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Characterization of cytotoxic proteins from mistletoe (Viscum album L.).

Proteins from a laboratory-made oak mistletoe extract and from the commercial mistletoe preparation Iscador Quercus were cytotoxic for leukemia Molt 4 cells in culture. A 50% growth inhibition was obtained with 0.1 microgram/ml proteins for the mistletoe extract and 0.025 microgram/ml for Iscador. On cation exchange chromatography, cytotoxic proteins from the mistletoe extract were mainly eluted at the same positions as purified lectins, while those of Iscador were eluted at the positions of viscotoxins. The data are discussed in relation to the pharmacological activities of the mistletoe protein complexes described in the literature.

Antineoplastic Agents, Phytogenic↗

Study of heterogeneity of lectins in mistletoe preparations by monoclonal antibodies to their A-subunits.

Monoclonal antibodies (monAbs) displaying diverse affinity to native and recombinant mistletoe lectins A-chains (ML I-A, ML II-A, ML III-A and rML-A) have been obtained. In accordance to specificity of monAb they have been classified into three groups: 1. monAb against MLI-A and MLII-A, 2. monAb against MLII-A and MLIII-A, 3. monAb against A-subunits of MLI, MLII and MLIII. The results indicate, that antigen determinants of mistletoe lectins recognized by monAb MNA4, MNA9 and MTC12 do not contain any carbohydrates. Assay of lectins in mistletoe preparations was based on enzyme-linked lectin assay to meet the requirements given in the guidelines for drug tests. In this paper a sandwich ELISA test-system is described which allows to identify each of three ML-toxins. With the detection limits below 3 ng/ml and a linear measuring range of 3-30 ng/ml, dosages of mistletoe lectins in therapeutic range can be assayed. The problems in mistletoe lectins determination, structural differences and nature of heterogeneity of this proteins are discussed.

Animals↗

Effects of a lectin- and a viscotoxin-rich mistletoe preparation on clinical and hematologic parameters: a placebo-controlled evaluation in healthy subjects.

BACKGROUND AND OBJECTIVES: Mistletoe preparations, which are widely used among patients with cancer in Germany, have immunomodulating properties in vitro and in vivo. The aim of this evaluation was to determine and compare the effects of a lectin-rich (Iscador Qu [IQ] special, Weleda Company, Schwäbisch, Gmünd, Germany.) and a lectin-poor but viscotoxin-rich (Iscador Pini [IP] Weleda Company) mistletoe preparation on clinical and hematologic parameters in healthy subjects. DESIGN: In a double-blinded study, 48 volunteers were randomized to one of three groups: 16 received IQ or IP in increasing doses or placebo twice per week subcutaneously for 12 weeks. The differential blood count and the acute phase markers haptoglobin and C-reactive protein were examined weekly and the symptoms were scored using standardized questionnaires. RESULTS: IQ resulted in significant eosinophilia (315 +/- 109) beginning at week 5 (until week 12) compared to IP (183 +/- 120) or placebo (200 +/- 179). Furthermore, the acute phase marker haptoglobin was significantly increased in the IQ group during week 4. Dose-dependent local reactions (LRs) at the injection site occurred in all subjects who received mistletoe preparations but were stronger in the IQ-treated subjects than in the IP-treated group. The LRs observed in the IQ-treated group were characterized by stronger itching and longer latency than LRs in the IP-treated group (p < 0.05). Severe side-effects did not occur in any of the probands. CONCLUSIONS: IQ but not IP can induce eosinophilia in healthy individuals, and this may be related to its content of mistletoe lectins. In contrast, exposure to the viscotoxin-enriched extract IP did not result in specific changes of hematologic parameters. Furthermore, intensity and time course of local reactions seemed to depend on the concentration of mistletoe lectins in those extracts.

Adjuvants, Immunologic↗

Donor-dependent and dose-dependent variation in the induction of T lymphocyte locomotion in a three-dimensional collagen matrix system by a mistletoe preparation (Iscador).

Controlled activation of non-specific and specific immune defence mechanisms can beneficially manipulate the host's ability to attack malignant cells. In this context, migration and tissue distribution of immunocompetent cells may be prerequisites for an efficient immune surveillance. The effect of various non-cytotoxic concentrations of the Viscum album L. (mistletoe) preparation Iscadore QuFrF on the locomotory activity of immunomagnetically isolated human CD4+ and CD8+ T lymphocytes from healthy donors was investigated. Cellular migration was examined within a three-dimensional collagen matrix. Donor-dependent variations in baseline activities of spontaneously locomoting T cells were accompanied by individual response patterns of T cells from different donors in the presence of various concentrations of mistletoe preparation (0.25-2.5 micrograms/ml). Using the three-dimensional collagen matrix assay an induction of locomotory activity was detected in a highly reproducible fashion although the optimal concentration of mistletoe preparation and the time point of maximal response were individual for each donor. Our data suggest that the direct stimulation of T-cell migration by mistletoe components may modulate the system of immune surveillance and recognition in patients under mistletoe therapy.

Antineoplastic Agents, Phytogenic↗

Contrasting cascades: insectivorous birds increase pine but not parasitic mistletoe growth.

1. Intraguild predation occurs when top predators feed upon both intermediate predators and herbivores. Intraguild predators may thus have little net impact on herbivore abundance. Variation among communities in the strength of trophic cascades (the indirect effects of predators on plants) may be due to differing frequencies of intraguild predation. Less is known about the influence of variation within communities in predator-predator interactions upon trophic cascade strength. 2. We compared the effects of a single predator community between two sympatric plants and two herbivore guilds. We excluded insectivorous birds with cages from ponderosa pine Pinus ponderosa trees parasitized by dwarf mistletoe Arceuthobium vaginatum. For 3 years we monitored caged and control trees for predatory arthropods that moved between the two plants, foliage-feeding caterpillars and sap-feeding hemipterans that were host-specific, and plant damage and growth. 3. Excluding birds increased the abundance of ant-tended aphids on pine and resulted in an 11% reduction in pine woody growth. Mutualist ants protected pine-feeding aphids from predatory arthropods, allowing aphid populations to burgeon in cages even though predatory arthropods also increased in cages. By protecting pine-feeding aphids from predatory arthropods but not birds, mutualist ants created a three-tiered linear food chain where bird effects cascaded to pine growth via aphids. 4. In contrast to the results for tended aphids on pine, bird exclusion had no net effects on untended pine herbivores, the proportion of pine foliage damaged by pine-feeding caterpillars, or the proportion of mistletoe plants damaged by mistletoe-feeding caterpillars. These results suggest that arthropod predators, which were more abundant in cages as compared with control trees, compensated for bird predation of untended pine and mistletoe herbivores. 5. These contrasting effects of bird exclusion support food web theory: where birds were connected to pine by a linear food chain, a trophic cascade occurred. Where birds fed as intraguild predators, the reticulate food webs linking birds to pine and mistletoe resulted in no net effects on herbivores or plant biomass. Our study shows that this variation in food web structure occurred between sympatric plants and within plants between differing herbivore guilds.

Animals↗

[Development of lymphocyte subsets in tumor patients after subcutaneous administration of mistletoe extracts].

OBJECTIVE: In order to exclude the possibility that mistletoe therapy may result in immunosuppression, as indicated by a significant reduction of defined lymphocyte subsets, PATIENTS AND METHODS: peripheral blood cells of 23 tumour patients were treated subcutaneously with increasing concentrations of aqueous mistletoe extracts (Helixor(R)). RESULTS AND CONCLUSIONS: Within an observation period of 7 months, the relative amount of lymphocytes and the number of natural killer (NK) cells increased while the number of lymphocyte subsets (i. e. CD19+ B cells, CD4+ T helper cells, CD8+ CD28- suppressor cells, CD8+ CD28+ cytotoxic cells) and the proportion of CD25+ (activated) cells within T cells showed a statistically remarkable trend; due to the multiple test problem of statistical evaluation this trend is not allowed to be termed significant. The leucocytes decreased insignificantly within the observation period. However, we were unable to verify a suggested increase of defined lymphocyte subsets within 2-3 months after the onset of mistletoe treatment. Nevertheless, for the parameters CD19+ B cells, CD4+ T helper cells, CD8+ cells, CD8+ CD28+ cytotoxic cells and CD16+/CD56+ NK cells we observed statistically remarkable peaks within die 2nd and 3rd month of therapy, confirming the hypothesis. The responses to the extracts were obviously interindividually different; the immune responses especially of patients with a lower number of peripheral T cells were less significant as compared to those of patients with adequate T cell numbers. Surprisingly, even an increase of the drug concentration >3 ng mistletoe lectin (as determined within the whole plant extract) per kg body weight enhanced the number of CD4+ T helper cells. A decreased immunological reaction on mistletoe extracts was shown especially for patients with a reduced number of peripheral T cells, whereas patients with normal T-cell number were more reactive.

Adult↗

[Problems of randomized studies in complementary medicine demonstrated in a study on mistletoe treatment of patients with breast cancer].

BACKGROUND: Prospective randomized studies on mistletoe therapy repeatedly demonstrated that there is a basic problem in the matter of enrolling the appropriate number of patients within a reasonable amount of time. Most studies have to face this problem. However, recent experience suggests that this problem is more pronounced in the case of mistletoe treatment of cancer patients. OBJECTIVE: Possibility of recruitment and randomization of breast cancer patients for a mistletoe study. PATIENTS: During a period of 28 months every patient was registered who was admitted to the Gynecological Hospital of the University of Heidelberg because of suspected cancer. RESULTS: Out of 1,922 patients who were operated on for breast tumor, 521 first met the inclusion and exclusion criteria. 154 out of these 521 patients agreed to take part in the study. After availability of the final results on tumor staging and the therapy plan for conventional treatment, 80 out of the 154 women had to be excluded from the study. From the remaining 74 patients (48%), however, only 29 (39%) would have agreed to take part in a randomized mistletoe study. CONCLUSIONS: This confirms our suspicion that the difficulties of enrollment and randomization in the case of mistletoe studies exceed those of studies conducted in conventional oncology. The reasons for this dramatic effect and the possibility of alternative study designs are discussed.

Antineoplastic Agents, Phytogenic↗

Randomized and double-blind studies--demands and reality as demonstrated by two examples of mistletoe research.

BACKGROUND: Two examples of clinical research with mistletoe extracts were used to demonstrate essential difficulties in carrying out randomized and placebo-controlled trials. STUDY 1: In a randomized, placebo-controlled, double-blind study investigating the immunological effects of mistletoe extract, healthy subjects were asked to state whether, in their estimation, they had been treated with verum or a placebo. Due to the intrinsic effects of the mistletoe therapy--local inflammatory reactions at the injection site--100% of the subjects treated with verum and 77% of those treated with a placebo made a correct assessment of their therapy. Although double-blind trials are preferable from the methodological point of view--above all in QoL research--this study shows that double blinding is barely achievable when the investigated therapy has obvious (side) effects. STUDY 2: A prospective, randomized, multicenter study of a mistletoe therapy complementary to chemotherapy treatment of breast cancer had to be stopped after a period of 28 months, because it proved impossible to recruit more than 16 patients in six large study centers. With regard to this example and to other failed, GCP-compliant clinical trials on mistletoe therapy we describe which factors interfere with successful clinical trials. One important point, especially in the investigation of complementary cancer treatments, is that cancer patients are unwilling to have their treatment determined by randomization. Many cancer patients in Germany have their own point of view, as to whether a complementary treatment could be of benefit to them or not. Faced with a life-threatening disease they wish to determine this part of their treatment themselves. CONCLUSION: This background elucidates the need for improving the methodology of non-randomized trials to obtain objective and reliable results even in these fields of clinical research.

Breast Neoplasms↗

Recent studies on the anticancer activities of mistletoe (Viscum album) and its alkaloids.

Detailed methods for in vitro/in vivo evaluation of anticancer drugs, with special reference to mistletoe extracts, have been reviewed. Mistletoe extracts have been shown to possess significant antitumor activity, in vivo, against murine tumors, Lewis lung carcinoma, colon adenocarcinoma 38 and C3H mammary adenocarcinoma 16/C. Methods for the extraction of biologically active alkaloids from mistletoe and their anticancer activities are presented. The possible origin of alkaloids in mistletoe plants, and their contributions towards a mechanism of anticancer activities of mistletoe extracts, are proposed.

Alkaloids↗

Effects of a mistletoe preparation with defined lectin content on chronic hepatitis C: an individually controlled cohort study.

Despite advances in the therapy of chronic hepatitis C for some hepatitis C virus (HCV) genotypes interferon and ribavirin combination therapy is effective in less than 50% of patients. Abnobaviscum Quercus (AQ) is a mistletoe preparation containing defined amounts of mistletoe lectins (ML). It has shown immunomodulatory properties in vitro and in vivo. In small clinical trials AQ resulted, within an anthroposophical treatment concept, in a biochemical or virological response in up to 40% of patients with chronic hepatitis C. In order to evaluate the effect of this preparation we conducted an individually controlled cohort study. 25 patients with chronic hepatitis C (mean duration 147 +/- 80 months) and elevated alanine aminotransferase (ALT) levels were included in the study. As control they were observed for 6 months pre-treatment. This pre-treatment period was followed by 6 months of active treatment in which the mistletoe preparation was subcutaneously injected three times a week. Main outcome parameters were normalization of ALT and viral load. Hepatitis C associated signs and symptoms like tiredness, fullness in the right upper abdomen and musculoskeletal pain were assessed monthly in a standardized questionnaire. All 25 patients completed the study and most of the patients wanted to continue treatment. Mean duration of treatment was 9.1 months. None of the patients had complete or partial normalization of ALT or HCV RNA levels during pre-treatment or treatment period. Mean ALT did not change during the study. Tiredness, fullness in the right upper abdomen and musculoskeletal pain were present in 18, 8 and 4 patients respectively. They significantly improved within two months of treatment. A significant eosinophilia (p=0.0001) occurred between month 2 and 6 during treatment. 9 month treatment with a ML containing mistletoe preparation has no effect on viral load or ALT as markers of activity in patients with chronic hepatitis C. However, frequency and intensity of clinical signs and symptoms in our patients decreased significantly, similar to reports of improved quality of life in tumour patients treated with such preparations. A significant eosinophilia suggests that ML containing mistletoe preparations induce a T-helper 2 immune response.

Adjuvants, Immunologic↗

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.

Adjuvants, Immunologic↗

Stimulation of the maturation of dendritic cells in vitro by a fermented mistletoe extract.

BACKGROUND: Dendritic cells (DC) play a key role during the initiation of specific immune responses. In cancer patients, however, an alteration of their function was observed. In our investigation we analysed the influence of a fermented mistletoe extract often used for adjuvant treatment of cancer patients on the generation and maturation of DC. MATERIALS AND METHODS: Monocytes from healthy individuals were incubated with a fermented mistletoe extract in the presence or absence of GM-CSF/IL-4. Surface marker expression was measured by flow cytometry. RESULTS: While there was no relevant effect on the generation of DC in the absence or presence of GM-CSF/IL-4 in 5-day cultures, the mistletoe extract significantly stimulated the maturation of pre-generated immature DC, as evidenced by a heightened expression of CD83. Like the positive control TNF-alpha, the mistletoe extract significantly activated CD80 and CD86 as well as HLA class I and II molecules on these cells. CONCLUSION: Our data clearly demonstrate an influence of the mistletoe extract on the maturation of DC, but it remains to be elucidated whether the function of DC is also activated and, especially, whether this effect can be observed in tumour patients as well.

ADP-ribosyl Cyclase↗

Adjuvant intravesical treatment with a standardized mistletoe extract to prevent recurrence of superficial urinary bladder cancer.

BACKGROUND: Adjuvant intravesical Bacillus Calmette-Guerin (BCG) treatment after resection of non invasive superficial bladder cancer has been shown to significantly decrease tumor recurrence. However, the serious local and systemic side-effects of this treatment have promoted the use of other immunoactive substances, which, to date, have all failed to show efficacy equal to BCG therapy. PATIENTS AND METHODS: In the present phase I/II clinical trial, an aqueous mistletoe extract, standardized to mistletoe lectin, was applied intravesically to 30 patients with superficial urothelial bladder carcinomas of stages pTa and pT1, grades 1 to 2. After transurethral resection, each patient received 6 instillations at weekly intervals of 50 ml of the extract with mistletoe lectin concentrations between 10 ng/ml and 5000 ng/ml. This was retained in the bladder for 2 hours. Three patients per group received a dose, which was then doubled in the next group. The clinical follow-up consisted of examinations by cystoscopy, cytology and random biopsies. RESULTS: Within the observation time of 12 months, 9 patients had tumor recurrence, while 21 patients remained tumor-free. This recurrence rate was comparable to that of local historical controls with superficial bladder cancer of the same stages and grades that had been treated with adjuvant BCG. The tolerability of the intravesically-administered mistletoe extract was very good. None of the study patients had local or systemic side-effects according to the WHO classification 1-4. CONCLUSION: From these results, it is concluded that the standardized mistletoe extract could be a potential adjuvant therapy for superficial bladder cancer. Further studies may show the optimal intravesical treatment regimen.

Administration, Intravesical↗

[Detection and quantitative determination of lectins and viscotoxins in mistletoe preparations].

Mistletoe lectins and viscotoxins, which up to now have been isolated only from plant material, were detected and quantitatively determined in the mistletoe preparation Iscador and in a fermented mistletoe extract. Lectins were isolated by affinity chromatography and analyzed by isoelectric focussing. Thus, in Iscador and in the fermented mistletoe extract only the mistletoe lectins ML II/III were found whereas typical proteins of the ML I complex were missing. The lectin content of the preparation and the extract was determined by "single radial immunodiffusion" (SRID). For identification and quantitative determination of viscotoxins, a HPLC method was designed.

Chromatography, Affinity↗

Differences in the in vitro effectiveness of preparations produced from mistletoes of various host trees.

The in vitro effectiveness of three Helixor preparations produced from mistletoes of different host trees on suspension cell cultures of the human leukemia cell line Molt 4 has been compared by means of dose-response investigations. After 72 h treatment the preparation produced from mistletoes of the appletree (Malus) shows the strongest effect on the growth and viability of the cell cultures. The preparation produced from mistletoes of the fir (Abies) shows also an evident but much weaker effect, whereas the preparation produced from mistletoes of the pine (Pinus) causes a weak effect only at a very high dosage. As shown in 24-h incorporation experiments with 3H-labelled DNA-, RNA- and protein precursors the preparation produced from mistletoes of the apple-tree reduces the protein synthesis of the cells to a greater extent than the DNA-and RNA-synthesis.

Cell Line↗

Immunoprotective activity of the galactoside-specific lectin from mistletoe after tumor destructive therapy in glioma patients.

35 patients suffering from malignant stage III/IV glioma were enrolled into a prospectively randomized clinical trial. All patients were provided with standard oncologic treatment (neurosurgery, radiation, basic clinical care according to protocol and indication) and randomly divided into a) treatment group: receiving subcutaneous injections of a ML (a galactoside-specific lectin from mistletoe) standardized mistletoe extract, 1 ng ML-1/kg BW, twice a week for 3 months, starting on day 1 post surgery, b) control group: without additional complementary treatment. Immunophenotyping of peripheral blood leukocytes was done by flow cytometry (pre surgery; day 1, week 1, month 3 and 6 post surgery) to evaluate the immunomodulating capacity of ML-1 standardized mistletoe extract. Standard tumor destructive treatment of glioma proved to be suppressive for peripheral blood lymphocytes, since all subsets tested revealed statistically significant down regulation. Unlike the lymphocyte counts and activities of patients from the control group who gave preoperative values after 3-6 months), mistletoe treatment induced a statistically significant up regulation of cell counts (CD-3, CD4, CD-8 cells) and activities (CD-25, HLA/DR positive cells) after 3 months, as compared to preoperative values. Obviously, a strong immunoprotective/immunostimulatory effect was induced by the treatment of glioma patients with ML-1 standardized mistletoe extract which correlated with an improved quality of life, as determined by a standard questionnaire (Spitzer).

Brain Neoplasms↗

Modulation of the cellular and humoral immune responses of tumor patients by mistletoe therapy.

There is evidence from recent data that mistletoe extracts exert immunostimulatory properties which could explain their therapeutic effects observed in some tumor patients. Aim of our study was, therefore, to investigate the effect of a subcutaneous 16-weeks therapy with a mistletoe extract (ABNOBAviscum Mali, AM) on the cellular and humoral immune responses in eight breast cancer patients. Mistletoe therapy induced a strong initial proliferation of peripheral blood mononuclear cells (PBMC) in all individuals, which, however, decreased in six patients during the observation period, indicating that not only activating but also inhibitory mechanisms have been induced. In all supernatants of AM-stimulated cell cultures TNF-alpha or IL-6 were found, indicating the activation of cells of the monocyte-/macrophage lineage by mistletoe extracts. Further analyses revealed, that AM induced in vitro also the release of low amounts of IFN-gamma and IL-4 with individual variations. At the end of the therapy, a shift to Th1- related cytokines could be observed in the in vitro cell culture system. All patients produced anti-mistletoe lectin 1 antibodies of the IgG-type during therapy and in four of them additionally antibodies of the IgE-type were found. It, therefore, seems that AM can influence the Th1/Th2 balance and, in case of a Th1 shift, this may favourably influence the tumor growth.

Adjuvants, Immunologic↗

Effects of a standardized mistletoe preparation on metastatic B16 melanoma colonization in murine lungs.

The immune response-modifying drug Lektinol is a mistletoe preparation which is standardized with respect to bioactive viscum album agglutinin, the most active component of mistletoe. The present study was designed to evaluate the antimetastatic effects of this preparation following intravenous injection of B16 melanoma cells into mice. The standardized mistletoe extract was administered intravenously in doses of 100, 1000 or 5000 microliters/kg (equivalent to 3, 30 or 150 ng/kg of viscum album agglutinin) once daily for three weeks. An inhibition of mean pulmonary metastatic colonization of 58 to 95%, as measured by the number of melanoma cells on lung tissue slides, and a significant decrease of percentage of bronchoalveolar lavage pigmented cells were observed. In addition, a correlation of this antimetastatic activity with cellular immune parameters was investigated. In lavage fluids from the tumor-bearing mice, there was a 5 to 6-fold significant increase in the percentage of MAC-1+ (CD11b/CD18) immunocompetent macrophages in comparison with cells from vehicle-treated animals. The percentages of double-positive immature CD4+8+ thymocytes were significantly increased in animals treated with the standardized mistletoe extract. There were no signs of treatment-related toxicity. The results of this study indicate that the standardized mistletoe extract shows antimetastatic activity against B16 melanoma lung colonization.

Animals↗