December 12, 2016

By: List Labs

By: Suzanne Canada, Ph.D.

Diphtheria toxin is an important tool used for selective killing (ablation) of cells for research purposes.  Using this technique, dubbed “toxin receptor–mediated cell knockout” when it was first used [1], researchers can selectively remove a specific type of cell in a live mouse without having to generate transgenic “knockout” animals, which can be more time-consuming.  The animals are engineered to express a diphtheria toxin (DT) receptor on the surface of a specific cell type.  These animals are normal until exposed to DT, which acts as a potent inhibitor of protein synthesis and kills only those cells that express the DT receptor.  This technique is a powerful tool to explore the role of specific cell types in disease, and is being used to study both the recovery of pituitary cells and the role of T-cells in inflammatory colitis.

Applications in Pituitary Cell Regeneration

The pituitary gland plays an important role in the endocrine system, which presides over growth and development, stress response (adrenal glands), and metabolism (thyroid gland).  Willems and colleagues [2] have been studying the regeneration of the pituitary—research that could lead to methods or therapies to heal pituitary deficiencies.  Transgenic mice that express a DT receptor on the membrane of the growth hormone (GH) cells were treated with DT, which selectively killed those cells.  The researchers then monitored the ability of these ablated cells to regenerate.  Using this technique, they found that stem cells in the pituitary participate in the regeneration process.  Younger mice had a greater ability to recover from injury to the pituitary than older mice.  However, if the injury was prolonged (11 days compared with 3 days) the ability for stem cells to react and aid in recovery could be delayed or even blocked. These researchers may find how stem cells could be activated to boost regeneration of a damaged pituitary gland.

Insights into T-Cell Regulation in Inflammatory Diseases

Cell regeneration also plays an important role in the digestive system: researchers are studying how T-cells regulate inflammation in the gut.  Increases in activated T-cells are associated with active flare-ups of ulcerative colitis and Crohn’s disease [Kappler, 2000].  To that end, Boschetti and colleagues [3] used DT to selectively deplete CD4+, CD25+, and Foxp3+ regulatory T-cells [T-regs] in the gut of transgenic mice.  Using that process, the researchers were able to ablate >95% of the T-regs. The proliferation and recovery of the various T-cell subsets in the lymph nodes and colon was monitored using flow cytometry.  By monitoring the recovery of the T-regs, the researchers found that inflammation causes regulatory T-cells to move to the colon lamina propria, and that those cells could suppress proliferation of CD4+ effector cells in vitro.  Although the Foxp3+ T-regs could not completely prevent colitis in the mice, they did reduce the severity of inflammation in the gut.

This technique is a powerful approach to selectively remove certain cells in mice and other model systems where the animals do not naturally have a DT receptor.  The DT from List Labs is recommended for this purpose because its high purity produces the best desired effect.

To read about even more uses of Diphtheria Toxin and other List Labs products, browse our Citations page. Contact us today with any questions or inquiries regarding our CDMO products and services.

 

References

1. Michiko Saito , Takao Iwawaki , Choji Taya , Hiromichi Yonekawa , Munehiro Noda , Yoshiaki Inui , Eisuke Mekada , Yukio Kimata , Akio Tsuru & Kenji Kohno (2001) Diphtheria toxin receptor|[ndash]|mediated conditional and targeted cell ablation in transgenic mice. Nature Biotechnology 19, 746–750. PMID: 11479567
2. Willems, C; Fu, Q; Roose, H; Mertens, F; Cox, B; Chen, J; Vankelecom, H (2016) Regeneration in the Pituitary After Cell-Ablation Injury: Time-Related Aspects and Molecular Analysis.  Endocrinology 157 705-21. PMID: 26653762
3. Boschetti, G; Kanjarawi, R; Bardel, E; Collardeau-Frachon, S; Duclaux-Loras, R; Moro-Sibilot, L; Almeras, T; Flourié, B; Nancey, S; Kaiserlian, D (2016) Gut Inflammation in Mice Triggers Proliferation and Function of Mucosal Foxp3+ Regulatory T Cells but Impairs Their Conversion from CD4+ T Cells. J Crohn’s and Colitis advanced access publication 30 June 2016.  PMID: 27364948 
4. Kappeler A1, Mueller C. (2000)The role of activated cytotoxic T cells in inflammatory bowel disease.  Histol Histopathol. 2000 Jan;15(1):167-72. PMID: 10668207