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In this study, we demonstrate the role of M cells in uptake of poly(D-L-lactic-co-glycolic acid) (PLGA) microspheres and transport into rabbit Peyer's patches. Microspheres 1 to 10 microns in diameter composed of 50:50 lactic acid:glycolic acid were instilled into intestinal segments containing jejunal or ileal Peyer's patches, and uptake by M cells was examined by electron microscopy. PLGA microspheres visualized as electron-lucent, spherical particles were taken up by M cells by pseudopod-like extensions of the M cell apical membrane and translocated to the pocket region containing mononuclear leukocytes within 60 min. These results indicate that PLGA microspheres can be directed to M cell apical surfaces for delivery to immunocompetent cells in gut-associated lymphoid tissues.

Freshly ejaculated bull semen was centrifuged and spermatozoa were resuspended in modified sperm TALP. Bovine uterine tube epithelial cell monolayers (BUTC) were obtained from cows in the periovulatory phase of estrus. In Experiment 1, sperm aliquots were assigned to culture wells containing either BUTC, BUTC-conditioned TALP, or control TALP. Sperm heads attached to the monolayers within 1 h of co-culture. Attached spermatozoa showed vigorous tail motion. At 5, 8 and 11 h of incubation at 39 degrees C, the percentage of unattached sperm cells with intact acrosome membranes and percentage of motility of these cells was measured. Sperm-BUTC co-cultures were also fixed in situ for electron microscopy. Unattached spermatozoa in co-culture had more (P<0.05) acrosomal membrane loss, showed hyperactive motion and had an overall decrease in motility as compared to sperm cells in control or conditioned medium. Evaluation by electron microscopy showed BUTC attached spermatozoa to behave in the co-culture system similar to reports for spermatozoa found in uterine tubes in vivo. Microvilli of the BUTC appeared to actively entrap the spermatozoa. Mucus-type granules could be seen on acrosomal regions and vesiculation of acrosomal membranes was seen in some cells. In Experiment 2, 43% of the 12 x 10(6) sperm cells added to 2-cm(2) BUTC bound within 4 h of co-culture. By 7 h of co-culture 19% of the previously bound sperm cells had been released from the BUTC. Released cells had limited motility and were mostly dead (73%). Sperm cells remaining on the monolayer at 7 h showed vigorous tail motion and were gradually released from the BUTC over 48 h. Spermatozoa in co-culture interacted with the BUTC in a manner much like that seen in vivo, and sperm capacitation changes were stimulated by this interaction.

Neoplastic canine keratinocytes derived from a spontaneous oral squamous cell carcinoma were maintained in culture for more than 45 passages. The presence of desmosomes and keratin filaments was demonstrated by electron microscopy and immunohistochemistry. The keratinocytes were grown in two different culture conditions to induce variations in the stage of differentiation, i.e., in submerged cultures and at the air-liquid interface. For comparison, normal canine keratinocytes were grown under the same conditions. Anisocytosis was present in neoplastic cultures grown submerged in medium. Grown at the air-liquid interface, neoplastic keratinocytes differentiated into a well-organized, multilayered stratified squamous epithelium analogous to normal keratinocytes. Rare areas of irregular growth and formation of whorls were detected. Expression of lectin binding sites and specific cell surface antigens of neoplastic and normal keratinocytes demonstrated marked similarities between the two cell lines. Neoplastic cells lacked certain surface antigens that are present on normal cells. Squamous cell carcinoma cells grew faster than normal canine keratinocytes as demonstrated by growth curve evaluation. Neoplastic keratinocytes responded to growth stimulation by epidermal growth factor and cholera toxin as do normal keratinocytes. Neoplastic cells grown in medium lacking these factors proliferated faster than growth factor stimulated normal keratinocytes.

Pemphigus antigens were localized, by use fo immunoelectron microscopy, on canine keratinocytes in vivo on esophageal mucosa and in vitro on established cultured keratinocytes. Convalescent sera from a human being with pemphigus vulgaris and a human being with pemphigus foliaceus reacted with the interdesmosomal cytoplasmic keratinocyte membrane of canine esophagus. Cultured canine keratinocytes expressed the pemphigus vulgaris antigen in a similar pattern, but did not carry the pemphigus foliaceus antigen. The differential presence of cell surface antigens and its relation to various forms of the disease are discussed.

Three experimental approaches were used to study transmission of blue tongue (BT), infectious bovine rhinotracheitis (IBR) and bovine virus diarrhoea (BVD) viruses. These were insemination with contaminated semen, experimental infection of embryo donor cows, or transfer of embryos experimentally exposed to virus in vitro to normal recipients. Parameters assessed included number and quality of embryos produced, virus detection (isolation and electron microscopy), serology and histopathology. All superovulated sesceptible cows inseminated with semen containing blue tongue virus (BTV) (n = 2) or infectious bovine rhinotracheitis virus (IBRV) (n = 2) became infected. One cow inseminated with semen containing BTV produced seven virus-free seven-day-old embryos; the second cow failed to produce any embryos. One of two cows inseminated with semen containing IBRV produced two underdeveloped, virus-free embryos while no embryos were produced by the second cow. One of two cows inseminated with semen containing bovine viral diarrhoea virus (BVDV) became infected. Two poorly developed, virus-free seven-day-old embryos were recovered from one of these cows. Superovulated susceptible cows inoculated either intramuscularly with BTV (n = 3) or intranasally with IBR virus (n = 2) became infected. Virus was isolated from some tissues of two BTV-infected cows, neither of which produced embryos. A third BTV-infected cow produced two virus-free embryos collected at necropsy five days after inoculation. One of two cows experimentally infected with IBR virus, produced three embryos but virus was not detected either by electron microscopy (1 embryo) or in cell culture by cytopathic alterations (1 embryo).(ABSTRACT TRUNCATED AT 400 WORDS)

We report on application of flow cytometric and immunogold labeling techniques to purify and identify two types of murine epidermal dendritic cells: Langerhans cells (LC) and Thy-1-positive dendritic epidermal cells (Thy 1+-dEC). After density centrifugation of epidermal cell (EC) suspensions through Ficoll gradients. IA-positive LC and Thy 1+-dEC are labeled with monoclonal antibodies (fluorescein-conjugated anti-IAd for LC and anti-Thy 1.2-biotin, followed by avidin-phycoerythrin, for Thy 1+-dEC). The fluorescence-activated cell sorter (FACS) is then used to obtain 95-98% pure populations of these dendritic cells with a yield of 2-4 X 10(6) cells and a viability of 80-90%. A post-fixation, pre-embedding immunogold labeling technique using 15 nm and 40 nm colloidal gold particles is employed to identify LC and Thy 1+-dEC, respectively, to confirm the purity of the sorting and to estimate the number of IA antigenic sites per LC. With transmission electron microscopy, ultrastructural morphology of sorted LC is preserved; however, Birbeck granules are markedly diminished compared to the pre-sorted population of LC. In contrast, characteristic dense-core granules are readily visualized in sorted Thy 1+-dEC. Purification of epidermal dendritic cells by flow cytometry may be a useful technique to employ in functional studies of epidermal dendritic cells.

A cholangiocarcinoma was diagnosed in a 4-month-old double yellow-cheeked Amazon parrot (Amazona autumnalis). Histologically, neoplastic cells effaced hepatocellular architecture. Ultrastructurally, variably dilated bile ductules were lined by simple cuboidal to low columnar ciliated epithelial cells with sparse cellular organelles and prominent junctional complexes. Elongated single cilia arising from basal bodies in the apical cytoplasm were occasionally evident. The presence of such a neoplasm in a 4-month-old parrot is extremely unusual.

Keratoconjunctivitis sicca was produced experimentally in 16 beagles by bilateral surgical removal of the lacrimal and nictitans glands; four dogs were not treated, and 12 received tear-replacement therapy on post-operative days 7 through 28. Keratoconjunctivitis sicca was verified by reduction in Schirmer tear test values by post-operative day 6, and there was no response on day 28 to tear-replacement therapy. Corneas of both normal and tear-deficient dogs had polygonal squamous epithelial cells of light and dark electron density by scanning electron microscopy. Light cells had more microvilli and microplicae than dark cells. Conjunctivae were similar to corneas, except for numerous goblet cells on the surface. Corneal dark-cell density and goblet cell density were not different between groups. Goblet cells most often occurred singly in normal dogs, while they were in clusters in tear-deficient dogs. A hypothesis that petrolatum/mineral oil ointment should provide more effective artificial tear replacement than hydroxymethylcellulose drops for tear-deficient dogs could not be confirmed by objective analysis of corneal dark-cell density or conjunctival goblet cell density.

A new method is described for preparing thin sections for transmission electron microscopy of ciliated respiratory epithelial cells cultivated in vitro on Teflon membranes. Hamster tracheal explant cultures were treated with collagenase to promote epithelial migration. Patches of monolayer growth occurred in the vicinity of the explants when the latter were incubated on FEP-etched Teflon membranes in Chamber/Dishes. Cells in the monolayer maintained a high degree of differentiation as evidenced by active ciliary motion. The monolayer was fixed and processed in situ. During the embedding process the chamber was supported on a solid Teflon cylinder to keep the membrane taut as it was heated to polymerize the resin. The membrane was removed and the monolayered cells were re-embedded and sectioned. Electron microscopic examination of cultures revealed clear cell profiles on a flat, even basal plane. This method provides for a normal spatial configuration of cells in a monolayer format, and should prove useful for electron microscopy studies of any cells cultured on flexible films.

Langerhans cells (LC) are epidermal antigen-presenting cells capable of inducing allogenic, antigen-specific, and cytotoxic T cell proliferation. Previous studies have examined the dynamics of LC maintained in vitro in crude epidermal cell (EC) suspensions in which the major cell type is the keratinocyte (KC). To avoid the confounding effects of KC and other immunoregulatory cells on LC dynamics in vitro, highly enriched murine LC (85%) were studied, through 72 h of incubation in vitro, for their ability to present alloantigen (in a primary allogenic proliferation assay) and foreign antigen (in a secondary autologous proliferation assay). The results were compared to similar studies using crude EC suspensions. Freshly prepared LC are very poor stimulators of a primary allogenic proliferation response, with a 12- to 16-fold increase in stimulatory capacity by 72 h using panned-enriched and crude EC suspensions, respectively. Similarly, freshly prepared LC are weak stimulators of a secondary autologous proliferation response, with a 2.5- to 6-fold increase in immunostimulatory capability by 72 h. The overall increased stimulatory effect observed with the crude EC suspensions compared to highly enriched LC is most likely attributed to the effect of KC on T cell proliferation, rather than to a maturation effect of KC on LC during the 72 h of in vitro incubation. Using back-scattered electron imaging, the surface density of MHC-class II molecules (Ia) increased three- to fourfold through culture, which parallels the increase in functional ability. This study demonstrates that LC in either a crude or highly enriched cell suspension mature into potent immunostimulatory cells after incubation in vitro with an increased surface expression of Ia molecules. Keratinocytes are not necessary for LC maturation in vitro, but seem to exert some stimulatory effect by enhancing lymphocyte proliferation in the functional assay system.

Tracheal explants were used to evaluate the relative ciliostatic and cytotoxic potential of heavy metal salts (cadmium chloride, chromium chloride, nickel chloride, and copper sulfate). Explants from hamster, rat, and guinea pig were all sensitive to the metals, though guinea pig explants showed the greatest difference between the untreated and metal treated tissues. Dosage levels were 50, 100, and 500 microM, for 24 to 148 h. Cadmium caused the greatest degree of ciliostasis and cell necrosis. Copper was less toxic, and nickel and chromium caused marginal damage when tested at 100 microM or lower. In each instance, damage became detectable at approximately 24 to 48 h and was nearly stabilized by 72 h. A significant loss of ciliary motion was always accompanied by a decrease in metabolic activity (dehydrogenase activity and ATP content). Transmission and scanning electron microscopy revealed a severely necrotic epithelium after exposure to cadmium, with only subtle morphological alterations after exposure to other metals. With all of the treatments there was no overt structural damage to cilia and little alteration in membranes of cells remaining in the epithelium. Some coagulation or vacuolization was noted in cadmium and copper treated explants but most cellular organelles did not display obvious damage. The most significant changes in the tracheal epithelium exposed to heavy metal salts in vitro were a loss of ciliary motion and a decrease in total ATP content.