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author:("bacille, F")
1.  Soluble galectin‐3 is a strong, colonic epithelial‐cell‐derived, lamina propria fibroblast‐stimulating factor 
Gut  2006;56(1):43-51.
Background
Colonic lamina propria fibroblasts (CLPFs) play an important role in the pathogenesis of fibrosis and strictures in Crohn's disease.
Aim
To identify colonic epithelial cell (CEC)‐derived factors that activate CLPFs.
Methods
Primary human CECs and CLPFs were isolated from control mucosa and interleukin 8 (IL8) of CLPF cultures was quantified by ELISA. Activation of nuclear factor κB (NF‐κB) was shown, and translocation of NF‐κB was inhibited by a dominant‐negative IκB‐expressing adenovirus. The major CLPF‐activating and IL8 inducing protein was purified using fast‐performance liquid chromatography (HiPrep 16/60 Sephacryl S‐200 High Resolution Column) and sodium dodecyl sulphate gel electrophoresis.
Results
A considerable increase in IL8 secretion by CLPFs cultured in CEC‐conditioned media compared with that in unconditioned media (155.00 (10.00) pg/µg v 1.434 (0.695) pg/µg) was found. The effect of CEC‐conditioned media on CLPF IL8 secretion was NF‐κB dependent. A protein or DNA array confirmed the involvement of NF‐κB and activator protein‐1. Purification of a candidate band isolated with the use of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and subsequent sequencing showed soluble galectin‐3 to be a strong CLPF‐activating factor. Depletion of galectin‐3 from conditioned media by immunoprecipitation abolished the CLPF stimulatory effect.
Conclusions
Using a classical biochemical approach, soluble galectin‐3 was identified as a strong activator of CLPFs produced by CEC. Galectin‐3 induced NF‐κB activation and IL8 secretion in these cells and may be a target for future therapeutic approaches to reduce or avoid stricture formation.
doi:10.1136/gut.2005.081646
PMCID: PMC1856646  PMID: 16709662
2.  Prognostic relevance of P‐cadherin expression in melanocytic skin tumours analysed by high‐throughput tissue microarrays 
Journal of Clinical Pathology  2006;59(7):699-705.
Aim
To investigate whether protein expression or cellular localisation of P‐cadherin is associated with clinicopathological characteristics in benign and malignant melanocytic skin tumours.
Experimental design
P‐cadherin expression and the Ki‐67 labelling index were analysed immunohistochemically by using tissue microarrays (TMAs). Membranous and cytoplasmic expression was scored semiquantitatively (0 to 2+).
Results
P‐cadherin protein expression of any intensity (1+ to 2+) was detected in the membrane in 41.5% (132/318) and in the cytoplasm in 64.2% (204/318) of patients. In general, P‐cadherin expression was significantly reduced in malignant melanomas (p<0.001) and melanoma metastases (p<0.001), compared with benign nevi. Additionally, loss of membranous P‐cadherin was associated with Clark level (p = 0.011) and tumour thickness (p<0.001). Interestingly, a significantly lower P‐cadherin expression was shown by dermal nevi than by compound and junctional nevi (p = 0.005; p = 0.025). In primary melanomas, a Ki‐67 labelling index <5% was not associated with P‐cadherin protein expression, suggesting that loss of P‐cadherin expression was not associated with proliferation. None of the other clinical and histological factors analysed was significantly related to P‐cadherin expression. Low cytoplasmic P‐cadherin expression was associated with tumour recurrence (p = 0.03) in all the patients who were analysed. After testing various multivariate Cox regression models, loss of cytoplasmic P‐cadherin expression remained a highly significant adverse risk factor for tumour recurrence in patients with tumours <2 mm.
Conclusions
Loss of cytoplasmic P‐cadherin expression is common in advanced melanomas and can be a prognostic marker of progression in patients with melanoma, most useful in patients with primary tumours <2 mm in thickness.
doi:10.1136/jcp.2005.034538
PMCID: PMC1860409  PMID: 16565225
3.  Prognostic impact of syndecan-1 expression in invasive ductal breast carcinomas 
British Journal of Cancer  2008;98(12):1993-1998.
Carcinoma cells lack syndecan-1 expression when they are transiting from an epithelial to a less-differentiated mesenchymal phenotype (epithelial–mesenchymal transition, EMT). Furthermore, a shift of syndecan-1 expression from malignant epithelial cells to reactive stromal cells has also been observed during progression of many carcinomas. Finally, epithelial and/or stromal syndecan-1 expression is of prognostic value in many carcinomas. Because recent results are contradictory in breast carcinomas, we have re-evaluated the prognostic significance of syndecan-1 expression in a cohort of 80 patients with invasive ductal breast carcinomas. The tumours from 80 patients diagnosed with invasive ductal breast carcinomas were used to construct a tissue microarray, which was stained with syndecan-1 by immunohistochemistry. We correlated syndecan-1 expression with clinicopathologic parameters and relapse-free survival (RFS). Exclusive epithelial expression of syndecan-1 is observed in 61.25% of the patients, whereas exclusive stromal expression is observed in 30% of the patients. Only 8.75% of the patients had both stromal and epithelial expressions of syndecan-1. A significant correlation was found between the loss of syndecan-1 epithelial expression and the syndecan-1 stromal expression with high grade of malignancy (P=0.011). The loss of syndecan-1 epithelial expression is correlated with RFS (P=0.001). Using multivariate Cox analysis, loss of epithelial syndecan-1 expression was the only prognostic indicator (P<0.001). We concluded that the loss of syndecan-1 epithelial expression was of strong prognostic value in breast carcinomas.
doi:10.1038/sj.bjc.6604400
PMCID: PMC2441962  PMID: 18542065
syndecan-1; invasive ductal carcinoma; epithelial–mesenchymal transition; stroma; prognostic factor
4.  Glycoprotein (gp) 96 expression: induced during differentiation of intestinal macrophages but impaired in Crohn’s disease 
Gut  2005;54(7):935-943.
Background: The glycoprotein (gp) 96 links the adaptive with the innate immune system. It is a chaperone with a binding domain for peptides generated by proteasomal degradation. During cellular stress, peptide loaded gp96 can be released and presented to T cells by antigen presenting cells (APCs).
Methods: mRNAs from in vitro differentiated macrophages (iv mac) and normal intestinal macrophages (IMACs) were compared by subtractive hybridisation and Affymetrix GeneChip analysis. Differentiation induced expression of gp96 was investigated in the multicellular spheroid (MCS) model. In vivo gp96 protein expression was detected by double labelling immunohistochemistry of human colon and in the CD4+ CD62L+ T cell transfer mouse model.
Results: Five of 76 clones obtained by subtractive hybridisation revealed >99% sequence homology to gp96. Affymetrix GeneChip analysis confirmed induction of gp96 in IMACs. Gp96 mRNA was detected in IMACs from normal and intestinal bowel disease mucosa. Induction of gp96 protein was observed after seven days in the MCS model of IMAC differentiation. Immunohistochemistry confirmed the presence of gp96 protein in IMACs in normal mucosa as well as in mucosa from patients with ulcerative colitis and diverticulitis. In mucosa from Crohn’s disease (CD) patients, gp96 protein was not detectable. In the CD4+ CD62L+ T cell transfer mouse model, gp96 was verifiable in non-activated IMACs.
Conclusion: Gp96 is induced during differentiation of normal IMACs but is not detected in IMACs in CD mucosa. As gp96 has been described as having a role in tolerance induction, this may be relevant for loss of tolerance against luminal bacteria found in CD patients.
doi:10.1136/gut.2004.053116
PMCID: PMC1774602  PMID: 15951537
Crohn’s disease; heat shock proteins; intestinal macrophages
5.  Morphological characterisation of Crohn’s disease fistulae 
Gut  2004;53(9):1314-1321.
Background: Fistulae are a common complication in up to 35% of all patients with Crohn’s disease. Their therapy is difficult and frequently unsatisfactory. To date, no histological comparison of Crohn’s disease fistulae with non-inflammatory bowel disease fistulae has been performed. In addition, Crohn’s disease fistulae have not been well characterised morphologically.
Methods: Eighty four fistulae from Crohn’s disease patients were compared with 13 fistulae from controls. Haematoxylin-eosin staining, electron microscopy, and immunohistochemistry for panCytokeratin (epithelial cells), CD20 (B cells), CD45R0 (T cells), and CD68 (macrophages) were performed according to standard techniques. In addition, histopathological findings were compared with clinical and laboratory data.
Results: In 31.0% of controls and 27.4% of Crohn’s disease specimens, fistulae had a lining of flattened intestinal epithelium without goblet cells or, in the case of cutaneous/perianal disease, narrow squamous epithelium. Non-epithelialised fistulae were covered by a thin layer of (myo)fibroblasts, focally forming a new basement membrane, as demonstrated by electron microscopy. All fistulae were surrounded by granulation tissue. Crohn’s disease fistulae presented with central infiltration by CD45R0+ T cells, followed by a small band of CD68+ macrophages and dense accumulation of CD20+ B cells. In contrast, in controls, there was dense infiltration by CD68+ macrophages with only few CD20+ B cells and CD45R0+ T lymphocytes.
Conclusions: Fistulae in Crohn’s disease differ markedly from non-Crohn’s disease fistulae with regard to their cellular composition. The presence of an epithelial lining in a subgroup of fistulae may be important for the therapeutic approach and healing process.
doi:10.1136/gut.2003.038208
PMCID: PMC1774207  PMID: 15306592
Crohn’s disease; fistulae; histology; immunohistochemistry; electron microscopy
6.  Alterations in p53 predict response to preoperative high dose chemotherapy in patients with gastric cancer 
Molecular Pathology  2003;56(5):286-292.
Aims: To evaluate the usefulness of molecular markers in predicting histopathological and clinical response to preoperative high dose chemotherapy (HDCT) and survival of patients with advanced gastric cancer.
Methods: In a phase II trial, 25 patients with metastatic gastric cancer received preoperative tandem HDCT consisting of etoposide, cisplatin, and mitomycin, followed by autologous bone marrow transplantation to achieve surgical resectability. Samples before and after treatment, from normal and tumour tissue, were characterised histopathologically, and both p53 and BAX expression was analysed by immunohistochemistry. Pretreatment formalin fixed, paraffin wax embedded samples from normal and tumour tissue were microdissected, and the extracted DNA was preamplified using improved primer extension preamplification polymerase chain reaction. Detection of microsatellite instability (MSI) or loss of heterozygosity (LOH) was performed using markers for p53, BAX, BAT25, BAT26, D2S123, D17S250, and APC. Exons 5–9 of the p53 gene were sequenced directly on ABI 373.
Results: Four parameters were significantly associated with response to chemotherapy and prolonged overall survival: positive p53 immunostaining, positive p53 mutation status before chemotherapy, strong histological regression induced by preoperative HDCT, and surgical treatment. Patients’s sex or age, tumour location or stage, lymph node status, Lauren classification, MSI, or LOH did not influence duration of survival significantly in this high risk population.
Conclusion: Positive p53 immunostaining and p53 mutation status in pretreatment tumour biopsies might be useful molecular predictors of response and prognosis in patients with advanced gastric cancer treated by preoperative HDCT.
PMCID: PMC1187340  PMID: 14514923
gastric cancer; preoperative high dose chemotherapy; molecular parameters; histological regression; p53

Results 1-6 (6)