Antiproliferative and immunomodulative potential of Citrullus colocynthis and its bioactive compounds in human lymphocytes and lung cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Citrullus colocynthis (L.) Schrad is a member of the Cucurbitaceae plant family which has been used in traditional medicine for the treatment of lung diseases such as asthma and bronchitis. AIM OF THE STUDY: The study was conducted to investigate antiproliferative and immunomodulating effects of C. colocynthis and isolated cucurbitacins on human T lymphocytes and lung epithelial cells in order to evaluate their potential in the treatment of airway diseases. MATERIALS AND METHODS: Different concentrations of an ethanolic extract of C. colocynthis fruits and cucurbitacins B (CuB), E (CuE) and E-glucopyranoside (CuE-Glu) were analysed for their cytotoxicity and immunomodulatory potential on Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors and on the epithelial lung cancer cell line A549. Viability and proliferation were tested using WST1 and CFSE assays. Flow cytometric analysis of AnnexinV/PI staining was used to investigate cell death through apoptosis/necrosis. Effects on regulatory mechanisms of T lymphocytes, such as CD69 and CD25 marker activation, cytokine production of the cytokines interleukin 2 (IL2), tumor necrosis factor α (TNFα) and interferon γ (IFNy) were also analysed via flow cytometry. Influences on the activator protein 1 (AP1), nuclear factor of activated T-cells (NFAT) or nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) pathways were analysed in the Jurkat reporter cell line. Cytokine secretion in A549 cells stimulated with virus-like particles was analysed using the bead-based Legendplex™ assay. RESULTS: Non-toxic concentrations of C. colocynthis and CuE-Glu showed dose-dependent effects on viability and proliferation in both T lymphocytes and A549 cells. The extracts inhibited lymphocyte activation and suppressed T cell effector functions, which was also shown by lower production of cytokines IL2, TNFα and IFNy. A dose dependent inhibition of the pathways NFκB, NFAT and AP1 in Jurkat cells could be observed. In A549 cells, especially CuE and CuE-Glu showed inhibitory effects on cytokine production following a simulated viral infection. Unglycosylated cucurbitacins were more effective in suppressing the immune function in lymphocytes than glycosylated cucurbitacins, however this activity is limited to cytotoxic concentrations. CONCLUSION: In our study we could confirm the immunmodulating effect of C. colocynthis and cucurbitacins B, E and E-glucopyranoside in vitro by suppression of different pathways of inflammation and T cell proliferation. Activity in a lung cell model using a virus-like stimulation shows promise for further research regarding cucurbitacins in airway diseases.

A cross-sectional study of nutritional status in healthy, young, physically-active German omnivores, vegetarians and vegans reveals adequate vitamin B12 status in supplemented vegans.

BACKGROUND/OBJECTIVE: Plant-based diets reduce the risk of cardiovascular disease but also increase the risk of certain micronutrient deficiencies, particularly, of vitamin B12 (B12). The extent to which the unsupervised use of oral nutrient supplements is sufficient to prevent these deficiencies is not well established. We analyzed nutrient intake, laboratory biomarkers, supplementation behavior, and B12 status adequacy amongst young, healthy, physically active omnivores, lacto-ovo-vegetarians and vegans from Germany. METHODS: We recruited 115 participants (n = 40 omnivores; n = 37 lacto-ovo-vegetarians, and n = 38 vegans) with comparable age, sex, marital status, physical activity and educational levels through online advertisements and local newspapers in Freiburg, Germany. RESULTS: Energy intake and macronutrient distribution were comparable across diets. Major differences included intake of fiber, cholesterol, and several vitamins. Vegans had the lowest intake of B12 from foods (0.43 (0.58) µg/d), compared to omnivores (2.14 (2.29) µg/d) and lacto-ovo-vegetarians (0.98 (1.34) µg/day). Multivariate analysis of 36 blood biomarkers revealed that three major classes of biomarkers contributed the most to the clustering of individuals by dietary group, namely, biomarkers of B12 status (B12, holoTC, Hcy), iron (iron, ferritin, transferrin) and lipid metabolism (vitamin A, HDL, LDL, total cholesterol, TAG). This suggests that nutrients that modify the metabolic pathways represented by these biomarkers have the most penetrating effect on health status across diets. Analysis of B12 status (including 4cB12) revealed adequacy in omnivores and vegans, and a poorer B12 status amongst lacto-ovo-vegetarians. Fewer lacto-ovo-vegetarians used B12 supplements compared to vegans (51% versus 90%). CONCLUSIONS: Even amongst homogeneously healthy Germans, each diet manifested with measurable differences in dietary intakes and biomarkers of health. Plant-based diets, in particular the vegan diet, exhibited the most favorable patterns of lipid metabolism and glycemic control, but the lowest food intake of B12. Supplementation of healthy vegans with B12 (median 250 µg B12/day, over 2 years) secured an adequate B12 status that was comparable to that of healthy omnivores.Clinical Trial Registry: German Clinical Trial register number: DRKS00027425.

Plant-based diets, in particular the vegan diet, exhibited the most favorable patterns of lipid metabolism and glycemic control, but the lowest food intake of B12.Analysis of B12 status (including 4cB12) revealed adequacy in omnivores and vegans, and a poorer B12 status amongst lacto-ovo-vegetarians.Supplementation with B12 (median 250 µg B12/day, over 1 year) in healthy physically-active vegans secured an adequate B12 status that was comparable to that of healthy omnivores.

Mass Spectrometric Analysis of Cucurbitacins and Dihydrocucurbitacins from the Tuber of Citrullus naudinianus.

The vast pool of structurally and functionally distinct secondary metabolites (i.e., natural products (NPs)) is constantly being expanded, a process also driven by the rapid progress in the development of analytical techniques. Such NPs often show potent biological activities and are therefore prime candidates for drug development and medical applications. The ethyl acetate extract of the tuber of Citrullus naudinianus (C. naudinianus), an African melon with edible fruits and seeds, shows in vitro immunomodulatory activity presumably elicited by cucurbitacins that are known major constituents of this plant. Further potentially immunomodulatory cucurbitacins or cucurbitacin derivatives were assumed to be in the tuber. Given the typically high content of cucurbitacins with similar physicochemical features but often distinct bioactivities, an efficient and reliable separation process is a prerequisite for their detailed characterization and assessment in terms of bioactivity. We therefore developed a detection method to screen and differentiate cucurbitacins via high-performance liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (HPLC-QTOF-MS/MS). In order to confirm the identification, the fragmentation patterns of two cucurbitacins and one 23,24-dihydrocucurbitacin were also investigated. Six characteristic fragments were identified and three of them were employed for the identification of cucurbitacins and 23,24-dihydrocucurbitacins in the extract. As a result, in addition to eight previously reported cucurbitacins from this plant four distinct 23,24-dihydrocucurbitacins (B, D, E, and I) were putatively identified and newly found in the ethyl acetate extract of the tuber of C. naudinianus. The established methodology enables rapid and efficient LC-MS-based analysis and identification of cucurbitacins and 23,24-dihydrocucurbitacins in plant extracts.

Characterization of Viscum album L. Effect on Immune Escape Proteins PD-L1, PD-L2, and MHC-I in the Prostate, Colon, Lung, and Breast Cancer Cells.

BACKGROUND: Viscum album L. (VA) preparations possess immunomodulatory properties and are used in complementary medicine to support cancer therapy. It is unclear if there is an impact of VA on the expression of immune checkpoint proteins on the surface of cancer cells. This study was designed to investigate the role of commercially available VA preparations on checkpoint programmed death ligand 1, 2 (PD-L1, PD-L2) and on major histocompatibility complex class I (MHC-I). METHODS: Four human cancer cell lines (prostate, colon, lung, and breast) were assayed for their PD-L1, PD-L2, and MHC-I level after stimulation with interferon-gamma (IFN-γ). The toxicity of mistletoe preparations for the cells was analysed. Afterwards, the effect of mistletoe preparations on the PD ligands and MHC-I was investigated. RESULTS: Surface protein analysis demonstrated that all tested tumour cell lines increased the PD-L1, PD-L2, and MHC-I-expression, but to different extents, after IFN-γ stimulation. Treatment with VA extracts did not influence the viability of the cells. The expression of PD ligands and MHC-I was not affected by incubation with the VA preparations. CONCLUSION: Our investigation concludes that VA treatment does not interfere with the expression of PD ligands or MHC-I among selected cancer cells.Viscum album L. (VA)-Präparate besitzen immunmodulatorische Eigenschaften und werden in der Komplementärmedizin zur Unterstützung in der Krebstherapie eingesetzt. Es ist jedoch unklar, ob VA einen Einfluss auf die Expression von Immuncheckpoint-Proteinen auf Krebszellen hat. In der vorliegenden Arbeit wurde ein Einfluss von handelsüblichen VA-Präparaten auf die Checkpoint-Proteine programmed death ligand 1, 2 (PD-L1, PD-L2) und major histocompatibility complex class I (MHC-I) untersucht.Vier humane Krebszelllinien der Prostata, des Dickdarms, der Lunge und Brust wurden nach Stimulation mit Interferon-gamma (IFN-γ) auf ihre PD-L1, PD-L2 und MHC-I Konzentration untersucht. Zunächst wurde die Toxizität von Mistelpräparaten auf die Tumorzellen analysiert. Anschließend erfolgte eine Charakterisierung der Wirkung von Mistelpräparaten auf die PD-Liganden und MHC-I.Die Oberflächenproteinanalysen zeigten, dass alle getesteten Tumorzelllinien nach einer IFN-γ-Stimulation die PD-L1, PD-L2 und MHC-I Expression in unterschiedlichem Ausmaß erhöhten. Die Behandlung mit verschiedenen VA-Extrakten hatte keinen Einfluss auf die Viabilität der Zellen, sowie auf die Expression der PD-Liganden und MHC-I.Unsere Untersuchung kommt zu dem Schluss, dass eine VA-Behandlung die Expression von PD-Liganden oder MHC-I in den untersuchten Krebszellen nicht beeinflusst.

Immunosuppressive activity of non-psychoactive Cannabis sativa L. extract on the function of human T lymphocytes.

BACKGROUND: Cannabis sativa L. extracts (CSE) are used for treating inflammatory conditions, but little is known about their immunomodulatory effects. We investigated a novel CSE with high (14%) CBD and low (0.2%) THC concentration in comparison with pure CBD on primary human lymphocytes. METHODS: Proliferation, cell cycle distribution, apoptosis/necrosis and viability were analysed with standard methods. Genotoxicity was evaluated with the comet-assay. The effect on T lymphocyte activation was evaluated via CD25/CD69 marker expression, degranulation assays and the production of cytokines. The influence on the transcription factors was analysed using Jurkat reporter cell lines. Specific CB2 receptor antagonist SR144528 and TRPV1 receptor antagonist A78416B were used to study the involvement of CB2 or TRPV1 receptors. RESULTS: CSE inhibited the proliferation of activated T lymphocytes in a dose-dependent manner without inducing apoptosis, necrosis, or affecting cell viability and DNA integrity. The inhibitory effect was mediated via the suppression of T lymphocytes activation, particularly by the suppression of CD25 surface marker expression. Furthermore, CSE interferes with the functionality of the T lymphocytes, as indicated by inhibition of degranulation, IL-2, and IFN-γ production. AP-1-and-NFAT-reporter activation was reduced implicating an AP-1-and-NFAT-mediated mode of action. The effects were in part reversed by SR144528 and A78416B, showing that the effects were mainly mediated by CB2 and TRPV1 receptors. CONCLUSION: CSE and CBD have immunomodulatory effects and interfere with the activation and functionality of T lymphocytes. A comparison between CSE and CBD suggests that the immunosuppressive effect of CSE is mostly due to the effect of CBD.

A Vegan Diet Is Associated with a Significant Reduction in Dietary Acid Load: Post Hoc Analysis of a Randomized Controlled Trial in Healthy Individuals.

The composition of diet strongly affects acid-base homeostasis. Western diets abundant in acidogenic foods (meat and cheese) and deficient in alkalizing foods (fruits and vegetables) increase dietary acid load (DAL). A high DAL has been associated with numerous health repercussions, including cardiovascular disease and type-2-diabetes. Plant-based diets have been associated with a lower DAL; however, the number of trials exploring this association is limited. This randomized-controlled trial sought to examine whether an isocaloric vegan diet lowers DAL as compared to a meat-rich diet. Forty-five omnivorous individuals were randomly assigned to a vegan diet (n = 23) or a meat-rich diet (n = 22) for 4 weeks. DAL was determined using potential renal acid load (PRAL) and net endogenous acid production (NEAP) scores at baseline and after 3 and 4 weeks, respectively. After 3 weeks, median PRAL (-23.57 (23.87)) and mean NEAPR (12.85 ± 19.71) scores were significantly lower in the vegan group than in the meat-rich group (PRAL: 18.78 (21.04) and NEAPR: 60.93 ± 15.51, respectively). Effects were mediated by a lower phosphorus and protein intake in the vegan group. Our study suggests that a vegan diet is a potential means to reduce DAL, whereas a meat-rich diet substantially increases the DAL burden.

Yersinia pseudotuberculosis cytotoxic necrotizing factor interacts with glycosaminoglycans.

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.

Reversibility of citrate synthase allows autotrophic growth of a thermophilic bacterium.

Biological inorganic carbon fixation proceeds through a number of fundamentally different autotrophic pathways that are defined by specific key enzymatic reactions. Detection of the enzymatic genes in (meta)genomes is widely used to estimate the contribution of individual organisms or communities to primary production. Here we show that the sulfur-reducing anaerobic deltaproteobacterium Desulfurella acetivorans is capable of both acetate oxidation and autotrophic carbon fixation, with the tricarboxylic acid cycle operating either in the oxidative or reductive direction, respectively. Under autotrophic conditions, the enzyme citrate synthase cleaves citrate adenosine triphosphate independently into acetyl coenzyme A and oxaloacetate, a reaction that has been regarded as impossible under physiological conditions. Because this overlooked, energetically efficient carbon fixation pathway lacks key enzymes, it may function unnoticed in many organisms, making bioinformatical predictions difficult, if not impossible.