The role of interleukin-12 in the heavy metal-elicited immunomodulation: relevance of various evaluation methods
Identifieur interne : 000499 ( Pmc/Corpus ); précédent : 000498; suivant : 000500The role of interleukin-12 in the heavy metal-elicited immunomodulation: relevance of various evaluation methods
Auteurs : Nasr Ya HemdanSource :
- Journal of Occupational Medicine and Toxicology (London, England) [ 1745-6673 ] ; 2008.
Abstract
Increasing evidence exists that heavy metals modulate T helper cell (Th) responses and thereby elicit various pathological manifestation. Interleukin (IL)-12, a crucial innate cytokine, was found to be regulated by such xenobiotic agents. This study aimed at testing whether IL-12 profiles may be indicative of heavy metals-induced immunomodulation.
Human immunocompetent cells, activated either by monoclonal antibodies or heat-killed
Modulation of IL-12 profiles in Cd- or Hg-exposed human PBMC was dose-dependent and significantly related to IFN-γ levels as well as to the Th1- or Th2-polarized responses. Similarly, skewing the Th1/Th2 ratios
It can be inferred that: (i) IL-12 profiles alone may represent a relevant indicator of heavy metal-induced immune modulation; (ii) evaluating cytokine profiles by CBA is relevant and can adequately replace other methods such as ELISA and RT-PCR in basic research as well as in immune diagnostics; and (iii) targeting IL-12 in therapeutic approaches may be promising to modify Th1/Th2-associated immune disorders.
Url:
DOI: 10.1186/1745-6673-3-25
PubMed: 18990205
PubMed Central: 2585571
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PMC:2585571Le document en format XML
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<author><name sortKey="Hemdan, Nasr Ya" sort="Hemdan, Nasr Ya" uniqKey="Hemdan N" first="Nasr Ya" last="Hemdan">Nasr Ya Hemdan</name>
<affiliation><nlm:aff id="I1">Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="I2">Institute of Clinical Immunology and Transfusion Medicine – IKIT, Faculty of Medicine, University of Leipzig, Germany</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="I3">Department of Zoology, Faculty of Science, University of Alexandria, Egypt</nlm:aff>
</affiliation>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">The role of interleukin-12 in the heavy metal-elicited immunomodulation: relevance of various evaluation methods</title>
<author><name sortKey="Hemdan, Nasr Ya" sort="Hemdan, Nasr Ya" uniqKey="Hemdan N" first="Nasr Ya" last="Hemdan">Nasr Ya Hemdan</name>
<affiliation><nlm:aff id="I1">Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="I2">Institute of Clinical Immunology and Transfusion Medicine – IKIT, Faculty of Medicine, University of Leipzig, Germany</nlm:aff>
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<affiliation><nlm:aff id="I3">Department of Zoology, Faculty of Science, University of Alexandria, Egypt</nlm:aff>
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>Increasing evidence exists that heavy metals modulate T helper cell (Th) responses and thereby elicit various pathological manifestation. Interleukin (IL)-12, a crucial innate cytokine, was found to be regulated by such xenobiotic agents. This study aimed at testing whether IL-12 profiles may be indicative of heavy metals-induced immunomodulation.</p>
</sec>
<sec sec-type="methods"><title>Methods</title>
<p>Human immunocompetent cells, activated either by monoclonal antibodies or heat-killed <italic>Salmonella enterica</italic>
, were cultured in the absence or presence of cadmium (Cd) acetate or mercuric (Hg) chloride. <italic>In vivo </italic>
experiments were set up where BALB/c mice were exposed to sub-lethal doses of Cd or Hg salts for 3 or 5 weeks. Cytotoxicity was assessed by MTT-reduction assay. Modulation of cytokine profiles was evaluated by enzyme-linked immunosorbent assay (ELISA), cytometric bead-based array (CBA) and real-time polymerase chain reaction (RT-PCR); the relevance of these methods of cytokine quantification was explored.</p>
</sec>
<sec><title>Results</title>
<p>Modulation of IL-12 profiles in Cd- or Hg-exposed human PBMC was dose-dependent and significantly related to IFN-γ levels as well as to the Th1- or Th2-polarized responses. Similarly, skewing the Th1/Th2 ratios <italic>in vivo </italic>
correlated significantly with up- or down-regulation of IL-12 levels in both cases of investigated metals.</p>
</sec>
<sec><title>Conclusion</title>
<p>It can be inferred that: (i) IL-12 profiles alone may represent a relevant indicator of heavy metal-induced immune modulation; (ii) evaluating cytokine profiles by CBA is relevant and can adequately replace other methods such as ELISA and RT-PCR in basic research as well as in immune diagnostics; and (iii) targeting IL-12 in therapeutic approaches may be promising to modify Th1/Th2-associated immune disorders.</p>
</sec>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Occup Med Toxicol</journal-id>
<journal-title>Journal of Occupational Medicine and Toxicology (London, England)</journal-title>
<issn pub-type="epub">1745-6673</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">18990205</article-id>
<article-id pub-id-type="pmc">2585571</article-id>
<article-id pub-id-type="publisher-id">1745-6673-3-25</article-id>
<article-id pub-id-type="doi">10.1186/1745-6673-3-25</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research</subject>
</subj-group>
</article-categories>
<title-group><article-title>The role of interleukin-12 in the heavy metal-elicited immunomodulation: relevance of various evaluation methods</article-title>
</title-group>
<contrib-group><contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Hemdan</surname>
<given-names>Nasr YA</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<xref ref-type="aff" rid="I2">2</xref>
<xref ref-type="aff" rid="I3">3</xref>
<email>nasr.hemdan@izi.fraunhofer.de</email>
</contrib>
</contrib-group>
<aff id="I1"><label>1</label>
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany</aff>
<aff id="I2"><label>2</label>
Institute of Clinical Immunology and Transfusion Medicine – IKIT, Faculty of Medicine, University of Leipzig, Germany</aff>
<aff id="I3"><label>3</label>
Department of Zoology, Faculty of Science, University of Alexandria, Egypt</aff>
<pub-date pub-type="collection"><year>2008</year>
</pub-date>
<pub-date pub-type="epub"><day>6</day>
<month>11</month>
<year>2008</year>
</pub-date>
<volume>3</volume>
<fpage>25</fpage>
<lpage>25</lpage>
<ext-link ext-link-type="uri" xlink:href="http://www.occup-med.com/content/3/1/25"></ext-link>
<history><date date-type="received"><day>17</day>
<month>6</month>
<year>2008</year>
</date>
<date date-type="accepted"><day>6</day>
<month>11</month>
<year>2008</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2008 Hemdan; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2008</copyright-year>
<copyright-holder>Hemdan; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0"></ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
<pmc-comment>
Hemdan
YA
Nasr
nasr.hemdan@izi.fraunhofer.de
The role of interleukin-12 in the heavy metal-elicited immunomodulation: relevance of various evaluation methods
2008 Journal of Occupational Medicine and Toxicology 3(1): 25-. (2008) 1745-6673(2008)3:1<25> urn:ISSN:1745-6673 </pmc-comment>
</license>
</permissions>
<abstract><sec><title>Background</title>
<p>Increasing evidence exists that heavy metals modulate T helper cell (Th) responses and thereby elicit various pathological manifestation. Interleukin (IL)-12, a crucial innate cytokine, was found to be regulated by such xenobiotic agents. This study aimed at testing whether IL-12 profiles may be indicative of heavy metals-induced immunomodulation.</p>
</sec>
<sec sec-type="methods"><title>Methods</title>
<p>Human immunocompetent cells, activated either by monoclonal antibodies or heat-killed <italic>Salmonella enterica</italic>
, were cultured in the absence or presence of cadmium (Cd) acetate or mercuric (Hg) chloride. <italic>In vivo </italic>
experiments were set up where BALB/c mice were exposed to sub-lethal doses of Cd or Hg salts for 3 or 5 weeks. Cytotoxicity was assessed by MTT-reduction assay. Modulation of cytokine profiles was evaluated by enzyme-linked immunosorbent assay (ELISA), cytometric bead-based array (CBA) and real-time polymerase chain reaction (RT-PCR); the relevance of these methods of cytokine quantification was explored.</p>
</sec>
<sec><title>Results</title>
<p>Modulation of IL-12 profiles in Cd- or Hg-exposed human PBMC was dose-dependent and significantly related to IFN-γ levels as well as to the Th1- or Th2-polarized responses. Similarly, skewing the Th1/Th2 ratios <italic>in vivo </italic>
correlated significantly with up- or down-regulation of IL-12 levels in both cases of investigated metals.</p>
</sec>
<sec><title>Conclusion</title>
<p>It can be inferred that: (i) IL-12 profiles alone may represent a relevant indicator of heavy metal-induced immune modulation; (ii) evaluating cytokine profiles by CBA is relevant and can adequately replace other methods such as ELISA and RT-PCR in basic research as well as in immune diagnostics; and (iii) targeting IL-12 in therapeutic approaches may be promising to modify Th1/Th2-associated immune disorders.</p>
</sec>
</abstract>
</article-meta>
</front>
<body><sec><title>Background</title>
<p>Human activities have led to global dispersion of heavy metals like cadmium and mercury into the environment [<xref ref-type="bibr" rid="B1">1</xref>
-<xref ref-type="bibr" rid="B3">3</xref>
], and hence heavy metal pollution has attained high visibility in the public arena and increasingly become a major scientific concern. Most of the early studies addressed the effects of relative higher doses that are not relevant to most populations. Despite intensive recent studies with regard to the immune system as a target of heavy metals, some inconsistencies have been evolved especially regarding the effects of low-dose exposure. The reported impairments ranged from minor insidious or transitional changes up to occasional death of the exposed subjects [<xref ref-type="bibr" rid="B1">1</xref>
-<xref ref-type="bibr" rid="B8">8</xref>
]. This variation in susceptibility has been attributed to genetic variability, variety of methodological regimes such as the metal form, duration of exposure, the dosage applied as well as the activity of the cells [<xref ref-type="bibr" rid="B9">9</xref>
]. The biological indicator or the read-out system used to evaluate the changes elicited can not be ruled out.</p>
<p>Cytokines are very important agents to detect modulations of the immune system. Due to their sensitivity, they can be modulated at lower doses than other arms of the immune system [<xref ref-type="bibr" rid="B10">10</xref>
]. Measuring cytokines and other soluble mediators involved in immune regulation has been the focus of researchers for over three decades. Several reports on the quantification of ever-growing number of analytes in a single reaction vessel have been emerged using fluorescent-labeled microspheres, the protein bead array (PBA)-based assays [<xref ref-type="bibr" rid="B11">11</xref>
-<xref ref-type="bibr" rid="B17">17</xref>
]. The importance of assessing cytokines is evidenced by the fact that they form the basis of a sophisticated cellular communication network for normal as well as modulated immune responses. Experiments using isolated human cells or cytokine gene knock-out mice have been proven to be useful for evaluating the regulation of immunocompetent cells in response to infection or following exposure to heavy metals. Central to these regulatory agents are CD4<sup>+ </sup>
T helper (Th) cells, that are known to differentiate into at least two subsets, Th1 and Th2, both in mice [<xref ref-type="bibr" rid="B18">18</xref>
] and in humans [<xref ref-type="bibr" rid="B19">19</xref>
]. These cells secrete different but overlapping sets of cytokines: the common precursor Th0 secrete cytokines including IL-2, IL-4, and IFN-γ; Th1 cells produce IL-2, IFN-γ, TNF-β, and low levels of IL-10 (only in human); and Th2 cells IL-4, IL-5, IL-9, IL-10, IL-13 [<xref ref-type="bibr" rid="B20">20</xref>
-<xref ref-type="bibr" rid="B22">22</xref>
] and IL-6 [<xref ref-type="bibr" rid="B23">23</xref>
]. Differentiation of Th0 is believed to be a consequence of several cellular influences, such as the cytokine milieu. While differentiation into Th1 cells requires the presence of IL-12, Th2 cells response warrants the presence of IL-4 [<xref ref-type="bibr" rid="B24">24</xref>
]. Moreover, both types cross-regulate each other in a variety of ways [<xref ref-type="bibr" rid="B20">20</xref>
,<xref ref-type="bibr" rid="B24">24</xref>
,<xref ref-type="bibr" rid="B25">25</xref>
]. The dominance of one of these subsets results in either a predominantly cellular (Th1-mediated) or an antibody (Th2-mediated) response [<xref ref-type="bibr" rid="B9">9</xref>
]. Therefore, the inclination of the Th1/Th2 balance indicated by cytokine profiles changes and/or cytokine-dependent regulatory pathways have been often considered to evaluate heavy metal-mediated immunomodulation and in risk assessment studies [<xref ref-type="bibr" rid="B26">26</xref>
].</p>
<p>The recent years were fruitful with the definition of new Th subsets including the CXCR5<sup>+ </sup>
and CD4<sup>+</sup>
CD25<sup>+</sup>
T<sub>reg </sub>
cells [<xref ref-type="bibr" rid="B27">27</xref>
]. T<sub>reg </sub>
cells includes, among others, constitutive CD4<sup>+</sup>
CD25<sup>+ </sup>
FoxP3<sup>+ </sup>
T<sub>reg </sub>
cells, Type 1 T regulatory cells (Tr1) and Th3 cells, characterized by production of high levels of IL-10 and TGF-β [<xref ref-type="bibr" rid="B28">28</xref>
]. Most recently, studies of experimental autoimmune encephalomyelitis and adjuvant-induced arthritis have pointed to the importance of IL-17-producing (Th17) cells, [<xref ref-type="bibr" rid="B27">27</xref>
]. The optimal recipe to differentiate into Th17 remains so far unclear; yet, together with TGF-β [<xref ref-type="bibr" rid="B29">29</xref>
-<xref ref-type="bibr" rid="B31">31</xref>
], a group of cytokines produced by LPS-activated DC, namely IL-1, IL-6, and TNF-α favor Th17 differentiation [<xref ref-type="bibr" rid="B32">32</xref>
]. Other cytokines seem to share inducing or maintaining different Th cell subsets, e.g. IL-23, due to its p40 unit, just like IL-12, in addition to induction of Th1, is known to be important for the survival of Th17 cells [<xref ref-type="bibr" rid="B33">33</xref>
].</p>
<p>Interleukin-12 is a 70-kDa heterodimeric (composed of covalently linked p35/p40) pro-inflammatory cytokine produced mostly by phagocytic cells and to some degree by B cells. It is considered to date the most critical factor for skewing the immune response towards a Th1 type, and thereby exerts a substantial stimulatory influence on host responses to intracellular pathogens [<xref ref-type="bibr" rid="B34">34</xref>
-<xref ref-type="bibr" rid="B36">36</xref>
]. However, there are clues that IL-12, in synergy with IL-4, supports the long-term proliferation and maturation of resting neonatal CD4<sup>+ </sup>
T cells into IFN-γ – or IL-4-producing cells, and transiently increases the production of both cytokines by human Th2-like cell clones [<xref ref-type="bibr" rid="B37">37</xref>
]. The present study was conducted to examine the relationship between heavy metal-induced IL-12 profile modifications and the accompanying Th1/Th2-polarized responses of cultured human peripheral blood mononuclear cells (PBMC). To this end, two pathways of cell activation were adopted, either through monoclonal antibodies (mAb: anti-CD3/-CD28/-CD40) or heat-killed <italic>Salmonella enterica </italic>
serovar Enteritidis (hkSE). Furthermore, the association of IL-12 with a skewed <italic>in vivo </italic>
immune response was also investigated in BALB/c mice exposed to heavy metals in a pathogen-free environment. In order to test the metal effects at the protein as well as mRNA levels and to evaluate the relevance of various traditionally-used methods, cytokines were assessed by ELISA, cytometric bead-based array (CBA) and RT-PCR.</p>
</sec>
<sec sec-type="methods"><title>Methods</title>
<sec><title>Preparation of cells</title>
<p>Cells used in this study were isolated from buffy coats of healthy blood donors from the Blood Bank of Leipzig University Clinic, Germany. The experiments were approved by the local authorities and informed consents of participating subjects were obtained. Ficoll Paque (Amersham Biosciences, Freiburg, Germany) density gradient centrifugation at 22°C and 400× g [<xref ref-type="bibr" rid="B38">38</xref>
] was applied to separate PBMC. Cells were finally washed with isotonic phosphate-buffered saline (Invitrogen, Karlsruhe, Germany).</p>
</sec>
<sec><title>Phenotypes of PBMC</title>
<p>Analysis of the human PBMC subsets was performed using surface marker staining and flow cytometry as previously described [<xref ref-type="bibr" rid="B26">26</xref>
]. Briefly, sets of mAbs against surface antigens were used (BD Biosciences, Heidelberg, Germany): Simultest™ CD3/CD8, CD3/CD4, CD3/CD19, CD3/CD16CD56, Simultest™ Leucogate™ (CD45/CD14) and Simultest™ Control γ1/γ2a (IgG<sub>1 </sub>
FITC/IgG<sub>2a </sub>
PE). Lymphocytes were gated in the forward-side scatter plot and various cell subsets were estimated.</p>
</sec>
<sec><title>Cell cultures</title>
<p>Isolated human PBMC were suspended in HybridoMed DIF 1000 medium (Biochrom, Berlin, Germany) containing 10 μg/mL gentamycin, 100 μg/mL streptomycin, 100 U/mL penicillin and 10% FCS (HyClone Laboratories, Logan, UT, USA) and incubated at 37°C/5% CO<sub>2 </sub>
and finally cultured (1 × 10<sup>6 </sup>
cells/1 mL/well) in 48-well microtiter plates (Greiner Bio-one GmbH, Nürtingen, Germany). Cells were activated with agonistic CD3 (OKT3, mouse IgG1, Ortho Biotech, Bridgewater, NJ, USA), CD28 (clone CD28.2, mouse IgG1. Beckman-Coulter, Krefeld, Germany) and CD40 (clone B-B20, Trinova Biochem, Gießen, Germany) mAb, 100 ng/mL each, or with hkSE (1.25 × 10<sup>5 </sup>
CFU/mL; ade<sup>-</sup>
, his<sup>-</sup>
, SALMOVAC SE<sup>®</sup>
, Impfstoffwerk Dessau-Tornau, Rosslau, Germany).</p>
</sec>
<sec><title>Application of heavy metals</title>
<p>Cd acetate and Hg chloride (Sigma, Steinheim, Germany) were dissolved in de-ionized water (stock solution = 10 g/L). Immediately before application, serial dilutions were made using the same culture medium and added to the culture plates to constitute a final concentration of Cd or Hg ranged from 0.5 ng to 50 μg/mL. Control samples were established, where cells received only either mAb or hkSE.</p>
</sec>
<sec><title>Cell vitality assay</title>
<p>Vitality response of human PBMC to mAb or hkSE was evaluated by 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazoliumbromide (MTT)-reduction test as previously reported [<xref ref-type="bibr" rid="B26">26</xref>
].</p>
</sec>
<sec><title>Detection of cytokine release by ELISA</title>
<p>Following 24-hr incubation, cytokine levels were determined in culture media by commercially available ELISA kits for IL-1β, IL-10, IL-12p70, IL-4, IL-6, IFN-γ, and TNF-α (OptEIA™ Kits; BD Biosciences) with a lower detection limit of 4 pg/mL, as previously described [<xref ref-type="bibr" rid="B26">26</xref>
,<xref ref-type="bibr" rid="B39">39</xref>
].</p>
</sec>
<sec><title>Animal model</title>
<p>Female wild-type BALB/c mice (9–12-week old, 23–27 g; purchased from Charles River, Sulzfeld, Germany), were allocated to this study. The animals were handled according to the animal protection laws of local authorities on the use of animals in research. Upon receipt from the vendor, mice were quarantined and acclimatized for 2 weeks prior to use. Animals were placed in filter-topped plastic cages (6–8 per cage) in exposure rooms with automatic 12:00-hr light 12:00-hr dark cycle, and allowed free access to food and water. Rooms were maintained at 22°C and 40–60% humidity.</p>
<p>Mice were assigned in groups of 12 mice and were injected intraperitoneally every third day with isotonic NaCl solution (controls) or with Cd or Hg (1.25 mg/kg body weight). Mice were monitored daily for food and water consumption and for signs of morbidity. Control mice were sacrificed at day 21 and other mice were sacrificed following 3 or 5 weeks of exposure to the heavy metal. At sacrifice, blood samples were collected and sera were separated by centrifuging the samples at 2,000 × g/37°C for 10 min.</p>
</sec>
<sec><title>Cytokine analysis using cytometric bead array</title>
<p>Serum cytokine levels were determined using CBA. The procedure was carried out according to the manufacturer's instruction (CBA™, BD Biosciences, San Jose, CA), modified by Tarnok <italic>et al</italic>
. [<xref ref-type="bibr" rid="B40">40</xref>
] to measure the cytokines using 25 μL serum. Here, the test samples were further reduced to use 20 μL of 1:2 diluted sera. Briefly, 4 μL of each mouse capture bead suspension were mixed for each sample, and 20 μL of mixed beads were transferred to each assay tube. Standard dilutions or test samples were added to the appropriate tubes (20 μL/tube), PE detection reagent (20 μL) was added and the tubes were incubated for 2 hr in dark at RT. Samples were washed with 1 mL wash buffer and centrifuged at 200 × g for 5 min. Finally, test buffer (250 mL) was added, and samples were analyzed on FACSCalibur (BD Biosciences) using the supplied cytometer setup beads and the CellQuest™ Software.</p>
</sec>
<sec><title>Evaluating Cytokines mRNA Expression</title>
<sec><title>Isolation of RNA and digestion of genomic DNA</title>
<p>Following collection of organs, 1/4 spleen was transferred into 1 mL RNA <italic>later</italic>
<sup>® </sup>
(Ambion, Germany), preserved overnight at 4°C and thereafter at -20°C. About 50 mg spleen was homogenized in 1 mL TriFast reagent (peqlab Biotechnologie, Erlangen, Germany) and RNA was separated according to the manufacturer's instruction. RNA probes were treated with DNA-free™ reagent (Ambion, Dresden, Germany) to eliminate genomic DNA.</p>
</sec>
<sec><title>Reverse transcription and purification of cDNA</title>
<p>Reverse transcription of RNA was conducted using AMV reverse transcriptase (Promega, Madison, USA), and cDNA probes were purified using QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instruction and were stored at -80°C.</p>
</sec>
<sec><title>Real-time PCR</title>
<p>Amplification of cDNA was performed on LightCycler using LightCycler-FastStart DNA Master<sup>PLUS </sup>
SYBR Green I<sup>® </sup>
(Roche, Mannheim, Germany). A 20-μL reaction mixture including 4 μL water, 2 μL primers, 9 μL DNA Master Mix and 5 μL (~0.2 μg) cDNA template was applied into the capillary and shortly centrifuged (3,000 × g). Reactions started by an initial activation step for 10 min at 95°C, and each following cycle started by a denaturation step for 15 s at 94°C. Specific cycle conditions were applied to validate the specificity for each primer pair. Products were controlled by SYBR Green dissociation curves, by agarose gel electrophoresis and via DNA-sequencing of the PCR products to ensure that only a single target-specific product of the appropriate length was amplified. GAPDH was chosen as a reference house-keeping gene as it showed amplification efficiency similar to those of other cytokine genes. The sequences of sense (s) and antisense (as) mouse primers were: (i) <italic>GAPDH </italic>
(NM_008084): s: 5'-CCC ACT AAC ATC AAA TGG GG-3'; as: 5'-CCT TCC ACA ATG CCA AAG TT-3'; (ii) <italic>IFN-γ </italic>
(NM_008337): s: 5'-AGC GGC TGA CTG AAC TCA GAT TGA AG-3', as: 5'-GTC ACA GTT TTC AGC TGT ATA GGG-3'; (iii) <italic>IL-12p40 </italic>
(NM_008352): s: 5'-GGA AGC ACG GCA GCA GAA TA-3', as: 5'-AAC TTG AGG GAG AAG TAG GAA TGG-3' and (iv) <italic>IL-4 </italic>
(NM_021283): s: 5'-TCA ACC CCC AGC TAG TTG TC-3' and as: 5'-TCT GTG GTG TTC TTC GTT GC-3'. The crossing point for each reaction was determined using the Second Derivative Maximum algorithm and the arithmetic baseline adjustment using the LightCycler software. REST<sup>© </sup>
software (downloaded from <ext-link ext-link-type="uri" xlink:href="http://www.gene-quantification.info/"></ext-link>
) was used to estimate cytokine mRNA expression as well as the up- or down-regulation factor for each gene relative to controls and based on an efficiency corrected mathematical model and a pair-wise fixed reallocation randomization test [<xref ref-type="bibr" rid="B41">41</xref>
].</p>
</sec>
</sec>
<sec><title>Statistical analysis</title>
<p>Experiments were carried out in triplicates. Data analysis was performed using Statistica 5.1 software (Statsoft, Hamburg, Germany). Variations among cytokine profiles of different donors were tested using a nonparametric ANOVA, the Kruskal-Wallis test, followed by Dunn's post-test. Wilcoxon's rank test for paired samples was used to analyze the differences between controls and heavy metal-treated cells in human PBMC cultures. The correlation between cytokine production and heavy metal doses a well as between cytokine pairs was analyzed using Spearman's rank correlation test. Unless otherwise indicated, significance was determined at p < 0.05.</p>
</sec>
</sec>
<sec><title>Results</title>
<sec><title>Distribution of different cell subsets</title>
<p>Isolated human PBMC were used in the <italic>in vitro </italic>
studies. Of the total cells, the percentages of lymphocytes, monocytes, granulocytes as well as the lymphocyte subpopulations were in the normal range as previously reported [<xref ref-type="bibr" rid="B26">26</xref>
].</p>
</sec>
<sec><title>Exposure to heavy metals significantly modulates IL-12 profiles of human PBMC</title>
<p>Profiling IL-12 of mAb-stimulated PBMC revealed that 24-hr exposure to Cd significantly decreased IL-12p70 release (p < 0.01, Wilcoxon's test) at all tested doses from 0.5 ng/mL to 50 μg/mL (Fig. <xref ref-type="fig" rid="F1">1A</xref>
); additional file <xref ref-type="supplementary-material" rid="S1">1</xref>
. Values of IL-12p70 in the supernatants of mAb-stimulated control cells ranged from 26 to 616 pg/mL with a mean value of 124 pg/mL. The inhibition of IL-12p70 levels was dose-dependent in the 14 subjects tested with a Spearman's r values ranged between -0.72 and -0.99 (p < 0.001).</p>
<fig position="float" id="F1"><label>Figure 1</label>
<caption><p><bold>Levels of IL-12p70 released by human PBMC exposed to Cd acetate for 24 hr</bold>
. Human PBMC were stimulated either by mAb (anti-CD3/-CD28/-CD40) (A) or heat-killed <italic>Salmonella enterica </italic>
(hkSE) (B). Data represent the values of 14 and 10 samples. For each donor, the mean values of 3 replicates were used to estimate the percentage relative to control cells (assigned to 100%). The horizontal lines represent the medians. Symbols above each plot show whether, cytokine release was significantly stimulated (#) or suppressed (*) compared to controls (Wilcoxon's Rank Sum test for paired samples; p < 0.05).</p>
</caption>
<graphic xlink:href="1745-6673-3-25-1"></graphic>
</fig>
<p>On the other hand, activating cells with hkSE has significantly induced production of IL-12p70 at Cd doses ranged from 0.5 to 50 ng/mL (Fig <xref ref-type="fig" rid="F1">1B</xref>
); cytokine levels tended to decrease with the increase in Cd levels as previously reported by our group [<xref ref-type="bibr" rid="B42">42</xref>
]. Considering the mean of the tested subjects (n = 14), the increase in IL-12p70 levels revealed a strong negative correlation with Cd doses from 0.5 to 50 ng/mL (Spearman's r value = -0.72; p <0.01). Control cells activated by hkSE revealed IL-12p70 values ranged between 22 and 413 pg/mL (mean = 127).</p>
<p>Samples of the same blood volunteers were used to evaluate the levels of Th1, Th2 as well as pro-inflammatory cytokine TNF-α, IL-1β and IL-6 following exposure to Cd at low and moderate doses. Results demonstrated that IFN-γ levels increased significantly up to toxic doses, and then declined with the increase of Cd toxicity as recently reported [<xref ref-type="bibr" rid="B42">42</xref>
]. Supernatants of mAb- or hkSE-activated control cells revealed values of IFN-γ ranged from 50 to 15900 pg/mL (mean = 3310) and from 56 to 2120 pg/mL (mean = 589), respectively.</p>
<p>Similarly, exposure to Hg at doses ranged from 15 pg to 50 μg/mL significantly decreased IL-12p70 release (Fig. <xref ref-type="fig" rid="F2">2</xref>
), also in a dose-dependent manner (Spearman's r range of -0.64 to -0.97; p < 0.01); additional file <xref ref-type="supplementary-material" rid="S2">2</xref>
. In hkSE-stimulated cells, however, the release of IL-12p70 was significantly increased at Hg doses from 15 pg/mL to 0.5 μg/mL, but decreased again with the increase of Hg toxicity. Again, the behavior of IL-12p70 secretion was positively correlated to the levels of IFN-γ previously reported by our group [<xref ref-type="bibr" rid="B26">26</xref>
].</p>
<fig position="float" id="F2"><label>Figure 2</label>
<caption><p><bold>Levels of IL-12p70 released by human PBMC exposedto HgCl<sub>2 </sub>
for 24 hr</bold>
. Cells were stimulated either by mAb (anti-CD3/anti-CD28/anti-CD40) (A) or hkSE (B). Data represent the values of 12 and 10 donors in both panels respectively; the horizontal lines represent the medians. Symbols above each plot show whether, cytokine release was significantly stimulated (#) or suppressed (*) compared to the controls (Wilcoxon's Rank Sum test for paired samples; p < 0.05).</p>
</caption>
<graphic xlink:href="1745-6673-3-25-2"></graphic>
</fig>
</sec>
<sec><title>Correlation between IL-12 and IFN-γ levels <italic>in vitro</italic>
</title>
<p>In both cases of cell activation, testing the relationship between the levels of IL-12p70 and IFN-γ released by cells exposed to either Cd or Hg indicates that they are positively correlated (Table <xref ref-type="table" rid="T1">1</xref>
). In cases where a significant correlation was evident, correlation test revealed Spearman's r values ranging from 0.63 to 0.97 and from 0.7 to 0.9 under stimulation by mAb or hkSE, respectively. Considering the mean values of the measured cytokines, r values ranged from 0.93 to 1 and from 0.59 to 0.71 have been emerged indicating a strong positive correlation between levels of both cytokines.</p>
<table-wrap position="float" id="T1"><label>Table 1</label>
<caption><p>The correlation between IFN-γ and IL-12p70 released by human PBMC.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><td></td>
<td align="left" colspan="4"><bold><italic>Cadmium acetate</italic>
</bold>
</td>
<td align="left" colspan="4"><bold><italic>Mercuric chloride</italic>
</bold>
</td>
</tr>
<tr><td></td>
<td colspan="8"><hr></hr>
</td>
</tr>
<tr><td></td>
<td align="left" colspan="2"><underline>mAb-stimulation</underline>
</td>
<td align="left" colspan="2"><underline>hkSE-stimulation</underline>
</td>
<td align="left" colspan="2"><underline>mAb-stimulation</underline>
</td>
<td align="left" colspan="2"><underline>hkSE-stimulation</underline>
</td>
</tr>
<tr><td align="left">Donor</td>
<td align="left">r</td>
<td align="left">p</td>
<td align="left">r</td>
<td align="left">p</td>
<td align="left">r</td>
<td align="left">p</td>
<td align="left">r</td>
<td align="left">p</td>
</tr>
</thead>
<tbody><tr><td align="left">1</td>
<td align="left">0.84</td>
<td align="left">0.0006</td>
<td align="left">0.41</td>
<td align="left">0.1826</td>
<td align="left">0.85</td>
<td align="left"><0.0001</td>
<td align="left">0.72</td>
<td align="left">0.0024</td>
</tr>
<tr><td align="left">2</td>
<td align="left">0.83</td>
<td align="left">0.001</td>
<td align="left">0.51</td>
<td align="left">0.0893</td>
<td align="left">0.96</td>
<td align="left"><0.0001</td>
<td align="left">0.73</td>
<td align="left">0.0022</td>
</tr>
<tr><td align="left">3</td>
<td align="left">0.95</td>
<td align="left"><0.0001</td>
<td align="left">0.06</td>
<td align="left">0.8629</td>
<td align="left">0.94</td>
<td align="left"><0.0001</td>
<td align="left">0.80</td>
<td align="left">0.0004</td>
</tr>
<tr><td align="left">4</td>
<td align="left">0.83</td>
<td align="left">0.001</td>
<td align="left">0.70</td>
<td align="left">0.0114</td>
<td align="left">0.89</td>
<td align="left"><0.0001</td>
<td align="left">0.70</td>
<td align="left">0.0034</td>
</tr>
<tr><td align="left">5</td>
<td align="left">0.90</td>
<td align="left"><0.0001</td>
<td align="left">0.86</td>
<td align="left">0.0003</td>
<td align="left">0.27</td>
<td align="left">0.3278</td>
<td align="left">0.87</td>
<td align="left"><0.0001</td>
</tr>
<tr><td align="left">6</td>
<td align="left">0.90</td>
<td align="left"><0.0001</td>
<td align="left">0.72</td>
<td align="left">0.0082</td>
<td align="left">0.96</td>
<td align="left"><0.0001</td>
<td align="left">0.85</td>
<td align="left"><0.0001</td>
</tr>
<tr><td align="left">7</td>
<td align="left">0.64</td>
<td align="left">0.0261</td>
<td align="left">0.50</td>
<td align="left">0.1006</td>
<td align="left">0.91</td>
<td align="left"><0.0001</td>
<td align="left">0.44</td>
<td align="left">0.0983</td>
</tr>
<tr><td align="left">8</td>
<td align="left">0.90</td>
<td align="left"><0.0001</td>
<td align="left">0.87</td>
<td align="left">0.0003</td>
<td align="left">0.63</td>
<td align="left">0.0121</td>
<td align="left">0.86</td>
<td align="left"><0.0001</td>
</tr>
<tr><td align="left">9</td>
<td align="left">0.93</td>
<td align="left"><0.0001</td>
<td align="left">0.76</td>
<td align="left">0.0045</td>
<td align="left">0.16</td>
<td align="left">0.5499</td>
<td align="left">0.26</td>
<td align="left">0.3549</td>
</tr>
<tr><td align="left">10</td>
<td align="left">0.63</td>
<td align="left">0.0283</td>
<td align="left">0.83</td>
<td align="left">0.001</td>
<td align="left">0.84</td>
<td align="left"><0.0001</td>
<td align="left">0.90</td>
<td align="left"><0.0001</td>
</tr>
<tr><td align="left">11</td>
<td align="left">0.97</td>
<td align="left"><0.0001</td>
<td></td>
<td></td>
<td align="left">0.23</td>
<td align="left">0.4201</td>
<td></td>
<td></td>
</tr>
<tr><td align="left">12</td>
<td align="left">0.94</td>
<td align="left"><0.0001</td>
<td></td>
<td></td>
<td align="left">0.78</td>
<td align="left">0.0007</td>
<td></td>
<td></td>
</tr>
<tr><td align="left">13</td>
<td align="left">0.80</td>
<td align="left">0.0016</td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr><td align="left">14</td>
<td align="left">0.94</td>
<td align="left"><0.0001</td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr><td colspan="9"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Mean</italic>
</td>
<td align="left"><italic>1</italic>
</td>
<td align="left"><italic><0.0001</italic>
</td>
<td align="left"><italic>0.59</italic>
</td>
<td align="left"><italic>0.0415</italic>
</td>
<td align="left"><italic>0.93</italic>
</td>
<td align="left"><italic><0.0001</italic>
</td>
<td align="left"><italic>0.71</italic>
</td>
<td align="left"><italic>0.0032</italic>
</td>
</tr>
</tbody>
</table>
<table-wrap-foot><p>Human peripheral blood mononuclear cells (PBMC) were cultured for 24 hr in absence or presence of cadmium acetate or mercuric chloride and stimulated either by monoclonal antibodies (mAb: anti-CD3/anti-CD28/anti-CD40) or heat-killed <italic>Salmonella enterica </italic>
serovar Enteritidis (hkSE). Cytokine values were evaluated in cell supernatants using ELISA; r represents Spearman correlation values estimated for each sample. The last raw represents the Spearman correlation between the mean values of IFN-γ and IL-12p70 of all investigated donors.</p>
</table-wrap-foot>
</table-wrap>
</sec>
<sec><title>Serum cytokine profiles of Cd- and Hg-exposed mice</title>
<p>Figure <xref ref-type="fig" rid="F3">3</xref>
shows serum profiles of IL-12p70 and IFN-γ analyzed by CBA in sera of mice exposed intraperitoneally to Cd acetate or Hg chloride for 3 or 5 weeks. Representative plots of control (PBS-treated) and of Cd-exposed mice are represented in figure <xref ref-type="fig" rid="F4">4</xref>
. The results demonstrate that following exposure to Cd for 5 weeks, the levels of IL-12p70 were positively correlated to the up- or down-regulation of the Th1 cytokine IFN-γ (r = 0.81, p < 0.01), which was in turn negatively correlated to the Th2 cytokines IL-4 (r value = -0.74, p < 0.01) and IL-5 (r value= -0.61, p < 0.01); data not shown. Although the increase in IL-12p70 serum profile of mice exposed for 3 weeks to Cd was not significant in comparison to control mice, exposure to Cd for 5 weeks significantly increased the levels of serum IFN-γ that coincided with the increase in IL-12p70 levels. However, in 3 out of a total of 12 examined mice, where IL-12 release was induced, as in case of 5-week exposure to Cd acetate (Fig. <xref ref-type="fig" rid="F4">4C</xref>
), both of Th1 and Th2 cytokines were also elevated comparable to control mice. In case of Hg-exposure, on the other hand, the decrease in IFN-γ levels, due to the progressive exposure to the metal beyond the first three weeks, was significantly correlated to the decrease in IL-12p70 throughout the same exposure period (r = 0.89, p < 0.01).</p>
<fig position="float" id="F3"><label>Figure 3</label>
<caption><p><bold>Serum profiles of IL-12p70 and IFN-γ in BALB/c mice exposed to heavy metals</bold>
. Data represent cytokines levels following 3- (3-w) or 5-week (5-w) exposure to cadmium (Cd) acetate or mercuric (Hg) chloride as evaluated by cytometric bead array. The horizontal bars represent the medians of 12 samples. The dotted line represents the control level.</p>
</caption>
<graphic xlink:href="1745-6673-3-25-3"></graphic>
</fig>
<fig position="float" id="F4"><label>Figure 4</label>
<caption><p><bold>Serum cytokine levels as evaluated by cytometric bead array (CBA)</bold>
. Representative plots show serum cytokine levels of control mice injected every other day for 3 weeks with isotonic saline solution (A) and following 3-week (B) or 5-week (C) exposure to cadmium acetate. Animals were sacrificed and blood samples were collected by heart puncture. Following separation of sera, cytokine content was evaluated by CBA as described in Materials and Methods.</p>
</caption>
<graphic xlink:href="1745-6673-3-25-4"></graphic>
</fig>
</sec>
<sec><title>Cytokine gene expression of Cd- and Hg-exposed mice</title>
<p>Results of RT-PCR revealed that following exposure of BALB/c mice to salts of Cd or Hg, mRNA expression of IL-12p40, IL-4 and IFN-γ has been significantly modified relative to control mice (Fig. <xref ref-type="fig" rid="F5">5</xref>
). Exposure to Cd for 5 weeks resulted in a significant 74-fold increase in IL-12p40 gene expression, whilst 5-week exposure to Hg decreased its expression 3 folds relative to controls; significant differences between the levels of IL-12p40 in both metals and during both time lapses have been indicated.</p>
<fig position="float" id="F5"><label>Figure 5</label>
<caption><p><bold>Relative cytokine mRNA expression in splenocytes of heavy metal-exposed BALB/c mice</bold>
. Data represent mRNA relative expression values ± SEM in spleen cells of mice exposed to Cd acetate or Hg chloride for 3 or 5 weeks (3-w, 5-w). Data were analyzed using the REST<sup>© </sup>
software as described in Materials and Methods. Control values were assigned to 1, values of relative gene expression > or < 1 indicate the up- or down-regulation of this gene relative to control mice. The asterisks (*) indicate significance relative to controls as obtained by the pair-wise fixed reallocation randomization test at p < 0.05; the horizontal lines indicate significant differences between the connected groups.</p>
</caption>
<graphic xlink:href="1745-6673-3-25-5"></graphic>
</fig>
<p>As an indicator for both of Th1 and Th2 responses, expression of IFN-γ and IL-4 mRNA was assessed and compared with the serum proteins. In concordance with the increase in IL-12p40 mRNA expression ratios, exposure to Cd resulted in 14- and 684-fold increase in IFN-γ following 3- or 5-week exposition, respectively. Similarly, following 5-week exposure to Cd, the relative expression of IL-12p40 mRNA was positively correlated to that of IFN-γ mRNA (r value = 0.98, p < 0.001).</p>
<p>In case of Hg-exposed mice, on the other hand, 5-week exposition yielded 15-fold decrease in IFN-γ mRNA. The increase in IFN-γ mRNA expression relative to control mice was also correlated to the increase in IL-12p40 mRNA expression. Here, recalling the decrease in IFN-γ mRNA in the 5-week Hg-group relative to the 3-week group, the exposure period constituted a significant factor. Furthermore, it is evident that both metals behave differently. Interesting was the expression of IL-4 mRNA, where a 4-fold increase of this cytokine gene was evident following 5-week exposure to Cd. Exposure to Hg either for 3 or 5 weeks caused 43- or 24-fold increase in IL-4 mRNA expression relative to control mice, respectively. Following 5-week exposure to Hg, IL-12p40 mRNA expression ratios were positively correlated to those of IFN-γ (r value = 0.7, p < 0.001) and negatively correlated to those of IL-4 mRNA (r value = 0.85, p < 0.0001).</p>
</sec>
</sec>
<sec><title>Discussion</title>
<p>To extend our knowledge on heavy metal-elicited immune modulation, the current study was conducted to address the modification of IL-12 profile and its relation to the Th1/Th2-polarized immune responses. Previous reports on this issue indicated that Cd- or Hg-induced immunomodulation was dependent on cell stimulation pathways [<xref ref-type="bibr" rid="B26">26</xref>
,<xref ref-type="bibr" rid="B42">42</xref>
]. Moreover, <italic>in vivo </italic>
data using BALB/c mice revealed a distinct Th1/Th2 pattern following exposure to sub-lethal doses of Cd acetate or Hg chloride for different periods [<xref ref-type="bibr" rid="B43">43</xref>
]. These effects were evident even at low concentrations of both metals, at which only insignificant changes in cell vitality were detected, indicating that "cytokines", may serve as more sensitive indicators for heavy metal-induced immune modulation than other parameters such as vitality response. The current data indicates the synergism between IL-12 and IFN-γ and reveals distinct patterns of response to heavy metals depending on the metal ion itself as well as duration of exposure.</p>
<p>In this study, cytokines were quantified by ELISA, CBA, and RT-PCR. In accordance with previous reports [<xref ref-type="bibr" rid="B14">14</xref>
,<xref ref-type="bibr" rid="B26">26</xref>
,<xref ref-type="bibr" rid="B40">40</xref>
], the three methods revealed comparable results indicating their relevance to evaluate the immune response. Comparing the serum cytokine profiles of heavy metal-exposed mice with mRNA expression ratios revealed an overall concordance between them (spearman's r value in all cases ≥ 0.81, p < 0.01). This infers that these metals exert a modulatory effect at the pre-transcriptional stages of cytokine production and indicates the relevance of CBA and RT-PCR to evaluate such modulation. However, evaluating cytokines by the PBA-based assays offers definitive advantages including being quantitative. RT-PCR is quantitative as well [<xref ref-type="bibr" rid="B41">41</xref>
], but only using other reference house-keeping gene for calibration, which can be even affected by the treatment. Another advantage of the PBA over enzyme immunoassays (EIAs) or ELISA and RT-PCR is that it is performed with a smaller sample volume to measure multiple analytes simultaneously in a single sample [<xref ref-type="bibr" rid="B12">12</xref>
,<xref ref-type="bibr" rid="B14">14</xref>
-<xref ref-type="bibr" rid="B16">16</xref>
,<xref ref-type="bibr" rid="B40">40</xref>
]. This factor reduces execution time and costs, even adds more precision to the assay and is an advantage when the sample volume is limited as in pediatrics, disease surveillance or in experiments using animal models [<xref ref-type="bibr" rid="B12">12</xref>
,<xref ref-type="bibr" rid="B16">16</xref>
,<xref ref-type="bibr" rid="B40">40</xref>
]. Therefore, and because of the longer processing time and the elaborate sample preparation as well as the costs of RT-PCR techniques, quantifying cytokines using the PBA offers a great ease of performance. In comparison with ELISA, the wide calibration range of the PBA offers a great flexibility [<xref ref-type="bibr" rid="B40">40</xref>
]. Moreover, analyzing the performance of an established HIV-1 PBA for detection of plasma Abs revealed its advantage in the resolution of weakly reactive samples comparable to two EIAs and Western blot technique [<xref ref-type="bibr" rid="B16">16</xref>
], the former being able to offer the high throughput of EIA combined with the individual protein testing capacity of the Western blotting.</p>
<p>However, PBA, like other assays, has its limitations. One of the most consistent observations is that, although it is more flexible, user friendly and cost-effective than ELISA, both still detect non-overlapping parameter populations. Another limitation may be the interference with some serum proteins as some samples revealed different results when measured with different titrations. Replacing such <italic>low-sensitive </italic>
parameters with more reliable agents and applying sample dilutions may relieve such problems. Here, the use of 20 μL of 1:2 diluted serum samples allowed quantification of cytokines adequately. Taken together, the PBA may be ranked as the most relevant to quantify cytokines in animal experiments and for immune surveillance in heavy metal-exposed subjects. Optimizing the assay may include acquiring a large number of individual bead measurements at low ligand concentrations [<xref ref-type="bibr" rid="B13">13</xref>
]. This will improve cost benefits and raise the reproducibility of PBA. A recently established analytical workflow [<xref ref-type="bibr" rid="B17">17</xref>
] may offer the possibility for a subsequent population-level analysis to test hypotheses generated by donor-profile analysis and thereby adding scientific insights to allow the practical distinguishing between different cohorts of the same population using such a promising tool of immune detection.</p>
<p>The results described here are consistent with previous reports and support the reciprocal interplay between Th1 and Th2 immune responses. The early preference of the Th differentiation was believed to depend on the balance between IL-12 (Th1 responses) and IL-4 (Th2 responses) profiles [<xref ref-type="bibr" rid="B44">44</xref>
]. Infection models of <italic>Listeria</italic>
, <italic>Leishmania</italic>
, <italic>Schistosoma </italic>
and <italic>Toxoplasma </italic>
inferred that IL-12 induced Th1 immune responses and inhibited Th2 cells differentiation [<xref ref-type="bibr" rid="B45">45</xref>
,<xref ref-type="bibr" rid="B46">46</xref>
]. Similarly, in peripheral type 1 response, IL-12 induced the synthesis of IFN-γ, IL-2, and TNF-α [<xref ref-type="bibr" rid="B47">47</xref>
]; TNF-α implicated in mediating the effects of IL-12 on NK cells. Furthermore, IL-12 and TNF-α were found to activate IFN-γ-producing NK and T cells [<xref ref-type="bibr" rid="B34">34</xref>
,<xref ref-type="bibr" rid="B48">48</xref>
]. Therefore, administration of IL-12 reversed the Th2-dominant heavy metal-induced inhibition of host defense and boosted the resistance of untreated mice marked by elevation of IFN-γ profiles [<xref ref-type="bibr" rid="B35">35</xref>
]. Similar effects were elucidated in wild-type as well as in IL-12 KO and IL-4 KO BALB/c mice [<xref ref-type="bibr" rid="B49">49</xref>
,<xref ref-type="bibr" rid="B50">50</xref>
], where IL-12 was indispensable for the induction of a protective Th1 response against <italic>Salmonella </italic>
infection. The possible promotion of IL-12 in Th1 effector function was confirmed by the observation that fully differentiated Th1 cells continue to express functional IL-12 receptor [<xref ref-type="bibr" rid="B51">51</xref>
]. The loss of the transcription factor GATA-3 expression by developing Th1 cells [<xref ref-type="bibr" rid="B52">52</xref>
], an augmenting Th2-cell-specific factor [<xref ref-type="bibr" rid="B53">53</xref>
], constitute an additional evidence for the requirement of a continued IL-12 signaling in the maintenance of a Th1 response.</p>
<p>However, there is evidence that diminishes the role of IL-12 in favoring a Th1-type immune response and indicates that the differentiation into either Th1 or Th2 phenotype does not constitute a simple dichotomy. For example, although <italic>Leishmania major </italic>
evokes an IL-4 response in IL-12p40 KO mice [<xref ref-type="bibr" rid="B54">54</xref>
], the diminished Th1 response observed in STAT4-deficient mice following infection with <italic>L. major </italic>
or <italic>Toxoplasma gondii </italic>
was not accompanied by a compensatory Th2 response [<xref ref-type="bibr" rid="B55">55</xref>
]. Furthermore, neither the depletion of IFN-γ in IL-12 KO mice nor the neutralization of IL-12 in IFN-γ R KO mice affected the corona virus-elicited type 1 cytokine pattern [<xref ref-type="bibr" rid="B56">56</xref>
], and hence IL-12 was proposed not to be essential for the generation of a Th1-polarized response and that viruses may selectively induce IFN-γ production even in the absence of IL-12. Other cytokines such as IFN-α [<xref ref-type="bibr" rid="B57">57</xref>
] or other host factors or signaling elements, e.g. MyD88 [<xref ref-type="bibr" rid="B58">58</xref>
], may compensate for the lack of endogenous IL-12 or IFN-γ in determining Th cell differentiation in such viral infections. Recently, it has been found that bacterially-stimulated DC can not produce IL-12 unless the cultures also contain other cells or cytokines such as IFN-γ [<xref ref-type="bibr" rid="B59">59</xref>
]. Taken together, these data recall the importance of IL-12 for Th1 competence rather than cell priming <italic>per se </italic>
[<xref ref-type="bibr" rid="B60">60</xref>
], i.e. it is involved in the maintenance of an ongoing Th1 response rather than part of the initial immunological decision-making process. In the current study, the finding that production of IL-12 depends on cytokines from previously activated T cells may indicate a direct effect of heavy metals on Th1- or Th2-priming cytokine milieu (e.g. IFN-γ or IL-4). According to the current results inferring the close association between IL-12p70 and the induction of Th1 response, it seems that IL-12 may play a central role in attaining a polarized Th1/Th2 response in heavy metal-exposed individuals even in the absence of pathogenic antigens. This may raise the importance of IL-12 as a target for therapeutic approaches to re-stabilize the immune response in heavy metal-exposed individuals.</p>
</sec>
<sec><title>Conclusion</title>
<p>Overall, consistent with previous reports, the results described herein that elucidate the synergism between IL-12 and the Th1 cytokine IFN-γ, which in its turn was shown to be reciprocally regulated by IL-4 (a key Th2-cytokine), may dominate IL-12 as a relevant indicator for heavy metal-induced immune modulation. Moreover, the association of modulating Th cell differentiation and IL-12 levels support the use of this cytokine as a target for therapeutic approaches to re-establish a normal balance that may modify Th1- or Th2-dependent diseases. Finally, measuring cytokines by CBA seems to be the most relevant and cost-effective methodology to evaluate immune modulation.</p>
</sec>
<sec><title>Competing interests</title>
<p>The author declares that they have no competing interests.</p>
</sec>
<sec><title>Authors' contributions</title>
<p>NYAH is the sole author and is responsible for the entire manuscript.</p>
</sec>
<sec sec-type="supplementary-material"><title>Supplementary Material</title>
<supplementary-material content-type="local-data" id="S1"><caption><title>Additional file 1</title>
<p><bold>ELISA_IL12_CdAc.</bold>
This sheet represents ELISA raw data of IL-12p70 (pg/mL) released by human PBMC following exposure to serial concentrations of cadmium acetate.</p>
</caption>
<media xlink:href="1745-6673-3-25-S1.xls" mimetype="application" mime-subtype="vnd.ms-excel"><caption><p>Click here for file</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="S2"><caption><title>Additional file 2</title>
<p><bold>ELISA_IL12_HgCl2.</bold>
This sheet represents ELISA raw data of IL-12p70 (pg/mL) released by human PBMC following exposure to serial concentrations of mercuric chloride.</p>
</caption>
<media xlink:href="1745-6673-3-25-S2.xls" mimetype="application" mime-subtype="vnd.ms-excel"><caption><p>Click here for file</p>
</caption>
</media>
</supplementary-material>
</sec>
</body>
<back><ack><sec><title>Acknowledgements</title>
<p>My great indebtedness is directed to Professor Ulrich Sack (IKIT, University of Leipzig, Germany) for providing the materials required for this study, for his supervision and reviewing the manuscript. For their substantial experimental support, thanks are also directed to Dr. Irina Lehmann (UFZ, Dept. of Environmental Immunology, Leipzig-Halle), Dr. Gunnar Wichmann (HNO Laboratories, University Hospital, Leipzig) and Dr. Jörg Lehmann (IZI, Leipzig). Dr. J. Lehmann has kindly provided the <italic>Salmonella </italic>
strain used in this study. NYAH was supported by the German Academic Exchange Service – DAAD and the Egyptian Ministry of Higher Education.</p>
</sec>
</ack>
<ref-list><ref id="B1"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Bertin</surname>
<given-names>G</given-names>
</name>
<name><surname>Averbeck</surname>
<given-names>D</given-names>
</name>
</person-group>
<article-title>Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review)</article-title>
<source>Biochimie</source>
<year>2006</year>
<volume>88</volume>
<fpage>1549</fpage>
<lpage>1559</lpage>
<pub-id pub-id-type="pmid">17070979</pub-id>
<pub-id pub-id-type="doi">10.1016/j.biochi.2006.10.001</pub-id>
</citation>
</ref>
<ref id="B2"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Guzzi</surname>
<given-names>G</given-names>
</name>
<name><surname>La Porta</surname>
<given-names>CA</given-names>
</name>
</person-group>
<article-title>Molecular mechanisms triggered by mercury</article-title>
<source>Toxicology</source>
<year>2008</year>
<volume>244</volume>
<fpage>1</fpage>
<lpage>12</lpage>
<pub-id pub-id-type="pmid">18077077</pub-id>
<pub-id pub-id-type="doi">10.1016/j.tox.2007.11.002</pub-id>
</citation>
</ref>
<ref id="B3"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Clarkson</surname>
<given-names>TW</given-names>
</name>
<name><surname>Vyas</surname>
<given-names>JB</given-names>
</name>
<name><surname>Ballatori</surname>
<given-names>N</given-names>
</name>
</person-group>
<article-title>Mechanisms of mercury disposition in the body</article-title>
<source>Am J Ind Med</source>
<year>2007</year>
<volume>50</volume>
<fpage>757</fpage>
<lpage>764</lpage>
<pub-id pub-id-type="pmid">17477364</pub-id>
<pub-id pub-id-type="doi">10.1002/ajim.20476</pub-id>
</citation>
</ref>
<ref id="B4"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Clarkson</surname>
<given-names>TW</given-names>
</name>
<name><surname>Magos</surname>
<given-names>L</given-names>
</name>
</person-group>
<article-title>The toxicology of mercury and its chemical compounds</article-title>
<source>Crit Rev Toxicol</source>
<year>2006</year>
<volume>36</volume>
<fpage>609</fpage>
<lpage>662</lpage>
<pub-id pub-id-type="pmid">16973445</pub-id>
<pub-id pub-id-type="doi">10.1080/10408440600845619</pub-id>
</citation>
</ref>
<ref id="B5"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Naz</surname>
<given-names>H</given-names>
</name>
<name><surname>Baig</surname>
<given-names>N</given-names>
</name>
<name><surname>Haider</surname>
<given-names>S</given-names>
</name>
<name><surname>Haleem</surname>
<given-names>DJ</given-names>
</name>
</person-group>
<article-title>Subchronic treatment with mercuric chloride suppresses immune response, elicits behavioral deficits and increases brain serotonin and dopamine metabolism in rats</article-title>
<source>Pak J Pharm Sci</source>
<year>2008</year>
<volume>21</volume>
<fpage>7</fpage>
<lpage>11</lpage>
<pub-id pub-id-type="pmid">18166511</pub-id>
</citation>
</ref>
<ref id="B6"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Houston</surname>
<given-names>MC</given-names>
</name>
</person-group>
<article-title>The role of mercury and cadmium heavy metals in vascular disease, hypertension, coronary heart disease, and myocardial infarction</article-title>
<source>Altern Ther Health Med</source>
<year>2007</year>
<volume>13</volume>
<fpage>S128</fpage>
<lpage>S133</lpage>
<pub-id pub-id-type="pmid">17405690</pub-id>
</citation>
</ref>
<ref id="B7"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Thompson</surname>
<given-names>J</given-names>
</name>
<name><surname>Bannigan</surname>
<given-names>J</given-names>
</name>
</person-group>
<article-title>Cadmium: toxic effects on the reproductive system and the embryo</article-title>
<source>Reprod Toxicol</source>
<year>2008</year>
<volume>25</volume>
<fpage>304</fpage>
<lpage>315</lpage>
<pub-id pub-id-type="pmid">18367374</pub-id>
<pub-id pub-id-type="doi">10.1016/j.reprotox.2008.02.001</pub-id>
</citation>
</ref>
<ref id="B8"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Huff</surname>
<given-names>J</given-names>
</name>
<name><surname>Lunn</surname>
<given-names>RM</given-names>
</name>
<name><surname>Waalkes</surname>
<given-names>MP</given-names>
</name>
<name><surname>Tomatis</surname>
<given-names>L</given-names>
</name>
<name><surname>Infante</surname>
<given-names>PF</given-names>
</name>
</person-group>
<article-title>Cadmium-induced cancers in animals and in humans</article-title>
<source>Int J Occup Environ Health</source>
<year>2007</year>
<volume>13</volume>
<fpage>202</fpage>
<lpage>212</lpage>
<pub-id pub-id-type="pmid">17718178</pub-id>
</citation>
</ref>
<ref id="B9"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tchounwou</surname>
<given-names>PB</given-names>
</name>
<name><surname>Ayensu</surname>
<given-names>WK</given-names>
</name>
<name><surname>Ninashvili</surname>
<given-names>N</given-names>
</name>
<name><surname>Sutton</surname>
<given-names>D</given-names>
</name>
</person-group>
<article-title>Environmental exposure to mercury and its toxicopathologic implications for public health</article-title>
<source>Environ Toxicol</source>
<year>2003</year>
<volume>18</volume>
<fpage>149</fpage>
<lpage>175</lpage>
<pub-id pub-id-type="pmid">12740802</pub-id>
<pub-id pub-id-type="doi">10.1002/tox.10116</pub-id>
</citation>
</ref>
<ref id="B10"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Shen</surname>
<given-names>X</given-names>
</name>
<name><surname>Lee</surname>
<given-names>K</given-names>
</name>
<name><surname>Konig</surname>
<given-names>R</given-names>
</name>
</person-group>
<article-title>Effects of heavy metal ions on resting and antigen-activated CD4(+) T cells</article-title>
<source>Toxicology</source>
<year>2001</year>
<volume>169</volume>
<fpage>67</fpage>
<lpage>80</lpage>
<pub-id pub-id-type="pmid">11696410</pub-id>
<pub-id pub-id-type="doi">10.1016/S0300-483X(01)00483-8</pub-id>
</citation>
</ref>
<ref id="B11"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tarnok</surname>
<given-names>A</given-names>
</name>
<name><surname>Emmrich</surname>
<given-names>F</given-names>
</name>
</person-group>
<article-title>Immune consequences of pediatric and adult cardiovascular surgery: report of the 7th Leipzig workshop</article-title>
<source>Cytometry B Clin Cytom</source>
<year>2003</year>
<volume>54</volume>
<fpage>54</fpage>
<lpage>57</lpage>
<pub-id pub-id-type="pmid">12827668</pub-id>
<pub-id pub-id-type="doi">10.1002/cyto.b.10018</pub-id>
</citation>
</ref>
<ref id="B12"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Morgan</surname>
<given-names>E</given-names>
</name>
<name><surname>Varro</surname>
<given-names>R</given-names>
</name>
<name><surname>Sepulveda</surname>
<given-names>H</given-names>
</name>
<name><surname>Ember</surname>
<given-names>JA</given-names>
</name>
<name><surname>Apgar</surname>
<given-names>J</given-names>
</name>
<name><surname>Wilson</surname>
<given-names>J</given-names>
</name>
<etal></etal>
</person-group>
<article-title>Cytometric bead array: a multiplexed assay platform with applications in various areas of biology</article-title>
<source>Clinical Immunology</source>
<year>2004</year>
<volume>110</volume>
<fpage>252</fpage>
<lpage>266</lpage>
<pub-id pub-id-type="pmid">15047203</pub-id>
<pub-id pub-id-type="doi">10.1016/j.clim.2003.11.017</pub-id>
</citation>
</ref>
<ref id="B13"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Jacobson</surname>
<given-names>JW</given-names>
</name>
<name><surname>Oliver</surname>
<given-names>KG</given-names>
</name>
<name><surname>Weiss</surname>
<given-names>C</given-names>
</name>
<name><surname>Kettman</surname>
<given-names>J</given-names>
</name>
</person-group>
<article-title>Analysis of individual data from bead-based assays ("bead arrays")</article-title>
<source>Cytometry A</source>
<year>2006</year>
<volume>69</volume>
<fpage>384</fpage>
<lpage>390</lpage>
<pub-id pub-id-type="pmid">16615139</pub-id>
</citation>
</ref>
<ref id="B14"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Prunet</surname>
<given-names>C</given-names>
</name>
<name><surname>Montange</surname>
<given-names>T</given-names>
</name>
<name><surname>Vejux</surname>
<given-names>A</given-names>
</name>
<name><surname>Laubriet</surname>
<given-names>A</given-names>
</name>
<name><surname>Rohmer</surname>
<given-names>JF</given-names>
</name>
<name><surname>Riedinger</surname>
<given-names>JM</given-names>
</name>
<etal></etal>
</person-group>
<article-title>Multiplexed flow cytometric analyses of pro- and anti-inflammatory cytokines in the culture media of oxysterol-treated human monocytic cells and in the sera of atherosclerotic patients</article-title>
<source>Cytometry A</source>
<year>2006</year>
<volume>69</volume>
<fpage>359</fpage>
<lpage>373</lpage>
<pub-id pub-id-type="pmid">16604541</pub-id>
</citation>
</ref>
<ref id="B15"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Sack</surname>
<given-names>U</given-names>
</name>
<name><surname>Scheibe</surname>
<given-names>R</given-names>
</name>
<name><surname>Wotzel</surname>
<given-names>M</given-names>
</name>
<name><surname>Hammerschmidt</surname>
<given-names>S</given-names>
</name>
<name><surname>Kuhn</surname>
<given-names>H</given-names>
</name>
<name><surname>Emmrich</surname>
<given-names>F</given-names>
</name>
<etal></etal>
</person-group>
<article-title>Multiplex analysis of cytokines in exhaled breath condensate</article-title>
<source>Cytometry A</source>
<year>2006</year>
<volume>69</volume>
<fpage>169</fpage>
<lpage>172</lpage>
<pub-id pub-id-type="pmid">16496377</pub-id>
</citation>
</ref>
<ref id="B16"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Faucher</surname>
<given-names>S</given-names>
</name>
<name><surname>Martel</surname>
<given-names>A</given-names>
</name>
<name><surname>Sherring</surname>
<given-names>A</given-names>
</name>
<name><surname>Bogdanovic</surname>
<given-names>D</given-names>
</name>
<name><surname>Malloch</surname>
<given-names>L</given-names>
</name>
<name><surname>Kim</surname>
<given-names>JE</given-names>
</name>
<etal></etal>
</person-group>
<article-title>A combined HIV-1 protein bead array for serology assay and T-cell subset immunophenotyping with a hybrid flow cytometer: a step in the direction of a comprehensive multitasking instrument platform for infectious disease diagnosis and monitoring</article-title>
<source>Cytometry B Clin Cytom</source>
<year>2006</year>
<volume>70</volume>
<fpage>179</fpage>
<lpage>188</lpage>
<pub-id pub-id-type="pmid">16615079</pub-id>
</citation>
</ref>
<ref id="B17"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Siebert</surname>
<given-names>JC</given-names>
</name>
<name><surname>Inokuma</surname>
<given-names>M</given-names>
</name>
<name><surname>Waid</surname>
<given-names>DM</given-names>
</name>
<name><surname>Pennock</surname>
<given-names>ND</given-names>
</name>
<name><surname>Vaitaitis</surname>
<given-names>GM</given-names>
</name>
<name><surname>Disis</surname>
<given-names>ML</given-names>
</name>
<etal></etal>
</person-group>
<article-title>An analytical workflow for investigating cytokine profiles</article-title>
<source>Cytometry A</source>
<year>2008</year>
<volume>73</volume>
<fpage>289</fpage>
<lpage>298</lpage>
<pub-id pub-id-type="pmid">18163472</pub-id>
</citation>
</ref>
<ref id="B18"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mosmann</surname>
<given-names>TR</given-names>
</name>
<name><surname>Sad</surname>
<given-names>S</given-names>
</name>
</person-group>
<article-title>The expanding universe of T-cell subsets: Th1, Th2 and more</article-title>
<source>Immunology Today</source>
<year>1996</year>
<volume>17</volume>
<fpage>138</fpage>
<lpage>146</lpage>
<pub-id pub-id-type="pmid">8820272</pub-id>
<pub-id pub-id-type="doi">10.1016/0167-5699(96)80606-2</pub-id>
</citation>
</ref>
<ref id="B19"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Romagnani</surname>
<given-names>S</given-names>
</name>
</person-group>
<article-title>Human TH1 and TH2 subsets: doubt no more</article-title>
<source>Immunol Today</source>
<year>1991</year>
<volume>12</volume>
<fpage>256</fpage>
<lpage>257</lpage>
<pub-id pub-id-type="pmid">1680337</pub-id>
<pub-id pub-id-type="doi">10.1016/0167-5699(91)90120-I</pub-id>
</citation>
</ref>
<ref id="B20"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mosmann</surname>
<given-names>TR</given-names>
</name>
<name><surname>Coffman</surname>
<given-names>RL</given-names>
</name>
</person-group>
<article-title>TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties</article-title>
<source>Annu Rev Immunol</source>
<year>1989</year>
<volume>7</volume>
<fpage>145</fpage>
<lpage>173</lpage>
<pub-id pub-id-type="pmid">2523712</pub-id>
<pub-id pub-id-type="doi">10.1146/annurev.iy.07.040189.001045</pub-id>
</citation>
</ref>
<ref id="B21"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mosmann</surname>
<given-names>TR</given-names>
</name>
<name><surname>Moore</surname>
<given-names>KW</given-names>
</name>
</person-group>
<article-title>The role of IL-10 in crossregulation of TH1 and TH2 responses</article-title>
<source>Immunol Today</source>
<year>1991</year>
<volume>12</volume>
<fpage>A49</fpage>
<lpage>A53</lpage>
<pub-id pub-id-type="pmid">1648926</pub-id>
<pub-id pub-id-type="doi">10.1016/S0167-5699(05)80015-5</pub-id>
</citation>
</ref>
<ref id="B22"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Cherwinski</surname>
<given-names>HM</given-names>
</name>
<name><surname>Schumacher</surname>
<given-names>JH</given-names>
</name>
<name><surname>Brown</surname>
<given-names>KD</given-names>
</name>
<name><surname>Mosmann</surname>
<given-names>TR</given-names>
</name>
</person-group>
<article-title>Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies</article-title>
<source>J Exp Med</source>
<year>1987</year>
<volume>166</volume>
<fpage>1229</fpage>
<lpage>1244</lpage>
<pub-id pub-id-type="pmid">2960769</pub-id>
<pub-id pub-id-type="doi">10.1084/jem.166.5.1229</pub-id>
</citation>
</ref>
<ref id="B23"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yamamoto</surname>
<given-names>M</given-names>
</name>
<name><surname>Yoshizaki</surname>
<given-names>K</given-names>
</name>
<name><surname>Kishimoto</surname>
<given-names>T</given-names>
</name>
<name><surname>Ito</surname>
<given-names>H</given-names>
</name>
</person-group>
<article-title>IL-6 Is Required for the Development of Th1 Cell-Mediated Murine Colitis</article-title>
<source>J Immunol</source>
<year>2000</year>
<volume>164</volume>
<fpage>4878</fpage>
<lpage>4882</lpage>
<pub-id pub-id-type="pmid">10779797</pub-id>
</citation>
</ref>
<ref id="B24"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Seder</surname>
<given-names>RA</given-names>
</name>
<name><surname>Paul</surname>
<given-names>WE</given-names>
</name>
<name><surname>Davis</surname>
<given-names>MM</given-names>
</name>
<name><surname>Fazekas de St Groth </surname>
<given-names>B</given-names>
</name>
</person-group>
<article-title>The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice</article-title>
<source>J Exp Med</source>
<year>1992</year>
<volume>176</volume>
<fpage>1091</fpage>
<lpage>1098</lpage>
<pub-id pub-id-type="pmid">1328464</pub-id>
<pub-id pub-id-type="doi">10.1084/jem.176.4.1091</pub-id>
</citation>
</ref>
<ref id="B25"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Abbas</surname>
<given-names>AK</given-names>
</name>
<name><surname>Murphy</surname>
<given-names>KM</given-names>
</name>
<name><surname>Sher</surname>
<given-names>A</given-names>
</name>
</person-group>
<article-title>Functional diversity of helper T lymphocytes</article-title>
<source>Nature</source>
<year>1996</year>
<volume>383</volume>
<fpage>787</fpage>
<lpage>793</lpage>
<pub-id pub-id-type="pmid">8893001</pub-id>
<pub-id pub-id-type="doi">10.1038/383787a0</pub-id>
</citation>
</ref>
<ref id="B26"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hemdan</surname>
<given-names>NY</given-names>
</name>
<name><surname>Lehmann</surname>
<given-names>I</given-names>
</name>
<name><surname>Wichmann</surname>
<given-names>G</given-names>
</name>
<name><surname>Lehmann</surname>
<given-names>J</given-names>
</name>
<name><surname>Emmrich</surname>
<given-names>F</given-names>
</name>
<name><surname>Sack</surname>
<given-names>U</given-names>
</name>
</person-group>
<article-title>Immunomodulation by mercuric chloride in vitro: application of different cell activation pathways</article-title>
<source>Clin Exp Immunol</source>
<year>2007</year>
<volume>148</volume>
<fpage>325</fpage>
<lpage>337</lpage>
<pub-id pub-id-type="pmid">17302730</pub-id>
<pub-id pub-id-type="doi">10.1111/j.1365-2249.2007.03338.x</pub-id>
</citation>
</ref>
<ref id="B27"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Alber</surname>
<given-names>G</given-names>
</name>
<name><surname>Kamradt</surname>
<given-names>T</given-names>
</name>
</person-group>
<article-title>Regulation of Protective and Pathogenic Th17 Responses</article-title>
<source>curr immunol rev</source>
<year>2007</year>
<volume>3</volume>
<fpage>3</fpage>
<lpage>16</lpage>
<pub-id pub-id-type="doi">10.2174/157339507779802223</pub-id>
</citation>
</ref>
<ref id="B28"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Taylor</surname>
<given-names>A</given-names>
</name>
<name><surname>Verhagen</surname>
<given-names>J</given-names>
</name>
<name><surname>Blaser</surname>
<given-names>K</given-names>
</name>
<name><surname>Akdis</surname>
<given-names>M</given-names>
</name>
<name><surname>Akdis</surname>
<given-names>CA</given-names>
</name>
</person-group>
<article-title>Mechanisms of immune suppression by interleukin-10 and transforming growth factor-beta: the role of T regulatory cells</article-title>
<source>Immunology</source>
<year>2006</year>
<volume>117</volume>
<fpage>433</fpage>
<lpage>442</lpage>
<pub-id pub-id-type="pmid">16556256</pub-id>
<pub-id pub-id-type="doi">10.1111/j.1365-2567.2006.02321.x</pub-id>
</citation>
</ref>
<ref id="B29"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Cua</surname>
<given-names>DJ</given-names>
</name>
<name><surname>Kastelein</surname>
<given-names>RA</given-names>
</name>
</person-group>
<article-title>TGF-[beta], a 'double agent' in the immune pathology war</article-title>
<source>Nat Immunol</source>
<year>2006</year>
<volume>7</volume>
<fpage>557</fpage>
<lpage>559</lpage>
<pub-id pub-id-type="pmid">16715066</pub-id>
<pub-id pub-id-type="doi">10.1038/ni0606-557</pub-id>
</citation>
</ref>
<ref id="B30"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Veldhoen</surname>
<given-names>M</given-names>
</name>
<name><surname>Stockinger</surname>
<given-names>B</given-names>
</name>
</person-group>
<article-title>TGFbeta1, a "Jack of all trades": the link with pro-inflammatory IL-17-producing T cells</article-title>
<source>Trends Immunol</source>
<year>2006</year>
<volume>27</volume>
<fpage>358</fpage>
<lpage>361</lpage>
<pub-id pub-id-type="pmid">16793343</pub-id>
<pub-id pub-id-type="doi">10.1016/j.it.2006.06.001</pub-id>
</citation>
</ref>
<ref id="B31"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Veldhoen</surname>
<given-names>M</given-names>
</name>
<name><surname>Hocking</surname>
<given-names>RJ</given-names>
</name>
<name><surname>Atkins</surname>
<given-names>CJ</given-names>
</name>
<name><surname>Locksley</surname>
<given-names>RM</given-names>
</name>
<name><surname>Stockinger</surname>
<given-names>B</given-names>
</name>
</person-group>
<article-title>TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells</article-title>
<source>Immunity</source>
<year>2006</year>
<volume>24</volume>
<fpage>179</fpage>
<lpage>189</lpage>
<pub-id pub-id-type="pmid">16473830</pub-id>
<pub-id pub-id-type="doi">10.1016/j.immuni.2006.01.001</pub-id>
</citation>
</ref>
<ref id="B32"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Infante-Duarte</surname>
<given-names>C</given-names>
</name>
<name><surname>Horton</surname>
<given-names>HF</given-names>
</name>
<name><surname>Byrne</surname>
<given-names>MC</given-names>
</name>
<name><surname>Kamradt</surname>
<given-names>T</given-names>
</name>
</person-group>
<article-title>Microbial lipopeptides induce the production of IL-17 in Th cells</article-title>
<source>J Immunol</source>
<year>2000</year>
<volume>165</volume>
<fpage>6107</fpage>
<lpage>6115</lpage>
<pub-id pub-id-type="pmid">11086043</pub-id>
</citation>
</ref>
<ref id="B33"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tato</surname>
<given-names>CM</given-names>
</name>
<name><surname>Laurence</surname>
<given-names>A</given-names>
</name>
<name><surname>O'Shea</surname>
<given-names>JJ</given-names>
</name>
</person-group>
<article-title>Helper T cell differentiation enters a new era: Le Roi est mort; vive le Roi!</article-title>
<source>J Exp Med</source>
<year>2006</year>
<volume>203</volume>
<fpage>809</fpage>
<lpage>812</lpage>
<pub-id pub-id-type="pmid">16606679</pub-id>
<pub-id pub-id-type="doi">10.1084/jem.20060522</pub-id>
</citation>
</ref>
<ref id="B34"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Heufler</surname>
<given-names>C</given-names>
</name>
<name><surname>Koch</surname>
<given-names>F</given-names>
</name>
<name><surname>Stanzl</surname>
<given-names>U</given-names>
</name>
<name><surname>Topar</surname>
<given-names>G</given-names>
</name>
<name><surname>Wysocka</surname>
<given-names>M</given-names>
</name>
<name><surname>Trinchieri</surname>
<given-names>G</given-names>
</name>
<etal></etal>
</person-group>
<article-title>Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-gamma production by T helper 1 cells</article-title>
<source>Eur J Immunol</source>
<year>1996</year>
<volume>26</volume>
<fpage>659</fpage>
<lpage>668</lpage>
<pub-id pub-id-type="pmid">8605935</pub-id>
<pub-id pub-id-type="doi">10.1002/eji.1830260323</pub-id>
</citation>
</ref>
<ref id="B35"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kishikawa</surname>
<given-names>H</given-names>
</name>
<name><surname>Song</surname>
<given-names>R</given-names>
</name>
<name><surname>Lawrence</surname>
<given-names>DA</given-names>
</name>
</person-group>
<article-title>Interleukin-12 promotes enhanced resistance to Listeria monocytogenes infection of lead-exposed mice</article-title>
<source>Toxicol Appl Pharmacol</source>
<year>1997</year>
<volume>147</volume>
<fpage>180</fpage>
<lpage>189</lpage>
<pub-id pub-id-type="pmid">9439714</pub-id>
<pub-id pub-id-type="doi">10.1006/taap.1997.8308</pub-id>
</citation>
</ref>
<ref id="B36"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Sathiyaseelan</surname>
<given-names>J</given-names>
</name>
<name><surname>Goenka</surname>
<given-names>R</given-names>
</name>
<name><surname>Parent</surname>
<given-names>M</given-names>
</name>
<name><surname>Benson</surname>
<given-names>RM</given-names>
</name>
<name><surname>Murphy</surname>
<given-names>EA</given-names>
</name>
<name><surname>Fernandes</surname>
<given-names>DM</given-names>
</name>
<etal></etal>
</person-group>
<article-title>Treatment of Brucella-susceptible mice with IL-12 increases primary and secondary immunity</article-title>
<source>Cellular Immunology</source>
<year>2006</year>
<volume>243</volume>
<fpage>1</fpage>
<lpage>9</lpage>
<pub-id pub-id-type="pmid">17184756</pub-id>
<pub-id pub-id-type="doi">10.1016/j.cellimm.2006.10.003</pub-id>
</citation>
</ref>
<ref id="B37"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Delespesse</surname>
<given-names>G</given-names>
</name>
<name><surname>Yang</surname>
<given-names>LP</given-names>
</name>
<name><surname>Shu</surname>
<given-names>U</given-names>
</name>
<name><surname>Byun</surname>
<given-names>DG</given-names>
</name>
<name><surname>Demeure</surname>
<given-names>CE</given-names>
</name>
<name><surname>Ohshima</surname>
<given-names>Y</given-names>
</name>
<etal></etal>
</person-group>
<article-title>Role of interleukin-12 in the maturation of naive human CD4 T cells</article-title>
<source>Ann N Y Acad Sci</source>
<year>1996</year>
<volume>795</volume>
<fpage>196</fpage>
<lpage>201</lpage>
<pub-id pub-id-type="pmid">8958930</pub-id>
<pub-id pub-id-type="doi">10.1111/j.1749-6632.1996.tb52668.x</pub-id>
</citation>
</ref>
<ref id="B38"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ruitenberg</surname>
<given-names>JJ</given-names>
</name>
<name><surname>Mulder</surname>
<given-names>CB</given-names>
</name>
<name><surname>Maino</surname>
<given-names>VC</given-names>
</name>
<name><surname>Landay</surname>
<given-names>AL</given-names>
</name>
<name><surname>Ghanekar</surname>
<given-names>SA</given-names>
</name>
</person-group>
<article-title>VACUTAINER CPT and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples</article-title>
<source>BMC Immunol</source>
<year>2006</year>
<volume>7</volume>
<fpage>11</fpage>
<pub-id pub-id-type="pmid">16725038</pub-id>
<pub-id pub-id-type="doi">10.1186/1471-2172-7-11</pub-id>
</citation>
</ref>
<ref id="B39"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Schwenk</surname>
<given-names>M</given-names>
</name>
<name><surname>Klein</surname>
<given-names>R</given-names>
</name>
<name><surname>Templeton</surname>
<given-names>DM</given-names>
</name>
</person-group>
<article-title>Lymphocyte subpopulations in human exposure to metals (IUPAC Technical Report)</article-title>
<source>Pure and Applied Chemistry</source>
<year>2008</year>
<volume>80</volume>
<fpage>1349</fpage>
<lpage>1364</lpage>
<pub-id pub-id-type="doi">10.1351/pac200880061349</pub-id>
</citation>
</ref>
<ref id="B40"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tarnok</surname>
<given-names>A</given-names>
</name>
<name><surname>Hambsch</surname>
<given-names>J</given-names>
</name>
<name><surname>Chen</surname>
<given-names>R</given-names>
</name>
<name><surname>Varro</surname>
<given-names>R</given-names>
</name>
</person-group>
<article-title>Cytometric bead array to measure six cytokines in twenty-five microliters of serum</article-title>
<source>Clin Chem</source>
<year>2003</year>
<volume>49</volume>
<fpage>1000</fpage>
<lpage>1002</lpage>
<pub-id pub-id-type="pmid">12766012</pub-id>
<pub-id pub-id-type="doi">10.1373/49.6.1000</pub-id>
</citation>
</ref>
<ref id="B41"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Pfaffl</surname>
<given-names>MW</given-names>
</name>
<name><surname>Horgan</surname>
<given-names>GW</given-names>
</name>
<name><surname>Dempfle</surname>
<given-names>L</given-names>
</name>
</person-group>
<article-title>Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR</article-title>
<source>Nucleic Acids Res</source>
<year>2002</year>
<volume>30</volume>
<fpage>e36</fpage>
<pub-id pub-id-type="pmid">11972351</pub-id>
<pub-id pub-id-type="doi">10.1093/nar/30.9.e36</pub-id>
</citation>
</ref>
<ref id="B42"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hemdan</surname>
<given-names>NY</given-names>
</name>
<name><surname>Emmrich</surname>
<given-names>F</given-names>
</name>
<name><surname>Sack</surname>
<given-names>U</given-names>
</name>
<name><surname>Wichmann</surname>
<given-names>G</given-names>
</name>
<name><surname>Lehmann</surname>
<given-names>J</given-names>
</name>
<name><surname>Adham</surname>
<given-names>K</given-names>
</name>
<etal></etal>
</person-group>
<article-title>The in vitro immune modulation by cadmium depends on the way of cell activation</article-title>
<source>Toxicology</source>
<year>2006</year>
<volume>222</volume>
<fpage>37</fpage>
<lpage>45</lpage>
<pub-id pub-id-type="pmid">16510232</pub-id>
<pub-id pub-id-type="doi">10.1016/j.tox.2006.01.026</pub-id>
</citation>
</ref>
<ref id="B43"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hemdan</surname>
<given-names>NY</given-names>
</name>
<name><surname>Emmrich</surname>
<given-names>F</given-names>
</name>
<name><surname>Faber</surname>
<given-names>S</given-names>
</name>
<name><surname>Lehmann</surname>
<given-names>J</given-names>
</name>
<name><surname>Sack</surname>
<given-names>U</given-names>
</name>
</person-group>
<article-title>Alterations of Th1/Th2 Reactivity by Heavy Metals. Possible Consequences Include Induction of Autoimmune Diseases</article-title>
<source>Ann NY Acad Sci</source>
<year>2007</year>
<volume>1109</volume>
<fpage>129</fpage>
<lpage>137</lpage>
<pub-id pub-id-type="pmid">17785298</pub-id>
<pub-id pub-id-type="doi">10.1196/annals.1398.015</pub-id>
</citation>
</ref>
<ref id="B44"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Trinchieri</surname>
<given-names>G</given-names>
</name>
</person-group>
<article-title>Interleukin-12: A Proinflammatory Cytokine with Immunoregulatory Functions that Bridge Innate Resistance and Antigen-Specific Adaptive Immunity</article-title>
<source>Annu Rev Immunol</source>
<year>1995</year>
<volume>13</volume>
<fpage>251</fpage>
<lpage>276</lpage>
<pub-id pub-id-type="pmid">7612223</pub-id>
</citation>
</ref>
<ref id="B45"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Trinchieri</surname>
<given-names>G</given-names>
</name>
</person-group>
<article-title>Role of interleukin-12 in human Th1 response</article-title>
<source>Chem Immunol</source>
<year>1996</year>
<volume>63</volume>
<fpage>14</fpage>
<lpage>29</lpage>
<pub-id pub-id-type="pmid">8934829</pub-id>
</citation>
</ref>
<ref id="B46"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mencacci</surname>
<given-names>A</given-names>
</name>
<name><surname>Cenci</surname>
<given-names>E</given-names>
</name>
<name><surname>Del Sero</surname>
<given-names>G</given-names>
</name>
<name><surname> Fé d'Ostiani</surname>
<given-names>C</given-names>
</name>
<name><surname>Mosci</surname>
<given-names>P</given-names>
</name>
<name><surname>Trinchieri</surname>
<given-names>G</given-names>
</name>
<etal></etal>
</person-group>
<article-title>IL-10 is required for development of protective Th1 responses in IL-12-deficient mice upon Candida albicans infection</article-title>
<source>J Immunol</source>
<year>1998</year>
<volume>161</volume>
<fpage>6228</fpage>
<lpage>6237</lpage>
<pub-id pub-id-type="pmid">9834110</pub-id>
</citation>
</ref>
<ref id="B47"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>McCabe</surname>
<given-names>MJ</given-names>
<suffix>Jr</suffix>
</name>
<name><surname>Singh</surname>
<given-names>KP</given-names>
</name>
<name><surname>Reiners</surname>
<given-names>JJ</given-names>
<suffix>Jr</suffix>
</name>
</person-group>
<article-title>Lead intoxication impairs the generation of a delayed type hypersensitivity response</article-title>
<source>Toxicology</source>
<year>1999</year>
<volume>139</volume>
<fpage>255</fpage>
<lpage>264</lpage>
<pub-id pub-id-type="pmid">10647925</pub-id>
<pub-id pub-id-type="doi">10.1016/S0300-483X(99)00147-X</pub-id>
</citation>
</ref>
<ref id="B48"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Une</surname>
<given-names>C</given-names>
</name>
<name><surname>Andersson</surname>
<given-names>J</given-names>
</name>
<name><surname>Orn</surname>
<given-names>A</given-names>
</name>
</person-group>
<article-title>Role of IFN-alpha/beta and IL-12 in the activation of natural killer cells and interferon-gamma production during experimental infection with Trypanosoma cruzi</article-title>
<source>Clinical and Experimental Immunology</source>
<year>2003</year>
<volume>134</volume>
<fpage>195</fpage>
<lpage>201</lpage>
<pub-id pub-id-type="pmid">14616777</pub-id>
<pub-id pub-id-type="doi">10.1046/j.1365-2249.2003.02294.x</pub-id>
</citation>
</ref>
<ref id="B49"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Lehmann</surname>
<given-names>J</given-names>
</name>
<name><surname>Bellmann</surname>
<given-names>S</given-names>
</name>
<name><surname>Werner</surname>
<given-names>C</given-names>
</name>
<name><surname>Schroder</surname>
<given-names>R</given-names>
</name>
<name><surname>Schutze</surname>
<given-names>N</given-names>
</name>
<name><surname>Alber</surname>
<given-names>G</given-names>
</name>
</person-group>
<article-title>IL-12p40-dependent agonistic effects on the development of protective innate and adaptive immunity against Salmonella enteritidis</article-title>
<source>J Immunol</source>
<year>2001</year>
<volume>167</volume>
<fpage>5304</fpage>
<lpage>5315</lpage>
<pub-id pub-id-type="pmid">11673546</pub-id>
</citation>
</ref>
<ref id="B50"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Lehmann</surname>
<given-names>J</given-names>
</name>
<name><surname>Springer</surname>
<given-names>S</given-names>
</name>
<name><surname>Werner</surname>
<given-names>CE</given-names>
</name>
<name><surname>Lindner</surname>
<given-names>T</given-names>
</name>
<name><surname>Bellmann</surname>
<given-names>S</given-names>
</name>
<name><surname>Straubinger</surname>
<given-names>RK</given-names>
</name>
<etal></etal>
</person-group>
<article-title>Immunity induced with a Salmonella enterica serovar Enteritidis live vaccine is regulated by Th1-cell-dependent cellular and humoral effector mechanisms in susceptible BALB/c mice</article-title>
<source>Vaccine</source>
<year>2006</year>
<volume>24</volume>
<fpage>4779</fpage>
<lpage>4793</lpage>
<pub-id pub-id-type="pmid">16647169</pub-id>
<pub-id pub-id-type="doi">10.1016/j.vaccine.2006.03.024</pub-id>
</citation>
</ref>
<ref id="B51"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Sallusto</surname>
<given-names>F</given-names>
</name>
<name><surname>Lenig</surname>
<given-names>D</given-names>
</name>
<name><surname>Forster</surname>
<given-names>R</given-names>
</name>
<name><surname>Lipp</surname>
<given-names>M</given-names>
</name>
<name><surname>Lanzavecchia</surname>
<given-names>A</given-names>
</name>
</person-group>
<article-title>Two subsets of memory T lymphocytes with distinct homing potentials and effector functions</article-title>
<source>Nature</source>
<year>1999</year>
<volume>401</volume>
<fpage>708</fpage>
<lpage>712</lpage>
<pub-id pub-id-type="pmid">10537110</pub-id>
<pub-id pub-id-type="doi">10.1038/44385</pub-id>
</citation>
</ref>
<ref id="B52"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ouyang</surname>
<given-names>W</given-names>
</name>
<name><surname>Ranganath</surname>
<given-names>SH</given-names>
</name>
<name><surname>Weindel</surname>
<given-names>K</given-names>
</name>
<name><surname>Bhattacharya</surname>
<given-names>D</given-names>
</name>
<name><surname>Murphy</surname>
<given-names>TL</given-names>
</name>
<name><surname>Sha</surname>
<given-names>WC</given-names>
</name>
<etal></etal>
</person-group>
<article-title>Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism</article-title>
<source>Immunity</source>
<year>1998</year>
<volume>9</volume>
<fpage>745</fpage>
<lpage>755</lpage>
<pub-id pub-id-type="pmid">9846495</pub-id>
<pub-id pub-id-type="doi">10.1016/S1074-7613(00)80671-8</pub-id>
</citation>
</ref>
<ref id="B53"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Zheng</surname>
<given-names>W</given-names>
</name>
<name><surname>Flavell</surname>
<given-names>RA</given-names>
</name>
</person-group>
<article-title>The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells</article-title>
<source>Cell</source>
<year>1997</year>
<volume>89</volume>
<fpage>587</fpage>
<lpage>596</lpage>
<pub-id pub-id-type="pmid">9160750</pub-id>
<pub-id pub-id-type="doi">10.1016/S0092-8674(00)80240-8</pub-id>
</citation>
</ref>
<ref id="B54"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Bix</surname>
<given-names>M</given-names>
</name>
<name><surname>Locksley</surname>
<given-names>RM</given-names>
</name>
</person-group>
<article-title>Independent and epigenetic regulation of the interleukin-4 alleles in CD4+ T cells</article-title>
<source>Science</source>
<year>1998</year>
<volume>281</volume>
<fpage>1352</fpage>
<lpage>1354</lpage>
<pub-id pub-id-type="pmid">9721100</pub-id>
<pub-id pub-id-type="doi">10.1126/science.281.5381.1352</pub-id>
</citation>
</ref>
<ref id="B55"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Fitzpatrick</surname>
<given-names>DR</given-names>
</name>
<name><surname>Shirley</surname>
<given-names>KM</given-names>
</name>
<name><surname>Kelso</surname>
<given-names>A</given-names>
</name>
</person-group>
<article-title>Cutting edge: stable epigenetic inheritance of regional IFN-gamma promoter demethylation in CD44highCD8+ T lymphocytes</article-title>
<source>J Immunol</source>
<year>1999</year>
<volume>162</volume>
<fpage>5053</fpage>
<lpage>5057</lpage>
<pub-id pub-id-type="pmid">10227972</pub-id>
</citation>
</ref>
<ref id="B56"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Schijns</surname>
<given-names>VE</given-names>
</name>
<name><surname>Haagmans</surname>
<given-names>BL</given-names>
</name>
<name><surname>Wierda</surname>
<given-names>CM</given-names>
</name>
<name><surname>Kruithof</surname>
<given-names>B</given-names>
</name>
<name><surname>Heijnen</surname>
<given-names>IA</given-names>
</name>
<name><surname>Alber</surname>
<given-names>G</given-names>
</name>
<etal></etal>
</person-group>
<article-title>Mice lacking IL-12 develop polarized Th1 cells during viral infection</article-title>
<source>J Immunol</source>
<year>1998</year>
<volume>160</volume>
<fpage>3958</fpage>
<lpage>3964</lpage>
<pub-id pub-id-type="pmid">9558103</pub-id>
</citation>
</ref>
<ref id="B57"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Belardelli</surname>
<given-names>F</given-names>
</name>
<name><surname>Gresser</surname>
<given-names>I</given-names>
</name>
</person-group>
<article-title>The neglected role of type I interferon in the T-cell response: implications for its clinical use</article-title>
<source>Immunol Today</source>
<year>1996</year>
<volume>17</volume>
<fpage>369</fpage>
<lpage>372</lpage>
<pub-id pub-id-type="pmid">8783497</pub-id>
<pub-id pub-id-type="doi">10.1016/0167-5699(96)10027-X</pub-id>
</citation>
</ref>
<ref id="B58"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Shi</surname>
<given-names>S</given-names>
</name>
<name><surname>Nathan</surname>
<given-names>C</given-names>
</name>
<name><surname>Schnappinger</surname>
<given-names>D</given-names>
</name>
<name><surname>Drenkow</surname>
<given-names>J</given-names>
</name>
<name><surname>Fuortes</surname>
<given-names>M</given-names>
</name>
<name><surname>Block</surname>
<given-names>E</given-names>
</name>
<etal></etal>
</person-group>
<article-title>MyD88 primes macrophages for full-scale activation by interferon-gamma yet mediates few responses to Mycobacterium tuberculosis</article-title>
<source>J Exp Med</source>
<year>2003</year>
<volume>198</volume>
<fpage>987</fpage>
<lpage>997</lpage>
<pub-id pub-id-type="pmid">14517275</pub-id>
<pub-id pub-id-type="doi">10.1084/jem.20030603</pub-id>
</citation>
</ref>
<ref id="B59"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Abdi</surname>
<given-names>K</given-names>
</name>
<name><surname>Singh</surname>
<given-names>N</given-names>
</name>
<name><surname>Matzinger</surname>
<given-names>P</given-names>
</name>
</person-group>
<article-title>T-cell Control of IL-12p75 Production</article-title>
<source>Scandinavian Journal of Immunology</source>
<year>2006</year>
<volume>64</volume>
<fpage>83</fpage>
<lpage>92</lpage>
<pub-id pub-id-type="pmid">16867152</pub-id>
<pub-id pub-id-type="doi">10.1111/j.1365-3083.2006.01767.x</pub-id>
</citation>
</ref>
<ref id="B60"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Jankovic</surname>
<given-names>D</given-names>
</name>
<name><surname>Kullberg</surname>
<given-names>MC</given-names>
</name>
<name><surname>Hieny</surname>
<given-names>S</given-names>
</name>
<name><surname>Caspar</surname>
<given-names>P</given-names>
</name>
<name><surname>Collazo</surname>
<given-names>CM</given-names>
</name>
<name><surname>Sher</surname>
<given-names>A</given-names>
</name>
</person-group>
<article-title>In the absence of IL-12, CD4(+) T cell responses to intracellular pathogens fail to default to a Th2 pattern and are host protective in an IL-10(-/-) setting</article-title>
<source>Immunity</source>
<year>2002</year>
<volume>16</volume>
<fpage>429</fpage>
<lpage>439</lpage>
<pub-id pub-id-type="pmid">11911827</pub-id>
<pub-id pub-id-type="doi">10.1016/S1074-7613(02)00278-9</pub-id>
</citation>
</ref>
</ref-list>
</back>
</pmc>
</record>
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