established. To clarify the PG synthetic capacity of the blastocyst and the various types of cells in the uterus at implantation, we have studied the immunohistochemical localization of PG synthase in the rat blastocyst on Days 5 to 7 and uterus on Days 1, 4, 5, 6, and 7 of pregnancy. Labeling of PG synthase was negligible in the uterus on Day 1 of pregnancy. On Day 4, there was increased labeling in the luminal and glandular epithelium, in stromal cells adjacent to the luminal epithelium, and in blood vessels and some leukocytes. PG synthase was detected in the blastocysts on Days 5 to 7, but there was a gradual loss of label in the luminal and glandular epithelial cells during this period. Early differentiating stromal cells adjacent to the luminal epithelium in the implantation site on Day 5 showed bright labeling, whereas peripheral stromal cells were only slightly labeled. By Day 7, the differentiated cells of the primary decidual zone showed little or no label, but cells in the secondary decidual zone were brightly labeled. These results indicate that PG synthase is present in the rat blastocyst and in several kinds of uterine cells, and that its localization in uterine cells changed markedly during the implantation process.

Mice were immunized with a protein antigen, horse ferritin, by eight different routes and the immune responses in the reproductive tract were compared by measuring specific IgA and IgG in vaginal fluid and by localizing anti-ferritin plasma cells in uterine horns, cervix and vagina. The eight routes of immunization were: subcutaneous with Freund's adjuvant (s.c.), intragastric (i.g.), intravaginal (i.v.), s.c.-i.g., s.c.-i.v., i.g.-i.v., i.v.-i.v. and s.c.-i.g.-i.v. The largest overall response, considering both IgA and IgG antibodies, was obtained by s.c. priming with ferritin in adjuvant followed by i.v. boosting. Intravaginal immunization also boosted priming by the i.g., s.c.-i.g. and i.v. routes, but the response to i.v. immunization alone was weak. All i.v. immunizations stimulated mainly IgA antibody responses in vaginal fluid. Specific plasma cells, mostly of the IgG isotype, were present in the vaginal fornix of several mice in the s.c.-i.v. and s.c.-i.g.-i.v. groups, but none were detected there in any other group and they were only rarely observed in the uterine horns. The results provide data on the relative effectiveness of different routes of immunization in producing a humoral immune response in vaginal fluid against a non-replicating antigen.

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.

The rat primary decidual zone (PDZ) is a transitory avascular region of transformed fibroblasts surrounding the implanting embryo. Studies using fluorescein-labeled tracers have shown that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight. In the present study we investigated the morphologic basis of the permeability barrier. Horseradish peroxidase (HRP) or HRP-labeled immunoglobulin G (IgG-HRP) was administered i.v. to rats on Day 7 of pregnancy, and the animals were killed 30 min to 2 h later. The reaction product of HRP was the same density in uterine blood vessels as in the intercellular spaces of the endometrium and PDZ at 30 min and 1 h after administration. Two hours after administration, the reaction product of IgG-HRP was dense in uterine blood vessels, much less dense in the interstitial spaces of the endometrium, and was not detected in the PDZ. There was an abrupt change in the density of the IgG-HRP reaction product at the intercellular clefts between endothelial cells, where cellular junctions were observed in control tissue. This suggests that the passage of large macromolecules from blood to the implantation chamber is limited initially by cellular junctions between capillary endothelial cells. The exclusion of IgG-HRP from the PDZ indicates that an additional barrier(s) to macromolecules in this region. Lanthanum nitrate tracer was uniformly present throughout the intercellular spaces of the PDZ except at tight junctions between decidual cells. Freeze-fracture replicas of the PDZ showed tight junctions that varied from single strands to interconnected networks of strands oriented mainly parallel to the long axis of the PDZ. Some strands were discontinuous. The tight junctions between decidual cells appear to be functionally discontinuous because HRP readily penetrated the PDZ, but such junctions may retard larger macromolecules such as IgG-HRP. The biological significance of the permeability barrier of the PDZ is discussed.

The luminal surfaces of non-ciliated uterine epithelial cells of 17 rabbits on Days 4--6 of pregnancy were studied histologically with light microscopy, transmission and scanning electron microscopy. The luminal surfaces of most epithelial cells exhibited short microvilli and did not project into the uterine lumen. However, the surfaces of some cells showed fewer microvilli and bulged out into the uterine lumen, giving the cells a dome-like appearance. The domes appeared most frequently on Day 6 of pregnancy and contained a few ribosomes, mitochondria, and membranous elements; large vacuoles and granules were absent. The domes were not involved in endocytosis. Ferritin introduced into the uterine lumen was incorporated into the cells by coated micropinocytotic invaginations at the base of the microvilli, rather than by any activity of the domes. There was also no indication that the domes pinched off to produce an apocrine secretion. The size and frequency of domes were, however, clearly related to the fixation procedure. Epithelial cells from uteri fixed by vascular perfusion showed fewer and less prominent domes, while cells from uteri fixed by immersion displayed a larger number of domes that projected further into the uterine lumen. The domes in the rabbit epithelium therefore differ structurally and functionally from the apical projections (pinopods) that occur in the uterine epithelium of the rat and mouse during the peri-implantation period.

Immunoglobulins A and G were localized by immunoperoxidase labelling in uteri of ovariectomized mice treated with oestradiol-17 beta and progesterone. The administration of oestradiol or progesterone alone to ovariectomized mice for 3 days increased the number of IgA plasma cells from about 1 to 14 per histological section. When the two hormones were administered simultaneously for 3 days the number of plasma cells per section was equal to or greater than with either hormone alone. Treatment with oestradiol followed by progesterone in a sequence that prepares the uterus for implantation resulted in about 31 IgA plasma cells per section. Counts of IgG plasma cells showed similar trends but the numbers were smaller. The results indicate that progesterone increases rather than decreases the number of plasma cells in the mouse uterus. This is consistent with observations on intact mice during oestrus and pregnancy and suggests that the marked increase in endometrial plasma cells at the time of implantation in mice is a response to progesterone acting on an oestrogen-primed uterus.

The present investigation studied the effects of sperm immunization in the gastrointestinal tract on anti-sperm antibody production and fertility in female mice. For comparative purposes, mice were also immunized with sperm intraperitoneally. Intraperitoneal immunization with 5 X 10(6) washed epididymal and vas deferens sperm 3 times per week for 7 wk produced anti-sperm IgG in plasma at 1:20,000 and in vaginal washings at 1:100 as determined by ELISA. Such mice have been shown previously to have reduced fertility. In comparison, mice immunized intragastrically with 5 X 10(6) sperm once per week for 11-14 wk had anti-sperm IgA in vaginal washings at only about 1:8 as determined by ELISA. After mating at the 14th wk these mice delivered 6.5 +/- 1.4 pups, which was not significantly different from the 7.1 +/- 1.1 pups delivered by an untreated control group. Mice immunized twice intragastrically and once intravaginally during a 25-day period had no detectable anti-sperm IgA in vaginal washings by ELISA. These mice delivered 9.7 +/- 1.2 pups after mating beginning on day 32, as compared to 9.7 +/- 0.8 pups in a PBS-sham immunized group. Mice immunized once intraduodenally and then once intraperitoneally 14 days later delivered 10.4 +/- 0.9 pups after mating 10-14 days after the second immunization, while a similar group of mice whose primary sperm immunization was directly into Peyer's patches delivered 9.0 +/- 1.4 pups. We could not detect anti-sperm IgG or IgA bound to sperm in the uterine or oviduct lumen using immunohistochemical labeling after any of the groups of immunized mice were mated.(ABSTRACT TRUNCATED AT 250 WORDS)

Throughout the second half of pregnancy in mice there were many plasma cells containing immunoglobulins A (IgA) and G (IgG) in the uterine endometrium. There was intense staining of IgA in uterine glands at all stages, but little staining of IgG. The staining of both immunoglobulins (Igs) in the luminal epithelium was moderate to dark on day 11, slight on day 14, and increased from day 16 to term. From day 14 to term the endometrium exhibited folds or villi around each placenta. The cores of the villi contained many plasma cells of both isotypes, and the staining of extracellular Igs in the villous cores was darker than in nonvillous endometrium. Both Igs were detected in the uterine lumen, and in visceral and parietal yolk sac endoderm cells at all stages. Near term, the staining of Igs in the visceral yolk sac was darkest in the peripheral villous portion adjacent to the endometrial villi. From day 14 to term IgG was present in the visceral yolk sac mesenchyme and embryo, consistent with its transfer from the uterine lumen to the embryo via the vitelline circulation. In contrast, IgA was not detected in yolk sac mesenchyme until day 19, when only slight staining was observed, and IgA was never detected in the embryo. Most trophoblast giant cells contained both Igs on day 11. During the remainder of pregnancy, there was staining of both Igs in labyrinthine trophoblast and in a few giant cells adjacent to the parietal yolk sac on the placenta, but there was negligible staining in the spongiotrophoblast region. Our observations suggest that the local immune system in the mouse uterus may protect the embryo during the second half of pregnancy by secreting anti-microbial immunoglobulins A and G into the uterine lumen surrounding the visceral yolk sac, and may at the same time contribute to the transfer of maternal IgG to the embryo via the yolk sac and vitelline circulation.

By 20 min after intravenous injection of horseradish peroxidase on Day 5 of pregnancy the tracer was present between the lateral epithelial cell membranes, in coated and non-coated invaginations in the lateral and basal membranes of epithelial cells, and in pinocytotic vesicles at the periphery of epithelial cells. By 60 min after injection the tracer was also distributed in vesicles throughout the epithelial cell cytoplasm. By 2 h the tracer was present only in a few vesicles in the apical region of epithelial cells, and some of these vesicles appeared to be fusing with the apical cell membrane. Pinocytotic invaginations in the basal membrane of uterine epithelial cells were 4--5-fold more numerous on Day 5 of pregnancy than on Days 1--4 and 7, suggesting a specific role for this activity during early pregnancy. The observations show that uterine epithelial cells can pinocytose substances derived from blood, transport them to the apical cytoplasm, and release them into the uterine lumen by exocytosis.