Tibor Hajto

Effect of a Recombinant Lectin, Viscum album Agglutinin on the Secretion of Interleukin-12 in Cultured Human Peripheral Blood Mononuclear Cells and on NK-Cell-Mediated Cytotoxicity of Rat Splenocytes in vitro and in vivo

A plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to increase the number and cytotoxic activity of natural killer (NK) cells in animal models, but the mechanisms underlying these effects are poorly understood. We investigated the effects of the recombinant form of this lectin (rVAA) on secretion of interleukin (IL)-12 and on NK-mediated cytotoxicity against K562 target cells in cultures of human peripheral blood mononuclear cells (PBMC) as well as against YAC-1 target cells in cultured rat spleen cells. In 24-hour cultures of PBMC, 10 ng/ml plant VAA-I and 50 ng/ml rVAA induced significant increases in the secretion of total IL-12. Its biologically active heterodimeric form, p70, was also significantly induced by rVAA. Preincubation of PBMC or splenocytes for 48 h with rVAA in concentrations ranging between 10 pg/ml and 100 pg/ml resulted in moderate enhancements of NK-mediated cytotoxicity. However, coincubation of a low dose of rVAA (100 pg/ml) together with IL-2 and IL-12 (60 U/ml and 2 U/ml, respectively) led to additive stimulation of NK activity. In in vivo experiments, rVAA showed an enhancing effect on NK activity with a bell-shaped curve of efficacy. Forty-eight hours after a single intravenous injection of its most effective doses, 0.5 and 1 ng/kg, into Wistar rats, the NK cytotoxicity of splenocytes against YAC-1 targets doubled, and the frequency of large granular lymphocytes in peripheral blood showed 2.1- and 3-fold increases as compared to control animals. Twenty-four hours following these low lectin doses, the number of large granular lymphocytes was also significantly elevated. After 48 h, 0.5 ng/kg rVAA induced a significant augmentation in the percentage of peripheral Mac-1+ mononuclear cells, including activated monocytes and NK cells. The present results suggest that rVAA augments the secretion of an active form of IL-12 and potentiates the cytokine-induced NK activation. These effects of rVAA may be related to its stimulatory effects on MHC-unrestricted cytotoxicity in vivo.

Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases are involved in induction of apoptosis

The plant lectin Viscum album agglutinin‐I (VAA‐I) was recently found to modulate protein synthesis and to induce apoptosis in various cells of immune origin. We found that VAA‐I induces de novo protein synthesis of metabolically 35S‐labeled human neutrophils when used at low concentrations (<100 ng/mL) but acts as an inhibitor at higher concentrations. Using both flow cytometry (FITC‐Annexin‐V/PI labeling) and cytology (Diff‐Quick staining) approaches, we found that VAA‐I could not modulate neutrophil apoptosis at low concentrations but could induce it in >98% of cells at 500 and 1000 ng/mL. VAA‐I was also found to reverse the delaying effect of GM‐CSF on neutrophil apoptosis and to inhibit GM‐CSF‐inducedde novo protein synthesis. In contrast to GM‐CSF, VAA‐I does not induce tyrosine phosphorylation by itself and does not alter the GM‐CSF‐induced response. Among the inhibitors used, genistein, pertussis toxin, staurosporine, H7, Calphostin C, manoalide, BpB, quinacrine HA‐1077, and z‐VAD‐FMK, only the latter (inhibitor of caspases‐1, ‐3, ‐4, and ‐7) was found to inhibit VAA‐I‐induced neutrophil apoptosis as the percentage of apoptotic cells decrease from 98 ± 1.3 to 54 ± 3.2% (n =4). Furthermore, we confirm that caspases are involved in VAA‐I‐induced neutrophil apoptosis as we have observed the fragmentation of the cytoskeletal gelsolin protein that is known to be caspase‐3‐dependent. Such degradation was reversed by the z‐VAD‐FMK inhibitor. We conclude that induction of neutrophil apoptosis by VAA‐I is a caspase‐dependent mechanism that does not involve tyrosine phosphorylation events, G‐proteins, PKCs, and PLA2. In addition, we conclude that at least caspase‐3 is involved. Correlation between VAA‐I‐induced neutrophil apoptosis and VAA‐I‐induced inhibition of de novo protein synthesis is discussed.

Oncopharmacological Perspectives of a Plant Lectin (Viscum album Agglutinin-I): Overview of Recent Results from In vitro Experiments and In vivo Animal Models, and Their Possible Relevance for Clinical Applications

An old goal of natural complementary medical therapy has been to aim at a long-term stimulation of natural resistance in order to restrain cancer progression or improve defective immunological conditions without toxic side effects. Mistletoe extracts were applied to a large number of cancer patients because of their modulatory effect on the natural immune system. By carefully removing lectins, an essential group of components, from mistletoe extracts, a significant reduction of their effectiveness on several cellular immune parameters could be observed in vivo (1). That is the reason why, for the last 14 years, biological research of mistletoe extracts has focused on lectins. Meanwhile, the quantitatively dominant lectin, Viscum album agglutinin (VAA)-I has become available in a recombinant form (rVAA). Other constitiuents of plant extracts such as viscotoxins (2,3), poly- and oligosaccharides (4), flavonoids (5,6), chitin-binding mistletoe lectin (7) and arginine have also been investigated in connection with the effects of mistletoe extracts on the host defense. However, little evidence has been found that these substances contribute to the effects of mistletoe in vivo (8).

Lectin-induced increase in clonogenic growth of haematopoietic progenitor cells

Abstract: The galactoside‐specific plant lectin, Viscum album agglutinin (VAA‐I) increases cellular parameters of natural host defence. It also binds to a variety of haematopoietic cells, including progenitors. We investigated whether VAA‐I has a stimulatory effect on haematopoietic progenitor cells. Peripheral blood progenitor cells from 7 healthy volunteers were cultured in a colony assay with VAA‐I plus erythropoietin (EPO) and stem cell factor (SCF). At 50 pg/ml VAA‐I induced a significant increase in the cytokine‐dependent clonogenic growth (52% in median, p<0.05). In another set of experiments purified CD34+ cells were isolated from the bone marrow aspirate of 4 patients with non‐metastatic breast cancer using fluorescence‐activated cell sorting. Binding to CD34+ cells was demonstrated by using directly fluorescence‐conjugated VAA‐I. Co‐incubation with d‐galactose significantly abrogated this effect. CD34+ cells were cultured in the presence of EPO, SCF, interleukin‐3, granulocyte/monocyte colony‐stimulating factor and granulocyte colony‐stimulating factor. VAA‐I alone had no measurable effect on the clonogenic growth of the isolated cells. However, at concentrations of 100 and 250 pg/ml VAA‐I increased the cytokine‐dependent proliferation and differentiation of CD34+ cells by a median of 75 and 85%, respectively. The results show that VAA‐I binds to haematopoietic progenitor cells and has a co‐stimulatory effect on their proliferation.

Mistletoe lectins as immunostimulants (chemistry, pharmacology and clinic)

The European mistletoe Viscum album L. has attracted special interest in folklore and ethnomedicine throughout the centuries. The Celtic druids used it already in their ceremonies more than 2000 years ago. Later on it was thought to be potent against epilepsy “since the mistletoe did not fall down – neither would the patient” and this belief was carried on through the middle ages. In the 16th century mistletoe was commonly used against the falling sickness as well as against illnesses of the blood circulation, heart failure, dropsy, asthma and nervous diseases [1, 2].

Effects of Mistletoe Extract on Murine Thymocytes in vivo and on Glucocorticoid-Induced Cell Count Reduction

Background: Mistletoe extracts are widely used in cancer patients due to their cytostatic and immunomodulatory effects. Essential components include mistletoe lectins which act as biomodulators with proinflammatory and apoptosisinducing effects. This study investigates the acute and longterm effects of standardized mistletoe extract (Iscador® M spec 5 mg) on thymocyte subpopulations and peripheral T-cells using a murine (Balb/c) model. Materials and Methods: Using cell surface CD4/CD8 staining and flow cytometry, we followed the changes in CD4-CD8- double-negative (DN), CD4+CD8+ double-positive (DP) and CD4+ or CD8+ single-positive (SP) T-cells 24 h after single or repeated injections of 3 different dilutions (1:12, 1:60, 1:300) corresponding to 2.1, 0.42 and 0.08 mg/kg of Iscador. Thymocyte apoptosis was detected by flow cytometry using Annexin V and propidium iodide. Results: 24 h after a single injection of the 2 lower doses, the number of DN thymocytes increased significantly with an enhanced ratio of apoptotic cells. Following administration of the lowest dose, in peripheral blood the CD4+/CD8+ ratio was elevated. In the long-term trial, Balb/c mice were treated twice a week with 3 different doses of Iscador +/- 20 mg/kg of dexamethasone (DX), resulting in significantly enhanced DN thymocytes and elevated levels of apoptotic cells after treatment with the 2 lower doses. Iscador also inhibited the DX-induced reduction in the thymic DN cell count, as well as the DX-induced decrease in the CD4+/CD8+ ratio and CD4+ in the peripheral blood. Conclusion: Our results suggest that standardized mistletoe extract modulates proliferation and apoptosis of thymocytes in a dose-dependent manner and may act lymphoprotective during DX treatment.