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.

The terminal web. A reevaluation of its structure and function.

The apical cytoplasm of epithelial cells of the small and large intestines has been examined by freeze-etch techniques as well as conventional and high voltage electron microscopy of sectioned material to gain a better understanding of the fine structural organization of the terminal web region. In the small intestine the terminal web exhibits a distinct stratification caused by the association of different sets of filaments with the three members of the junctional complex. Individual filaments of this network are closely associated with the sealing elements of the tight junctions, the surface of the core microfilament bundles, and the intermicrovillar plasma membrane. This region of the terminal web is the apical zone. The adherens zone appears as a band of interwoven filaments of two different diameters extending across the cytoplasm at the level of the intermediate junction. Within this region of the terminal web, individual 60-70 A actin-like filaments separate from the bundles of core microfilaments to interact with one another and with filaments of similar diameter from the zonula adherens. 100 A tonofilaments also contribute to the adherens zone, presumably stabilizing the orientation of the actin-like filaments. The basal zone which underlies the adherens zone consists of closely interwoven bundles of tonofilaments that are anchored to and interconnect the spot desmosomes. Within the large intestine the cytoplasmic microfilaments form a looser and less clearly stratified network which nevertheless retains the same basic organization found in the small intestine. Transmembrane linkers appear to originate within the cytoplasmic plaques of the spot desmosomes, pass through the plasma membranes, and meet in a staggered configuration in the intercellular space; these linkers may thus mediate the actual mechanical coupling between the cytoskeletal networks of tonofilament bundles of adjacent cells. This integrated system of cytoplasmic filaments and intercellular junctions endows the apical cytoplasm with both the flexibility and the stability necessary for the normal functioning of the epithelium.

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.

The mathematics of SELEX against complex targets.

We have developed a computer model for the simulation of simultaneous SELEX against multiple targets. The model assumes equilibrium behavior for the formation of binary ligand:target complexes, and that there is no ligand:ligand or target:target interaction. Target concentrations, ligand concentrations, and affinity distributions of the initial ligand pool for each individual target may be set by the user. We have used this program to gain an understanding of how the presence of multiple targets affects the selection process. In most cases, we find that SELEX is capable of generating different ligands for the different targets in a heterogeneous mixture, regardless of large variations in target concentrations and ligand:target affinities. A low relative partitioning efficiency (the efficiency with which ligands complexed with a target are separated from free ligands) for a target in a mixture gives a greatly reduced rate of selection of high-affinity ligands to that target. The ratio of each high-affinity ligand to its individual target within a pool of ligands selected for binding against a mixture of targets is approximately proportional to the concentration of the target multiplied by the ligand:target partitioning efficiency.

Structural integration of skin equivalents grafted to Lewis and Sprague-Dawley rats.

Bilayered skin equivalents, composed of a sheet of epidermal cells overlying a collagen lattice populated with fibroblasts, quickly become structurally integrated with the surrounding host skin after grafting to Lewis rats. Three days after transplantation, the skin equivalent lies on a bed of host granulation tissue and is loosely attached to the adjoining host dermis. Blood vessels begin to invade the collagen lattice by 5 days after grafting. By the 7th day a fully keratinized, hypertrophic epidermis covers the surface of the graft and blood vessels penetrate the lattice to the base of the epidermis. Vascularization of the graft is accompanied by activation and proliferation of the fibroblasts and by a condensation of the collagen matrix. During the 2nd week after grafting, the collagen fibrils become organized into thin fibers that show a basketweave pattern of birefringence when examined using polarized light. By 1 month the structure of the skin equivalent has become stabilized. The fibroblasts now resemble the quiescent fibrocytes of normal, resting dermis and the epidermis remains moderately hypertrophic. One to two years after grafting to Sprague-Dawley rats, the skin equivalents do not appear hypertrophic. The graft lacks secondary derivatives such as hair follicles and sweat glands, presumably because the stem cells are lost during the isolation of the epidermal cells. Grafts that are prepared using epidermal cells overlying a collagen gel without fibroblasts give rise to raised, linear scars within 2 weeks.

Fibroblasts in isogeneic skin equivalents persist for long periods after grafting.

We have fabricated skin equivalents by combining fibroblasts from female Fischer rats with collagen to form a lattice and overlaying the lattice with a suspension of epidermal cells. The epidermal cells attach and form a sheet which differentiates. These skin equivalents were then grafted to male Fischer rats in order to follow the fate of the fibroblasts after implantation. Biopsies of the skin equivalent were taken between 9 days and 13 months after grafting and examined histologically or placed in tissue culture to permit karyotyping of the resident fibroblasts. Approximately 82% of the fibroblasts from the graft biopsied at 9 days were female, with this proportion decreasing sharply to 50% at 2 weeks and 60-64% at 1 month. At 1 month, this initial sharp drop is followed by a slow, linear decline which continues through the 13th month when 42% of the fibroblasts are female. We conclude that fibroblasts of the grafted skin equivalent become permanent residents of the skin of the host rat.

The reconstitution of living skin.

A living-skin equivalent useful as a skin replacement and as a model system for basic studies has been fabricated and tested extensively. It consists of two components: (1) a dermal equivalent made up of fibroblasts in a collagen matrix that is contracted and modified by the resident cells, and (2) an epidermis that develops from keratinocytes "plated" on the dermal equivalent. A multilayered keratinizing epidermis with desmosomes, tonofilaments, and hemidesmosomes forms. Basement lamella formation occurs within 2 weeks in vitro when rat cells are used. With human cells, crypt or pseudofollicular morphogenesis is observed in vitro within 3 weeks after plating cells on the dermal equivalent. Autografts and isografts of rat-skin equivalents made with cultured cells from biopsies are rapidly vascularized, block wound contraction, and persist essentially for the lifespan of the host. Seven to 9 days after grafting, donor cells become activated biosynthetically and mitotically. By 1 year, the dermal population decreases to a normal level and the matrix has been extensively remodeled. The grafts remain free of hair and sebaceous glands. Grafts to rats have been in place for over 2 years. Now, allografts of dermal equivalents have been made across a major histocompatibility barrier and are not rejected. The persistence of cellular elements of the grafts is monitored by use of a genetic marker. Challenge of the allograft with a second skin-equivalent graft after 1 month does not result in rejection of the original graft or of the second skin-equivalent graft. We propose that allografts of tissue equivalents are tolerated because cells with class II antigens are selected against during in vitro cultivation and are excluded from the graft. Thus the fabrication of skin-equivalent tissues or of other equivalent tissues with parenchymal cells that do not bear class II antigens may render transplants of such tissues immunologically acceptable despite the presence of allogeneic cells. The capacity to graft across major histocompatibility barriers using living tissue equivalents may have important clinical significance.

Characterization of label-retaining cells in the epidermis of a human skin equivalent.

The human skin equivalent (HSE) is an in vitro reconstructed model that resembles skin morphologically and biochemically. The HSE is formed by overlaying a fibroblast-populated collagen matrix with a suspension of epidermal cells. Basal keratinocytes attach to the dermal equivalent via a newly formed basement membrane and multiply to form a stratified, differentiated epidermis. The aim of the studies described here was to characterize the basal cells of the HSE in terms of their cell cycling potential. The experiments utilized long-term labelling of the cells with tritiated thymidine ([3H]dT), followed by irradiation with ultraviolet light. [3H]dT incorporation was analysed via routine autoradiography. Irradiation with 100 J/m2 UV light increased the number of labelled basal cells by 58% over the control, the maximal stimulation observed. Decreased numbers of labelled basal cells were observed at doses of UV light greater than 100 J/m2. The maximal number of labelled basal cells was observed on day 14 and decreased over time; the number of labelled suprabasal cells increased concomitantly. Label-retaining cells (12%) persisted in the stratum basale of control HSEs after 32 days in culture. Labelled cells were observed in the apical layers of the stratum granulosum of control HSEs after 22 days in culture. These data suggest that the stratum basale of the HSE contains a population of slow-cycling cells whose characteristics resemble a subpopulation of slowly cycling cells found in normal human skin.

A study of HTLV-I and its associated risk factors in Trinidad and Tobago.

Seroprevalence of human T-lymphotropic virus type 1 (HTLV-I) among a sample of persons selected from a government register of businesses in Trinidad was 3.2% in 1,025 persons of African descent compared to 0.2% among 487 persons of Asian descent and 0% among 46 persons of European-descent. In Tobago, from a coastal village, among persons of African ancestry ascertained as part of a cardiovascular survey, the rate was 11.4%, which was significantly higher when corrected for age and race than the rate in Trinidad. The seroprevalence rate of antibodies to hepatitis A and B was also significantly elevated in Tobago compared to Trinidad. HTLV-I seroprevalence rates were higher in females than males while hepatitis A and B rates were not significantly different in the two sexes. For males, age was a significant determinant of HTLV-I seropositivity, while for females, age, markers of poor sanitation, and hepatitis B were each independently linked to HTLV-I seropositivity. The frequent occurrence of multiple infectious exposures in persons of lower socioeconomic circumstances in this tropical environment may result in immune activation that heightens susceptibility to HTLV-I infection.

Regulation of proliferation of fibroblasts of low and high population doubling levels grown in collagen lattices.

While IMR 90 and AG 1519 fibroblasts of low and high population doubling levels grow to confluency when plated on plastic surfaces, they cease to divide within four days when incorporated into collagen lattices. Growth inhibition in the lattices is not due to exhaustion of the medium or isotope, or to contact inhibition; nor is it due to impermeability of the lattice to the materials in the medium. While cells in a lattice arrest in G0, this state is reversible when cells are permitted to leave the lattice and populate a plastic substrate. We conclude that fibroblasts in tissue-like lattices may be responsive to some of the same controls as cells in connective tissues.

Acceptance of allogeneic fibroblasts in skin equivalent transplants.

Living skin equivalents (SE) were prepared by combining cultured fibroblasts with a collagen matrix and overlaying this lattice with keratinocytes. SEs prepared using allogeneic female rat fibroblasts or xenogeneic rabbit or human fibroblasts and keratinocytes isogeneic to the graft recipient were transplanted to recipient male rats. Biopsies of some of these SE grafts were examined histologically at intervals ranging from 5 days to 2 months. Biopsies of other grafts were done, and fibroblasts grown from them were karyotyped to determine the percentage of donor fibroblasts remaining in the graft. SEs containing xenogeneic fibroblasts were rejected. Allografted fibroblasts in SEs were accepted by recipient rats after a transient mononuclear cell response. A second SE allograft from the same donor strain did not provoke rejection either in the original allograft or in the challenge allograft. A secondary graft of allogeneic skin did not provoke rejection in the original SE graft, although the skin graft was rejected. Grafting the recipient first with allogeneic skin and then with the SE allograft led to rejection of the skin but not of the SE graft, ruling out the possibility that suppressor T cells were responsible for SE allograft acceptance. Allografted fibroblasts in SEs do not provoke a rejection response, even in presensitized animals, do not render the recipient tolerant to allogeneic skin, and do not act as targets when active rejection is taking place. We propose that cells bearing class I antigens may be acceptable graft constitutents if incorporated in a tissue equivalent excluding cells with class II antigens.

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.

Natural transovarial transmission of dengue 4 virus in Aedes aegypti in Trinidad.

The isolation of dengue 4 virus from adult Aedes aegypti, reared from eggs collected in nature, is reported for the first time. From the locality where the isolate was made, 25 pools consisting of 1,848 Ae. aegypti reared from eggs were processed. In this study, 10 different localities were sampled and a total of 10,957 Ae. aegypti adults, collected as eggs or larvae in nature, were processed for virus isolation. From a total of 158 mosquito pools tested, one recovery of dengue 4 virus was made. The isolation of dengue 4 virus from this field-collected material gives further evidence that transovarial transmission of dengue viruses occurs in nature.

The relative importance of rotavirus and other pathogens in the etiology of gastroenteritis in Trinidadian children.

Over a 2-year period, 300 infants less than 3 years old with gastroenteritis admitted to hospitals in Trinidad were investigated for the presence of certain microorganisms in the feces, along with an equal number of age- and sex-matched controls. Rotavirus was detected in 23% of cases and 1% of controls; Salmonella in 7% of cases and in 1% of controls; Shigella in 4% of cases and in no controls and two serotypes of enteropathogenic E. coli in 7% of cases and in 2% of controls. Campylobacter fetus subspecies jejuni was cultured from 7 out of 60 cases and from 1 of 60 controls. Enterotoxigenic E. coli, most strains of enteropathogenic E. coli, cytopathic enteroviruses and adenoviruses and fecal parasites were not significantly associated with diarrhea. Rotaviruses were detected throughout the year but were more prevalent in the dry than in the rainy season. They were found less often in children younger than 6 months than in those aged 6 to 35 months and were present in 6 of the 20 children who died.

Sylvatic yellow fever activity in Trinidad, 1988-1989.

Of a total of 18,068 mosquitoes (361 pools) collected in south-eastern Trinidad forests from December 1988 to May 1989, 47 species belonging to 14 genera were identified. Five yellow fever virus isolates were made from Haemagogus janthinomys and one from Sabethes chloropterus. All the other pools of mosquitoes examined were negative for the virus. The mosquito isolates were made in December and January. In addition, in late February and early March, 2 infected howler monkeys (Alouatta sp.) were detected. Since March, despite continued surveillance, no yellow fever virus has been detected in mosquitoes or monkeys. There has been no reported human infection.

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.

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.

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.

A mathematical model to measure the impact of the Measles Control Campaign on the potential for measles transmission in Australia.

BACKGROUND: The aims of this study were to determine the impact of the Australian Measles Control Campaign (MCC) on the transmission dynamics of measles by calculating the reproductive number (R) before and after the MCC, and to predict measles control in Australia in the future. METHODS: A national serosurvey was conducted before and after the MCC. Sera were tested for anti-measles IgG using enzyme immunoassay (EIA). A mathematical model, using serosurvey results and vaccine coverage estimates, was used to calculate the change in R after the MCC. RESULTS: The values of R calculated before and after the MCC were 0.90 and 0.57. At vaccine coverage levels indicated by the Australian Childhood Immunisation Register (ACIR), the value of R will exceed 1 (the epidemic threshold) in 2007-2008 nationally, and sooner in some regions of Australia. Coverage of at least 84% with two doses of MMR is required to sustain measles control. CONCLUSIONS: The Australian MCC had a significant impact on the transmission dynamics of measles. However, current vaccine coverage levels may result in indigenous measles transmission by 2007. Sustained efforts are required to improve coverage with two doses of MMR and to ensure elimination of indigenous measles transmission.