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.

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.

A mouse model for studies of mucosal immunity to vaginal infection by herpes simplex virus type 2.

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.

A possible role for the dimer ribbon state of scallop sarcoplasmic reticulum. Dimmer ribbons are associated with stabilization of the Ca(2+)-free Ca-ATPase.

The Ca-ATPase activity of membranous scallop sarcoplasmic reticulum was found to be unstable when the Ca(2+)-binding sites on the Ca-ATPase were unoccupied. The decay in activity could be slowed or halted by inclusion in the preincubation medium of Na+, K+, nucleotides, ethylene glycol, or high concentrations of choline chloride. Stabilization of the Ca(2+)-free Ca-ATPase by Na+ and K+ showed a markedly different concentration dependence to that seen with activation of the Ca(2+)-activated ATPase activity by the two ions. Examination in the electron microscope of scallop membranes negatively stained in the presence of EGTA under conditions where the enzyme had been stabilized against lack of Ca2+ always showed vesicles containing dimer ribbon structures, whereas unstabilized membranes did not show dimer ribbons. There was an association between the effectiveness of a medium in stabilizing the enzyme in the presence of EGTA and the extent and quality of the dimer arrays seen in the microscope. Comparison of the range of Ca2+ concentration over which the Ca(2+)-binding sites on the scallop Ca-ATPase titrated with the range over which the dimer ribbon structural state was lost indicated that the Ca(2+)-binding sites on the Ca-ATPase must be empty for dimer ribbon formation to occur. Previous studies (Franzini-Armstrong, C., Ferguson, D. G., Castellani, L., and Kenney, L. J. (1987) Ann. N. Y. Acad. Sci. 483, 44-56) have found that the Ca-ATPase molecules in scallop adductor muscle freeze-fractured after fixation under relaxing conditions are arranged in dimer ribbons. Thus, the association of stabilization of the Ca(2+)-free Ca-ATPase with the presence of dimer ribbons implies that one function of the dimer state may be to stabilize the scallop enzyme in situ, when the Ca2+ concentration in the sarcoplasm is low and the muscle is relaxed.

Localization of boar sperm proacrosin during spermatogenesis and during sperm maturation in the epididymis.

The localization of proacrosin was determined by using colloidal gold labeling and electron microscopy of boar germ cells during spermiogenesis to post-ejaculation. Proacrosin was first localized in round spermatids during the Golgi phase of spermiogenesis; it was associated with the electron-dense granule, or acrosomal granule that was conspicuous within the acrosome. It remained within the acrosomal granule during the cap and acrosome phases of spermiogenesis. At these stages, there was no apparent association of the proacrosin molecule with the acrosomal membranes. During the maturation phase of spermiogenesis, proacrosin was seen to become dispersed into all regions of the acrosome except the equatorial segment. When sperm from different segments of the epididymis and ejaculated sperm were examined, localization was observed throughout the acrosome except for the equatorial segment. Here proacrosin appeared to be localized on both the inner and outer acrosomal membranes as well as with the acrosomal matrix, although further studies are required to verify the membrane localization. No labeling was seen on the plasma membrane. These data suggest that the synthesis and movement of proacrosin to sites in the acrosome are controlled by an as yet unknown process. The absence of proacrosin on the plasma membrane of mature ejaculated sperm makes it unlikely that this enzyme plays a role in sperm-zona adhesion prior to capacitation.

Two-dimensional polyacrylamide gel electrophoresis characterization of APz, a sperm protein involved in zona binding in the pig and evidence for its binding to specific zona glycoproteins.

A boar sperm integral plasma membrane protein (APz) involved in the adhesion of uncapacitated and capacitated sperm to the porcine zona pellucida (ZP) has been characterized by two-dimensional polyacrylamide gel electrophoresis (PAGE) and tested for its ability to bind to various zona glycopeptides. APz shows microheterogeneity and focuses over a wide pH range, with predominant forms focusing above pH 7. The protein, when excised from nonreducing polyacrylamide gels, inhibited sperm-egg binding and bound heat-solubilized zonae preventing these zonae from blocking sperm binding to eggs. In an indirect assay, a polyclonal monovalent antibody, which blocks sperm-egg binding and which is absorbed by APz, was used to determine the ability of zona glycopeptides to prevent the sperm-egg blocking activity of the antibody from being absorbed by intact sperm. When whole heat-solubilized ZP was added to sperm at doses that block sperm-egg binding and the excess ZP was removed, the sperm-egg blocking activity of the antibody was not absorbed by these sperm, and antibody-containing supernatants blocked the binding of untreated sperm to eggs as effectively as antibody that was not mixed with fresh sperm. When alpha ZP3 was used in the same manner, sperm-egg blocking activity again was not absorbed by antibody-treated cells. Beta ZP3, however, failed to block sperm-egg binding and failed to absorb the sperm-egg blocking activity of the antibody. These findings support the argument that the action of APz is physiologically significant and involves specific binding sites on the ZP3 component of the ZP.

Secretory immune responses in the mouse vagina after parenteral or intravaginal immunization with an immunostimulating complex (ISCOM).

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.

Characterization of membrane-associated actin in boar spermatozoa.

Biochemical, immunological, and electron microscopic methods have been used to provide semi-quantitative estimates and to localize actin in membranes of boar spermatozoa. Immunoblots, using a monoclonal antibody raised against actin from chicken gizzard, detected the protein in caput and cauda sperm plasma membranes. Immunoassay indicated that approximately 1% of the total plasma membrane protein was actin. Monomeric actin accounted for more than one-half of the membrane actin. Approximately 30-40% of plasma membrane actin was insoluble in Triton X-100, and approximately 10% of the total actin remained insoluble after treatment with guanidine hydrochloride. The presence of F-actin in sperm plasma membranes and in plasma membrane detergent-insoluble proteins was detected by fluorescence microscopy using the specific probe NBD phallacidin. When S1 myosin subfragments attached to colloidal gold were used to localize F-actin by electron microscopy, the label was restricted to the outer acrosomal membrane of intact epididymal and ejaculated sperm. Filaments appeared in short arrays along the anterior region of the membrane. S1/gold labeled detergent-insoluble plasma membrane fractions but did not label the plasma membrane in intact sperm. Filaments were least prominent in intact caput spermatozoa and most prominent in ejaculated spermatozoa. We conclude that most actin associated with sperm membranes is in monomeric form in boar spermatozoa, but that actin filaments or protofilaments are components of the outer acrosomal membrane. These filaments may also associate with the plasma membrane overlying the acrosome.

Effect of phosphorylation on scallop sarcoplasmic reticulum.

Fragmented sarcoplasmic reticulum prepared from the cross-striated adductor muscle of the deep sea scallop (Placopecten magellanicus) was phosphorylated with inorganic phosphate to the E2P (ADP-insensitive) form. Negative staining of these preparations showed that the Ca-ATPase was organized into a quasi-crystalline array, which differed from the 'dimer ribbon' structure previously reported for the membrane under relaxing conditions (Castellani & Hardwicke, J. cell. Biol. 97 (1983) 557-61; Castellani et al., J. molec. Biol. 185 (1985) 579-94). In this new form there was only a single Ca-ATPase per unit cell. Dephosphorylation of the E2P membranes and incubation with substrate or substrate analogues in the absence of Ca2+ caused the 'dimer ribbon' structure to appear. These results imply that rotation of at least half of the Ca-ATPase subunits in the scallop sarcoplasmic reticulum may occur about an axis perpendicular to the plane of the membrane on conversion from the E2P state to the state corresponding to that existing in the relaxed muscle.

Fine structure of anterior terminus of apical sense organ in Macracanthorhynchus hirudinaceus (Acanthocephala).

The apex of the proboscis of Macracanthorhynchus hirudinaceus is crowned by a cone-shaped projection with a small opening in its center. The bottom of this opening is the anterior terminus of the apical sensory organ. When viewed in transverse section, the anterior terminus of this organ appears as a series of distinct layers that encircle a central cone enclosing a complex arrangement of nerves and a sensory support cell duct. Four membrane-defined layers encircle the cone area. The outermost glycocalyx is morphologically identical with that described on the metasoma. The second layer, or tegument, is similar in appearance to that observed on the trunk except for the greater abundance of keratinlike bundles throughout. These bundles are also organized into a loose network along the inner tegumental membrane. The third layer, a latticework of fine filaments containing few organelles, has an erratic boundary that occasionally extends into layer 4. The area adjacent to the inner and outer boundary contains numerous vesicles. Layer 4 has 2 distinct zones. The outer contains filaments arranged as in circular muscle; whereas, the medial lacks such filaments but consists of a finely grained matrix. Radiating throughout both zones are numerous osmiophilic bundles of fibers. The cone at this level contains 8 branches of the apical sensory nerves that interdigitate with the duct from the sensory support cell. Numerous filaments and vesicles are associated with this complex.

Ultrastructural study of biochemically modulated ADCC in HSV-1 infected and uninfected Chang liver cells.

The effect of cytochalasin B, ouabain and 25-OH cholesterol on specific lysis due to antibody dependent cellular cytotoxicity (ADCC) in allogeneic and xenogeneic systems was studied using Herpes simplex I infected Chang liver cells. Cytochalasin B reduced both cytotoxicity and lymphocyte/target (LT) binding in the allogeneic system whereas cytotoxicity but not LT binding was reduced in the xenogeneic system. Ouabain inhibited ADCC in both systems as well as LT binding in the allogeneic system; however, binding in the xenogeneic system was not significantly reduced. The 25-OH cholesterol produced a marked decrease in ADCC in both systems but had no significant effect on LT binding in either system. The biochemical and ultrastructural data suggest that the modulators act at different stages in the ADCC response and that there may be more than one mechanism of ADCC to handle different types of target antigens.

Ultrastructure of antibody dependent cellular cytotoxicity in HSV 1 infected and uninfected Chang liver cells.

The binding and ultrastructural features of antibody dependent cellular cytotoxicity (ADCC) mediated by human peripheral blood lymphocytes were studied in herpes simplex virus type I (HSV-1) infected Chang liver (CL) cells plus human anti-HSV-1 serum, and in uninfected CL cells plus guinea pig anti-CL antiserum. Non-cytolytic controls included target cells treated with normal serum in place of sensitized targets and heat shocked lymphocytes instead of normal lymphocytes. By transmission electron microscopy, target cell membranes were either broadly indented by effector cells or locally invaginated by means of effector cell filopodia. In neither case did the indentation appear to break the plasma membrane of the target. Control preparations showed only non-indented areas of simple membrane contact. By scanning electron microscopy, the effector lymphocytes in both the active ADCC and normal serum control preparations had a sparse distribution of short microvilli over their surfaces. The majority of heat shock control lymphocytes appeared normal, but 12-20% demonstrated surface patches devoid of microvilli. The hypothesis that ADCC may involve a three-step process is discussed.

The effect of aculeacin A and papulacandin B on morphology and cell wall ultrastructure in Candida albicans.

Transmission (TEM) and scanning electron microscopy (SEM) of Candida albicans cultures treated with the cell wall active antibiotics aculeacin A and papulacandin B (10 micrograms/mL) revealed highly distorted, wrinkled, and collapsed cells. Dividing cells failed to separate properly and aggregates of enlarged and elongated forms were often seen. TEM sections revealed thick and layered cell walls in the treated cultures and bud cross walls failed to segregate completely. Approximately 20% of the cells demonstrated complete cell necrosis accompanied with cytoplasmic deterioration, layered and distorted walls, and improperly formed buds and scars.

Virus-like particles and lytic plaque formation in lawns of Candida albicans.

The antifungal agent Aculeacin A at subinhibitory levels induced lytic plaques in lawns of Candida albicans. Electron microscopic examination of plaque lysates suspended in phosphotungstic acid revealed the presence of spherical particles 12, 18, and 28 to 30 nm in size. Particles were also found in ultrathin sections of treated C. albicans cells. The plaque lysate lost infectivity after treatment with UV light, heat treatment at 80 degrees C for 10 min, or being held at pH 2 for 30 min.

Localization of Streptococcus mutans GS-5 glucosyltransferases and intracellular invertase.

Antibodies directed against Streptococcus mutans GS-5 intracellular invertase and glucosyltransferase fractions capable of synthesizing primarily water-soluble or insoluble glucans were used to ultrastructurally localize the enzymes by means of the unlabeled antibody peroxidase-antiperoxidase method. This immunocytochemical procedure revealed that the intracellular invertase was associated primarily with the cytoplasmic membrane of the cariogenic organism. The glucosyltransferase complex responsible for insoluble glucan synthesis was localized as aggregates attached to the cell surface or extracellular polysaccharides of strain GS-5. In contrast, the glucosyltransferase activity synthesizing primarily water-soluble glucans was distributed uniformly over the cell surface or in association with extracellular polysaccharides. These results are discussed relative to the sucrose-metabolizing ability of Streptococcus mutans.

Comparison of the glucosyltransferases of Streptococcus mutans gs-5 to a non-cariogenic mutant.

The extracellular glucosyltransferase (GTF) enzymes of Streptococcus mutans GS-5 and avirulent mutant GS-511 were fractionated using agarose and DEAE-cellulose columns. GTF of GS-5 produced both water-soluble and -insoluble glucans, while those of GS-511 made soluble products almost exclusively. Nearly all (98%) of the small amount of insoluble GTF enzyme made by GS-511 was bound to the cell wall.

Ultrastructural localization of sucrases in Streptococcus mutans GS-5 and an extracellular polysaccharide mutant: a comparative cytochemical and immunocytochemical study.

Electron microscopy and cytochemical and immunocytochemical procedures were used to study the ultrastructural distribution of sucrase enzymes in two strains of Streptococcus mutans. In a strongly adherent and virulent parent strain, GS-5, most of the invertase and fructosyltransferase activities were demonstrated extracellularly or bound to the cell surfaces. Intracellularly, enzymatic sites were detected near the plasma membrane on the periphery of the nucleoid and central mesosome. In GS-511, a mutant of diminished virulence and adherence, most of the enzymatic activity was not located on the cell surfaces, but was found away from the cell walls and associated with extracellular polysaccharides. Intracellularly, GS-511 manifested the same distribution of invertase and fructosyltransferase as did GS-5; however, the close association of these enzymes with the plasma membrane was not shown in GS-511. In both strains, extracellular areas near regions associated with cross wall formation appeared to show localized concentrations of these sucrases. Antibodies against partially purified glucosyltransferase (GTF) enzymes from GS-5 were used to localize GTF by immunocytochemical techniques. Indirect ferritin localization procedures showed that the extracellular and cell-bound GTF enzymes were distributed in similar locations as the fructosyltransferase and invertase enzymes. By absorption of the antiserum with whole GS-511 cells, the location of extracellular GTF and surface antigens unique to GS-5 was demonstrated. The dramatically reduced levels of cell-bound sucrase activity in GS-511 indicates the significant role of these enzymes in adherence and cariogenicity.

Biochemical study of the relationship of extracellular glucan to adherence and cariogenicity in Streptococcus mutans and an extracellular polysaccharide mutant.

A mutant of Streptococcus mutans, GS-5, which differed in extracellular polysaccharide (EPS) produced from sucrose, was used to study the role of EPS in the production of dental caries. The mutant proved to be identical to the parent strain in sugar fermentation, growth rate, and serotype. Strain GS-5 synthesized an EPS, which in electron micrographs appeared to be of fibrillar structure, whereas the mutant produced no fibrillar material but only a globular EPS. Analysis of the EPS revealed that about 30% of the glucose units in the GS-5 polymer carried (1-3)-like bonds either as branch points or as part of the linear backbone and that the mutant material contained only about 3% of these linkages. When grown in sucrose broth, the proportion of the mutant culture adherent to the glass vessel was dramatically less than that of the parent strain. Caries scores produced in conventional rats by the mutant were significantly lower than those obtained with the parent strain. Since the only difference discovered between strain GS-5 and the mutant was the inability of the mutant to synthesize either a fibrillar EPS or an EPS with more than about 3% (1-3)-like linkages, it was concluded that the fibrillar EPS of strain GS-5 contained about 30% (1-3)-like linkages and was necessary for adherence of the bacteria to surfaces and for production of dental caries in test animals.

Morphological study of Streptococcus mutans and two extracellular polysaccharide mutants.

Two extracellular polysaccharide mutants of Streptococcus mutans GS-5 were obtained and examined. The mutants were distinguished by colonial morphology and by growth on and adherence to hard surfaces. A technique was devised which allowed these bacteria to be studied as they appeared when grown on a hard surface in liquid medium which contained sucrose. Negative stains, replicas, and scanning electron micrography clearly revealed differences in cellular aggregation due to the various extracellular polysaccharides produced. Comparison of sections of the adherent parent strain (GS-5) with those of the nonadherent mutant (GS-511) allowed the extracellular polysaccharide(s) responsible for adhesion to be visually localized.