Carmen Steinborn

Viscum album neutralizes tumor-induced immunosuppression in a human in vitro cell model

Tumor cells have the capacity to secrete immunosuppressive substances in order to diminish dendritic cell (DC) activity and thereby escape from immune responses. The impact of mistletoe (Viscum album) extracts (VAE), which are frequently used as an additive anti-cancer therapy to stimulate the immune response, is still unknown. Using a human cellular system, the impact of two different VAE (VAEA + VAEI) on the maturation of human dendritic cells and on T cell function has been investigated using flow cytometry, automated fluorescence microscopy and cytokine bead array assays. Furthermore, we examined whether VAEI was able to counteract tumor-induced immunosuppression within this cellular system using a renal cancer cell model. The role of mistletoe lectin (ML) was analyzed using ML-specific antibodies and ML-depleted VAEI. VAEI and VAEA augmented the maturation of dendritic cells. VAEI abrogated tumor-induced immunosuppression of dendritic cells and both processes were partially mediated by ML since ML-depleted VAEI and ML-specific antibodies almost neutralized the rehabilitative effects of VAEI on DC maturation. Using these settings, co-culture experiments with purified CD4+ T cells had no influence on T cell proliferation and activation but did have an impact on IFN-γ secretion. The study provides a potential mode-of-action of VAE as an additive cancer therapy based on immunomodulatory effects. However, the impact on the in vivo situation has to be evaluated in further studies.

Immunomodulatory effects of metal salts at sub-toxic concentrations.

Because different metals are used in complementary medicine for the treatment of diseases related to a dysfunction of the immune system, this study aimed at determining the immunomodulatory potential of Pb(NO3)2, AuCl3, Cu(NO3)2, HgCl2, AgNO3, SnCl2, AsCl3 and SbCl3 at sub‐toxic concentrations and at assessing possible toxic side effects of low‐concentrated metal preparations. The influence of the metal salts on primary human mononuclear cells was analyzed by measuring cell viability using the water‐soluble tetrazolium salt assay, apoptosis and necrosis induction by annexin V/propidium iodide staining and proliferation by carboxyfluorescein diacetate succinimidyl ester staining and flow cytometry. Effects on T‐cell activation were assessed with CD69 and CD25 expression using flow cytometry whereas CD83, CD86 and CD14 expression was measured to evaluate the influence on dendritic cell maturation. Alterations of interleukin‐2 and interferon‐γ secretion were detected by enzyme‐linked immunosorbent assay and genotoxic effects were analyzed using the comet assay. At sub‐toxic concentrations retardation of T‐cell proliferation was caused by Pb(NO3)2, AuCl3 and Cu(NO3)2 and inhibitory effects on interleukin‐2 secretion were measured after incubation with Pb(NO3)2, AuCl3, Cu(NO3)2, HgCl2 and AsCl3. Cu(NO3)2 had immunosuppressive activity at dosages within the serum reference range for copper. All other metal salts showed effects at dosages above upper serum limits of normal. Therefore, only low‐concentrated copper preparations are promising to have immunomodulatory potential. Toxic side effects of metal preparations used in complementary medicine are improbable because upper limits of metals set in the drinking water ordinance are either not exceeded or the duration of their application is limited. Copyright

Effects of extracts and compounds from Tricholoma populinum Lange on degranulation and IL-2/IL-8 secretion of immune cells

Abstract: Tricholoma populinum Lange is an edible basidiomycete from the family Tricholomataceae. Extracts, fractions, and different metabolites isolated from the fruiting bodies of this mushroom were tested for degranulation-inhibiting activities on RBL-2H3 cells (rat basophils). Dichloromethane extracts decreased degranulation significantly, as did a fraction after column chromatography. In addition, the extract decreased the IL-2 release from Jurkat T cells and the release of IL-8 from HMC-1 human mast cells. The results show the significant effects of extracts of T. populinum on cells of the innate (basophils and mast cells) and adaptive (T cells) immune system and indicate the influence of the mushroom on different immunological processes. As one fraction showed activity, it seems to be possible that it includes an active principle. The compounds responsible for this effect, however, could not be identified as the contents oleic acid (1), ergosterol peroxide (2), and 9,11-dehydroergosterol peroxide (3) showed no effects. Nevertheless, the mushroom could be used for supporting allergy treatment in future studies.

Impact of Green Tea Catechin ECG and Its Synthesized Fluorinated Analogue on Prostate Cancer Cells and Stimulated Immunocompetent Cells

Among the known or suspected risk factors, inflammation plays an important role in infectious and non-infectious pathways leading to cancer. Green tea polyphenols have been associated with reducing inflammation and protection against carcinogenesis, especially in prostate cancer. While most of the research in this field, so far, has focussed on epigallocatechin-3-O-gallate only, we studied epicatechin-3-O-gallate, the second most abundant green tea polyphenol with essential therapeutic potential, to obtain a more detailed understanding of its anti-tumor and anti-inflammatory action. Furthermore, to improve the bioactivity of (-)-epicatechin-3-O-gallate, we synthesized a difluoro analogue, called (-)-5,7-difluoro-epicatechin-3-O-gallate. Both compounds reduced cell proliferation of human primary inflammatory lymphocytes in an apoptosis-specific fashion, while (-)-5,7-difluoro-epicatechin-3-O-gallate had a significantly higher activity compared to the natural product (-)-epicatechin-3-O-gallate. Treatment of low-metastatic LNCaP and high-metastatic PC-3 prostate cancer cells with (-)-epicatechin-3-O-gallate and (-)-5,7-difluoro-epicatechin-3-O-gallate demonstrated a dose-dependent inhibition of cell viability in the low micromolar range. These effects suggest that (-)-epicatechin-3-O-gallate and the more effective (-)-5,7-difluoro-epicatechin-3-O-gallate could be therapeutically used to inhibit tumorigenesis during initiation, promotion, and progression by diminishing the amount of inflammation due to a reduction of inflammatory lymphocytes. Further studies are needed to prove this in in vivo experiments.

Investigations on the constitutional types under consideration of anthropometric data, autonomic regulation and immunological parameters

OBJECTIVES Over time different systems were developed for the characterization of individuals according to their physical and psycho-vegetative traits which until today play a role in complementary medicine. This pilot study aimed at investigating if the concepts of polar constitutional types of anthroposophic medicine and according to Kretschmer can be further clarified using empirical method. METHODS 96 participants, preselected by two polar body mass index (BMI) ranges (17-19.5 kg/m2 and 27-31 kg/m2), were categorized using both classification systems. Anthropometrical measurements were carried out and differences in the autonomic regulation were assessed using a questionnaire. From 12 participants showing a pronounced polar constitutional type, production of reactive oxygen species, proliferation, autophagy, and glucose uptake by lymphocytes, monocytes and granulocytes were measured in vitro. RESULTS Correlations between the BMI and the strength of constitutional classification were found for both classification systems. Additionally, a strong correlation between the two systems themselves could be seen. Analysis of the overall questionnaire score of autonomic regulation did not yield significant correlations. However, using a modified 11 item score, reliability (Cronbach α = 0.656) and a differentiation of polar constitutional types was demonstrated (p < 0.001). Regarding the immune function slightly varying levels of reactive oxygen species, autophagy in granulocytes and differences in the strength of inhibition of lymphocyte proliferation by dexamethasone and cyclosporine A were detected. However, most of these in vitro results did not reach significance. CONCLUSION This study represents a first empirical approach toward the classification of anthroposophic constitutional types.

Constituents from oak bark (Quercus robur L.) inhibit degranulation and allergic mediator release from basophils and mast cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE Oak bark has been used since ancient times in Europaen ethnomedicine because of its adstringent, antimicrobial and hemostatic features, e.g. as a remedy for the treatment of wounds and skin diseases. PURPOSE Oak bark tannins are considered as bioactive natural products, interacting with surface proteins of mucous membranes and might be beneficial for the treatment of allergic diseases. This study investigated the effect of an oak bark decoction (OBD) and isolated tannin fractions on the degranulation capacity and cytokine/chemokine release from rat basophilic cells and human mast cells in vitro, which are essential for the initiation of early- and late-phase allergic reactions. METHODS AND METHODS By chromatographic separation on Sephadex® LH-20 high- and low-molecular weight tannins were separated from OBD and the tannin composition analyzed by HPLC(DAD)-MSn. Then, the OBD and its fractions were tested in degranulation (β-hexosaminidase activity) of allergen-specific-activated basophilic cells in a photometric assay. RESULTS The OBD and the high-molecular tannin fraction showed a dose-dependent inhibition of cell degranulation. Furthermore, the OBD and particularly its high molecular weight tannin fraction exhibited an inhibitory activity on the IL-8-, IL-6- and TNF-α-secretion from stimulated human mast cells, detected and quantified by ELISA. CONCLUSION The OBD and its high-molecular weight tannins revealed an impact on allergic mediator release of basophilic cells and human mast cells and thereby provide a rationale for the topical treatment with OBD preparations.