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VI and VII
Discussion

RONALD SHIPPEE: This has been a great session, and I think that with the focus on immunosuppression we are seeing in our Special Operations folks (and I have been talking with some people in England and with other Special Operations researchers), we need to have a workshop just on immunology. I think the climate is right.

I have two general comments; Tim Kramer has convinced me that the way for us to go is to use whole blood [to assess immune status], and I have adopted that for my lab.

But Ficoll-Hypaque, Percoll, or some of the other gradients are not without their problems, also, and I think Doug Wilmore would agree that, in the burn situation, you can get a selective collection of cells during the different degrees of burn. There are two papers on exercise in which before and after exercise, you get a selection of different cells, so you have got to be careful with that. It is not a trivial issue. Obviously, whole blood is easier for us to manipulate, too, [in addition to preventing cell selection].



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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability VI and VII Discussion RONALD SHIPPEE: This has been a great session, and I think that with the focus on immunosuppression we are seeing in our Special Operations folks (and I have been talking with some people in England and with other Special Operations researchers), we need to have a workshop just on immunology. I think the climate is right. I have two general comments; Tim Kramer has convinced me that the way for us to go is to use whole blood [to assess immune status], and I have adopted that for my lab. But Ficoll-Hypaque, Percoll, or some of the other gradients are not without their problems, also, and I think Doug Wilmore would agree that, in the burn situation, you can get a selective collection of cells during the different degrees of burn. There are two papers on exercise in which before and after exercise, you get a selection of different cells, so you have got to be careful with that. It is not a trivial issue. Obviously, whole blood is easier for us to manipulate, too, [in addition to preventing cell selection].

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability I think you always have to remember, and I learned this from Dr. Good and Dr. Mason when I did 8 years in the burn unit, that when you take whole blood, you are looking at a window of the whole lymphoid system. You take 10 ccs out and that is what you are looking at right there. With a burned rat model, if you look at a 30-percent scald burn, the peripheral blood will say the rat is immunosuppressed, based on proliferation to concanavalin A. But the draining lymph node and the spleen will say the rat is hyperresponsive. When I look at the data from my subjects, and they increase their repertoire of immune responses, I think we always have to question whether we are trying to fix something that isn't broke here. We have to ask: are they supposed to look like this for a reason? ARTHUR ANDERSON: You raise an important point. One of the things that happens during the induction phase of immune response is that there is a fairly large amount of apoptosis of cells that is residual from previous responses. There is a big release of thymidine and other components of cells during this digestion of apoptopic cells. Free-thymidine could cause a cell to appear to be refractory to mitogen stimulation in vitro and, therefore, it would be unable to take up tritiated thymidine or other labels and would not score as an activated cell. That would not be something that would affect the CD25 assays that were discussed for interleukin-2 (IL-2) receptors. On the other hand, immunization will cause CD25+ cells to selectively compartmentalize out of the blood, and a drop in CD25 cells in the circulation would probably be reflected by a homing of CD25+ cells to an appropriate lymphoid tissue. GUY MILLER: I was wondering if you could comment or maybe I can take the opportunity to comment to the committee on the new class of compounds that has been rapidly identified, the meganins and defensins, and their whole role in immunological defense. Maybe I could just give a little introduction. When folks were looking at the immunosurveillance mechanisms that amphibians use to coat all their vulnerable tissues, it turns out that if you look at a frog, every single epithelial layer, from the oral cavity and the skin down through the entire GI tract, has the potential to secrete small peptides of about 25 amino acids in length. These compounds confer significant resistance to invading microorganisms. It links to some of the thoughts that Harris Lieberman has. These peptides are contiguous with the neuroendocrine system. They are elaborated by the neurological system and released on stimulation. In putting this little story together, you could see where the cytokines and other small peptides are also secreted in response, and one part of the response is a blood medi-

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability ated one. One is a local hormonal response, and the other one, which has not yet been studied well, is the meganin-defension system. I think there is a significant potential to look at these very small peptides, maybe 25 [amino acids] in length, and their role in gut immunity, and whether we need to focus time and energy also on looking at that system. ARTHUR ANDERSON: I appreciate those comments. I have seen those papers on the meganin-defensins system,1 and they are very intriguing. I have another similar observation. It was published in The Journal of Leukocyte Biology in April of this year,2 where we discovered that IL-8 is secreted in sweat collected from individuals who have run between 1 and 8 km with plastic bags taped around them. IL-8, as a terminal cytokine in this inflammatory cytokine system, and monocyte chemotactic protein 1, which is also a terminal cytokine, represent two parts of the family of cytokines called chemokines. They are all about 8 to 10 kD in size, and they all have very similar conformation and structure. Therefore, they would be very difficult to purify unless you use special techniques. But because they were shotgun cloned out of endotoxin-stimulated macrophages, we had libraries to work with. IL-8 is a neutrophil chemotactic factor. It is a keratinocyte chemotactic factor; it is made by keratinocytes. It has effects on fibroblastic cells and on endothelial cell angiogenesis. At lower concentrations, it tracks lymphocytes, so when you are sweating, you are basically allowing diffusion of IL-8 into the basement membrane of your skin. So if the skin is intact, lymphocytes are already beginning to accumulate in the skin as a kind of a sentinel population. If you get a scratch, there is a higher concentration in a focal area, so neutrophils will begin to migrate out even before bacteria go across the epithelium. This is apparently found to be true in most mucosal sites, that IL-8 is made by mucosal epithelial cells on contact between pathogenic organisms and the mucosal epithelium. It secretes in a polarized way a small puff of IL-8, which attracts neutrophils to the site before the bacterial invasion takes place. Like the defensins that you mentioned, IL-8 is part of this first line of defense. I was happy to hear that we normally do not have IL-8 in urine. There was a study published out of Walter Reed3 —the protocol for which I reviewed 1   B.S. Schonwetter, E.D. Stolzenberg, and M.A. Zasloff. 1995. Epithelial antibiotics induced at sites of inflammation. Science 267:1645-1648. 2   A.P. Jones, L.M.C. Webb, A.O. Anderson, E.J. Leonard, and A. Rot. 1995. Normal human sweat contains interleukin-8. J. Leukocyte Biol. 57:434-437.P.M. Murphy. 1995. Blood, sweat, and chemotactic cytokines [commentary]. J. Leukocyte Biol. 57:438-439. 3   K.H. Ramsey, H. Schneider, R.A. Kuschner, A.F. Trofa, A.S. Cross, and C.D. Deal. 1994. Inflammatory cytokine response to experimental human infection with Neisseria gonorrhoeae. Ann. N.Y. Acad. Sci. 730:322-325. C. Svanborg, W. Agace, S. Hedges, R. Lindsedt, and M.L. Svensson. 1994. Bacterial adherence and mucosal cytokine production. Ann. N.Y. Acad. Sci. 730:162-181.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability on the human use committee—where during a gonorrhea experiment, IL-8 increased tremendously in urine, showing that IL-8 in urine is probably a rational way of determining at least urinary tract infection. LYLE MOLDAWER: We have not seen it in hospitalized patients before surgery.