Biliary epithelial senescence and plasticity in acute cellular rejection.

Biliary epithelial cells (BEC) are important targets in some liver diseases, including acute allograft rejection. Although some injured BEC die, many can survive in function compromised states of senescence or phenotypic de-differentiation. This study was performed to examine changes in the phenotype of BEC during acute liver allograft rejection and the mechanism driving these changes. Liver allograft sections showed a positive correlation (p < 0.0013) between increasing T cell mediated acute rejection and the number of BEC expressing the senescence marker p21(WAF1/Cip) or the mesenchymal marker S100A4. This was modeled in vitro by examination of primary or immortalized BEC after acute oxidative stress. During the first 48 h, the expression of p21(WAF1/Cip) was increased transiently before returning to baseline. After this time BEC showed increased expression of mesenchymal proteins with a decrease in epithelial markers. Analysis of TGF-ß expression at mRNA and protein levels also showed a rapid increase in TGF-ß2 (p < 0.006) following oxidative stress. The epithelial de-differentiation observed in vitro was abrogated by pharmacological blockade of the ALK-5 component of the TGF-ß receptor. These data suggest that stress induced production of TGF-ß2 by BEC can modify liver allograft function by enhancing the de-differentiation of local epithelial cells.

Chronic allograft nephropathy: intraepithelial signals generated by transforming growth factor-beta and bone morphogenetic protein-7.

It has been suggested that TGFbeta can cause chronic allograft nephropathy by inducing epithelial to mesenchymal transition (EMT); some studies show a reverse transition can be produced by bone morphogenetic protein-7 (BMP7). The current study assessed the relative contribution of signals generated within tubular epithelial cells by TGFbeta and BMP7 in normal kidney and after transplantation. Epithelial cells in normal human kidneys expressed phosphorylated forms of both Smad2/3 and Smad1/5/8 within their nuclei; cell culture experiments showed that these signaling molecules were generated in response to TGFbeta and BMP7, respectively. Phospho(p)-Smad2/3 was expressed at increased levels by tubular epithelial cells during acute renal allograft rejection and chronic allograft nephropathy but pSmad1/5/8 was expressed at very low levels; this staining profile was associated with induction of the EMT marker, S100A4. Further in vitro experiments demonstrated that this pattern of Smad signaling was a consequence of the stimulation of tubular epithelial cells with TGFbeta in the absence of BMP7. Importantly, addition of BMP7 to TGFbeta-stimulated cells enhanced the expression of pSmad1/5/8 and reduced expression of S100A4. These results suggest that exogenous BMP7 could restore the homeostatic balance of pSmad signaling found in normal kidneys, thereby preventing or reversing the development of chronic allograft nephropathy.

Acid-etch patterns on the buccal surface of human permanent teeth.

Since the introduction of acid etching to aid adhesion to enamel, there has been much research into dental materials to improve bond strength, but little into the surface topography of etched enamel, particularly regarding possible variations between tooth types. This study was a systematic investigation into the quality and quantity of etch patterns found on the buccal surfaces of different human permanent teeth. Twenty-nine orthodontic patients had high-resolution silicone impressions taken of the buccal surface of incisor, canine, premolar and molar, upper and lower teeth, following etching for 30s with 37% phosphoric acid. Impressions (n=266) were replicated in epoxy resin and examined under high magnification in a scanning electron microscope. A modification of the classification of Galil and Wright was used, with histometric techniques, to quantify the quality of etch patterns on enamel surfaces where orthodontic brackets are typically bonded. There was no difference between right and left or between upper and lower teeth of the same type (P>0.05). There was a general trend toward the increasing occurrence of no etch (type D) from anterior to posterior teeth, and a trend toward fewer good-quality etches (types A and B) in the same direction. Etch types A and B were found to occupy the smallest area on the etched buccal surface enamel. The greatest amount of type A etch 'ideal' was found on the lower incisors, yet it occupied less than 5% of the etched buccal surface enamel. The greatest area of etched enamel surface was occupied by type C (etched, but enamel prisms not evident). It was concluded that there is a significant difference in the acid-etch patterns achieved on different tooth types, which suggests that bond-strength studies should be performed with a single tooth type or that an equal number of different tooth types be included.

Active TGF-beta1 expression in kidney transplantation: a comparative study of cyclosporin-A (CyA) and tacrolimus (FK506).

Chronic rejection is a major cause of graft dysfunction following kidney transplantation. This fibroproliferative disease may be promoted by overproduction of transforming growth factor beta (TGF-beta). Previous studies have suggested that cyclosporin-A (CyA) might increase production of this growth factor. The current study was designed to measure the expression of TGF-beta in renal transplant biopsies from patients immunosuppressed with either CyA or tacrolimus. Paraffin-embedded renal biopsies were sectioned, dewaxed and incubated with primary antibody against active TGF-beta1 antibody. After washing, the sections were treated with secondary antibody conjugated with fluorescein isothiocyanate (FITC). In each case the sections were assessed by semi-quantitative scanning laser confocal microscopy. Biopsies from patients receiving CyA expressed significantly more active TGF-beta1 than biopsies from patients receiving tacrolimus (P < 0.0001, Mann-Whitney test). The increased level of active TGF-beta1 expression in renal biopsies of patients receiving CyA may indicate a mechanism of chronic rejection.

TGF-beta expression in renal transplant biopsies: a comparative study between cyclosporin-A and tacrolimus.

BACKGROUND: Chronic rejection is a major cause of graft dysfunction after kidney transplantation. This fibroproliferative disease may be promoted by overproduction of transforming growth factor beta (TGF-beta). Previous studies have suggested that CsA might increase production of this growth factor. The current study was designed to measure the expression of TGF-beta(b) in renal transplant biopsy specimens from patients undergoing immunosuppressive therapy with either CsA or tacrolimus (FK506). METHOD: Paraffin-embedded renal biopsy specimens were sectioned, dewaxed, and incubated with primary antibody against TGF-beta(b)1 latency-associated protein and active TGF-beta(b1). After washing, the sections were treated with secondary antibody conjugated with FITC. In each case, the sections were assessed by semi-quantitative scanning laser confocal microscopic method. RESULTS: There was no significant difference in latent TGF-beta(b) expression between biopsy specimens from patients receiving CsA and patients receiving FK506. However, biopsy specimens from patients receiving CsA expressed significantly more active TGF-beta(b1) than biopsy specimens from patients receiving FK506 (P<0.0001, Mann-Whitney test). DISCUSSION: The increased level of active TGF-beta1 expression in renal biopsy specimens of patients receiving CsA may indicate a mechanism of chronic rejection. However, these biopsies were performed to assess deranged renal function; therefore, the specimens may reflect events rather than differences in medication.

Beta-chemokine expression and distribution in paraffin-embedded transplant renal biopsy sections: analysis by scanning laser confocal microscopy.

Previous immunohistochemical and in situ hybridisation studies have shown that, in tubulitis associated with acute cellular rejection of human renal allografts, intratubular T cells proliferate and are fully activated in situ. In the immunohistochemical study reported here we have attempted to establish some understanding of the involvement of the beta-chemokines RANTES, MCP-1, MIP-1alpha and MIP-1beta in recruiting T cells to the intratubular site. Paraffin-embedded routine biopsy sections were treated for conventional indirect immunofluorescence to detect the selected chemokines. Scanning laser confocal microscopy was used to provide a measure of fluorescence intensity resulting from binding of FITC-labelled secondary antibody. Cells expressing chemokines could be identified and, within the limits of the staining method, it was possible to obtain a semi-quantitative assessment of individual chemokine activity at different points in biopsy sections by constructing a profile of fluorescence intensity. High concentrations of chemokines (especially RANTES, MIP-1beta and/or MIP-1alpha) were localised to the basolateral surface of tubular epithelial cells (TEC). MCP-1 was also consistently present but at a lower level than RANTES except in one case identified as BANFF category 3. There was diffuse distribution of chemokines in the interstitial matrix and low intensity fluorescence outlined some endothelial cells of peritubular venules and interstitial fibroblast-like cells. Our results suggest a mechanism for specific chemotactic recruitment of inflammatory cells by TEC-produced chemokines.

Cell-contact-stimulated formation of filamentous appendages by Salmonella typhimurium does not depend on the type III secretion system encoded by Salmonella pathogenicity island 1.

The formation of filamentous appendages on Salmonella typhimurium has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717-724, 1994). We have examined the roles of cell contact and Salmonella pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of S. typhimurium strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type S. typhimurium bacteria adhering to both cultured epithelial cells and to murine Peyer's patch follicle-associated epithelia. Wild-type S. typhimurium adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient S. typhimurium strains carrying mutations in components of SPI1 (invA, invG, sspC, and prgH) exhibited filamentous appendages similar to those on wild-type S. typhimurium when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, an S. typhimurium invG mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on S. typhimurium strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.

Promotion of Salmonella typhimurium adherence and membrane ruffling in MDCK epithelia by staurosporine.

Infection of Madin-Darby canine kidney epithelial cell monolayers with Salmonella typhimurium SL1344 for 60 min results in widespread bacterial invasion which is associated with remodelling of the apical cell membrane to form "membrane ruffles'. Treatment of Madin-Darby canine kidney cell monolayers with the protein kinase inhibitor staurosporine resulted in a 12-fold increase in the number of adhered bacteria without significantly affecting bacterial invasion. Staurosporine treatment also significantly increased both the number and size of membrane ruffles. As S. typhimurium adhere preferentially to these areas of membrane lacking microvilli, the increased extent of membrane ruffling may explain the increased bacterial adherence. These data provide evidence that the propagation of membrane ruffles during S. typhimurium infection is modulated by changes in the phosphorylation state of host proteins.

Differential expression of lectin-binding sites defines mouse intestinal M-cells.

We investigated the binding of four lectins to the follicle-associated epithelium (FAE) overlying fixed mouse small intestinal Peyer's patches to identify M-cell-specific surface markers. Wheat germ agglutinin and peanut agglutinin displayed heterogeneous staining patterns, binding most avidly to the intestine goblet cells. In contrast, the lectins Ulex europaeus 1 (UEA 1) and Psophocarpus tetragonolobus (winged bean; WBA) were almost exclusively M-cell specific. When confocal laser scanning images of tissues stained with fluorescein isothiocyanate (FITC)-conjugated UEA1 or WBA were compared with the appearance of the same tissues under the scanning electron microscope (SEM), UEA1 strongly stained 97.2% (106/109) of M-cells, 0.6% (3/516) enterocytes, and 0% (0/28) goblet cells, whereas WBA stained 100% (83/83) M-cells, 1.7% (6/361) enterocytes, and 5.3% (1/19) goblet cells. The M-cell specificity of the lectin binding was further demonstrated by localization of horseradish peroxidase (HRP)-conjugated lectins under the transmission electron microscope (TEM). This is the first demonstration of carbohydrates in the glycocalyx of M-cells that are not expressed elsewhere on the FAE surface. These carbohydrates not only provide a means to identify mouse M-cells by LM but may also contribute to the occurrence of specific interactions between microorganisms and the M-cell apical membrane.