Perforin-expressing granulated metrial gland cells in murine deciduoma.

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.

Purification and measurement of secretory IgA in mouse milk.

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.

Uptake of immunoglobulins and other proteins from serum into epithelial cells of the mouse uterus and oviduct.

The transport of immunoglobulins into the lumen of the female reproductive tract is not well understood, especially in the case of IgG. In mice, there are conflicting reports concerning the presence of immunoglobulins in uterine luminal and glandular epithelial cells, and immunoglobulins have not been detected in the luminal epithelial cells of the oviduct. In the present study we detected both IgA and IgG in uterine luminal and glandular epithelial cells on day 1 of pregnancy by immunolabeling. Also, we observed that fluorescein-conjugated mouse and bovine IgG and other proteins were taken up into vesicles in uterine luminal and glandular epithelial cells after intravenous administration. These observations indicate that both kinds of epithelial cells take up immunoglobulins from the interstitial fluid on day 1 of pregnancy, and that the cells may therefore be involved in the transport of immunoglobulins and other proteins to the uterine lumen at that time. In the oviduct, we detected IgA and IgG in vesicles in the luminal epithelial cells of the preampulla by immunolabeling, and we observed fluorescein-conjugated IgA and IgG in similar vesicles after intravenous administration. The presence of IgA and IgG in vesicles in the epithelial cells of the preampulla, together with the previous demonstration of plasma cells of both isotypes and large amounts of interstitial immunoglobulins in the lamina propria of this segment, suggests that the preampulla of the oviduct may be an important site for the local immune system in the mouse female genital tract.

The effect of sperm immunization in the gastrointestinal tract on anti-sperm antibody production and fertility in female mice.

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)

Protective immunity against HSV-2 in the mouse vagina.

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.

Secretory immunoglobulin binding to bacteria in the mouse uterus after mating.

Bacteria of several species were present in the mouse uterus on the morning after mating, as demonstrated by bacterial cultures and microscopic examination of Gram-stained uterine luminal contents. The similarity between bacteria cultured from the vagina before mating and from the uterus after mating suggested that bacteria were introduced into the uterus from the vagina, possibly by coitus. The bacteria were cleared from the uterus about two days after mating. Immunohistochemical labeling of smears of the luminal contents on the morning after mating demonstrated IgA, IgG, and possibly IgM bound to many of the bacteria. The bacteria were often agglutinated, and there was a correlation between the intensity of immunoglobulin labeling on bacteria and the extent of agglutination. The amount of antibody bound to bacteria in multiparous mice was about the same as in mice that had not been mated previously. We observed both IgG and IgA on bacteria when organisms from vaginal cultures were incubated for 60 min in the uteri of estrogen-primed, virgin, female mice. This indicated that the uterus was the source of at least part of the immunoglobulins bound to bacteria. We did not demonstrate that the immunoglobulins bound to bacteria were specific anti-bacterial antibodies, but the binding persisted through three washing steps and there was no immunoglobulin binding to sperm in the same preparations. Neutrophils in the uterine lumen on the day after mating contained phagocytosed bacteria. These results suggest that the secretory immune system in the female mouse reproductive tract may play a role in returning the uterus to an aseptic state after mating by at least three mechanisms: direct blocking of attachment sites involved in bacterial binding to mucosal epithelium, agglutination of bacteria and thus reduction in the number of organisms available for binding to the epithelium, and opsonization of bacteria for phagocytosis by neutrophils.

Localization of immunoglobulins in the mouse uterus, embryo, and placenta during the second half of pregnancy.

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.

Deposition of C3 on bacteria in the mouse uterus after mating.

A previous study demonstrated that several species of bacteria were present in the mouse uterine lumen on the day after mating, and that many of these bacteria had immunoglobulins bound to their surface. Neutrophilic leukocytes containing phagocytosed bacteria were also present in the lumen. Since bacteria are phagocytosed efficiently only when they are opsonized by the binding of specific antibody or C3b or both to their surface, we investigated whether the uterine bacteria were coated with C3. Immunolabeling demonstrated that an antigenic portion of C3, possibly C3b, was bound to many of the uterine bacteria. This observation suggests that bacteria in the mouse uterus after mating may be opsonized by both antibody and complement and that phagocytosis of these bacteria by neutrophils may play an important role in returning the uterus to an aseptic state before implantation.

Antigen recognition in the female reproductive tract: I. Uptake of intraluminal protein tracers in the mouse vagina.

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.

Anti-bacterial IgA and IgG in mouse uterine luminal fluid, vaginal washings and serum.

A previous study demonstrated that mouse uterine horns contained bacteria of several species on the morning after mating, and immunolabeling showed that many of these bacteria were coated with immunoglobulins. In the present study we used an ELISA technique to detect naturally-occurring antibodies against bacteria in mouse uterine luminal fluid, vaginal washings and serum. Each fluid contained specific IgA and/or IgG antibodies to five of the six bacterial species recovered from the uterus after mating. The uterine fluid antibodies that bound to the bacteria were mainly IgA molecules, while those in the serum were mainly IgG. Naturally-occurring bacterial antibodies in mouse uterine luminal fluid may play a role in protecting the endometrium against microbial infections.

A comparison of specific antibody responses in mouse vaginal fluid after immunization by several routes.

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.

Immunity to vaginal infection by herpes simplex virus type 2 in adult mice: characterization of the immunoglobulins in vaginal mucus.

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.

The effect of adjuvants on antibody titers in mouse vaginal fluid after intravaginal immunization.

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.

Synthesis and granular localization of tumor necrosis factor-alpha in activated NK cells in the pregnant mouse uterus.

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.

Observations on recovery from and recurrence of HSV-2 infections in adult mice that were rescued from lethal vaginal infection by antiviral therapy.

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.

Endocytosis at the basal and lateral membranes of rat uterine epithelial cells during early pregnancy.

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.

The changing role of advanced practice nursing in a managed care environment.

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.

Relationship of uterine closure to ovarian hormones and endocytosis in the rat.

Ovariectomized rats were treated with various hormonal regimens. The first stage of uterine closure and epithelial cell endocytosis were observed in ovariectomized rats treated with progesterone or progesterone after oestrogen priming but not in hormonally untreated rats or those treated with oestrogen alone. The simultaneous appearance of closure and endocytosis in response to the same physiological conditions is consistent with the proposal that the endocytotic activity in the uterine epithelial cells may remove fluid from the uterine lumen and thus mediate the first stage of uterine closure.

Effects of ovarian hormones on endocytosis at the basal membranes of rat uterine epithelial cells.

Ovariectomized rats treated with progesterone and estradiol-17 beta in sequences that prepared the uterus for implantation showed marked increases in the number of endocytotic invaginations in the basal membranes of luminal epithelial cells and in the amount of intravenously administered horseradish peroxidase taken into the cells. Tight junctions between the epithelial cells blocked the direct passage of horseradish peroxidase into the uterine lumen. The number of basal pinocytotic invaginations in glandular epithelial cells was relatively small and did not increase following hormonal stimulation. The probable occurrence of a pathway for the transepithelial transport of macromolecules from the blood or stroma into the uterine lumen, and its sensitivity to stimulation by ovarian hormones, suggests a likely mechanism for altering the molecular environment of the embryo during the implantation period.