Fig. 4.16 shows the binding of soluble H7Fc, H9Fc and of FcATG to cryosections of the respiratory epithelium of chicken, turkey and swine trachea as well as to the bronchi of the porcine lung. To obtain binding on chicken and turkey 400 pmol of soluble protein was used. For the porcine samples 600 pmol were applied. Binding was detected using anti-human-IgG-FITC. Nuclei were stained with DAPI and cell boundaries and the epithelial surface were visualized with Phalloidine-rhodamine, which stains F-actin.
This staining was also useful to assess the quality of the sections as a disrupted ep-ithelium can be recognized by distorted F-actin staining.
On all epithelia analysed, no FcATG binding could be observed, showing the speci-ficity of the solHA binding. The solHas of avian origin, H7Fc and H9Fc, bound well to the respiratory epithelium of the turkey and chicken trachea. Binding was observed over the whole surface with some larger patches probably from residual mucus. H9Fc also bound in both chicken and turkey to submucosal cells of the trachea (fig. 4.16, asterisks). On porcine tracheal and bronchial epithelium both solHAs showed weaker binding. H9Fc here bound better than H7Fc and gave clear signals in the trachea and the bronchus. But the binding was not as distributed over the whole surface as found in the section of the avian trachea: sites with bound proteins are indicated with white arrow heads. H7Fc only showed weak binding to swine tracheal epithelium. In the bronchus no H7Fc binding could be observed.
Figure 4.16:Binding of H7Fc, H9Fc and FcATG to the respiratory epithelium of chicken, turkey and swine trachea as well as the porcine bronchus. Binding on chicken turkey was achieved using 400 pmol of solHAs on the larger swine samples 600 pmol protein were used.
Soluble protein binding was visualized using anti-human-FITC (green), nuclei were stained with DAPI (blue). To visualize cell boundaries and epithelial surface and to assess quality of the sections phalloidine-rhodamine was used to stain F-Actin (red). Arrow heads show smaller areas of HA binding compared to binding on the whole surface. Asterisks indicate binding in submucosal tissue. Pictures were taken by CLSM.
Again the following solHAs were only tested once for binding on cryosections as work with those constructs just started in the last month of the thesis.
Binding was tested on chicken, turkey and swine trachea and on swine lung tissue for H5Fc, H1_2009Fc, H1_1918Fc and H1_WFc (fig. 4.17) with the same amounts as before. H5Fc bound very well on the avian tracheal surface. H5Fc also appears in the submucosal layers of the epithelium (asterisks). It also was found on the porcine trachea on both surface and submucosal cells. In the porcine lung binding was weaker
and less frequent. Again some binding in the submucosa was detected.
The human H1_2009Fc hardly bound at all in the chicken trachea. Some of the few observed fluorescent signals may result from antibody aggregates. Clear conclusions can only be drawn when the experiment has been repeated. The same is true for the binding of H1_1918Fc on chicken trachea.
On turkey both constructs bound very well on the epithelial surface as well as in deeper epithelial layers down to the submucosa. In the porcine trachea binding can be found as a fine line on the epithelial surface. H1_2009Fc binding also was found in deeper layers of the epithelium but not at all in the porcine lung. Here on the other hand H1_1918Fc showed binding to single cells of the epithelium.
The porcine origin H1_WFc hardly bound to chicken trachea, like the human H1 solHAs and only a little better to turkey trachea. But H1_WFc showed binding in both porcine trachea and bronchus. In the trachea this binding was distributed over the surface whereas in the bronchus single cells seemed to be detected by the soluble HA.
For overview of the solHA binding to different tissues see table 4.5. As these results have not yet been confirmed they should be interpreted with caution.
Figure 4.17:Binding of solHAs to the respiratory epithelium of chicken, turkey and swine trachea as well as the porcine bronchus. Binding on chicken turkey was achieved using 400 pmol of solHAs on the larger swine samples 600 pmol protein were used. Soluble protein binding was visualized using anti-human-FITC (green), nuclei were stained with DAPI (blue). To visualize cell boundaries and epithelial surface and to assess quality of the sections phalloidine-rhodamine was used to stain F-Actin (red). Arrow heads show smaller areas of HA binding compared to binding on the whole surface. Asterisks indicate binding in submucosal tissue.
Pictures were taken by CLSM.
Table 4.5: Scoring of solHA binding on tissue sections. - = no binding, + = binding, ++ = good binding, +++ = strong binding
ck trachea tk trachea sw trachea sw lung
H7Fc +++ +++ +
-H9Fc +++ +++ ++ +
H5Fc +++ +++ ++ +
H1_2009Fc (+) +++ ++
-H1_1918Fc (+) +++ ++ ++
H1_WFc (+) (+) ++ ++
Respiratory tissue sections were also analysed with lectins (fig. 4.18). Using MAAII-Biotin + Streptavidin-Cy3 and SNA-FITC, both chicken and turkey trachea showed a clear predominance of α2,3-linked sialic acids. In chicken SNA staining was hardly found but in the turkey trachea α2,6-linked sialic acids was detected on the surface and in the submucosa (hollow arrow heads). In the porcine trachea and lung mainly α2,6-linked sialic acids were found in the epithelium. MAAII staining was observed in the submucosa in both porcine tissues analysed. In the trachea and the lung of pigs α2,3-linked sialic acids were also detected on the epithelium, but not as abundant as α2,6-linked sialic acids. Especially in the porcine lung, it appeared as single cells were expressing this kind of Sias (white arrow heads). Co-staining with tubulin and mucin antibodies was applied to visualize ciliated and mucus producing cells. But so far this has not led to satisfying results because of cross-reactivity of antibodies from different species or background by secondary antibodies.
Figure 4.18: Lectin staining of the respiratory epithelium of chicken, turkey and swine trachea as well as the porcine bronchus. MAAII-Biotin + Streptavidin-Cy3 was used to stain α2,3 linked sialic acids (red) and SNA-FITC to detect α2,6-linked sialic acids (green). White arrow heads show MAAII staining in the porcine samples, hollow arrow heads SNA staining in avian tissues.