Development of cytochrome b558 and oxidative metabolism in human granulocytes, monocytes and during differentiation of HL-60 and U 937 cells

The development of cytochrome b558 (Cyt b) as determined spectrophotometrically, was investigated in human polymorphonuclear neutrophils (PMN), monocytes (MN) and during differentiation of HL-60 and U 937 cells induced by retinoic acid (RA) alone or in combination with IFN gamma. O2- release in response to a panel of stimulating agents, ie latex particles, opsonised zymosan, PMA, Con A and fMLP, was monitored by lucigenin-amplified chemiluminescence (CL). In parallel the expression of myeloperoxidase (MPO) was investigated and its catalytic activity on H2O2 related to luminol-amplified CL responses. In mature PMN and MN phagocytes, regardless of the stimulating agent, the O2- production is closely related to Cyt b but not to MPO specific contents. In differentiated HL-60 and U 937 cells, the oxidative metabolism increases in parallel with Cyt b specific contents, both being enhanced by the addition of IFN gamma to the RA treatment. However, marked differences in the O2- production intensities are observed depending on the stimulating agent tested and the state of differentiation considered. The PMA-stimulated O2- production is rather low ie 100 and 20 times less in granulocytic HL-60 and monocyto-macrophagic U 937 cells than in PMN and MN respectively. Latex, zymosan and Con A stimulated responses are close to those of MN, in monocyte-macrophagic U 937 cells. In conclusion, these data show that during differentiation; 1), Cyt b plays a critical role in O2- production; 2), the pathways leading to NADPH oxidase activation are diversely modulated following phagocyte differentiation with IFN gamma and/or with RA.     

Interferon gamma encapsulated into liposomes enhances the activity of monocytes and natural killer cells and has antiproliferative effects on tumor cells in vitro

The effects of human interferon gamma (IFN gamma) encapsulated into liposomes were investigated in vitro. Monocytes were induced to release a cytotoxic factor with either IFN gamma encapsulated into liposomes, free IFN gamma or lipopolysaccharide (LPS). If IFN gamma was applied in the liposomal form, less IFN activity was required to stimulate monocytes. Most of the cytotoxic factor was secreted during the first 4 h of stimulation. The cytotoxic factor in supernatants from PMNLs was completely neutralized by a monospecific polyclonal antiserum to tumor necrosis factor (TNF). Combining subthreshold doses of IFN gamma liposomes or IFN gamma with lipopolysaccharide synergistically enhanced the release of TNF. In fluorescence analysis, altered expression of the class II HLA-DR antigen on LeuM3 positive monocytes was induced with IFN gamma liposomes as well as with IFN gamma. Not only monocytes but also natural killer (NK) cells were stimulated to higher cytotoxicity by IFN gamma liposomes in a dose-dependent manner. In comparison with IFN gamma, the same amount of activity was necessary for adequate stimulation of NK-cells against the K562 target cells. Furthermore, the antiproliferative effects of IFN gamma liposomes and free IFN gamma on several human tumor cell lines was compared. Among several cell lines tested, U937 and A549 turned out to be sensitive to IFN gamma, and both cell lines reacted with 50% growth inhibition at a lower amount of gamma presented by liposomes than in the free form. These data show production of IFN gamma liposomes which possess immunomodulatory and antiproliferative activity in vitro. In several of the test systems studied, liposome-encapsulated IFN gamma was more effective than free IFN gamma.     

Effect of interferon on human neutrophilic granulocytes

The in vitro influence of interferon (IFN) on various functions of human neutrophilic granulocytes was investigated. It was observed that the attachment and engulfment of opsonized yeast particles by human neutrophilic granulocytes were enhanced after preincubation in vitro with IFN for 30 min. The same result was obtained whether the particles were opsonized with fresh normal serum (complement) or with specific antibodies. However, after incubation of the granulocytes with IFN for 3 h the phagocytosis rate was somewhat decreased. Nitroblue tetrazolium (NBT) reduction by resting granulocytes was slightly, although not significantly, increased by preincubation with IFN for 30 min, but their NBT reduction during phagocytosis of E. coli was significantly increased. No major effects of preincubation with IFN were observed on spontaneous or random migration of granulocytes.     

Effect of interferon on human neutrophilic granulocytes

Summary The in vitro influence of interferon (IFN) on various functions of human neutrophilic granulocytes was investigated. It was observed that the attachment and engulfment of opsonized yeast particles by human neutrophilic granulocytes were enhanced after preincubation in vitro with IFN for 30 min. The same result was obtained whether the particles were opsonized with fresh normal serum (complement) or with specific antibodies. However, after incubation of the granulocytes with IFN for 3 h the phagocytosis rate was somewhat decreased. Nitroblue tetrazolium (NBT) reduction by resting granulocytes was slightly, although not significantly, increased by preincubation with IFN for 30 min, but their NBT reduction during phagocytosis of E. coli was significantly increased. No major effects of preincubation with IFN were observed on spontaneous or random migration of granulocytes.     

Characterization of small and large human peripheral blood monocytes: effects of in vitro maturation on hydrogen peroxide release and on the response to macrophage activators

Human peripheral blood monocytes (HPBM) isolated from normal donors by centrifugal elutriation were divided into two populations according to volume. (Median volumes of small monocytes (SM) and large monocytes (LM) were 255 micron and 280 micron, respectively.) H2O2 production was determined during in vitro culture and in response to bacterial lipopolysaccharide (LPS), and to recombinant human interferon-gamma (rIFN-gamma). On day 1, H2O2 production by LM was significantly greater than that by SM. In vitro culture of SM resulted in an augmented ability to produce H2O2. By day 3, SM were the major H2O2 producers. Freshly isolated SM and LM, exposed for 24 hr to LPS and rIFN-gamma, showed different patterns of activation. Both SM and LM responded to LPS, with LM responding maximally at lower doses than SM. Only SM showed a significant augmentation of H2O2 production with rIFN-gamma treatment. We also assessed the effect of in vitro culture with activation. SM but not LM showed an increased H2O2 to LPS and rIFN-gamma after 7 days in culture. Continuous exposure of SM to rIFN-gamma resulted in maximal H2O2 production at day 3 of culture; this pattern was not seen for LPS. The production of H2O2 by HPBM is related to in vitro maturation. The enhanced H2O2 production by HPBM upon exposure to rIFN-gamma may be related to the induction of in vitro maturation.     

Functional antagonism between type I and type II interferons on human macrophages

A three-day treatment with IFN-gamma enhanced up to 300% the capacity of human monocytes and macrophages to produce H2O2 during the respiratory burst. IFN-alpha or -beta (type I IFNs), which did not by themselves influence the burst, were found to antagonize the enhancing effect of IFN-gamma (type II IFN). The antagonism was concentration-dependent and required the presence of type I IFNs during the whole period of IFN-gamma pretreatment. These results suggest that the host defense function of mononuclear phagocytes may be controlled by the relative local concentrations of type I and type II IFNs.     

Interferon γ stimulates prostaglandin E2 production by mouse Kupffer cells

When mononuclear phagocytes, including Kupffer cells, are activated by various agents, they synthesize and release arachidonic acid metabolites, prostaglandins (PGs) and leukotrienes (LTs). In this study, we examined the effect of in vitro Kupffer cell activation with recombinant murine IFN gamma on PGE2 and LTB4 secretion. IFN gamma enhanced PGE2 secretion, and this effect of IFN gamma was stronger than that of IL-1 or TNF. Moreover, IFN gamma promoted LTB4 release especially in the absence of PGs. On the other hand, dexamethasone and indomethacin inhibited and, EGTA and TMB-8, which reduce intracellular Ca++ Levels, blocked IFN gamma induced PGE2 production, which suggested that the activation of phospholipase A2 and cyclooxygenase in Kupffer cells requires the elevation of intracellular Ca++ levels.     

The enhancing effect of interferon-beta and -gamma on the killing of Leishmania tropica major in human mononuclear phagocytes in vitro

Both native human IFN-beta or -gamma added to human monocytes in culture increased their leishmaniacidal effect on intracellular Leishmania tropica major (L. major) amastigotes. This effect was dose-dependent, and was apparent if the IFN was added either before or after infection of the monocyte cultures with the promastigote form of the parasite. Compared on the basis of antiviral activity, IFN-gamma was shown to have a leishmaniacidal effect approximately three times greater than IFN-beta. Recombinant IFN preparations showed similar effects. In addition, IFN-gamma increased H2O2 production from human monocytes in culture in a dose-dependent manner. Monoclonal antibody to IFN-gamma abrogated both its effect on the leishmaniacidal capacity and on H2O2 production by the monocytes. These results suggest that IFN-gamma may be of therapeutic value in cutaneous leishmaniasis.     

In vitro and in vivo activation of human mononuclear phagocytes by interferon-gamma. Studies with normal and AIDS monocytes

To determine the potential immunotherapeutic role of interferon-gamma (IFN-gamma) as a mononuclear phagocyte-activating agent, we examined the effector cell function of peripheral blood monocytes from healthy donors and acquired immunodeficiency syndrome (AIDS) patients after either in vitro and/or in vivo treatment with recombinant (r) IFN-gamma. When assayed immediately after a 24-hr in vitro pulse with 300 U/ml, normal and AIDS monocytes behaved similarly with little augmentation of their intrinsically high levels of H2O2 release and activity against Toxoplasma gondii; in contrast, activity toward the more resistant intracellular pathogen, Leishmania donovani, was appreciably enhanced by rIFN-gamma. In addition, upon testing 4 to 6 days after in vitro pulsing, both normal and AIDS monocytes showed clear evidence of persistent activation in all three assays. The capacity of IFN-gamma to similarly activate monocytes in vivo was confirmed in all ten treated AIDS patients by examining cells before and after 24-hr infusions of 0.03 and 0.5 mg of rIFN-gamma/square meter (M2) of body surface area. For postinfusion monocytes tested after 1 day in culture, H2O2 release and antitoxoplasma activity were essentially unchanged, but antileishmanial effects were augmented. After 5 to 7 days in culture, monocytes from treated patients showed 3.2- to 5.9-fold increases in H2O2-releasing capacity and increases of 49 to 68% and 35 to 61% in intracellular activity against T. gondii and L. donovani, respectively. These results indicate that the human monocyte can be induced by rIFN-gamma to express signs of both immediate and persistent activation and suggest that, as a direct activator of mononuclear phagocytes, rIFN-gamma may also have potential as an immunotherapeutic agent for patients with intracellular infections.     

The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.     

The role of monocytes in the induction and regulation of IFN-gamma production by lectin-activated human T lymphocytes.

The data presented show that the production of interferon gamma (IFN-gamma) by pokeweed mitogen (PWM)-activated T lymphocytes requires monocytes and that the amount of lymphokine produced depends on the number of monocytes present in the culture. Accessory function of monocytes was independent from their ability to secrete IL-1 but required cell-cell contact, since blocking of adhesion molecules reduced the IFN-gamma production. Furthermore, production of IFN by lectin-preactivated T lymphocytes could not be triggered by IL-2 but also required monocyte-T cell interaction.     

Phagocytic activity and protein content of human monocytes cultivated in the presence of various homologous interferon preparations

We have studied the effect of different types of interferons (IFN) on phagocytic activity and protein content when present during in vitro cultivation of human blood monocytes. Recombinant IFN-alpha and partially and highly purified leukocyte IFN preparations blocked the increase in phagocytic activity and protein content that occurs during in vitro cultivation of human monocytes. Fibroblast IFN blocked the increase in protein content, but did not significantly alter the phagocytic activity. IFN-gamma slightly enhanced phagocytic activity and protein content, while lymphoblastoid IFN preparations had no effect. The phagocytic activity and protein content of monocytes matured in vitro without IFN and then treated with IFN for 24 h was also tested. Phagocytosis via the non-specific receptors and the protein content was reduced by treatment of these cells with the IFN-alpha preparations. On the other hand Fc-receptor mediated phagocytosis was stimulated by IFN-gamma. Our data indicate that IFN effects on monocytes in culture varies depending on type and possibly subtypes of IFNs, and also on the timing of the treatment.     

In vivo and in vitro macrophage activation induced by IFN gamma spontaneously released by spleen cells from tumor bearing mice.

Peritoneal macrophages obtained from Lewis Lung carcinoma (3LL) tumor bearing mice release high amounts of soluble factors such as C3,H2O2 and lysosomal enzymes but fail to exert cytotoxic activity on tumor cells. In the present work we show that they acquire this property and become fully activated after in vitro incubation with supernatants derived from cultures of splenocytes from tumor bearing syngeneic mice. The presence of IFN gamma in the above supernatants was detected by immunoblotting analysis and by bioassay. The role played by IFN gamma in macrophage activation was investigated.     

The suppressive effects of recombinant human tumor necrosis factor-alpha on normal and malignant myelopoiesis: synergism with interferon-gamma

The modulation of growth of normal and leukemic myeloid progenitor cells in soft agar cultures by recombinant human tumor necrosis factor-alpha (TNF alpha) and recombinant human interferon-gamma (IFN gamma) was investigated. TNF alpha inhibited colony formation of all colony types representing different maturational stages of normal progenitor cells committed to the myeloid lineage with different orders of sensitivity. Blast-type colonies derived from patients with acute myelogenous leukemia were more sensitive to TNF alpha inhibition than progenitor cells purified from normal bone marrow or bone marrow from patients with stable-phase chronic myelogenous leukemia. The response of most colony types to IFN gamma was poor. However, when IFN gamma was administered together with TNF alpha, synergistically enhanced antiproliferative effects were detected in all colony types tested. The antiproliferative action of IFN gamma on myelopoiesis was enhanced in culture by the presence of autologous monocytes, presumedly by inducing endogenous production of TNF alpha. However, TNF alpha seemed to act directly on the progenitor cells themselves to suppress their clonal growth, rather than involving accessory marrow elements such as monocytes and/or T lymphocytes.     

Trace levels of bacterial lipopolysaccharide prevent interferon-gamma or tumor necrosis factor-alpha from enhancing mouse peritoneal macrophage respiratory burst capacity

Preexposure of resident mouse peritoneal macrophages for 1 hr to traces of bacterial lipopolysaccharide (LPS) (less than or equal to 1 ng/ml) rendered the cells refractory to activation by recombinant interferon-gamma (rIFN gamma) or recombinant tumor necrosis factor-alpha (rTNF alpha), as evaluated by release of H2O2 upon stimulation with phorbol myristate acetate. Inhibition persisted for at least 4 days. Fifty percent inhibition of activation mediated by rIFN gamma followed 1 hr exposure to 10 pg/ml LPS. Fifty percent inhibition of activation mediated by rTNF alpha was achieved with 1 hr exposure to 1 pg/ml LPS. Such low levels LPS exposures (concentration X time) are far below those reported for many other actions of LPS on host cells. Inhibition was partially prevented by the cyclooxygenase inhibitors indomethacin, ibuprofen, and acetylsalicylic acid. Exogenous prostaglandins PGE1 and PGE2, and the 3',5'-cyclic adenosine monophosphate analog dibutyryl cyclic adenosine monophosphate (cAMP), mimicked the inhibitory effect of LPS in a dose-dependent manner, consistent with the hypothesis that formation of endogenous cyclooxygenase products in response to LPS may elevate intracellular cAMP and that the latter may mediate the observed inhibition. In addition, neutralizing antibody against IFN alpha and IFN beta selectively prevented LPS inhibition of activation mediated by rIFN gamma, but not by rTNF alpha. This suggests that IFN alpha and/or IFN beta induced by LPS also contributed to inhibition of activation by rIFN gamma. Thus, release of LPS may afford microorganisms a means by which to interfere with immunologically mediated enhancement of the respiratory burst-dependent antimicrobial capacity of macrophages.     

Participation of macrophages in enhanced in vitro immune interferon (IFN gamma) production with mouse spleen cells

IFN gamma production in cultures of spleen cells obtained from mice sensitized with TH69, a live Streptococcus faecalis preparation, was examined to determine how macrophages participate. It was demonstrated that sensitized spleen macrophages participated in enhanced IFN gamma production by T cells at an early stage (0-6 hr) of incubation, and that this production is mainly dependent on Ia-bearing macrophages In the reconstitution experiments where different combinations of spleen macrophages and T cells obtained from mice sensitized with TH69, OK-432, and BCG were used, T cells required that the identity between the sensitizing organisms in vivo and the stimulating organisms in vitro be the same for enhanced IFN gamma production while macrophages did not. Macrophage-mediated production of IFN gamma appears to be genetically restricted because IFN gamma was only produced in cultures where the H-2 region of macrophages and T cells matched. Further examination revealed that for macrophages to participate in enhanced IFN gamma production, first contact between cycloheximide-treated macrophages and T cells was required. Second, enhanced IFN gamma production occurred when culture supernatants of macrophages obtained from sensitized spleen cells were added to T cells. However, the addition of culture supernatant obtained from sensitized peritoneal macrophages resulted in inhibition of IFN gamma production. These results clearly showed the crucial role of macrophages in enhanced IFN gamma production by spleen T cells in vitro.     

Enhancement of human monocyte tumoricidal activity by recombinant M-CSF

Activated monocytes are an important component of immunologic defense against neoplastic disease. A variety of agents capable of inducing tumoricidal activity have been described, including bacterial LPS, IFN-gamma, IL-1, IL-2, TNF, and GM-CSF. We now show that pretreatment of monocytes with recombinant human macrophage-specific colony stimulating factor (M-CSF) augments the tumoricidal activity of human peripheral blood monocytes induced by other activating agents. Monocytes were preincubated for three days with M-CSF at 10(3) U/ml, washed, and treated for an additional two days with secondary activators. Tumoricidal activity was measured in a 6-h 51Cr-release assay using NK-resistant WEHI 164 cells that had been treated with actinomycin D. Pretreatment of monocytes with M-CSF significantly increased tumoricidal activity induced by LPS, IFN gamma, LPS plus IFN gamma, and LPS plus PMA. Pretreatment with IL-1, IL-2, IL-3, IL-4, or GM-CSF was not as effective as M-CSF in increasing tumoricidal activity. Enhanced tumoricidal activity was directly correlated to the increased TNF production resulting from M-CSF pretreatment. TNF antiserum completely blocked tumoricidal activity, demonstrating that TNF was responsible for the M-CSF-mediated increase in tumor cell lysis. M-CSF pretreatment also enhanced non-TNF mediated tumoricidal activity by monocytes, as seen by increased killing of the TNF-resistant target P815. This study demonstrated that in addition to the role of M-CSF in the proliferation and differentiation of monocyte/macrophage precursors, M-CSF also augments an effector function of mature blood monocytes.     

Current understanding of macrophage type 1 cytokine responses during intracellular infections

Macrophages are important effector cells in cell-mediated immunity against intracellular infection. Among cytokines that macrophages are able to release are IL-12 and TNF alpha. IL-12 is a critical linker between the innate and adaptive cell-mediated immunity, capable of Th1 differentiation and IFN gamma release by T and NK cells. IFN gamma is critically required for the activation of macrophage bactericidal activities. Recently emerging evidence suggests that macrophages are able to release not only IL-12 and TNF alpha but also IFN gamma. However, the mechanisms that control the release of each of these type 1 cytokines in macrophages appear different. While macrophages release TNF alpha in an indiscriminate and IL-12-independent way, the release of IL-12, particularly bioactive IL-12 p70, and IFN gamma is under tight control. We are just beginning to understand what controls the release of IL-12 p70, a question of fundamental importance to understanding the mechanisms underlying the initiation of cell-mediated immunity. Our recent findings have shed more insights into the regulatory mechanisms of macrophage IFN gamma responses. It has become evident that IL-12 is required not only for Th1 differentiation but also for IFN gamma responses by both T cells and macrophages during intracellular infection. In this overview, we have discussed about the current understanding of the regulation of macrophage type 1 cytokine responses during intracellular infection, based upon the recent findings from us and others.     

Effect of catalase on the proliferation of human lymphocytes to phorbol myristate acetate

Phorbol esters have been documented to stimulate the proliferation of human blood mononuclear cell cultures. In addition, these agents are also known to stimulate the production and release of reactive oxygen species by monocytes. We demonstrated previously that H2O2, one of these oxygen metabolites, impairs the proliferative capacity of human blood lymphocytes. Therefore, in these experiments, we determined whether or not the H2O2 released by monocytes after activation by PMA modifies the proliferation of lymphocytes to this agent. Human blood mononuclear cells (80% lymphocytes and 20% monocytes) were incubated with PMA, and lymphoblastic transformation (LBT) was quantitated at 3 and 5 days by pulsing the cultures with thymidine. Initial experiments established that the concentration of PMA required for optimal LBT was 50 ng/ml. We then demonstrated that this concentration of PMA also induces a burst in hexose monophosphate shunt activity and H2O2 production of mononuclear cells as indicated by the enhanced oxidation of 14C-glucose and 14C-formate, respectively. The amount of H2O2 released into the medium was substantial. Our measurements indicate that the concentration of H2O2 could reach values as high as 0.008 mM during the first 2 hr of the cultures. The addition of catalase to PMA-treated cultures in concentrations sufficient to scavenge the H2O2 released by the monocytes was associated with an enhanced thymidine uptake (mean 79%). These results indicate that the hydrogen peroxide released by the monocytes modifies the response of lymphocytes to the PMA. Paradoxically, mononuclear cell cultures depleted of monocytes also had a lower proliferation to PMA than mononuclear cell cultures. This observation indicates that monocytes also produce factors required for lymphocyte proliferation to PMA such as an interleukin. In contrast, to PMA cultures, catalase did not alter the proliferation of mononuclear cell cultures stimulated by PHA. We previously documented that PHA does not stimulate an immediate burst in the oxidative metabolism of mononuclear cultures. Therefore, the effect of catalase in these two culture systems appears to correlate with the capacity of the mitogen to stimulate the oxidative metabolism of mononuclear cells. These observations suggest that the release of reactive oxygen species by monocytes may modify the response of lymphocytes to antigens both in vitro and in vivo.     

The in vitro development of cytotoxicity in response to granulocyte/macrophage-colony-stimulating factor or interferon γ in the peripheral blood monocytes of patients with solid tumors: Modulation by arachidonic acid metabolic inhibitors

Summary The capacity of granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interferon (IFN ) to elicit monocyte cytotoxicity in vitro in the peripheral blood monocytes of patients with solid tumors was investigated. The cytotoxicity elicited by IFN was significantly reduced in cancer patient monocytes compared to normal monocytes. The cytotoxicity elicited by GM-CSF, however, was comparable between cancer patient monocytes and normal monocytes, but was lower than that induced by IFN . Indomethacin, a cyclooxygenase inhibitor, significantly augmented IFN -elicited cytotoxicity in cancer patient monocytes, but not in normal monocytes. In contrast, indomethacin augmented GM-CSF-elicited cytotoxicity in both cancer patient monocytes and normal monocytes. Nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, was found to suppress cytotoxicity in response to IFN and GM-CSF in both cancer patient monocytes and normal monocytes. The addition of leukotrienes to NDGA-treated cultures restored the development of cytotoxicity. Thus there are differences in the in vitro response of cancer patient monocytes and normal monocytes to distinct biological activators. Furthermore, these responses can be manipulated by agents that modulate arachidonic acid metabolism.Supported in part by PHS grant CA 41741Fellow of the A. Onassis Foundation