Metabolomic workflow for the accurate and high-throughput exploration of the pathways of tryptophan, tyrosine, phenylalanine, and branched-chain amino acids in human biofluids

10openInternationalInternational coauthor/editorThe modulation of host and dietary metabolites by gut microbiota (GM) is important for maintaining correct host physiology and in the onset of various pathologies. An ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the targeted quantitation in human plasma, serum, and urine of 89 metabolites resulting from human-GM cometabolism of dietary essential amino acids tryptophan, tyrosine, and phenylalanine as well as branched-chain amino acids. Ninety-six-well plate hybrid-SPE enables fast clean-up of plasma and serum. Urine was diluted and filtered. A 15 min cycle enabled the acquisition of 96 samples per day, with most of the metabolites stable in aqueous solution for up to 72 h. Calibration curves were specifically optimized to cover expected concentrations in biological fluids, and limits of detection were at the order of ppb. Matrix effects were in acceptable ranges, and analytical recoveries were in general greater than 80%. Inter and intraday precision and accuracy were satisfactory. We demonstrated its application in plasma and urine samples obtained from the same individual in the frame of an interventional study, allowing the quantitation of 51 metabolites. The method could be considered the reference for deciphering changes in human-gut microbial cometabolism in health and disease. Data are available via Metabolights with the identifier MTBLS4399.openAnesi, Andrea; Berding, Kirsten; Clarke, Gerard; Stanton, Catherine; Cryan, John F.; Caplice, Noel; Ross, R. Paul; Doolan, Andrea; Vrhovsek, Urska; Mattivi, FulvioAnesi, A.; Berding, K.; Clarke, G.; Stanton, C.; Cryan, J.F.; Caplice, N.; Ross, R.P.; Doolan, A.; Vrhovsek, U.; Mattivi, F

Rubus idaeus extract improves symptoms in knee osteoarthritis patients: results from a phase II double-blind randomized controlled trial.

peer reviewed[en] BACKGROUND: Osteoarthritis (OA) is the most frequent cause of disability in elderly people. In daily practice, the main objective of the physician is to reduce patient symptoms using treatments without adverse effects. However, the most prescribed treatment to manage OA symptoms remains nonsteroidal anti-inflammatory drugs which are associated with severe adverse effects. Therefore, we need a safe alternative to managing OA. One candidate is Rubus idaeus leaf extracts known to inhibit inflammatory responses. OBJECTIVE: This study aimed to evaluate the effects of a 12-weeks intervention with an ethanolic extract from Rubus idaeus leaf on symptoms of knee osteoarthritis. METHOD: The study was a randomized, double-blind, placebo-controlled, monocentric trial of 198 participants with femorotibial osteoarthritis. Participants were randomized equally to receive one daily during 3 months either 1 capsule of Rubus idaeus leaf extract 400 mg, 1 capsule of Rubus idaeus leaf extract 200 mg, or 1 capsule of placebo. The participants were assessed at baseline and after one and three months of treatment. The primary endpoint was an absolute change of the Western Ontario McMaster osteoarthritis index (WOMAC) pain subscale. The secondary endpoints were WOMAC global score, stiffness and function sub-scales, knee pain VAS score at walking, the Short Form (SF)-36, the Short Physical Performance Battery (SPPB), the 20-m walk test, and the International Physical Activity Questionnaire (IPAQ) and Outcome Measures in Rheumatology Clinical Trials and Osteoarthritis Research Society International (OMERACT-OARSI) responders rate. Statistical analyses were conducted on the intent-to-treat (ITT) population. RESULTS: In the Intention-to-treat population, WOMAC pain was not significantly modified by Rubus idaeus leaf extract compared to placebo. In contrast, Rubus idaeus leaf extract 400 mg after 12 weeks of treatment significantly reduced pain measured by the VAS. The mean pain decrease induced by Rubus ideaus leaf extract was over -7 mm which is clinically relevant and reached a clinically statistical difference compared to placebo with the highest dose. Rubus Ideaus was not significantly more efficient than the placebo on WOMAC global score, stiffness, and physical function subscores, IPAQ, SF-36, walking distance in treadmill test, SPPB, and evaluation of associated treatments needed to manage OA. CONCLUSION: Rubus idaeus leaf extract was well tolerated and effective to relieve pain in a patient with knee osteoarthritis. TRIAL REGISTRATION: NCT03703024  (11/10/2018)

Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

Background The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8+ and CD4+ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects. Methodology/Principal Findings To establish the basis for a SAPN-based vaccine, B- and CD8+ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ+, IL-2+) long-lived central memory CD8+ T-cells. Furthermore, we demonstrated that these Ab or CD8+ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites. Conclusion The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP

IgG2 Antibodies against a Clinical Grade Plasmodium falciparum CSP Vaccine Antigen Associate with Protection against Transgenic Sporozoite Challenge in Mice

The availability of a highly purified and well characterized circumsporozoite protein (CSP) is essential to improve upon the partial success of recombinant CSP-based malaria vaccine candidates. Soluble, near full-length, Plasmodium falciparum CSP vaccine antigen (CS/D) was produced in E. coli under bio-production conditions that comply with current Good Manufacturing Practices (cGMP). A mouse immunogenicity study was conducted using a stable oil-in-water emulsion (SE) of CS/D in combination with the Toll-Like Receptor 4 (TLR4) agonist Glucopyranosyl Lipid A (GLA/SE), or one of two TLR7/8 agonists: R848 (un-conjugated) or 3M-051 (covalently conjugated). Compared to Alum and SE, GLA/SE induced higher CS/D specific antibody response in Balb/c mice. Subclass analysis showed higher IgG2:IgG1 ratio of GLA/SE induced antibodies as compared to Alum and SE. TLR synergy was not observed when soluble R848 was mixed with GLA/SE. Antibody response of 3M051 formulations in Balb/c was similar to GLA/SE, except for the higher IgG2:IgG1 ratio and a trend towards higher T cell responses in 3M051 containing groups. However, no synergistic enhancement of antibody and T cell response was evident when 3M051 conjugate was mixed with GLA/SE. In C57Bl/6 mice, CS/D adjuvanted with 3M051/SE or GLA/SE induced higher CSP repeat specific titers compared to SE. While, 3M051 induced antibodies had high IgG2c:IgG1 ratio, GLA/SE promoted high levels of both IgG1 and IgG2c. GLA/SE also induced more potent T-cell responses compared to SE in two independent C57/BL6 vaccination studies, suggesting a balanced and productive TH1/TH2 response. GLA and 3M-051 similarly enhanced the protective efficacy of CS/D against challenge with a transgenic P. berghei parasite and most importantly, high levels of cytophilic IgG2 antibodies were associated with protection in this model. Our data indicated that the cGMP-grade, soluble CS/D antigen combined with the TLR4-containing adjuvant GLA/SE warrants further evaluation for protective responses in humans

Dendritic Cells and Hepatocytes Use Distinct Pathways to Process Protective Antigen from Plasmodium in vivo

Malaria-protective CD8+ T cells specific for the circumsporozoite (CS) protein are primed by dendritic cells (DCs) after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2Kb epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d) and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase

Vaccine Platforms Combining Circumsporozoite Protein and Potent Immune Modulators, rEA or EAT-2, Paradoxically Result in Opposing Immune Responses

Malaria greatly impacts the health and wellbeing of over half of the world's population. Promising malaria vaccine candidates have attempted to induce adaptive immune responses to Circumsporozoite (CS) protein. Despite the inclusion of potent adjuvants, these vaccines have limited protective efficacy. Conventional recombinant adenovirus (rAd) based vaccines expressing CS protein can induce CS protein specific immune responses, but these are essentially equivalent to those generated after use of the CS protein subunit based vaccines. In this study we combined the use of rAds expressing CS protein along with rAds expressing novel innate immune response modulating proteins in an attempt to significantly improve the induction of CS protein specific cell mediated immune (CMI) responses.BALB/cJ mice were co-vaccinated with a rAd vectors expressing CS protein simultaneous with a rAd expressing either TLR agonist (rEA) or SLAM receptors adaptor protein (EAT-2). Paradoxically, expression of the TLR agonist uncovered a potent immunosuppressive activity inherent to the combined expression of the CS protein and rEA. Fortunately, use of the rAd vaccine expressing EAT-2 circumvented CS protein's suppressive activity, and generated a fivefold increase in the number of CS protein responsive, IFNγ secreting splenocytes, as well as increased the breadth of T cells responsive to peptides present in the CS protein. These improvements were positively correlated with the induction of a fourfold improvement in CS protein specific CTL functional activity in vivo.Our results emphasize the need for caution when incorporating CS protein into malaria vaccine platforms expressing or containing other immunostimulatory compounds, as the immunological outcomes may be unanticipated and/or counter-productive. However, expressing the SLAM receptors derived signaling adaptor EAT-2 at the same time of vaccination with CS protein can overcome these concerns, as well as significantly improve the induction of malaria antigen specific adaptive immune responses in vivo

Illuminating the New Standards with Learning Style: Striking a Perfect Match

Dedicated educators have long been guiding their students toward the highest levesl of excellence and attainment. The new standards movement has emerged in response to a commonly perceived need to raise even further the expectations, quality of teaching, and levels considered as satisfactory achievement for all students in the United States. The standards movement today largely sterns from the National Commision on Excellence in Education\u27s (1983) pubication of A Nation at Risk: The Imperative for Educational Reform. The report indicated that the academic performance of students was far below that of students in countries considered to be our economic competitors. The report also held that our society\u27s educational foundations were being undermined by a rising current of commonness. Schools should have high expectations for all learners, according to the report\u27s writers, and should assist learners in every possible way to reach those expectations

The effect of Lactobacillus reuteri supplementation in Helicobacter pylori infection: a placebo-controlled, single-blind study

Abstract Background Helicobacter pylori is the major cause of chronic gastritis, and considered as a risk factor for peptic ulcer and gastric cancer. The H. pylori standard antibiotic therapy fails in about 25–30% of cases, particularly because of the increasing occurrence of resistance to antibiotics. The aim of the current study was to investigate whether the strain Lactobacillus reuteri DSM17648 which has been previously shown to reduce Helicobacter pylori load additionally improves gastrointestinal symptoms in H. pylori positive subjects when used in a 28 days supplementation. Methods In a single-blinded, placebo controlled study 24 H. pylori-positive adults (13 females, 11 males; median age: 43.5) with mild dyspepsia (mean GSRS score: 11.82) received placebo for 28 days followed by Pylopass™ containing the L. reuteri DSM 17648 (2 × 1010 cells per day) for the following 28 days. After 28 days of Pylopass™ supplementation the change in H. pylori load was measured by 13C urea breath test (13C-UBT) and the change in symptoms were determined by the Gastrointestinal Symptom Rating Scale (GSRS). In addition, blood assessments were conducted to measure the physiological changes relevant in terms of safety. Results After a 28-day supplementation phase with Pylopass™ there was a trend for reduction of H. pylori load in 62.5% of the subjects and for the overall GSRS scores in 66.7% of subjects. The overall GSRS scores from baseline to day 56 following all 24 subjects undergoing the placebo phase followed by the Pylopass™ phase was significantly decreased (p = 0.005). The mean 13C-UBT δ value decreased by 22.5% during the Pylopass™ supplementation phase (− 3.14), while the mean 13C-UBT δ increased by 37.3% (+ 3.79) in the placebo phase. No side effects were reported in either study phase. Conclusion The results demonstrated that L. reuteri DSM17648 has the potential to suppress H. pylori infection, and may lead to an improvement of H. pylori-associated gastro intestinal symptoms. Further studies with adequate power should be performed. Trial registration Clinicaltrials.gov: NCT02051348 (January 30, 2014)

Impact of Learning-Style Instructional Strategies on Students\u27 Achievement and Attitudes: Perceptions of Educators in Diverse Institutions

Educators from various institutions responded to key questions concerning learning style as it was implemented in their institutions and communities. Among those items explored were the impact of learning styles on teaching practices, syllabi, and values and whether the construct improved instruction or student outcomes, how it improved students\u27 perceptions of their learning outcomes, and how it contributed to the profession of education

DNA prime/Adenovirus boost malaria vaccine encoding P. falciparum CSP and AMA1 induces sterile protection associated with cell-mediated immunity

Contains fulltext : 118242.pdf (publisher's version ) (Open Access)BACKGROUND: Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection. METHODOLOGY/PRINCIPAL FINDINGS: The vaccine regimen was three monthly doses of two DNA plasmids (DNA) followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad). The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea), possibly related to immunization, was severe (Grade 3), preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27%) were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44-817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5-102) and were not associated with protection. Ex vivo IFN-gamma ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13-408; AMA1 348, range 88-1270) and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019). Flow cytometry identified predominant IFN-gamma mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant. SIGNIFICANCE: The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%). Protection was associated with cell-mediated immunity to AMA1, with CSP probably contributing. Substituting a low seroprevalence vector for Ad5 and supplementing CSP/AMA1 with additional antigens may improve protection. TRIAL REGISTRATION: ClinicalTrials.govNCT00870987