Authors' reply

We very much appreciate the interest of Drs Ballinger and Azooz in our work.

In 1997, we discovered that calpain inhibitor I exerted potent anti-inflammatory effects in rats with endotoxin shock.¹ We therefore speculated that calpain inhibitor I may reduce the degree of inflammation and tissue injury associated with other models of inflammation, including colitis. The model of hapten induced colitis used by us is similar, but not identical, to the one introduced by John Wallace and colleagues in 1989 (Gut 2001;48:478–8) in which instillation of a solution containing a “barrier breaker” (0.25 ml of 50% ethanol) and the hapten 2,4,6-trinitrobenzene sulphonic acid (TNBS 5–30 mg) are used to cause colonic ulceration and inflammation in the rat. Clearly, the severity of colitis caused and the mortality of any given model may vary with the experimental design used. We would like to propose that the high mortality associated with our model of hapten induced colitis is a reflection of the fact that our animals were exposed to the hapten for a relatively long period. As stated in the methods section of our article, adult male Wistar rats were anaesthetised with isoflurane and received intrarectal administration of 25 mg of dinitrobenzene sulphonic acid (DNBS) for a period of 15 minutes.² Exposure of the colon of the mouse to 1 mg of TNBS even for very short periods (30 seconds) also results in a substantial degree of mortality³ but mortality may vary between studies even if the same experimental protocol is used by the same investigator.⁴ When investigating the effects of insulin-like growth factor 1 on the linear growth retardation associated with colitis,⁵ Ballinger et al exposed prepuberal rats (26 days old, anaesthetised with Hypnorm) of both sexes to 8 mg of TNBS in 40% ethanol for an unspecified period of time. When investigating the role of neuropeptide Y on the weight loss associated with experimental colitis, investigators from this group used intrarectal administration of 20 mg of TNBS (for an unspecified period of time) to cause colitis in male Wistar rats.⁶ Unfortunately, neither study provides any histological evidence of the severity of the colitis or any mortality data.^{5, 6} We understand from Dr Ballinger that the mortality of 8% cited in her letter is a summary of experiments carried out by her group over the last 4–5 years. These mortality values have not been published.

We apologise if the number of animals studied was less clear than we thought. All of the data outlined in figures 1, 3, 5, 6, and 9 were, indeed, based on results obtained from 10 animals (survivors). The n numbers of the individual groups at the beginning of the experiments were the following: sham operated treated with vehicle, n=10; sham operated animals treated with calpain inhibitor I, n=10; DNBS control, n=18; DNBS rats treated with calpain inhibitor I, n=13.

In the final paragraph of our article (Gut 2001;48:478–88), we stated that our findings suggest that calpain inhibitor I may be useful in conditions associated with local or systemic inflammation, including inflammatory bowel disease. In the last few months, we have reported that calpain inhibitor I also attenuates the multiple organ injury and dysfunction associated with haemorrhagic shock⁷ as well as the tissue injury associated with pleuritis (induced with carrageenan) and arthritis (induced by collagen).⁸ All of these findings support the view¹ that calpain inhibitor I exerts potent anti-inflammatory effects in vivo.