Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78ngmL and the CCß to be 1ngmL. Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC-MS/MS showed a high correlation (R=0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1ngmL, and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1ngmL. This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1µgL. This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.

Molecularly Imprinted Polymers for the isolation of bioactive naphthoquinones from plant extracts

Molecularly Imprinted Polymers (MIPs) targeting shikonin, a potent antioxidant and wound healing agent, have been prepared using methacrylic acid (MAA) and 2-diethylaminoethyl methacrylate (DEAEMA) as functional monomers. An investigation of solution association between shikonin and both acidic and basic functional monomers by UV-Vis titrations, suggested stronger affinity towards the basic functionality. Strong inhibition of the co-polymerisation reaction of such basic monomers was observed, but was overcome by reduction of the amount of template used during polymer synthesis. Polymer morphology was severely impacted by the template’s radical scavenging behaviour as demonstrated by solid state NMR spectroscopy measurements. HPLC evaluation of the final materials in polar conditions revealed limited imprinting effects and selectivity, with the MAA polymers exhibiting marginally better performance. During application of the polymers as MI-SPE sorbents in non-polar solvents it was found that the DEAEMA based polymer was more selective towards shikonin compared to the MAA counterpart, while shikonin recoveries of up to 72% were achieved from hexane solutions of a commercial sample of shikonin, hexane extract of Alkanna tinctoria roots and a commercial pharmaceutical ointment.

Development and validation of the first high performance-lateral flow immunoassay (HP-LFIA) for the rapid screening of domoic acid from shellfish extracts

A lateral flow immunoassay (LFIA) has been developed and fully validated to detect the primary amnesic shellfish poisoning (ASP) toxin, domoic acid (DA). The performance characteristics of two versions of the test were investigated using spiked and naturally contaminated shellfish (mussels, scallops, oysters, clams, and cockles). The tests provide a qualitative result, to indicate the absence or presence of DA in extracts of shellfish tissues, at concentrations that are relevant to regulatory limits. The new rapid assay (LFIA version 2) was designed to overcome the performance limitations identified in the first version of the assay. The improved test uses an electronic reader to remove the subjective nature of the generated results, and the positive cut-off for screening of DA in shellfish was increased from 10 ppm (version 1) to 17.5 ppm (version 2). A simple extraction and test procedure was employed, which required minimal equipment and materials; results were available 15 min after sample preparation. Stability of the aqueous extracts at room temperature (22 C) at four time points (up to 245 min after extraction) and across a range of DA concentrations was 100.3±1.3% and 98.8±2.4% for pre- and post-buffered extracts, respectively. The assay can be used both within laboratory settings and in remote locations. The accuracy of the new assay, to indicate negative results at or below 10 ppm DA, and positive results at or above 17.5 ppm, was 99.5% (n=216 tests). Validation data were obtained from a 2-day, randomised, blind study consisting of multiple LFIA lots (n=3), readers (n=3) and operators (n=3), carrying out multiple extractions of mussel tissue (n=3) at each concentration (0, 10, 17.5, and 20 ppm). No matrix effects were observed on the performance of the assay with different species (mussels, scallops, oysters, clams, and cockles). There was no impact on accuracy or interference from other phycotoxins, glutamic acid or glutamine with various strip incubations (8, 10, and 12 min). The accuracy of the assay, using naturally contaminated samples to indicate negative results at or below 12.5 ppm and positive results at or above 17.5 ppm, was 100%. Variability between three LFIA lots across a range of DA concentrations, expressed as coefficient of variation (% CV), was 1.1±0.4% (n=2 days) based on quantitative readings from the electronic reader. During an 8 week stability study, accuracy of the method with test strips stored at various temperatures (6, 22, 37 and 50 C) was 100%. Validation for both versions included comparisons with results obtained using reference LC-UV methods. © 2013 Elsevier B.V.

Effects of Undaria pinnatifida, Himanthalia elongata and Porphyra umbilicalis extracts on in vitro α-glucosidase activity and glucose diffusion

Background: Seaweeds are good sources of dietary fibre, which can influence glucose uptake and glycemic control.Objective: To investigate and compare the in vitro inhibitory activity of different extracts from Undaria pinnatifida (Wakame), Himanthalia elongata (Sea spaghetti) and Porphyra umbilicalis (Nori) on α-glucosidase activity and glucose diffusion.Methods: The in vitro effects chloroform-, ethanol- and water-soluble extracts of the three algae were assayed on α- glucosidase activity and glucose diffusion through membrane. Principal Components Analysis (PCA) was applied to identify patterns in the data and to discriminate which extract will show the most proper effect.Results: Only water extracts of Sea spaghetti possessed significant in vitro inhibitory effects on α-glucosidase activity (26.2% less mmol/L glucose production than control, p < 0.05) at 75 min. PCA distinguished Sea spaghetti effects, supporting that soluble fibre and polyphenols were involved. After 6 h, Ethanol-Sea spaghetti and water-Wakame extracts exerted the highest inhibitory effects on glucose diffusion (65.0% and 60.2% vs control, respectively). This extracts displayed the lowest slopes for glucose diffusion-time lineal adjustments (68.2% and 62.8% vs control, respectively).Conclusions: The seaweed hypoglycemic effects appear multi-faceted and not necessarily concatenated. According to present results, ethanol and water extracts of Sea spaghetti, and water extracts of Wakame could be useful for the development of functional foods with specific hypoglycemic properties.

Characterisation of polyacetylenes in carrot extracts using electrospray ionisation quadrupole time of flight mass spectrometry.

Rai, D., Koidis, A., Rawson, A., McLouglin, P., Brunton, N. (2010). Characterisation of polyacetylenes in carrot extracts using electrospray ionisation quadrupole time of flight mass spectrometry. Delegate CD-Rom PS2.8, EFFoST Annual Meeting, Dublin, Ireland, 10 -12/11/2010 (Poster).

Development and Validation of a Novel Lateral Flow Immunoassay (LFIA) for the Rapid Screening of Paralytic Shellfish Toxins (PSTs) from Shellfish Extracts

A single-step lateral flow immunoassay (LFIA) was developed and validated for the rapid screening of paralytic shellfish toxins (PSTs) from a variety of shellfish species, at concentrations relevant to regulatory limits of 800 μg STX-diHCl equivalents/kg shellfish meat. A simple aqueous extraction protocol was performed within several minutes from sample homogenate. The qualitative result was generated after a 5 min run time using a portable reader which removed subjectivity from data interpretation. The test was designed to generate noncompliant results with samples containing approximately 800 μg of STX-diHCl/kg. The cross-reactivities in relation to STX, expressed as mean ± SD, were as follows: NEO: 128.9% ± 29%; GTX1&4: 5.7% ± 1.5%; GTX2&3: 23.4% ± 10.4%; dcSTX: 55.6% ± 10.9%; dcNEO: 28.0% ± 8.9%; dcGTX2&3: 8.3% ± 2.7%; C1&C2: 3.1% ± 1.2%; GTX5: 23.3% ± 14.4% (n = 5 LFIA lots). There were no indications of matrix effects from the different samples evaluated (mussels, scallops, oysters, clams, cockles) nor interference from other shellfish toxins (domoic acid, okadaic acid group). Naturally contaminated sample evaluations showed no false negative results were generated from a variety of different samples and profiles (n = 23), in comparison to reference methods (MBA method 959.08, LC-FD method 2005.06). External laboratory evaluations of naturally contaminated samples (n = 39) indicated good correlation with reference methods (MBA, LC-FD). This is the first LFIA which has been shown, through rigorous validation, to have the ability to detect most major PSTs in a reliable manner and will be a huge benefit to both industry and regulators, who need to perform rapid and reliable testing to ensure shellfish are safe to eat.

Identification of arsenic species in sheep-wool extracts by different chromatographic methods

Sheep on the island of North Ronaldsay (Orkney, UK) feed mostly on seaweed, which contains high concentrations of dimethylated arsenoribosides. Wool of these sheep contains dimethylated, monomethylated and inorganic arsenic, in addition to unidentified arsenic species in unbound and complexed form. Chromatographic techniques using different separation mechanisms and detectors enabled us to identify five arsenic species in water extracts of wool. The wool contained 5.2 ± 2.3 μg arsenic per gram wool. About 80% of the arsenic in wool was extracted by boiling the wool with water. The main species is dimethylarsenic, which accounted for about 75 to 85%, monomethylated arsenic at about 5% and the rest is inorganic arsenic. Depending on the separation method and condition, the chromatographic recovery of arsenic species was between 45% for the anion exchange column, 68% for the size exclusion chromatography (SEC) and 82% for the cation exchange column. The SEC revealed the occurrence of two unknown arsenic compounds, of which one was probably a high molecular mass species. Since chromatographic recovery can be improved by either treating the extract with CuCl/HCl (CAT: 90%) or longer storage of the sample (CAT: 105%), in particular for methylated arsenic species, it can be assumed that labile arsenic -protein-like coordination species occur in the extract, which cannot be speciated with conventional chromatographic methods. It is clear from our study of sheep wool that there can be different kinds of 'hidden' arsenic in biological matrices, depending on the extraction, separation and detection methods used. Hidden species can be defined as species that are not recordable by the detection system, not extractable or do not elute from chromatographic columns. Copyright © 2003 John Wiley & Sons, Ltd.

Development and Validation of a Lateral Flow Immunoassay for the Rapid Screening of Okadaic Acid and All Dinophysis Toxins from Shellfish Extracts

A single-step lateral flow immunoassay was developed and validated to detect okadaic acid (OA) and dinophysis toxins (DTXs), which cause diarrhetic shellfish poisoning. The performance characteristics of the test were investigated, in comparison to reference methods (liquid chromatography tandem mass spectrometry and/or bioassay), using both spiked and naturally contaminated shellfish. A portable reader was used to generate a qualitative result, indicating the absence or presence of OA-group toxins, at concentrations relevant to the maximum permitted level (MPL). Sample homogenates could be screened in 20 min (including extraction and assay time) for the presence of free toxins (OA, DTX1, DTX2). DTX3 detection could be included with the addition of a hydrolysis procedure. No matrix effects were observed from the species evaluated (mussels, scallops, oysters, and clams). Results from naturally contaminated samples (n = 72) indicated no false compliant results and no false noncompliant results at <50% MPL. Thus, the development of a new low-cost but highly effective tool for monitoring a range of important phycotoxins has been demonstrated.

Potential health benefits of Artemisia gorgonum extracts

Relevância etnofarmacológica: Artemisia gorgonum (Asteraceae), conhecida como “losna ou lorna”, é usada em Cabo Verde na medicina tradicional para o tratamento de inflamações, febre e gastroenterites. Estudos recentes sugerem que artimetina, isolada a partir de Artemisa gorgonum, poderia ser usada para o tratamento da malária devido à sua atividade antiplasmodial. Objetivo do estudo: Avaliação in vitro da atividade anti-microbiana e sinergética dos extratos hidroetanol (70%) e metanol de A. gorgonum (EHAG e EMAG) em bactérias do trato urinário e uma espécie de fungo. A atividade antioxidante dos extratos de hidroetanol (70%), metanol, clorofórmio e clorofórmio-metanol (1:2), e o efeito protetor de EHAG contra lesões hepáticas em ratos induzidos com CCl4 também foram analisados. Material e métodos: A atividade antimicrobiana dos extratos de A. gorgonum foi testada in vitro contra sete estirpes de microrganismos, incluindo bactérias Gram-positivas, Gram-negativas e uma espécie de fungo. O método DAA (Decimal assay for additivity) foi determinado para atividade antibacteriana do EHAG contra Pseudomonas aeruginosa. O efeito antioxidante in vitro de vários extratos de A. gorgonum foi analisado pelo método DPPH. A lesão hepática foi induzida por injeção intrapeitoral do CCl4. Seguidamente, os ratos foram administrados oralmente com EHAG, diariamente, por um período de 7 dias. Resultados e Discussão: Foi observada atividade antibacteriana dos extratos de EHAG e EMAG contra todos os microrganismos usados neste estudo. O crescimento das estirpes de Escherichia coli e Pseudomonas aeruginosa foi o mais inibido por ambos os extratos, apresentando valores significativos, enquanto o crescimento das estirpes S. aureus e Klebsiella spp. foi o menos afetado. Candida albicans foi inibida fortemente pelo EMAG. As combinações de extrato hidroetanólico com antibióticos demonstraram atividade antibacteriana sinergética contra todos os patogénicos testados. Em contrapartida, a combinação de extrato metanólico com antibióticos permitiu observar efeitos antagónicos contra todas as bactérias, exceto Klebsiella spp. que apresentou atividade sinérgica. O EHAG e EMAG mostraram efeito significativo na eliminação do radical DPPH. A atividade hepatoprotetora foi observada em ratos previamente administrados com CCl4. Estes estudos evidenciam os potenciais benefícios de A. gorgonum.

Evaluation of the in vitro biological activities of extracts from carob tree and mediterranean oaks

Dissertação mest., Engenharia Biológica, Universidade do Algarve, 2009