Roberta Sferra

Increased proliferation and apoptosis of colonic epithelial cells in dextran sulfate sodium-induced colitis in rats

We have evaluated morphologic alterations and epithelial cell apoptosis and proliferation of colonic mucosa in the acute and chronic phases of DSS-induced colitis. Colitis was induced in Sprague-Dawley rats by 7 days of 4% DSS oral administration followed by 7 days of tap water for one, two, and three cycles. Control rats receved tap water only. Morphological changes in colonic mucosa were evaluated and scored by light and scanning electron microscopy. Apoptosis was studied by TUNEL assay and cell proliferation by Ki-67 immunoreaction. The expression of both proapoptotic (Fas, FasL, Bax, p53) and antiapoptotic (Bcl2) cellular proteins was determined by immunohistochemistry. Morphologic assessment showed the most severe colonic epithelial lesions and inflammation in the distal colon with a trend to increasing severity from the first to the third DSS cycle. In DSS rats, the epithelial apoptotic index increased 20-fold after the first cycle and 120-fold after the second and third cycles compared with the controls; in the same way, the expression index of proapoptotic proteins (Fas, FasL, Bax, p53) dramatically increased. The proliferative index increased about 40 to 60-fold compared to controls, with no difference among the three DSS cycles. In conclusion, DSS-induced colitis in rats, which has many structural and ultrastructural features similar to those seen in human ulcerative colitis, is a suitable model for studying increased epithelial apoptosis and proliferation. Further studies employing this model will permitt two hypotheses to be tested. (1) Increased apoptosis may lead to a breakdown of the epithelial barrier function and facilitate the mucosal invasion of intraluminal microorganisms and/or antigens. (2) Abnormal and persistent epithelial hyperproliferation could be causally related to the development of colorectal cancers in the setting of chronic colonic inflammation.

Targeted disruption of Smad3 confers resistance to the development of dimethylnitrosamine-induced hepatic fibrosis in mice

Background: Hepatic fibrosis is characterized by a progressive accumulation of fibrillar extracellular matrix (ECM) proteins including collagen, which occurs in most types of chronic liver diseases. Transforming growth factor‐β (TGF‐β)/Smad3 signalling plays a central role in tissue fibrogenesis, acting as a potent stimulus of ECM accumulation.

Prevention of colonic fibrosis by Boswellia and Scutellaria extracts in rats with colitis induced by 2,4,5-trinitrobenzene sulphonic acid

Background  Currently, no effective preventive measures or medical therapies are available for intestinal fibrosis and, thus, surgery remains the only available strategy in the management of fibrostenotic enteropathies, especially Crohns disease. The aim of this study was to evaluate the efficacy of a combined therapy of anti‐inflammatory Boswellia and antifibrotic Scutellaria extracts on the development of colonic fibrosis in rats.

H3 histamine receptor-mediated activation of protein kinase Cα inhibits the growth of cholangiocarcinoma in vitro and in vivo

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The d-myo-inositol 1,4,5-trisphosphate (IP3)/Ca2+/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(α)-(−)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP3 and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKCα was visualized by immunofluorescence in cell smears and immunoblotting for PKCα in cytosol and membrane fractions. Following knockdown of PKCα, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal–regulated kinase (ERK)-1/2 phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKCα, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP3 levels and PKCα phosphorylation and decreased ERK1/2 phosphorylation. RAMH induced a shift in the localization of PKCα expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKCα prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKCα expression was increased. RAMH inhibits cholangiocarcinoma growth by PKCα-dependent ERK1/2 dephosphorylation. Modulation of PKCα by histamine receptors may be important in regulating cholangiocarcinoma growth. (Mol Cancer Res 2009;7(10):1704–13)

Smad3 loss confers resistance to the development of trinitrobenzene sulfonic acid–induced colorectal fibrosis

Background  Transforming growth factor‐β (TGF‐β)/Smad3 signalling plays a central role in tissue fibrogenesis, acting as a potent stimulus of extracellular matrix (ECM) protein accumulation. The aim of this study was to evaluate the potential role of Smad3 in the pathogenesis of colonic fibrosis induced by trinitrobenzene sulfonic acid (TNBS) in Smad3 null mice.

Catheter-Related Candidemia Caused by Candida lipolytica in a Patient Receiving Allogeneic Bone Marrow Transplantation

ABSTRACT Candida lipolytica was recovered from the blood and the central venous catheter in a patient receiving allogeneic bone marrow transplantation. Two C. lipolytica strains from different geographical areas and the ATCC 9773 strain of C. lipolytica were used as controls. C. lipolytica was identified by standard methods. MICs indicated antifungal susceptibilities to amphotericin B, fluconazole, and itraconazole for all strains. In vitro testing and scanning electron microscopy showed that C. lipolytica was capable of producing large amounts of viscid slime material in glucose-containing solution, likely responsible for the ability of the yeast to adhere to catheter surfaces. Restriction fragment length polymorphisms revealed an identical profile for all clinical isolates, unrelated to those observed for the control strains. This finding suggested the absence of microevolutionary changes in the population of the infecting strain, despite the length of the sepsis and the potential selective pressure of amphotericin B, which had been administered to the patient for about 20 days. The genomic differences that emerged between the isolates and the control strains were indicative of a certain degree of genetic diversity between C. lipolytica isolates from different geographical areas.

Liver Metabolic Zonation and Hepatic Microcirculation in Carbon Tetrachloride-Induced Experimental Cirrhosis

The exact cause of the hepatic failure in livercirrhosis is currently unclear, and two main theorieshave been proposed: the first is based on the alteredhepatocyte function (sick hepatocyte hypothesis); the second on the abnormal hepatic architecture(intact hepatocyte hypothesis). Moreover, themicrocirculation, a fundamental component in liverstructure, shows dramatic changes in cirrhosis thatwould heavily influence the development of thedisease. In order to determine the importance of themicrovascular alterations on liver morphofunctionalfeatures in experimentally induced cirrhosis, theirrelationships with structural, ultrastructural, andhistoenzymological hepatocyte modifications wereinvestigated. Experimental cirrhosis was induced withcontrolled intragastric CCl4 administration.Scanning electron microscopy of the vascular corrosion casttechnique, associated with light microscopy,transmission electron microscopy, and histoenzymologytechniques were employed. The results demonstrated acharacteristic micronodular cirrhosis in all the liversstudied; the microcirculation displayed the presence ofnewly formed perinodular plexus. Inside the nodule,areas with two or more hepatocyte-thick laminae were present. Moreover, a rearrangement of thehepatocyte quantitative ultrastructure without realpathological changes and a loss of normal metaboliclobular zonation were noted in the liver parenchyma.These findings support the concept that theprogressive modifications of the microcirculation duringexperimental CCl4 cirrhosis modify not onlythe normal blood flow direction, but also the normalhepatic metabolic gradient with a loss of the normal hepatocyticzonation.

Novel PPARγ Modulator GED-0507-34 Levo Ameliorates Inflammation-driven Intestinal Fibrosis.

Background:Intestinal fibrosis is mainly associated with Crohns disease and is defined as a progressive and excessive deposition of extracellular matrix components. No specific antifibrotic therapies are available. In this study, we evaluate the antifibrotic effect of a novel 5-ASA analog able to activate the peroxisome proliferator-activated receptor &ggr;, named GED-0507-34 Levo. Methods:Colonic fibrosis was induced in 110 C57BL/6 mice by 3 cycles of 2.5% (wt/vol) dextran sulfate sodium administration for 6 weeks. The preventive effects of oral daily GED (30 mg·kg−1·d−1) administration were evaluated using a macroscopic and histological score and also through biological endpoints. Expression of main markers of myofibroblasts activation was determined in transforming growth factor (TGF-&bgr;)–stimulated intestinal fibroblasts and epithelial cells. Results:GED improved macroscopic and microscopic intestinal lesions in dextran sulfate sodium-treated animals and reduced the profibrotic gene expression of Acta2, COL1a1, and Fn1 by 1.48-folds (P < 0.05), 1.93-folds (P < 0.005), and 1.03-fold (P < 0.05), respectively. It reduced protein levels of main markers of fibrosis (&agr;-SMA and Collagen I-II) and the main TGF-&bgr;/Smad pathway components. GED also decreased the interleukin-13 and connective tissue growth factor expression by 1.89-folds (P < 0.05) and 2.2-folds (P < 0.005), respectively. GED inhibited TGF-&bgr;–induced activation of both fibroblast and intestinal epithelial cell lines, by regulating mRNA expression of &agr;-SMA and fibronectin, and restoring the TGF-&bgr;–induced loss of intestinal epithelial cell markers. GED treatment also reduced the TGF-&bgr; and ACTA1 expression in primary human intestinal fibroblasts from ulcerative colitis patients. Conclusions:GED ameliorates intestinal fibrosis in dextran sulfate sodium-induced chronic colitis in mice and regulates major profibrotic cellular and molecular mechanisms.

Peyer's patches epithelium in the rat: a morphological, immunohistochemical, and morphometrical study.

The epithelial layer covering lymphoid follicles of Peyers patches consists of cells with a different surface morphology. Some of these cells have been described as a distinct cytotype, the so-called M cells. In order to resolve the controversy on the specific morphological and biochemical markers of M cells, structural, ultrastructural, and morphometrical study of the epithelium covering the rat Peyers patches were performed. Peyers patches from healthy rats were processed for light microscopy, immunohistochemistry, in situ nick-end labeling (TUNEL), and scanning and transmission electron microscopy. A morphometric study was also performed to evaluate microvillus density, length, and number of lysosomes in different areas of the epithelium. Peyers patches were covered by simple columnar/cubical dome epithelium (DE). Scarce goblet cells and a large number of enterocytes were observed. Ultrastructural observations revealed that the DE showed cells with different morphology. The density and length of microvilli and the lysosome number varied along the whole dome without significant differences. The DE cells characterized by short and disorganized microvilli appeared always in close spatial relationship with lymphocytes. In conclusion, the concept that distinct cell types (enterocytes and M cells) can be identified in the rat DE does not appear to be valid based on morphological criteria. It seems correct to consider that in rat Peyers patches the presence of scarce goblet cells and a large number of enterocytes showing dynamic morphofunctional modifications is related to the functional state and/or to cell cycle.

Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). Cyclin D1 deregulated expression in PCa is associated with a more aggressive disease: however its role in radioresistance has not been determined. Cyclin D1 levels in the androgen-independent PC3 and 22Rv1 PCa cells were stably inhibited by infecting with cyclin D1-shRNA. Tumorigenicity and radiosensitivity were investigated using in vitro and in vivo experimental assays. Cyclin D1 silencing interfered with PCa oncogenic phenotype by inducing growth arrest in the G1 phase of cell cycle and reducing soft agar colony formation, migration, invasion in vitro and tumor formation and neo-angiogenesis in vivo. Depletion of cyclin D1 significantly radiosensitizes PCa cells by increasing the RT-induced DNA damages by affecting the NHEJ and HR pathways responsible of the DNA double-strand break repair. Following treatment of cells with RT the abundance of a biomarker of DNA damage, γ-H2AX, was dramatically increased in sh-cyclin D1 treated cells compared to shRNA control. Concordant with these observations DNA-PKcs-activation and RAD51-accumulation, part of the DNA double-strand break repair machinery, were reduced in shRNA-cyclin D1 treated cells compared to shRNA control. We further demonstrate the physical interaction between CCND1 with activated-ATM, -DNA-PKcs and RAD51 is enhanced by RT. Finally, siRNA-mediated silencing experiments indicated DNA-PKcs and RAD51 are downstream targets of CCND1-mediated PCa cells radioresistance. In summary, these observations suggest that CCND1 is a key mediator of PCa radioresistance and could represent a potential target for radioresistant hormone-resistant PCa.