Identification of histamine receptors in the canine gastrointestinal tract.

The important role of histamine in chronic gastrointestinal diseases has been increasingly recognized over the last two decades in human medicine. Histamine is released following mast cell activation and exerts its action through binding to four different histamine receptors (H1, H2, H3, and H4). Histamine receptors are dispersed throughout the body, and each different receptor mediates a unique response. Documentation of the presence and type of histamine receptors in the differing sections of the canine gastrointestinal tract will provide additional research opportunities to further explore the role of histamine and its receptors in chronic canine enteropathies, as well as potential therapeutic options. Full thickness gastric, duodenal, jejunal, ileal, and colonic biopsies were obtained from 6 clinically normal adult dogs immediately after humane euthanasia. Commercially available histamine receptor antibodies predicted to react with canine tissues were applied to paraffin-embedded tissue sections using standard immunohistochemistry techniques to identify different histamine receptors. Staining intensity was graded from negative to strong, and the specificity of each antibody was evaluated with western blot. The presence and distribution of histamine receptors varied by anatomic site and histologic level within sections of the canine gastrointestinal tract. All 4 histamine receptors were readily identified, although the distribution of H4 receptors was decreased in comparison to the other histamine receptors. The distribution of the various histamine receptors was similar to that seen in the normal human gastrointestinal tract. H1 receptors were located in the stomach, lymphoid tissue of the ileum and colon, and the smooth muscle and ganglia of all sections. H2 receptors were located in all sections of the gastrointestinal tract, with greatest staining intensity in the gastric mucosa. H3 receptors were located in the stomach and colonic mucosa, smooth muscle and ganglia of all sections, and ileal and colonic lymphoid tissue. H4 receptors were located in the ganglia and smooth muscle of the gastrointestinal tract, as well as the gastric and colonic mucosal and ileal lymphoid tissue. Western blot demonstrated both specific and non-specific staining with the H1 and H3 receptor antibody, but good specificity with the H4 receptor antibody. The H2 receptor antibody was not compatible with western blot techniques, despite excellent immunohistochemical specificity and consistency. Further studies to compare the density and distribution of the various histamine receptors in dogs with gastrointestinal disease are warranted.

Synthesis of a covalent gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic and its cytotoxic anti-neoplastic activity against chemotherapeutic-resistant SKBr-3 mammary carcinoma.

UNLABELLED: Gemcitabine is a potent chemotherapeutic that exerts cytotoxic activity against several leukemias and a wide spectrum of carcinomas. A brief plasma half-life in part due to rapid deamination and chemotherapeutic-resistance frequently limit the utility of gemcitabine in clinical oncology. Selective 'targeted' delivery of gemcitabine represents a potential molecular strategy for simultaneously prolonging its plasma half-life and minimizing exposure of innocent tissues and organ systems. MATERIALS AND METHODS: Gemcitabine was combined in molar excess with N-[p-maleimidophenyl]-isocyanate (PMPI) so that the isocyanate moiety of PMPI which exclusively reacts with hydroxyl groups preferentially created a carbamate covalent bond at the terminal C(5)-methylhydroxy group of gemcitabine. Monoclonal immunoglobulin with binding-avidity specifically for HER2/neu was thiolated with 2-iminothiolane at the terminal ε-amine group of lysine amino acid residues. The gemcitabine-(carbamate)-PMPI intermediate with a maleimide moiety that exclusively reacts with reduced sulfhydryl groups was then combined with thiolated anti-HER2/neu monoclonal immunoglobulin. Western-blot analysis was utilized to delineate the molecular weight profile for gemcitabine-(carbamate)-[anti-HER2/neu] while cell binding characteristics were determined by cell-ELISA utilizing SKBr-3 mammary carcinoma which highly over-expresses HER2/neu receptors. Cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] between the gemcitabine-equivalent concentrations of 10(-12) and 10(-6)M was determined utilizing vitality staining analysis of chemotherapeutic-resistant SKBr-3 mammary carcinoma. RESULTS: Gemcitabine-(carbamate)-[anti-HER2/neu] was synthesized at a molar incorporation index of 1:1.1 (110%) and had a molecular weight of 150kDa that was indistinguishable from reference control immunoglobulin fractions. Cell-ELISA detected progressive increases in SKBr-3 mammary carcinoma associated immunoglobulin with corresponding increases in covalent gemcitabine immunochemotherapeutic concentrations. The in vitro cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] was approximately 20% and 32% at 10(-7) and 10(-6)M (gemcitabine-equivalent concentrations) after a 182-h incubation period. DISCUSSION: The investigations describes for the first time a methodology for synthesizing a gemcitabine anti-HER2/neu immunochemotherapeutic by creating a covalent bond structure between the C(5)-methylhydroxy group of gemcitabine and thiolated lysine amino acid residues of monoclonal antibody or other biologically active protein fractions. Gemcitabine-(carbamate)-[anti-HER2/neu] possessed binding-avidity at HER2/neu receptors highly over-expressed by chemotherapeutic-resistant SKBr-3 mammary carcinoma. Alternatively, gemcitabine can be covalently linked at its C(5)-methylhydroxy group to monoclonal immunoglobulin fractions that possess binding-avidity for other receptors and membrane complexes uniquely highly over-expressed by a variety of neoplastic cell types. Compared to chemotherapeutic-resistant SKBr-3 mammary carcinoma, gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic is anticipated to exert higher levels of cytotoxic anti-neoplastic potency against other neoplastic cell types like pancreatic carcinoma, small-cell lung carcinoma, neuroblastoma, glioblastoma, oral squamous cell carcinoma, cervical epithelioid carcinoma, or leukemia/lymphoid neoplastic cell types based on their reportedly greater sensitivity to gemcitabine and gemcitabine covalent conjugates.