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Evaluation of the Eects of Nutrients on Immune FunctionSUSANNACUNNINGHAM-RUNDLES

Immunology Research Laboratory, Division of Hematology and Oncology,Department of Pediatrics, New or! Presbyterian Hospital, "ornell #niversity $eill %edical "ollege, &'(( or! )venue, New or!, N &((*&, #+)

Introduction and OverviewNutrients are primary factors in the regulation of the human immuneresponse. Both macronutrients and micronutrients derived from the diet aectimmune-system function through actions at several levels in the gastrointestinaltract, thymus, spleen, regional lymph nodes and immune cells of the circulatingblood (Chandra, 199! Cunningham-"undles and #in, 199$! %allace et al, &'''! Cunningham-"undles, &''1. )ects at one level may be opposedor modi*ed at another level. +hus, the development of an eperimentalapproach capable of revealing critical interactions reuires study of more thanone aspect of immune function (Cunningham-"undles, 199! /uga and

0rider, 1999! Beisel, &'''. +he eect of any single nutrient is dependentupon concentration, interactions ith other 2ey nutrients, host genetic epressionand internal environmental conditions. 3n situations of nutrient imbalance,duration of the altered condition and age of the host are also oftencritical factors (Cunningham-"undles and Cervia, 1994! 5irve and 0anatra,199! /iles et al, &''1.Nutrients aect speci*c immune6cell types dierently through in7uencingintrinsic cell function and by in7uencing cell6cell interactions. /uch of thecritical action appears to occur in the local microenvironment during theresponse to antigen. Classically, the immune system has been considered asan operational duality divided into an innate system, mediating immunereactions that do not functionally change ith re-eposure to signal, and anadaptive immune system, hich is capable of developing the response to

antigen encounter and evolving ith re-eposure. 8daptive immunity hasbeen further characteried according to cell type, as the response of bonemarro-derived B-cells of the humoral immune system and thymus-derived +-cells of the cellular immune system. +his rather static picture of compartmentaliedfunction is changing. No, it is increasingly clear that signi*cant +-© CABInternational 2002.Nutrition and Immune -unction

(eds P.C. Calder, C.J. Field and H.S. Gill) 21

cell dierentiation does occur independently of the thymus 6 for eample, inthe gastrointestinal tract. Current studies also sho that the innate immunesystem, mediated by such cells as natural 2iller (N: and N: +-cells, monocytesand dendritic cells, in7uences the nature of cyto2ine production by theadaptive immune system. +his occurs through secretion of cyto2ines byinnate immune cells into the microenvironment (;oherty et al, 1999! 0arciaet al, 1999! see also ;evereu, Chapter 1, this volume. +he eect of themicroenvironment is to drive the immune response toards either a +-helpertype 1 (+h1 or a +-helper type & (+h& response (see ;evereu, Chapter 1,this volume. /icronutrients, such as trace elements and vitamins, are presentin the local environment and have important regulatory eects on adaptiveimmune-cell function. <or eample, the trace element inc supports a +h1response, hereas vitamin 8 appears to produce a +h& response(<ran2enburg et al, 199$! =han2ar and >rasad, 199$. +hus the neimmunology provides a more 7uid representation of a potentially evolvingprocess that presents as a de*ned pattern according to an environmentaldynamic rather than a static programme that is derived from *ed cellularcharacteristics. +he basic elements are shon in <ig. &.1.



Fig. 2.1. Microenvironment of immune response. APC, antigen-presenting cell; IFN- ? ,

interferon- ? ; IL, interleukin; MHC, major histocompatibility complex.8ge of the host or developmental stage is often a critical variable. 8ntigenspeci*chumoral and cellular immunity are central to the adaptive immuneresponse generated in the adult host. 3n contrast, neonates and infants rely primarilyon innate immunity, speci*cally complement, maternal antibody, circulatingmediators of the in7ammatory response and phagocytes (seeBrandtaeg, Chapter 1@, this volume. 5oever, many of the components of innate immunity are not as functional in young children as in adults (3nsoft et al, 1994! see also Chapter 1@. )ncounters ith potential pathogens, such asparasitic infections or viruses, may easily compromise these resources. =tudyof this permits a glimpse of ho the naive immune system copes ith the suddenin7u of signals, ne antigens and potential pathogens. %hen malnutritionis present, the overall development and epression of the immune

response are signi*cantly impaired (Cunningham-"undles et al, &''', &''&!see Chandra, Chapter , this volume. =imilarly, the ageing process aectsnutrient needs and the immune response in an interactive fashion. +he eectof ageing on the response to immuniation and the enhancing eects of micronutrients are ell 2non (#esourd, 199! >allast et al, 1999! see#esourd et al, Chapter 1, this volume. 3n addition, there are fundamentalage-related changes, hich may re7ect in7ammatory processes (see Chapter1, such as the report that plasma levels of certain adhesion moleculesincrease ith age and appear to in7uence the impact of dietary *sh oil supplementation(/iles et al, &''1.8ssessment of ho nutrients may interact in human immune function is acomple underta2ing, more diAcult than the assay of the response to a speci*cantigen of interest 6 for eample, the serological antibody response to a virus.3n the latter case, it is usually possible to 2no hat level of response correlatesith protection. Because of the great speci*city and sensitivity of this information,



some of the best data regarding nutrient interaction ith the humanimmune system have been based on the use of response to speci*c pathogensas the point of reference. 5oever, etrapolation from speci*c settings may behaardous. 3t is seldom clear that immune de*ciencies in vitro ill predictimmune de*ciency in vivo. +herefore, investigators often see2 to strengtheninferences by inclusion of in vivo tests, such as delayed-type hypersensitivity

measured by s2in testing, and by assessment of the humoral immune responsethrough assay of speci*c antibodies arising in response to primary or secondary(booster immuniation. Consistency of an altered immune response in theabsence of acute clinical presentation continues to serve as the benchmar2indicator of a putative intrinsic immune defect. By analogy, repeated studies inthe absence of the acute clinical process are crucial for the study of immunechanges secondary to chronic malnutrition.0eneral assessment of the anatomy of the immune system in humansincludes measurement of serum immunoglobulins and complement and theevaluation of lymphocyte subsets by immunophenotyping. 8nalytical studiesreuire selection among a ide range of tests that measure immune function invitro or e. vivo as a re7ection of the immune response in vivo (:ramer andBurri, 199! aye et al, 199$! Cunningham-"undles, 1999! Berguist et al,

&'''. 8 basic panel of tests is also reuired to reveal ho the overall balanceNutrients and Immune -unction 23

of the immune system has been aected. 3mmune studies are often based onlimited studies of immune-cell subsets, serum or plasma concentrations of cyto2ines or the functional response of mononuclear cells cultured in highlystandardied systems, using a chosen stimulus and often a single end-point.Neer methods have made it possible to assess dierentiation in antigenepression on peripheral-blood mononuclear cells in response to activation, tostudy early events in the activation pathay and to analyse gene activation. +he development of cyto2ine biology has provided a critical means of clarifyingthe fundamental impact of nutrients on immune response. 3n general,nutrients appear to aect the immune system most profoundly through regulatorymechanisms aecting the epression and production of cyto2ines (e.g.=avendahl and nderood, 199! "in2 and :irchner, &'''. =ince the type of cyto2ine pattern produced is crucial for the response to infectious pathogens,serious nutrient imbalance ill ultimately compromise the development of thefuture immune response. 5oever, hile malnutrition promotes susceptibilityto pathogens, even subclinical infections directly aect nutrient inta2e andmetabolism. =evere, acute infection ill have a very strong impact. +he factthat cyto2ine production during the acute-phase response to generalied sepsiscan lead to loss of lean tissue and body fat is ell 2non (#in et al, 199$.3nterestingly, this cascade of events can be altered by nutritional intervention(eevanandam et al, 1999. 3mmune de*ciency and susceptibility to infectionare often directly lin2ed ith malnutrition, hich as the leading cause of acuired immune de*ciency before the appearance of the human immunode*ciency

virus (53D. /alnutrition is also a maEor factor contributing to the progressionof 53D infection, especially in less developed countries. =incemalnutrition and 53D aect the host in similar ays, the combination is particularlydevastating. /any of the infections observed in human protein6energymalnutrition (>)/, such as tuberculosis, herpes, Pneumocystis carinii pneumoniaand measles, are caused by intracellular pathogens, indicating that thecellular immune system is particularly aected (:eusch, 199! see Chandra,Chapter , this volume.%hile the eects of infection and malnutrition on the immune response areinteractive, the eects of each upon immune response are also independent. 8recent eamination by /ishra et al (199$ of graded >)/ in children in relationshipto tuberculosis infection and response to a s2in-test anergy panel,including puri*ed protein derivative of % tuberculosis (>>;, has shon that

impaired cellular immunity as observable in all grades of malnutrition, eceptfor response to >>; in grade 3, and that infection did not aect this.



;ierentiation of lymphocyte subpopulations is also directly aected bymalnutrition. =tudies sho that +-cells from children ith severe >)/ areimmature, compared ith those from ell-nourished children, and that thedegree of immaturity is directly associated ith thymic involution, as measuredby echo radiography (>arent et al, 199@. %hile nutritional repletionaected anthropometric measures ithin 1 month, regroth of the thymus

too2 longer (Chevalier et al, 1994, 199$. +he long-term conseuences of slo thymic regroth are un2non. +hese studies underscore the importanceof longitudinal studies."esponse to certain pathogens may actually be enhanced in some states of malnutrition. 0enton et al (199$ assessed the incidence of malaria in children in>apua Ne 0uinea, and found that increased height-for-eight at baseline (anindicator of a better nutritional state predicted susceptibility to malaria during theyear of study and that the lymphocyte response to malarial antigens as loeramong the less asted children. <urthermore, cyto2ine production toardsmalarial antigens as greater among malnourished children, suggesting that afavourable cyto2ine regulatory shift might be the basis of improved responseamong stunted, but not asted, children. =tunting has often been considered asan adaptive and partially protective host response to prolonged nutrient deprivation.

"i2imaru et al (199$ evaluated lymphocyte subpopulations andimmunoglobulins among healthy children and children ith 2ashior2or, marasmusand marasmic 2ashior2or in 0hana. 3nterestingly, immunoglobulin 8 (3g8and C@ ere higher, hereas C and relative B-cell percentage ere loer, in theseverely malnourished groups. +hese studies demonstrate the advantages of using lin2ed measurements to develop a full immunological pro*le.3n summary, the study of nutrient immune interaction reuires considerationof the setting and a design that includes evaluation of possible complementaryeects at more than one level. #ongitudinal studies are often usefuland permit assessment of the evolution of the immune response and characteriationof donstream eects, hich may modulate outcome.

Evaluation of Human Immune Response

ntil recently, methods for evaluating the human immune system ere derivedlargely from eperimental approaches designed to analyse de*cits in hostdefence in speci*c clinical settings. %ith the advent of molecular approaches,immune function has been studied more directly and has led to clari*cation of speci*c pathays. 8s a result, the molecular basis of primary and acuiredimmune de*ciency syndromes is better understood. 3n addition, the developmentof vaccines and the study of the natural response to infectious eposurehave epanded eponentially in the a2e of the 53D crisis, leading to thedevelopment of increasingly targeted methods of measuring the immuneresponse. %hile assessment of the humoral immune response at the level of speci*c antibody is no ell standardied and often routine, evaluation of thecomple interactions that are needed to produce speci*c antibody and the idiotypicinteractions that govern this remains a specialied research endeavour.

+he study of the cellular immune response as a hole continues to remainlargely a research activity, although this is beginning to change. +his discussionill focus on methods that have been applied to the study of nutrients, and illinclude approaches that have led to ne discoveries in other areas. +he most idely applied methods of evaluating + lymphocyte activationhave used peripheral-blood mononuclear cells, isolated by density-gradientcentrifugation and cultured ith plant lectins (mitogens, or bacterial or viralactivators, or antigens, hich elicit a secondary response that depends uponprior priming or natural eposure in vitro (>aton et al, &''1. +he typical

Nutrients and Immune -unction 25

mononuclear-cell culture contains a miture of +-cells, B-cells and monocytes.8fter several days in culture, the cells are pulse-labelled ith a radioactive precursor(usually thymidine, and incorporation is measured by assessing incorporation

into ;N8. +he amount of incorporated tracer is closely related to theamount of ;N8 synthesis and ensuing cell division. +he use of hole blood



diluted and cultured in the presence of activators also provides an inde of mononuclear-cell response but is fundamentally dierent, since the concentrationof cells is not standardied, as it is hen mononuclear cells are isolatedfrom hole blood. 5oever, the advantage of this 2ind of e. vivo test is thatplasma proteins and soluble factors present in blood are not removed (=ottonget al, &'''. <urther, the interrelationships among cell types are preserved.

+he development of monoclonal antibodies directed against cell-surfacedeterminants has evolved from the detection of lymphocyte-subset dierentiationantigens de*ning +-cells, B-cells and N: cells to the elucidation of criticalreceptors, such as cyto2ine and groth-factor receptors, as ell as many moleculesinvolved in the activation, dierentiation and dissemination of immuneresponse. +hese methods are applicable to a ide range of studies(Cunningham-"undles, 199$. )amples include monoclonal antibodies recogniingintracellular cyto2ines, adhesion molecules and early surface mar2ersproduced in response to antigen. <lo cytometry provides a means of studyinglymphocyte-subset activation ithout resort to the use of radioactive tracers. 3nthe folloing section, eamples from current or2 ill be discussed.Overall designNutrition research oers a very interesting and potentially novel ay to study

the human immune system, and provides an important counterpart to thestudy of the immune response in primary or secondary immune de*ciencyhere infection, autoimmunity or malignancy are manifest at clinical presentation.%hile it is clear that there is substantial variation in the normal immuneresponse, the basis of this dierence, hether genetic or environmental,remains to be determined. <undamental studies are needed to determine honutrient status may in7uence the development and epression of host genesinvolved in the immune response. Bendich (199F has proposed that tests of immune function should be considered in determining the recommended dailyalloance (";8 of certain nutrients, since the levels of several micronutrientsneeded to support optimal immune function are often higher than those levelsneeded to ualify as clinical nutrient de*ciency, hich are usually de*ned inassociation ith secondary clinical presentation. %hile there is good evidencethat reduced immune function as measured in vitro or e. vivo is lin2ed to ris2of infection or to the development of tumours in vivo, tests of immune functionare not speci*c for speci*c nutrients. 8 valid test of the eect of nutrient de*ciencyon immune function ould probably reuire that repletion be proved tocorrect the defect induced by depletion. +his has been achieved for inc by>rasad (&''', ho has demonstrated that eperimental human inc depletionby dietary means leads to reduced levels of +h1 cyto2ines.)vidence that nutrients have direct eects on human host defence hascome mainly from clinical observations and *eld studies in settings of severe orchronic nutrient de*ciency. +hese investigations are often complicated by hostenvironmental factors or by eposure to toins, carcinogens, pathogens orendemic infection (Blot et al, 199! Ghang et al, 199F! 0iuliano et al, 199!

;ai and %al2er, 1999. %hile many studies have described interesting andpotentially critical associations, fe have identi*ed causal relationships. No singleinvestigational design is necessarily capable of revealing the causal lin2s thatgovern these intricate relationships. +he choice of study population is fundamental and this directly aects the2inds of controls that are needed. #aboratory controls are highly informativefor internal technical uality if run in parallel ith subEect studies. 3n somecases, this can be achieved by using aliuots of froen cells from the samedonor, but this has the disadvantage of not providing information concerningthe normal range. >arallel controls should include fresh samples from subEectsmatched for age, se and clinical status. #ongitudinal studies may be crucialand, in some cases, may enable the use of each subEect as hisHher on control.%hen the study design is observational and a nutrient or immune abnormality

is 2non or suspected, study of other potentially related immune-functionvariables becomes critical. <or eample, both +h1 and +h& cyto2inesshould be measured hen a +h1 de*ciency is suspected. 3n the contet of



intervention studies, reliable data can be obtained using dierent designs, suchas placebo-controlled, double-blind and crossover. 3nferences may also bedran from some single-arm studies ith unambiguous and uanti*able endpoints.3n some cases, it has been possible to use eperimental depletion andrepletion of the same study group. 3n other cases, lingering eects have blurreddistinctions. <or greater stringency, it may be necessary to include several repletion

arms at graded doses and to follo changes for a length of time, since theimmune system often shos a transient rebound eect that is not seen at latertime points. 3t is also essential to measure other nutrient levels that are positivelyor negatively regulated by the nutrient under study.Eperimental approachImmune activators3mmune activation reuires a signal hen circulating blood is used as the cellsource, since the peripheral-blood lymphocyte is a resting cell. +his signal isoften a plant lectin, or another signal, such as certain divalent cations, calciumionophores or surface-reactive molecules, including monoclonal antibodies toC;, hich provide a non-antigenic stimulus that activates + lymphocytesindependently of antigenic history. 3mpaired response to mitogens in humansettings of >)/ may or may not be accompanied by loss of response to

pathogens. )amples include the study of response to >>; in malnourishedchildren at ris2 of tuberculosis and the eect of stunting on the response tomalarial antigens (discussed above. 3t is ell 2non that infections ith even

Nutrients and Immune -unction 27

relatively non-pathogenic viruses, such as measles, are often fatal in childrenith >)/, because measles-virus infection causes a serious but usually transientsuppression of the cellular immune response (=chlender et al, 1994! 3to et al, 199, hich, in the malnourished host, may continue to prevent immuneclearance. #ongitudinal studies are often essential to demonstrate long-termeects, such as the lingering eect of vitamin 8 de*ciency, hich increasesmortality from infections (%est et al, 1999. Current studies suggest that thespeci*city of the response, de*ned as a +h1 or +h& cyto2ine-pattern, to aspeci*c microbe is critically associated ith host defence. =tudy designs thatincorporate antigens that are actually being encountered at the time of study orthat focus on the type of cyto2ine production may therefore provide importantand uniue information.+election of methodology 0ood study design is based on the formulation of a clear uestion thataddresses a critical issue. +able &.1 illustrates ho the integration of the studydesign ith a ell-chosen methodology can lead to informative results in dierentareas of research. 8 balance of human and eperimental animal-modelstudies is presented, since development of this *eld has depended upon both. +he study of hole foods, fats and certain micronutrients and ho thesecould in7uence immune function is currently under development.<undamental observation of human >)/ has shon that generalied malnutrition

leads to impaired immune response and susceptibility to infection (seeChandra, Chapter , this volume. 5oever, direct eamination of hodietary inta2e of any particular nutrient aects the immune response is acomple underta2ing. +able &.1 includes four studies on dietary inta2e.#abeta et al (&''' addressed the fundamental uestion of ho human mil2might activate the neonatal immune system by molecular mimicry throughthe isolation and seuencing of a relevant polypeptide. <ai et al (&'''focused on ho a hole food, speci*cally tomatoes, may protect againstmorbidity and mortality, an idea that has come from studies implicatingantioidants as improving immune function (see 5ughes, Chapter 9, this volume. +he relationship held true even ith correction for total vitamin 8 level(<ai et al, &'''. +he strength of this study is derived from the large scale6 more than &$,''' infants studied 6 and the careful surveys conducted,

combined ith ecellent statistical analysis.%hen stress is added to the euation, the nutrient reuirements for



immune response are further altered, and development of hypotheses oftenreuires more than one approach. <or eample, there are etensive observationsshoing that total parenteral nutrition suppresses immune response in thesurgical patient and related studies shoing that glutamine becomes a conditionalessential amino acid during metabolic stress (Calder and Iaoob, 1999!JK<laherty and Bouchier-5ayes, 1999! see also Calder and Nesholme,

Chapter 4, this volume. +hese observations have led to the discovery thatnutrients provide an essential stimulus for the induction, dierentiation andmaintenance of the mucosal immune system. #ac2 of enteral dietary inta2e

!a"le 2.1. Experimental approach to nutrient–immune function interaction.

impairs mucosal 3g8 and secretory-component production, the number of 3g8containingcells and the level of 3g0 and promotes mucosal groth (5eel et al,199$! :uds2 et al, &'''. )ven foods such as indigestible saccharides canhave a stimulating eect upon the immune system (:udoh et al, 199$. +hestudy of :uds2 et al (&''', included in +able &.1, has added signi*cantly tothis *eld, shoing speci*c dierences among animals fed on laboratory food,by total parenteral nutrition and by parenteral nutrition supplemented ith glutamine

on the pattern of cyto2ine and 3g8 production. #oss of nutrient stimulationled to loss of total lymphocyte number in >eyerKs patches, in theintraepithelial layer and in the lamina propria, a reduced C;@L +-cell to C;$L +-cell ratio and a reduced intestinal level of 3g8 (:uds2 et al, &'''.<urthermore, lac2 of enteral nutrition may signal increased neutrophil recruitmentthrough up-regulation of the intercellular adhesion molecule 1 (3C8/-1,causing increased leucocyte binding in the intestine (<u2atsu et al, 1999. +hese studies indicate ho the immune response during stress may be modulatedeperimentally by speci*c amino acids in the diet. +he study of lipids provides great challenges for study design, since incorporationinto membranes, as ell as direct eects on metabolic pathays, mustbe considered. +here is increasing evidence that increase in fat inta2e mayimpair immune function, as ell as leading to obesity (Nieman et al, 1994. 8

relationship beteen fat inta2e and cancer ris2 has been indicated ("isch et al,199@, but the mechanisms remain unclear. "ecent data demonstrate that thefatty-acid composition of cellular membranes can cause immune perturbation.



/echanisms of action include modulation of adhesion-molecule epression(/iles et al, &''' and are apparently related to speci*c fatty-acid composition. +he activation state of the cell is a determining factor in ho fatty acidsaect the immune response (%allace et al, &'''. +his topic has beenaddressed by %allace et al (&''1 in a thorough study in hich mice ere fedlo-fat diets or high-fat diets, containing either saturated or unsaturated fats.

Both n- and n-4 polyunsaturated fatty acids ere used, permitting distinctionof their eects on cyto2ine production. ;ata shoed that n- fatty acids erestrongly suppressive of +h1 cyto2ines (see also Calder and <ield, Chapter @,this volume. +his classic feeding study included measurement of fatty-acidincorporation, cyto2ine secretion and cyto2ine m"N8 production.Jther or2 has shon ho emerging information about the humangenome may be used to study basic mechanisms. <or eample, the discoveryof the gene 5<) has revealed that the molecular basis of hereditaryhaemochromatosis, hich involves increased iron upta2e from the gastrointestinaltract, can be attributed to homoygous inheritance of one mutation(<eder et al, 199$! 0ross et al, 199$. 5<) regulates the metabolism and distributionof iron by aecting the binding of iron to transferrin, is a maEor histocompatibilitycomple (/5C class 3 protein and is also non-covalently

associated ith ? &-microglobulin (? &m. +he signi*cance of this physical associationis unclear. )cess iron has been observed in association ith loss of C;$L + lymphocytes in the ? &m-2noc2out mouse. C;$L +-cells are alsoreduced in a subgroup of haemochromatosis patients ho sho an increasedrate of iron loading (>orto et al, 199. +he lo-C;$ phenotype is alsoobserved in a subset of patients ith transfusion-related iron overload(Cunningham-"undles et al, &'''. 3nterestingly, studies in compound mutantmice lac2ing both 5<) and ? &m have shon that more iron as deposited invarious tissues than as observed in mice ith either mutation alone (#evy et al, &'''. 5oever, studies in genetic-deletion models (e.g. the or2 of Bahram et al, 1999 indicate that the basis of a putative lin2 beteen immunefunction and iron handling remains unresolved.0ood study design is critically important for studies in comple settings,such as 53D infection, here nutrient imbalance is fundamentally lin2ed toinfection but hard to study in a clear-cut manner. %eight loss is a commonoccurrence in general chronic viral illness and, in the case of 53D infection, canevolve into a asting condition, hich may become intractable ith failure of antiretroviral therapy. 3nfection-induced malnutrition, as discussed above, is primarilycyto2ine-mediated and is associated ith the acute-phase response. +hisis accompanied by multiple eects on metabolism, such as altered 7ues of ironand inc and loss of nitrogen, potassium, magnesium, phosphate, inc and vitamins. +his process is accompanied by retention of salt and ater. /alnutritionmay also present during the asymptomatic phase of 53D infection (Niyongaboet al, 199! >eters et al, 199$. /any studies have shon that micronutrientstatus is profoundly aected in 53D infection, but the aetiological signi*cance of

these changes has been diAcult to demonstrate (Cunningham-"undles, &'''. +herefore, the or2 of Campa et al (1999, included in +able &.1, has providedan important advance. sing careful longitudinal studies and good statisticaldesign, this group as able to establish that selenium de*ciency in childrenith acuired immune de*ciency syndrome (83;= as independently associatedith mortality.Immune assessment Ne assay methods have enabled the design of eperiments addressing dierentstages involved in immune-cell activation and the study of eects on signallingpathays, hich may then lead to the characteriation of causalrelationships. +able &.& outlines some of the types of methods currently in use./ost investigations begin ith a general assessment of ho a nutrient oraltered nutritional state aects the general parameters of the immune system,

immune-cell subsets and function. /easurement of changes in freuency andnumber of circulating lymphocyte subpopulations in the course of observationor dietary intervention is no accepted as a useful and idely comparable procedure,



but attention must be given to the issue of controls +his analysis shouldinclude standardied performance of immunophenotyping, using correction forpurity of the gating region, uantitative recovery of the cell type and positiveidenti*cation of cellular subsets. <or human studies, a complete blood countand dierential are needed to uantify eects on absolute numbers of cells.8lthough there is freuently a limitation on blood to be dran for nutritional

studies, it is essential that the baseline evaluation includes parallel studies providinga complete blood count, haematological analysis of haemoglobin,haematocrit, etc., on an aliuot of the same specimen of blood.

!a"le 2.2. Assessment of functional immune response.

3n addition to assessment of relative percentages of +-cells, B-cells and N: cells, immunophenotyping for activation-antigen epression (e.g. C;49, coepressionof critical molecules involved in cell6cell interaction (e.g. C;&$, +-cellreceptor (+C" changes and percentages of naive and memory cells may beinformative. <unctional studies should be carried out on fresh anticoagulatedblood henever possible (or blood stored at room temperature in the dar2 forunder &@ h before mononuclear cells are isolated. %hen blood is being sent byair or transported to a distant laboratory, it is etremely important to include acontrol specimen dran in parallel to serve as an internal standard for the shippingprocess. 3n addition, the type of tube chosen to dra the blood is important.#ithium heparin- or ethylenediamine tetra-acetic acid ();+8-containingtubes cannot be used for functional studies. =odium heparin (preservative-freeor acid citrate detrose (8C; tubes should be used and consistency of tubetype is important. +here may be dierences beteen venous and arterialblood. +he uestion of hen the blood should be dran is important. 3n general,most data have been obtained ith blood dran in the morning and thereare circadian eects on hormones and immune-cell phenotypes that may in7uenceresults. %hen this cannot be done, it is helpful to continue to maintain auniformity of draing time for an individual subEect or group. Concurrent controlblood must be dran to ensure that technical performance standards are

met. 3t is important that positive and negative (normal range and abnormalrange controls be included. ;ouble-baseline studies 6 as a minimum, beforeand after intervention is underta2en 6 are recommended.



=tudies of immune function usually start ith a general assessment of mononuclear response in vitro to a mitogen, to another non-speci*c activator orto antigen, as discussed above. +hese methods are generally based on assay of cell division at the pea2 of response folloing microtitre plate culture for severaldays. Culture methods profoundly aect results, and conditions need to be optimiedaccording to the 2inetics of the response. "esponses measured under most

conditions favour +-cell proliferation, as the +-cell is the most prevalent lymphocytein peripheral blood. +he elicited composite response is highly uantitativehen radioactive tracers 6 usually thymidine 6 are used. "ecently, hole-bloodmethodology has been introduced as an alternative e. vivo method that canre7ect potential response in vivo (=ottong et al, &'''! this method correlatesith the level of ;N8 synthesis found hen isolated mononuclear cells are culturedunder optimal standard conditions. Comparative studies have also shonthat there is a signi*cant correlation beteen the hole-blood method and isolatedmononuclear cells for cyto2ine production (Iaoob et al, 1999. =ome laboratorieshave replaced thymidine incorporation assays ith a combination of cell-surface mar2er-induction assays and a measurement of the percentage of cells in various phases of the cell cycle folloing activation. ;yes have beendeveloped that stably integrate into the membranes of live lymphocytes, such

that, ith each successive division, the amount of dye per cell is decreased.<luorescence can be used to measure the number of cell divisions. Jther assaysbased on hole blood measure early responses of cells selected through adherenceto magnetic beads to hich monoclonal antibodies recogniing cells of particularinterest are attached. 8ssessment is achieved by an assay of adenosinetriphosphate (8+> production by the luciferinHluciferase reaction (=ottong et al,&'''. 8ssays such as this may provide accurate assessment of in vivo responsein vitro +his method may be combined ith a uantitative measure of speci*clymphocyte subsets by 7o cytometry for eamination of response per cell.Jther approaches use measurement of cyto2ine response, receptor up-regulationor activation antigen to assess initial immune response, rather than thesecondary response of cells recruited in the ampli*ed reaction. 8lso, in vivo regulationof the immune response can be assessed through evaluation of unstimulatedlevels of secreted products hen the producer-cell source of these productsand normal levels are 2non. /ethods measuring early events in + lymphocyteactivation may or may not correlate ith cell division, since cell division is onlyone aspect of the immune response. Jne of the earliest events that occurs folloing +-cell activation is the rapid increase in intracellular free calcium. +his isfolloed by a change in p5 and changes in the membrane potential. 8ll of theseeects can be measured by 7o cytometry, using functional probes. <olloing +cellactivation via C;H+C" or via C;& (the alternate +-cell activation pathay,the *rst measurable surface mar2er induced is C;49. +his mar2er is a disulphide-lin2ed homodimer that is present on &'6'M of normal thymocytes, buthich is not epressed on resting peripheral-blood lymphocytes. C;49 reachespea2 levels ithin 1$6&@ h and declines if the stimulus is removed. sing 7o

cytometry, it is possible to measure increase in C;49 epression on speci*c lymphocytesubsets. 3t is apparent that C;49 induction is not part of the pathayleading to cell division, as induction of C;49 can occur ithout subseuent cellproliferation. 8 good ay to measure C;49 epression is to consider the relativeepression of this mar2er on the subpopulation of interest, as this removes theconfounding eect of subpopulation sie. Jther cell-surface mar2ers appear onactivated +-cells at variable times folloing activation, including C;&F (the ? chain of the interleu2in-& (3#-& receptor and the transferrin receptor C;1(both ithin &@6@$ h and human leucocyte antigen (5#8-;" (after @$ h.<inally, statistical evaluation is crucial to all of the studies described here. +hisincludes evaluation of both the internal and the study-group controls. =tudies of certain types may be suitable for the collection and ban2ing of specimens prior toassay, such as cyto2ine supernatants. +his may be helpful in giving a homogeneous

data set ith a lo coeAcient of variation, as long as controls and eperimentalspecimens are run simultaneously. 0ood design is often based on internalcross-chec2s, hich can be developed from the or2ing hypothesis and hich



allo for dierent elements in the same pathay to be considered.3n summary, the emerging *eld of nutritional immunology has bene*tedfrom the evolution of cellular and molecular immunology. Ne approacheshave provided a strong foundation for eperimental design and oer a choiceof analytical methods for approaching hypothesis testing. +he 2ey to anyspeci*c investigation is the identi*cation of clear uestions and the choice of

relevant and practical methods. +hese methods then need to be tested in apilot study, before launching the investigation. +he use of an integrated design,including biostatistical considerations and complementary assays, is importantin the development of meaningful data and of critical 2noledge.

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evaluation of the effects of

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