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Dr Beck: We are doing some of those studies now. We thought initially that other chemokines might be up-regulated. We looked at MIP-1-, MCP-1, and RANTES. They are the same in the affected knockout animals as in the nonknockout animals, whether or not they were fed a deficient diet. The obvious answer is that an increase in chemokines did not occur. We are now going back to look at some other cytokines, because something else is being up-regulated in those animals to cause the inflammation; it is not chemokines.
Dr Bistrian: Was there any evidence in the Cuban population that mutated virus went into the more normally nourished people to produce the same visual problems?
Dr Beck: No, but we want to look at the nutritional records more clearly. It was a widespread epidemic with widespread nutritional deficiency. A lot of people who were nutritionally deficient weren't sick, and there were some with nutritional deficiencies from whom we could not isolate the virus. So there is some overlap.
Dr Bistrian: Heavy cigar smoking was also a factor. I'm also interested in fish oil. I think that when you say fish oil causes vitamin E loss, the effect may well be due to the fact that, although most oils containing polyunsaturated fatty acids have amounts of vitamin E or other antioxidants in proportion to their content of polyunsaturated fatty acids, there are 2 fats to which this general rule does not apply. One is olive oil, which has a large amount of polyphenols, which are important antioxidants. The other is fish oils, which have a very low content of vitamin E but a high content of polyunsaturated fatty acids. Some of the adverse effects shown by Dr Meydani's group that were thought to be due to fish oil were prevented if enough vitamin E was included in the diet.
Dr Milner: In the older literature, high intakes of selenium above the required level reduced the virulence of Epstein-Barr viruses and various other types of viruses. Are you assuming that a similar kind of mechanism occurs in both cases?
Dr Beck: It is difficult to know for sure. We are looking at a deficiency. In our case, I think it has something to do with the nature of a selection process owing to the increased oxidative stress. With an RNA virus, there is not one particular genotype. Every time the virus replicates, errors occur in the virus. If you increase the replication rate, you increase the error rate. I think we are selecting out a more virulent virus from the large number of mutations that occur ordinarily with an RNA virus.
Dr Milner: Is it an all-or-nothing phenomenon? If you can achieve protection against some virally induced tumors with high selenium intake, could there be a range of responses between deficient and excessive?
Dr Beck: We don't know.
Dr Milner: If, in the knockout mouse, you get rid of all the glutathione peroxidase, why shouldn't 100% of the animals develop myocarditis rather than only 50%? Does that mean that free radical generation accounts for only half of the effect?
Dr Beck: We were wondering that, too. Something else must be going on.
Dr Hathcock: With regard to the high selenium versus deficient selenium intake and virally induced tumors, I was wondering about glutathione peroxidase activity in relation to anticarcinogenicity studies in animals. Is the implication that the amounts of selenium needed for major anticarcinogenic impact are higher than the amounts needed to maintain high glutathione peroxidase activity?
Also, is it possible that, in human clinical trials, there is a viral connection with higher than nutritionally sufficient selenium levels, that is, nutritionally sufficient as measured by glutathione peroxidase activity?
Dr Beck: When we compared our model of the selenium-deficient animal with the glutathione peroxidase knockout mouse, the immune responses were not the same. Only half of the knockout animals developed disease. Something else seems to be going on with selenium that's not just related to glutathione peroxidase. I don't know what that would be.
Dr Milner: In the studies done by Clark and Combs [Clark LC, Combs GF Jr, Tumbull BW, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA 1996;276:1957–63], fairly high quantities of selenium were used to get the apparent anticarcinogenesis protection. You have already maximized glutathione peroxidase well above their level. If there is any added benefit, it cannot be explained on the basis of glutathione peroxidase.
Dr Hathcock: Are the animal data consistent with that?
Dr Milner: Yes.
Dr Green: Before the West entered China to look at Keshan disease, treatment, according to the physicians I know in China, was intravenous vitamin C. Apparently, it was the standard treatment and an effective one. Is it worthwhile to look at animal models that don't synthesize vitamin C and include vitamin C as a variable to study?
Dr Beck: I am working with Oliver Smithies and Nobuyo Maeda's laboratory at the University of North Carolina. They have just made a knockout mouse model for vitamin C deficiency. We are going to use these knockout mice to study the impact of vitamin C deficiency on virus-induced pathogenicity.
Dr Field: Could your oxidative stress theory for increasing mutations go the other way around? If an organism were infected with a really virulent virus, might that actually decrease the virulence of the mutation?
Dr Beck: We have just worked with the coxsackievirus field. We are now trying to expand and look at things such as influenza, rotavirus, and other RNA viruses to see what the effect might be.
The only thing we did that bears on your question was to look at the virulent strain, coxsackievirus (CV) B3/20. These deficient animals got even sicker. Thus, a normally virulent virus, CVB3/20, became even more virulent in a nutritionally deficient host.
Because of the way we do the experiment, we are taking the virus at the time of peak virulence, so we are actually selecting for the more virulent mutations. There is no reason to believe that the mutations always have to be driven toward virulence. They could just as easily be driven toward an avirulent state.
With regard to the coxsackievirus, the virulent virus may be more stable than the avirulent one, so we are driving it toward the more stable form, which in this case happens to be the virulent virus. That might not be the case with something like influenza or some other viruses.
Dr Crowell: With regard to the Clark and Combs study, the population studied was in the lower range of normal plasma levels reported in the United States. Clark and Combs gave 200 µg, which is enough to achieve a plateau for glutathione peroxidase. Selenium goes many other places—into methionine, cysteine, dimethyl selenide—so there are other possible mechanisms for selenium effects.