Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

The effect of apathy and compulsivity on planning and stopping in sequential decision-making

Real-life decision-making often comprises sequences of successive decisions about whether to take opportunities as they are encountered or keep searching for better ones instead. We investigated individual differences related to such sequential decision-making and link them especially to apathy and compulsivity in a large online sample (discovery sample: n = 449 and confirmation sample: n = 756). Our cognitive model revealed distinct changes in the way participants evaluated their environments and planned their own future behaviour. Apathy was linked to decision inertia, i.e., automatically persisting with a sequence of searches for longer than appropriate given the value of searching. Thus, despite being less motivated, they did not avoid the effort associated with longer searches. In contrast, compulsivity was linked to self-reported insensitivity to the cost of continuing with a sequence of searches. The objective measures of behavioural cost insensitivity were clearly linked to compulsivity only in the discovery sample. While the confirmation sample showed a similar effect, it did not reach significance. Nevertheless, in both samples, participants reported awareness of such bias (experienced as “overchasing”). In addition, this awareness made them report preemptively avoiding situations related to the bias. However, we found no evidence of them actually preempting more in the task, which might mean a misalignment of their metacognitive beliefs or that our behavioural measures were incomplete. In summary, individual variation in distinct, fundamental aspects of sequential decision-making can be linked to variation in 2 measures of behavioural traits associated with psychological illness in the normal population.

This preregistered study investigated individual differences in a sequential decision making task. The cognitive model revealed distinct differences in the way participants evaluated their environments and planned their own future behaviour connected to clinical dimensions; apathy was linked to decision inertia and compulsivity to perceived insensitivity to the cost of continuing with a sequence of searches, but also attempts of pre-emptive avoidance.     

Structure and permeability of goblet cell tight junctions in rat small intestine

Summary Two major cell types, goblet and absorptive cells, dominate the epithelial lining of small intestinal villi. We used freezefracture replicas of rat ileal mucosa to examine the possibility that tight junction structure, known to relate to transepithelial resistance, might vary with cell type. Tight junctions between absorptive cells were uniform in structure while those associated with villus goblet cells displayed structural variability. In 23% of villus goblet cell tight junctions the strand count was less than 4 and in 30% the depth was less than 200 nm. In contrast, only 4% of absorptive cell tight junctions had less than 4 strands and only 9% had depth measurements less than 200 nm. Other structural features commonly associated with villus goblet cell tight junctions but less commonly with absorptive cell tight junctions were: deficient strand cross-linking, free-ending abluminal strands, and highly fragmented strands. Bothin vivo ileal segments and everted loops were exposed to ionic lanthanum. Dense lanthanum precipitates in tight junctions and paracellular spaces were restricted to a subpopulation of villus goblet cells and were not found between villus absorptive cells. After exposure of prefixed ileal loops to lanthanum for 1 hour, faint precipitates of lanthanum were found in 14% of tight junctions and paracellular spaces between absorptive cells compared to 42% of tight junctions and paracellular spaces adjacent to villus goblet cells. When tested in Ussing chambers, the methods used for lanthanum exposure did not lower transepithelial resistance. Everted loops exposed to ionic barium and examined by light microscopy showed dense barium precipitates in the junctional zone and region of the paracellular space of villus goblet cells but not in these regions between absorptive cells. However, the macromolecular tracers, microperoxidase, cytochromec and horseradish peroxidase, were excluded from both villus goblet cell and absorptive cell paracellular spaces inin vivo segments. These findings suggest that a subpopulation of villus goblet cells may serve as focal sites of high ionic permeability and contribute to the relatively low resistance to ionic flow which characterizes the small intestinal epithelium.     

STRUCTURAL FEATURES OF THE EPITHELIO-MESENCHYMAL INTERFACE OF RAT DUODENAL MUCOSA DURING DEVELOPMENT

In fetal rats 5–7 days before birth, the duodenal epithelium is separated from mesenchymal cells by a well-defined basal lamina. By 3–4 days before birth, when small rudimentary villi are first seen, direct contact between epithelial and mesenchymal cells occurs by means of epithelial cell cytoplasmic processes which project through gaps in the basal lamina into the lamina propria. At contact sites, the epithelial and mesenchymal cell plasma membranes were less than 100 A apart but membrane fusion was not seen. In number and size these epithelial cell processes increase strikingly during the last 2 days of gestation, and they persist in large numbers until 7–10 days after birth. Thereafter, they decrease gradually in both number and size until 3–4 wk after birth, when the morphology of the epithelio-mesenchymal interface resembles that seen in adult rats, i.e., there are only rare epithelial cell processes which penetrate deeply into the lamina propria. The presence of a large number of epithelio-mesenchymal contact sites during the period of rapid growth and differentiation of duodenal mucosa may reflect epithelio-mesenchymal cell interactions which may facilitate the maturation of the duodenal mucosa.     

Epithelial cell proliferation in the intestine of the winter flounder,Pseudopleuronectes americanus

Summary To study epithelial cell proliferation in the North American flounder (Pseudopleuronectes americanus), fed and fasted fish received intravenous injections of ³H-thymidine and were killed 11/2 to 2 h later. Radioautographs of proximal, middle, and distal intestinal segments revealed proliferating epithelial cells at all levels of intestinal folds including the crest although labelled nuclei were most abundant in the epithelial cells on the lower half of folds and between folds. Mature appearing goblet cells with labelled nuclei were observed at all levels of the folds. The mean labelling index was greater in the epithelium of fed than fasted flounder. In fed flounder the mean labelling index was greatest in the proximal segment and least in the distal segment; no substantive differences in mean labelling indices were observed in the various segments of intestine from fasted fish. Electron microscopy revealed no major structural differences among epithelial cells along the base of folds compared to cells near the crest of folds. These findings indicate that 1) epithelial cell proliferation occurs at all levels of the folds of flounder intestine and is not compartmentalized to the base of the folds and interfold epithelium as reported in other teleosts, and 2) epithelial cell proliferation in the flounder intestine varies with feeding status.Supported be research grants AM 17537 and RR 05764 from the National Institutes of Health, Bethesda, Maryland and grant DEB7826821 AO1 from the National Science Foundation, Washington, D.C.The authors are grateful to Dr. Michael Field for stimulating discussions and suggestions and for providing facilities for collecting material from fish     

Dermal substitution in acute burns and reconstructive surgery: a subjective and objective long-term follow-up.

Tissue engineering and dermal substitution are currently prominent topics of wound-healing research. However, no extensive clinical trials with objective evaluation criteria have been published so far that support the clinical effectiveness of dermal equivalents in the long term. The dermal substitute that is discussed here is derived from bovine collagen and elastin-hydrolysate and has been shown to improve skin elasticity during a short-term clinical follow-up of scar reconstructions. In this study we will present the long-term outcome by means of objective and subjective scar assessment tools for dermal substitution in acute burn wounds and scar reconstructions.In a clinical trial, an intraindividual comparison was performed between the conventional split-thickness autograft and a combination of the collagen/elastin substitute with an autograft. After 1 year, scars were evaluated by the Cutometer SEM 474 for objective elasticity measurements and by planimetry to establish scar contraction. An independent observer subjected scars to a generally accepted clinical scar assessment tool: the Vancouver Scar Scale. In addition, patients gave their impression of the outcome. Forty-two paired burn wounds and 44 paired scar reconstructions were included and evaluated 1 year after surgery.Although substituted scar reconstructions demonstrated an elasticity improvement of approximately 20 percent compared with control wounds, no statistically significant differences were found for skin elasticity, scar contraction, Vancouver Scar Scale, and patient's impression in both categories after 1 year. An extensive long-term follow-up shows that the dermal substitute, which was proven effective in a clinical trial on a short-term basis, did not yield statistical evidence for a long-term clinical effectiveness of dermal substitution.     

A basophil-neuronal axis promotes itch

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Identification and mapping of a linear epitope of centromere protein F using monoclonal antibodies

Autoantibodies against centromere protein ‐F have been reported to be associated with various types of cancer with poor prognosis. The characterization of these autoantibody specificities is important in both diagnostics and basic research. In this study, we mapped the epitope (NELSRIRSEKA) of two monoclonal centromere protein F antibodies. The epitope was localized by screening of overlapping peptides followed by a fast and efficient estimation of the minimal peptide length required for antibody recognition, based on the screening of terminally truncated resin‐bound peptide analogs. The epitope was determined through competitive inhibition assays of systematically truncated free peptides. In addition, the importance of the involved amino acid side chains of the identified epitope was determined through competitive inhibition assays using alanine‐substituted analogs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

RhoA is down-regulated at cell–cell contacts via p190RhoGAP-B in response to tensional homeostasis

Breast epithelial cells cultured in three-dimensional (3D) collagen gels undergo ductal morphogenesis when the gel is compliant and they can achieve tensional homeostasis. We previously showed that this process requires down-regulation of Rho in compliant collagen gels, but the mechanism remains undefined. In this study, we find that p190RhoGAP-B, but not p190RhoGAP-A, mediates down-regulation of RhoA activity and ductal morphogenesis in T47D cells cultured in compliant 3D collagen gels. In addition, both RhoA and p190RhoGAP-B colocalize with p120-catenin at sites of cell–cell contact. The association between p190RhoGAP-B and p120-catenin is regulated by matrix compliance such that it increases in compliant vs. rigid collagen gels. Furthermore, knockdown of p120-catenin disrupts ductal morphogenesis, disregulates RhoA activity, and results in loss of p190B at cell–cell contacts. Consistent with these findings, using a RhoA-specific FRET biosensor (RhoA-FLARE.sc), we determined spatial RhoA activity to be significantly decreased at cell–cell contacts versus cell–ECM adhesions, and, of importance, spatial RhoA activity is regulated by p190B. This finding suggests that RhoA exists as an inactive pool at cell–cell contacts and is recruited to cell–ECM contacts within stiff matrices. Overall, these results demonstrate that RhoA is down-regulated at cell–cell contacts through p190RhoGAP-B, which is localized to cell–cell contacts by association with p120-catenin that is regulated by tensional homeostasis.