[Rehabilitation in persons with schizophrenic spectrum disorders: the impact of cognition and cognitive remediation therapy]. / Rehabilitation von Menschen mit schizophrenen Psychosen: Die Bedeutung von Kognition und Training kognitiver Funktionen.

Schizophrenia is a chronic disorder, which severely limits the social and occupational functioning. Employment, education, relationships, housing and health are among the most frequently stated life and treatment goals among persons suffering from schizophrenia. Rehabilitation for persons with schizophrenia aims at preservation and improvement of psychosocial functions in areas such as work, social relationship and independent living skills, promotes recovery-oriented interventions and, therefore, serves the central goals of affected persons. Cognitive functioning, education, negative symptoms, social support and skills, age, work history, and rehabilitation service to restore community functioning have proven to be strong predictors for successful psychiatric rehabilitation. It makes sense to concentrate on these predictors when improvement of psychiatric rehabilitation is targeted. Cognitive remediation produces moderate improvements in cognitive performance and, when combined with functional training and embedded in comprehensive psychiatric rehabilitation, also enhances functional outcome. Germany provides a highly differentiated system of psychosocial support for schizophrenic patients. However, the "German disease" with different care providers being in charge in subsequent stages of recovery hampers efficient organisation of psychiatric rehabilitation. Improvement of overall organisation, i.e., configuration of interfaces, understanding of the complex interactions of measures, design of disease specific programmes, research and economic evaluation constitute major challenges in the field of psychiatric rehabilitation.

Memory performance in acute and weight-restored anorexia nervosa patients.

BACKGROUND: Anorexia nervosa (AN), at the stage of starvation and emaciation, is characterized by abnormalities in cognitive function, including memory performance. It is unclear whether memory impairment persists or is reversible following weight restoration, and whether memory function differs between AN subtypes. The aim of the present study was to investigate general memory performance in currently ill and fully weight-restored patients of different AN subtypes. METHOD: Memory performance was assessed using the Wechsler Memory Scale-Revised (WMS-R) in a total of 99 participants, including 34 restricting-type AN patients (AN-RESTR), 19 binge-eating/purging-type AN patients (AN-PURGE), 16 weight-restored AN patients (AN-W-R) and 30 healthy controls (CONTROL). Cognitive evaluation included a battery of standardized neuropsychological tasks for validating the findings on memory function. RESULTS: Deficits were found with respect to immediate and delayed story recall in currently ill AN patients irrespective of AN subtype. These deficits persisted in weight-restored AN patients. Currently ill and weight-restored AN patients did not differ significantly from healthy controls with respect to working memory or other measures of neuropsychological functioning. CONCLUSIONS: The findings suggest that impaired memory performance is either a stable trait characteristic or a scar effect of chronic starvation that may play a role in the development and/or persistence of the disorder.

Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I.

The carbohydrate specificity of mistletoe toxic lectin-I (ML-I) was studied by haemagglutination-inhibition assay. The results indicated that ML-I has a broad range of affinity for Gal alpha,beta linked sequences. The galabiose (E, Gal alpha 1----4Gal) sequence, a receptor of the uropathogenic E. coli ligand, was one of the best disaccharide inhibitors tested. The lectin also exhibits affinity for Lac(Gal beta 1----4Glc), T(Gal beta 1----3GalNAc), I/II(Gal beta 1----3/4GlcNAc) and B(Gal alpha 1----3Gal) sequences. Gal alpha 1----4Gal and Gal beta 1----4Glc are frequently occurring sequences of many glycosphingolipids located at the mammalian cell membranes, such as intestinal and red blood cell membranes, for ligand binding and toxin attachment. This finding provides important information concerning the possible mechanism of intoxication of cells by the mistletoe preparation.

A sheep hydatid cyst glycoprotein as receptors for three toxic lectins, as well as Abrus precatorius and Ricinus communis agglutinins.

The binding properties of a glycoprotein with blood group P1 specificity isolated from sheep hydatid cyst fluid with Gal and GalNAc specific lectins was investigated by quantitative precipitin and precipitin inhibition assays. The glycoprotein completely precipitated Ricinus communis agglutinin (RCA1), Abrus precatorius agglutinin (APA) and Mistletoe toxic lectin-I (ML-I). Only 1.0 microgram of P1 glycoprotein was required to precipitate 50% of 5.1 micrograms ML-I nitrogen. It also reacted well with abrin-a and ricin, precipitating over 73% of the lectin nitrogen added, but poorly or weakly with Dolichos biflorus (DBL), Vicia villosa (VVL, a mixture of A4, A2B2 and B4), VVL-B4, Arachis hypogaea (PNA), Maclura pomifera (MPL), Bauchinia purpurea alba (BPL) and Wistaria floribunda (WFL) lectins. When an inhibition assay in the range of 5.1 micrograms N to 5.9 micrograms N of lectins (ML-I, abrin-a; ricin, RCA1, and APA, and 10 micrograms P1 active glycoprotein interaction was performed; from 76 to 100% of the precipitations were inhibited by 0.44 and 0.52 mumol of Gal alpha 1-->4Gal and Gal beta 1-->4GlcNAc, respectively, but not or insignificantly with 1.72 mumol of GlcNAc. The Gal alpha 1-->4Gal disaccharide found in this P1 active glycoprotein is a frequently occurring sequence of many glycosphingolipids located at the surface of mammalian cell membranes, especially human erythrocytes and intestinal cells for ligand binding and microbial toxin attachment. The present finding suggests that the Gal alpha 1-->4Gal beta 1-->4GlcNAc sequence in this P1 active glycoprotein is one of the best glycoprotein receptors for three toxic lectins (ricin, abrin-a, and ML-I) as well as for APA, and RCA1, and the result of inhibition assay implies that these lectins are recognizing part or all of the Gal alpha 1-->4Gal beta 1-->4GlcNAc sequence in the P1 active glycoprotein.

Characterisation of granulocyte stimulation by thionins from European mistletoe and from wheat.

Thionins are small basic peptides found in different plant species, which are known to exert cytotoxic properties. In addition, previous data indicated an activation of human granulocytes by thionins from European mistletoe (viscotoxins, VT). To extend these latter findings, we investigated the influence of VT and from thionins from wheat flour (purothionin) on human granulocytes by flow cytometry and tried to characterise the involved molecular structures and mechanisms. Phagocytosis was determined by incorporation of FITC-labelled Escherichia coli and respiratory burst by oxidation of dihydrorhodamine 123 to rhodamine 123. VT and purothionin significantly enhanced E. coli-stimulated phagocytosis and respiratory burst at 25 and 250 microgram/ml. Phagocytosis of damaged lymphocytes by granulocytes was detected by electron microscopy in the VT-stimulated (100 microgram/ml) but not in the control cultures. The poly-cationic structure of the intact molecule seems to be crucial, as evidenced by comparison of the burst and phagocytosis-enhancing effects induced by other poly-cationic (protamine sulphate, histone, poly-l-arginine, poly-l-lysine) and poly-anionic (poly-l-glutamic acid) peptides, while pore forming due to amphipathic properties seems to be less important. Ca2+ and Mg2+ could not inhibit VT-enhanced phagocytosis and, thus, could not inhibit binding of VT to granulocytes. In addition, verapamil at low concentrations inhibited VT activity, suggesting the involvement of Ca2+ channels for granulocyte activation by the VT. Similarly, thionins and histones in contrast to protamine sulphate induced cell death of granulocytes at 250 microgram/ml as demonstrated by an enhanced release of reactive oxygen intermediates in unstimulated granulocytes. From these data one may suggest that activity of VT is induced by strong unspecific ionic binding, probably followed by specific receptor binding, and thionins exhibit stimulatory and cytotoxic effects on immune cells, which have to be further characterised.

Selective killing of CD8+ cells with a 'memory' phenotype (CD62Llo) by the N-acetyl-D-galactosamine-specific lectin from Viscum album L.

As reported previously by our group, among the toxic proteins from Viscum album L. only the mistletoe lectins (MLs) induce the apoptotic killing pathway in human lymphocytes. Although one may expect a homogenous distribution of carbohydrate domains on cell surface receptors for the carbohydrate binding B chains of the toxic protein, the sensitivity of cells to these B chains obviously differ. Here we report a selective killing of CD8+ CD62Llo cells from healthy individuals by the galNAc-specific ML III (and RCA60, which binds to gal and galNAc), while the gal-specific ML I was less effective. This selective killing is not sufficiently explained by protein synthesis inhibition alone, since this subset was not affected by other ribosome inhibiting proteins such as the lectin from Ricinus communis (RCA120), lectin from Abrus precatorus (APA), abrin A, and inhibitors of RNA, DNA and/or protein synthesis such as actinomycin D, mitomycin C, and cycloheximide. We conclude that CD8+ cells with 'memory' phenotype (CD62Llo) are more sensitive to the ML III-mediated killing than their CD8+ CD62Lhi counterparts, CD4+ T cells, and CD19+ B cells. These cells probably express a distinct receptor with galNAc domains that is missing or not active on CD8+ cells with a 'naive' phenotype.

Crystallization of the ribosome inactivating protein ML1 from Viscum album (mistletoe) complexed with beta-D-galactose.

A ribosome inactivating protein (ML1) from the mistletoe plant (Viscum album) has been crystallized. The crystals, grown in the presence of beta-D-galactose, are hexagonal, space group P6(1)22 or P6(5)22, a = b = 111.0 A, c = 309.3 A with 24 molecules per unit cell (assuming 33% solvent by weight). The protein of molecular mass 63 kDa is a heterodimer consisting of two chains, A and B, joined by a disulfide bond. The A-chain, 29 kDa, inhibits protein synthesis by depurinating an adenine residue (A4324) in a highly conserved RNA loop of the 28 S ribosomal subunit. The toxicity of the protein is mediated by the B-chain, 34 kDa, which has lectin activity, interacting with sugar residues of glycoproteins and glycolipids on the surface of target cells.

Induction of apoptosis by mistletoe lectin I and its subunits. No evidence for cytotoxic effects caused by isolated A- and B-chains.

Type II ribosome inactivating proteins (RIP II) are generally known to induce apoptosis in human cells by the inhibition of protein biosynthesis. Recent data from mistletoe RIP II proteins (eg. mistletoe lectin I; ML1) suggest an additional mode of apoptosis induction through the binding of their lectin part to certain cell surface receptors as is known for some human galectins. In order to clarify this possibility, we used highly sensitive flow cytometric apoptosis assays and mistletoe hololectin subunits of proven purity to show that neither human lymphocytes nor Molt-4 cells undergo apoptosis after treatment with isolated lectin-type B-chains. In contrast to earlier investigations, only the hololectin was able to induce apoptosis in these assays. We conclude that direct apoptosis induction by mistletoe lectins occurs only after uptake of the molecules into the cell due to the action of the ribosome inactivating A-chain.

Isolation and partial characterization of a small chitin-binding lectin from mistletoe (Viscum album).

A novel lectin, called VisalbCBA, was isolated from European mistletoe (Viscum album). This lectin differs completely from the classical galactose/N-acetylgalactosamine-binding mistletoe lectins MLI, MLII and MLIII. Biochemical analyses indicated that VisalbCBA is a dimeric protein composed of two identical subunits of approx. 10 kDa. VisalbCBA exhibits specificity towards oligomers of N-acetylglucosamine and shows sequence homology to the previously isolated chitin-binding plant proteins. Although VisalbCBA is less toxic than the other mistletoe lectins, it definitely exhibits cytotoxic properties. The possible involvement of VisalbCBA in the biological and therapeutic effects of mistletoe is discussed.

Comparison of properties of mistletoe lectin I A-chain and ricin B-chain conjugate with native toxins.

Chimeric toxin protein was prepared from the mistletoe lectin I A-chain and ricin B-chain by using the disulfide exchange reaction. Ricin and chimeric protein were indistinguishable in binding to immobilized asialofetuin in ELISA. Chimeric protein was more toxic to Jurkat cells than native mistletoe lectin I, but not so effective as native ricin. In the presence of NH4Cl, which enhances the toxicity of some toxins and immunotoxins, but does not influence ricin toxicity, both ricin and chimeric toxin had equal cytotoxic activity. The possibility is discussed that the ricin B-chain protects the ricin A-chain (RTA) from degradation during delivering RTA from the cell surface to the place where RTA is translocated into the cytosol.

Recognition of different antigens of mistletoe extracts by anti-mistletoe lectin antibodies.

Anti-mistletoe lectin-1 (ML-1) antibodies are produced during treatment of cancer patients with mistletoe extracts. However, little is known about their ability to recognise distinct epitopes present in mistletoe extracts. To estimate this, ML-1, ML-2 and ML-3 were analysed by Western blot analysis using high titred anti-ML antibody positive sera from cancer patients treated with different mistletoe extracts. In these experiments we could clearly demonstrate that anti-ML antibodies bind to ML-1 A- and B-chains and, in addition, that they recognised a spectrum of other antigens. This kind of immunological response varied from one individual to another and was not influenced by the different mistletoe extracts. Elution studies showed that anti-ML-1 A-chain or B-chain specific antibodies cross-reacted with A- or B-chains of the other lectins indicating homologies between these molecules (probably in the glycosylated side chain). However, the unglycosylated ML-3 A-chain was only detectable by antibodies specific for the ML-3 A-chain. From our data it has to be concluded that different epitopes of the mistletoe extracts are involved in the induction of the humoral immune response during mistletoe therapy and also that cross-reactivity between the different ML exist.

Induction of apoptosis by the N-acetyl-galactosamine-specific toxic lectin from Viscum album L. is associated with a decrease of nuclear p53 and Bcl-2 proteins and induction of telomeric associations.

The ribosome-inhibiting proteins from Viscum album L., i.e. the mistletoe lectins (ML), were recognized to induce apoptosis in various tumour cell lines and human lymphocytes. However, several aspects of ML-induced cell death are unclear. We report that the galNAc-binding ML III incubated with human lymphocytes mediates a very effective death signal resulting in the binding of Annexin-V and expression of mitochondrial membrane proteins Apo2.7, but also in an influx of the DNA intercalating dye propidium iodide. The addition of the ribosome-inhibiting protein Volkensin also induced Apo2.7 molecules, while Momordin, lacking a carbohydrate-binding chain, did not enter the cell membrane and thus did not affect the cells. However, we observed ML III to preferentially affect CD8+ cells with a memory phenotype (CD62L(lo)) as compared to their CD8+ CD62L(hi) counterparts, CD4+ T cells and CD19+ B cells. Furthermore, ML III did not induce sister chromatid exchange-inducing DNA lesions but reduced the intensity of telomeric signals, increased the frequencies of telomeric associations and C-anaphases and reduced nuclear Bcl-2 and p53 proteins. Whatever the exact mechanisms are, our results provide strong evidence that the ML III-mediated cytotoxicity involves distinct killing pathways, i.e. (1) primary cell death via an induction of apoptosis which may not be dependent on protein and/or RNA synthesis and may not involve p53 and Bcl-2 proteins and (2) a loss of telomeres resulting in chromosomal instability in the surviving cells which is incompatible with life. However, we cannot exclude the possibility that this effect is due to a decrease in nuclear p53 proteins.

Induction of mitochondrial Apo2.7 molecules and generation of reactive oxygen-intermediates in cultured lymphocytes by the toxic proteins from Viscum album L.

We analysed mitochondrial alterations in human lymphocytes incubated with toxins exerting RNA and/or protein synthesis/transport inhibitory activity. We found that all toxins known to affect macromolecule synthesis, such as ricin from Ricinus communis, mistletoe lectin I (ML I) from Viscum album, cycloheximide, actinomycin D, and brefeldin A but also the thionins from Viscum album (viscotoxins; VT) generated reactive oxygen intermediates (ROI) and induced expression of newly described mitochondrial membrane proteins Apo2.7, however, with different kinetics. Apart from a rapid permeabilisation of cell membranes by the VT with swelling of mitochondria, loss of their cristae and ROI generation within 2-4 h, the majority of the cells may have received a distinct 'death signal' resulting in an induction of Apo2.7 molecules within 24 h. In contrast, protein synthesis/transport inhibition may signal for apoptosis within 24 h by decreasing distinct 'survival promotors' which remain to be characterised.

Induction of apoptosis in human lymphocytes treated with Viscum album L. is mediated by the mistletoe lectins.

Viscum album L. (VAL) is a phytopreparation used in adjuvant cancer therapy with both immunostimulatory and DNA stabilizing properties at low drug concentrations and cytostatic/cytotoxic properties at higher concentrations. The present work examines the cytotoxic effects of VAL extracts produced from mistletoes grown on different host trees and of purified toxic proteins from VAL, such as the D-galactose-specific lectin I (ML I), the N-acetyl-D-galactosamine-specific ML II and ML III, and crude viscotoxins towards cultured human lymphocytes. The decrease in the number of cultured lymphocytes and blast cells treated with whole plant extracts from VAL was host tree-specific. Nevertheless, there was no close correlation to the content of MLs or viscotoxins. Using the purified proteins, it became obvious that the cell killing was mediated by the induction of apoptosis, as measured by the appearance of a hypodiploid DNA peak using flow cytometry. ML III was the most effective to induce apoptosis, followed by ML II and ML I, while the viscotoxins and oligosaccharides from VAL did not. By measuring the surface expression of IL-2R alpha chains, transferrin receptors and APO-1/Fas molecules on non-apoptotic T cells, no significant changes were observed at low ML concentrations (1 ng/ml), but their decrease at higher ones. Our findings suggest that there might be at least two different ways of cell killing operative in VAL-mediated cytotoxicity: (a) the typical apoptotic cell death with the appearance of hypo-diploid nuclei, and (b) a direct or indirect killing by damaging the cell membrane with subsequent influx of Ca2+ and of the DNA intercalating dye propidium iodide and cell shrinkage. These effects might not be exclusive, as they probably occur simultaneously.

Is the binding of mistletoe lectins I and III a useful prognostic indicator in colorectal carcinoma?

A retrospective study on 127 patients with colorectal carcinoma was performed to evaluate the use of mistletoe lectins I and III as possible prognostic indicators. Tissue sections were stained by a histochemical technique using these lectins and the staining results correlated with survival. No correlation between survival and the presence of lectin binding or non-binding was found. A lectin with the same monosaccharide specificity as ML-III, Helix pomatia agglutinin, which was of prognostic significance in the same patient cohort in another study showed on Western blot analysis a slightly different lectin binding pattern towards glycoproteins than the MLs. The results of this study indicate that even subtle differences in the carbohydrate composition of glycoconjugates which can be differentiated by lectins are of biological significance in terms of the metastatic spread of tumours.

Separation of mistletoe lectins based on the degree of glycosylation using boronate affinity chromatography.

A mixture of two mistletoe lectins (MLs) has been separated according to the degree of glycosylation using boronate affinity chromatography. The mistletoe lectins, mistletoe lectin I (MLI) and mistletoe lectin III (MLIII) with degrees of glycosylation of 6.1 and 3.8%, respectively, were used in the investigation. MLI exhibited a higher retention time than MLIII due to its higher degree of glycosylation. Separation was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The developed method may lead to new applications for the boronate affinity technique, as well as provide an alternative separation method for MLs.

Generation of reactive oxygen intermediates (ROI) by the thionins from Viscum album L.

BACKGROUND: Extracts from Viscum album L. are widely used as an adjuvant in complementary cancer therapy. While the mechanisms of mistletoe lectin (ML) cytotoxicity are well described, the viscotoxin (VT) effects are unclear at present. Thus, we treated human lymphocytes with VT and measured cell death-associated changes by flow cytometry. RESULTS: Treatment of lymphocytes with VT for 2 hours resulted in the binding of Annexin-V, permeabilisation of cell membranes, and generation of ROI. Apart from interindividual differences in response to VT, the number of intracellulary Bcl-2 proteins increased only marginally. The VT A1, A2, A3 and 1-PS were similar in their ROI-inducing potencies, while other cationic and/or amphipathic substances such protamine sulfate, VT B, purothionin or wasp venom peptides mastoparan I and II were less effective. However, the cytotoxic properties of VT-rich whole plant extracts from mistletoe grown on different host trees (Iscador), correlated with the content of ML rather than VT. CONCLUSIONS: Permeabilisation of lymphocytes cell membranes by VT was associated with generation of ROI within 2 hours indicating accidental cell death. VT cannot be ignored any longer as they posses both immunomodulating and cytotoxic properties, which both might be of clinical relevance.

Viscotoxin-free aqueous extracts from European mistletoe (Viscum album L.) stimulate activity of human granulocytes.

BACKGROUND: Extracts from European mistletoe (Viscum album L., VAL) are used for complementary cancer treatment. Viscotoxins (VT) and whole plant extracts with high amounts of the VT have been shown to stimulate functional activity of granulocytes. MATERIALS AND METHODS: We stimulated neutrophils from healthy donors in vitro with aqueous VT-free VAL extracts and mistletoe lectins (ML) in the presence of E.coli and studied phagocytosis (via incorporation of FITC-labelled E.coli) and respiratory burst (via oxidation of dihydrorhodamine 123 to rhodamine 123) by flow cytometry. RESULTS: The VT-free VAL extract significantly stimulated granulocyte activity, and this effect correlated with the content of the ML, although the ML exerted no influence at relevant concentrations. Co-incubation of the cells with VAL in the presence of VT further increased granulocyte response. CONCLUSIONS: From these data it is suggested that (1) a non-VT non-ML component of the VAL extracts activated granulocytes and (2) different activation pathways may be involved in the stimulation by the whole plant extract and the VT.

Toxic proteins from European mistletoe (Viscum album L.): increase of intracellular IL-4 but decrease of IFN-gamma in apoptotic cells.

BACKGROUND: Mistletoe lectins (ML), the major biologically active components of mistletoe extracts, which are used for adjuvant cancer therapy, induce apoptosis in lymphocytes and tumor cells. In addition, ML at toxic concentrations induce the release of cytokines, but it remains unclear as to whether dying or activated cells are responsible. MATERIALS AND METHODS: By flow cytometry, expression of IFN-gamma, IL-4, apoptosis marker Apo2.7 and anti-apoptotic Bcl-2 proteins were analyzed in response to ML or viscotoxins (VT) in PBMC from controls and plasmocytoma cells (U-266). RESULTS: While ML inhibited PMA/Ca-ionophore/monensin co-stimulated IFN-gamma production, they increased IL-4 expression in CD8+ and CD4+ T-cells. Thereby, IL-4 was mainly expressed in apoptotic cells with a low level of Bcl-2 proteins. In contrast, the cell membrane permeabilising VT induced complete loss of Bcl-2 proteins but did not stimulate IL-4 production within 24 hours, indicating that IL-4 expression is related to apoptosis but not to necrosis. CONCLUSION: Despite the role of IL-4 during activation of type2 T-helper cells, IL-4 expression may play an important yet undefined role during apoptosis of normal and tumor cells.

Intracellular expression of IL-4 and inhibition of IFN-gamma by extracts from European mistletoe is related to induction of apoptosis.

BACKGROUND: Extracts from European mistletoe are used for adjuvant cancer treatment. Their influence on the intracellular expression of cytokines of the T-helper cells type-1 (Th1; IFN-gamma) or type-2 (Th2; IL-4) is still unknown. MATERIALS AND METHODS: Lymphocytes from controls were incubated with mistletoe extracts (ME) and mistletoe lectins (ML) for 24 hours and co-stimulated with PMA/Ca-ionophore/monensin during the last 6 hours. Apoptosis and intracellular cytokine expression were detected by flow cytometry, the cytokine release into the supernatants by ELISA. RESULTS: ME and ML significantly inhibited intracellular expression of IFN-gamma but stimulated IL-4. Thereby, IL-4 was mainly expressed in apoptotic (Apo2.7+) cells. However, IFN-gamma secretion into the supernatants of the cells was dose-dependently inhibited by ME and ML, while IL-4 was not detected at all. CONCLUSION: The intracellular expression of the 'Th2-cytokine' IL-4 in ME- and ML-exposed cells may not be related to a typical Th2-response but rather to cell death. This effect might be of great relevance e.g. after intratumoural injection of the mistletoe extracts and, in general, for the inhibition of an inflammatory response during apoptosis.