Pnina Fishman

Evidence for involvement of Wnt signaling pathway in IB-MECA mediated suppression of melanoma cells

The A3 adenosine receptor, A3AR, belongs to the family of Gi proteins, which upon induction, suppresses the formation of cAMP and its downstream effectors. Recent studies have indicated that activation of A3AR by its agonist, IB-MECA, results in growth inhibition of malignant cells. Here we demonstrate the ability of IB-MECA to decrease the levels of protein kinase A, a downstream effector of cAMP, and protein kinase B/Akt in melanoma cells. Examination of glycogen synthase kinase 3β, GSK-3β, whose phosphorylation is controlled by protein kinase A and B, showed a substantial decrease in the levels of its phosphorylated form and an increase in total GSK-3β levels in IB-MECA treated melanoma cells. This observation suggests that the treatment of cells with IB-MECA augments the activity of GSK-3β in the cells. Evaluation of β-catenin, a key component of Wnt signaling pathway which, upon phosphorylation by GSK-3β rapidly ubiquitinates, showed a substantial decrease in its level after IB-MECA treatment. Accordingly, the level of β-catenin responsive cell growth regulatory genes including c-myc and cyclin D1 was severely declined upon treatment of the cells with IB-MECA. These observations which link cAMP to the Wnt signaling pathway provide mechanistic evidence for the involvement of Wnt pathway via its key elements GSK-3β and β-catenin in the anti-tumor activity of A3AR agonists.

The PI3K–NF-κB signal transduction pathway is involved in mediating the anti-inflammatory effect of IB-MECA in adjuvant-induced arthritis

The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A3 adenosine receptor (A3AR). The aim of the present study was to decipher the molecular mechanism involved with the inhibitory effect of IB-MECA, an A3AR agonist, on adjuvant-induced arthritis.The adjuvant-induced arthritis rats responded to IB-MECA treatment with a decrease in the clinical score and the pathological score of the disease. The response to IB-MECA was neutralized by the antagonist MRS 1220, confirming that the efficacy of the synthetic agonist was A3AR mediated.The A3AR protein expression level was highly expressed in the synovia, in the peripheral blood mononuclear cells and in the drain lymph node (DLN) tissues of adjuvant-induced arthritis rats in comparison with naïve animals. Downregulation of A3AR expression was noted upon treatment with IB-MECA. Analysis of synovia and DLN protein extracts revealed a decreased expression level of PI3K, PKB/Akt, IKK, NF-κB and tumor necrosis factor alpha, known to affect survival and apoptosis of inflammatory cells, whereas the caspase-3 level was upregulated.Taken together, high A3AR expression is found in the synovia, in the immune cells in the DLN and in peripheral blood mononuclear cells. IB-MECA, an orally bioavailable molecule, activates the A3AR, inducing receptor downregulation and the initiation of a molecular mechanism that involves de-regulation of the PI3K–NF-κB signaling pathway. As a result, a potent anti-inflammatory effect manifested in the improvement of the disease clinical score and pathological score occurs. The finding that the A3AR expression level in the peripheral blood mononuclear cells and in the DLN reflects the receptor status in the remote inflammatory site suggests use of the A3AR as a follow-up biomarker.

Increased interleukin-1 and interleukin-3 like activity in schizophrenic patients

1. The interleukins play an important role in the development and maintenance of the immune system 2. Decreased cell mediated immunity measures were found in schizophrenic patients. 3. The purpose of the present study was to study the spontaneous production of interleukin-1 (IL-1) and interleukin-3 like activity (IL-3-LA) by human mononuclear cells from schizophrenic patients in comparison to healthy individuals. 4. Interleukin-1 was increased significant by schizophrenic patients as compared to controls. 5. Interleukin-3 like activity was slightly elevated in schizophrenic patients as compared to controls. 6. These findings support the hypothesis of an autoimmune dysfunction in some schizophrenic patients.

Inhibition of experimental auto-immune uveitis by the A3 adenosine receptor agonist CF101.

Uveitis is an inflammation of the middle layer of the eye with a high risk of blindness. The Gi protein associated A3 adenosine receptor (A3AR) is highly expressed in inflammatory cells whereas low expression is found in normal cells. CF101 is a highly specific agonist at the A3AR known to induce a robust anti-inflammatory effect in different experimental animal models. The CF101 mechanism of action entails down-regulation of the NF-κB-TNF-α signaling pathway, resulting in inhibition of pro-inflammatory cytokine production and apoptosis of inflammatory cells. In this study the effect of CF101 on the development of retinal antigen interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveitis (EAU) was assessed. Oral treatment with CF101 (10 µg/kg, twice daily), initiated upon disease onset, improved uveitis clinical score measured by fundoscopy and ameliorated the pathological manifestations of the disease. Shortly after treatment with CF101 A3AR expression levels were down-regulated in the lymph node and spleen cells pointing towards receptor activation. Downstream events included a decrease in PI3K and STAT-1 and proliferation inhibition of IRPB auto-reactive T cells ex vivo. Inhibition of interleukin-2, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production and up-regulation of interleukin-10 was found in cultured splenocytes derived from CF101-treated animals. Overall, the present study data point towards a marked anti-inflammatory effect of CF101 in EAU and support further exploration of this small molecule drug for the treatment of uveitis.

Chloramphenicol Induced Inhibition of Platelet Protein Synthesis: in Vitro and in Vivo Studies

Chloramphenicol (CAP), an antibiotic which causes various blood dyscrasias, was shown to inhibit in vitro protein synthesis of human blood platelets. The effect is dose‐ and time‐dependent, it is reversible after incubation for 2 h, and is comparable with the suppression achieved by cycloheximide (CXM). Electron microscopic examination revealed swelling and destruction of mitochondria.

Putative role of the adenosine A 3 receptor in the antiproliferative action of N 6 -(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N6-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A3 receptor (hA3R) ligands with affinities in the high nanomolar range (Ki values of 159 and 649xa0nM, respectively). These values were comparable to the observed Ki value of adenosine on hA3R, which was 847xa0nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A3R. In a functional assay in Chinese hamster ovary cells transfected with hA3R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A3R antagonist VUF5574. Both IPA and reference A3R agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A3R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A3R-independent mechanism, as was previously reported for other A3R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A3R.

Scanning and Transmission Electron Microscopy Study on the Plasma Cells of a Patient with Multiple Myeloma

The ultrastructural features of the plasma cells of a 32-year-old patient suffering from multiple myeloma are described. The high percentage (90%) of plasma cells in the bone marrow aspirate permitted the examination of an almost homogenous population. The appearance of the plasma cells seen with the transmission electron microscope did not differ from that described in other reports. The surface architecture of the plasma cells, such as revealed by the scanning electron microscope, differed from that of the normal and pathological white blood cells. Of particular interest were the membrane-bound portions of the cytoplasm seen as buddings, or round bodies in the vicinity of the plasma cells which contained most probably pathological proteins.

Generation of Superoxide Anions during Phagocytosis by Monocytes of Uremic Patients

The phagocytic activity of monocytes from 15 uremic patients was estimated by the superoxide anion test. There was a significant decrease in the ability of monocytes from these patients to produce superoxide anions in comparison with monocytes obtained from healthy subjects. Addition of urea to monocytes from healthy subjects caused a marked reduction in their ability to produce superoxide anions, while addition of creatinine did not cause any effect. Monocytes from patients with renal failure of glomerular origin were found to produce less superoxide anions than those from patients with other types of renal diseases.

Ultrastructural and Functional Studies on Human Platelets Incubated with Diclofenac Sodium (Voltaren)

The in vitro effect of diclofenac sodium (Voltaren) on the ultrastructure of healthy donors platelets was examined and compared with those induced by aspirin (ASA). In distinction to ASA, which causes loss of platelet pseudopodia, Voltaren induced an increase and marked elongation of these pseudopodia. The implication of this finding in the explanation of the decreased platelet aggregation caused by the drug is discussed. Voltaren increased the phagocytotic activity of the individual platelet, although the overall ability of the cells to phagocytize latex particles was not markedly increased. Platelets incubated with Voltaren showed a decrease in their total protein synthesizing capacity. Voltaren did not exert any effect on the internal ultrastructure, platelet factor 3, and calcium content of the incubated platelets.

Ultrastructural Study of the Erythropoietic Events in Human Embryonic Livers

The ultrastructural features of the erythropoietic events in 5- to 9-week embryonic livers are described. By the 5th week of gestation, the human embryonic liver becomes an active site of erythropoiesis represented by the formation of reticular-mesenchyme cells and hemocytoblasts. At the 6--7th week, the first proerythroblasts and islands of polychromatophilic and orthochromatic erythroblasts can be detected. From this stage, by the 8th week of embryonic development, the liver assumes its function as a fully developed embryonic organ. In the embryonic livers studied there were no signs of granuloor lymphopoiesis. Although megakaryocytes were found in the 8-week embryonic liver, there is no evidence that these cells are produced in the liver. Macrophages were found in the 5-week embryonic liver.