Automated image analysis in the study of collagenous colitis

Anne-Marie Kanstrup Fiehn,1,2 Martin Kristensson,3 Ulla Engel,4 Lars Kristian Munck,5,6 Susanne Holck,4 Peter Johan Heiberg Engel1,6 1Department of Pathology, Roskilde Hospital, Roskilde, 2Department of Pathology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, 3Digital Pathology, Visiopharm, Hørsholm, 4Department of Pathology, Copenhagen University Hospital, Hvidovre, 5Department of Gastroenterology, Koege Hospital, Koege, 6Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Purpose: The aim of this study was to develop an automated image analysis software to measure the thickness of the subepithelial collagenous band in colon biopsies with collagenous colitis (CC) and incomplete CC (CCi). The software measures the thickness of the collagenous band on microscopic slides stained with Van Gieson (VG). Patients and methods: A training set consisting of ten biopsies diagnosed as CC, CCi, and normal colon mucosa was used to develop the automated image analysis (VG app) to match the assessment by a pathologist. The study set consisted of biopsies from 75 patients. Twenty-five cases were primarily diagnosed as CC, 25 as CCi, and 25 as normal or near-normal colonic mucosa. Four pathologists individually reassessed the biopsies and categorized all into one of the abovementioned three categories. The result of the VG app was correlated with the diagnosis provided by the four pathologists. Results: The interobserver agreement for each pair of pathologists ranged from Κ-values of 0.56–0.81, while the Κ-value for the VG app vs each of the pathologists varied from 0.63 to 0.79. The overall agreement between the four pathologists was Κ=0.69, while the overall agreement between the four pathologists and the VG app was Κ=0.71. Conclusion: In conclusion, the Visiopharm VG app is able to measure the thickness of a subepithelial collagenous band in colon biopsies with an accuracy comparable to the performance of a pathologist and thereby provides a promising supplementary tool for the diagnosis of CC and CCi and in particular for research. Keywords: microscopic colitis, collagenous colitis, incomplete collagenous colitis, automated image analysi

Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2

The human orthologue of the H+-coupled amino acid transporter (hPAT1) was cloned from the human intestinal cell line Caco-2 and its functional characteristics evaluated in a mammalian cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the intestinal tract. When expressed heterologously in mammalian cells, hPAT1 mediated the transport of α-(methylamino)isobutyric acid (MeAIB). The cDNA-induced transport was Na+-independent, but was energized by an inwardly directed H+ gradient. hPAT1 interacted with glycine, l-alanine, l-proline, α-aminoisobutyrate (AIB) and γ-aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the d-isomers of alanine and proline. With serine and cysteine, though the l-isomers did not interact with hPAT1 to any significant extent, the corresponding d-isomers were recognized as substrates. With proline and alanine, the affinity was similar for l- and d-isomers. However, with cysteine and serine, the d-isomers showed 6- to 8-fold higher affinity for hPAT1 than the corresponding l-isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H+-coupled amino acid transport system in Caco-2 cells. Furthermore, there was a high degree of correlation (r2 = 0.93) between the relative potencies of various amino acids to inhibit the H+-coupled MeAIB transport measured with native Caco-2 cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco-2 cells. These data suggest that hPAT1 is responsible for the H+-coupled amino acid transport expressed in the apical membrane of Caco-2 cells

New recipe for targeting resistance

The rapid spread of antibiotic-resistant bacteria demands novel treatment approaches that delay or even reverse the evolution of resistance. A new screening strategy identifies two compounds that select against a common tetracycline-resistance gene in Escherichia coli