Aryltetralin lignans from Hyptis brachiata inhibiting T lymphocyte proliferation

Increased activation and proliferation of T lymphocytes plays an essential role in the development of chronic inflammation and autoimmune diseases. Currently used immunosuppressive drugs often do not provide long-lasting relief of symptoms and show a gradual loss of efficacy over time, and are accompanied by various side effects. Therefore, novel immunosuppressive lead substances are needed. For this purpose, an in-house library consisting of 600 extracts of plants from Panama was screened for inhibition of human T lymphocyte proliferation. As one of the hits, an ethyl acetate extract from the aerial parts of Hyptis brachiata (Lamiaceae) exhibited strong inhibitory effects. Subsequent investigation resulted in the isolation of seven aryltetralin lignans, five arylnaphthalene lignans, two flavonoids, three triterpenes, and cinnamyl cinnamate. Aryltetralin lignans inhibited T lymphocyte proliferation in a concentration-dependent manner without induction of apoptosis. No relevant inhibition was observed for the arylnaphthalene lignans, flavonoids, and triterpenes. Additional cell cycle arrest investigations revealed that isolated aryltetralin lignans potently inhibited cell division in G2/M phase similarly to podophyllotoxin. Multifluorescence panel analyses of the extract also showed weak suppressive effects on the production of IL-2 and TNF-α. Therefore, preparations made out of H. brachiata could be further explored as an interesting herbal alternative in the treatment of autoimmune diseases

Effects of selected natural products on human immunocompetent cells

The identification of new lead compounds, and the development of novel drugs for the treatment of autoimmune diseases, are of great importance, since today’s available pharmaceuticals often have substantial limitations. Glucocorticoids and drugs that inhibit the deoxyribonucleic acid (DNA) synthesis (e.g. cyclophosphamide) often cause severe side effects, while state-of-the-art biologicals usually impose a heavy financial burden. Plant extracts are a good starting point for the development of immunosuppressive leads, since they are evolutionarily optimized to serve numerous biological functions. The track record of natural product drug discovery for immunosuppressive leads has been distinguished by blockbuster drugs, such as cyclosporin A or tacrolimus; however, a well-planned, multidisciplinary research approach is required for screening plant extracts, characterizing their effects, clarifying targets, and isolating bioactive compounds. Enhanced T cell proliferation is a feature of autoimmune diseases such as rheumatoid arthritis or multiple sclerosis; therefore, as a starting point, this study investigated the T cell proliferation inhibitory potential of a library of 435 extracts, prepared from plants used in traditional Chinese medicine (TCM). The immunosuppressive activity of the extracts was assessed by a proliferation-based assay utilizing physiologically-relevant anti-CD3 and anti-CD28 stimulated primary human T lymphocytes. It showed that an Artemisia argyi (Asteraceae, A. argyi) ethyl acetate extract and a Boswellia carteri (Burseraceae, B. carteri) dichloromethane (DCM) extract were active, reflected by a half maximal inhibitory concentration (IC50) of 16.2 µg/mL for the A. argyi extract and 27.0 µg/mL for B. carteri extract. The observed inhibitory effect on T cell proliferation was based on a specific intervention of T cell signaling via an interleukin-2 (IL-2)-dependent mechanism, rather than induced apoptosis or necrosis. Further characterizations revealed a reduced expression of the T cell activation markers CD25 and CD69, as well as a decreased production of IL-2 and interferon-γ (IFN-γ), by the A. argyi extract; the B. carteri extract also suppressed the IL-2 and IFN-γ secretion. Moreover, treatment with B. carteri extract resulted in a reduced degranulation capacity of stimulated T cells. Both extracts were subjected to high-performance liquid chromatography (HPLC)-mass spectrometry (MS)-based activity profiling. A T cell proliferation assay identified 8-acetyl-artanomaloide, arteglasin A, jaceosidin, 1R-canin, and (4S,5S,6S,7S)- and (4R,5R,6S,7S)-seco-tanapartholides as active constituents of A. argyi. The proliferation assay showed that for B. carteri, 3-O-acetyl-8,24-dienetirucallic acid, 3-O-acetyl-7,24-dienetirucallic acid, 3-oxo-8,24-dienetirucallic acid and 3-O-acetyl-α-boswellic acid suppressed the proliferation of stimulated T lymphocytes. To validate the target of the active A. argyi and B. carteri compounds, monitoring of the T cell signaling cascade was performed, starting with the IL-2 transcription factor activator protein 1 (AP-1), the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and the nuclear factor of activated T cells (NFAT). Suppression of the NF-κB and NFAT activity, with IC50 values between 2.0 and 9.3 µM for NF-κB and 1.6 and 9.3 µM for NFAT, was detected for the A. argyi sesquiterpene lactones. 3-O-acetyl-alpha-boswellic acid was found to be the most promising candidate among the B. carteri compounds, as reflected by an IC50 value of 5.6 µM for NFAT activity suppression. For A. argyi the T cell signaling cascade monitoring was extended to the calcium flux in anti-CD3 stimulated Jurkat T cells. The results indicated a suppression of the calcium flux by 30 µg/mL A. argyi extract; however, no influence on the calcium flux of stimulated Jurkat T cells could be shown for the A. argyi compounds, suggesting that the crude plant extract may affect the signaling on a more upstream level than the single compounds, isolated thus far. This study also evaluated the potential wound healing and immune modulating capacities of extracts from nine plants that are traditionally used in Nepal to improve wound healing. An ethyl acetate extract of Gmelina arborea (Lamiaceae, G. arborea) positively influenced the wound-healing capacity of human keratinocytes and fibroblasts. For satisfactory wound healing, a balance between pathogen clearance by inflammatory feedback loops, and regulatory mechanisms to prevent fatal inflammatory responses, is essential; thus, the influence of the extracts on inflammation parameters was addressed. The G. arborea ethyl acetate extract, and an ethyl acetate extract from Bassia longifolia (Sapotaceae, B. longifolia), concentration-dependently inhibited the proliferation of stimulated T cells. This proliferation inhibition was not related to induced apoptosis or necrosis. The observed suppression of T cell proliferation could be linked to a decreased secretion of IL-2, which is essential for the proliferation and differentiation of T lymphocytes. Furthermore, the degranulation capacity of stimulated T cells was shown to decrease in response to treatment with either B. longifolia or G. arborea extract, emphasizing the anti-inflammatory potential of both extracts. Dendritic cells (DCs) play an important role in wound closure, since they increase the cell migration rate of keratinocytes by secreting interleukin-8 (IL-8). A slightly enhanced IL-8 secretion by DCs was detected after treatment with ethyl acetate extracts of either G. arborea or B. longifolia

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

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.; 96 participants, preselected by two polar body mass index (BMI) ranges (17-19.5 kg/m; 2; and 27-31 kg/m; 2; ), 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.; 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.; This study represents a first empirical approach toward the classification of anthroposophic constitutional types

Ipecac root extracts and isolated circular peptides differentially suppress inflammatory immune response characterised by proliferation, activation and degranulation capacity of human lymphocytes in vitro

Circular peptides are attractive lead compounds for drug development; this study investigates the immunomodulatory effects of defined root powder extracts and isolated peptides (called cyclotides) from Carapichea ipecacuanha (Brot.) L. Andersson ('ipecac'). Changes in the viability, proliferation and function of activated human primary T cells were analysed using flow cytometry-based assays. Three distinct peptide-enriched extracts of pulverised ipecac root material were prepared via C; 18; solid-phase extraction and analysed by reversed-phase HPLC and mass spectrometry. These extracts induced caspase 3/7 dependent apoptosis, thus leading to a suppressed proliferation of activated T cells and a reduction of the number of cells in the G2 phase. Furthermore, the stimulated T cells had a lower activation potential and a reduced degranulation capacity after treatment with ipecac extracts. Six different cyclotides were isolated from C. ipecacuanha and an T cell proliferation inhibiting effect was determined. Furthermore, the degranulation capacity of the T cells was diminished specifically by some cyclotides. In contrast to kalata B1 and its analog T20K, secretion of IL-2 and IFN- γ was not affected by any of the caripe cyclotides. The findings add to our increased understanding of the immunomodulating effects of cyclotides, and may provide a basis for the use of ipecac extracts for immunomodulation in conditions associated with an exessive immune responses

Hapalindoles from the Cyanobacterium Hapalosiphon sp. Inhibit T Cell Proliferation

Novel immunomodulating agents are currently sought after for the treatment of autoimmune diseases and cancers. In this context, a screening campaign of a collection of 575 cyanobacteria extracts for immunomodulatory effects has been conducted. The screening resulted in several active extracts. Here we report the results of subsequent studies on an extract from the cyanobacterium; Hapalosiphon; sp. CBT1235. We identified 5 hapalindoles as the compounds responsible for the observed immunomodulatory effect. These indole alkaloids are produced by several strains of the cyanobacterial family Hapalosiphonaceae. They are known for their anti-infective, cytotoxic, and other bioactivities. Modulation of the activity of human immune cells has not yet been described. The immunomodulatory activity of the hapalindoles was characterized; in vitro; using flow cytometry-based measurements of T cell proliferation after carboxyfluorescein diacetate succinimidyl ester staining, and apoptosis and necrosis induction after annexin V/propidium iodide staining. The most potent compound, hapalindole A, reduced T cell proliferation with an IC; 50; of 1.56 µM, while relevant levels of apoptosis were measurable only at 10-fold higher concentrations. Hapalindole A-formamide and hapalindole J-formamide, isolated for the first time from a natural source, had much lower activity than the nonformylated derivatives while, at the same time, being less selective for antiproliferative over apoptotic effects

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

Vitamin B12 Status Upon Short-Term Intervention with a Vegan Diet-A Randomized Controlled Trial in Healthy Participants

Vegans are at an increased risk for certain micronutrient deficiencies, foremost of vitamin B; 12; . Little is known about the short-term effects of dietary change to plant-based nutrition on vitamin B; 12; metabolism. Systemic biomarkers of vitamin B; 12; status, namely, serum vitamin B; 12; and holotranscobalamin, may respond quickly to a reduced intake of vitamin B; 12; . To test this hypothesis, 53 healthy omnivore subjects were randomized to a controlled unsupplemented vegan diet (VD,; n; = 26) or meat-rich diet (MD,; n; = 27) for 4 weeks. Vitamin B; 12; status was examined by measurement of serum vitamin B; 12; , holotranscobalamin (holo-TC), methylmalonic acid (MMA) and total plasma homocysteine (tHcy). Holo-TC decreased significantly in the VD compared to the MD group after four weeks of intervention, whereas metabolites MMA and tHcy were unaffected. Body weight remained stable in both groups. VD intervention led to a significant reduction of cholesterol intake, and adequate profiles of nutrient and micronutrient status. Lower intake of vitamin B; 12; was observed in VD, which was mirrored by a lower concentration of serum vitamin B; 12; and reduced holo-TC after 4 weeks. Plasma holo-TC may be a fast-responding biomarker to monitor adequate supply of vitamin B; 12; in plant-based individuals

Vegan diet reduces neutrophils, monocytes and platelets related to branched-chain amino acids - A randomized, controlled trial

Vegan diet (VD) has improved inflammatory activity in patients with rheumatoid arthritis (RA) in several small controlled trials. The underlying mechanism remains widely unclear. We investigated the effect of a VD in comparison to a meat-rich diet (MD) on markers of inflammation (which have been shown to be relevant in patients with RA) in healthy volunteers.; 53 healthy, omnivore subjects were randomized to a controlled VD (n = 26) or MD (n = 27) for 4 weeks following a pre-treatment phase of a one week controlled mixed diet. Primary parameters of interest were sialylation of immunoglobulins, percentage of regulatory T-cells and level of interleukin 10 (IL10). Usual care immune parameters used in patients with RA and amino acid serum levels as well as granulocytes and monocytes colony stimulating factor (GM-CSF) serum levels were secondary parameters.; In the VD group, total leukocyte, neutrophil, monocyte and platelet counts decreased and after four weeks they were significantly lower compared to the MD group (ANCOVA: leukocytes p = 0.003, neutrophils p = 0.001, monocytes p = 0.032, platelets p = 0.004). Leukocytes, neutrophils, monocytes, and platelets correlated with each other and likewise conform with serum levels of branched-chain amino acids, which were significantly lower in the VD compared to the MD group. The primary parameters did not differ between the groups and BMI remained stable in the two groups.; Four weeks of a controlled VD affected the number of neutrophils, monocytes and platelets but not the number or function of lymphocytes. The relation with branched-chain amino acids and GM-CSF suggests a mode of action via the mTOR signaling pathway. REGISTERED AT: http://www.drks.de (German Clinical Trial register) at DRKS00011963

Influence of VAEA on DC maturation and cytokine secretion.

<p>CD14⁺ monocytes were matured to immature DC and incubated with medium (DC), maturation cocktail (DC Stim; 500 ng/mL LPS; 50 ng/mL TNF-alpha and 10 ng/mL IL-1beta), or VAEA (DC + VAEA; abnobaVISCUM® Fraxini; 0.06 μg/ml). After cultivation flow cytometric analysis of CD83 <b>(A),</b> CD86 <b>(B)</b> and HLA-DR <b>(C)</b> expression was carried out. High CD83, CD86 and HLA-DR levels indicate DC maturation. MFI = Mean fluorescence intensity. Data and mean of 16 (DC Stim, CD83 and CD86) and 6 (DC + VAEA, CD83 and CD86) or 5 (HLA-DR) individual experiments are presented in relation to untreated cells (DC = 100%). <b>(D)</b> The mediators IL-12p70, IL-6, IL-8 and TNFα were detected in the supernatants of cultured cells of 6 independent experiments and analyzed using cytokine bead array assay. Asterisks indicate significant differences between the groups (*P < 0.05, **P < 0.01, ***P < 0.001).</p