The efficacy of a DNA vaccine encoding herpes simplex virus type 1 (HSV-1) glycoprotein D in decreasing ocular disease severity following corneal HSV-1 challenge.

Antiviral effects of a DNA vaccine against herpes simplex virus 1 (HSV-1) glycoprotein D (gD) were evaluated in eight week-old female BALB/c mice. The nuclease-insensitive construct (gD-ASOR) consisted of an HSV-1 gD encoding plasmid coupled to asialo orosomucoid (ASOR), targeting it to cells bearing ASOR receptors. Mice were immunized on day 0 and 7 with 10 microg doses of gD-ASOR or control substances. Fourteen days later, mice were infected by the corneal route with 10(5) pfu or 10(6) pfu HSV-1, strain 17syn+. Immunized mice showed a significant decrease in ocular disease severity over a 21-day observation period following infection compared to sham-immunized mice. Acute replication kinetic assays demonstrated a 100-fold decrease in viral titers on day 6 in trigeminal ganglia from immunized BALB/c mice compared to sham-immunized mice. Immunized mice showed a significant increase in numbers of CD4(+)T cells infiltrating the trigeminal ganglia at day 6 post infection compared to sham-immunized mice. Significant differences were not seen in latent viral reservoir between immunized and unimmunized mouse groups. Immunization with gD-ASOR decreased the severity of acute ocular HSV-1 infection, induced a CD4(+) T cell response, decreased the viral load in the trigeminal ganglia, but did not diminish viral latency.

Murine response to DNA encoding herpes simplex virus type-1 glycoprotein D targeted to the liver.

Plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) was complexed with asialoorosomucoid conjugated to poly-L-lysine. Following its intravenous injection into BALB/c mice, this complex was targeted to the liver. Liver cells expressing gD-1 were detected immunohistochemically through day 6 post-immunization, while gD-1 DNA was detectable through 14 days post-immunization. Decline of gD-1 expression and detectable gD-1 DNA in the liver correlated with influx of T cells, predominantly CD4(+). The ASOR-poly-L-lysine DNA carrier system promotes hepatic expression of gD-1 and may be useful in vaccination against herpes simplex virus type-1.

Targeted delivery of DNA encoding herpes simplex virus type-1 glycoprotein D enhances the cellular response to primary viral challenge.

Intravenous injection of plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) complexed with asialoorosomucoid-poly-L-lysine (gD-ASOR) targets foreign DNA to the liver, leading to hepatic expression of gD-1. BALB/c mice were given two intravenous injections of gD-ASOR, pBK-ASOR (plasmid lacking the gD-1 gene but complexed with ASOR), or PBS. The skin was inoculated with 1 x 10(4) PFU of HSV-1 or sham-inoculated, and analyzed for infectious virus and cellular infiltration 1, 3, and 5 days after inoculation. Prior immunization with gD-ASOR led to significantly lower (P < 0.05) viral titers in the skin 5 days after inoculation compared with controls. Infiltration of the skin at the site of inoculation by polymorphonuclear neutrophils (PMNs), T cells, B cells, dendritic cells, and macrophages was monitored immunohistochemically. Significantly higher numbers (P < 0.05) of CD4+ and CD8+ T cells, dendritic cells, and macrophages responded to HSV-1 challenge in mice immunized with gD-ASOR than in mice immunized with pBK-ASOR or PBS. The response by PMNs and B cells was indistinguishable among the treatment groups. These results suggest that BALB/c mice sensitized to gD-1 following gD-ASOR immunization develop an enhanced T-cell response to primary HSV-1 infection.

Differential response of basal keratinocytes in a human skin equivalent to ultraviolet irradiation.

The human skin equivalent (HSE) provides a convenient model system for studying the cellular responses of basal keratinocytes to UV irradiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were raised to an air-liquid interface to promote epidermal differentiation. HSEs were exposed to ultraviolet radiation from a 500-W Hot Quartz Hanovia therapeutic sunlamp, at a total dose of 100 J/m2. The HSEs were then frozen every 4 h over a 48-h period and cryosectioned. For each time period, the expression of beta1 integrin and cyclin E, p53, or Bcl-2 were quantified using dual immunolocalization. Basal cells expressing beta1 integrin were divided into two subpopulations, denoted beta1high or beta1low. The proportion of beta1high keratinocytes expressing Bcl-2 and cyclin E increased significantly 4 and 8 h, respectively, after exposure to UV; during the subsequent 16 h, this basal cell subpopulation expressed p53. By contrast, significant numbers of beta1low basal keratinocytes expressed p53, but not Bcl-2. These results suggest that beta1high and beta1low populations of basal epidermal cells in HSEs respond differently to UV irradiation.

Production of IL-1 alpha and IL-1 beta by human skin equivalents parasitized by Sarcoptes scabiei.

Human skin equivalents (HSEs) were used as a model to investigate interleukin (IL)-1 alpha and IL-1 beta secretions by keratinocytes stimulated by Sarcoptes scabiei (SS). SS mites burrowed into the stratum corneum when placed on the surface of cultured HSEs. Mites lived for 14 days. Mites and mite products induced cells in the HSEs to secrete IL-1 alpha and IL-1 beta within 16 hr. Scabies mites induced production of greater amounts of IL-1 alpha than IL-1 beta. Hepatocyte growth factor in the culture medium at 3 and 30 ng/ml upregulated the secretions of both IL-1 alpha and IL-1 beta by mite-infested skin equivalents, whereas 10 ng/ml of IL-6 upregulated production of only IL-1 beta. Therefore, these cytokines were important immunomodulating factors influencing keratinocyte secretion of IL-1 alpha and IL-1 beta in vitro. The results of this study provide the first evidence that keratinocytes (possibly fibroblasts) in the skin produce these cytokines in response to scabies mites or other ectoparasitic arthropods. Because IL-1 alpha and IL-1 beta are potent inducers of inflammation and keratinocytes are among the first effector cells to encounter scabies mites and their products, these cells may be key initiators of the inflammatory/immune reaction to scabies.

Long term effects of diaminophenoxypentane in the rat retina: protection against light damage.

The retinotoxic drug diaminophenoxypentane (DAPP) was administered to rats to determine its long term effects on rhodopsin levels, retinal morphology and the retina's susceptibility to damage from visible light. In rats given 2 intraperitoneal injections of DAPP at doses of 65 mg/kg body wt, there was a dramatic and sustained loss of rhodopsin. One wk later visual pigment levels were 41% lower than in comparable dark maintained rats injected with saline. Rhodopsin levels in the DAPP treated rats remained lower than in control animals for the 13 wk period of the study. Morphologically, the ROS of rats 1-2 wks after DAPP treatment exhibited some disorganization and shortening; the RPE was unremarkable. Seven wks after DAPP treatment an occasional focal area of damage was seen in the RPE. Similarly, focal areas of degeneration were seen in the outer nuclear layer between the rows of photoreceptor cells. As determined by photoreceptor cell nuclear counts, the retinotoxic effect of DAPP persisted long after drug administration. In the treated rats the loss of visual cell nuclei was 11% at 7 wks; it was 22% 13 wks after DAPP treatment. Immediately after exposure to intense visible light, damage was seen in both the photoreceptor cells and RPE of DAPP treated rats. However, the effects of exposure in the treated rats were less dramatic than in retinas from rats without DAPP treatment. Following a 2 wk dark recovery period, the DAPP treated rats had a normal appearing retinal morphology and an intact RPE layer. The retinas of rats without DAPP treatment showed extensive visual cell and RPE loss.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of ultraviolet radiation-induced damage to keratinocytes in a skin equivalent in vitro.

The human skin equivalent (HSE) provides a convenient model for studying the dermatological effects of exposure to ultraviolet (UV) radiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were maintained submerged for 1 week after the addition of epidermal cells and then raised to the air-liquid interface for an additional 3 weeks. HSEs were exposed to sublethal doses of UV radiation ranging from 0 to 500 J/m2, incubated up to 48 h in medium containing 3H-thymidine and fixed for ultrastructural and autoradiographic analysis. Exposure to radiation doses greater than 50 J/m2 led to vacuolation of the cornified envelopes and enlargement of intercellular spaces. These doses also led to the formation of dense cytoplasmic bodies, and separation and vesiculation of the nuclear envelope in the basal cells. DNA synthesis in the basal cells was analyzed autoradiographically. Maximal numbers of labeled basal cells were observed 24 h after exposure to UV radiation at 50 J/m2. Although the proportions of labeled cells varied among different epidermal donors, the maximal responses and time-course of 3H-thymidine incorporation remained consistent, supporting the usefulness of the HSE in studying the effects of UV irradiation on human skin.

Cornification and basement membrane formation in a bilayered human skin equivalent maintained at an air-liquid interface.

Human keratinocytes that were grown in a skin equivalent at an air-liquid interface were analyzed morphologically and biochemically to demonstrate differentiation approaching that of human skin. Within 3 weeks of growth at the interface, cuboidal basal cells, distinct spinous and granular zones, and a fully developed cornified layer of enucleated cells formed the multilayered epidermis. Ultrastructurally, the keratinocytes in the upper granular layer contain tonofilament bundles and membrane-coating granules. These cells form cornified squames that are resistant to degradation by sodium dodecyl sulfate/dithiothreitol. Basal cells are attached to a developing basement membrane with hemidesmosomes. Immunogold silver staining analysis with monoclonal antibodies demonstrated the expression of basement membrane collagens IV and VII. This level of differentiation might improve "take" of human grafts and provides a useful system with which to study topical carcinogens and tumor promoters in vitro.

Coverage of full-thickness burns with bilayered skin equivalents: a preliminary clinical trial.

A total of six patients have received bilayered skin-equivalent coverage of full-thickness burns, with takes of 50% to 70% in the later patients. These skin-equivalent grafts are constructed by combining allogeneic fibroblasts with collagen to form a sheet and adding a suspension of autologous epidermal cells to the surface of the collagen matrix. These bilayered skin-equivalent grafts have provided an expansion of at least fifteenfold to twentyfold for the area covered by the donor epidermis. By 8 months after grafting, the skin-equivalent grafts appeared smooth and approximated the color of normal skin. Long-term problems associated with hypertrophic scarring or graft fragility have not developed during the 18-month period of follow-up.

Replacement of full-thickness burns on mice with isogeneic skin equivalents.

A copper slug heated with a soldering iron was used to produce full- and partial-thickness burns on the backs of mice. The untreated partial-thickness burns healed by outgrowth of epidermal cells from the hair follicles and adjoining skin, and the full-thickness burns formed linear scars. Skin equivalents containing isogeneic fibroblasts and epidermal cells were used to replace full-thickness burns; these grafts were fully vascularized and covered with a cornified epidermis within 2 weeks. The grafts maintained 34% of their original area at 180 days, but the full-thickness burns retained only 4.5% of the initial area. For the first 2 weeks, the splenic index in animals that received burns followed by surgical excision and grafting was significantly greater than in the animals that had burns not followed by excision, but the difference was no longer significant by 21 days.

Structural characterization of a rat acinar cell tumor.

A transplantable acinar cell tumor of the rat pancreas has been examined by light and electron microscopy. The tumor cells, though highly cytodifferentiated and characterized by the presence of abundant rough-surfaced endoplasmic reticulum, elements of the Golgi complex, and zymogen granules, undergo mitosis in a manner similar to that seen in the developing pancreas. Cells in the parenchyma of the tumor grow as disarrayed cords and sheets, are randomly oriented with respect to each other, and do not form acinar structures. However, when in contact with the adventitial surface of blood vessels, the tumor cells palisade and form a polarized layer of cells with their zymogen granule-rich poles oriented away from the vessel lumen. Only in this area of the tumor is a basal lamina present that underlies the basal plasmalemma of the reoriented epithelial cells. Freeze-fracture electron microscopy of tumor cells in the parenchyma shows extensive disruption of tight junctions whose sealing strands are randomly distributed over the entire plasmalemma. Gap junctions are infrequent and when present are often enclosed by tight-junctional strands. Intramembrane particles are randomly distributed over the cell surface. Both the absence of basal lamina and derangement of the junctional complexes may account in part for the altered morphogenesis of this tumor.

Cell surface properties of normal, differentiating, and neoplastic pancreatic acinar cells.

Acinar, centroacinar, and endocrine cells of the adult rat pancreas each exhibit distinctive cell-surface glycoconjugate patterns as detected by binding of a battery of lectin-ferritin conjugates. Acquisition of these unique glycoconjugate patterns appears to be developmentally regulated, as studies on embryonic rat pancreases at days 15, 17, and 19 of gestation indicate. Further, the three cell types appear to arise from a common stem cell(s) with surface glycoconjugate properties similar to those of the adult centroacinar cell. STudies on the cell-surface properties of a rat acinar cell tumor indicate that the neoplastic acinar cells are likely to be arrested at a developmental stage equivalent to acinar cells of the day 19 embryonic pancreas and are characterized by absence of detectable basal lamina. We hypothesize that pancreatic cancers may arise from the equivalent of the undifferentiated embryonic stem cell(s) and that the morphologic features of the tumor depend on the extent of cell differentiation, including expression of cell-surface glycoproteins and extracellular matrix, prior to neoplastic proliferation.

Functional significance of the variations in the geometrical organization of tight junction networks.

Using freeze-fracture techniques, we have examined the morpholog of tight junction networks found along the length of the alimentary tract of Xenopus laevis before and after metamorphosis. We have developed the hypothesis, based on these observations, that the geometrical organization of the network determined by the stress-induced shape changes normally experienced by the cells linked by the network. Consistent with this theory, tight junctions can be classified into two distinct types of network organization which differ in their response normal and experimentally induced stress conditions: (a) loosely interconnected networks which can stretch or compress extensively under tension, thereby adapting to stress changes in the tissue; and (b) evenly cross-linked networks which retain their basic morphology under normal stress conditions. The absorptive cells of the large intestine as well as the mucous cells of the gastrointestine or stomach are sealed by the first, flexible type of tight junction. The second type of junctional organization, the evenly cross-connected network, is found between absorptive cells of the small intestine and ciliated cells of the esophagus, and reflects in its constant morphology the relative stability of the apical region of both of these cell types. Networks intermediate between these two types arise when a cell which would normally form a lossely interconnected network borders a cell which tends to form a more evenly cross-linked network, as is found in the esophagus where ciliated and goblet cells adjoin. Despite the change in the animal's diet during metamorphosis from herbivorous to carnivorous, the basic gemetrical organization of the networks associated with each tissue of the alimentary tract remains the same.