European Neuropsychopharmacology (2007) 17, 774–780 w w w. e l s e v i e r. c o m / l o c a t e / e u r o n e u r o Immunomodulatory effect of selective serotonin reuptake inhibitors (SSRIs) on human T lymphocyte function and gene expression Michal Taler a,⁎, Irit Gil-Ad a , Liat Lomnitski b , Inna Korov a , Ehud Baharav c , Meytal Bar a , Amichay Zolokov a , Abraham Weizman a,d a Laboratory of Biological Psychiatry, Tel Aviv University, Israel b Perrigo Israel Pharmaceuticals LTD, c Department of Medicine B, Rabin Felsenstein Medical Research Center, d Research Unit, Geha Mental Health Felsenstein Medical Research Center and Sackler Faculty of Medicine, Israel Medical Center, and Laboratory of Physiopathology of Joints and Inflammation, Petah Tiqva, Israel Center, Israel Received 13 February 2007; received in revised form 12 March 2007; accepted 29 March 2007 KEYWORDS SSRI; Immune system; T cells; Cytokines Abstract Antidepressants have an antiproliferative effect in some cell lines. Depression may be associated with activation of some pro-inflammatory cytokines. Therefore, we evaluated the ex-vivo immunomodulatory effect of selective serotonin reuptake inhibitors (SSRIs) in T cells. We found that the SSRIs, paroxetine and sertraline decreased T-cell viability with IC50 around 10 μM. The inhibition obtained with exposure to the SSRIs was more pronounced than that achieved with dexamethasone. Moreover, these SSRIs inhibit the secretion of the TH1 factor-tumor necrosis factor(TNF)α from the cells. On the molecular level, the SSRIs suppressed signal transducer and activator of transcription 3 (Stat3) and cyclooxygenase(Cox)2 protein expression. The inhibitory effects were accompanied by alterations in gene expression as assessed in the gene array. These findings reveal an immunomodulatory effect of the SSRIs paroxetine and sertraline in human T cells. The clinical implications of our findings merit further investigation. © 2007 Elsevier B.V. and ECNP. All rights reserved. 1. Introduction Antidepressants are widely used drugs for treating mood disorders, particularly depression and anxiety disorders. Selective serotonin reuptake inhibitors (SSRIs) are among the ⁎ Corresponding author. Laboratory of Biological Psychiatry, Felsenstein Medical Research Center, Beilinson Campus, Petah Tikva 49100, Israel. Tel.: +972 3 9376783; fax: +972 3 9211478. E-mail address: michalt@post.tau.ac.il (M. Taler). most commonly used antidepressants, due to their efficacy, safety and tolerability (Stafford et al., 2001). SSRIs block the reuptake of serotonin into the presynaptic nerve terminals, resulting in enhanced synaptic serotonin levels (Anderson et al., 2002). A recent report showed that human peripheral lymphocytes possess serotonin transporter, and might be directly affected by antidepressants, especially by SSRIs. (Barkan et al., 2004). Previous studies (Xia et al., 1997), showed that antidepressants, such as the tricyclics imipramine and clomipramine, induced apoptosis in human 0924-977X/$ - see front matter © 2007 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2007.03.010 Immunomodulatory effect of SSRIs on human T lymphocyte function and gene expression peripheral lymphocytes with IC50 range of 20–50 μM. Moreover, these agents induced apoptosis accompanied by caspase 3 activation in myeloid leukemia HL-60 cells (Xia et al., 1999). Furthermore, SSRIs induced rapid and marked apoptosis in Burkitt lymphoma cells and reversed the overexpression of the antiapoptotic protooncogene Bcl2 (Serafeim et al., 2003). In addition, depression is associated with alterations in the immune system. The “macrophage theory of depression” suggests that secretion of pro-inflammatory cytokines such as interleukin(IL)-1, tumor necrosis factor (TNF)α and interferon(INF)γ is associated with major depression (Smith, 1991). Depression has also been associated with hyper-activation of the immune system resulting in increased TH1 cytokines (Sedgwick and Czerkinsky, 1992; Maes, 1995) A recent study has demonstrated increased mRNA expressions of the cytokines IL-1β, IL-6, IFNγ, and TNFα in depressed patients compared to healthy controls. This amplification was attenuated following fluoxetine treatment (Tsao et al., 2006). Furthermore, suppression of pro-inflammatory cytokines was absent in depressed patients who failed to respond to SSRI treatment (O'Brien et al., 2006). These reports imply a possible relationship between major depression, SSRIs and the immune system. Among the key players of transcription factors affecting the immune system, is the signal transducer and activator of transcription 3 (STAT3), which is a member of the STAT family. The STAT proteins are a family of transcription factors latent in the cytoplasm that consists of seven different members (Darnell, 1997). These transcription factors are activated by a series of extra-cellular signaling proteins such as cytokines, growth factors, and hormones that bind to specific cell-surface receptors. The resulting signal transduction pathways permit them to play different roles in cell processes, such as differentiation, proliferation, apoptosis, and angiogenesis (Horvath, 2000) STAT3 is a key regulator of both cell growth and differentiation (Hauser et al., 1998). In the immune system, STAT3 is activated by IL-6 and other cytokines and therefore plays a role in the inflammatory process (Yoshimura, 2006). In addition, activation of STAT3 may contribute to depression, since transfection with dominant negative STAT3 significantly blocks 5-HT1A-receptor-mediated neuronal outgrowth. (Fricker et al., 2005). Another important component of the immune system is cyclooxygenase2 (COX2). This enzyme is usually absent in basal conditions, but is inducible by various cytokines, growth factors and mitogens (Williams et al., 1999). COX2 metabolizes arachidonic acid to a family of bioactive fatty acids designated prostaglandins (PGs) (Breyer and Harris, 2001). PGs mediate common inflammatory manifestations, such as swelling, pain, and fever, revealing that the COX2 enzyme seems to be an important mediator in inflammation (Williams et al., 1999). Interestingly, recent evidence has shown that treating depressed patients with the COX2 inhibitor celecoxib, in addition to antidepressants, resulted in the improvement in their depressive condition (Müller et al., 2006). Clinical and laboratory findings demonstrate that the combination of SSRI and antiinflammatory agents accelerate the antidepressants' activity (Brunello et al., 2006). Differences in gene expression establish the basis for phenotype changes that occur as a result of treatment. Genomic and proteomic technologies have already begun to 775 uncover novel functional pathways and therapeutic targets in the immune system. Moreover, bioinformatic approaches have highlighted several key targets and functional networks in this system. (Sioud, 2006). In the present study our aims were to evaluate the effect of two widely used SSRIs, namely, sertraline and paroxetine, on mitogen-induced proliferation and TNFα secretion in human T-lymphocytes, and to determine the gene expression profile accompanying this process by using a gene array (Affimetrix) methodology. 2. Experimental procedures 2.1. Ethics The study was approved by the Institutional Ethical Committee for Animal Experimentation at Felsenstein Research Medical Center, Petah Tikva and Tel-Aviv University, Israel. 2.2. Human lymphocyte primary culture Human lymphocytes were obtained from whole blood of healthy volunteers (Blood Services Center, Tel-Hashomer, Israel) using density gradient separation by UNI-SEP MAXI (NOVAmed, Jerusalem, Israel) following the manufacturer's instructions. The separated lymphocytes were then removed to nylon wool columns (NOVAmed, Jerusalem, Israel) in order to get T cells enriched population. The procedure was done using the manufacturer's protocol. The pellets were suspended in RPMI 1640 medium supplemented with 1% glutamine, 1% antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin) and 10% heat-inactivated fetal calf serum (Beit Haemek, Israel) and incubated for 24 h at 37 °C in humidified air containing 5% CO2. T-lymphocytes were then plated at a concentration of 2 × 106 cells/well in 1 ml medium. The relevant drugs were added to triplicate wells in addition to the mitogens PHA (10 μg/ml; Sigma Israel) or anti-CD3 (10 μg/ml; Serotec, Kidlington, UK). They were then incubated for 48 h at 37 °C in humidified air containing 5% CO2. 2.3. Cell viability assay — Alamar Blue Alamar blue is a nontoxic reagent, which incorporates a redox indicator that changes color in response to metabolic activity. The reduction-induced color change varies proportionately with cell number and time (Ahmed et al., 1994). Cells were incubated for 48 h with or without the drugs; a 100 μl/well sample of the cell suspension at final concentration of 1 × 105 cells/ml was seeded in triplicate in a 96 well microtiter plate. A 10 μl of AlamarBlue™ (Serotec, Kidlington, UK) was added to each well. The plates were incubated at 37 °C in a humidified 5% CO2 incubator for 3 h. Alamar blue fluorescence was measured at an excitation wavelength of 544 nm and an emission wavelength of 590 mm (FLUOstar BMG Lab Techonologies, Offenburg, Germany). 2.4. ELISA for cytokines detection After 48 h of incubation, lymphocyte cultures were centrifuged at 500 ×g for 8 min at 25 °C. The supernatants were collected and stored at −70 °C until the assay performance. TNFα levels were determined using a recombinant human TNFα enzyme-linked immunosorbent assaysELISA kit (CytoLab Ltd, Rehovot, Israel). The assay employed cytokine standard, capture and detection biotinylated antibodies and streptavidin–horseradish peroxidase. Color intensity induced by the enzymatic reaction was positively related to the cytokine concentration, and determined by reading sample in ELISA reader set to 405 nm. 776 M. Taler et al. 2.7. Statistical analysis Student's t-test and analysis of variance (ANOVA) including Student– Neuman–Keuls post-hoc test were performed as appropriate. Significance was set to p b 0.05. 3. Results 3.1. The effect of antidepressant treatment on the viability of human T cells Figure 1 A. Concentration-dependent effect of paroxetine on human T-cells viability assessed by Alamar blue induced with PHA (5 μM) 48 h after exposure to the drug. Each point is the mean ± SEM of 3 independent determinations. B. Concentrationdependent effect of paroxetine and sertraline in comparison to dexamethasone on human T-cells viability (Alamar blue) induced with PHA (5 μM) 48 h after exposure to the drugs. Each point is the mean ± SEM of 3 independent determinations. 2.5. Western blot analysis Total protein lysates from human lymphocytes were extracted with lysis buffer (150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM Na2VO4, 200 mM Hepes and 50 mMNaF). Cells were incubated for 60 min at 4 °C. The proteins were cleared by centrifugation at 14,000 ×g for 20 min at 4 °C. Protein concentration was determined by BCA kit (PIERCE, Rockford, USA). For Western blot analysis, 30 μg protein from each sample was resolved in 7.5% SDS-PAGE. Electroblotted proteins were detected using polyoclonal antibody anti-STAT3 (Santa Cruz cat.no, sc-7179,) polyoclonal antibody anti-COX-2 (Cayman Chemical Company, Michigan, USA) or monoclonal antibody anti-Actin (Biomeda, Burlingame, USA). Bound antibodies were visualized using chemiluminescence reaction (PIERCE, Rockford, USA). Several antidepressants, mainly SSRIs and some TCAs, have been shown to possess potent pro-apoptotic activity in different cell lines. In order to investigate the effect of antidepressants on human T-cells proliferation, culture of human T cells was prepared from healthy human lymphocytes. Cell cultures were treated ‘ex-vivo’ with different antidepressants for 48 h. By using cell viability test (AB) we found a consistent pattern in which IC50 of both paroxetine and sertraline in PHA-activated human T cells, was around 10 μM (Fig. 1A and C). The effect of proliferation suppression caused by paroxetine was much more prominent than that of dexamethasone treatment (Fig. 1B). 3.2. The effect of paroxetine and sertraline on TNFα secretion from anti-CD3 activated human T cells Several clinical studies have indicated that depressive illness is associated with activation of the pro-inflammatory response system resulting in increased circulating concentrations of monocyte and T-cell-derived pro-inflammatory cytokines (Maes et al., 1994). In the second part of our study we aimed to examine whether the antidepressant treatment suppressing the proliferation of T cells (Fig. 1) would lead to the same effect on TNFα secretion from anti-CD3 activated human T-cell culture. The cultures were treated ‘ex-vivo’ with paroxetine and sertraline at different concentrations (0–30 μM) for 48 h after anti-CD3 activation. The level of TNFα secretion was detected by ELISA. Both drugs suppressed TNFα secretion, indicating treatment's suppressive effect on the activity of inflammatory cells activity (Fig. 2). 2.6. RNA isolation and gene array Total RNA was extracted from 10 μM paroxetine treated and untreated primary lymphocytes cultures (5 samples each), using TRI reagent (Molecular Research Center, ICN Cincinnati USA) and protocols provided as previously described (Wilfinger et al., 1997). All samples were analyzed by using Affymetrix Human Genome Focus Array chip which represents over 8500 genes as described by the Affymetrix human data sheet .pdf (http://www.affymetrix. com/support/technical/datasheets/human_datasheet.pdf) (Affymetrix, Santa Clara, CA, http://www.affymetrix.com/index.affx). The hybridization reaction was done in the “Bioinformatics and Biological Computing Unit in Weizmann Institute of Science Israel” according to the Affymetrix Expression Manual. Analysis of the results was done according to NetAffex Analysis Center instructions (https://www.affymetrix.com/site/login/login.affx). A meaningful change was considered a change of at least 2 fold in transcript level between a baseline array and an experiment array. Figure 2 Human T cells were exposed for 48 h to paroxetine or sertraline at different concentrations. The levels of TNF-α secretion were determined by ELISA kit. Each point is the mean ±SEM of 3 independent determinations. Immunomodulatory effect of SSRIs on human T lymphocyte function and gene expression 3.3. The effect of paroxetine on the expression level of Stat3 and Cox2 proteins Stat3 is a downstream effector of both cytokines and growth factors (Aaronson and Horvath, 2002). Stat3 induces genes that encode proteins involved in various cellular processes, one of them being cell proliferation (Bromberg et al., 1999). Our results demonstrate that treating activated human T cells with paroxetine for 48 h decreased the Stat3 protein expression level, similar to the effect achieved with dexamethasone treatment (Fig. 3, upper panel). Cox1 and Cox2 catalyzed the rate-limiting step in prostaglandin synthesis, the conversion of arachidonic acid to prostaglandin H2 (PGH2). The Cox2 enzyme could be induced in most tissues in response to inflammatory and hormonal signals and has a wider range of substrates (Dubois et al., 1998). Recent studies have pointed out that Stat3 dominant negative (DN) blocked IL-10-mediated inhibition of Cox2 expression (Williams et al., 2004) Therefore, we wished to examine whether the suppressive effect of paroxetine on Stat3 expression would be accompanied by a similar decrease in Cox2 expression. Fig. 3 demonstrates that treating T cells with paroxetine caused marked suppression of Cox2 expression (Fig. 3 middle panel). From this data we may conclude that paroxetine has a suppressive effect on both proliferative and inflammatory genes in human T cells. Table 1 Comparative gene expression profiles of cell populations originate from human T-lymphocytes Category Gene title Gene symbol Down regulated genes — proliferation and cell cycle Cell division cycle 2, G1 to S and G2 to M Cell division cycle 6 homolog (S. cerevisiae) Tumor necrosis factor (ligand) superfamily, member 8 Interleukin 2 receptor, alpha Transcription factor Dp1 Lectin, mannosebinding, 1 Signal transducer and activator of transcription 1, 91 kDa Solute carrier family 16 (monocarboxylic acid transporters), member 1 MAD2 mitotic arrest deficient-like 1 (yeast) mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) Actin, beta Cathepsin D (lysosomal aspartyl peptidase) Integrin, alpha 5 (fibronectin receptor, alpha polypeptide) CDC2 Down regulated genes — inflammation Down regulated genes — cancer 3.4. Differences in gene expression in response to paroxetine treatment in human T lymphocytes Affymetrix human focus oligonucleotide arrays (Santa Clara, CA,) were used to determine the gene expression profile of the human lymphocytes treated with paroxetine compared to untreated cells. We assessed 5 different independent pairs of T lymphocytes treated with 10 μM paroxetine and untreated T lymphocytes extracted from the same blood sample. Differences in genes expression were revealed. Table 1 summarizes the up-regulated and down-regulated genes that were found in at least 3 samples which are presented in clusters according to the biological function of their proteins. As shown, inhibition of gene expression 777 Up-regulated genes — apoptosis CDC6 TNFSF8 IL2RA TFDP1 LMAN1 STAT1 SLC16A1 MAD2L1 MSH2 BUB1 ACTB CTSD ITGA5 Genes downregulated and up-regulated by more than twofold in treated cells compared with untreated cells in more than 3 independent experiments. involved in the proliferative processes was detected. Consistent down-regulation of Cdc6, which is critical for DNA synthesis, was revealed in all samples (see Table 1). 4. Discussion Figure 3 Whole cell lysates were prepared from human T cells which were left untreated (lane1) or were treated only with 5 μM PHA (lane 2), with PHA and paroxetine 0.5 μM (lane 3), PHA and dexamethasone 0.05 μM (lane 4). Proteins were resolved in a 7.5% SDS-PAGE and were then reacted with anti-Stat3 (upper panel), anti-Cox2 (middle panel) or anti-actin (lower panel) using Western blot analysis. There is considerable evidence indicating a continuous interaction between the brain and the immune system. Both stress and psychiatric disorders are associated with alterations in the immune function (Stein et al., 1991). A role of immune activation for depressive symptoms comes from studies showing a reduction in pro-inflammatory, but increase in antiinflammatory cytokine secretion by various antidepressants, and in particular those promoting serotonergic transmission. As pro-inflammatory cytokines can reduce serotonin levels as well as tryptophan availability by increasing the serotonin metabolism, it is intriguing that 778 SSRI have been shown to reduce immunotherapy-induced depression (Maes et al., 1994). Antidepressant therapy has been shown to be associated with an immunomodulatory effect (Crowson and Magro, 1995). We have recently reported that phenothiazines possess an antiproliferative effect on several cell lines (Gil-Ad et al., 2006). The neurotransmitter 5-HT can induce apoptosis in established Burkitt lymphoma lines (Serafeim et al., 2002). Moreover, the SSRI have been shown to lead to apoptotic cell death in those cells (Serafeim et al., 2003). In our present study the SSRIs were shown to cause dosedependent reductions in the viability of mitogen-stimulated lymphocytes. Moreover, they were shown to attenuate mitogen-stimulated increases in the immunoreactive protein content of Stat3, an important signal-induced transcription factor within lymphocytes, and COX2, an enzyme involved in prostaglandin biosynthesis and a marker of the inflammatory response of lymphocytes. Paroxetine and sertraline also reduced the secretion of TNFα, an important mediator of inflammation, from lymphocytes activated by antibody to CD3 on their cell surface. Moreover, paroxetine downregulated expression of mRNA for a specific protein that is involved in lymphocyte proliferation. Our findings warrant the exploration of the nature of the relationship between antidepressants and the immune system may be of interest. In the current work we demonstrated the modulatory effect of antidepressants on the immunocompetent cells. Major depressive disorders have been associated with the suppression of various components of the immune response, including a reduction of lymphocyte proliferation in response to mitogens, as well as a reduction of natural killer cells activity (Irwin, 1995). Fluoxetine, a prototype of SSRIs, was found to suppress lymphocyte proliferation in a dose-dependent manner (Berkelley et al., 1994). In the present study, our results indicated that paroxetine and sertraline significantly decrease the proliferation of human T cells. This inhibition might be a result of the up-regulation in genes involved in the apoptotic pathway on the one hand and downregulation of genes responsible for cell proliferation on the other. A previous study has shown that SSRIs might interfere with DNA synthesis in Burkitt lymphoma cells. The SSRI-induced inhibition of S-phase entry correlated with a relative accumulation of cells in the G0/G1 phase of cell cycle (Serafeim et al., 2003). Our results concerning gene expression are consistent with these data, showing that all the SSRI treated cells exhibited a significant decrease in the Cdc6 gene expression compared to the untreated ones. DNA replication is highly conserved in eukaryotes. Cdc6 gene function is required for the initiation of DNA replication and is a key regulatory protein during cell cycle progression (Luo et al., 2006). Immunodepletion of Cdc6 in human cells by microinjection of anti-Cdc6 antibody could block the initiation of DNA replication, which suggests that Cdc6 is a limiting factor in DNA synthesis in human cells (Hateboer et al., 1998). Therefore, the decreased level of this gene expression together with the up-regulation of genes involved in cell death probably play a part in the mechanisms which lead to cell cycle arrest and inhibition of proliferation, as demonstrated in the cell viability decrease (Fig. 1). The reduction of cell numbers and the inhibition of the immunocompetent cells' activity, are among the mechanisms M. Taler et al. that regulate the immune system function. Tumor necrosis factor-α (TNF-α) is a potent cytokine with a wide range of pro-inflammatory activities. It is secreted by T cells and other immune cells (Cenci et al., 2000). Several clinical studies indicate that depressive illness is associated with activation of the inflammatory response system resulting in increased circulating levels of monocyte and T-cell-derived pro-inflammatory cytokines such as TNF-α (Hestad et al., 2003). We found that the SSRIs in addition to the suppression of cell proliferation also inhibited TNFα secretion, leading to the attenuation of pro-inflammatory activity. SSRIs inhibit the reuptake of serotonin, raising the possibility of the involvement of serotonin in the immunomodulatory mechanism of sertraline and paroxetine. A recent study has shown antidepressant-induced suppression of cell proliferation and cytokine secretion, independent of their antagonistic activity in the serotonin or norepinephrine transporters (Diamond et al., 2006). In addition, the non-serotonergic effects of fluoxetine have been reported (Garcia-Colunga et al., 1997) suggesting the possible involvement of other mediators in the immunomodulatory effect of some SSRIs. This issue merits further investigation. It is of note that the concentrations of SSRIs shown to be active in human T cells (IC50 = 10 μM) were 10-fold higher than those associated with their antidepressant therapeutic effects. (b 1 μM) (Kirchherr and Kühn-Velten, 2006) indicating that the cellular mechanisms are more complex than the monoamine transport blockade. Nonetheless, because of the wide safety margin of SSRIs, it is not inconceivable to consider expanded indications for these medications, prescribed at higher doses, for lymphoproliferative diseases and malignancies. Overall, the data generated in this study indicate that SSRIs have a modulatory effect on some components of the human immune system. The phenotypic effect is characterized by the inhibition of T-cell proliferation and TNFα secretion. These effects seem to be related to the suppressive effect of SSRIs on the expression of genes involved in cell proliferation (most consistent Cdc6). In addition, STAT3 and COX2 protein expression was suppressed, apparently by the SSRIs. In summary, the data show that SSRIs may diminish the proliferative and inflammatory responses of lymphocytes; the mechanism could include a cascade of gene expression secondary to effects on the serotonin transporter that is expressed on the surface of lymphocytes and suppression of Stat3 expression. Alternatively, these effects may have little to do with serotonin per se and the mechanism of the antidepressant actions of SSRIs. The data suggest that SSRIs may have a therapeutic role to play in the treatment of at least some lymphoproliferative diseases and malignancies, apart from any primary effect on mood. In conclusion, it seems that antidepressants from the SSRIs family have an in vitro suppressive immunomodulatory influence. This effect appears to be mediated by changes in relevant gene expression. Further studies are needed to evaluate the complex cellular and molecular mechanisms of the pharmacological immunomodulation and its clinical relevance. Role of the funding source The Nofar Foundation, The Chief Scientists Office, The Israel Ministry of Commerce and Industry gave financial support for this research. Immunomodulatory effect of SSRIs on human T lymphocyte function and gene expression Contributors Michal Taler (author) designed the study, conducted most of the procedures and wrote the manuscript. Ehud Baharav helped with conducting the cytokine measurements. Inna Korov performed most of the immunological measurements. Meytal Bar helped with some of the immunological measurements. Amichay Zolokov helped with some of the immunological measurements. Liat Lomnitski helped to design the study protocol and attained funding. Irit Gil-Ad helped to design the study protocol and supervised the performance of the assays. Abraham Weizman (head of the lab) helped to design the study protocol and the writing of the manuscript. All authors contributed to the study and have approved the final draft of the manuscript. Conflict of interest There is no conflict of interest for Taler Michal, Gil-Ad Irit, L, Korov Inna, Baharav Ehud, Bar Meytal, Zolokov Amichay and Weizman Abraham. Liat Lomnitski is an employee of Perrigo Israel Pharmaceuticals Ltd. Acknowledgement The authors thank Shirley Horn-Saban from the DNA Array Unit in the Weizmann Institute for Science for the gene array assays. References Ahmed, S.A., Gogal, R.M., Walsh, J.E., 1994. A new rapid and simple non-radioactive assay to monitor and determine proliferation of lymphocytes: an alternative to {3H}thymidine incorporation assay. J. Immunol. Methods 170, 211–224. Anderson, G.M., Benett, A.J., Weld, K.P., Pushas, J.G., Ocame, D.M., Higley, J.D., 2002. Serotonin in cisternal cerebrospinal fluid of rhesus monkeys: basal levels and effects of sertraline administration. Psychopharmacology (Berl) 161 (1), 95–99. Aaronson, D.S., Horvath, C.M., 2002. A road map for those who know JAK-STAT. Science 296, 1653–1655. 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