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Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses.

Nicholls JM, Bourne AJ, Chen H, Guan Y, Peiris JS - Respir. Res. (2007)

Bottom Line: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract.In addition we found that there were differences in tissue distribution of the alpha2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used.This finding is important if conclusions about the potential binding sites of SAalpha2,3 binding viruses, such as influenza or human parainfluenza are to be made.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pathology Department, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong SAR. nicholls@pathology.hku.hk

ABSTRACT

Background: Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of alpha2,3-linked and alpha2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAalpha2,6Gal and Maackia amurensis agglutinin (MAA) for SAalpha2,3Gal in the respiratory tract of normal adults and children.

Methods: We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers.

Results: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the alpha2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs.

Conclusion: The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAalpha2,3 binding viruses, such as influenza or human parainfluenza are to be made.

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Effect of different retrieval techniques on lectin binding. Single labelling of paediatric respiratory mucosa by FITC- conjugated Sambucus nigra agglutinin (SNA) (A-F) and Maackia amurensis agglutinin (MAA) (G-J) using different methods of retrieval. No antigen retrieval, (A) and (F), Citrate buffer (B) and (G), EDTA (C) and (H), Pronase (D) and (I), and Trypsin (E) and (J). Stain intensity was graded as strong (++) and a weaker pattern as +. In the absence of unmasking techniques there was minimal to weak (-/+) SNA binding and weak (+) MAA binding in the basal epithelium and epithelial cells of the bronchial mucosa of paediatric tissues. All forms of retrieval enhanced the lectin staining of the surface epithelial cells and mucus containing cells for both SNA and MAA. Examination with dual FITC/Rhodamine filter. Magnification × 200.
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Figure 1: Effect of different retrieval techniques on lectin binding. Single labelling of paediatric respiratory mucosa by FITC- conjugated Sambucus nigra agglutinin (SNA) (A-F) and Maackia amurensis agglutinin (MAA) (G-J) using different methods of retrieval. No antigen retrieval, (A) and (F), Citrate buffer (B) and (G), EDTA (C) and (H), Pronase (D) and (I), and Trypsin (E) and (J). Stain intensity was graded as strong (++) and a weaker pattern as +. In the absence of unmasking techniques there was minimal to weak (-/+) SNA binding and weak (+) MAA binding in the basal epithelium and epithelial cells of the bronchial mucosa of paediatric tissues. All forms of retrieval enhanced the lectin staining of the surface epithelial cells and mucus containing cells for both SNA and MAA. Examination with dual FITC/Rhodamine filter. Magnification × 200.

Mentions: In the absence of unmasking techniques and using the lectins SNA and MAA from EY Labs, there was minimal to weak (-/+) SNA binding and weak (+) MAA binding in the basal epithelium and epithelial cells of the bronchial mucosa of paediatric tissues. All forms of retrieval enhanced the lectin binding to the surface epithelial cells and mucus containing cells for both SNA and MAA (Figure 1) and this appeared to be most prominent in the submucous glands. Trypsin and pronase digestion also produced increased binding, but it also tended to result in more surface epithelial denudation of cells from the section. When microwave heating was used the increase in staining was maximal after 15 minutes. As citrate buffer heating appeared to just as beneficial as other forms of retrieval this was chosen as the preferred method of unmasking for future work. Tissue sections of duck intestine was used as a control and this showed MAA binding on the surface of the columnar cells in keeping with its SAα2,3Gal moiety.


Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses.

Nicholls JM, Bourne AJ, Chen H, Guan Y, Peiris JS - Respir. Res. (2007)

Effect of different retrieval techniques on lectin binding. Single labelling of paediatric respiratory mucosa by FITC- conjugated Sambucus nigra agglutinin (SNA) (A-F) and Maackia amurensis agglutinin (MAA) (G-J) using different methods of retrieval. No antigen retrieval, (A) and (F), Citrate buffer (B) and (G), EDTA (C) and (H), Pronase (D) and (I), and Trypsin (E) and (J). Stain intensity was graded as strong (++) and a weaker pattern as +. In the absence of unmasking techniques there was minimal to weak (-/+) SNA binding and weak (+) MAA binding in the basal epithelium and epithelial cells of the bronchial mucosa of paediatric tissues. All forms of retrieval enhanced the lectin staining of the surface epithelial cells and mucus containing cells for both SNA and MAA. Examination with dual FITC/Rhodamine filter. Magnification × 200.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2169242&req=5

Figure 1: Effect of different retrieval techniques on lectin binding. Single labelling of paediatric respiratory mucosa by FITC- conjugated Sambucus nigra agglutinin (SNA) (A-F) and Maackia amurensis agglutinin (MAA) (G-J) using different methods of retrieval. No antigen retrieval, (A) and (F), Citrate buffer (B) and (G), EDTA (C) and (H), Pronase (D) and (I), and Trypsin (E) and (J). Stain intensity was graded as strong (++) and a weaker pattern as +. In the absence of unmasking techniques there was minimal to weak (-/+) SNA binding and weak (+) MAA binding in the basal epithelium and epithelial cells of the bronchial mucosa of paediatric tissues. All forms of retrieval enhanced the lectin staining of the surface epithelial cells and mucus containing cells for both SNA and MAA. Examination with dual FITC/Rhodamine filter. Magnification × 200.
Mentions: In the absence of unmasking techniques and using the lectins SNA and MAA from EY Labs, there was minimal to weak (-/+) SNA binding and weak (+) MAA binding in the basal epithelium and epithelial cells of the bronchial mucosa of paediatric tissues. All forms of retrieval enhanced the lectin binding to the surface epithelial cells and mucus containing cells for both SNA and MAA (Figure 1) and this appeared to be most prominent in the submucous glands. Trypsin and pronase digestion also produced increased binding, but it also tended to result in more surface epithelial denudation of cells from the section. When microwave heating was used the increase in staining was maximal after 15 minutes. As citrate buffer heating appeared to just as beneficial as other forms of retrieval this was chosen as the preferred method of unmasking for future work. Tissue sections of duck intestine was used as a control and this showed MAA binding on the surface of the columnar cells in keeping with its SAα2,3Gal moiety.

Bottom Line: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract.In addition we found that there were differences in tissue distribution of the alpha2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used.This finding is important if conclusions about the potential binding sites of SAalpha2,3 binding viruses, such as influenza or human parainfluenza are to be made.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pathology Department, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong SAR. nicholls@pathology.hku.hk

ABSTRACT

Background: Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of alpha2,3-linked and alpha2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAalpha2,6Gal and Maackia amurensis agglutinin (MAA) for SAalpha2,3Gal in the respiratory tract of normal adults and children.

Methods: We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers.

Results: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the alpha2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs.

Conclusion: The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAalpha2,3 binding viruses, such as influenza or human parainfluenza are to be made.

Show MeSH
Related in: MedlinePlus