Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells

Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed, at least in part, to the presence of oleuropein and hydroxytyrosol. In this study, oleuropein and hydroxytyrosol, major phenolic compound of olive oil, was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay), cell viability (Guava ViaCount assay), cell apoptosis, cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation, and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 μg/mL of oleuropein or 50 μg/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G₁ to S phase transition manifested by the increase of cell number in G₀/G₁ phase.     

Activity and location of olive oil phenolic antioxidants in liposomes

The antioxidant activity of hydroxytyrosol, hydroxytyrosol acetate, oleuropein, 3,4-dihydroxyphenylelenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylelenolic acid dialdehyde (3,4-DHPEA-EDA) towards oxidation initiated by 2,2'-azobis(2-amidinopropane) hydrochloride in a soybean phospholipid liposome system was studied. The antioxidant activity of these olive oil phenols was similar and the duration of the lag phase was almost twice that of alpha-tocopherol. Trolox, a water-soluble analogue of alpha-tocopherol, showed the worst antioxidant activity. However, oxidation before the end of the lag phase was inhibited less effectively by the olive oil phenols than by alpha-tocopherol and Trolox. Synergistic effects (11-20% increase in lag phase) were observed in the antioxidant activity of combinations of alpha-tocopherol with olive oil phenols both with and without ascorbic acid. Fluorescence anisotropy of probes and fluorescence quenching studies showed that the olive oil phenols did not penetrate into the membrane, but their effectiveness as antioxidants showed they were associated with the surface of the phospholipid bilayer.     

Hydroxytyrosol and oleuropein of olive oil inhibit mast cell degranulation induced by immune and non-immune pathways

The aim of this study was to determine whether hydroxytyrosol and oleuropein, the major phenols found in olives and olive oil, inhibit mast cell activation induced by immune and non-immune pathways. Purified peritoneal mast cells were preincubated in the presence of test compounds (hydroxytyrosol or oleuropein), before incubation with concanavalin A, compound 48/80 or calcium ionophore A23187. Dose–response and time-dependence studies were carried out. Comparative studies with sodium cromoglycate, a classical mast cell stabilizer, were also made. After incubation the supernatants and pellets were used to determine the β-hexosaminidase content by colorimetric reaction. The percentage of β-hexosaminidase release in each tube was calculated and taken as a measure of mast cell activation. Other samples of cell pellets were used for cell viability studies by the trypan blue dye exclusion test, or fixed for light and electron microscopy. Biochemical and morphological findings of the present study showed for the first time that hydroxytyrosol and oleuropein inhibit mast cell degranulation induced by both immune and non-immune pathways. These results suggest that olive phenols, particularly hydroxytyrosol and oleuropein, may provide insights into the development of useful tools for the prevention and treatment of mast cell-mediated disorders.     

Influence of olive oil and its components on mesenchymal stem cell biology

Extra virgin olive oil is characterized by its high content of unsaturated fatty acid residues in triglycerides, mainly oleic acid, and the presence of bioactive and antioxidant compounds. Its consumption is associated with lower risk of suffering chronic diseases and unwanted processes linked to aging, due to the antioxidant capacity and capability of its components to modulate cellular signaling pathways. Consumption of olive oil can alter the physiology of mesenchymal stem cells(MSCs). This may explain part of the healthy effects of olive oil consumption, such as prevention of unwanted aging processes. To date,there are no specific studies on the action of olive oil on MSCs, but effects of many components of such food on cell viability and differentiation have been evaluated. The objective of this article is to review existing literature on how different compounds of extra virgin olive oil, including residues of fatty acids,vitamins, squalene, triterpenes, pigments and phenols, affect MSC maintenance and differentiation, in order to provide a better understanding of the healthy effects of this food. Interestingly, most studies have shown a positive effect of these compounds on MSCs. The collective findings support the hypothesis that at least part of the beneficial effects of extra virgin olive oil consumption on health may be mediated by its effects on MSCs.     

Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells

3,4-dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco-2 cell monolayers as the model system. The kinetic data demonstrate that [(14)C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3-hydroxy-4-methoxyphenylethanol, the product of catechol-O-methyltransferase; when DPE is assayed in vitro with the purified enzyme a K(m) value of 40 microM has been calculated.     

Effect of different olive oil-derived antioxidants (hydroxytyrosol and 3,4-dihydroxyphenylglycol) on the quality of frozen-thawed ram sperm

The aim of the present study was to evaluate the effect of the addition of different concentrations of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during the freezing-thawing process. Sperm was collected, pooled and diluted with commercial extenders and then divided into aliquots supplemented with different concentrations (10 μg/ml, 30 μg/ml, 50 μg/ml and 70 μg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. A control group, without antioxidant, was also prepared. Sperm motility, viability, acrosome integrity, mitochondrial membrane potential and lipid peroxidation (LPO) were assessed. The results showed that frozen-thawed ram spermatozoa exhibited lower values for motility, membrane integrity, acrosome and mitochondrial membrane potential than fresh samples (P ≤ 0.01). However, when antioxidants were added, thawed spermatozoa exhibited relatively low LPO, recording values similar to fresh spermatozoa; by contrast, the control group of frozen-thawed spermatozoa without antioxidants exhibited significantly higher LPO (P ≤ 0.01). The addition of a HT+DHPG mixture (MIX) had a negative impact on sperm membrane and acrosome integrity, suggesting that a pure antioxidant supplementation has the potential to offer superior results. In conclusion, HT and DHPG exhibited a positive effect on the frozen-thawed spermatozoa inasmuch as they reduced the LPO. These olive oil-derived antioxidants have the potential to improve frozen-thawed sperm quality, although further studies should be carried out to analyse the antioxidant effect at different times after thawing.     

The enantioselective immunoaffinity extraction of an optically active ibuprofen-modified peptide fragment

Recent in vitro studies have demonstrated antioxidant properties of some virgin olive oil phenolic compounds. One of the prerequisites to extrapolate these data to an in vivo situation is the knowledge of their bioavailability in humans. In the present work we describe an analytical method which enables us to perform hydroxytyrosol and tyrosol quantitative determinations in human urine. This method was successfully used in bioavailability studies of both phenolic compounds after acute olive oil administration. Virgin olive oil was administered to healthy volunteers after a low phenolic diet. The dose administered of both phenolic compounds was estimated in reference to free forms of hydroxytyrosol and tyrosol present in virgin olive oil extracts before and after being submitted to hydrolytic conditions. These conditions mimic those occurring during digestion. Urine samples were collected before and after acute olive oil intake and analyzed by capillary gas chromatography-mass spectrometry. Hydroxytyrosol and tyrosol urinary recovery increased in response to olive oil administration, obtaining maximal values in the first 4 h. Our results further indicate that hydroxytyrosol and tyrosol are mainly excreted in conjugated form, since only 5.9 +/- 1.4% (hydroxytyrosol) and 13.8 +/- 5.4% (tyrosol) of the total amounts excreted in urine were in free form.     

Hydroxytyrosol from olive fruits prevents blue-light-induced damage in human keratinocytes and fibroblasts

Skin aging is a complex biological process influenced by a combination of endogenous or intrinsic and exogenous or extrinsic factors due to environmental damage. The primary environmental factor that causes human skin aging is the ultraviolet irradiation from the sun. Recently, it was established that the long-term exposure to light-emitting-diode-generated blue light (LED-BL) from electronic devices seems to have a relevant implication in the molecular mechanisms of premature photoaging. BL irradiation induces changes in the synthesis of various skin structures through DNA damage and overproduction of reactive oxygen species (ROS), matrix metalloproteinase-1 and -12, which are responsible for the loss of the main components of the extracellular matrix of skin like collagen type I and elastin. In the current study, using human keratinocytes and fibroblasts exposed to specific LED-BL radiation doses (45 and 15 J/cm ²), we produced an in vitro model of skin photoaging. We verified that, compared with untreated controls, the treatment with LED-BL irradiation results in the alteration of metalloprotease-1 (collagenase), metalloprotease-12 (elastase), 8-dihydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and collagen type I. Moreover, we showed that the photoaging prevention is possible via the use of hydroxytyrosol extracted from olive fruits, well known for antioxidant properties. Our results demonstrated that hydroxytyrosol protects keratinocytes and fibroblasts from LED-BL-induced damage. Thus, hydroxytyrosol might be proposed as an encouraging candidate for the prevention of BL-induced premature photoaging.     

Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects

We investigated the anti-proliferative effects of an olive oil polyphenolic extract on human colon adenocarcinoma cells. Analysis indicated that the extract contained hydroxytyrosol, tyrosol and the various secoiridoid derivatives, including oleuropein. This extract exerted a strong inhibitory effect on cancer cell proliferation, which was linked to the induction of a G2/M phase cell cycle block. Following treatment with the extract (50 microg/ml) the number of cells in the G2/M phase increased to 51.82+/-2.69% relative to control cells (15.1+/-2.5%). This G2/M block was mediated by the ability of olive oil polyphenols (50 microg/ml) to exert rapid inhibition of p38 (38.7+/-4.7%) and CREB (28.6+/-5.5%) phosphorylation which led to a downstream reduction in COX-2 expression (56.9+/-9.3%). Our data suggest that olive oil polyphenols may exert chemopreventative effects in the large intestine by interacting with signalling pathways responsible for colorectal cancer development.     

Major phenolic compounds in olive oil: metabolism and health effects

It has been postulated that the components in olive oil in the Mediterranean diet, a diet which is largely vegetarian in nature, can contribute to the lower incidence of coronary heart disease and prostate and colon cancers. The Mediterranean diet includes the consumption of large amounts of olive oil. Olive oil is a source of at least 30 phenolic compounds. The major phenolic compounds in olive oil are oleuropein, hydroxytyrosol and tyrosol. Recently there has been a surge in the number of publications that has investigated their biological properties. The phenolic compounds present in olive oil are strong antioxidants and radical scavengers. Olive "waste water" also possesses compounds which are strong antioxidant and radical scavengers. Typically, hydroxytyrosol is a superior antioxidant and radical scavenger to oleuropein and tyrosol. Hydroxytyrosol and oleuropein have antimicrobial activity against ATTC bacterial strains and clinical bacterial strains. Recent syntheses of labeled and unlabelled hydroxytyrosol coupled with superior analytical techniques have enabled its absorption and metabolism to be studied. It has recently been found that hydroxytyosol is renally excreted unchanged and as the following metabolites as its glucuronide conjugate, sulfate conjugate, homovanillic acid, homovanillic alcohol, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylacetaldehyde. Studies with tyrosol have shown that it is excreted unchanged and as its conjugates. This review summarizes the antioxidant abilities; the scavenging abilities and the biological fates of hydroxytyrosol, oleuropein and tyrosol which have been published in recent years.     

Antioxidant/prooxidant effects of dietary non-flavonoid phenols on the Cu2+-induced oxidation of human low-density lipoprotein (LDL)

A central role in the oxidative development of atherosclerotic lesions has been ascribed to the peroxidation of plasma low-density lipoprotein (LDL). Dietary supplementation with virgin olive oils increases the total plasma antioxidant status and the resistance of low-density lipoprotein to ex vivo oxidation. We have studied the effects of some dietary non-flavonoid phenols from Olea europaea L., both in purified form or in complex mixtures obtained by biotransformation of olive leaf extracts, on the LDL oxidation induced by Cu2+ ions. Cu2+-Induced LDL oxidation is inhibited by oleuropein and hydroxytyrosol in the initiation phase of the reaction at concentrations of phenols higher than that of Cu2+ ions. Interestingly, at lower concentration, both phenols anticipated the initiation process of LDL oxidation, thus exerting prooxidant capacities. Although similar effects are already described for flavonoids, such as quercetin, rutin, and apigenin, it is the first time that a prooxidant effect of dietary non-flavonoid phenols, such as oleuropein and hydroxytyrosol, on the LDL oxidation is reported. Our results show that a net effect of oleuropein and hydroxytyrosol on Cu2+-induced LDL peroxidation is determined by a balance of their pro- and antioxidant capacities. It is worth to underline that, during Cu2+-induced LDL oxidation in the presence of bioreactor eluates, we have evidence of a synergistic effect among phenolic compounds that enhance their antioxidant capacities so avoiding the prooxidant effects.     

Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by long-wave ultraviolet radiation in melanoma cells

Previous studies showed that long-wave ultraviolet (UVA) radiation induces severe skin damage through the generation of reactive oxygen species and the depletion of endogenous antioxidant systems. Recent results from our laboratory indicate a dramatic increase of both lipid peroxidation products (TBARS) and abnormal L-isoaspartyl residues, marker of protein damage, in UVA-irradiated human melanoma cells. In this study, the effects of hydroxytyrosol (DOPET), the major antioxidant compound present in olive oil, on UVA-induced cell damages, have been investigated, using a human melanoma cell line (M14) as a model system. In UVA-irradiated M14 cells, a protective effect of DOPET in preventing the uprise of typical markers of oxidative stress, such as TBARS and 2'7'-dichlorofluorescein (DCF) fluorescence intensity, was observed. In addition, DOPET prevents the increase of altered L-isoAsp residues induced by UVA irradiation. These protective effects are dose dependent, reaching the maximum at 400 microM DOPET. At higher concentrations, DOPET causes an arrest of M14 cell proliferation and acts as a proapoptotic stimulus by activating caspase-3 activity. In the investigated model system, DOPET is quantitatively converted into its methylated derivative, endowed with a radical scavenging ability comparable to that of its parent compound. These findings are in line with the hypothesis that the oxidative stress plays a major role in mediating the UVA-induced protein damage. Results suggest that DOPET may exerts differential effects on melanoma cells according to the dose employed and this must always be taken into account when olive oil-derived large consumer products, including cosmetics and functional foods, are employed.     

Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage

Radiation-induced cellular injury is attributed primarily to the harmful effects of free radicals, which play a key role in irradiation-induced apoptosis. In this study, we investigated the radioprotective efficacy of edaravone, a licensed clinical drug and a powerful free radical scavenger that has been tested against γ-irradiation-induced cellular damage in cultured human peripheral blood lymphocytes in studies of various diseases. Edaravone was pre-incubated with lymphocytes for 2 h prior to γ-irradiation. It was found that pretreatment with edaravone increased cell viability and inhibited generation of γ-radiation-induced reactive oxygen species (ROS) in lymphocytes exposed to 3 Gy γ-radiation. In addition, γ-radiation decreased antioxidant enzymatic activity, such as superoxide dismutase and glutathione peroxidase, as well as the level of reduced glutathione. Conversely, treatment with 100 μM edaravone prior to irradiation improved antioxidant enzyme activity and increased reduced glutathione levels in irradiated lymphocytes. Importantly, we also report that edaravone reduced γ-irradiation-induced apoptosis through downregulation of Bax, upregulation of Bcl-2, and consequent reduction of the Bax:Bcl-2 ratio. The current study shows edaravone to be an effective radioprotector against γ-irradiation-induced cellular damage in lymphocytes in vitro. Finally, edaravone pretreatment significantly reduced DNA damage in γ-irradiated lymphocytes, as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment) (p < 0.05). Thus, the current study indicates that edaravone offers protection from radiation-induced cytogenetic alterations.     

羟基酪醇作为线粒体营养素的调控机制

地中海饮食,特别是橄榄油,赋予了地中海周边国家人民对于退行性疾病的强力抵抗,尤其是心血管疾病和肿瘤的发生率以及致死率相对更低。羟基酪醇是橄榄油中的多酚类化合物之一,在防治紫外辐射、糖尿病、老年性视网膜黄斑病变、心血管疾病以及肿瘤等方面都具有重要的生物学效应。将具体阐述羟基酪醇作为一种线粒体营养素（如通过调节线粒体的动态变化以及Nrf2介导的抗氧化酶的诱导等）如何促进其有益功能。     

Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol

Hydroxytyrosol is one of the o-diphenolic compounds in extra virgin olive oil and has been suggested to be a potent antioxidant. The superoxide radical (O2*-) and nitric oxide (NO*) can react very rapidly to form peroxynitrite (ONOO ), a reactive tissue damaging species thought to be involved in the pathology of several chronic diseases. Hydroxytyrosol was highly protective against the peroxynitrite-dependent nitration of tyrosine and DNA damage by peroxynitrite in vitro. Given that extra virgin olive oil is consumed daily by many humans, hydroxytyrosol derived from this diet could conceivably provide a defense against damage by oxidants in vivo. The biological activity of hydroxytyrosol in vivo will depend on its intake, uptake and access to cellular compartments.     

Olive oil hydroxytyrosol protects human erythrocytes against oxidative damages

Hydroxytyrosol, the major representative phenolic compound of virgin olive oil, is a dietary component. Its possible protective effect on hydrogen peroxide (H(2)O(2))-induced oxidative alterations was investigated in human erythrocytes. Cells were pretreated with micromolar hydroxytyrosol concentrations and then exposed to H(2)O(2) over different time intervals. Subsequently, erythrocytes were analyzed for oxidative hemolysis and lipid peroxidation. Our data demonstrate that hydroxytyrosol prevents both oxidative alterations, therefore, providing protection against peroxide-induced cytotoxicity in erythrocytes. The effect of oxidative stress on erythrocyte membrane transport systems, as well as the protective role of hydroxytyrosol, also were investigated in conditions of nonhemolytic mild H(2)O(2) treatment. Under these experimental conditions, a marked decrease in the energy-dependent methionine and leucine transport is observable; this alteration is quantitatively prevented by hydroxytyrosol pretreatment. On the other hand, the energy-independent glucose transport is not affected by the oxidative treatment. The reported data give new experimental support to the hypothesis of a protective role played by nonvitamin antioxidant components of virgin olive oil on oxidative stress in human systems.     

Effects of virgin olive oil phenolics on scavenging of reactive nitrogen species and upon nitrergic neurotransmission

The major phenolics from the polar fraction of virgin olive oil (caffeic acid, oleuropein, tyrosol and hydroxytyrosol) have well-established antioxidant activities but their effects on reactive nitrogen species and nitrergic neurotransmission have not been fully investigated. The three catechol compounds were active as scavengers of nitric oxide generated spontaneously from the decomposition of sodium nitroprusside (approximately 50% inhibition achieved at 75 microM), and had similar ability to scavenge chemically generated peroxynitrite, as determined by an alpha1-antiproteinase inactivation assay (67.2%-92.4% reduction when added at 1 mM). Tyrosol was less active in these tests, but does not possess the catechol functionality. Despite their ability to interact with chemically prepared nitric oxide, neither oleuropein nor hydroxytyrosol at 5 microM altered NO*-mediated relaxations of the nerve-stimulated rat anococcygeus preparation, but this may be because the nitrergic transmitter is protected from the effects of externally applied scavengers. In conclusion, the phenolics found in virgin olive oil possess ability to scavenge reactive oxygen and nitrogen species that are implicated in human pathologies, but their impact may be restricted to those species present in the extracellular environment.     

Hydroxytyrosol, as a component of olive mill waste water, is dose- dependently absorbed and increases the antioxidant capacity of rat plasma

Hydroxytyrosol is the most potent phenolic antioxidant of olive oil and olive mill waste water (OMWW) and its biological activities have stimulated research on its potential role in cardiovascular protection. However, evidence of the absorption of OMWW phenolics and on their possible in vivo activity has, until now, never been provided. Three groups male Sprague-Dawley rats were administered 1, 5, or 10 mg/Kg of the OMWW extract, respectively, providing 41.4, 207, and 414 μg/Kg of hydroxytyrosol, respectively. Urine was collected for 24 h and the urinary levels of hydroxytyrosol were quantified by mass spectrometry. Hydroxytyrosol was dose-dependently (R²=0.95) absorbed and excreted in the urines mostly as a glucuronide conjugate. Further, the administration of an hydroxytyrosol-rich OMWW extract (10 mg/kg) to the rats was also associated with an increase of their plasma antioxidant capacity. Future experiments will eventually further clarify its metabolic fate and its in vivo actions.     

A novel tubulin polymerization inhibitor,MPT0B206, downregulates Bcr-Abl expression and induces apoptosis in imatinib-sensitive and imatinib-resistant CML cells

Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.     

IL-16 signaling specifically induces STAT6 activation through CD4

Biologic activities of IL-16 have been well described (e.g., chemotaxis of CD4+ cells, CD25 upregulation, secretion of IL-1b, IL-4 and TNF-a secretion) but very few signaling events have been described. To gain a better understanding of how the biologic activities of IL-16 are regulated following receptor engagement (CD4) we have analyzed the activation state of numerous STAT proteins in primary human peripheral blood mononuclear cells (PBMCs) and the human monocytic cell line THP-1 following IL-16 stimulation. Of the four STAT proteins tested, only STAT6 was activated (phosphorylated) in a dose-dependant manner by IL-16. The activation of STAT6 was completely abolished when IL-16 was pre-incubated with soluble CD4 (the IL-16 cell surface receptor), demonstrating the need for CD4 engagement in STAT6 activation. These results are the first to demonstrate a link between IL-16 and STAT6 activation.