John J. Bozzola

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.

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.

Conventional Specimen Preparation Techniques for Transmission Electron Microscopy of Cultured Cells

This chapter covers the conventional methods and considerations for preparing cultured cells for examination in the transmission electron microscope. Techniques for handling cells grown in liquid culture, as well as on substrates such as culture dishes or agar, are discussed. Directions are given on how to prepare the most commonly used buffers, fixatives, enrobement media, and embedding resins. These methods may be applied to most cultured organisms, from bacteria to mammalian 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.

Conventional Specimen Preparation Techniques for Scanning Electron Microscopy of Biological Specimens

In this chapter, methods are described for preparing biological specimens for examination in the scanning electron microscope. Conventional procedures are described for handling cells grown in liquid culture as well as on substrates such as culture dishes, slide culture chambers, or agar. These protocols may be used to process not only cultured organisms but also larger botanical and zoological specimens.

Protein transport and organization of the developing mammalian sperm acrosome.

Experiments indicate that the mammalian acrosome develops as a result of a time-dependent sequence of events which involves protein incorporation into distinct regions or acrosomal domains. These domains can be characterized by electron microscopy and their isolation and partial purification are being accomplished. Recent success in isolating and characterizing major proteins that compromise the Golgi apparatus should accelerate knowledge of the interaction of the Golgi with the developing acrosome. Progress in this area is reviewed with the view that understanding the events involved in the transport of proteins from the Golgi apparatus to the acrosome and the mechanisms involved in positioning and modifying these proteins during spermiogenesis should provide a clearer understanding of how the acrosome develops in preparation for its role in fertilization.

Processing Biological Tissues for Ultrastructural Study

Biological tissues are passed through numerous procedures before they can be studied at the ultrastructural level with the electron microscope. Chemical fixation is widely used as a method for preserving structural detail and can be performed by simple immersion or total body vascular perfusion. A 2 to 4% solution of glutaraldehyde buffered with 0.1 M sodium phosphate, or a combination of similarly buffered glutaraldehyde and paraformaldehyde, can be used successfully to preserve the fine structure of biological tissues. The material next is washed briefly in the buffer vehicle and then secondarily fixed in 1% osmium tetroxide (osmic acid), which also is buffered with sodium phosphate. The tissue then is thoroughly dehydrated in solutions of ethanol at increasing concentrations of 50%, 70%, 95%, and 100%. After dehydration, tissues are infiltrated for a prescribed time interval with an epoxy embedding medium. After infiltration, specimens are transferred into fresh epoxy resin and polymerized at 60 to 70 degrees C for several hours. This orderly process ultimately yields fixed tissues that are encased in hardened blocks that can be thin-sectioned with an ultramicrotome. The thin sections are counterstained with solutions of heavy metals to add contrast. The material then can be subjected to the electron beam in an electron microscope to produce useful images for ultrastructural study. This overall procedure has been used successfully since the advent of biological electron microscopy to define the minute details of cells and tissues.

Effect of phosphorylation on scallop sarcoplasmic reticulum

SummaryFragmented 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; Castellaniet 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.

In situ antigen localization with fluorescamine-conjugated antibodies: A new method

Abstract A simple and rapid procedure for the preparation of fluorescent antibodies with fluorescamine is described. The major advantage of the method is that labeled antibody requires no further purification following conjugation. The utility of the method is illustrated by fluorescent staining of virus-specific cytoplasmic and nuclear antigens, those of vesicular stomatitis virus and adenovirus, respectively. The intensity of fluorescence permits short photographic exposure times, and the stained preparations have retained their fluorescence for more than 9 months.

Animate Protocells from Inanimate Thermal Proteins

Seven thermal protein complexes and the dialyzed products from one of these produced typical protocells (0.1–10 µm in diameter) on their surfaces when moistened with water. Protocells (about 0.5 µm in diameter) were visualized (800–1200x): almost instantly at 60 C; within a few min at 23 C; and, after about 15 min at 4 C. Protocells of about 3.0 µm diameter were observed associated with the thermal protein surfaces: within 30 sec to a few min at 60 C; after 4–10 min at 23 C; and, after 9 hr at 4 C. In all cases, the small protocells were free or in loose aggregates. The large protocells were often: free; linked in chains (filaments) or dendritic structures (5–15 protocells in the branched structures); or, more rarely, multi-linked protocellular clusters (15 or more protocells). This method of observing protocell formation provides opportunities for the study of factors involved in the self-assembly process and in protocell survival (e. g., effects of nutritional requirements for growth, differentiation, and reproduction).