Cesonia A. Martinusso

Intraperitoneal but Not Intravenous Cryopreserved Mesenchymal Stromal Cells Home to the Inflamed Colon and Ameliorate Experimental Colitis

Background and Aims Mesenchymal stromal cells (MSCs) were shown to have immunomodulatory activity and have been applied for treating immune-mediated disorders. We compared the homing and therapeutic action of cryopreserved subcutaneous adipose tissue (AT-MSCs) and bone marrow-derived mesenchymal stromal cells (BM-MSCs) in rats with trinitrobenzene sulfonic acid (TNBS)–induced colitis. Methods After colonoscopic detection of inflammation AT-MSCs or BM-MSCs were injected intraperitoneally. Colonoscopic and histologic scores were obtained. Density of collagen fibres and apoptotic rates were evaluated. Cytokine levels were measured in supernatants of colon explants. For cell migration studies MSCs and skin fibroblasts were labelled with Tc-99m or CM-DiI and injected intraperitonealy or intravenously. Results Intraperitoneal injection of AT-MSCs or BM-MSCs reduced the endoscopic and histopathologic severity of colitis, the collagen deposition, and the epithelial apoptosis. Levels of TNF-α and interleukin-1β decreased, while VEGF and TGF-β did not change following cell-therapy. Scintigraphy showed that MSCs migrated towards the inflamed colon and the uptake increased from 0.5 to 24 h. Tc-99m-MSCs injected intravenously distributed into various organs, but not the colon. Cm-DiI-positive MSCs were detected throughout the colon wall 72 h after inoculation, predominantly in the submucosa and muscular layer of inflamed areas. Conclusions Intraperitoneally injected cryopreserved MSCs home to and engraft into the inflamed colon and ameliorate TNBS-colitis.

Effects of Mycophenolate Mofetil and Lisinopril on Collagen Deposition in Unilateral Ureteral Obstruction in Rats

Background/Aims: Unilateral ureteral obstruction (UUO) in rats has been used as a model of renal interstitial fibrosis, in which therapeutic trials can be of important clinical relevance. In this study, we investigated the effects of mycophenolate mofetil (MMF), the angiotensin-converting enzyme (ACE) inhibitor lisinopril (L), and the combination of both drugs, given daily for 14 days to UUO rats, on the renal fibrogenic process triggered by UUO. Methods: Rats underwent surgical UUO, followed by treatment with daily doses of either MMF, lisinopril, or both, and were then sacrificed after 14 days. Kidney fragments were fixed for histopathological examination (hematoxylin-eosin and periodic acid-Schiff reactive) and immunohistochemistry for myofibroblasts (α-smooth muscle actin; α-SMA) and macrophages (ED-1). Histomorphometrical analysis of collagen was performed with Sirius red staining, and collagen content was assessed by the amount of hydroxyproline. Cortex and medulla were analyzed separately. Results: MMF, lisinopril and MMF+L reduced the density of α-SMA- and ED-1-positive cells (p < 0.05), interstitial volume (p < 0.05) and decreased Sirius-red-stained areas by 54.6, 35.6 and 58.0%, and hydroxyproline content by 60.1, 49.7 and 62.7%, respectively. No differences were observed among treated groups. Conclusion: MMF and the ACE inhibitor lisinopril attenuated the progression of the fibrogenic process of UUO in an equivalent manner. The combination of both drugs did not add any further improvement in the collagen content.

Hedgehog pathway signaling regulates human colon carcinoma HT-29 epithelial cell line apoptosis and cytokine secretion.

The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon carcinoma cell lines to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components and related genes and proteins were assessed by real-time PCR and immunofluorescence. Viability, apoptosis and cell proliferation were measured by the MTT assay, Annexin-V/7-AAD staining and BrdU uptake, respectively. Chemokines production was measured by ELISA in culture supernatants. Indian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine or GANT61. BMP4 and BMP7 expression increased after stimulation with Hh agonists. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted β-catenin reduction and subcellular redistribution. Levels of IL-8 and MCP-1 decreased upon exposure to Hh agonists compared to Hh antagonists, LPS, IFN-γ or EGF. Monocyte chemotaxis decreased upon exposure to supernatants of HT-29 cells treated with Shh compared to Hh antagonists, LPS and IFN-γ. Cellular incorporation of BrdU and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Hh pathway is a key controller of colon cancer cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response and in epithelial barrier integrity, with potential homeostatic implications for the development of colonic inflammation and malignancies.

Effects of ethanol on gut-associated lymphoid tissues in a model of bacterial translocation: a possible role of apoptosis ☆

Chronic ethanol intake has been shown to be associated with immune suppression and impairment of epithelial barrier function. We investigated the effects of ethanol on intestinal immunity and its relation to bacterial translocation (BT). Male Wistar rats were assigned to one of three groups and received respective diets for 28 days. The ethanol-fed group [(EG); n=11] received a liquid diet containing 5% [volume/volume (vol./vol.)] ethanol; a pair-fed group [(PFG); n=11] received an isocaloric diet without ethanol; and a third (control) group [(CG); n=11] received water and chow ad libitum. On experimental day 29, animals in the EG and the PFG underwent distal ileum ligature and small intestine inoculation of a tetracycline-resistant Escherichia coli strain (TcR E. coli R6), by means of gastric intubation, followed by duodenal ligature. One hour after inoculation, mesenteric lymph nodes, right lobe of liver, spleen, and left kidney were excised for bacterial studies. Sections of jejunum and colon were immunostained, with the use of antibodies against immunoglobulin (Ig) A, T cells, macrophages, and proliferating cell nuclear antigen (PCNA). Apoptosis was determined by the terminal deoxynucleotidyltransferase TdT-mediated dUDP-biotin nick end labeling (TUNEL) method. Bacterial translocation rates were greater in the PFG compared with findings for the EG. Lamina propria of the jejunum of the EG showed a reduction in the densities of IgA+ cells and T cells compared with findings for the PFG and the CG. Colonic lamina propria of the EG showed reduced densities of IgA+ cells and macrophages compared with findings for the PFG and the CG. Apoptotic index was increased in the EG compared with findings for the PFG and the CG, in both jejunum and colon. Proliferation index was not significantly different among groups. Results of the current study show that chronic ethanol ingestion led to a reduction of cellular and humoral components of the intestinal mucosa, possibly by cell loss as a result of ethanol-induced apoptosis. The reduced rates of BT observed after chronic ethanol intake seem to indicate that factors irrespective of immune function might be involved in BT inhibition.

Análise do lavado broncoalveolar em vítimas de queimaduras faciais graves

Objective: To analyze bronchoalveolar lavage (BAL) specimens of burn victims who inhaled smoke, in order to identify alterations associated with mortality or survival. Methods: Eighteen victims of facial burns were submitted to BAL up to 24 h after the event. We investigated cell and protein content, including TNF-α, HLA-DR, CD14, CD68 and iNOS. Results: Of the 18 patients submitted to bronchoscopy, 8 (44.4%) died during the follow-up period. The mean age of patients who died was significantly higher (44.7 vs. 31.5 years). On average, the patients who died had burns covering 60.1% of the total body surface area, compared with 26.1% in the survivors (p < 0.0001). Of the 18 patients submitted to bronchoscopy, 11 (61.1%) showed endoscopic signs of smoke inhalation injury, and 4 (36.4%) of those 11 died. Of the 7 patients with no signs of smoke inhalation injury, 4 (57.1%) died. The mean number of ciliated epithelial cells in the BAL fluid was significantly higher in the patients who died than in the survivors (6.6% vs. 1.4%; p = 0.03). There were no significant differences between the groups in terms of any of the other parameters evaluated. Conclusions: The total body surface area burned was a predictive factor for mortality. Increased numbers of ciliated epithelial cells in the BAL fluid, denoting bronchial epithelial desquamation, were associated with higher mortality in patients with facial burns.

T1769 Disruption of the Hedgehog Signaling Pathway in Crohn's Disease: A Novel Role in Intestinal Inflammation

Background and aims: Mucosal immunity and epithelial integrity are interconnected factors involved in intestinal homeostasis. The hedgehog (Hh) family of morphogens is essential in embryogenesis, but it has been also associated with inflammation and tissue repair. We hypothesized that the Hh-pathway could affect the inflammatory response and cell survival, crucial factors in IBD pathogenesis. Methods: Endoscopic biopsies were obtained from the inflamed colon of 14 patients with Crohns disease (CD), 12 ulcerative colitis (UC), and 15 controls. Frozen sections of mucosal samples were analyzed by immunohistochemistry using antibodies against Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Gli-1. Double immunofluorescence was used for co-localization studies. RT-PCR was used for analyzing the expression of Gli-1 mRNA. Tissue cultures of colon samples were performed during 24h in the presence of Shh peptide, anti-Shh antibody, cyclopamine, a specific Hh inhibitor, or diluent. Supernatants were analyzed for cytokine production, by ELISA, and total protein was extracted from tissue for caspase-3 activity assay. Results: CD colonic mucosa showed a lower expression of Gli-1 mRNA compared to UC and controls (p=0.03). Hh-proteins were characteristically expressed at the superficial epithelium, and a marked reduction was observed in CD compared to UC (p=0.04), and controls (p=0.001). In the lamina propria, low densities of Hh-family members basically do not co-localize with infiltrating T-cells or macrophages. Baseline levels of cytokines in culture supernatants were higher in IBD compared to controls. In CD colon explants, treatment with Shh peptide resulted in a significant decrease in the levels of TNF-alpha (p=0.04), IL-17 (p=0.01), and TGF-beta (p=0.03). Caspase-3 activity was higher in CD and significantly decreased after blockade of Hh, either with anti-Shh antibody(p=0.04) or with cyclopamine (p=0.03), in contrast to UC and controls. Levels of Gli-1 negatively correlated to caspase-3 activity (CC -0.75, p=0.01). Conclusion: Normal colonic mucosa constitutively expresses Hh-proteins, which appear to constitute a key pathway for intestinal homeostasis. Hh-signaling affects cytokine production and the control of apoptosis within the colonic mucosa. Disruption of the intestinal Hhpathway might be implicated in the pathogenesis of CD.