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1.  Immunosuppressive activity of an aqueous Viola tricolor herbal extract 
Journal of ethnopharmacology  2013;151(1):299-306.
Ethnopharmacological relevance
Heartsease (Viola tricolor L.), a member of the Violaceae family, has a long history as a medicinal plant and has been documented in the Pharmacopoeia of Europe. Due to its anti-inflammatory properties it is regarded as a traditional remedy against skin diseases, for example for the treatment of scabs, itching, ulcers, eczema or psoriasis, and it is also used in the treatment of inflammation of the lungs and chest such as bronchitis or asthma. Because T-cells play an important role in the pathological process of inflammatory diseases we investigated the effect of an aqueous Viola extract on lymphocyte functions and explored the ‘active’ principle of the extract using bioactivity-guided fractionation.
Material and Methods
An aqueous Viola extract was prepared by C18 solid-phase extraction. Effects on proliferation of activated lymphocytes (using the cell membrane permeable fluorescein dye CFSE), apoptosis and necrosis (using annexin V and propidium iodide staining), interleukin-2 (IL-2) receptor expression (using fluorochrome-conjugated antibodies) and IL-2 cytokine secretion (using an ELISA-based bead array system) were measured by flow cytometry. Influence on lymphocyte polyfunctionality was characterized by Viola extract-induced production of IFN-γ and TNF-α, as well as its influence on lymphocyte degranulation activity. Fractionation and phytochemical analysis of the extract were performed by RP-HPLC and mass spectrometry.
Results
The aqueous Viola extract inhibited proliferation of activated lymphocytes by reducing IL-2 cytokine secretion without affecting IL-2 receptor expression. Similarly, effector functions were affected as indicated by the reduction of IFN-γ and TNF-α production; degranulation capacity of activated lymphocytes remained unaffected. Bioassay-guided fractionation and phytochemical analysis of the extract led to identification of circular plant peptides, so called cyclotides, as bioactive components.
Conclusion
An aqueous Viola extract contains bioactive cyclotides, which inhibit proliferation of activated lymphocytes in an IL-2 dependent manner. The findings provide a rationale for use of herbal Viola preparations in the therapy of disorders related to an overactive immune system. However, further studies to evaluate its clinical potency and potential risks have to be performed.
doi:10.1016/j.jep.2013.10.044
PMCID: PMC3918579  PMID: 24216163
Violaceae; Viola tricolor L.; Immunosuppression; Cyclotides; Psoriasis; Anthroposophical medicine; Phytotherapy
2.  Preclinical Evaluation of 4-Methylthiobutyl Isothiocyanate on Liver Cancer and Cancer Stem Cells with Different p53 Status 
PLoS ONE  2013;8(8):e70846.
Isothiocyanates from plants of the order Brassicales are considered promising cancer chemotherapeutic phytochemicals. However, their selective cytotoxicity on liver cancer has been barely researched. Therefore, in the present study, we systematically studied the chemotherapeutic potency of 4-methylthiobutyl isothiocyanate (MTBITC). Selective toxicity was investigated by comparing its effect on liver cancer cells and their chemoresistant subpopulations to normal primary hepatocytes and liver tissue slices. Additionally, in a first assessment, the in vivo tolerability of MTBITC was investigated in mice. Growth arrest at G2/M and apoptosis induction was evident in all in vitro cancer models treated with MTBITC, including populations with cancer initiating characteristics. This was found independent from TP53; however cell death was delayed in p53 compromised cells as compared to wt-p53 cells which was probably due to differential BH3 only gene regulation i. e. Noxa and its antagonist A1. In normal hepatocytes, no apoptosis or necrosis could be detected after repeated administration of up to 50 µM MTBITC. In mice, orally applied MTBITC was well tolerated over 18 days of treatment for up to 50 mg/kg/day, the highest dose tested. In conclusion, we could show here that the killing effect of MTBITC has a definite selectivity for cancer cells over normal liver cells and its cytotoxicity even applies for chemoresistant cancer initiating cells. Our study could serve for a better understanding of the chemotherapeutic properties of isothiocyanates on human liver-derived cancer cells.
doi:10.1371/journal.pone.0070846
PMCID: PMC3732292  PMID: 23936472
3.  Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism 
PLoS ONE  2013;8(6):e68016.
Cyclotides are a diverse and abundant group of ribosomally synthesized plant peptides containing a unique cyclic cystine-knotted topology that confers them with remarkable stability. Kalata B1, a representative member of this family of mini-proteins, has been found to inhibit the proliferation of human peripheral blood mononuclear cells. Analysis of T-cell proliferation upon treatment with chemically synthesized kalata B1 mutants revealed a region comprising inter-cysteine loops 1 and 2 of the cyclotide framework to be important for biological activity. Cytokine signaling analysis using an ‘active’ kalata B1 mutant [T20K], and the reference drug cyclosporin A (CsA) demonstrated that treatment of activated T-lymphocytes with these compounds decreased the expression of the interleukin-2 (IL-2) surface receptor as well as IL-2 cytokine secretion and IL-2 gene expression, whereas the ‘inactive’ kalata B1 mutant [V10K] did not cause any effects. The anti-proliferative activity of [T20K] kalata B1 was antagonized by addition of exogenous IL-2. Furthermore, treatment with [T20K] kalata B1 led to an initial reduction of the effector function, as indicated by the reduced IFN-γ and TNF-α production, but the levels of both cytokines stabilized over time and returned to their normal levels. On the other hand, the degranulation activity remained reduced. This indicated that cyclotides interfere with T-cell polyfunctionality and arrest the proliferation of immune-competent cells through inhibiting IL-2 biology at more than one site. The results open new avenues to utilize native and synthetically-optimized cyclotides for applications in immune-related disorders and as immunosuppressant peptides.
doi:10.1371/journal.pone.0068016
PMCID: PMC3694003  PMID: 23840803
cyclotide; lymphocytes; proliferation; immunosuppression; plant; peptide; cytokine
4.  Quality of life, immunomodulation and safety of adjuvant mistletoe treatment in patients with gastric carcinoma – a randomized, controlled pilot study 
Background
Mistletoe (Viscum album L.) extracts are widely used in complementary cancer therapy. Aim of this study was to evaluate safety and efficacy of a standardized mistletoe extract (abnobaVISCUM® Quercus, aVQ) in patients with gastric cancer.
Patients and Methods
32 operated gastric cancer patients (stage Ib or II) who were waiting for oral chemotherapy with the 5-FU prodrug doxifluridine were randomized 1:1 to receive additional therapy with aVQ or no additional therapy. aVQ was injected subcutaneously three times per week from postoperative day 7 to week 24 in increasing doses. EORTC QLQ-C30 and -STO22 Quality of Life questionnaire, differential blood count, liver function tests, various cytokine levels (tumor necrosis factor (TNF)-alpha, interleukin (IL)-2), CD 16+/CD56+ and CD 19+ lymphocytes were analyzed at baseline and 8, 16 and 24 weeks later.
Results
Global health status (p <0.01), leukocyte- and eosinophil counts (p ≤0.01) increased significantly in the treatment group compared to the control group. Diarrhea was less frequently reported (7% vs. 50%, p=0.014) in the intervention group. There was no significant treatment effect on levels of TNF-alpha, IL-2, CD16+/CD56+ and CD 19+ lymphocytes and liver function tests measured by ANOVA.
Conclusion
Additional treatment with aVQ is safe and was associated with improved QoL of gastric cancer patients. ClinicalTrials.Gov Registration number NCT01401075.
doi:10.1186/1472-6882-12-172
PMCID: PMC3488325  PMID: 23033982
Qol; EORTC QLQ-C30; QLQ-STO22; 5-FU; Viscum album
5.  Do Plant Cyclotides Have Potential As Immunosuppressant Peptides? 
Journal of natural products  2012;75(2):167-174.
Cyclotides are an abundant and diverse group of ribosomally synthesized plant peptides containing a cyclic cystine-knotted structure that confers them with remarkable stability. They are explored for their distribution in plants, although little is known about the individual peptide content of a single species. Therefore, we chemically analyzed the crude extract of the coffee-family plant Oldenlandia affinis using a rapid peptidomics workflow utilizing nano-LC-MS, peptide reconstruct with database identification, and MS/MS automated sequence analysis to determine its cyclotide content. Biologically, cyclotides are mainly explored for applications in agriculture and drug design; here we report their growth-inhibiting effects on primary cells of the human immune system using biological and immunological end points in cell-based test systems. LC-MS quantification of the active O. affinis plant extract triggered the characterization of the antiproliferative activity of kalata B1, one of the most abundant cyclotides in this extract, on primary activated human lymphocytes. The effect has a defined concentration range and was not due to cytotoxicity, thus opening a new avenue to utilize native and synthetically optimized plant cyclotides for applications in immune-related disorders and as immunosuppressant peptides.
doi:10.1021/np200722w
PMCID: PMC3399773  PMID: 22272797
6.  A biochemical marker panel in MRI-proven hyperacute ischemic stroke-a prospective study 
BMC Neurology  2012;12:14.
Background
Computer tomography (CT) is still the fastest and most robust technique to rule out ICH in acute stroke. However CT-sensitivity for detection of ischemic stroke in the hyperacute phase is still relatively low. Moreover the validity of pure clinical judgment is diminished by several stroke imitating diseases (mimics). The "Triage® Stroke Panel", a biochemical multimarker assay, detects Brain Natriuretic Peptide (BNP), D-Dimers (DD), Matrix-Metalloproteinase-9 (MMP-9), and S100B protein and promptly generates a Multimarkerindex of these values (MMX). This index has been licensed for diagnostic purposes as it might increase the validity of the clinical diagnosis to differentiate between stroke imitating diseases and true ischemic strokes. Our aim was to prove whether the panel is a reliable indicating device for the diagnosis of ischemic stroke in a time window of 6 h to fasten the pre- and intrahospital pathway to fibrinolysis.
Methods
We investigated all consecutive patients admitted to our stroke unit during a time period of 5 months. Only patients with clinical investigation, blood sample collection and MRI within six hours from symptom onset were included. Values of biochemical markers were analyzed according to the results of diffusion weighted MR-imaging. In addition MMX-values in ischemic strokes were correlated with the TOAST-criteria. For statistical analysis the SAS Analyst software was used. Correlation coefficients were analyzed and comparison tests for two or more groups were performed. Statistical significance was assumed in case of p < 0.05. Finally a ROC-analysis was performed for the MMX-Index.
Results
In total 174 patients were included into this study (n = 100 strokes, n = 49 mimics, n = 25 transitoric ischemic attacks). In patients with ischemic strokes the mean NIHSS was 7.6 ± 6.2, while the mean DWI-lesion volume was 20.6 ml (range 186.9 to 4.2 ml). According to the MMX or the individual markers there was no statistically significant difference between the group of ischemic strokes and the group of mimics. Moreover the correlation of the index and the DWI-lesion-volume was poor (p = 0.2).
Conclusions
In our setting of acute MRI-proven ischemic stroke the used multimarker-assay (Triage® Stroke Panel) was not of diagnostic validity. We do not recommend to perform this assay as this might lead to a unjustified time delay.
doi:10.1186/1471-2377-12-14
PMCID: PMC3380723  PMID: 22400994
Stroke; Biochemical marker; Brain natriuretic peptide; D-dimers; Matrix-metalloproteinase-9
7.  Safety and effects of two mistletoe preparations on production of Interleukin-6 and other immune parameters - a placebo controlled clinical trial in healthy subjects 
Background
In Germany, Iscucin® Populi (IP), a preparation from mistletoe growing on the poplar tree, is used in cancer therapy while Viscum Mali e planta tota (VM), a preparation from mistletoe growing on the apple tree, is used in patients with osteoarthritis. Since mistletoe preparations are suspected to induce production of potentially tumor promoting cytokines like interleukin (IL)-6, further studies on the immunological effects are of interest.
Methods
In this 3-armed randomized, double blind clinical trial healthy volunteers received increasing doses of either IP (strength F, 0.0125%, G, 0.25% and H, 5%, each for 4 weeks), or VM (1:1000 [D3], 1:100 [D2] and 2% each for 4 weeks) or placebo (isotonic solution) subcutaneously twice per week over a period of 12 weeks. Physical examination was performed weekly. Routine laboratory parameters and immunological parameters (C-reactive protein (CRP), differential blood count, lymphocyte subsets, immunoglobulins, IL-6 and tumor necrosis factor (TNF)-α) were analysed every 4 weeks.
Results
71 subjects were included in the study (IP = 30, VM = 21, placebo = 20) of whom 69 concluded it according to protocol. Application of IP strengths G and H caused strong local reactions at the site of injection. In parallel, a distinct eosinophilia (p < 0.001 compared to placebo) occurred. Furthermore, application of all IP concentrations resulted in an increase of CD4 cell counts (p < 0.05) compared to placebo. Stimulation of IL-6 production, CRP or relevant deviations in other laboratory parameters were not observed. Because of local reactions, IP strengths G and H were considered less tolerable than placebo. VM 2% was slightly less tolerable than placebo, caused only mild local reactions and an only small increase in eosinophile counts.
Conclusion
Treatment with IP results in eosinophilia and an increase of CD4 cells but not in an increase of IL-6 or CRP. No safety concerns regarding the two mistletoe preparations have been raised by this study. EudraCT-Number 2007-002166-35.
Trial registration
ClinicalTrials.gov: NCT01378702
doi:10.1186/1472-6882-11-116
PMCID: PMC3257204  PMID: 22114899
8.  Fibrinolysis and Beyond: Bridging the Gap between Local and Systemic Clot Removal 
Recanalization methods in ischemic stroke have been progressively expanded over the past years. In addition the continuous development of specialized mechanical devices for thrombectomy a broad spectrum of new drugs has been tested: Both options, novel drugs as well as new devices, can be employed independently of each other, but in most cases a combination of the two with the standard treatment of intravenous fibrinolysis is applied. Until recently, a large number of case series have been performed to investigate the effects of various drugs and interventions, but only a few trials have been conducted to determine the optimal conditions for combining both procedures. This review surveys the different systemic and endovascular vessel reopening practices and their major bridging techniques.
doi:10.3389/fneur.2011.00007
PMCID: PMC3044492  PMID: 21373206
fibrinolysis; bridging; thrombectomy; stroke
9.  Effects of abdominal hot compresses on indocyanine green elimination – a randomized cross over study in healthy subjects 
BMC Gastroenterology  2007;7:27.
Background
Hot compresses on the right upper abdomen are used as support for patients with liver diseases in Germany. The study was designed to determine, whether they affect hepatic blood flow.
Methods
Single dose kinetics of indocyanine green (ICG) were studied in 13 healthy subjects with or without hot compresses on the right upper abdomen over 40 minutes. The time interval between the investigations was 8 days, the sequence was randomly assigned.
Results
Within non-linear kinetic analyses the area under the curves (AUC) were 23% (95% confidence interval [CI]: 5–37%) lower with hot compresses. In the initial phase, however, no differences were detected (p = 0.295). The differences occurred only in the late phase after 30–40 minutes, when the genuine ICG is eliminated from the plasma and only the degradation product remains.
Conclusion
Hot compresses have no effect on ICG elimination in healthy subjects but seem to affect the elimination of ICG metabolites.
Trial registration
ClinicalTrials.gov NCT00484913
doi:10.1186/1471-230X-7-27
PMCID: PMC1931598  PMID: 17623067

Results 1-9 (9)