Rabbit Anti-Human SNAP25 (C-terminal) Polyclonal antibody

Brush cells have recently been classified as solitary chemosensory cells. However, tracheal brush cells have not been morphologically and immunohistochemically characterized yet. In the present study, the morphological and immunohistochemical characteristics of tracheal brush cells were analyzed using immunohistochemistry and scanning, and transmission electron microscopies. Brush cells in the tracheal epithelium were barrel-like or columnar in shape and were immunoreactive for villin. Scanning and transmission electron microscopies revealed densely arranged thick microvilli on the apical surface of tracheal brush cells and tubular membranous elements and/or vesicular formations in the supranuclear region. A morphometrical analysis of tracheal wholemount preparations showed that the density of brush cells was greater in the cranial third and the mucosa on the annular ligament. Double immunofluorescence revealed that the morphology of villin-immunoreactive brush cells was distinct from other non-ciliated cells in the tracheal epithelium, i.e., MUC5AC-immunoreactive mucous cells, SNAP25-immunoreactive neuroendocrine cells, and GNAT3-immunoreactive solitary chemosensory cells. On the other hand, tracheal brush cells were immunoreactive for the marker proteins for intestinal brush cells, CK18, DCLK1, and Cox1; however, these antibodies also recognized cells other than brush cells. Furthermore, immunoreactivity for PKD2L1, a cation channel subunit, was detected in brush cells. The present results demonstrated that tracheal brush cells are independent cell types. These brush cells may be activated by acid and the secretion of prostaglandins. In conclusion, the present study revealed that tracheal brush cells are independent cell types based on the morphological and immunohistochemical characteristics.

The current treatment of botulism is to administer animal-derived antitoxin, which frequently causes severe adverse reactions in the recipients. In this study, a heavy chain antibody fragment (VH/VHH) phage display library was constructed by amplification of the immunoglobulin genes of a nonimmune camel, Camelus dromedarius, using primers specific to human VH gene segments. A recombinant light chain of type A botulinumtoxin, BoTxA/LC, with zinc endoprotease activity was used in phage bio-panning to select phage clones displaying BoTxA/LC-bound VH/VHH. Soluble VH/VHH were produced and purified from 10 VH/VHH phagemid-transformed E. coli clones. Complementary determining regions (CDRs) and immunoglobulin frameworks (FRs) of the 10 camel VH/VHH-deduced amino acid sequences were determined. FR2 sequences of two clones showed a hallmark of camel VHH, i.e. (F/Y)(ER50)-E-42-R-49(G/F)(52). The remaining eight clones had an FR2 amino acid tetrad of conventional VH, i.e. V(42)G(49)L(50)W(52). VHH of one clone (V(H)H17) neutralized the SNAP25 hydrolytic activity of BoTxA/LC, whereas mouse polyclonal anti-BoTxA/LC did not have such activity. Mimotope sequences of V(H)H17 matched with the 194-206 amino acid residues of BoTxA/LC, which are located near the S'1 subsite of the catalytic cleft of the enzyme. Molecular docking revealed that CDR3 of the V(H)H17 bound to epitope in the toxin enzymatic cleft. Therefore, the BoTxA/LC neutralization by the V(H)H17 should be due to the VHH insertion into the enzymatic cleft of the toxin, which is usually inaccessible to a conventional antibody molecule. This antibody fragment warrants further development as a therapeutic agent for botulism.