Jean-François Mosnier

Vγ9Vδ2 T Cell Response to Colon Carcinoma Cells

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vγ9Vδ2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-α and IFN-γ secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vγ9Vδ2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vγ9Vδ2 T cells of various origins, and Vγ9Vδ2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vγ9Vδ2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vγ9Vδ2 T cell agonists in immunotherapies targeting colon tumors.

Phosphohistone H3 labelling for histoprognostic grading of breast adenocarcinomas and computer-assisted determination of mitotic index

Background: Microscopic evaluation of mitotic figures is a routine procedure in the assessment of the histoprognostic grade of tumours. Nevertheless, their count may be fraught with difficulties. As histone H3 phosphorylation at serine 10 is closely linked to chromosomal condensation, a new monoclonal antibody directed to phosphorylated histone H3 (PPH3) was recently proposed to detect mitotic cells. Aim: To test the reliability of this antibody in detecting and counting mitotic figures in sections of breast adenocarcinomas, because of the importance of mitotic count in histoprognostic grading. Methods: The pattern of PPH3 staining in formalin-fixed paraffin wax-embedded tissues, including normal tissues and a series of 39 breast adenocarcinomas, was examined. A new computer-assisted method was also developed for determining the mitotic index. Results and conclusions: In all tissues tested, PPH3-labelled mitotic figures were easily detected, allowing a rapid identification of the area of highest mitotic activity. In breast carcinomas, a strong correlation was observed between PPH3-stained and haematoxylin and eosin-stained mitotic counts (r = 0.86, p<0.0001). Counting of prophase nuclei that coexpress cyclin B1, a marker of the G2/M phase, was possible by PPH3 staining; its accuracy led us to reconsider the tumour grade in three cases. Finally, an automatic computer-assisted method was designed for assessing mitotic index with confocal microscopy and image-analysis software.

Integrin up-regulation in chronic liver disease: relationship with inflammation and fibrosis in chronic hepatitis C.

In 94 patients with chronic hepatitis C, the pattern of integrin expression was correlated with firstly, the histological activity index, necro‐inflammatory grade, and stage of fibrosis; secondly, the expression of inflammatory markers including ICAM‐1; and thirdly, the extent and intensity of laminin deposition in the perisinusoidal matrix. Immunohistochemical results were evaluated according to a semi‐quantitative scoring system or by image analysis. Increased β1 expression was observed in 88.2% of cases. The expression of α1 and α5 was increased in 55% and 58.5% of cases, respectively. α6 chain was detected in 78.7% of cases. There were no statistically significant differences in integrin expression level according to Knodells score, inflammatory grade, or stage of fibrosis. ICAM‐1 expression was higher in patients with high scores for β1 expression, but the differences were not statistically significant. There were significantly more patients with high scores for β1 expression among those with continuous perisinusoidal deposition of laminin. Moreover, a close statistical correlation was observed between α6 induction and perisinusoidal laminin deposition (p<0.001). The results suggest that integrin up‐regulation in chronic hepatitis C is more closely related to the fibrotic process than to the inflammatory lesions. This reinforces the idea that integrin induction in chronic liver disease is part of a coordinated process involved in the progression of liver fibrosis. Copyright

ADAM15 upregulation and interaction with multiple binding partners in inflammatory bowel disease

A disintegrin and metalloproteinase (ADAM)15 is upregulated in some tissues undergoing remodeling. This glycoprotein is characterized by adhesive function through its interaction with members of the integrin family and protease properties. The goal of this work was to describe the tissue distribution of ADAM15 and its spatial relationship with its known binding partners in inflammatory bowel disease. ADAM15 expression was examined using frozen tissues from eight patients with ulcerative colitis or Crohns disease and four normal colons by immunohistochemistry, immunoblotting and quantitative reverse transcription-polymerase chain reaction. In addition expression of α5β1- and αvβ3-integrins, VE-cadherin, α-smooth mucle actin (α-SMA) and collagen IV was examined using immunohistochemistry and confocal microscopy. In the normal colon, ADAM15 was expressed by all epithelial cells throughout the crypt and by pericryptic myofibroblasts coexpressing α-SMA and collagen IV. ADAM15 was also expressed by endothelial cells and vascular myocytes in all layers of the intestinal wall as well as by nonvascular myocytes of the muscularis mucosae and muscularis propria. In inflammatory bowel diseases, ADAM15 was strongly upregulated at the mRNA level and expressed only as an active form as shown by immunoblotting analysis. Parallel to its upregulation, ADAM15 expression was found both at the plasma membrane and in the cytoplasm of epithelial cells in acute attacks of the disease. In the crypt abcesses, ADAM15-positive epithelial cells were in close contact with α5β1-integrin-positive leukocytes localized between these cells and in the crypt lumen. In the regenerative areas, ADAM15-positive epithelial cells were in close contact with α5β1- and αvβ3-positive pericryptic myofibroblasts. In endothelial cells, VE-cadherin was decreased. In contrast, ADAM15 was strongly expressed by endothelial cells and was in close contact with α5β1-positive leukocytes. There is a differential expression of ADAM15 in epithelial cells during inflammatory bowel disease compared with the normal colon. In addition, the spatial relationships with its binding partners suggest a role for ADAM15 in the differentiation of regenerative colonic mucosa as well as in leukocyte transmigration across epithelial and endothelial barriers.

Immunostaining for membrane attack complex of complement is related to cell necrosis in fulminant and acute hepatitis.

BACKGROUND/AIMS Complement activation is one of the mechanisms involved in inflammatory lesions. Initiation of the complement terminal pathway at a cell surface leads to the formation of a cytolytic membrane attack complex. Our study assess whether a membrane attack complex-associated mechanism is involved in liver cell necrosis of fulminant and subfulminant hepatitis. METHODS Immunostaining for membrane attack complex was compared with immunostaining for cytokeratin and complement inhibitory proteins such as membrane cofactor protein, decay-accelerating factor, and homologous restriction factor in 15 patients with fulminant hepatitis and 5 patients with nonfulminant acute hepatitis. RESULTS In all patients, hepatocytes surrounding necrotic areas, but not those at a distance, were stained for membrane attack complex, whereas the opposite staining pattern for membrane cofactor protein was observed. In controls, no hepatocyte staining for membrane attack complex was observed, whereas membrane cofactor protein, but not decay-accelerating factor or homologous restriction factor, was detected on hepatocytes. CONCLUSIONS Complement activation by antibody-dependent or non-antibody-dependent mechanisms might be involved in the pathogenesis of either fulminant or acute hepatitis. Modulation of membrane cofactor protein expression on hepatocytes might contribute to the sensitivity of hepatocytes to membrane attack complex and subsequent cell lysis.

Human colonic myocytes are involved in postischemic inflammation through ADAM17-dependent TNFα production

The aim of this study was to identify human colonic resident cells able to initiate an inflammatory response in postischemic injury. Postischemic colonic injury, a condition relevant to various clinical settings, involves an inflammatory cascade in intestinal tissues through the recruitment of circulating inflammatory cells. However, there is no information on the nature of resident cells of the different intestinal layers able to initiate a postischemic inflammatory response. It is however an important issue in the context of a pharmacological approach of the early phase of intestinal ischemia. We reasoned that maintaining the different colonic layers as explant cultures in an oxygenated medium immediately after colonic resection, that is, after an ischemic period, would allow one to identify the resident cells able to initiate an inflammatory cascade, without interference of recruited inflammatory/immune cells. To this end, we designed an explant culture system that operationally defines three compartments in surgical specimens of the human colon, based on the microdissected layers, that is, mucosa, submucosa (containing muscularis mucosae) and muscularis propria. To validate the results obtained in explant cultures in the clinical setting of ischemic colitis, eight cases of sigmoid volvulus were examined. Only the myocytes‐containing explants produced tumor necrosis factor alpha (TNFα), via an ADAM17 (a disintegrin and metalloproteinase‐17)‐dependent pathway, as shown by the abrogation of TNFα production by the inhibitor Tapi‐2. Immunofluorescence studies identified nonvascular and vascular myocytes as resident cells coexpressing TNFα and ADAM17, both in our postischemic explant system and in surgical specimens from ischemic colitis patients. Finally, time‐course experiments on explanted tissues showed that TNFα production by myocytes was an early event triggered by a postischemic oxidative stress involving nuclear factor kappa B (NF‐κB). In conclusion, this study identifies human intestinal myocytes as resident cells able to initiate an inflammatory reaction through TNFα production in postischemic conditions, and delineates two points of control in TNFα production, NF‐κB and ADAM17, which can be targeted by pharmacological manipulation.

Over-expression of neurotensin high-affinity receptor 1 (NTS1) in relation with its ligand neurotensin (NT) and nuclear ß-catenin in inflammatory bowel disease-related oncogenesis

We investigated the expression of the neurotensin high-affinity receptor 1 (NTS1) during inflammatory bowel disease (IBD)-related colorectal oncogenesis, in colonic samples from 30 patients with IBD-related adenocarcinomas, dysplasias, and inflammatory mucosa (IM). The percentage of NTS1-positive epithelial cells progressively increased from the inflammatory condition to adenocarcinoma and was significantly higher in adenocarcinomas than in IM (p=0.0169). In parallel, the percentage of neurotensin (NT)-positive epithelial cells increased during the IBD-related oncogenesis. Finally, as NTS1 is a ss-catenin inducible gene, we found that a number of preneoplastic lesions and adenocarcinomas co-expressed NTS1 and beta-catenin without NT expression. Therefore, this study suggests two pathways of NTS1 overexpression during IBD-related oncogenesis: one triggered by NT overexpression, and a second associated with an activation of the APC/beta-catenin pathway, these two pathways being not mutually exclusive.

Restriction of V beta gene usage of liver-derived lymphocytes in chronic hepatitis B and C

T lymphocytes have been reported to be the predominant inflammatory cells in the liver of patients with chronic viral hepatitis. Their presence may reflect either nonspecific inflammation or a virus-specific immune response. To assess the repertoire of intra-hepatic T cells, we investigated the TCR V beta gene usage of T cells in 10 patients with chronic hepatitis B and 15 with chronic hepatitis C. Liver-derived lymphocytes and peripheral blood lymphocytes were analyzed by flow cytometry. Five out of the 10 hepatitis B patients were found to have an accumulation of certain V beta T cells in the liver (V beta 6.7; V beta 6.7; V beta 3.1, V beta 5.1, and V beta 6.7; V beta 3.1; V beta 12.1, respectively). Four out of the 15 hepatitis C patients were found to have an accumulation of certain V beta T cells in the liver (V beta 5.1; V beta 8 and V beta 5.2 and 5.3; V beta 3.1 and V beta 5.2 and 5.3; V beta 3.1 and V beta 12.1, respectively). Despite a limited screening of V beta subfamilies, this study indicates that, in patients with chronic hepatitis B and C, T cells using a certain V beta gene may accumulate in the liver. This suggests that intra-hepatic T cells are oligoclonal and possibly virus specific. Our results argue against the role of a superantigen in perpetuating liver disease. In addition, this study supports a role for T lymphocytes in the pathogenesis of chronic hepatitis C.

Gastric carcinogenesis and Helicobacter pylori infection.

Helicobacter pylori is the most frequent cause of infection-induced cancer worldwide. Gastric carcino-genesis is the consequence of the important and life-long inflammation induced by H. pylori in the stomach. Gastric carcinogenesis, can be studied in many ways. In this chapter, we focus on some aspects concerning the bacteria, and others concerning the host. On the bacterial side, the methods exploring the presence of the cag pathogenicity island including cagA and the consequences on epithelial cells are presented. On the host side, tissue microarray, immunohistochemistry and chromogenic in situ hybridization (CISH) are described.

Plasma cell variant of Castleman's disease localized to the liver: a case report.

Localized plasma cell variant of Castlemans disease is an uncommon lymphoproliferative disorder. It is characterized by numerous, evenly distributed germinal centres with moderate or extensive sheets of plasma cells in between. It may be associated with a wide variety of systemic manifestations that disappear after removal of lesion. Rare cases of Castlemans disease have been reported in extranodal locations, i.e. muscle, larynx, vulva, lung, pericardium and cranium. We report the first the case of a liver tumour with clinico-pathological features of the plasma cell variant of Castlemans disease. This tumour differs from the hepatic inflammatory pseudotumour, which is frequently associated with infectious conditions.