Preparation of an engineered safer immunotoxin against colon carcinoma based on the ribotoxin hirsutellin A

Immunotoxins are chimeric proteins composed of an antibody domain that specifically directs the action of the toxic domain, resulting in the death of the targeted cells. Over recent years, immunotoxins have been widely studied and the number of different constructions has increased exponentially. Protein engineering has allowed the design of optimized versions of immunotoxins with an improved tumor binding affinity, stability or cytotoxic efficacy, although sometimes this has compromised the safety of the patient in terms of undesirable adverse secondary reactions. A triple mutant at three Trp residues (HtA3DW) of the ribotoxin hirsutellin A retains its specific ribonucleolytic activity, although cell internalization capacity is lacking.This toxin variant has been fused to the single chain variable fragment A33 (scFvA33). This immunoconjugate (IMTXA33HtA3DW) was produced in the methylotrophic yeast Pichia pastoris and purified using nickelnitrilotriacetic acid affinity chromatography. Both target and toxic domains were characterized. The immunotoxin showed an exquisite specific binding against GPA33-positive culture cells, which results in the death of the targeted cells because of specific ribonucleolytic activity against ribosomes of the engineered hirsutellin A variant. IMTXA33HtA3DW represents a promising structure in the search for an improved immunotoxin without compromising the safety of patients.

Thermal reference points as an index for monitoring body temperature in marine mammals

BACKGROUND Monitoring body temperature is essential in veterinary care as minor variations may indicate dysfunction. Rectal temperature is widely used as a proxy for body temperature, but measuring it requires special equipment, training or restraining, and it potentially stresses animals. Infrared thermography is an alternative that reduces handling stress, is safer for technicians and works well for untrained animals. This study analysed thermal reference points in five marine mammal species: bottlenose dolphin (Tursiops truncatus); beluga whale (Delphinapterus leucas); Patagonian sea lion (Otaria flavescens); harbour seal (Phoca vitulina); and Pacific walrus (Odobenus rosmarus divergens). RESULTS The thermogram analysis revealed that the internal blowhole mucosa temperature is the most reliable indicator of body temperature in cetaceans. The temperatures taken during voluntary breathing with a camera held perpendicularly were practically identical to the rectal temperature in bottlenose dolphins and were only 1 °C lower than the rectal temperature in beluga whales. In pinnipeds, eye temperature appears the best parameter for temperature control. In these animals, the average times required for temperatures to stabilise after hauling out, and the average steady-state temperature values, differed according to species: Patagonian sea lions, 10 min, 31.13 °C; harbour seals, 10 min, 32.27 °C; Pacific walruses, 5 min, 29.93 °C. CONCLUSIONS The best thermographic and most stable reference points for monitoring body temperature in marine mammals are open blowhole in cetaceans and eyes in pinnipeds.