Critical Evaluation of Specific Efficacy of Preparations Produced According to European Pharmacopeia Monograph 2371.

European Pharmacopoeia monograph 2371 describes the production of homeopathic preparations. A specific efficacy of these preparations in high dilution levels is questionable in view of basic scientific principles. There is empirical evidence for such effects, for example in a Lemna-intoxication bioassay published 2010. To test the replicability and robustness of this bioassay, we conducted two experimental series (five independent blinded and randomised experiments each). The specimen of Lemna gibba L., clone-number 9352, were stressed in arsenic solution for 48 h (158 mg/L AsNa2HO4 (250 mg/L in series 2)), then grew in either As2O3 preparations produced according to Eu. Pharm. Monogr. 2371 or control solution. Comparing the area-related relative growth rate of day 3-9 (rgr 3-9) between treatment and control groups for each series showed differences that were not significant in series 1 (p = 0.10), significant in series 2 (p = 0.04) and significant in the pooled data of both series (p < 0.01). The effect direction (rgr 3-9 increase) was comparable to experiments of 2010, but the effect size was smaller, likely due to a changed light cycle. These results are not compatible with the hypothesis that the application of European Pharmacopoeia monograph 2371 results in pharmaceutical preparations without specific effects. Further studies are needed to investigate a potential mode of action explaining these effects

Systematic Review of Plant-Based Homeopathic Basic Research: An Update.

BACKGROUND Plant-based test systems have been described as a useful tool for investigating possible effects of homeopathic preparations. The last reviews of this research field were published in 2009/2011. Due to recent developments in the field, an update is warranted. Publications on plant-based test systems were analysed with regard to publication quality, reproducibility and potential for further research. METHODS A literature search was conducted in online databases and specific journals, including publications from 2008 to 2017 dealing with plant-based test systems in homeopathic basic research. To be included, they had to contain statistical analysis and fulfil quality criteria according to a pre-defined manuscript information score (MIS). Publications scoring at least 5 points (maximum 10 points) were assumed to be adequate. They were analysed for the use of adequate controls, outcome and reproducibility. RESULTS Seventy-four publications on plant-based test systems were found. Thirty-nine publications were either abstracts or proceedings of conferences and were excluded. From the remaining 35 publications, 26 reached a score of 5 or higher in the MIS. Adequate controls were used in 13 of these publications. All of them described specific effects of homeopathic preparations. The publication quality still varied: a substantial number of publications (23%) did not adequately document the methods used. Four reported on replication trials. One replication trial found effects of homeopathic preparations comparable to the original study. Three replication trials failed to confirm the original study but identified possible external influencing factors. Five publications described novel plant-based test systems. Eight trials used systematic negative control experiments to document test system stability. CONCLUSIONS Regarding research design, future trials should implement adequate controls to identify specific effects of homeopathic preparations and include systematic negative control experiments. Further external and internal replication trials, and control of influencing factors, are needed to verify results. Standardised test systems should be developed

Influence of traditionally used Nepalese plants on wound healing and immunological properties using primary human cells in vitro

Ethnopharmacological relevance: The improvement of wound healing has always been an important issue for both ethnopharmacological and modern medical research. In this study, we used state-of-the-art methods to investigate extracts of plants used traditionally in Nepal for more than 1000 years to treat inflammatory injuries. Aim of the study: We focused on the potential of the plant extracts to ameliorate wound healing and to influence immune modulatory properties. Materials and methods: Nine Nepalese plant extracts in three different solvents (methanol, ethyl acetate, petroleum ether) were immunologically characterised. Water-soluble tetrazolium (WST-1) assays and scratch assays were performed to determine their impact on viability and wound healing capacity of human keratinocytes and fibroblasts. Effects on proliferation, viability and function of physiologically relevant anti-CD3 and anti-CD28 stimulated primary human T lymphocytes were assessed using carboxyfluorescein succinimidyl ester (CFSE), annexin V/propidium iodide staining assays and flow cytometry-based surface receptor characterisation. The secretion level of interleukin-2 (IL-2) was analysed with the ELISA technique. Dendritic cells were generated out of peripheral blood mononuclear cells (PBMC) by CD14(+) magnetic bead selection. Flow cytometry-based surface receptor characterisation and ELISA-based technique were used to evaluate the DC activation state and the interleukin-8 (IL-8) secretion level. Results: We demonstrate that an ethyl acetate extract of Bassia longifolia and of Gmelina arborea have anti-inflammatory capacities, indicated by reduced proliferation, inhibition of IL-2 secretion and degranulation capacity of activated human T cells, when compared with adequate concentrations of synthetic positive drug controls. Furthermore, Gmelina arborea improved the wound healing of keratinocytes and fibroblasts and has tendency to increase the secretion of IL-8 by human primary dendritic cells. Conclusion: With this preliminary screening, we offer a scientific basis for the immunomodulatory properties of the two Nepalese medicinal plants Bassia longifolia and Gmelina arborea. However, further detailed studies regarding the responsible compounds are necessary

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

Effects of VAEI-treated DC on T cells.

<p>Purified human CD4⁺ T lymphocytes were cultured in the presence of medium (NC) or stimulated with phytohemagglutinin-L (PC; 10 μg/ml). Unstimulated T cells were further co-cultivated with immature DC (DC, CD4⁺), DC treated with a maturation cocktail (DC Stim, CD4⁺; 500 ng/mL LPS; 50 ng/mL TNF-alpha and 10 ng/mL IL-1beta), VAEI-treated DC (DC + VAEI, CD4⁺; Iscador® Qu Spez; 0.5 μg/ml), DC incubated with ML-depleted VAEI (DC + VAEI-ML, CD4⁺; Iscador®; 0.66 μg/ml) or VAEI and anti-ML antibody (DC + VAEI + aML-Ab, CD4⁺; 2.5 μg/ml). <b>(A)</b> Cell proliferation analysis was done using CFSE staining and flow cytometry. <b>(B)</b> CD25 surface marker expression was analyzed as a second indicator for T cell activation. Data of 6 individual experiments are presented as mean ± SD in relation to untreated T cells (NC) or untreated T cells co-cultured with immature DC (DC, CD4⁺). <b>(C)</b> IFN-γ release by T cells was analyzed in the supernatants of the co-cultures of 6 independent experiments using a cytokine bead array assay. Asterisks indicate significant differences between the groups (*P < 0.05, **P < 0.01, ***P < 0.001). Data points with values below the detection limit are not pictured.</p

Effects of VAE on tumor-induced suppression of DC maturation.

<p>Dendritic cells derived from CD14⁺ monocytes were stimulated with maturation cocktail (DC Stim; 500 ng/mL LPS; 50 ng/mL TNF-alpha and 10 ng/mL IL-1beta) and incubated with IL-6 as an inhibition positive control (DC Stim + IL-6; 12.5 ng/ml). Unstimulated DC were further treated with 10% RPMI 1640 medium (DC + Medium), 10% tumor supernatant of an RCC line (DC + TS) or 10% tumor supernatant and VAEI (DC + TS + VAEI; Iscador® Qu Spez; 0.5 μg/ml) or VAEA (DC + TS + VAEA; abnobaVISCUM® Fraxini; 0.06 μg/ml). DC maturation was assessed by flow cytometric analysis of CD83 <b>(A)</b> and CD86 <b>(B)</b> surface marker expression. MFI = Mean fluorescence intensity. Data and mean of 7 (IL-6 and DC + TS + VAEA), 16 (DC + TS) or 9 (DC + TS + VAEI) individual experiments are presented in relation to stimulated cells (DC Stim = 100%), stimulated DC cultured in medium alone (DC + Medium = 100%) or stimulated cells cultured with tumor supernatant (DC + TS = 100%). Asterisks indicate significant differences between the groups (*P < 0.05, **P < 0.01, ***P < 0.001).</p

Impact of mistletoe lectins on the restoration of DC maturation with VAEI after tumor-induced immunosuppression.

<p>Dendritic cells derived from CD14⁺ monocytes were incubated with 10% tumor supernatant (DC + TS). Further treatment of DC + TS was carried out with VAEI (DC + TS + VAEI; Iscador® Qu Spez; 0.5 μg/ml), ML-depleted VAEI (DC + TS + VAEI-ML; Iscador®; 0.66 μg/ml), or VAEI and anti-ML antibody (DC + TS + VAEI + aML-Ab; 2.5 μg/ml). DC maturation was assessed by flow cytometric analysis of CD83 <b>(A),</b> CD86 <b>(B)</b> and HLA-DR <b>(C)</b> expression. MFI = Mean fluorescence intensity. Data and mean of 12 (DC + TS + VAEI), 3 (DC + TS + VAEI-ML and DC + TS + VAEI + aML-Ab) or 5 (HLA-DR) individual experiments are presented in relation to stimulated and TS-treated cells (DC + TS = 100%), or TS and VAEI-treated DC (DC + TS + VAEI = 100%). Asterisks indicate significant differences between the groups (*P < 0.05, **P < 0.01, ***P < 0.001).</p