Topical hemostatic powder promotes reepithelialization and reduces scar formation after extensive esophageal mucosal resection.

The development of techniques for endoscopic resection has provided new strategies for radical conservative treatment of superficial esophageal neoplasms, even those that are circumferential, such as Barrett's neoplasia. However, it is necessary to prevent the formation of scar tissue that can be responsible for esophageal strictures following circumferential resection. Preliminary data have suggested the possible efficacy of a hemostatic powder in the promotion of wound healing. The study aims to assess the effectiveness of Hemospray (Cook Medical) in a swine model of post-endoscopic esophageal stricture. Our prospective controlled study included 21 pigs. A 6-cm circumferential submucosal dissection of the esophagus (CESD) was performed in each pig. Group 1 (n = 11) only underwent CESD and Group 2 (n = 10) had repeated Hemospray applications after CESD. Clinical, endoscopic, and radiological monitoring were performed, blood levels of four inflammatory or pro-fibrotic cytokines were assessed, and histological analysis was performed. Median esophageal diameter was greater in the group treated with Hemospray (2 mm [1-3] vs. 3 mm [2-4], P = 0.01), and the rate of symptomatic esophageal stricture was 100% and 60% in Groups 1 and 2, respectively (P = 0.09). The thicknesses of esophageal fibrosis and inflammatory cell infiltrate were significantly lower in Group 2 than in Group 1 (P = 0.002 and 0.0003, respectively). The length of the neoepithelium was greater in Group 2 than in Group 1 (P = 0.0004). Transforming growth factor-ß levels were significantly lower in Group 2 than in Group 1 (P = 0.01). The application of Hemospray after esophageal CESD reduces scar tissue formation and promotes reepithelialization, and therefore is a promising therapeutic approach in the prevention of post-endoscopic esophageal stricture.

Tumor invasion induced by oxidative stress is dependent on membrane ADAM 9 protein and its secreted form.

Oxidative stress plays a role in the regulation of cancer cell metastasis which involves cell invasion and adhesion that could be supported by ADAM proteins through the activities of their metalloprotease and disintegrin domains. We hypothesized that oxidative stress could act through the induction of ADAM9 protein in some cancer cells. Indeed, Western blot analysis for ADAM9 performed on A549 cells exposed to H(2) O(2) reveals a dose-dependent induction of two proteins (80 and 68 kDa) correlated with a sharp increase of the ADAM protease activity measured in supernatant while the activity measured on the cell layer was slightly affected. The 80kDa protein corresponds to the mature form of ADAM9. Immunoprecipitation analysis performed on concentrated supernatants revealed that the 68 kDa protein is a secreted form of ADAM9. When exposed to H(2) O(2) , A549 cells cocultured with confluent endothelial vascular cells resulted in a 5.5 fold (p < 0.001) increase in the number of adherent cells. Similarly, matrigel assay revealed a 3.25 fold (p < 0.01) increase in the number of invasive cells. The suppression of ADAM9 expression by specific small interfering RNA reduced oxidative stress-induced invasiveness and adhesiveness. These functions could be mediated by an interaction between ADAM9 and ß1 integrin because each of them were inhibited when the experiment is performed in presence of mAbs targeting ADAM9 ectodomain or ß1-integrin. These results emphasize the importance of oxidative stress in the regulation of cancer cell metastasis and suggest that ADAM9 and its secreted isoform can be important determinants in the ability of cancer cells to disseminate.

The oxidation induced by antimyeloperoxidase antibodies triggers fibrosis in microscopic polyangiitis.

Lung fibrosis is considered a severe manifestation of microscopic polyangiitis (MPA). Antimyeloperoxidase (anti-MPO) antibodies in MPA patients' sera can activate MPO and lead to the production of reactive oxygen species (ROS). While high levels of ROS are cytotoxic, low levels can induce fibroblast proliferation. Therefore, we hypothesised that the oxidative stress induced by anti-MPO antibodies could contribute to lung fibrosis. 24 MPA patients (45 sera) were enrolled in the study, including nine patients (22 sera) with lung fibrosis. Serum advanced oxidation protein products (AOPP), MPO-induced hypochlorous acid (HOCl) and serum-induced fibroblast proliferation were assayed. AOPP levels, MPO-induced HOCl production and serum-induced fibroblast proliferation were higher in patients than in healthy controls (p<0.0001, p=0.0001 and p=0.0005, respectively). Increased HOCl production was associated with active disease (p=0.002). Serum AOPP levels and serum-induced fibroblast proliferation were higher in patients with active MPA and lung fibrosis (p<0.0001). A significant linear relationship between fibroblast proliferation, AOPP levels and HOCl production was observed only in patients with lung fibrosis. Oxidative stress, in particular the production of HOCl through the interaction of MPO with anti-MPO antibodies, could trigger the fibrotic process observed in MPA.

The role of natural IgM in the hyperacute rejection of discordant heart xenografts.

The mechanism of xenograft hyperacute rejection in discordant species combinations remains controversial. The purpose of this work was to study the role of natural antibodies in the hyperacute rejection of guinea pig hearts transplanted into rats, a highly discordant combination. This study was conducted in vitro, ex vivo, and in vivo. The endothelial cells of the graft being the first targets damaged in the process of hyperacute rejection, the binding of rat natural antibodies to guinea pig endothelial cells was studied by immunofluorescence. The study was carried out in vitro on guinea pig endothelial cells in culture, and ex vivo on isolated guinea pig hearts perfused with either rat serum or immunoglobulins or immunoglobulin fragments bearing the antigen-binding site. In vitro and ex vivo, rat natural IgM were found to bind specifically to guinea pig endothelial cells, since IgM fragments bearing the antigen-binding site (Fab mu and Fab' mu) could be detected on these cells. IgM fragments were able to inhibit the fixation of native IgM molecules. In contrast, rat IgG only bound to endothelial cells through Fc portions. Thus rat natural IgM might play a role in hyperacute rejection by binding to the graft endothelial cells and triggering the complement cascade activation. In order to test the role of natural IgM in vivo, isolated guinea pig hearts were first perfused with rat Fab' mu, which inhibit the binding of IgM and are unable to activate the complement cascade. These hearts were then transplanted into Lewis rats. The rejection time of Fab' mu-perfused guinea pig hearts was prolonged compared with hearts perfused with buffer or IgG F(ab')2. Therefore, in the guinea pig to rat combination, preventing the binding of the recipient's natural IgM to the graft endothelium delays the hyperacute rejection. In addition, natural IgM are likely to play a greater role than natural IgG.

Target antigens of hyperacute xenogeneic rejection in the rat to guinea pig to rat discordant combinations.

The increasing shortage in allografts has led to a renewed interest in xenogeneic transplantation. Discordant combinations are characterized by hyperacute rejection partly due to the presence of natural antixenogeneic antibodies in the recipient. The aim of this work was to characterize the target antigens, using 2 discordant models. In the rat into guinea pig model, analysis of organ homogenates by immunoblotting revealed numerous bands. Some of these bands were organ specific, whereas others, namely in the 55-kDa region, were detected in liver, heart, lung, and kidney. Using membrane extracts of liver cells or of aortic endothelial cells, only bands of 55 kDa were revealed. No band could be seen using extracts of isolated hepatocytes. Two bands of 55 kDa disappeared after preabsorption of guinea pig sera on the various rat tissue homogenates, suggesting that they represent xenoantigens common to these tissues. In order to investigate the in vivo relevance of these 55-kDa antigens, isolated rat livers were perfused with decomplemented guinea pig sera. Eluates revealed one single print of 55 kDa on rat tissue homogenates. Finally, preincubation of rat mononuclear cells with various xenogeneic sera did not inhibit the binding of mAb specific for rat class I or class II MHC antigens, suggesting that the latter are not recognized by natural xenoantibodies. In the guinea pig to rat model, the antigens detected had a molecular mass ranging from 95 to 110 kDa. Absorption and perfusion experiments also showed that these antigens were common to various tissues and involved in the binding of rat natural antibodies ex vivo. In conclusion, our results indicate that rat xenoantigens of about 55 kDa are recognized by guinea pig natural antibodies, while guinea pig xenoantigens of 95-110 kDa are bound by rat natural antibodies. These antigens are common to liver, heart, lung, and kidney, are borne by endothelial cells, and cannot be found on hepatocytes.

Comparison between aortic and sinusoidal liver endothelial cells as targets of hyperacute xenogeneic rejection in the pig to human combination.

Endothelial cells of aortic origin are usually used in vitro as targets of hyperacute xenogeneic rejection, although endothelial cells from organs may have different properties. The sensitivities of aortic and liver endothelial cells to hyperacute xenogeneic rejection were compared in the pig to human combination. Sinusoidal liver endothelial cells were isolated and purified by collagenase perfusion of pig livers, sedimentation on a percoll gradient and selective adherence. Purity and viability of isolated liver endothelial cells after adherence were 85+/-6% and >95%, respectively. Endothelial cells from pig aortae (purity and viability >95%) were isolated by scraping. Immunoblotting analysis of xenoantigens on liver and aortic endothelial cell membranes preparations showed identical patterns. The strongest bands revealed by human IgM were located between 110 and 135 kD, while human IgG detected two major bands at 115 and 75kD. The membrane expression of xenoantigens recognized by human sera, analyzed by flow cytometry, was significantly lower on liver than on aortic endothelial cells (IgM: P=0.0006; IgG: P=0.0009). However, the complement-dependent cytotoxic activity of human sera was the same whether liver (54.5+/-1.4%) or aortic endothelial cells (50.0+/-4.2%) were used as targets. Taken together, those results allow the use of aortic instead of sinusoidal liver endothelial cells in the characterization of pig antigens recognized by human natural antibodies.

Hyperacute xenograft rejection in the swine-to-human donor-recipient combination. In vitro analysis of complement activation.

Complement activation is central to the rejection of discordant xenografts. In order to assess the respective roles of direct and alternative pathways, an in vitro model of hyperacute rejection in the swine-to-human donor-recipient combination was designed, using a complement-dependent cytotoxicity test with swine endothelial cells in culture as targets, and fresh human serum as the source of xenogeneic antibodies and complement. The cytotoxic activity of the sera was evaluated by a colorimetric assay using (3-[4,5-dimethyldiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). Pure human serum lysed 58 +/- 5% of swine endothelial cells. Selective inhibition of the direct pathway by adding EGTA to the serum reduced cytolysis to 51 +/- 2% (P < 0.01 versus normal serum). Similarly, when using C1q-deficient human sera, only 37 +/- 7% of swine endothelial cells were killed (P < 0.001 versus normal serum). When the alternative pathway was selectively inhibited by heating for 20 min at 50 degrees C, the lytic activity of human serum dropped to 42 +/- 5% (P < 0.001 versus normal serum). Factor B-deficient human serum could only lyse 42 +/- 10% of porcine endothelial cells (P < 0.001 versus normal serum). Syngeneic normal swine serum and heat-inactivated serum were not cytotoxic. Mixing serum with deficient direct pathway and serum with deficient alternative pathway restored the cytotoxicity to normal levels. Similarly, the cytotoxic activity of deficient serum supplemented with purified C1q or factor B at physiological concentrations reached that of normal human serum. In this model of in vitro hyperacute rejection, both pathways of complement activation are involved, suggesting that regimens designed to inhibit hyperacute rejection of swine xenografts into humans should take into account the dual activation of complement in this donor-recipient combination.

[Superacute xenogenic rejection: attempted treatment with anti-idiotypic antibodies]. / Rejet hyperaigu xénogénique: tentative de traitement à l'aide d'anticorps anti-idiotypiques.

Heart xenotransplantation in the guinea-pig to rat model, a highly discordant combination, is invariably followed by a hyperacute rejection of the graft. Preformed IgMs were shown to be responsible for the destruction of the graft by stimulating the complement cascade. We tried to inhibit preformed antibodies, before transplantation, by inducing anti-idiotypic antibodies using autologous Fab' mu specific for guinea-pig endothelial cells. This treatment seems to exert a neutralizing effect on xenogeneic hyperimmunization, against guinea-pig endothelial cells but not against preformed anti-guinea-pig antibodies.

Role of the CXCL12-CXCR4 axis in the development of deep rectal endometriosis.

Immunological and angiogenetic factors enhance the implantation of endometrial cells in the peritoneal cavity. The aim of this work was to determine the role of the CXCL12-CXCR4 axis in the attraction and the peritoneal implantation of endometriotic stromal cells in deep infiltrating endometriosis (DIE). Biopsies of DIE nodules were obtained from 14 patients undergoing surgical treatment for DIE with low rectal involvement and from 12 patients without macroscopic endometriosis undergoing laparoscopy. CXCR4 expression was evaluated by Western blot analysis and flow cytometry in eutopic endometrial cells and DIE stromal cells in primary cultures derived from the biopsies. CXCL12-induced migration of DIE eutopic endometrial stromal cells was evaluated by transwell migration. CXCL12 was assayed in peritoneal fluids by ELISA. CXCR4 expression was higher in eutopic endometrial stromal cells than in control endometrial cells (p<0.05) and in DIE stromal cells (p<0.05). Eutopic endometrial stromal cells were more attracted by CXCL12 than control cells (p<0.01). CXCL12 was higher in DIE peritoneal fluids than in controls (p<0.05). CXCR4 was down-regulated in deep infiltrating endometriotic stromal cells. The CXCL12-CXCR4 axis plays a role in the attraction of eutopic endometrial cells into the peritoneal cavity, and the down-regulation of CXCR4 in resident endometriotic cells could cause their arrest in situ.

The organotelluride catalyst LAB027 prevents colon cancer growth in the mice.

Organotellurides are newly described redox-catalyst molecules with original pro-oxidative properties. We have investigated the in vitro and in vivo antitumoral effects of the organotelluride catalyst LAB027 in a mouse model of colon cancer and determined its profile of toxicity in vivo. LAB027 induced an overproduction of H(2)O(2) by both human HT29 and murine CT26 colon cancer cell lines in vitro. This oxidative stress was associated with a decrease in proliferation and survival rates of the two cell lines. LAB027 triggered a caspase-independent, ROS-mediated cell death by necrosis associated with mitochondrial damages and autophagy. LAB027 also synergized with the cytotoxic drug oxaliplatin to augment its cytostatic and cytotoxic effects on colon cancer cell lines but not on normal fibroblasts. The opposite effects of LAB027 on tumor and on non-transformed cells were linked to differences in the modulation of reduced glutathione metabolism between the two types of cells. In mice grafted with CT26 tumor cells, LAB027 alone decreased tumor growth compared with untreated mice, and synergized with oxaliplatin to further decrease tumor development compared with mice treated with oxaliplatin alone. LAB027 an organotelluride catalyst compound synergized with oxaliplatin to prevent both in vitro and in vivo colon cancer cell proliferation while decreasing the in vivo toxicity of oxaliplatin. No in vivo adverse effect of LAB027 was observed in this model.

Radical oxygen species production induced by advanced oxidation protein products predicts clinical evolution and response to treatment in systemic sclerosis.

OBJECTIVES: To investigate the role of reactive oxygen species (ROS) in the development of the various patterns of systemic sclerosis (SSc) and the mechanisms of ROS production by endothelial cells and fibroblasts. METHODS: Production of hydrogen peroxide (H(2)O(2)), nitric oxide (NO) and cellular proliferation were determined following incubation of endothelial cells and fibroblasts with 56 SSc and 30 healthy sera. Correlations were established between those markers, the type and the severity of the clinical involvements, and the response to treatment. The factors leading to ROS production were determined. RESULTS: H(2)O(2) production by endothelial cells and fibroblasts was higher after incubation with SSc sera than with normal sera (p<0.001) and with sera from SSc patients with severe complications than sera from other patients (p<0.05). Sera from patients with lung fibrosis triggered the proliferation of fibroblasts more than other SSc sera (p<0.001), whereas sera from patients with vascular complications exerted no proliferative effect on fibroblasts, but inhibited endothelial cell growth (p<0.05) and induced NO overproduction (p<0.05). Bosentan reduced NO release by 32%, whereas N-acetylcystein potentiated 5-fluorouracil (5FU) to inhibit fibroblast proliferation by 78%. Those serum-mediated effects did not involve antibodies but advanced oxidation protein products that selectively triggered cells to produce H(2)O(2) or NO. CONCLUSIONS: SSc sera induce the production of different types of ROS that selectively activate endothelial cells or fibroblasts, leading to vascular or fibrotic complications. Assaying serum-induced ROS production allows clinical activity of the disease to be followed and appropriate treatments to be selected.

Effect of cholestasis and bile acids on interferon-induced 2',5'-adenylate synthetase and NK cell activities.

BACKGROUND/AIMS: The mechanisms involved in resistance to interferon alfa in patients with chronic hepatitis C are unclear. Both cirrhosis and cholestasis have been shown to be predictive of resistance. The aim of this study was to evaluate the influence of cholestasis and bile acids on 2',5'-oligoadenylate synthetase and natural killer activities, which are both involved in the antiviral activity of interferon. METHODS: 2',5'-Oligoadenylate synthetase activity was evaluated in spleen, liver, and isolated hepatocytes from bile duct-ligated rats, and the effect of bile acids in vitro on interferon-induced 2',5'-oligoadenylate synthetase and natural killer activities was examined in fresh mononuclear cells from healthy subjects. RESULTS: Cholestasis had a time-dependent inhibitory effect on 2',5'-oligoadenylate synthetase activity in liver, spleen, and isolated hepatocytes from cholestatic rats (-70%, 86%, and 70% relative to baseline, respectively). In vitro, endogenous bile acids had a concentration-dependent inhibitory effect on interferon-induced 2',5'-oligoadenylate synthetase and natural killer activities, which was related to their structure. This inhibitory effect correlated with the surface activity index. CONCLUSIONS: Cholestasis and bile acids diminish the biological activity of interferon and natural killer activity. The results suggest a decrease in the antiviral defenses in cholestatic conditions.