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--> C Overview of Immune Assessment Tests Basic Tests of Immune Status The Agency for Toxic Substances and Disease Registry (ATSDR) basic panel of immune function tests consists of a group of assessments that are performed with serum and several that are performed on whole blood. Assays of serum include the following: C-reactive protein (CRP), a marker of acute-phase reactions and a sensitive indicator of tissue damage from a variety of causes; immunoglobulins (Ig) G and M, indicators of humoral immune status; IgA, an indicator of mucosal humoral immunity; antinuclear antibody, an indicator of autoantibodies; and total protein, used to correct the concentration of immunoglobulins for concentration of blood proteins.
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--> Whole blood measures include the following: a complete blood count with a five-part differential to determine total white cells, total lymphocytes, and total eosinophils; and number of CD4+ lymphocytes and the CD4+:CD8+ ratio, by lymphocyte phenotyping. CD4+ lymphocytes are helper–inducer T-lymphocytes, whereas CD8+ cells are suppressor or cytotoxic T-cells. A decrease in CD4+ and a CD4+:CD8+ ratio of less than 1.5 is correlated with immune impairment and increased susceptibility to infection (Bloom et al., 1992). Although lymphocyte phenotyping with a complete panel of major phenotypes is recommended by the ATSDR as the most predictive test for immunotoxicity in animals (Luster et al., 1992, 1993) and can be performed with small samples of whole blood (making the tests amenable to field studies), these tests are not able to evaluate functional status. Lymphocyte subpopulations vary over time and between individuals. In addition, peripheral immune cells and compartments, although easy to sample, may not reflect immune function in the lymphoid tissues where the most significant changes in lymphocyte subpopulations are likely to occur, and samples drawn from peripheral pools may be more reflective of one part of the immune system than the other (humoral versus systemic) (see Susanna Cunningham-Rundles, see Chapter 9). Monoclonal antibodies (Mabs) that identify cell-surface markers associated with T-cell activation (such as CD25, the interleukin-2 (IL-2) receptor) are now becoming available and may alleviate some of the problems mentioned. Focused Tests of Immune (Deficiency) Function For individuals whose basic test results provide evidence of immune deficiency, a panel of more specific tests may be performed. Tests of Serum Secondary humoral response (IgM and IgG responses to an antigen to which the subject has been exposed previously) can be assessed in samples of serum drawn before and after immunization with the recall antigen, making this a suitable technique for use in the field (Straight et al., 1994). Factors that must be considered include the timing of blood sampling (to take into account variations in immune function resulting from biological rhythms [see Erhard Haus, Chapter 20]) and the background (''before'') antibody titer expected. An additional consideration is the great degree of variability observed in response to immunization, including a fairly high percentage of nonresponders. Antibody response to primary immunization with a novel antigen may be measured as an alternative. This test requires identification of an antigen to which the subject has not been exposed previously (see Cunningham-Rundles, Chapter 9).
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--> A specific antibody response can also be assessed by measuring the titer of isohemagglutinins, the antibodies to blood type A or B antigens present in everyone except those with type AB blood (Straight et al., 1994). Complement assays (CH50 is the most common) can detect deficiencies in components of the complement system, which can manifest as recurrent infections with normal concentrations of plasma IgG (Straight et al, 1994). Advanced Immune Function Assessments Advanced immune function assessments are performed in whole blood or in cultures of cells isolated from blood. Granulocyte nitroblue tetrazolium dye reduction provides an assessment of granulocyte function but should be replaced eventually by lymphocyte phenotyping. Studies of lymphocyte proliferation or function are in vitro assays of the ability of plant mitogens, lymphokines, recall antigens, or allogeneic cells to stimulate lymphocyte blastogenesis and proliferation. The use of a recall antigen permits the evaluation of immunologic memory as well as proliferation (Straight et al., 1994). Measurable indicators of proliferation include [3H]thymidine incorporation into DNA (see Tim R. Kramer, Chapter 10), expression of cell-surface activation antigens such as CD25, release of cytokines, and shedding of soluble receptors for the cytokine IL-2 (Straight et al., 1994). Functional assays such as measurement of IL-2 release (via enzyme-linked immunosorbent assay [ELISA]) and IL-2 receptor expression (CD25) in cell culture supernatants may be a more physiological as well as reliable index of T-helper 1 (Th1) cell proliferation and would obviate the need for radioisotopes. The measure of Th1 proliferation in this way has been shown to be associated with stress levels and with the frequency of upper respiratory infections (URIs), although there is disagreement regarding the use of cytokine release as an index of proliferation. A number of factors must be considered with regard to tests of lymphocyte stimulation by mitogens. One factor is that not all plant lectin mitogens are specific for T-cells; some have B-cell stimulatory properties as well. Also, mitogens are polyclonal activators that bypass the early events in lymphocyte stimulation. Thus, they may not produce the same response as a defined antigen. Plant lectins are not often encountered in vivo by the immune systems of most people (Shephard et al., 1994). Thus, any inferences regarding the type of cell responding must be made with caution, particularly since it may not be possible to account for differential changes in various subpopulations of cells due to illness or other stressors. In addition to tests on serum and whole blood, ATSDR includes delayed hypersensitivity skin testing in its second tier of assays. Using a panel of four to five antigens such as mumps and diphtheria, this test measures functional T-cell-mediated immunity. However, the test is difficult to perform in large population-based studies, in part because it requires two visits (48 h apart) to the clinic.
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--> Assays of Circulating Cytokines and Soluble Receptors A large number of cytokines, including at least 18 identifiable interleukins and 3 interferons, have been characterized thus far. All can be measured by immunoassay, and many can also be measured by bioassay. Many methodological issues must be considered in planning a strategy for the assessment of cytokines, their receptors, and inhibitors. A prime factor, which will not be discussed here, is the cost of individual assays. A second factor, or group of factors, of considerable importance for field studies is the proper means of sample collection, storage, and shipping; this is discussed below. A third and somewhat related factor is the inaccuracy that may be introduced by the assay of the plasma or serum rather than the supernatant of cultured lymphocytes. In his discussion of on the use of whole blood cultures to measure lymphocyte proliferation, Kramer (Chapter 10) described attempts of his laboratory to identify cytokines whose production in response to phytohemagglutinin (PHA)-stimulation not only paralleled [3H]thymidine incorporation but also would be measurable in blood samples. Currently, such a parallel substance would obviate the need to use tritiated thymidine and would permit actual analyses to be performed in the field. Kramer and coworkers compared the parameters of [3H]thymidine incorporation to those of immunoassayable production and release of the soluble receptor for IL-2 (sIL-2r), as well as tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ) and IL-10, in supernatants from whole blood. When the coefficients of variation (CVs) were compared between whole blood and isolated cell supernatants for measurements of TNF, sIL-2r, IFN-γ, and IL-10, the CVs for TNF and sIL-2r compared well with that for [3H]thymidine, but those for IFN-γ and IL-10 did not; Kramer indicated that this was an area of ongoing investigation. Two possible explanations for the results were offered by J.G. Cannon (Pennsylvania State University, University Park, personal communication, 1996), who emphasized the need to correlate cytokine production by whole blood with that of isolated blood cells. In a study by his group (Nerad et al., 1992), it was observed that when whole blood cultures were stimulated by lectins, IL-1β production compared favorably with that in standard peripheral blood mononuclear cell (PBMC) cultures. In contrast, because neutrophils in blood synthesize large numbers of TNF receptors as well as TNF itself, there was a reduction in measurable TNF compared with that in standard cultures. Cannon also mentioned that commercial assays, developed for measurement of cytokines in cell culture supernatants, can be unpredictable in measuring factors in samples that contain serum or plasma. Cannon et al. (1993) reviewed the factors that must be considered in the measurement of circulating cytokines. With respect to the samples themselves, they pointed out that differences in plasma and serum affect assays in ways that must be tested; that white blood cells (WBCs) collected in blood samples are, themselves, a source of cytokines; and that contaminants on some types of assay
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--> tubes are capable of stimulating the ex vivo synthesis of cytokines by these WBCs. Assays of circulating cytokines are affected by three types of cytokine-binding proteins and by cytokine antagonists found in plasma (Cannon et al., 1993). The binding proteins include immunoglobulins, α2-macroglobulin, and soluble receptors. All three types of proteins are affected by changes in physiological status, not necessarily in parallel with changes in the cytokines themselves. In addition, although most soluble receptors inhibit the effects of the cytokines that bind to them, the soluble receptor for IL-6 actually increases the activity of the cytokine. Cytokine antagonists inhibit the effects of cytokines by competing with them for binding to receptors. The assessment of cytokine values in urine, where circulating receptors are less prevalent, is suggested by Virella et al. (1997) and Moldawer (1997). These authors caution, however, that the effect of hydration status (diuresis) must be corrected for by the concurrent assessment of urinary creatinine or a similar substance. Another potential problem is that urinary concentrations of the cytokine of interest may be too low to detect changes (G. Sonnenfeld, University of Kentucky, Louisville, personal communication, 1997). Other considerations in the assay of cytokines include the low sensitivity of most immunoassays. Concentrations of cytokines in blood and urine are often near the lower limit of detection for many assays in current use. Although bioassays are much more sensitive, they are far less specific. Antibody-based assays, such as ELISA, detect the presence of the epitope to which the antibody is directed, which may or may not assess biological activity. In addition, combinations of cytokines can cause synergistic effects. Thus, it is necessary to perform numerous assays with the specific types of samples and assay of interest, including many types of controls and standards. An additional problem with cytokine assays is that the correlation between circulating levels of cytokines or their membrane receptors and functional immune status is still not well characterized (G. Sonnenfeld, University of Kentucky, Louisville, personal communication, 1997). This problem is compounded by the possible lack of relevance of circulating levels of cytokines compared to those in the lymphoid tissues of origin or other cytokine-producing cells. According to Cannon and coworkers (1993), factors that affect the measurement of cytokines also affect their biological activity in vivo. Thus, for example, the measurement of a cytokine without measurement of its antagonist gives a false picture of the role of the cytokine in a physiological process or disease state. Binding proteins can influence the half-life of cytokines as well as their distribution to other tissue compartments and their association with cell-surface receptors. In addition, because some cytokines remain primarily cell associated, they may never be detected in plasma, or their presence in plasma may not be meaningful. Finally, cytokines rarely act alone, but instead function in synergy with or opposition to others. Thus, it is important, but not always possible, to know how the concentration of one cytokine relates to that of others.
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--> Nevertheless, Sonnenberg and coworkers include assays of IL-4, IL-6, IL-12, TNF-α and β, and IFN-γ in PBMC supernatants of shuttle astronauts (G. Sonnenfeld, University of Kentucky, Louisville, personal communication, 1997). The influence of exercise on IL-1 and IL-2 and on IL-2 receptors has been reviewed by Shephard and coworkers (1995). Natural Killer Cell Activity Natural killer cytolytic activity is assessed by measuring the in vivo release of radiolabeled chromium from target cells, usually a human myeloid tumor cell line. References Bloom, B.R., R.L. Modlin, and P. Salgame. 1992. Stigma variations: observations on suppressor T cells and leprosy. Annu. Rev. Immunol. 10:453-88. Cannon, J.G., J.L. Nerad, D.D. Poutsiaka, C.A. Dinarello. 1993. Measuring circulating cytokines. J. Appl. Physiol. 75(4):1897-902. Luster, M.I., C. Portier, D.G. Pait, G.J. Rosenthal, D.R. Germolec, E. Corsini, B.L. Blaylock, P. Pollock, Y. Kouchi, and W. Craig. 1993. Risk assessment in immunotoxicology. II. Relationships between immune and host resistance tests. Fundam. Appl. Toxicol. 21(1):71-82. Luster, M.I., C. Portier, D.G. Pait, K.L. White Jr., C. Gennings, A.E. Munson, G.J. Rosenthal. 1992. Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fundam. Appl. Toxicol. 18(2):200-210. Moldawer, L.L. 1997. The validity of blood and urinary cytokine measurements for detecting the presence of inflammation. Pp. 417-430 in Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability, S.J. Carlson-Newberry and R.B. Costello, eds. Committee on Military Nutrition Research, Food and Nutrition Board. Washington, D.C.: National Academy Press. Nerad, J.L., J.K. Griffiths, J.W. Van der Meer, S. Endres, D.D. Poutsiaka, G.T. Keusch, M. Bennish, M.A. Salam, C.A. Dinarello, and J.G. Cannon. 1992. Interleukin-1 beta (IL-1 beta), IL-1 receptor antagonist, and TNF alpha production in whole blood. J. Leukoc. Biol. 52(6):687-692. Shephard, R.J., S. Rhind, and P.N. Shek. 1994a. Exercise and the immune system. Natural killer cells, interleukins and related responses. Sports Med. 18(5):340-69. Shephard, R.J., and P.N. Shek. 1995. Exercise, aging and immune function. Int. J. Sports Med. 16(1):1-6. Straight, J.M., H.M. Kipen, R.F. Vogt, and R.W. Amler. 1994. Immune Function Test Batteries for Use in Environmental Health Studies. U.S. Department of Health and Human Services, Public Health Service. Publication Number: PB94-204328. Virella, G., C. Enockson, and M. La Via. 1997. New approaches to the study of abnormal immune function. Pp. 431-450 in Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability, S.J. Carlson-Newberry and R.B. Costello, eds. Committee on Military Nutrition Research, Food and Nutrition Board. Washington, D.C.: National Academy Press.
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