Selection of methodology

Good study design is based on the formulation of a clear question that addresses a critical issue. Table 2.1 illustrates how the integration of the study design with a well-chosen methodology can lead to informative results in different areas of research. A balance of human and experimental animal-model studies is presented, since development of this field has depended upon both.

The study of whole foods, fats and certain micronutrients and how these could influence immune function is currently under development. Fundamental observation of human PEM has shown that generalized malnutrition leads to impaired immune response and susceptibility to infection (see Chandra, Chapter 3, this volume). However, direct examination of how dietary intake of any particular nutrient affects the immune response is a complex undertaking. Table 2.1 includes four studies on dietary intake. Labeta et al. (2000) addressed the fundamental question of how human milk might activate the neonatal immune system by molecular mimicry through the isolation and sequencing of a relevant polypeptide. Fawzi et al. (2000) focused on how a whole food, specifically tomatoes, may protect against morbidity and mortality, an idea that has come from studies implicating antioxidants as improving immune function (see Hughes, Chapter 9, this volume). The relationship held true even with correction for total vitamin A level (Fawzi et al., 2000). The strength of this study is derived from the large scale - more than 28,000 infants studied - and the careful surveys conducted, combined with excellent statistical analysis.

When stress is added to the equation, the nutrient requirements for immune response are further altered, and development of hypotheses often requires more than one approach. For example, there are extensive observations showing that total parenteral nutrition suppresses immune response in the surgical patient and related studies showing that glutamine becomes a conditional essential amino acid during metabolic stress (Calder and Yaqoob, 1999; O'Flaherty and Bouchier-Hayes, 1999; see also Calder and Newsholme, Chapter 6, this volume). These observations have led to the discovery that nutrients provide an essential stimulus for the induction, differentiation and maintenance of the mucosal immune system. Lack of enteral dietary intake

Table 2.1. Experimental approach to nutrient-immune function interaction.

Study

Design

Methods

Findings

Reference

Effect of glutamine on mucosal immunity

Randomized study; groups of rats given food, total parenteral nutrition (TPN) or isocaloric/isonitrogenous TPN with 2% glutamine

Respiratory tract and intestinal washings obtained for IgA and Th2 cytokines measured

TPN decreased IgA, IL-4 and IL-10; supplementation with 2% glutamine enhanced levels of IgA and Th2 cytokines

Effect of gene for haemochromatosis (HFE) on iron metabolism and immunity

Dietary fats and immune response

Innate immunity to bacteria after birth

Tomato intake and morbidity/mortality

Risk of mortality in selenium-deficient HIV+ children

Deletion of HFE a1 and a2 putative ligand-binding domains in vivo

Mice fed high-fat (saturated, n-6 or n-3) or low-fat diet

Identification of bacterial-type pattern in human milk protein

Large-scale longitudinal study of infants in Sudan

Perinatally exposed children enrolled over a 3-month period studied for 5 years

HFE-deficient animals were analysed for a comprehensive set of metabolic and immune parameters

Fatty-acid composition, spleen lymphocyte proliferation, Th1 and Th2 cytokine profile of spleen cells measured

Isolation of human milk polypeptide - studied by mass spectrometry and sequencing

Morbidity/mortality from diarrhoeal/respiratory disease and intake of tomatoes during previous 2-3 days

CD4 cell count and nutritional status were studied; regression models were used to test relationship to survival

Plasma iron, transferrin saturation and hepatic iron were increased - traceable to augmented duodenal iron absorption; no obvious effect on immune system

Polyunsaturated but not saturated fatty acids decreased Th1 cytokine production; little effect on Th2 cytokines; effect shown at level of mRNA

Confirmed protein to be a soluble form of the bacterial pattern-recognition receptor CD14 (sCD14)

Intake linked to reduced mortality after adjustment for total vitamin A intake, inversely correlated with diarrhoeal incidence, respiratory infection

Low plasma selenium and CD4+ T-cell number below 200 linked independently with mortality

Bahram et al. (1999)

Wallace et al. (2001)

Labeta et al. (2000)

IL4, interleukin; IgA, immunoglobulin A.

impairs mucosal IgA and secretory-component production, the number of IgA-containing cells and the level of IgG and promotes mucosal growth (Heel et al., 1998; Kudsk et al., 2000). Even foods such as indigestible saccharides can have a stimulating effect upon the immune system (Kudoh et al., 1998). The study of Kudsk et al. (2000), included in Table 2.1, has added significantly to this field, showing specific differences among animals fed on laboratory food, by total parenteral nutrition and by parenteral nutrition supplemented with glut-amine on the pattern of cytokine and IgA production. Loss of nutrient stimulation led to loss of total lymphocyte number in Peyer's patches, in the intraepithelial layer and in the lamina propria, a reduced CD4+ T-cell to CD8+ T-cell ratio and a reduced intestinal level of IgA (Kudsk et al., 2000). Furthermore, lack of enteral nutrition may signal increased neutrophil recruitment through up-regulation of the intercellular adhesion molecule 1 (ICAM-1), causing increased leucocyte binding in the intestine (Fukatsu et al., 1999). These studies indicate how the immune response during stress may be modulated experimentally by specific amino acids in the diet.

The study of lipids provides great challenges for study design, since incorporation into membranes, as well as direct effects on metabolic pathways, must be considered. There is increasing evidence that increase in fat intake may impair immune function, as well as leading to obesity (Nieman et al., 1996). A relationship between fat intake and cancer risk has been indicated (Risch et al., 1994), but the mechanisms remain unclear. Recent data demonstrate that the fatty-acid composition of cellular membranes can cause immune perturbation. Mechanisms of action include modulation of adhesion-molecule expression (Miles et al., 2000) and are apparently related to specific fatty-acid composition. The activation state of the cell is a determining factor in how fatty acids affect the immune response (Wallace et al., 2000). This topic has been addressed by Wallace et al. (2001) in a thorough study in which mice were fed low-fat diets or high-fat diets, containing either saturated or unsaturated fats. Both n-3 and n-6 polyunsaturated fatty acids were used, permitting distinction of their effects on cytokine production. Data showed that n-3 fatty acids were strongly suppressive of Th1 cytokines (see also Calder and Field, Chapter 4, this volume). This classic feeding study included measurement of fatty-acid incorporation, cytokine secretion and cytokine mRNA production.

Other work has shown how emerging information about the human genome may be used to study basic mechanisms. For example, the discovery of the gene HFE has revealed that the molecular basis of hereditary haemochromatosis, which involves increased iron uptake from the gastrointestinal tract, can be attributed to homozygous inheritance of one mutation (Feder et al., 1998; Gross et al., 1998). HFE regulates the metabolism and distribution of iron by affecting the binding of iron to transferrin, is a major histocompatibility complex (MHC) class I protein and is also non-covalently associated with ^-microglobulin (^m). The significance of this physical association is unclear. Excess iron has been observed in association with loss of CD8+ T lymphocytes in the ^m-knockout mouse. CD8+ T-cells are also reduced in a subgroup of haemochromatosis patients who show an increased rate of iron loading (Porto et al., 1997). The low-CD8 phenotype is also observed in a subset of patients with transfusion-related iron overload (Cunningham-Rundles et a¡., 2000). Interestingly, studies in compound mutant mice lacking both HFE and ^m have shown that more iron was deposited in various tissues than was observed in mice with either mutation alone (Levy et a¡., 2000). However, studies in genetic-deletion models (e.g. the work of Bahram et a¡., 1999) indicate that the basis of a putative link between immune function and iron handling remains unresolved.

Good study design is critically important for studies in complex settings, such as HIV infection, where nutrient imbalance is fundamentally linked to infection but hard to study in a clear-cut manner. Weight loss is a common occurrence in general chronic viral illness and, in the case of HIV infection, can evolve into a wasting condition, which may become intractable with failure of antiretroviral therapy. Infection-induced malnutrition, as discussed above, is primarily cytokine-mediated and is associated with the acute-phase response. This is accompanied by multiple effects on metabolism, such as altered fluxes of iron and zinc and loss of nitrogen, potassium, magnesium, phosphate, zinc and vitamins. This process is accompanied by retention of salt and water. Malnutrition may also present during the asymptomatic phase of HIV infection (Niyongabo et a¡., 1997; Peters et a¡., 1998). Many studies have shown that micronutrient status is profoundly affected in HIV infection, but the aetiological significance of these changes has been difficult to demonstrate (Cunningham-Rundles, 2000). Therefore, the work of Campa et a¡. (1999), included in Table 2.1, has provided an important advance. Using careful longitudinal studies and good statistical design, this group was able to establish that selenium deficiency in children with acquired immune deficiency syndrome (AIDS) was independently associated with mortality.

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