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Muc5b is the major polymeric mucin in mucus from thoroughbred horses with and without airway mucus accumulation.

Rousseau K, Cardwell JM, Humphrey E, Newton R, Knight D, Clegg P, Thornton DJ - PLoS ONE (2011)

Bottom Line: Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells.The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus.In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Mucus accumulation is a feature of inflammatory airway disease in the horse and has been associated with reduced performance in racehorses. In this study, we have analysed the two major airways gel-forming mucins Muc5b and Muc5ac in respect of their site of synthesis, their biochemical properties, and their amounts in mucus from healthy horses and from horses with signs of airway mucus accumulation. Polyclonal antisera directed against equine Muc5b and Muc5ac were raised and characterised. Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells. Western blotting after agarose gel electrophoresis of airway mucus from healthy horses, and horses with mucus accumulation, was used to determine the amounts of these two mucins in tracheal wash samples. The results showed that in healthy horses Muc5b was the predominant mucin with small amounts of Muc5ac. The amounts of Muc5b and Muc5ac were both dramatically increased in samples collected from horses with high mucus scores as determined visually at the time of endoscopy and that this increase also correlated with increase number of bacteria present in the sample. The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus. In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.

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Related in: MedlinePlus

Histological and immunohistochemical staining of normal equine tracheal tissue.5 µm sections of tracheal tissue were stained with (panels A and D) PAS, (panels B and E) MANeq5ac-I and (panels C and F) MANeq5b-I. In panels B, C, E and F the sections were reduced and carboxymethylated prior to staining with protein-A purified antibodies. Panels A, B and C show the staining of the surface epithelium while panels D, E and F show staining of the submucosal glands. Panels G and H show the quantitation of staining for PAS and the two antibodies. The analysis of the staining was performed on three different horses. For the surface epithelium the goblet cell staining was determined for 9×750 µm lengths of epithelium. For submucosal gland staining a minimum of 6 glands per horse were analysed. The red line represents the average and the green line the median.
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pone-0019678-g005: Histological and immunohistochemical staining of normal equine tracheal tissue.5 µm sections of tracheal tissue were stained with (panels A and D) PAS, (panels B and E) MANeq5ac-I and (panels C and F) MANeq5b-I. In panels B, C, E and F the sections were reduced and carboxymethylated prior to staining with protein-A purified antibodies. Panels A, B and C show the staining of the surface epithelium while panels D, E and F show staining of the submucosal glands. Panels G and H show the quantitation of staining for PAS and the two antibodies. The analysis of the staining was performed on three different horses. For the surface epithelium the goblet cell staining was determined for 9×750 µm lengths of epithelium. For submucosal gland staining a minimum of 6 glands per horse were analysed. The red line represents the average and the green line the median.

Mentions: Immunohistochemical analysis of gastric and salivary mucins, as well as the biochemical analysis of the mucins, has demonstrated the specificity of the two polyclonal antisera. Therefore, we used the two mucin-probes to analyse the sites of synthesis of mucin production. Staining of normal tracheal tissue with PAS-AB (Figure 5A) showed individual epithelial goblet cells stained both pink (neutral mucins) and/or blue (acidic mucins). Within the glands the PAS-AB staining is predominantly blue, indicating that the mucins are mainly acidic (Figure 5D). Serial sections stained with protein-A purified MANeq5ac-I (Figure 5B and 5E) and MANeq5b-I (Figure 5C and 5F) showed that both mucins are expressed in epithelial goblet cells (Figure 5B and 5C) and submucosal gland cells (Figure 5E and 5F). Analysis of the staining showed that the surface epithelium contained more goblet cells positive for Muc5b than Muc5ac (approx. 1 cell staining for Muc5b and approx. 0.5 cell staining for Muc5ac per 30 µm of surface epithelium, p = 0.0003, Figure 5G). Furthermore, a higher percentage area of the glands stained with Muc5ac than Muc5b (21% and 11.5% respectively, p = 0.0009, Figure 5H). It is not clear whether both mucins are present in the same cells. For both mucins, the antibody staining was abolished by inhibition with the peptide used for immunisation.


Muc5b is the major polymeric mucin in mucus from thoroughbred horses with and without airway mucus accumulation.

Rousseau K, Cardwell JM, Humphrey E, Newton R, Knight D, Clegg P, Thornton DJ - PLoS ONE (2011)

Histological and immunohistochemical staining of normal equine tracheal tissue.5 µm sections of tracheal tissue were stained with (panels A and D) PAS, (panels B and E) MANeq5ac-I and (panels C and F) MANeq5b-I. In panels B, C, E and F the sections were reduced and carboxymethylated prior to staining with protein-A purified antibodies. Panels A, B and C show the staining of the surface epithelium while panels D, E and F show staining of the submucosal glands. Panels G and H show the quantitation of staining for PAS and the two antibodies. The analysis of the staining was performed on three different horses. For the surface epithelium the goblet cell staining was determined for 9×750 µm lengths of epithelium. For submucosal gland staining a minimum of 6 glands per horse were analysed. The red line represents the average and the green line the median.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3094342&req=5

pone-0019678-g005: Histological and immunohistochemical staining of normal equine tracheal tissue.5 µm sections of tracheal tissue were stained with (panels A and D) PAS, (panels B and E) MANeq5ac-I and (panels C and F) MANeq5b-I. In panels B, C, E and F the sections were reduced and carboxymethylated prior to staining with protein-A purified antibodies. Panels A, B and C show the staining of the surface epithelium while panels D, E and F show staining of the submucosal glands. Panels G and H show the quantitation of staining for PAS and the two antibodies. The analysis of the staining was performed on three different horses. For the surface epithelium the goblet cell staining was determined for 9×750 µm lengths of epithelium. For submucosal gland staining a minimum of 6 glands per horse were analysed. The red line represents the average and the green line the median.
Mentions: Immunohistochemical analysis of gastric and salivary mucins, as well as the biochemical analysis of the mucins, has demonstrated the specificity of the two polyclonal antisera. Therefore, we used the two mucin-probes to analyse the sites of synthesis of mucin production. Staining of normal tracheal tissue with PAS-AB (Figure 5A) showed individual epithelial goblet cells stained both pink (neutral mucins) and/or blue (acidic mucins). Within the glands the PAS-AB staining is predominantly blue, indicating that the mucins are mainly acidic (Figure 5D). Serial sections stained with protein-A purified MANeq5ac-I (Figure 5B and 5E) and MANeq5b-I (Figure 5C and 5F) showed that both mucins are expressed in epithelial goblet cells (Figure 5B and 5C) and submucosal gland cells (Figure 5E and 5F). Analysis of the staining showed that the surface epithelium contained more goblet cells positive for Muc5b than Muc5ac (approx. 1 cell staining for Muc5b and approx. 0.5 cell staining for Muc5ac per 30 µm of surface epithelium, p = 0.0003, Figure 5G). Furthermore, a higher percentage area of the glands stained with Muc5ac than Muc5b (21% and 11.5% respectively, p = 0.0009, Figure 5H). It is not clear whether both mucins are present in the same cells. For both mucins, the antibody staining was abolished by inhibition with the peptide used for immunisation.

Bottom Line: Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells.The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus.In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Mucus accumulation is a feature of inflammatory airway disease in the horse and has been associated with reduced performance in racehorses. In this study, we have analysed the two major airways gel-forming mucins Muc5b and Muc5ac in respect of their site of synthesis, their biochemical properties, and their amounts in mucus from healthy horses and from horses with signs of airway mucus accumulation. Polyclonal antisera directed against equine Muc5b and Muc5ac were raised and characterised. Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells. Western blotting after agarose gel electrophoresis of airway mucus from healthy horses, and horses with mucus accumulation, was used to determine the amounts of these two mucins in tracheal wash samples. The results showed that in healthy horses Muc5b was the predominant mucin with small amounts of Muc5ac. The amounts of Muc5b and Muc5ac were both dramatically increased in samples collected from horses with high mucus scores as determined visually at the time of endoscopy and that this increase also correlated with increase number of bacteria present in the sample. The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus. In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.

Show MeSH
Related in: MedlinePlus