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Cocoa Procyanidins and Human Cytokine Transcription and Secretion^{1 ,2}

Tin Mao^*, Judy Van de Water^*, Carl L. Keen, Harold H. Schmitz^** and M. Eric Gershwin^*,3

^* Division of Rheumatology, Allergy and Clinical Immunology, and Department of Nutrition, University of California at Davis, Davis, California 95616 and ^** Analytical and Applied Sciences, Mars, Incorporated, Hackettstown, New Jersey 07840

	ABSTRACT

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We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1ß and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1ß concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1ß expression by 7–19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71–86%. This observation at the transcription level for IL-1ß was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61–73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

KEY WORDS: • cocoa • procyanidin • cytokines

	INTRODUCTION

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Cocoa is ingested by many cultures, and the discovery of its residue in ancient Mayan vessels suggests that humans have been consuming it, in some form, since at least 480 A.D. (Seligson et al. 1994 ). Common components of fresh cocoa beans (cotyledons) include theobromine, caffeine, polyphenols and saturated and monounsaturated fatty acids (Hoskin 1994 ). However, when ingested in cocoa, these constituents often have biological activities that differ from those of the isolated molecules. Thus, cocoa butter does not raise concentrations of total serum cholesterol, although this would be expected from its rich content of saturated fatty acids (Denke 1994 ). A further example is seen with theobromine (3,7-dimethylxanthine), which is a purine alkaloid related to caffeine (1,3,7-trimethylxanthine) and is toxic to many laboratory species, including rat (Wang and Waller 1994 ), dog (Strachan and Bennet 1994 ) and rabbit (Soffietti et al. 1989 ). Although a recent study suggested that commonly consumed cocoa products contain pharmacologically active doses of both theobromine and caffeine (Mumford et al. 1994 ), Wang and Waller (1994 ) have shown that theobromine, at a level that is toxic in isolation, has no effect on rats when ingested as part of the cocoa extract. In contrast to the negative health consequences often associated with these compounds, cocoa is also known to contain polyphenols, which are postulated to have positive health benefits.

Indeed, Sanbongi et al. (1997 ) demonstrated that cacao liquor polyphenols inhibit reactive oxygen species and reduce the expression of interleukin (IL)⁴ -2 mRNA in human lymphocytes. Polyphenols from other plant sources also inhibit the cellular expression of IL-8 and monocyte chemoattractant-1 when induced by the proinflammatory cytokine tumor necrosis factor- (Sato et al. 1997 ). Procyanidins have been identified as the primary polyphenol in Theobroma cacao (Jalal and Collin 1977 , Quesnel 1968 ). This class of polyphenolic compounds is present in plant species as individual monomers and as oligomeric units (Porter et al. 1991 ). Studies have shown that the degree of polymerization of procyanidins can ultimately determine their effectiveness on a wide range of properties. According to Dauer et al. (1998 ), the antimutagenic effect is augmented by an increasing degree of polymerization in the proanthocyanidins. In another study, vascular activity in porcine coronary arteries was dependent on the relative molecular masses of procyanidins, with effectiveness decreasing with size (Melzer et al. 1991 ). In addition, dimeric and trimeric procyanidins, purified from Douglas fir bark, were found to enhance the inhibition of a potent tumor promoter relative to their monomeric fraction (Gali et al. 1994 ).

Excluding the observations introduced by Sanbongi et al. (1997 ), there has been a paucity of information available regarding the potential immunoregulatory effects of cocoa procyanidins on human peripheral blood mononuclear cells (PBMC). These recent findings, suggesting possible immunomodulatory functions, prompted us to examine the effects of cocoa procyanidins on the modulation of cytokines. In the present study, we consider the effects of cocoa procyanidins, in the form of purified oligomers, on both mRNA expression and protein secretion of cytokines (IL-1ß, -2 and -4) from unstimulated and stimulated PBMC.

IL-1ß is a multifunctional cytokine that acts on nearly every cell type and is central to the early onset of inflammation in humans (Dinarello 1998 ). There is conclusive evidence that control of IL-1ß production can take place at the level of either gene transcription or mRNA translation (Dinarello 1997 , Schindler et al. 1990 ). Moreover, there is a narrow margin between levels of IL-1ß that are physiological and those that are inflammatory (Dinarello 1998 ), so moderate alterations in its production are important. IL-4 is a cytokine that also affects a variety of target cells in multiple ways, although it has anti-inflammatory properties (Brown and Hural 1997 ). IL-4–mediated effects include the enhancement of IgE production by B cells, hematopoiesis and the development of effector T-cell responses. Aberrant production of IL-4 has been implicated in allergy, autocrine growth of tumors and susceptibility to some infectious diseases (Brown and Hural 1997 ). IL-2 is involved in the control of T-cell expansion and activation (Leonard et al. 1985 , Smith 1988 ). Thus, the regulation of IL-2 production is critical for initiating an immune response.

	METHODS

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Cocoa fraction preparation.

Water-soluble procyanidin (phenolic) fractions were prepared from Cocoapro cocoa (M&M/Mars, Elizabethtown, PA) after acetone/water extraction. The fractions were purified from the crude extract using HPLC methodology according to Adamson et al. (1999 ). Purified fractions of monomers through decamers were investigated. These purified procyanidin fractions contained <0.5% (total, w/w) of total alkaloids (theobromine and caffeine). The procyanidin composition, estimated by HPLC and molecular weights of these preparations, is shown in Table 1 . In addition, (+)-catechin and (-)-epicatechin (Sigma Chemical Co., St. Louis, MO) were investigated because these are the two molecules that make up the monomeric fraction present in cocoa. All samples were suspended in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Norcross, GA). They were then diluted with the same medium to final concentrations of 25 µg/ml for IL-1ß and IL-4 investigations and 50 µg/ml for the IL-2 analysis. Note that the dimer, trimer and tetramer fractions were not tested for IL-2 gene expression.

Peripheral blood from healthy volunteers was collected into sodium citrate–containing tubes and mixed 1:1 with Hanks’ balanced salt solution (HBSS; GIBCO BRL) without calcium chloride, magnesium chloride or magnesium sulfate. The diluted blood was then layered over an Accu-Paque gradient (Accurate Chemical & Scientific Corp., Westbury, NY) and centrifuged at 500 x g for 30 min at room temperature. PBMC were harvested from the interface layer, washed twice with HBSS and then counted. The cells were resuspended in RPMI 1640 containing 10% fetal bovine serum and supplemented with 0.1% of 50 mg/ml gentamicin (GIBCO BRL). PBMC concentration was adjusted to 2–2.5 x 10⁶ viable cells/ml after estimation of viability by trypan blue exclusion assay. Viability was consistently >96%.

Culture of PBMC with cocoa fractions.

For the cytokine expression assay, 200 µl of a 5.0 x 10⁵ cell suspension was cultured with an equal volume of the various cocoa treatments for 8 h at 37°C with 5% CO₂ on 48-well plates. PBMC were incubated with individual cocoa fractions at 25 µg/ml, and the transcription of IL-1ß, IL-2 and IL-4 was analyzed. Each cocoa-stimulated fraction was compared with control cultures treated with medium alone. In addition, PBMC were stimulated with 25 µg/ml phytohemagglutinin (PHA) (Sigma Chemical Co.) along with each cocoa fraction at 25 µg/ml.

For the protein secretion assay, 500 µl of a 1.0 x 10⁶ cell suspension were cultured with 500 µl of the various cocoa treatments for 72 h at 37°C with 5% CO₂ on 48-well plates. Individual cocoa fractions at 25 µg/ml were incubated in the presence and absence of PHA at 25 µg/ml.

Reverse transcriptase–polymerase chain reaction.

Cells were harvested at 8 h and transferred into 1.5-ml RNase-free Eppendorf tubes. Total cellular RNA was immediately extracted from cells using TRIzol Reagent (GIBCO BRL). Briefly, PBMC pellets were homogenized with 250 µl TRIzol, and chloroform (50 µl) was then added. After vigorously shaking of the tubes for 15 s, the cells were incubated for 3 min at room temperature and then centrifuged at 12,000 x g for 15 min at 4°C. The aqueous phase was transferred into another tube containing 125 µl isopropanol and 10 µg carrier tRNA (yeast tRNA; GIBCO BRL). The samples were then incubated at room temperature for 10 min and again centrifuged at 12,000 x g for 10 min at 4°C. The supernatant was removed, and the RNA pellet was washed with 250 µl of 75% ethanol. The sample was mixed by vortexing and then centrifuged at 7500 x g for 5 min at 4°C before drying. The RNA pellet was dissolved in 12 µl of diethylpyrocarbonate-treated H₂O and stored at -80°C for up to 4 wk without significant deterioration in message amplification (data not shown).

The RNA was then subjected to first-strand synthesis at 42°C for 50 min in a 20-µl reaction mixture containing 1 µg RNA (5 µl), 25 mM Tris-HCl (pH 8.3, at room temperature), 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 20 U RNasin (Promega, Madison, WI), 1 µl dNTP mix (10 mM concentration of each of dATP, dCTP, dGTP and dTTP) (Pharmacia Biotech, Uppsala, Sweden), 0.5 µg oligo(dT)s and 200 U Superscript II (GIBCO BRL). After the completion of first-strand synthesis, the cDNA was diluted 1:10 with diethylpyrocarbonate-treated H₂O.

IL-1ß and IL-2 gene expression was evaluated using standard polymerase chain reaction (PCR) where the total number of cycles (32) analyzed was determined to fall within the linear range of amplification. Primers for either IL-1ß or IL-2 was coamplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene. We used 2 µl of the diluted cDNA template for PCR. The final PCR mixture (25 µl) contained a 0.2 mM concentration of each dNTP, 25 ng of each specific primer, 1 U of AmpliTaq Gold (Perkin–Elmer Cetus, Foster City, CA) and 2.5 µl of 10x reaction buffer containing 15 mM MgCl₂ (Perkin–Elmer Cetus). Specific primer sequences were chosen to cross introns to avoid amplifying genomic DNA (Mao et al. 1999 and 2000 ). The PCR product was mixed thoroughly with 5 µl of 10x loading buffer (20% Ficoll 400, 0.1 M Na₂EDTA, 1.0% SDS, 0.25% bromophenol blue and 0.25% xylene cyanol). Then, 10 µl of the mixture was then carefully loaded onto a well of a 1.8% agarose LE (Boehringer-Mannheim, Indianapolis, IN) gel prepared with TAE buffer containing 0.4 µg/ml ethidium bromide. The gels were electrophoresed in TAE buffer at 80 V for 60 min. The bands were visualized on a UV light box and photographed using Polaroid film (type 667). The positive image was computer scanned using Adobe Photoshop (Adobe Systems Inc., San Jose, CA). The intensity readings of each band, which correlate with the amount of cytokine, were then calculated with NIH Image 1.57.

For IL-4, real-time PCR was used in which the final PCR mixture (50 µl) contained a 0.2 mM concentration of each dNTP, 0.3 µM concentration of each specific primer, 1 U of AmpliTaq Gold (Perkin–Elmer Cetus), 3.0 mM MgCl₂, 1x reaction buffer (Perkin–Elmer Cetus) and 5 µl of the diluted cDNA template. Again, specific primer sequences were chosen to cross introns to avoid amplifying genomic DNA (Mao et al. unpublished observations). In general, an external control was constructed consisting of a plasmid standard for IL-4 and GAPDH. Total RNA was extracted from PBMC, and desired cDNA fragments were generated by reverse transcription (RT)–PCR with the same primers as described previously (Mao et al. unpublished observations). The amplicon was then cloned into pCR 2.1-TOPO vector (TOPO TA Cloning Kit; Invitrogen, Carlsbad, CA). The ligated fragments were transformed into competent Escherichia coli. Plasmid DNA was then isolated and confirmed by DNA sequencing. The concentration was measured with optical density spectrophotometry, and serial dilutions were used as standard curves, each containing a known amount of plasmid DNA.

IL-1ß,4 secretion assays (enzyme-linked immunosorbent assays).

Culture supernatant fractions were harvested after 72 h and were stored at -20°C until analysis by enzyme-linked immunosorbent assay (ELISA). Protein levels were measured in supernatants from 1.0 x 10⁶ cells/ml stimulated with cocoa fractions in the presence or absence of PHA. The Quantikine Human IL-1ß ELISA kit and Quantikine High Sensitivity Human IL-4 ELISA kit (R&D Systems, Minneapolis, MN) were used. The lower limits of detection for the ELISA systems were 3.9 and 0.25 pg/ml for IL-1ß and IL-4, respectively.

Quantification.

The results from the cytokine expression assay are presented as percent change compared with baseline (i.e., control values without cocoa). Furthermore, to eliminate variations caused by different yields of cDNA, results were normalized against an endogenous reference (i.e., GAPDH, a housekeeping gene). Hence, the amount of cytokine and GAPDH in each experimental sample was determined and the level of cytokine was divided by the level of GAPDH (endogenous reference) to obtain a normalized sample value. To generate relative expression values, the normalized sample value was divided by the normalized baseline value.

Statistical analysis.

ELISA results induced by cocoa were compared with control values (i.e., cells treated without cocoa) using a paired t test with a two-tailed P-value. Percentage changes from PCR analysis were compared with a theoretical value of 0 (i.e., control value without cocoa) using a one-sample t test with a two-tailed P-value. In both cases, significance was taken as P < 0.05.

	RESULTS

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IL-1ß.

Transcription of IL-1ß was assessed in resting and PHA-stimulated PBMC from five subjects after an 8-h treatment with 25 µg/ml concentration of the individual cocoa fractions. Their effects on constitutive IL-1ß gene expression relative to media control were variable among the subjects tested (Fig. 1 ). The heptamer did significantly (P = 0.018) induce gene expression by 16 ± 4% (means ± SEM; n = 5). When stimulated with PHA at 25 µg/ml, small (less than or equal to tetramer) and large (more than or equal to pentamer) cocoa fractions showed contrasting effects on the production of IL-1ß transcripts (Fig. 2 ). The small procyanidins slightly suppressed PHA-induced expression of IL-1ß, whereas the larger procyanidins continually augmented IL-1ß expression in stimulated PBMC cultures from 7 to 19%. However, statistical significance was detected only with the trimer, octamer and nonamer (P = 0.021, 0.005 and 0.0008, respectively).

View larger version (25K): [in this window] [in a new window]   Figure 1. The effect of cocoa procyanidins on IL-1ß gene expression in resting cells. PBMC were incubated with 25 µg/ml concentration of the 10 cocoa fractions for 8 h before total RNA was isolated for subsequent RT-PCR analysis. Data represent percent difference relative to media control (means ± SEM of five subjects). Percentage changes were compared with 0 (i.e., control value without cocoa) using a one-sample t test with a two-tailed P-value. Significance (*) was taken as P < 0.05.

View larger version (30K): [in this window] [in a new window]   Figure 2. The effect of cocoa procyanidins on PHA-induced gene expression of IL-1ß. PBMC were stimulated with PHA (25 µg/ml) in the presence of the 20 cocoa fractions (25 µg/ml) for 8 h before total RNA was isolated for subsequent RT-PCR. Data represent the percent difference relative to PHA control (means ± SEM of five subjects). Percentage changes were compared with 0 (i.e., control value without cocoa) using a one-sample t test with a two-tailed P-value. Significance (*) was taken as P < 0.05.

In our analysis of IL-2, cocoa alone was unable to stimulate constitutive gene expression. However, the pentamer, hexamer and heptamer (at 50 µg/ml) effectively inhibited IL-2 gene expression in PHA-stimulated cells by 61% (P = 0.002), 63% (P = 0.011) and 73% (P = 0.0005), respectively (Fig. 3 ). The monomeric fraction showed little modulation in mitogen-induced production of IL-2 transcripts.

View larger version (52K): [in this window] [in a new window]   Figure 3. The effect of cocoa procyanidins on PHA-induced gene expression of IL-2. PBMC were stimulated with PHA (20 µg/ml) in the presence of each oligomeric cocoa fraction (50 µg/ml) for 8 h before total RNA was isolated for subsequent RT-PCR. Data are compared with PHA alone and are expressed as percent inhibition of control (means ± SEM of five subjects). Percentage changes were compared with 0 (i.e., control value without cocoa) using a one-sample t test with a two-tailed P-value. Significance (*) was taken as P < 0.05.

The samples used for the IL-1ß investigation were also used for the IL-4 analysis. The effects of the cocoa fractions on the constitutive gene expression of IL-4 at 8 h are shown in Table 3 where data represent percent differences relative to media control (means ± SEM; n = 5). Responses varied markedly among the volunteers tested, and of the 10 fractions analyzed, only the pentameric fraction significantly altered IL-4 expression, reducing transcript levels by 65 ± 10% (P = 0.003; Table 3 ). In addition, modulation in IL-4 gene expression was assessed in PHA-stimulated cells. PHA alone markedly augmented the gene expression of IL-4 transcripts relative to media control (300 ± 65%; data not shown). The dimeric and trimeric fractions inhibited PHA-induced expression of IL-4 by 46 ± 7% and 40 ± 8%, respectively (P = 0.003 and P = 0.008, respectively; Table 3 ). The hexameric through decameric procyanidin fractions enhanced this effect by reducing IL-4 expression by 76 ± 7% (P = 0.0003), 71 ± 11% (P = 0.003), 85 ± 5% (P < 0.0001), 86 ± 4% (P < 0.0001) and 79 ± 12% (P = 0.003), respectively. The monomer, tetramer and pentamer exhibited fluctuating levels of IL-4 transcripts when coincubated with PHA.

	DISCUSSION

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Our analysis of IL-1ß suggests that the incubation of PBMC with any one of the cocoa procyanidin fractions can effectively modulate constitutive gene expression of this cytokine. However, the magnitude of this stimulation was observed to vary between test subjects. This variation at the transcript level of IL-1ß in healthy volunteers is expected. It is possible that the stability of the mRNA may have been a factor in the variability of IL-1ß transcription. One study estimated the half-life of IL-1ß mRNA to be 2 h in the absence and 4.5 h in the presence of lipopolysaccharide, a potent stimulator of IL-1ß production (Schindler et al. 1990 ). In addition, our investigation did not focus on kinetic responses of cocoa procyanidins, nor did we vary their dosage. Therefore, fluctuating levels of IL-1ß transcripts are conceivable. Moreover, adherence of PBMC to the plastic culture plates, which has been reported to induce IL-1ß mRNA expression (Schindler et al. 1990 ), could have masked any minor effects of the procyanidins. Thus, it appears as though small-molecular-weight cocoa fractions (monomers through tetramers) are responsible for the down-regulation of IL-1ß production in stimulated PBMC, whereas the larger oligomers (hexamers though decamers) increase synthesis.

T-cell activation is an important step in the initiation of an immunological response. Normally, resting T cells do not contain constitutive levels of IL-2 (Smith 1988 ). However, stimulation of T cells by mitogenic lectins (i.e., PHA) activates a cascade of signaling events, including the up-regulation of transcription factors (i.e., nuclear factor-B, activator protein-1 and nuclear factor-AT), all leading to the transcription and secretion of IL-2 (Han et al. 1998 , Zhao et al. 1999 ). The eventual interaction between IL-2 and its receptor promotes T cells to undergo cell cycle progression (Crabtree 1989 ). Thus, the regulation of IL-2 at the level of transcription is critically involved in the control of T-cell expansion and the normal immune response (Mao et al. 1999 , Smith 1988 ).

None of the cocoa fractions tested in our study were able to stimulate transcription of IL-2 in resting PBMC. However, when comparing the oligomeric procyanidins with its monomer fraction, we observed markedly different effects in stimulated PBMC. The larger oligomers (heptamer, octamers and decamers) were shown to be cytotoxic to the cells in a majority of the cell cultures. In contrast, the pentamer, hexamer, and heptamer fractions displayed an average inhibition that slightly increased with degree of polymerization, although the individual values from each subject did not all correlate to this proposed interpretation (data not shown). The monomeric fraction demonstrated an overall effect of 7 ± 15% inhibition that is not statistically significant (P = 0.65). Thus, it appears from this work as though procyanidin oligomers (pentamers–heptamers) are responsible for the inhibition of PHA-induced stimulation of IL-2 in PBMC.

In this work, there was some suggestion that the larger fractions (octamer–decamer) may stimulate IL-4 secretion, but this was not supported by the gene expression data in which, in general, decreases in the constitutive IL-4 expression were observed in the presence of oligomeric procyanidins. Nevertheless, the overall effects of procyanidin fractions on constitutive IL-4 secretion were of small magnitude with protein levels not reaching 2 pg/ml. In contrast, PHA-stimulated cells (which secreted >20 pg/ml IL-4) had their gene expression and secretion of IL-4 inhibited by oligomeric procyanidins. Again, similar to our previous findings (Mao et al. 1999 , Mao et al. unpublished observations), the hexamer, heptamer and octamer fractions were most active, whereas data were inconsistent for pentamer (gene expression) and nonamer and decamer (protein secretion) fractions. These latter observations may in part be due to the difficulty in obtaining ultrapure fractions of nonamers and decamers, which, in this study, were only 60 and 40% pure, respectively. It is unclear why results with pentameric procyanidins were inconsistent, at least for gene expression, but it may be that the intracellular activity of these phenolic fractions requires them to be small enough for effective cellular uptake but large enough for efficient scavenging of reactive oxygen species. Nevertheless, given the relatively consistent findings among studies that procyanidin fractions between pentamers and octamers inhibit cytokine release of stimulated cells, these fractions should now be tested in an in vivo model.

The mechanism of action of polyphenols in inhibiting cytokine transcription is not clear. However, studies have so far identified a reduction in intracellular reactive oxygen species, which activate nuclear transcription factor-B, and an inhibition of cytoplasmic calcium ions in response to these polyphenols (Rotondo et al. 1998 , Sato et al. 1997 ). Intracellular reactive oxygen species may activate nuclear factor-B (Rotondo et al. 1998 ), which in turn mediates transcription and secretion of many cytokines. Hence, the inhibition of intracellular reactive oxygen species by procyanidins could lead to a reduction in gene transcription and protein synthesis of a number of cytokines. Such a mechanism may explain the apparently disparate biological properties of dietary polyphenols; the scavenging of intracellular free radicals could inhibit the transcription of nuclear transcription factors, thereby altering cellular cytokine profiles and affecting responses to carcinogens and inflammatory mediators.

The identification of a polyphenolic fraction that modulates cytokine production is important because this may have implications for other ingested plant products and could even allow the isolation of this fraction for pharmacological studies. Nevertheless, we studied the individual procyanidin fractions to delineate which of these was responsible for the observed inhibition. Considering that it is difficult to reconcile the efficacy of these oligomeric procyanidins, we did not investigate synergy of the individual species. The implications of these data are that cocoa, as a potential immune modulator, may have therapeutic advantages in human disease that involve activation of the immune system, such as eczema and arthritis. These possibilities should be investigated using further in vitro and select in vivo models.

	FOOTNOTES

 
¹ Presented at the symposium "Chocolate: Modern Science Investigates an Ancient Medicine," held February 19, 2000, during the 2000 Annual Meeting and Science Innovation Exposition at the American Association for the Advancement of Science in Washington, D.C. Published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were John W. Erdman, Jr., University of Illinois at Urbana-Champaign; Jo Wills, Mars, United Kingdom and D’Ann Finley, University of California, Davis.

² This work was supported in part by grants from the National Institutes of Health (DK-35747) and Mars Incorporated.

³ To whom reprint requests should be addressed.

⁴ Abbreviations used: IL, interleukin; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBSS, Hanks’ balanced salt solution; PBMC, peripheral blood mononuclear cell(s); PCR, polymerase chain reaction; PHA, phytohemagglutinin; RT, reverse transcription.

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