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Editor,—We read with considerable interest the excellent article on Helicobacter pyloricolonisation with MUC5AC in the human stomach by Van den Brinket al (
). The authors used antibodies against gastric mucin core proteins (anti-MUC6 and MUC5AC (45M1)) in conjunction with antibody against H pyloriand demonstrated attachment of H pylori to gastric surface mucous cells and the presence of H pylori in extracellular mucins derived from gastric surface mucous cells.
These finding are similar to those previously reported by us1 using histochemical staining specific for gastric mucins (dual staining consisting of galactose-oxidase cold thionine Schiff reaction (GOTS) and paradoxical concanavalin A staining (PCS))2, and immunostaining for H pylori. GOTS recognises galactose orN-acetyl galactosamine residues of gastric surface mucous cell mucins and stains gastric surface mucous cell mucins blue (fig 1A). Histochemical reactivity of GOTS is identical to that of immunostaining with anti-MUC5AC (45M1) (fig 1B). PCS recognises the specific sugar residues with peripheralN-acetyl glucosamine in gastric gland mucous cells (cardiac gland cells, mucous neck cells, and pyloric gland cells) and stains gastric gland mucous cell mucins brown (fig 1A). We used tissue sections from surgically resected stomachs fixed in Carnoy's solution which has the advantage of fixing the gastric surface mucous gel layer (SMGL) in paraffin embedded tissue sections.3
Dual staining of GOTS-PCS of Carnoy fixed gastric mucosa withoutH pylori infection showed that SMGL had a characteristic structure consisting of laminating layers of two types of mucins: the surface mucous cell type and the gland mucous cell type (fig 1C).1 3 Dual staining of GOTS-PCS in conjunction with immunostaining for H pylori in Carnoy fixed stomachs with H pylori infection revealed that H pylori characteristically existed on and between the surface mucous cells and in the SMGL (fig1D).1 In the SMGL, this organism was most often associated with the layer of surface mucous cell type mucins (fig 1D, E).1 Receptors responsible for adherence ofH pylori might exist only on the plasma membrane of surface mucous cells. In the SMGL, these receptors appear to be mixed with the surface mucous cell type mucins. The SMGL inhabited by H pylori revealed marked derangement of the multilaminating structure with fragmentation of the mucin layers and formation of vacuoles (fig 1D, E).1 After eradication of H pylori, the SMGL regained the laminating structure.1 Alteration of the SMGL found inH pylori infected stomachs suggests destruction of the SMGL by bacterial lipase and protease fromH pylori. 4 5
H pylori in the SMGL was more abundant than that attached to the surface mucous cells (fig 1D, E).1The SMGL appears to be the major site of H pylori colonisation and may serve as a vehicle for diffusion ofH pylori to other sites in the stomach and duodenum.
Editor,—I thank Dr Ota et al for their interest in our work. I was already familiar with the exquisite pictures of the laminated structure of gastric mucus by these authors. The present new data as shown, in conjunction with our previous data, shed more light on the phenomenon ofHelicobacter pylori attachment in the stomach. Although I generally support the conclusions of Otaet al, the following remarks are in order.
It seems very likely from figure 1A and 1B of their letter that the surface mucous cells that produce MUC5AC (by 45M1 immunostaining) were stained blue on galactose-oxidase cold thionine Schiff reaction (GOTS) staining. This implies that the blue sublayers within the laminated extracellular mucus would also be composed of MUC5AC. Also, as the authors show in fig 1 (D, E), H pylori was specifically localised to: (1) the blue layers within the mucus layer (that is, most likely MUC5AC; fig. 1D); and (2) the blue stained cells (that is, most likely producing MUC5AC; fig 1D, E). Implicit in our collective data is that the brown staining layers, as demonstrated by paradoxical concanavalin A staining (PCS), are probably composed of MUC6. Collectively, these data corroborate our previous conclusion that MUC5AC and MUC5AC producing cells (and not MUC6) are the main attachment sites for H pylori in the stomach. Yet final prove awaits immunohistochemical co-localisation of MUC5AC, MUC6, and H pylori in the mucus layer. As Carnoy's solution may not allow simultaneous immunochemical detection of these components, technical problems presently hamper final conclusions. In pursuing this, I think it is extremely important to concentrate on the primary gene products—that is, localisation of MUC5AC and MUC6 proteins—rather than on carbohydrates on complex molecules. Glycosylation is a complex process that may easily become disturbed in H pylori infection, for example. Thus as glycosylation may change under pathological conditions, I would urge the authors and other workers in the field to concentrate on the invariant part of the mucins: the non-O-glycosylated parts of the protein, as discussed by us previously.1-1
One of the issues addressed by Ota et al is the correlation of H pylori infection with disturbance of the laminated gastric mucus layer, as demonstrated in fig 1C versus fig 1D. In common with the authors, it is tempting to speculate that H pylori has a direct role in this disturbance. Similar to most bacteria, H pylori produces enzymes that in principle can degrade mucins, such as proteases and “mucinases”, as referred to by the authors. I personally doubt whether H pylori is directly responsible for disturbance of the mucus layer for two reasons. Firstly, as the bacterium resides in the mucus layer, what would be the benefit of destroying its own milieu? Secondly, there are data by many authors to indicate that the tissue dynamics of the gastric mucosa is altered by H pyloriinfection. Accompanying the inflammatory response, epithelial turnover is enhanced, and there are profound shifts in cell populations within the epithelium. As a result, which was also mentioned in our previousGut article, MUC6 may be over expressed inH pylori infected stomach at the expense of MUC5AC expression. From our own work, and that of Byrd and colleagues,1-2 it appears that the number of cells expressing MUC5AC declines and that concomitantly the relative number of MUC6 producing cells rises. Meanwhile, we have further data to corroborate this notion, and it appears that there is a statistically highly significant decrease in MUC5AC producing cells (Van de Bovenkamp JHB, Korteland-Van Male A, Büller HA, et al, unpublished data). Thus shifts in expression levels of individual mucins that constitute the mucus layer may easily explain the disturbance of the gastric mucus layer. Obviously, this type of discussion calls for a more dynamic approach to this problem. Only a detailed study of mucin biosynthesis (at the mRNA and protein levels and rate of secretion) may reveal the truth behind my speculations. At this point we will have to make do with an educated guess.
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