Review of existing experimental methods for assessing the outcome of plant food supplementation on immune function

Plant food supplements (PFS) have become increasingly popular with respect to their consumption for improving human immune function. Despite this popularity, critical review is lacking regarding the analytical methods used to assess PFS outcome. The suitability of such methods for clinical-based studies remains particularly unclear.We undertook a literature-based review of the methods used to assess PFS outcome in immune function, to identify and assess the relevance of different technologies. Most methods described in this review adequately measured the functions of innate and adaptive immunity, were applicable to both healthy and diseased subjects, and were appropriate for assessing the benefit claims of PFS on immune function. However, the design and reporting quality of studies varied widely across trials, in some cases potentially impacting negatively on the outcomes and interpretations. Several strategies to enhance study robustness and quality were outlined, to improve the validity of the data generated in the field

Discovering new transmission-blocking antimalarial compounds : challenges and opportunities

The ability to target human-mosquito parasite transmission challenges global malaria elimination. However, it is not obvious what a transmission-blocking drug will look like; should it 1) target only parasite transmission stages; 2) be combined with a partner drug killing the pathogenic asexual stages or 3) kill both the sexual and asexual blood stages, preferably displaying polypharmacology. The development of transmission-blocking anti-malarials requires objective analyses of the current strategies. Here, pertinent issues and unanswered questions regarding the target candidate profile of a transmission-blocking compound, and its role in malaria elimination strategies are highlighted and novel perspectives proposed. The essential role of a test cascade that integrates screening and validation strategies to identify next generation transmission-blocking anti-malarials is emphasised.Koen Dechering (TROPIQ, The Netherlands), Francesco Silvestrini (Istituto Superiore di Sanità, Rome, Italy), Sarah D’Alessandro and Donatella Taramelli (University of Milan, Italy), Robert Sauerwein (University of Nijmegen, The Netherlands) and Omar Vandal (The Bill & Melinda Gates Foundation) are acknowledged for their contributions towards the screening cascade, which they developed in the course of the Bill and Melinda Gates Foundation Project OPP1040394 ‘Gametocyte Assays for Malaria (GAM) for novel transmission blocking drugs’, coordinated by PA. The South African Transmission-blocking Consortium is funded by the Medical Research Council of South Africa as a Strategic Health Innovation Partnership (MRC SHIP) project.http://www.journals.elsevier.com/trends-in-parasitology2017-09-30hb2016Biochemistr

Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum</it>, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting <it>P. falciparum </it>spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level.</p> <p>Results</p> <p>Morphological analysis of <it>P. falciparum </it>3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels.</p> <p>Conclusions</p> <p>This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in <it>P. falciparum</it>, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.</p

Genetic variability of histamine receptors in patients with Parkinson's disease

<p>Abstract</p> <p>Background</p> <p>Changes in the density and expression of histamine receptors (HRH) have been detected in Parkinson's disease (PD) patients, and HRH antagonists bring about improvements in motor and other symptoms, thus suggesting that HRH play a role in the clinical response of PD patients. This study is aimed to analyse polymorphic variations of HRH in patients with PD.</p> <p>Methods</p> <p>Leukocytary DNA from 195 PD patients and a control group of 231 unrelated healthy individuals was studied for the nonsynonymous HRH1Leu449Ser and the promoter HRH2G-1018A polymorphisms by using amplification-restriction analyses.</p> <p>Results</p> <p>The HRH1Leu449Ser amino acid substitution was identified in two women with late-onset PD whereas it was not observed among healthy subjects. The HRH2G-1018A polymorphism was observed with allele frequencies = 3.59 (95% CI = 1.74–5.44) and 5.0 (95% CI = 3.00–6.96) for patients with PD and healthy controls, respectively. These frequencies were independent of gender and age of onset of the disease. Multiple comparison analyses revealed that differences were not statistically significant.</p> <p>Conclusion</p> <p>These results indicate that the polymorphisms analyzed are not a major risk factor for PD, although the HRH1Leu449Ser amino acid substitution might be related to PD.</p

Thermo-responsive non-woven scaffolds for "smart" 3D cell culture

The thermo-responsive polymer poly(N-isopropylacrylamide) has received widespread attention for its in vitro application in the non-invasive, non-destructive release of adherent cells on two dimensional surfaces. In this study, 3D non-woven scaffolds fabricated from poly(propylene) (PP), poly(ethylene terephthalate) (PET) and nylon that had been grafted with PNIPAAm were tested for their ability to support the proliferation and subsequent thermal release of HC04 and HepG2 hepatocytes. Hepatocyte viability and proliferation was estimated using the Alamar Blue assay and Hoechst 33258 total DNA quantification. The assays revealed that the pure and grafted non-woven scaffolds maintained the hepatocytes within the matrix and promoted 3D proliferation comparable to that of the commercially available AlgimatrixTM alginate scaffold. Albumin production and selected cytochrome P450 genes expression was found to be superior in cells growing on pure and grafted non-woven PP scaffolds as compared to cells grown as a 2D monolayer. Two scaffolds, namely, PP-g- PNIPAAm-A and PP-g-PNIPAAm-B were identified as having far superior thermal release capabilities; releasing the majority of the cells from the matrices within 2 h. This is the first report for the development of 3D non-woven, thermo-responsive scaffolds able to release cells from the matrix without the use of any enzymatic assistance or scaffold degradation.The Council for Scientific and Industrial Researchhttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-0290hb2016Biochemistr

In vitro anti-plasmodial activity of Dicoma anomala subsp. gerrardii (Asteraceae): identification of its main active constituent, structure-activity relationship studies and gene expression profiling

<p>Abstract</p> <p>Background</p> <p>Anti-malarial drug resistance threatens to undermine efforts to eliminate this deadly disease. The resulting omnipresent requirement for drugs with novel modes of action prompted a national consortium initiative to discover new anti-plasmodial agents from South African medicinal plants. One of the plants selected for investigation was <it>Dicoma anomala </it>subsp. <it>gerrardii</it>, based on its ethnomedicinal profile.</p> <p>Methods</p> <p>Standard phytochemical analysis techniques, including solvent-solvent extraction, thin-layer- and column chromatography, were used to isolate the main active constituent of <it>Dicoma anomala </it>subsp. <it>gerrardii</it>. The crystallized pure compound was identified using nuclear magnetic resonance spectroscopy, mass spectrometry and X-ray crystallography. The compound was tested <it>in vitro </it>on <it>Plasmodium falciparum </it>cultures using the parasite lactate dehydrogenase (pLDH) assay and was found to have anti-malarial activity. To determine the functional groups responsible for the activity, a small collection of synthetic analogues was generated - the aim being to vary features proposed as likely to be related to the anti-malarial activity and to quantify the effect of the modifications <it>in vitro </it>using the pLDH assay. The effects of the pure compound on the <it>P. falciparum </it>transcriptome were subsequently investigated by treating ring-stage parasites (alongside untreated controls), followed by oligonucleotide microarray- and data analysis.</p> <p>Results</p> <p>The main active constituent was identified as dehydrobrachylaenolide, a eudesmanolide-type sesquiterpene lactone. The compound demonstrated an <it>in vitro </it>IC₅₀of 1.865 μM against a chloroquine-sensitive strain (D10) of <it>P. falciparum</it>. Synthetic analogues of the compound confirmed an absolute requirement that the α-methylene lactone be present in the eudesmanolide before significant anti-malarial activity was observed. This feature is absent in the artemisinins and suggests a different mode of action. Microarray data analysis identified 572 unique genes that were differentially expressed as a result of the treatment and gene ontology analysis identified various biological processes and molecular functions that were significantly affected. Comparison of the dehydrobrachylaenolide treatment transcriptional dataset with a published artesunate (also a sesquiterpene lactone) dataset revealed little overlap. These results strengthen the notion that the isolated compound and the artemisinins have differentiated modes of action.</p> <p>Conclusions</p> <p>The novel mode of action of dehydrobrachylaenolide, detected during these studies, will play an ongoing role in advancing anti-plasmodial drug discovery efforts.</p

Development of thermoresponsive poly(propylene-g-N-isopropylacrylamide) non-woven 3D scaffold for smart cell culture using oxyfluorination-assisted graft polymerisation

Growing cells on 3D scaffolds is far superior to the conventional 2D monolayer culture method. In this study, a novel 3D thermoresponsive poly(propylene-g-N-isopropylacrylamide) (PP-g-PNIPAAm) nonwoven fabric (gNWF) was developed for cell culture using oxyfluorination-assisted graft polymerisation (OAGP). New polar functional groups were detected on the oxyfluorinated NWF (oNWF), and PNIPAAm was confirmed in the gNWF by attentuated total-reflectance Fourier transform infrared (ATR-FTIR) and scanning X-ray photoelectron spectroscopy (S-XPS). Scanning electron microscopy (SEM) revealed a rough surface morphology and confinement of the PNIPAAm graft layer to the surface of the fibres in the gNWF. The OAGP method did not affect the crystalline phase of bulk PP, however, twin-melting thermal peaks were detected for the oNWF and gNWF indicating crystal defects. Contact angle studies showed that the surface of the gNWF exhibited a thermoresponsive behaviour. Hepatocyte cells attached onto gNWF disks in a bioreactor at 37 ◦C and remained viable for 10 days in culture. Upon cooling the cell culture media to 20 ◦C, cells were spontaneously released as 3D multi-cellular constructs without requiring destructive enzymes. The development of 3D thermoresponsive scaffolds capable of non-invasive 3D cell culture could provide a more reliable in vitro model for cells.http://www.elsevier.com/locate/colsurfahb201

In vitro anti-malarial interaction and gametocytocidal activity of cryptolepine

YesBackground: Discovery of novel gametocytocidal molecules is a major pharmacological strategy in the elimination and eradication of malaria. The high patronage of the aqueous root extract of the popular West African anti-malarial plant Cryptolepis sanguinolenta (Periplocaceae) in traditional and hospital settings in Ghana has directed this study investigating the gametocytocidal activity of the plant and its major alkaloid, cryptolepine. This study also investigates the anti-malarial interaction of cryptolepine with standard anti-malarials, as the search for new anti-malarial combinations continues. Methods: The resazurin-based assay was employed in evaluating the gametocytocidal properties of C. sanguinolenta and cryptolepine against the late stage (IV/V) gametocytes of Plasmodium falciparum (NF54). A fixed ratio method based on the SYBR Green I fluorescence-based assay was used to build isobolograms from a combination of cryptolepine with four standard anti-malarial drugs in vitro using the chloroquine sensitive strain 3D7. Results: Cryptolepis sanguinolenta ( IC50 = 49.65 nM) and its major alkaloid, cryptolepine ( IC50 = 1965 nM), showed high inhibitory activity against the late stage gametocytes of P. falciparum (NF54). In the interaction assays in asexual stage, cryptolepine showed an additive effect with both lumefantrine and chloroquine with mean ΣFIC50s of 1.017 ± 0.06 and 1.465 ± 0.17, respectively. Cryptolepine combination with amodiaquine at therapeutically relevant concentration ratios showed a synergistic effect (mean ΣFIC50 = 0.287 ± 0.10) whereas an antagonistic activity (mean ΣFIC50 = 4.182 ± 0.99) was seen with mefloquine. Conclusions: The findings of this study shed light on the high gametocytocidal properties of C. sanguinolenta and cryptolepine attributing their potent anti-malarial activity mainly to their effect on both the sexual and asexual stages of the parasite. Amodiaquine is a potential drug partner for cryptolepine in the development of novel fixed dose combinations

Nowhere to hide : interrogating different metabolic parameters of Plasmodium falciparum gametocytes in a transmission blocking drug discovery pipeline towards malaria elimination

BACKGROUND : The discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions. METHODS : Gametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable. RESULTS : A highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4 %. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident. CONCLUSIONS : With this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broaddiversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.South African Medical Research Council Strategic Health Initiatives Partnerships with the Medicines for Malaria Venture as well as the Council for Scientific and Industrial Research, and the 3R Foundation (project 118–10).http://www.malariajournal.comhb201