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10
Application of Whole-Blood Cultures to Field Study Measurements of Cellular Immune Function In Vitro

Tim R. Kramer1

This chapter describes the use of whole-blood cultures in evaluating the functional activity of blood lymphoid cells in vitro, with emphasis on the feasibility of their use in field studies. Results are presented from experiments to establish optimal mitogenic lymphocyte proliferation, interleukin production, and release of interleukin receptors in whole-blood cultures.

Background

In vitro measurements of mitogen- and antigen-induced blood lymphocyte proliferation, interleukin production, and release of soluble interleukin receptors are commonly used to determine the functional activity of T-lymphocytes. Such measurements are routine when the volume of blood is abundant, the number of samples is small, and the blood can be processed by an experienced worker in a well-equipped laboratory. They are not routine when a large number of blood samples must be processed on a given day, when blood volume is limited, or

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Tim R. Kramer, U.S. Department of Agriculture-Agricultural Research Service, Beltsville Human Nutrition Research Center, Phytonutrients Laboratory, Beltsville, MD 20705-2350



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--> 10 Application of Whole-Blood Cultures to Field Study Measurements of Cellular Immune Function In Vitro Tim R. Kramer1 This chapter describes the use of whole-blood cultures in evaluating the functional activity of blood lymphoid cells in vitro, with emphasis on the feasibility of their use in field studies. Results are presented from experiments to establish optimal mitogenic lymphocyte proliferation, interleukin production, and release of interleukin receptors in whole-blood cultures. Background In vitro measurements of mitogen- and antigen-induced blood lymphocyte proliferation, interleukin production, and release of soluble interleukin receptors are commonly used to determine the functional activity of T-lymphocytes. Such measurements are routine when the volume of blood is abundant, the number of samples is small, and the blood can be processed by an experienced worker in a well-equipped laboratory. They are not routine when a large number of blood samples must be processed on a given day, when blood volume is limited, or 1   Tim R. Kramer, U.S. Department of Agriculture-Agricultural Research Service, Beltsville Human Nutrition Research Center, Phytonutrients Laboratory, Beltsville, MD 20705-2350

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--> when facilities and expertise are limited. Each of these scenarios independently can prohibit the traditional use of density gradient-separated (Bøyum, 1977) peripheral blood mononuclear cells (PBMC) for measurement of T-lymphocyte functions in vitro. Preparation of a PBMC suspension requires an abundance of blood and well-trained laboratory workers, and it is time consuming. In an effort to demonstrate various effects on the cellular immune system of humans, several investigators have established the use of whole-blood cultures for measurement in vitro of lymphocyte proliferation (Bloemena et al., 1989; Bocchieri et al., 1995; Fletcher et al., 1992; Fritze and Dystant, 1984; Kramer and Burri, 1997; Kramer et al., 1997, in press; Leroux et al., 1985; Zhang et al., 1995) and interleukin production (DeForge and Remick, 1991; DeGroote et al., 1992; Desch et al., 1989; Ellaurie et al., 1991; Elsässer-Beile et al., 1993; Kirchner et al., 1982; Lyte, 1987; Nerad et al., 1992; Oliver et al., 1993). The use of whole-blood cultures offers four advantages: (1) only small amounts of blood are required; (2) many samples can be processed at once; (3) samples contain their own natural milieu of blood components, both cellular and humoral (fluid components of blood); and (4) whole-blood cultures are more cost effective than are cultures containing PBMC. Using whole-blood cultures has helped to establish associations between suppressed mitogenic proliferative responsiveness of T-lymphocytes in vitro and each of the following: marathon running (Eskola et al., 1978), intense treadmill exercise (Nieman et al., 1994), intense military training with reduced caloric intake (Kramer et al., 1997b), certain types of cancer (Fritze and Dystant, 1984), infection with human immunodeficiency virus (Bocchieri et al., 1995; Kramer and Chan, 1994), marginal zinc status (Kramer et al., 1990), and a diet low in carotene (Kramer and Burri, 1997). Whole-blood cultures have also been used to demonstrate the suppressive effects of treatment with oral corticosteroids on production of interferon-gamma (INF-γ) in response to dust mite antigen in vitro (Ellaurie et al., 1991), and to monitor the effects of biologic response modifiers on interleukin production by immune cells (Elsässer-Beile et al., 1993). Methods and Results Blood Donors Healthy adults donated blood for this series of experiments. Study of the donors was approved by the Institutional Review Board of Johns Hopkins University and by the U.S. Department of Agriculture Human Studies Review Committee. The project was conducted in accord with the Helsinki Declaration of 1975 as revised in 1983.

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--> Blood Collection Tests of leukocyte function, lymphocyte proliferation, interleukin production, and receptor release were conducted on 12-h fasting blood samples collected in sterile 3-ml VACUTAINER® tubes containing 45 USP units of sodium heparin (Becton Dickinson Co., Rutherford, N.J.). The blood was held at room temperature (20–22°C) until processed. Processing of blood for in vitro lymphocyte responses was started within 3 hours after collection. Lymphocyte Proliferation in Whole-Blood Cultures Through a series of experiments, the procedure described in Table 10-1 was established as optimal for this laboratory for maximum mitogenic proliferative responsiveness of blood lymphocytes in whole-blood cultures to phytohemagglutinin (PHA). Amount of Blood per Culture Maximum mitogenic proliferative responsiveness of lymphocytes to optimal-dose PHA in whole-blood cultures has been reported to occur in round-bottom microculture wells containing 12.5 μl of heparinized blood in a total volume of 200 μl of combined blood plus RPMI-1640 tissue culture medium (Kramer et al., in press). Table 10-2 lists results of a representative experiment to determine the optimal blood volume needed for maximum mitogenic proliferative responsiveness of blood lymphocytes to PHA in whole-blood cultures. As observed previously, cultures containing 12.5 μl of heparinized blood showed the highest mean level of maximum mitogenic proliferative responsiveness of lymphocytes to optimal-dose PHA in vitro. This amount of blood per microculture was obtained by the addition of 50 μl of 1:4 diluted blood into each culture well (Steps 1 and 2, Table 10-1). Cultures containing 8.3 and 6.25 μl of blood (blood-to-PRMI-1640 dilutions of 1:24 and 1:32, respectively) showed lower lymphocyte proliferation than did those with 12.5 μl of blood (a 1:16 dilution). Microcultures containing 25 μl of blood were too concentrated (1:8 final dilution) with erythrocytes for proper harvester collection of the whole-blood cultures (data not presented). Microcultures containing 6.25 and 12.5 μl of whole blood showed similar and lower intersubject (N = 11) coefficients of variation (CV) in lymphocyte proliferation than did cultures containing 8.3 μl of blood. Building on these results, the following experiments on lymphocyte proliferation were conducted with cultures containing 12.5 μl of heparinized blood.

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--> TABLE 10-1 Mitogenic Proliferative Responsiveness of Lymphocytes in Whole-Blood Cultures* Step Procedure 1 Place 400 mL heparinized blood into 1200 TL RPMI-1640.† 2 Add 50 mL 1:4 diluted blood to each culture well.‡ 3 Add 50 mL PHA at 1.25-80 μg/ml to each well.§ 4 Add 100 mL RPMI-1640 to each culture well.|| 5 Incubate in 5% CO2, 95% humidified air at 37°C for 96 hours. 6 Add 1 μCi 3H-thymidine to each culture.# 7 Harvest cultures onto fiberglass filters.** 8 Count 3H-thymidine incorporation by blood lymphocytes.†† 9 Tabulation of lymphocyte proliferative responsiveness.‡‡ * According to the method of Kramer et al. (in press). † Dilution prepared in 4.0 ml polystyrene tubes (FALCON®, Becton Dickinson Co., Rutherford, N.J.). The RPMI-1640 contains GlutaMAX-I (GibcoBRL, Grand Island, N.Y.) at 2.0 mmol/liter and penicillin streptomycin at 100,000 U/liter and 100 mg/liter, respectively; referred to herein as PRMI-1640. ‡ Typically, wells 1 to 12 of two consecutive rows of round-bottom, 96-well tissue culture plates (Corning Glass Works, Corning, N.Y.) prepared per diluted blood sample. § PHA (Sigma Chemical Co., St. Louis, Mo.) added at twofold concentrations to each set of triplicate cultures starting at 1.25 μg/ml. PRMI-1640 alone added to first set of triplicate for unstimulated responsiveness. || Each culture contains a final volume of 200 μl, with the final blood dilution at 1:16. # At 18 hours before termination of culture incubation. ** With a multicell harvester (Skatron Inc., Sterling, Va.). †† In a beta liquid scintillation counter (Beckman LS 3801) using a single-label disintegrations per minute (dpm) program with the activity reported in dpm per culture. ‡‡ The median value of each set of triplicate cultures is chosen as the activity for a given concentration of stimulant. Final presentation of the data is in becquerels (Bq, disintegrations per second) per culture or per lymphocyte, when lymphocyte numbers are available. Depending on the distribution of lymphocyte proliferation data among subjects, values may be transformed (log or square root) to conform with normality assumptions.

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--> TABLE 10-2 Volume of Blood Needed per Culture for Maximum Mitogenic Proliferative Responsiveness of Lymphocytes to PHA   Blood Quantity (μl)   6.25 8.3 12.5 Mean* 3,653 4,302 4,806 SD† 907 1,319 1,164 CV‡ 25 31 24 * N = 11. Maximum lymphocyte proliferative responsiveness (Bq) to PHA at twofold concentrations (starting with the lowest concentration), 1.25–80 µg/ml, 96-h CO2 incubation, 24-h label with 3H-thymidine. † Standard deviation. ‡ Intersubject coefficient of variation. Duration of Incubation of Whole-Blood Cultures Seventy-two hours of incubation is the standard for evaluating the mitogenic proliferative responsiveness of PBMC to PHA in vitro. PBMC cultures for lymphocyte proliferation are free of erythrocytes and isologous (syngeneic) plasma. Because whole-blood cultures contain both of these blood components, a series of experiments was conducted to determine the optimal duration of incubation for maximum mitogenic proliferative respon siveness of TABLE 10-3 Duration of Incubation for Mitogenic Proliferative Responsiveness of Lymphocytes to PHA in Whole-Blood Cultures   Duration (hours)   72 96 120 144 168 Mean* 1,954 4,501 6,087 4,217 2,120 SD† 510 1,157 1,283 1,272 1,254 CV‡ 26 26 21 30 59 * N = 8. Lymphocyte proliferative responsiveness (Bq) to PHA at twofold concentrations (starting with the lowest concentration), 2.5–10 µg/ml, 24-h label with 3H-thymidine. † Standard deviation. ‡ Intersubject coefficient of variation.

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--> lymphocytes to PHA in whole-blood microcultures containing 12.5 μl of heparinized blood. Table 10-3 presents the results of a representative experiment, which indicated that the optimal duration of incubation was 120 hours. Duration of 3H-Thymidine Label of Whole-Blood Cultures Estimates of DNA synthesis by mitogen-stimulated lymphocytes are frequently accomplished in microcultures containing 5 × 104 to 2 × 105 PBMC in a 200 μl volume labeled with 0.5 to 1.0 μCi of 3H-thymidine during the final 3 to 24 hours of incubation. Table 10-4 presents the results from a representative study to determine the optimal duration of labeling PHA-stimulated whole-blood cultures with 1.0 μCi (37 KBq)2 of 3H-thymidine ([methyl-3H], specific activity of 248 GBq [6.7 Ci]/mmol; Dupont NEN Products, Boston, Mass.) for maximum mitogenic proliferative responsiveness of blood lymphocytes cultured for a total of 120 hours ex vivo. PHA-stimulated whole-blood cultures labeled with 3H-thymidine during the final 18 hours of the 120-h incubation showed a higher mean 3H-thymidine incorporation than did those labeled for 3, 6, 12, and 24 hours. These cultures and those labeled with 3H-thymidine for only 3 hours showed the lowest intersubject CV. TABLE 10-4 Duration of 3H-Thymidine Label for Mitogenic Proliferative Responsiveness of Lymphocytes to PHA in Whole-Blood Cultures   Duration (hours)   3 6 12 18 24 Mean* 2,782 4,416 5,504 5,580 5,165 SD† 470 923 1,179 975 1,046 CV‡ 17 21 21 17 20 * N = 8. Lymphocyte proliferative responsiveness (Bq) to PHA at twofold concentrations (starting with the lowest concentration), 2.5-10 μg/ml, 120-h incubation. † Standard deviation. ‡ Intersubject coefficient of variation. 2   One Becquerel (Bq) is equal to 0.027 × 10-9 Curies (Ci) or 1 disintegration per second (dps); thus, 1 Ci equals 3.7 × 1010 dps and 2.2 × 1012 dpm.

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--> Finalized Durations of Cell Culture and 3H-Thymidine Labeling The results in Table 10-3 showed that 120-h incubation was optimal for maximum lymphocyte proliferation in response to PHA in whole-blood cultures. This differs from the current recommendation of 96 hours (Table 10-1; Kramer et al., in press). Because of the results presented in Tables 10-3 and 10-4, a follow-up experiment was conducted to determine whether maximum mitogenic proliferative responsiveness of lymphocytes to PHA in whole-blood cultures occurs in cultures incubated for 96 or 120 hours and labeled with 3H-thymidine during the final 18 or 24 hours. The results presented in Table 10-5 show that PHA-stimulated whole-blood cultures labeled with 3H-thymidine during the final 18 hours of a 96-h incubation had higher lymphocyte proliferation with lower intersubject CV than did those labeled for 24 hours and those cultured for 120 hours and labeled with 3H-thymidine for 18 and 24 hours. Data Presentation of Lymphocyte Proliferation in Whole-Blood Cultures Traditionally, in vitro lymphocyte proliferation data are presented as activity for a constant number of PBMCs per culture. Lymphocyte proliferation in whole-blood cultures is most frequently presented as becquerels of 3H-thymidine incorporated per culture, thus, per volume of blood. However, when the number of absolute lymphocytes is known by automated cell count, the proliferative responsiveness also can be presented as activity per TABLE 10-5 Optimized Duration of Cell Culture Incubation and 3H-Thymidine Labeling for Maximum Proliferative Responsiveness of Lymphocytes to PHA in Whole-Blood Cultures Hours of 3H-thymidine* Incubation† Mean‡ CV§ 18 96 5,961 13 24 96 5,050 16 18 120 4,815 19 24 120 4,672 23 * 1.0 µCi 3H-thymidine. † In 5% CO2 at 37°C. ‡ N = 8. Maximum proliferative responsiveness (Bq) to doses of PHA at twofold concentrations (starting with the lowest concentration), 1.25–80 µg/ml. § Intersubject coefficient of variation.

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--> TABLE 10-6 Lymphocyte Proliferative Responsiveness per Blood Volume and per Cell Unit Mean* SD† CV‡ Volume 4,368 786 18 Lymphocyte 0.204 0.045 22 * N = 15. Maximum proliferative responsiveness (Bq). † Standard deviation. ‡ Intersubject coefficient of variation. lymphocyte. As illustrated in Table 10-6, higher intersubject variation is routinely found when the proliferative activity is presented as the amount of 3H-thymidine incorporated per blood lymphocyte compared with blood volume. Whenever conditions and facilities permit, hematologic data are collected that allow the results to be presented in both forms: per blood volume and per lymphocyte. Effects of Holding and Shipping Blood on Whole-Blood Lymphocyte Proliferation The traditional practice of setting up cell cultures for lymphocyte proliferation within a few hours after blood collection has limited the use of this in vitro test in many studies. In efforts to expand the use of in vitro lymphocyte proliferation tests in field studies, this laboratory has studied the effects of 24-h holding and shipping of blood on PHA-induced lymphocyte proliferation in whole-blood cultures. In general, holding blood for 24 hours has presented mixed effects. Results presented in Table 10-7 show that holding blood at room TABLE 10-7 Effects of Holding Blood on Maximum Proliferative Responsiveness of Lymphocytes to PHA in Whole-Blood Cultures Holding time (hours) Mean* SD† CV‡ ~4 4,112 560 14 ~29 4,270 802 19 * N = 14. Maximum proliferative responsiveness (Bq). † Standard deviation. ‡ Intersubject coefficient of variation.

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--> temperature (22–25°C) for approximately 29 hours after blood collection did not cause a change in mean (N = 14) maximum proliferative responsiveness of blood lymphocytes to PHA in whole-blood cultures. There was, however, suppressed activity in cultures from the blood that had been held for 24 hours and stimulated with suboptimal concentrations of PHA for maximum lymphocyte proliferation in vitro (data not presented). As presented in Table 10-7, and routinely observed in other unpublished results, holding blood for 24 hours before setup in culture causes an increase in intersubject variation. This laboratory found that heparinized blood held up to 30 hours (total length of time from collection to setup in incubation) after collection at temperatures of 11 to 25°C (including time/temperature during air shipment) shows 0 to ±10 percent change in mean (N = 15) maximum proliferative responsiveness (unpublished data). Cytokine Production and Receptor Release In Vitro Table 10-8 shows the procedure currently used to measure release of tumor necrosis factor-alpha (TNF-α), INF-γ, interleukin-10 (IL-10), and the release of soluble receptor for IL-2 (sIL-2R) in response to PHA in whole-blood cultures. The procedure is based on the results presented in Table 10-9, using PHA as the TABLE 10-8 Cytokine Production and Release of sIL-2R Step Procedure 1 Place 200 mL heparinized blood into 2 sterile tubes.* 2 Add 200 mL RPMI-1640 to the control-culture.† 3 Add 200 mL RPMI-1640 with PHA to test-culture.†‡ 4 Incubate in 5% CO2 at 37°C for designated time.§ 5 Centrifuge culture tubes.|| 6 Collect supernatants and store at -70°C until analyzed.# * 4.0 ml polystyrene tubes (FALCON®, Becton Dickinson Co.). One tube serves as the unstimulated control; the other is the stimulated-test culture. † Total volume, 400 mL; final blood dilution, 1:2. ‡ PHA at appropriate concentration for designated cytokine test culture; suggested concentrations presented in Table 10-9. § Suggested times presented in Table 10-9. || 10 min at 1,000 × g in 10°C. # Measured according to directions of ELISA kit for each cytokine or receptor.

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--> stimulant. The use of other stimulants, especially specific antigens, will require changes in incubation time for production of the various cytokines and for the release of sIL-2R. Using the procedure outlined in Table 10-8, from 2.5 ml of heparinized whole-blood, cultures are prepared for the measurement of TNF-α, INF-γ, and IL-10 and the release of sIL-2R. This is in addition to preparation of 48 microcultures for lymphocyte proliferation in vitro, as described in Table 10-1. Concentration of Stimulant per Culture Based on results of preliminary experiments (data not presented), the durations of incubation and concentrations of PHA listed in Table 10-9 are chosen to determine the amounts of PHA stimulant needed per whole-blood culture, according to the procedure presented in Table 10-8, for optimal production of TNF-α, INF-γ, and IL-10 and for release of sIL-2R. The mean concentration of TNF-α was high in whole-blood cultures stimulated with 48 µg of PHA per culture for 24 hours (Table 10-9). The mean concentrations of INF-γ, IL-10, and sIL-2R were higher in whole-blood cultures stimulated with 16 µg than in those stimulated with 32 µg of PHA per culture. It is encouraging TABLE 10-9 Dose-Response Cytokine Production and Release of Soluble Interleukin Receptor Cytokine/Receptor Hours µg PHA* Mean† SD‡ CV§ TNF-α|| (pg/ml) 24 48 7,019 1,364 19 INF-γ# (pg/ml) 48 16 14,195 7,959 56   48 32 11,496 5,996 52 IL-10** (pg/ml) 96 16 8,967 3,607 40   96 32 7,061 2,423 34 sIL-2R†† (U/ml) 96 16 4,774 1,032 22   96 32 4,206 833 20 * Concentration per culture (value × 2.5 = µg/ml). † N = 13. ‡ Standard deviation. § Intersubject coefficient of variation. || Tumor necrosis factor-α (Medgenix Diagnostics, Belgium). # Interferon-γ (Endogen, Inc., Cambridge, Mass.). ** Interleukin-10 (Endogen, Inc., Cambridge, Mass.). †† Soluble interleukin-2 receptor (T-Cell Diagnostics/Endogen, Inc., Cambridge, Mass.).

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--> that the intersubject CV for TNF-α and sIL-2R was similar to that for PHA-stimulated lymphocyte proliferation per blood volume placed in culture within 6 hours after collection (Tables 10-6 and 10-8). However, it is of concern that intersubject variation is so high for supernatants evaluated for INF-γ and IL-10 content. Studies are currently under way to determine whether the variation can be reduced, even as cytokine production is maintained. Author's Conclusion As an alternative to the traditional use of PBMCs to determine the functional activity of blood lymphocytes in vitro, the use of whole-blood cultures presents several advantages: they require less blood, less laboratory work time, and less worker expertise, and thus they are more cost effective than are cultures of PBMCs. The use of whole-blood cultures is well established for measurement of lymphocyte proliferation. Less established is their use in the study of interleukins. This area, however, is currently receiving increased attention. The feasibility of conducting cost-effective cellular immunity tests in vitro is greatly increased by the use of whole-blood cultures in conjunction with overnight shipment of fresh blood to central laboratories for delayed processing or for study in remote field laboratories in partnership with a central laboratory. References Bloemena, E., M.T.L. Roos, J.L.A.M. Van Heijst, J.M.J.J. Vossen, and P.T.A. Schellekens. 1989. Whole-blood lymphocyte cultures. J. Immunol. Meth. 122:161-167. Bocchieri, M.H., M.A. Talle, L.M. Maltese, I.R. Ragucci, C.C. Hwang, and G. Goldstein. 1995. Whole blood culture for measuring mitogen induced T-cell proliferation provides superior correlations with disease state and T-cell phenotype in asymptomatic HIV-infected subjects. J. Immunol. Meth. 181:233-243. Bøyum, A. 1977. Separation of lymphocytes, lymphocyte subgroups and monocytes: A review. Lymphology 10:71-76. DeForge, L.E., and D.G. Remick. 1991. Kinetics of TNF, IL-6, and IL-8 gene expression in LPS-stimulated human whole blood. Biochem. Biophys. Res. Commun. 174:18-24. De Groote, D., P.F. Zangerle, Y. Gevaert, M.F. Fassotte, Y. Beguin, F. Noizat-Pirenne, J. Pirenne, R. Gathy, M. Lopez, I. Dehart, D. Igot, M. Baudrihaye, D. Delacroix, and P. Franchimont. 1992. Direct stimulation of cytokines (IL-1J, TNF-I, IL-6, IL-2, IFN-K and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation. Cytokine 4:239-248. Desch, C.E., N.L. Kovach, W. Present, C. Broyles, and J.M. Harlan. 1989. Production of human tumor necrosis factor from whole blood ex vivo. Lymphokine Res. 8:141-146. Ellaurie, M., S.L. Yost, and D.L. Rosenstreich. 1991. A simplified human whole blood assay for measurement of dust mite-specific gamma interferon production in vitro. Ann. Allergy 66:143-147. Elsässer-Beile, U., S. von Kleist, A. Lindenthal, R. Birken, H. Gallati, and J.S. Mönting. 1993. Cytokine production in whole blood cell cultures of patients undergoing

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--> therapy with biological response modifiers or 5-fluorouracil. Cancer Immunol. Immunother. 37:169-174. Eskola, J., O. Ruuskanen, E. Soppi, M.K. Viljanen, M. Järvinen, H. Toivonen, and K. Kouvalmnen. 1978. Effect of sport stress on lymphocyte transformation and antibody formation. Clin. Exp. Immunol. 32:339-345. Fletcher, M.A., N. Klimas, R. Morgan, and G. Gjerset. 1992. Lymphocyte proliferation. Pp. 213-219 in Manual of Clinical Laboratory Immunology, 4th ed., N.R. Rose, E. Conway DeMacario, J.L. Fahey, H. Friedman, and G.M. Penn, eds. Washington, D.C.: American Society for Microbiology. Fritze, D., and P. Dystant. 1984. Detection of impaired mitogen responses in autologous whole blood of cancer patients using an optimized method of lymphocyte stimulation. Immunol. Lett. 8:243-247. Kirchner, H., C. Kleinicke, and W. Digel. 1982. A whole-blood technique for testing production of human interferon by leukocytes . J. Immunol. Meth. 48:213-219. Kramer, T.R., and B.J. Burri. 1997. Modulated mitogenic proliferative responsiveness of lymphocytes in whole-blood cultures by low carotene diet and mixed-carotenoid supplementation of women. Am. J. Clin. Nutr. 65:871-875. Kramer, T.R., and M.M. Chan. 1994. Use of whole-blood cultures to monitor mitogenic responsiveness (MR) of lymphocytes in HIV + children. FASEB J. 8:A494. Kramer, T.R., R.J. Moore, R.L. Shippee, K.E. Friedl, L. Martinez-Lopez, M.M. Chan, and E.W. Askew. 1997. Effects of food restriction in military training on T-lymphocyte responses. Int. J. Sports Med. 18 (suppl. 1):S84-S90. Kramer, T.R., K. Praputpittaya, Y. Yuttabootr, R. Singkamani, and M. Trakultivakorn. 1990. The relationship between plasma zinc and cellular immunity to Candida albicans in young females of northern Thailand. Ann. N.Y. Acad. Sci. 587:300-302. Kramer, T.R., M.P. Howard, and J. Chittams. In Press. Mitogen-induced T-lymphocyte proliferation in whole-blood cultures: Optimization based on activity and variation. J. Nutr. Immunol. Leroux, M., L. Schindler, R. Braun, H.W. Doerr, H.P. Geisen, and H. Kirchner. 1985. A whole-blood lymphoproliferative assay for measuring cellular immunity against herpes viruses. J. Immunol. Meth. 79:251-262. Lyte, M. 1987. Generation and measurement of interleukin-1, interleukin-2, and mitogen levels in small volumes of whole blood. J. Clin. Lab. Anal. 1:83-88. Nerad, J.L., J.K. Griffiths, J.W.M. 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 1J (IL-1J), IL-1 receptor antagonist, and TNF-α production in whole blood. J. Leukoc. Biol. 52:687-692. Nieman, D.C., A.R. Miller, D.A. Henson, B.J. Warren, G. Gusewitch, R.L. Johnson, J.M. Davis, D.E. Butterworth, J.L. Herring, and S.L. Nehlsen-Cannarella. 1994. Effect of high- versus moderate-intensity exercise on lymphocyte subpopulations and proliferative response. Int. J. Sports Med. 15:199-206. Oliver J.C., L.A. Bland, C.W. Oettinger, M.J. Arduino, S. K. McAllister, S.M. Aguero, and M.S. Favero. 1993. Cytokine kinetics in an in vitro whole blood model following an endotoxin challenge. Lymphokine Cytokine Res. 12:115-120. Zhang, Y.H., T.R. Kramer, P.R. Taylor, J.Y. Li, W.J. Blot, C.C. Brown, W. Guo, S.M. Dawsey, and B. Li. 1995. Possible immunologic involvement of antioxidants in cancer prevention. Am. J. Clin. Nutr. 62 (suppl.):1477S-1482S.

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--> Discussion RONALD SHIPPEE: Do you supplement the whole-blood cultures with fetal bovine serum? TIM KRAMER: No. We had found that the addition of fetal bovine serum to whole-blood cultures decreased the mitogenic responsiveness of lymphocytes to phytohemagglutinin. SYDNE CARLSON-NEWBERRY: What is the number of peripheral blood lymphocytes needed to reliably measure the proliferative responsiveness of lymphocytes in whole-blood cultures? TIM KRAMER: We have not done a definitive study to determine the minimal absolute lymphocyte number needed for reliable lymphocyte proliferation results in whole-blood cultures. However, in collaboration with Dr. Maria M. Chan of Children's National Hospital in Washington, D.C., children being treated for bone marrow transplantation failed to show functional lymphocyte proliferation. JEFFREY ROSSIO: I think the issue of logistics in shipping blood overnight is very important. We have found that shipping blood when the temperature is very cold can be suppressive on the functional capacity of the blood. TIM KRAMER: I agree. We have found suppressed lymphocyte proliferation when the blood had been held overnight at near freezing or at 39°C. SEYMOUR REICHLIN: There are many places where immune function can be regulated in stress. You could have marginations so that part of the cell population of interest does not even appear in the blood anymore, and is stuck to the walls of the endothelium. That is a catecholamine-mediated function. You can have changes in the population of cells being synthesized in the thymus and spleen. You can have transient changes in glucocorticoids, growth hormone, and prolactin, all of which can change the activity of the cells at the time that they are exposed to this level of hormone when one takes a sample from a stressed individual; so, that was really my question. You are diluting the blood so that the steroid levels have been reduced. You are diluting the blood so that the growth hormone and prolactin levels have been reduced. You are only measuring the functions of the cells that appear in the blood, not the ones that are stuck in the tissues somewhere. I think that is the question. That is especially true in acute stress situations. If you look at the literature on short-term stress,

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--> for example, one-day stresses, [academic] examinations, as with the work of the Glasers at Ohio State, all of that is very short-term stuff and might not even be reflected in these kinds of studies. TIM KRAMER: I can totally relate to your concern about that. Except for probably the skin test response, the whole- blood culture system is probably the closest that we can get to what is going on in the body. This is one of the reasons that we have been interested in it, because it keeps the milieu constant.