Fig 1: Evaluation of cobalamin receptor abundances in canine ileal epithelium–Data acquisition and image analysis via confocal laser scanning microscopy.A: Immunofluorescence staining of the amnionless (AMN) subunit (green) in the cross-sectioned ileum of a dog with chronic inflammatory enteropathy (CIE). Nuclei are DAPI (diamidine phenylindole)-stained (blue). For quantification by photon counting, 12 regions of interest (ROI) each were set in the apical (pink circles) and basolateral part (turquoise circles) of the epithelial cells, and within the nuclei of subepithelial parenchymal cells (yellow circles; controls). Focal green staining within the lamina propria is elicited by nonspecific fluorescence of erythrocytes. B: Grayscale-converted label (frame in A) showing the highest (red voxel) and lowest photon counts (blue voxel) by thresholding the saturation points of the signal. ROI were set at the sites of highest photon counts within the apical and basolateral part of the enterocytes. C: Detail (frame in A) showing ROI within the nuclei of subepithelial parenchymal cells used for data normalization.
Fig 2: Correlation of ileal cobalamin receptor expression with clinical and clinicopathological patient variables.Groups of dogs are indicated by different colors: green = dogs with chronic inflammatory enteropathy (CIE) and normocobalaminemia, yellow = CIE dogs with suboptimal cobalamin status, and red = CIE dogs with severe hypocobalaminemia; and gray dots indicate the healthy control group. Each y-axis shows the corresponding normalized epithelial photon counts (nEPC). A: Correlation between apical amnionless (AMN) expression and the dogs’ age (ρ = 0.80, P<0.0001; including healthy controls: ρ = 0.61, P = 0.0003). B: Correlation between basolateral AMN expression and CCECAI scores (ρ = 0.42, P = 0.0491; only including dogs with CIE). C: No correlation was seen between the apical AMN expression and serum cobalamin concentrations (ρ = -0.23, P = 0.2963; only including CIE dogs). Dark gray area: reference interval for serum cobalamin concentration (251–908 ng/L; www.vetmed.tamu.edu/gilab). D: Correlation of the apical AMN expression with serum folate concentrations (ρ = -0.55, P = 0.0078, Pcorr = 0.0312; only CIE dogs). Dark gray: reference interval for serum folate concentration (7.7–24.4 μg/L, www.vetmed.tamu.edu/gilab).
Fig 3: Ileal cobalamin receptor expression in canine chronic inflammatory enteropathy (CIE) and in health.Quantitative confocal laser scanning microscopy analysis and comparison of (A) amnionless (AMN) and (B) cubilin (CUBN) in the apical and basolateral compartment of the intestinal epithelial cells among the four groups of dogs: healthy controls, and dogs with CIE that were either normocobalaminemic (serum cobalamin concentration 400–908 ng/L), have a suboptimal serum cobalamin status (251–400 ng/L) or are hypocobalaminemic (serum cobalamin concentration ≤250 ng/L). Cobalamin receptor expression was determined by computation of normalized epithelial photon counts (nEPC). Data showed increased levels of both receptor subunits in dogs with CIE, particularly hypocobalaminemic dogs, compared to healthy controls. Boxplots mark the median, quartiles, and range; outliers are indicated by a circle.
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