Conclusions: Militarily Important Issues Identified in this Report
William R. Beisel1
The workshop held on May 20–21, 1996, and this report provide a broad review of current concepts about nutritional immunology. The task of this chapter is to summarize militarily important issues about a complex subject. LTC Karl E. Friedl's chapter introduces Army concerns that multistressor components of rigorous military training induced weight loss, impairments in immunological indices, and at times, an increased incidence of infectious illnesses (see Chapter 4 in this volume). Because higher energy intakes appeared to preserve immune functions, even in the presence of multiple other military stresses, the Committee on Military Nutrition Research (CMNR) was asked to consider five complex questions:
- What methods for assessment of immune function are most appropriate in military nutrition laboratory research, and what methods are most appropriate for field research?
- What are the significant military hazards or operational settings most likely to compromise immune function in soldiers?
- The proinflammatory cytokines have been proposed to decrease lean body mass, mediate thermoregulatory mechanisms, and increase resistance to infectious disease by reducing metabolic activity in a way that is similar to the reduction seen in malnutrition and other catabolic conditions. Interventions to sustain immune function can alter the actions, nutritional costs, and potential changes in the levels of proinflammatory cytokines. What are the benefits and risks to soliders of such interventions?
- What are the important safety and regulatory considerations in the testing and use of nutrients or dietary supplements to sustain immune function under field conditions?
- Are there areas of investigation for the military nutrition research program that are likely to be fruitful in the sustainment of immune function in stressful conditions? Specifically, is there likely to be enough value added to justify adding to operational rations or including an additional component?
Ranjit Kumar Chandra, for decades a world leader and prolific writer in the field of nutritional immunology, discusses the role of malnutrition in diminishing resistance against infectious diseases and in creating dysfunctions in cell-mediated immunity, in humoral and secretory antibody production, in phagocyte function, in cytokine production, and in complement system effectiveness (see Chapter 7 in this volume).
These effects can be termed generalized or single-nutrient malnutrition, nutritionally acquired immune dysfunction syndromes, or NAIDS. NAIDS, in combination with various forms of infectious diseases, are the major cause of human mortality, leading to the deaths of over 20,000 children (largely in underdeveloped nations) each day (along with deaths of countless elderly individuals) and in modern medical centers, deaths of patients with severe medical illnesses and surgical interventions.
Chandra emphasizes the practical applications of nutritional supplementation in both preventive and therapeutic situations. The committee's task will be to translate Chandra's immunological data into meaningful recommendations concerning optimal nutritional support for healthy, well-conditioned, young adults who face the multiple, complex stresses associated with military operations in training situations as well as in operational missions that may include combat.
importance. Rossio begins with an overview of the cytokine system; note that almost every speaker at this workshop mentioned cytokines and their importance in nutritional immunology. Rossio brings the general topic of cytokines up-to-date in a chapter that extends and amplifies Lyle L. Moldawer's discussion of cytokine assay methods in an earlier CMNR report (Moldawer, 1997). Rossio defines five types of cytokine activities, and he reviews the complex interactions among cytokines. In discussing their highly intracoordinated controls, Rossio emphasizes the propensity for cytokines to overlap in their multiple activities and to regulate each other. The systems of checks and balances that maintain hormonal homeostasis are minuscule in comparison with the checks and balances that regulate the synthesis and cellular effects of various cytokines.
Cytokine actions on cells can be influenced by the production of cytokine receptors, the release of such receptors into plasma, and the inhibitory proteins that block the cell wall receptors. At the same time, cellular production of individual cytokines can be reduced by other inhibitory cytokines, by hormones, or by other biologically active molecules. Rossio suggests that an unusual profile of serum cytokines (and related molecules) might signify a departure from homeostasis. This concept bears further thought and research.
Rossio warns that scientists have viewed cytokines with individualized perspectives and that his perceptions are as an immunologist. However, from the perspectives of this report, the nutritional role of proinflammatory cytokines that this author's U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) team discovered a full quarter century ago must be emphasized (Pekarek and Beisel, 1971; Wannemacher et al., 1972). The newly discovered agents were called "leukocytic endogenous mediators," or LEMs, and it was a struggle for many years to gain the LEMs the recognition they deserved. LEMs were never accepted as hormones, although as Reichlin points out, they met all the definitions of hormones. Eventually, the words interleukin and cytokine were introduced. With new and acceptable immunological names, and the advances in biotechnology that allowed the production of sizable amounts of individual cytokines, the field experienced logarithmic growth. In any event, the immunological benefits and functions of the cytokines often have large nutritional costs for the host. Cytokine-induced "acute phase reactions" are accompanied by cytokine-induced malnutrition.
Rossio points out that cytokine responses might be used as surrogate markers of nutritional adequacy. In this regard, almost every essential nutrient is involved in the production of cytokines and/or in the diverse cellular activities they stimulate.
Reichlin discusses the neuroendocrine consequences of systemic inflammation (see Chapter 18 in this volume). Echoing the presentations of both Leonard P. Kapcala (see Chapter 19 in this volume) and Rossio, Reichlin also emphasizes the primary role of the flood of polypeptide cytokines that are released into the circulation by lymphocytes, monocytes, macrophages, and
endothelial cells. These pluripotent mediators induce the relatively stereotyped pathophysiological responses termed the acute phase reaction , which is characterized by fever; nutrient catabolism; changes in protein, carbohydrate, and lipid metabolism; and profound changes in hepatic functions and in all components of the endocrine system. Reichlin also points out that associated sickness behaviors during infection can proceed to cognitive deficits, delirium, stupor, and coma if the infection becomes severe.
Proinflammatory cytokines activate the hypothalamic-pituitary-adrenal axis and stimulate a release of growth hormone and prolactin. At the same time, they inhibit pituitary stimulation of both thyroidal and hypothalamic-gonadal functions. As Reichlin points out, certain of these cytokines, such as interleukin (IL)-1 and tumor necrosis factor (TNF), can transduce the endothelia of the blood-brain barrier to exert central nervous system (CNS) effects. Cytokines may also be produced within the CNS.
Militarily Useful Methods to Evaluate Immunological Functions
Susanna Cunningham-Rundles discusses measures needed to evaluate human immune functions (see Chapter 9 in this volume). She emphasizes tests of cell-mediated immunity, humoral immunity, cytokine expression, and phagocyte function. She also emphasizes the need to determine the body content of key nutrients, focusing on those that significantly affect immune functions (protein, energy, most vitamins especially A, C, B6, B12, and folic acid; and trace elements including Fe, Zn, Cu, and Se).
Tim R. Kramer follows up his previous comments before this committee (IOM, 1993; 1997) by discussing methods using whole blood samples collected during field studies to assess cell-mediated immunity and to study the production of cytokines and the cellular release of cytokine receptors (see Chapter 10 in this volume). Such large-scale testing is a military necessity.
As demonstrated by these two presentations and the discussions that followed at the May 1996 workshop, methodological concerns and data interpretation are highly important issues in all studies of nutritional immunology.
Health Status, Stress, and Immune Function
David C. Nieman describes the effects of both acute and long-term exercise on natural killer (NK) cells, neutrophils, and macrophage/monocytes, and the somewhat lesser effects on T- and B-lymphocytes (see Chapter 17 in this volume). Nieman cites evidence that neutrophil function is suppressed by strenuous physical training, and this may increase the risk for upper respiratory tract infections (URIs). In contrast, a limited number of studies suggest that URI
incidence may be reduced in those who engage in moderate level exercise. A recent paper by Peters et al. (1993) showed that vitamin C supplements could reduce the incidence of post-race URIs in ultramarathon runners.
Kapcala (see Chapter 19 in this volume) amplifies the views of Rossio and Reichlin (see Chapters 8 and 18 in this volume, respectively) by emphasizing the important bidirectional communication and interactions between the immune and neuroendocrine systems. Mediators for these interactions include cytokines, hormones, and neurotransmitters, with responding cells exhibiting the appropriate surface receptors. Stimulation of the hypothalamic-pituitary-adrenal axis by proinflammatory cytokines is of particular importance during inflammatory stresses. Kapcala points out the importance of the array of checks and balances provided by this bidirectionally interactive system, which he demonstrates clearly by studies in laboratory rats.
Kapcala's rat model demonstrates the potential benefits of cytokine release as well as the disastrous consequences of cytokine excesses. Benefits and detriments of cytokine activities are certainly seen during infections in human patients, but the role of glucocorticoids is often exaggerated in rodent models as compared with human disease. A full understanding of cytokine benefits and possible detriments is needed for an optimal management of battlefield casualties and infections seen in military personnel.
Although the CMNR has heard presentations on biological rhythms by speakers at earlier meetings, none were as clearly presented or as specifically focused toward nutritional concerns as the chapter by Erhard Haus (see Chapter 20 in this volume). The data shown by Haus clearly indicate the daily rhythmic changes in circulating lymphocyte numbers and in their subsets, as well as in their propensity to produce or respond to various cytokines. These cytokines also undergo circadian changes in concentration. The same seems to be true for certain soluble cytokine receptors and cytokine antagonists.
Lymphocyte activities and functions, including responses to antigens and antibody production, also exhibit circadian rhythms, a fact of potential military importance. Can responses to military vaccines be improved by administering them at just the right time of day? The same question can be asked about the deleterious effects of infections, or toxemias, if contracted at various times of day.
Abrupt changes in daily living and eating patterns, and in degrees of emotional and physical stress, are characteristic of military operations, as are intercontinental flights. It has long been recognized that military operations may disrupt circadian rhythms. However, relatively little is known about possible immunological rhythms having weekly, monthly, or yearly periodicity. In theory, such long-term rhythms would be less likely to have military relevance. Lymphocyte rhythms and their relationships to other body rhythms may be dramatically altered by the presence of chronic diseases, such as AIDS, or by changes in endocrine rhythms known to occur in acute febrile infections. One
might ask if immunological rhythms are also altered by military or by various forms of military stress.
Stephen Morse's discusses emerging infections, nutritional status, and immunity in Appendix A of this volume. As Morse notes, this was an appropriate topic to be presented in the USAMRIID building, the country's finest laboratory for studying newly emerged, highly dangerous infectious microorganisms. Morse points out that environmental and social changes, as well as antibiotic resistance will accelerate the ''microbial traffic'' that gives animal pathogens the potential chance to infect new host populations, including human beings.
Morse also emphasizes that malnutrition could have added or synergistic effects on these other factors. He points out the propensities for immunosuppression caused by malnutrition to allow pathogens to spread more rapidly through immunocompromised populations, especially those with high population densities. In making his point, Morse cites the collaborative research of Melinda A. Beck and Orville A. Levander, research which showed that an avirulent strain of Coxsackievirus underwent rapid genomic evolution to become virulent when the infection was initiated in selenium-deficient mice.
Pål Wiik (see Chapter 6 in this volume) describes immunological studies in Norwegian Rangers during 7-d training courses that included 90 percent starvation, sleep deprivation, and a variety of other severe military stresses. Although infections were not observed, these Rangers exhibited a two- to threefold increase in blood granulocytes and monocytes and a 30 to 40 percent reduction in B- and T-lymphocytes and in eosinophil numbers. Concentrations of serum immunoglobulins also decreased, and variable responses were detected in cytokine measurements.
Wiik's data confirm and extend observations made in U.S. Rangers, as reviewed in several earlier reports of the CMNR (IOM, 1992, 1993). It must be assumed that the immunological dysfunctions, which develop quickly during Ranger-type military training, will also develop during military combat scenarios. With their longer training exercises, U.S. Army Rangers did experience outbreaks of infectious diseases. It is a historical fact that during major, long-term wars, more casualties are caused by naturally occurring infections than by hostile enemy actions.
The CMNR report must ask and try to differentiate, if possible, the relevance of dietary diminutions during combat and severe military training operations, in contrast to the relevance of numerous other types of stress that might lead to immunological dysfunctions in the military forces.
Is it possible that extraordinary efforts to maintain an adequate dietary intake of energy and essential nutrients during combat situations might prevent these stress-associated immunological dysfunctions? In short, can dietary measures, or supplements, provide as much protection against infectious illness as can the long-term military strategy of using immunizations to provide
protection against selected infectious diseases? Wiik's chapter certainly raises some interesting questions.
New Research Directions
CMNR member Douglas W. Wilmore changes the direction of the chapters by discussing the potential nutrient needs for a single amino acid, glutamine, during periods of military stress (see Chapter 11 in this volume). As Wilmore points out, glutamine is of great importance in diverse biochemical reactions. It is a major substrate for gluconeogenesis, and a major fuel for lymphocytes and macrophages. The functional capacity of the immune system is related to the availability of glutamine in a dose-dependent manner.
Although not an essential amino acid under ordinary circumstances, glutamine's consumption in cells increases beyond its rates of synthesis during periods of malnutrition, injury, or infection. This creates a "relative" deficiency state in which glutamine supplementation is beneficial, as confirmed by clinical studies. Wilmore suggests that glutamine supplements may also be of value for military personnel exposed to radiation or chemical injury or to bacterial pathogens. He postulates that glutamine supplementation might benefit the performance of healthy soldiers by supporting muscle protein synthesis and glycogen stores and by stimulating the production of growth hormone as well as bicarbonate.
LTC Ronald L. Shippee (see Chapter 19 in this volume) follows on this same theme with data from recent studies in military personnel exposed to severe stresses, including dietary deprivation. Micronutrient intervention studies, which included glutamine and selected antioxidant vitamins and minerals, demonstrated the potential military significance of such supplements.
Laura C. Rall and Simin Nikbin Meydani (see Chapter 13 in this volume) amplify the points raised by LTC Shippee about the relationship of antioxidants to the immune system. They call attention to the importance of oxidant-antioxidant balance in maintaining integrity and functionality of membrane lipids, cellular proteins, and nucleic acids. A balanced need for antioxidants also contributes to the control of signal transduction and gene expression in immune cells. Meydani's data show the immunological value of antioxidant supplements in aged individuals and in laboratory animals exposed to the oxidative stress of environmental pollutants. These comments about the broad protective roles of antioxidant vitamins also relates to the immunological value of glutamine supplements and the importance of glutamine in the synthesis of glutathione, one of the major intracellular antioxidants, as pointed out by Wilmore.
The data presented by Wilmore, Shippee and Meydani at the May 1996 workshop regarding glutamine and antioxidant supplements will receive careful evaluation by this committee. It will also need to evaluate the possible needs for arginine supplements. Arginine is the sole source of nitric oxide (NO) (Stamler et al., 1992). NO is thought to have bactericidal powers as effective as those of
the better-known free oxygen radicals. The synthesis of NO is stimulated by proinflammatory cytokines, and gives rise to an excretion of nitrate (Wishnok et al., 1996
Single nutrients were also discussed by the last four speakers. The research studies of Richard D. Semba (see Chapter 12 in this volume) have contributed to a renewed interest in vitamin A during the past decade, in terms of its immunological and anti-infection value in malnourished children. Vitamin A undoubtedly provides considerable support for immune system functions. Programs for vitamin A supplementation are now reducing childhood morbidity and mortality throughout the world.
The CMNR must consider these data in terms of military needs. Military diets provide quite adequately for normal vitamin requirements. Healthy service personnel should therefore possess adequate stores of vitamin A in their bodies. It has been presumed that stores of vitamin A were sufficient to see soldiers through relatively short periods of severe military stress, even if those stresses include semistarvation. However, it must now be asked whether these stresses generate an increased degradation of vitamin A and other essential single nutrients. The military must also find out if the urinary excretion of vitamin A increases during military stress situations, as it does during infectious illnesses. The committee must also look into the unsettled question of whether betacarotene possesses immunostimulatory properties that are absent in vitamin A.
After reviewing the roles of zinc and copper in supporting the immune system, Beck describes her own studies of selenium deficiency in mice, work done in collaboration with Levander (see Chapter 16 in this volume). These scientists demonstrated changes in the infecting Coxsackievirus B3 genome in the presence of selenium deficiency in the mouse host. A genetic mutation involving six nucleotide changes caused the avirulent virus to become virulent. These surprising findings are of great biological significance, for, as pointed out earlier by Morse (see Appendix A in this volume), effects of malnutrition in the host could possibly allow newly virulent microorganisms to emerge as a threat to humans.
Several pertinent questions must now be asked. Is there any evidence of selenium deficiency in the military's well-nourished troops? Could selenium deficiency emerge as a problem as a result of military stress, including that of partial starvation? Moreover, would selenium supplements provide any benefits, including those ascribed to its antioxidant properties? Selenium supplements are reportedly widely used in Europe, with little, if any, apparent evidence of toxicity.
Similar questions can be asked about possible copper deficiency. Is it present in U.S. troops? Could it occur during stress or semistarvation?
Of greater concern is the possible zinc deficiency in military personnel. Although zinc is sequestered during cytokine-induced acute phase reactions, there is normally no pool of stored zinc in the body. Furthermore, losses of zinc from the body appear to parallel losses of nitrogen during infections. The
question arises, are losses of body zinc commonplace during military stresses that do not include infection? Nonetheless, there are concerns about zinc supplements, based on evidence that even slightly increased intakes of zinc might have immunosuppressive effects.
Darshan S. Kelley discusses interactions between essential polyunsaturated fatty acids (PUFAs) and the immune system (see Chapter 14 in this volume). He points out that lipid excesses as well as deficiencies can have immunosuppressive effects and that proper dietary balances between n-3 and n-6 PUFA are also important. The composition of cell wall lipids can be altered in animals and in humans, by dietary changes involving n-3 versus n-6 PUFA intakes.
More information is needed about the practical immunological importance of changes in cell wall lipids, as introduced by dietary manipulations. A diet high in n-3 PUFA can cause the cytokine-stimulated production of eicosanoids to shift to less-potent 5-series leukotrines and 3-series prostaglandins. The committee must ask if such dietary manipulations might have military relevance.
As an example, data have been obtained from horses fed supplements of n-3 PUFA containing linseed oil in an effort to induce the formation of eicosanoid mediators that might help resist endotoxin challenge. The concept worked fine when cells from n-3 PUFA-fed horses were tested in vitro against endotoxin (Morris et al., 1989), but in contrast, the donor horses supplemented by linseed oil retained their full, characteristically dangerous in vivo hypersensitivity to endotoxin (Henry et al., 1991). Although the composition of eicosanoid-generating cell wall PUFA can be altered by the manipulation of human diets, it is not yet known if such changes will affect the outcome of infectious illnesses.
Gerald T. Keusch rounds out the discussions on essential nutrients with a presentation on iron metabolism, microbial virulence, and host defense mechanisms (see Chapter 15 in this volume). Keusch points out that the normal state of iron nutrition in humans is one of significant physiological limitations in the availability of free iron. Iron is required by various microorganisms to support growth and replication. The possibility that an excess of iron (as related to the binding capacity of iron-transport proteins) could increase the severity of infectious illnesses has been an unsettled point for decades. Keusch's chapter, in agreement with newly published data (Gordeur et al., 1992; van Hensbroek et al., 1995), shows that the question is still unsettled, especially in regard to malaria. Oral iron therapy can be given safely to malarial patients who are receiving appropriate antimalarial drugs (van Hensbroek et al., 1995). At the same time, use of iron chelation therapy can hasten recovery from severe cerebral malaria. The military has poured much time, effort, and money into malaria research, including a 40-y screening program involving over 250,000 possible drugs, plus major vaccine development efforts (Wyler, 1992); thus, the potential contribution of iron is of considerable interest.
Iron nutrition is clearly a prominent concern, especially in female military personnel, whose physical performance is impaired in the presence of iron deficiency anemia. This committee has recently made formal recommendations concerning military problems of iron nutrition. As was summarized in a recent report from the CMNR (IOM, 1995b), 56 percent of women recruits enter basic training with low iron stores as assessed by serum ferritin, and 84 percent have low stores at the end of basic training. As a result, the CMNR recommended that women with reduced iron stores should not be enlisted until this problem has been corrected (IOM, 1995b). The prevalence of iron deficiency among men in the military is low. Nevertheless, because combat and even training can result in injuries causing blood loss, the role of iron deficiency in reducing immune function is of potential importance for men as well. Also, military personnel often are exposed to less than sanitary conditions and infectious organisms. Thus, the periodic monitoring of iron status is important for men and especially for women.
As the workshop and resulting report elaborate so clearly, a variety of nutritional deficiencies can initiate clinically apparent forms of NAIDS. These clinical forms of NAIDS result from deficiencies of protein, energy, vitamins A and C, zinc, and iron. An isolated deficiency of many other essential nutrients can induce NAIDS in experimental animals, and some nutrients—such as the B-group vitamins, vitamins D and E, copper, selenium, essential fatty acids, and amino acids such as glutamine and arginine—may be of value in providing additional dietary support for the human immune system.
Even short-term dietary restrictions during stressful military training operations can induce laboratory evidence of NAIDS while longer periods of military stress can also predispose soldiers to the development of infectious illnesses. It remains unclear if derangements in immunological indices observed during strenuous military exercises are due solely to dietary deprivation, to other military stresses, or to some combination of both.
Much additional research is needed to generate a full understanding of the genesis of NAIDS and its practical importance in military operations.
An array of difficult questions have been posed to the CMNR. Data presented at this workshop will assist the committee in formulating answers to the questions posed and in developing recommendations for future research and operational practices. Finally, the words of Hippocrates may still hold much truth, "Let your food be your medicine, and your medicine be your food."
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