Alastair Hansen

Glomerular endotheliosis in normal pregnancy and pre‐eclampsia

Objective To investigate the proportion of women with findings characteristic for pre‐eclampsia, as opposed to renal disease, in a controlled study of hypertensive pregnant women undergoing antepartum renal biopsy.

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.

Serum cystatin C reflects glomerular endotheliosis in normal, hypertensive and pre‐eclamptic pregnancies

Objective To study the correlation between serum cystatin C levels and renal structural changes in normal, hypertensive and pre‐eclamptic pregnancy to evaluate it as a marker of the degree of renal involvement in pre‐eclampsia.

Integrated genetic and epigenetic analysis of bladder cancer reveals an additive diagnostic value of FGFR3 mutations and hypermethylation events.

The bladder cancer genome harbors numerous oncogenic mutations and aberrantly methylated gene promoters. The aim of our study was to generate a profile of these alterations and investigate their use as biomarkers in urine sediments for noninvasive detection of bladder cancer. We systematically screened FGFR3, PIK3CA, TP53, HRAS, NRAS and KRAS for mutations and quantitatively assessed the methylation status of APC, ARF, DBC1, INK4A, RARB, RASSF1A, SFRP1, SFRP2, SFRP4, SFRP5 and WIF1 in a prospective series of tumor biopsies (N = 105) and urine samples (N = 113) from 118 bladder tumor patients. We also analyzed urine samples from 33 patients with noncancerous urinary lesions. A total of 95 oncogenic mutations and 189 hypermethylation events were detected in the 105 tumor biopsies. The total panel of markers provided a sensitivity of 93%, whereas mutation and methylation markers alone provided sensitivities of 72% and 70%, respectively. In urine samples, the sensitivity was 70% for all markers, 50% for mutation markers and 52% for methylation markers. FGFR3 mutations occurred more frequently in tumors with no methylation events than in tumors with one or more methylation events (78% vs. 33%; p < 0.0001). FGFR3 mutation in combination with three methylation markers (APC, RASSF1A and SFRP2) provided a sensitivity of 90% in tumors and 62% in urine with 100% specificity. These results suggest an inverse correlation between FGFR3 mutations and hypermethylation events, which may be used to improve noninvasive, DNA‐based detection of bladder cancer.

Expression and function of toll-like receptor 8 and Tollip in colonic epithelial cells from patients with inflammatory bowel disease.

Objective. Growing evidence indicates that innate immunity, including toll-like receptor (TLR) signalling, plays a role in inflammatory bowel disease (IBD). This may also apply in the case of TLR-8, which has recently been shown to reverse the immunosuppressive function of regulatory T cells. However, the role of TLR-8 in IBD is currently unknown, and therefore we investigated the expression of TLR-8 and its natural antagonist, Tollip, in normal and inflamed human gut, and examined whether the receptor is functionally active. Methods. TLR-8 and Tollip mRNA expression were measured in colonic epithelial cells (CEC) and lamina propria mononuclear cells (LPMNC) by quantitative polymerase chain reaction. TLR-8 protein expression was visualized in whole biopsy specimens by indirect immunofluorescence microscopy. Cellular localization of TLR-8 protein was assessed by immuno-electron microscopy. IL-8 secretion was measured by ELISA after stimulation with TLR-8 ligand. Results. TLR-8 mRNA and protein expression were substantially up-regulated in CEC from inflamed mucosa from patients with ulcerative colitis (∼350-fold, p<0.01) and Crohns disease (∼45-fold, p<0.05) compared to controls. TLR-8 proteins resided on the luminal surface membrane and in intracellular organelles. Tollip was not increased in CEC from IBD patients. CEC from normal mucosa responded to TLR-8 stimulation by secreting IL-8. TLR-8 was expressed only on the mRNA level in LPMNC with no differences between IBD patients and controls. Conclusion. Expression of TLR-8, but not Tollip, is highly up-regulated in the colonic epithelium from patients with active IBD. Since the receptor is functionally active, our data suggest that TLR-8 signalling is important in the pathogenesis of IBD.

Bladder pain syndrome/interstitial cystitis in a Danish population: a study using the 2008 criteria of the European Society for the Study of Interstitial Cystitis.

Study Type – Symptom prevalence (prospective cohort)
Level of Evidence 1b

YKL‐40 and mast cells are associated with detrusor fibrosis in patients diagnosed with bladder pain syndrome/interstitial cystitis according to the 2008 criteria of the European Society for the Study of Interstitial Cystitis

Richter B, Roslind A, Hesse U, Nordling J, Johansen J S, Horn T & Hansen A B
(2010) Histopathology 57, 371–383
YKL‐40 and mast cells are associated with detrusor fibrosis in patients diagnosed with bladder pain syndrome/interstitial cystitis according to the 2008 criteria of the European Society for the Study of Interstitial Cystitis

Influence of angiogenesis inhibitors on endothelial cell morphology in vitro

Human umbilical vein endothelial cells (HUVEC) propagated in co‐culture with fibroblasts form capillary‐like networks of tubes. Here we characterize the morphology and ultrastructure of HUVEC in such co‐cultures and investigate the influence of different angiogenesis inhibitors on endothelial cell morphology. Addition of angiogenesis inhibitors to the co‐culture disrupted endothelial network formation and influenced endothelial cell morphology in two distinct ways. Instead of characteristic capillary‐like networks, the endothelial cell morphology appeared as either short cords or compact cell clusters of variable size. Electron microscopy (EM) showed that in co‐culture untreated HUVEC formed capillary‐like tubes with lumina and retained important ultrastructural and physiological properties of endothelial cells in functional vessels as they contained both Weibel‐Palade bodies and transport vesicles. Immuno‐EM showed that the endothelial cell marker CD 31 stained endothelial membranes at cell‐cell contacts, and at the luminal and abluminal side of the capillary‐like tubes, although most abundantly at the luminal membranes. No ultrastructural signs of apoptosis were seen in HUVEC in inhibitor‐treated co‐cultures. Our results demonstrate that treatment with levamisole or anti‐VEGF inhibits endothelial cell differentiation into tubes or instead induces formation of compact endothelial cell clusters. Treatment with platelet factor 4, suramin and TNP‐470 results in formation of short endothelial cell cords. We discuss the implications of these findings.

AgNOR counts and histological grade in stage pTa bladder tumours: reproducibility and relation to recurrence pattern

Silver‐stained nucleolar organizer regions (AgNORs) were studied in 90 patients with a single, primary, non‐invasive papillary bladder tumour (stage pTa). All tumours were locally resected and patients were followed for 5 years (n= 68) or until the development of an invasive recurrence (n= 22).

A Simple Method to Establish Short-Term Cultures of Normal Human Colonic Epithelial Cells from Endoscopic Biopsy Specimens: Comparison of Isolation Methods, Assessment of Viability and Metabolic Activity

Background: Abnormalities in colonic epithelial cell function have been implicated in the pathogenesis of various intestinal disorders, especially inflammatory bowel disease (IBD). The mechanisms, however, remain obscure owing to the lack of representative human colonic epithelial cell models. The aim of this study was to develop and validate a method for establishment of short-term culture of normal human colonic epithelial cells from endoscopic biopsies. Methods: Epithelial cells were isolated from colonoscopic biopsies by means of ethylenediaminetetraacetic acid/ethylene glycol tetraacetic acid (EDTA/EGTA) (10 or 60 min) or by enzyme treatment and cultured in collagen-coated wells. Viability was measured with a methyltetrazoleum conversion assay, confocal laser, and electron microscopy. Metabolic function was measured by means of butyrate oxidation, 14C-leucine and 3H-glucosamine incorporation; DNA synthesis by means of 3H-thymidine incorporation, and apoptosis with an enzyme-linked immunosorbent assay (ELISA) for histone-associated DNA fragments. Cell types were identified by immunocytochemistry. Results: Ten minutes of EDTA/EGTA treatment released intact crypts and was superior to both the 60-min treatment and enzymatic treatment in terms of viability and nonepithelial cell contamination, respectively. Despite activation of detachment-induced apoptosis, a median 51% of the isolated cells was viable after 24 h of culture and metabolically active as judged by 3H-thymidine, 14C-leucine, and 3H-glucosamine incorporation. Butyrate oxidation followed more complex kinetics (substrate activation) than observed previously in other models. The apparent Km values (medians) were 0.7 mM and 4.5 mM in low and high concentration ranges, respectively. Conclusion: We report a simple method to establish culture of human colonic epithelial cells from endoscopically obtained biopsy specimens, producing sufficient viable cells to perform metabolic studies pertinent to the pathogenesis of IBD and related human disorders.