Significant human beta-cell turnover is limited to the first three decades of life as determined by in vivo thymidine analog incorporation and radiocarbon dating.

AIMS: Diabetes mellitus results from an absolute or relative deficiency of insulin-producing pancreatic ß-cells. The turnover rate of adult human ß-cells remains unknown. We employed two techniques to examine adult human islet ß-cell turnover and longevity in vivo. METHODS: Subjects enrolled in National Institutes of Health clinical trials received thymidine analogs [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8 d to 4 yr prior to death. Archival autopsy samples from 10 patients (aged 17-74 yr) were employed to assess ß-cell turnover by scoring nuclear analog labeling within insulin-staining cells. Human adult ß-cell longevity was determined by estimating the cells' genomic DNA integration of atmospheric (14)C. DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15-yr-old donor, and purified ß-cell DNA was obtained from two donors (ages 48 and 80 yr). (14)C levels were then determined using accelerator mass spectrometry. Cellular "birth date" was determined by comparing the subject's DNA (14)C content relative to a well-established (14)C atmospheric prevalence curve. RESULTS: In the two subjects less than 20 yr of age, 1-2% of the ß-cell nuclei costained for BrdU/IdU. No ß-cell nuclei costained in the eight patients more than 30 yr old. Consistent with the BrdU/IdU turnover data, ß-cell DNA (14)C content indicated that the "birth date" of cells occurred within the subject's first 30 yr of life. CONCLUSIONS: Under typical circumstances, human ß-cells and their cellular precursors are established by young adulthood.

[Retrospective study of malignant melanoma patients treated with mistletoe extracts]. / Retrospektive Untersuchung von Patienten mit malignem Melanom unter einer Misteltherapie.

OBJECTIVE: The aim of the present investigation was to analyze survival time and survival rate of all patients with malignant melanoma who had been counseled at the Tumorambulanz Herdecke of the Community Hospital Herdecke. PATIENTS AND METHODS: 284 melanoma patients were included in a retrospective questionnaire study. Only those patients were considered for analysis in whom the prognostic factors histology, tumor localization, and Clark level were known. The data of the study population were compared with patient data obtained from the literature. RESULTS: 94 patients were included in the analysis. 66 of whom had received and 7 had not received mistletoe treatment, in the remaining 21 patients there was no information whether or not mistletoe treatment had been given. Thus, we did our study without a clearly defined internal control group. The median survival time among patients treated with mistletoe had been 14.1 years. The 5- and 10-year survival rates were 80 and 68% for the mistletoe-treated patients, respectively. DISCUSSION: The 5-year survival rate of the mistletoe-treated patients is comparable to that of patients without mistletoe therapy while the 10-year survival rate is a little bit lower. This may be due to the fact that, in contrast to the patients from the relevant literature, 33.3% of the patients suffered from lymph node and/or distant metastases already before counseling the Tumorambulanz Herdecke. Moreover, 50% of our patients had melanoma of Clark level IV in contrast to 22.2% or 31% in the relevant literature. CONCLUSIONS: In spite of the theoretical reservations against mistletoe treatment in melanoma patients, our retrospective analysis did not show any clues about disadvantages of mistletoe treatment in melanoma patients. A controlled prospective study therefore should prove the efficacy of a mistletoe therapy in patients with malignant melanoma.

Induction of antibodies to viscotoxins A1, A2, A3, and B in tumour patients during therapy with an aqueous mistletoe extract.

Mistletoe extracts exert immunomodulatory properties on immunocompetent cells of the innate as well as the specific immune system. These effects have been mainly ascribed to mistletoe lectin 1 (ML-1) present in most of the extracts. However, it became evident that also other components of these extracts may induce immunological reactions, and especially viscotoxins (VT) may be of relevance. Aim of the study was, therefore, to evaluate whether VT like ML-1 could activate B-cells and lead to the production of VT-specific antibodies. Sera from 26 patients with different tumours who were treated with the mistletoe extract ABNOBAviscum Mali (AM) 4 for at least 18 weeks were analysed before therapy and after 3, 6, 9, 12, and 18 weeks. Sera were tested by ELISA against the four viscotoxins A1, A2, A3, B, as well as against ML-1. Within the observation period twenty-four (92%) of the 26 patients developed antibodies to at least one of the four VT and 25 (96%) to ML-1. In most instances, anti-VT antibodies appeared after 6-9 weeks of treatment. The antibodies were predominantly of the IgG type belonging preferentially to the IgG1 and IgG3 subclass. IgE antibodies were found only to VT-B and to ML-1. There was no relation between the development of antibodies to VT and ML-1, and also cross-reactivity could be excluded with high probability. These data indicate that not only ML-1 but also VT induce immunological responses in patients treated with mistletoe extracts. Whether there is any relationship to the postulated anti-tumour effect of mistletoe extracts has, however, still to be evaluated.

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.

Expression of interleukin-4 in apoptotic cells: stimulation of the type-2 cytokine by different toxins in human peripheral blood mononuclear and tumor cells.

BACKGROUND: Immunological reactivity is regulated by T-cell populations (type-1 and type-2 cells) via cytokine secretion, but their influence on apoptosis remains unclear. METHODS: Intracellular expression of type-1 (interferon [IFN]-gamma) and type-2 (interleukin [IL]-4) cytokines and apoptosis-related molecules (Apo2. 7, Bcl-2 protein) was studied by flow cytometry in human peripheral blood mononuclear cells (PBMC), myeloma (U-266), monocytic (THP-1), and T-leukemia cells (MOLT-4) in response to toxins, which act on different intracellular targets (actinomycin D, cycloheximide, the mistletoe lectins [ML]-1 and ML-3, brefeldin A, staurosporine). RESULTS: The apoptosis-inducing toxins stimulated intracellular IL-4 expression mainly in PBMC with high expression of the mitochondrial apoptosis marker, Apo2.7, but with decreased level of the anti-apoptotic Bcl-2 protein. Up-regulation of IL-4 coincided with a significant down-regulation of IFN-gamma in CD4(+) and CD8(+) cells. The inhibitor of oxidative phosphorylation, oligomycin, and the caspase inhibitor, z-VAD-fmk, abolished IL-4 expression and DNA fragmentation in the PBMC. Also in the myeloma, monocytic, and T-leukemia cells, IL-4 was mainly observed in the Apo2.7(+) apoptotic cells in response to the toxins. CONCLUSIONS: We suggest that the different apoptotic toxins activate a common pathway in which IL-4 production plays a yet unknown intracellular role further downstream during apoptosis.

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.

Apoptosis-associated generation of reactive oxygen intermediates and release of pro-inflammatory cytokines in human lymphocytes and granulocytes by extracts from the seeds of Acalypha wilkesiana.

Seeds from Acalypha wilkesiana (Euphorbiaceae) are an essential component of a complex plant mixture used empirically by traditional healers in Southwest Nigeria to treat breast tumours and inflammation. To investigate their biological properties, we incubated human lymphocytes and granulocytes with aqueous and ethanolic extracts of A. wilkesiana seeds (AWS). In lymphocytes, we observed an induction of apoptosis and generation of reactive oxygen intermediates (ROI), as measured by the oxidation of hydroethidine, within 2 h, while in granulocytes, an aqueous seed extract induced the oxidative burst and enhanced phagocytosis of Escherichia coli within 10-20 min. In the supernatants of 72-h cultured lymphocytes, AWS induced the release of the pro-inflammatory cytokines tumour necrosis factor-alpha and interleukin-6, and also T-cell-associated cytokines interleukin-5 and interferon-gamma. These preliminary results encourage further investigations of this drug with both cytotoxic and immunomodulating properties.

Induction of Fas ligand (CD95L) by the toxic mistletoe lectins in human lymphocytes.

BACKGROUND: Fas ligand (FasL, CD95L) predominantly expressed on activated cytotoxic T cells and NK cells triggers apoptosis in Fas receptor (Apo-1, CD95) positive target cells. We investigated the expression of FasL, Fas and tumor necrosis factor (TNF) receptor 1 (TNF-R1, CD120a) on cultured human lymphocytes and leukemic T and B cells. MATERIALS AND METHODS: Lymphocytes from six healthy individuals, from four patients with chronic lymphocytic T or B cell leukaemia, and leukemic Molt-4 cells were incubated with the apoptosis- inducing mistletoe lectins (ML I and ML III). RESULTS: Incubation of differentiated lymphocytes with the ML resulted in a significant upregulation of FasL in the surviving CD4+ T helper cells, CD8+ cells and CD19+ B cells. Similarly, the TNF receptor expression increased, while the Fas molecule decreased. In contrast, FasL was not induced in leukemic cells. CONCLUSIONS: Apart from a direct induction of apoptosis in response to an inhibition of protein synthesis by the enzymic ML A chain, ML treatment may indirectly induce apoptosis in Fas+ tumour cells through activated FasL+ lymphocytes.

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.

Thionins from Viscum album L: influence of the viscotoxins on the activation of granulocytes.

BACKGROUND: Extracts from European mistletoe (Viscum album L.) are applied in adjuvant cancer treatment, and some components, especially the mistletoe lectins (ML) have been immunologically characterised, but not the thionins, termed viscotoxins (VT). MATERIALS AND METHODS: The influence of the VT on human granulocytes was studied by flow cytometry: E.coli co-stimulated respiratory burst by oxidation of dihydrorhodamine 123 to rhodamine 123 and phagocytosis by ingestion of FITC-labelled E.coli. RESULTS: VT (25 and 250 micrograms/ml), in contrast to ML, significantly enhanced phagocytosis and burst activity. VT-rich mistletoe extracts also exerted significant effects. In addition, E.coli-activated granulocytes positively stain with Annexin-V and propidium iodide only due to 250 micrograms/ml VT incubation, suggesting that at this concentration burst activity was induced by the physiological activity of granulocytes after microbial ingestion and also by cytotoxic effects. CONCLUSION: Viscotoxins exert yet unknown strong immunomodulatory effects on human granulocytes, which might be of benefit for tumour patients, in addition to their cytotoxic properties.

Accidental cell death and generation of reactive oxygen intermediates in human lymphocytes induced by thionins from Viscum album L.

The cytotoxic mechanisms of thionins from Viscum album L., the viscotoxins, were investigated in human granulocytes and lymphocytes. The time course of viscotoxin effects indicate accidental cell death, i.e. membrane permeabilization, degradation of cytoplasm and chromatin, swelling of mitochondria with loss of their cristae, and generation of reactive oxygen intermediates within 1-2 h, followed by secondary apoptosis-associated events. The viscotoxin homologue purothionin from whole-wheat flour and viscotoxin B, however, did not induce cell death in cultured lymphocytes. Cytotoxicity of cationic and amphipathic viscotoxin was prevented only by cleavage of its disulphide bridges.

Characterisation of immunological reactivity of patients with adverse effects during therapy with an aqueous mistletoe extract.

Drugs, which are effective are also bond to exert adverse effects. This is also true for mistletoe extracts. Extracts from European mistletoe (Viscum album, VAL) belong to the complementary therapeutic regimens and are used for adjuvant cancer therapy. This study was performed to characterise immunological reactivity of patients with adverse effects during treatment with an aqueous VAL extract (Helixor). VAL-stimulated proliferation and cytokine release of peripheral blood mononuclear cells (PBMC) and anti-mistletoe lectin (ML)-1 antibody production were investigated. 34 patients with proven adverse effects due to VAL therapy (group 1) and 9 patients with unproven relation (group 2) were studied and compared to 14 tumour patients treated with VAL for more than 2 years without side effects (TTP). VAL-stimulated proliferation of PBMC of group 1 was significantly enhanced as compared to group 2 patients and TTP. PBMC from patients with local manifestations proliferated significantly stronger than those from patients with systemic symptoms. Anti-ML-1 antibodies of the IgE type were produced in patients with proven adverse effects but not in patients without adverse effects. Production of Th1 and Th2 specific cytokines varied considerably, indicating that different mechanisms were involved in the induction of adverse effects. In conclusion, our study provide evidence that adverse effects towards VAL (Helixor) are seldom and are dominated by an application site reaction suggesting the involvement of delayed type hypersensitivity (DTH) reactions.

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.

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.

Influence of polysaccharides from Viscum album L. on human lymphocytes, monocytes and granulocytes in vitro.

BACKGROUND: An acidic arabinogalactan from European mistletoe (Viscum album L, VAL; 1.34 x 10(6) Dalton) was studied in detail because its immunological properties are poorly characterised. MATERIALS AND METHODS: Flow cytometric studies focussed on PS-activated proliferation of human lymphocytes measured via incorporation of bromo-deoxyuridine (BrdU), granulocyte phagocytosis via ingestion of FITC-labelled E.coli, and respiratory burst via oxidation of dihydrorhodamine 123 to rhodamine 123. Cytokines were detected in the cell culture supernatants by ELISA. RESULTS: PS, in contrast to mistletoe lectins (ML), significantly stimulated proliferation of CD4+ T-cells but not CD8+ and CD19+ cells. However, ML influenced PS-mediated stimulation, with a synergistic effect in one and an inhibitory effect in another individual. Furthermore, IFN-gamma release was significantly enhanced by PS, favouring a T-helper cell type-1 cytokine pattern, further IL-6 was significantly stimulated, while granulocyte activity was not affected. CONCLUSIONS: VAL-PS exert yet unknown stimulatory activities, especially on specific CD4+ T-cells which may be influenced by other extract components like the ML. These components may contribute to the anti-tumour effect of VAL.

Release of interleukin-6 in cultured B-chronic lymphocytic leukaemia cells is associated with both activation and cell death via apoptosis.

BACKGROUND: There is growing evidence that some cytokines promote B cell survival, while others enhance cell death. Interleukin-6 was reported to induce proliferation of chronic lymphocytic leukaemia (B-CLL) cells, and to enhance survival of these cells through inhibition of spontaneous apoptosis. MATERIALS AND METHODS: To more clearly define the effects of an in vitro stimulation of B-CLL cells, lymphocytes from 13 patients with B-CLL and from 6 healthy individuals were incubated for 7 d with immunomodulators such as interleukin-6 (IL-6), pokeweed mitogen (PWM), lipopolysaccharides (LPS), and extracts from Viscum album L. (VAL; Helixor), which were recognised to induce apoptosis but also to induce a release of pro-inflammatory cytokines such as IL-1, IL-6, and tumour necrosis factor-alpha. RESULTS: Although both, IL-6 and PWM induced a release of IL-6 and expression of the activation markers CD25 and CD71 on the surface of B-CLL cells, IL-6 did not increase or accelerate the proliferation of these cells. In contrast, VAL extracts did not result in an upregulation of activation markers or proliferation of B-CLL cells but induced both, cell death via apoptosis and IL-6 release. In response to PWM, only few clones of leukemic B cells incorporated the thymidine-analogue 5-bromo-2'-deoxyuridine. Moreover, a remarkable response of B-CLL cells towards the immunomodulators was observed only in one patient with an advanced stage. CONCLUSIONS: These preliminary results do not support theoretical objections of a B-CLL stimulation via induction of IL-6 in vitro.

Differential binding of toxic lectins from Viscum album L., ML I and ML III, to human lymphocytes.

The killing capacity of extracts from Viscum album L., widely used as an adjuvant in complementary cancer therapy, is dependent on the content of toxic proteins, especially the mistletoe lectins (ML). Although one may expect a homogeneous distribution of 'receptors' for these proteins on the cell surface, the sensitivity of cells to the ML-mediated cytotoxicity obviously differs, as the galNAc-binding ML III in contrast to the gal-binding ML I selectively killed CD8+ lymphocytes with a 'memory' phenotype (CD62Llo), while CD19+ B cells remained almost unaffected. B cells hardly bind ML III but did bind the gal-specific ML I. In accordance with these observations, in leukaemic B cells from patients with B chronic lymphocytic leukaemia and the human IgE-secreting myeloma cell line U-266 a strong induction of apoptosis-associated mitochondrial Apo2.7 molecules was observed after treatment with ML I and less effectively by ML III, while in the leukaemic T cell line Molt-4 both ML were strong inductors of apoptosis. In the light of these findings, the possible impact of ML I- and ML III-rich mistletoe extracts in the treatment of B cell neoplasia has to be carefully investigated.

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.