Acetylated sialic acid residues and blood group antigens localise within the epithelium in microvillous atrophy indicating internal accumulation of the glycocalyx

Abstract

Background: Microvillous atrophy, a disorder of intractable diarrhoea in infancy, is characterised by the intestinal epithelial cell abnormalities of abnormal accumulation of periodic acid-Schiff (PAS) positive secretory granules within the apical cytoplasm and the presence of microvillous inclusions. The identity of the PAS positive material is not known, and the aim of this paper was to further investigate its composition.

Methods: Formaldehyde fixed sections were stained with alcian blue/PAS to identify the acidic or neutral nature of the material, phenylhydrazine blocking was employed to stain specifically for sialic acid, and saponification determined the presence of sialic acid acetylation. The specificity of sialic acid staining was tested by digestion with mild sulphuric acid. Expression of blood group related antigens was tested immunochemically.

Results: Alcian blue/PAS staining identified a closely apposed layer of acidic material on the otherwise neutral (PAS positive) brush border in controls. In microvillous atrophy, a triple layer was seen with an outer acidic layer, an unstained brush border region, and accumulation within the epithelium of a neutral glycosubstance that contained acetylated sialic acid. Blood group antigens were detected on the brush border, in mucus, and within goblet cells in controls. In microvillous atrophy they were additionally expressed within the apical cytoplasm of epithelial cells mirroring the PAS abnormality. Immuno electron microscopy localised expression to secretory granules.

Conclusions: A neutral, blood group antigen positive, glycosubstance that contains acetylated sialic acid accumulates in the epithelium in microvillous atrophy. Previous studies have demonstrated that the direct and indirect constitutive pathways are intact in this disorder and it is speculated that the abnormal staining pattern reflects accumulation of glycocalyx related material.

Figure 1

Alcian blue/periodic acid-Schiff staining. (A) Control (case 7). Magenta stained brush border, indicating the presence of neutral glycosubstances, with a tightly apposed purple stained luminal acidic layer, presumably attached mucus (arrow). Note purple goblet cells and mucus in lumen. (B) Microvillous atrophy (case 1). Magenta stained neutral glycosubstance accumulated within the apical cytoplasm. No magenta brush border stain but the tightly apposed luminal purple staining acidic layer remained, producing a triple layered sandwich effect (arrow).

Figure 2

Staining for sialic acid. Control (A) case 7 showed faint brush border and goblet cell stain for sialic acid. (B) Stain for acetylated sialic acid showing the same pattern as in (A) but more strongly stained. Microvillous atrophy (case 2). (C) Stain for sialic acid showing similar distribution to alcian blue/periodic acid-Schiff stain. (D) Positive staining for acetylated sialic acid in apical cytoplasm and goblet cells. (E) Reduced staining following sulphuric acid digestion confirming the presence of acetylated sialic acid.

Figure 3

Staining for sialic acid C₇/C₈ position of acetylation. (A) Control (case 7) showing brush border (arrowhead) and goblet cell (G) staining. (B) Microvillous atrophy (case 2) staining in upper crypt apical cytoplasm (arrowhead) and goblet cells (G).

Figure 4

Blood group staining. Lewis Le^a related antibody staining. Controls: (A) case 10 with positive fluorescence in brush border (arrow), goblet cells (G), and luminal mucus (M). Microvillous atrophy: (B) case 2 demonstrating positive staining within the apical cytoplasm (arrow) of upper crypt cells in a similar location to the abnormal accumulation of periodic acid-Schiff positive material, and goblet cells (G). Blood group A staining: controls: (C) case 14 showing brush border (arrow), goblet cell (G), and mucus (M) staining. Microvillous atrophy: (D) positive staining within the upper crypt epithelium (arrow) (case 2), (E) apical cytoplasm of low villous epithelium (arrow) (case 5), and (F) circular staining within surface epithelium (arrow) (case 3).

Figure 5

Immuno electron microscopy. Control (case 6): gold particles on (A) mucus within goblet cell and (B) microvilli. Microvillous atrophy (case 2): (C) view of microvilli and secretory granules. Rectangle marks inset (D) showing gold labelling present on secretory granules (arrows). Bar = 100 nm.