主题：【讨论】不明白转基因有啥好争论的 -- 没那么美好

Stanley Ewen and Arpad Pusztai (Oct 16, p 1353)1 raise intriguing questions about the potential of Galanthus nivalis agglutinin (GNA) to cause morphological alterations in the intestinal tract and suggest that the lectin's effects may be exacerbated in genetically modified potatoes. However, the ways in which Ewen and Pusztai assess the enteropathic properties of GNA mean that their findings must be interpreted with extreme caution.

The indices of crypt length and jejunal intraepithelial lymphocyte (IEL) count have been used to study intestinal immunopathology for 20 years or more2, 3 but several aspects of Ewen and Pusztai's methodology warrant attention. First, the study relies entirely on image analysis of formalin—fixed, paraffin-embedded sections, which are notoriously subject to shrinkage, distortion, and other fixation artifacts. These problems can be partly overcome by careful choice of well-oriented villuscrypt units combined with exhaustive measurement techniques, but Ewen and Pusztai do not indicate whether they did this. Errors created by measuring crypts in different planes of section on the sample could account for the high variation reported. The fact that the crypt lengths reported (60-90 μm in the jejunum) are much smaller than those normally found in the rat4 reinforces the view that the measurements may not have been accurate. More sensitive methods for processing and measuring intestinal tissues include non-formalin-based fixatives, microdissection, and direct morphometry of crypt and villus lengths.2—4 The enterocyte mitotic rate is probably the most sensitive index of intestinal pathology,2 and this can be measured easily by metaphase arrest or incorporation of bromodeoxyuridine.

Ewen and Pusztai use IEL counts to support their hypothesis that genetically modified potatoes cause jejunal lesions. They state that “IEL are known to increase when non-specific intestinal damage occurs”, but an increase in IEL count is specifically a feature of enteropathies associated with activated T lymphocytes.3 Thus an increased IEL count in animals receiving lectins could be compelling evidence for these materials inducing immunologically mediated damage to the gut. However, Ewen and Pusztai have not shown this conclusively. They do not seem to have counted IELs by a well-established method in which IEL are counted per 100 enterocyte nuclei or as an absolute number per length, volume or area of mucosa. That the technique they used is not ideal is underlined by the numbers of IEL they report, which are in the region of 7-11 per 48 villi. To reconcile these estimates, given that a single column of villus enterocytes in the rat jejunum contains 200-300 enterocytes and the density of IEL in the normal small intestine is 10-20 per 100 epithelial cells, is difficult. The low protein content of some of the diets, referred to by other commentators as a possible source of error,5 could account for some deviation of IEL numbers from normal but not for such a gross change. However, it is feasible that such a diet might have made the rats more susceptible to the intestinal infections known to cause the kind of changes in IEL and crypts3 noted here.

The speculation that the lectin caused jejunal crypt hyperplasia via a direct stimulatory effect on crypt cells cannot be substantiated by the data. Hyperplasia implies increased mitotic activity, which was not measured. Also, the time course for these changes is not described, and no parameters of villus pathology are provided. In the absence of this information, it is impossible to say whether the changes in crypt morphology are primary effects of the lectin or secondary to villus damage.

Interactions between lectins, intestinal epithelial cells, and the local immune apparatus is an important and poorly understood area. Appropriate methods for studying the enteropathic effects of lectins are available and are comparatively simple and inexpensive. Application of these techniques may help elucidate the issues raised by this provocative study.

While much of the debate has focused on the nature of the diets studied by Stanley Ewen and Arpad Pusztai1 and possible differences between them, one central question is the effects of these on cell proliferation in the gut. Ewen and Pusztai talk about “proliferative effects” when they have not measured intestinal cell proliferation but merely crypt depth. Crypt depth might reflect hypoplasia and hyperplasia but this has yet to be shown. Various methods can be used to measure intestinal epithelial cell proliferation, such as the numbers of dividing cells in optimally sectioned crypts, but for definitive conclusions we need measurements related to the rate of crypt or gland cell production;2 the size of the epithelial population also needs to be assessed appropriately. Perhaps the best way of doing this is to use metaphase arrest and the microdissection method,3 in which not only the rate of crypt cell production but also good measurements of crypt and villus size can be captured simultaneously.

Another point is that many such studies can be confounded by concomitant changes in the denominator,4, 5 and the data on intraepithelial lymphocytes, with sectioned villus as the denominator, could be subject to the same criticism.

We hope these comments will help to ensure that if these studies are repeated (as they should be), robust, rapid, and reliable methods for assessment of cell proliferation are used.

The Lancet criticised the Royal Society last May1 for its “breathtaking impertinence” in reviewing Arpad Pusztai's work before it had been published. Richard Horton now repeats that criticism.2 We commented on Pusztai's unpublished work because he himself had commented on it, so extensively that it had become a matter of public interest. Since a one-sided debate was raging on the back of unvalidated experimental data, the Royal Society had a duty to examine such evidence as it could secure from all sources, including Pusztai himself. That is impertinence only if you endorse scientists flouting normal practice and rushing to the press with unvalidated data and invalid conclusions.

In introducing the Ewen and Pusztai research letter3 Horton helpfully describes the ambivalence of the referees and emphasises the value of having the data out in the open. He also states that the data are nongeneralisable. It is therefore surprising that the journal allowed the paper to appear with two general conclusions in the final paragraph.

In the circumstances, Horton's comments on the “failure to understand the… dialogue of accountability” are somewhat ironic.