Until recently, methods for evaluating the human immune system were derived largely from experimental approaches designed to analyse deficits in host defence in specific clinical settings. With the advent of molecular approaches, immune function has been studied more directly and has led to clarification of specific pathways. As a result, the molecular basis of primary and acquired immune deficiency syndromes is better understood. In addition, the development of vaccines and the study of the natural response to infectious exposure have expanded exponentially in the wake of the HIV crisis, leading to the development of increasingly targeted methods of measuring the immune response. While assessment of the humoral immune response at the level of specific antibody is now well standardized and often routine, evaluation of the complex interactions that are needed to produce specific antibody and the idio-typic interactions that govern this remains a specialized research endeavour. The study of the cellular immune response as a whole continues to remain largely a research activity, although this is beginning to change. This discussion will focus on methods that have been applied to the study of nutrients, and will include approaches that have led to new discoveries in other areas.
The most widely applied methods of evaluating T lymphocyte activation have used peripheral-blood mononuclear cells, isolated by density-gradient centrifugation and cultured with plant lectins (mitogens), or bacterial or viral activators, or antigens, which elicit a secondary response that depends upon prior priming or natural exposure in vitro (Paxton et al., 2001). The typical mononuclear-cell culture contains a mixture of T-cells, B-cells and monocytes. After several days in culture, the cells are pulse-labelled with a radioactive precursor (usually thymidine), and incorporation is measured by assessing incorporation into DNA. The amount of incorporated tracer is closely related to the amount of DNA synthesis and ensuing cell division. The use of whole blood diluted and cultured in the presence of activators also provides an index of mononuclear-cell response but is fundamentally different, since the concentration of cells is not standardized, as it is when mononuclear cells are isolated from whole blood. However, the advantage of this kind of ex vivo test is that plasma proteins and soluble factors present in blood are not removed (Sottong et al., 2000). Further, the interrelationships among cell types are preserved.
The development of monoclonal antibodies directed against cell-surface determinants has evolved from the detection of lymphocyte-subset differentiation antigens defining T-cells, B-cells and NK cells to the elucidation of critical receptors, such as cytokine and growth-factor receptors, as well as many molecules involved in the activation, differentiation and dissemination of immune response. These methods are applicable to a wide range of studies (Cunningham-Rundles, 1998). Examples include monoclonal antibodies recognizing intracellular cytokines, adhesion molecules and early surface markers produced in response to antigen. Flow cytometry provides a means of studying lymphocyte-subset activation without resort to the use of radioactive tracers. In the following section, examples from current work will be discussed.
Nutrition research offers a very interesting and potentially novel way to study the human immune system, and provides an important counterpart to the study of the immune response in primary or secondary immune deficiency where infection, autoimmunity or malignancy are manifest at clinical presentation. While it is clear that there is substantial variation in the normal immune response, the basis of this difference, whether genetic or environmental, remains to be determined. Fundamental studies are needed to determine how nutrient status may influence the development and expression of host genes involved in the immune response. Bendich (1995) has proposed that tests of immune function should be considered in determining the recommended daily allowance (RDA) of certain nutrients, since the levels of several micronutrients needed to support optimal immune function are often higher than those levels needed to qualify as clinical nutrient deficiency, which are usually defined in association with secondary clinical presentation. While there is good evidence that reduced immune function as measured in vitro or ex vivo is linked to risk of infection or to the development of tumours in vivo, tests of immune function are not specific for specific nutrients. A valid test of the effect of nutrient deficiency on immune function would probably require that repletion be proved to correct the defect induced by depletion. This has been achieved for zinc by Prasad (2000), who has demonstrated that experimental human zinc depletion by dietary means leads to reduced levels of Th1 cytokines.
Evidence that nutrients have direct effects on human host defence has come mainly from clinical observations and field studies in settings of severe or chronic nutrient deficiency. These investigations are often complicated by host environmental factors or by exposure to toxins, carcinogens, pathogens or endemic infection (Blot et al., 1993; Zhang et al., 1995; Giuliano et al., 1997; Dai and Walker, 1999). While many studies have described interesting and potentially critical associations, few have identified causal relationships. No single investigational design is necessarily capable of revealing the causal links that govern these intricate relationships.
The choice of study population is fundamental and this directly affects the kinds of controls that are needed. Laboratory controls are highly informative for internal technical quality if run in parallel with subject studies. In some cases, this can be achieved by using aliquots of frozen cells from the same donor, but this has the disadvantage of not providing information concerning the normal range. Parallel controls should include fresh samples from subjects matched for age, sex and clinical status. Longitudinal studies may be crucial and, in some cases, may enable the use of each subject as his/her own control.
When the study design is observational and a nutrient or immune abnormality is known or suspected, study of other potentially related immune-function variables becomes critical. For example, both Th1 and Th2 cytokines should be measured when a Th1 deficiency is suspected. In the context of intervention studies, reliable data can be obtained using different designs, such as placebo-controlled, double-blind and crossover. Inferences may also be drawn from some single-arm studies with unambiguous and quantifiable endpoints. In some cases, it has been possible to use experimental depletion and repletion of the same study group. In other cases, lingering effects have blurred distinctions. For greater stringency, it may be necessary to include several repletion arms at graded doses and to follow changes for a length of time, since the immune system often shows a transient rebound effect that is not seen at later time points. It is also essential to measure other nutrient levels that are positively or negatively regulated by the nutrient under study.
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