Coupling of a Major Allergen to the Surface of Immune Cells for Use in Prophylactic Cell Therapy for the Prevention of IgE-Mediated Allergy.

Up to a third of the world's population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols.

Antiproliferative properties of Padma Lax and its components ginger and elecampane.

BACKGROUND: Padma Lax (PL) is a multi-component herbal laxative, derived from traditional Tibetan medicine. It has been used in the treatment of constipation dominant irritable bowel syndrome. Beyond its purgative and bowel-regulating properties we found it to exhibit antiproliferative properties. MATERIALS AND METHODS: C6 tumor cells were incubated with either an ethanolic or aqueous extract of PL. Cell proliferation, cell cycle, percentage of apoptotic cells, caspase-3/-7 activity as well as mitochondrial membrane potential were determined. RESULTS: Ethanolic extracts of PL inhibited cell proliferation in a dose- and time-dependent manner (half max concentration: 384.4 mug/ml after 48 h of incubation). Aqueous extracts were less effective. Ginger and elecampane were the active components of PL in respect to its antiproliferative action and were found to act synergistically. Supplementing the culture medium with polyamines could not override the cytostatic action of PL. Incubation of C6 cells with PL in the presence of catalase proved that the PL effect was specific and not due to oxidative stress. PL had no effects on the cell cycle at a low dose but arrested cells in G1 at high concentrations. Reduction of cell numbers was found to be due to apoptosis. The caspase- 3/-7 pathway was not involved in the PL-induced cell death. However, mitochondrial membrane potential was lost during the course of incubation with PL indicating a mitochondrial- but not caspase-mediated induction of apoptosis. CONCLUSION: PL exhibits antiproliferative properties which may be beneficial to prevent constipation-related cancer. This study may also contribute to a future development of a new herbal-based antiproliferative treatment.