Oral supplementation with fish cartilage hydrolysate in an adult population suffering from knee pain and function discomfort: results from an innovative approach combining an exploratory clinical study and an ex vivo clinical investigation.

BACKGROUND: Aging is frequently associated with impairments of the musculoskeletal system and many elderly people experience joint discomfort or pain which might reduce their ability to move and consequently alter their quality of life. A beneficial effect of fish cartilage hydrolysate (FCH) on pain and joint function has recently been shown in an ACLT/pMMx osteoarthritis rat model. METHODS: We therefore performed an exploratory, non-comparative, multi-centric clinical trial including 33 subjects with moderate knee joint discomfort and loss of functionality to investigate the efficacy of FCH on their algo-functional status. We further determined the potential health benefit of FCH in an original clinical ex vivo study investigating the role of FCH human metabolites on primary human chondrocytes. RESULTS: FCH significantly improved knee pain and function, as assessed by the Knee injury and Osteoarthritis Outcome Score (KOOS). Moreover, FCH significantly reduced pain at rest and while walking, and patient global assessment (PGA), as assessed by the Visual Analogue Scale (VAS), and improved patients' quality of life (SF-36). FCH metabolites decreased the synthesis of catabolic factors (MMP-13) and pro-inflammatory mediators (NO, PGE2) and limited the inhibitory effect of IL-1ß on the synthesis of cartilage matrix components (GAG and collagen). CONCLUSIONS: Thus, these data provide insights on the mode of action of FCH in humans and contribute to explain how FCH may relieve pain and improve joint function in subjects with knee discomfort. Although these preliminary data need to be confirmed in a randomized controlled trial, they strongly support the potential health benefit of such an active ingredient. TRIAL REGISTRATION: The study was registered on clinicaltrials.gov with the identifier NCT04420091 (09/06/2020).

NASAFYTOL® supplementation in adults hospitalized with COVID-19 infection: results from an exploratory open-label randomized controlled trial.

Objectives: The effect and safety of Nasafytol®, a food supplement combining curcumin, quercetin, and Vitamin D, on hospitalized COVID-19-positive patients as support to standard of care were to be assessed. Methods: This exploratory, open-label, randomized, controlled trial was carried out among hospitalized adults with COVID-19 infection. Participants were randomly assigned to receive Nasafytol® or Fultium® control. The improvement of the clinical condition and occurrence of (serious) adverse events were evaluated. The study was registered on clincaltrials.gov with the identifier NCT04844658. Results: Twenty-five patients received Nasafytol®, and 24 received Fultium®. Demographic characteristics were well balanced between the groups. On day 14 (or at hospital leave if < 14 days), no difference was observed between groups regarding their clinical condition, fever, or the need of oxygen therapy. At day 7, however, 19 participants had been discharged from the hospital in the Nasafytol® arm compared to 10 participants in the Fultium® arm. No participants were transferred to the ICU or died in the Nasafytol® arm, vs. 4 transfers and 1 death in the Fultium® arm. The clinical condition of participants in the Nasafytol® arm had improved, as evidenced by a decrease in the COVID-19 WHO score. Interestingly, five SAEs occurred with Fultium®, while no SAE was observed with Nasafytol®. Conclusion: Supplementation with Nasafytol®, in addition to standard-of-care treatment, led to a faster discharge from the hospital, improved clinical conditions of participants, and a reduced risk of serious outcomes, including transfer to the intensive care unit or death, in patients hospitalized with COVID-19.

Multipotent mesenchymal stromal cells as treatment for poor graft function after allogeneic hematopoietic cell transplantation: A multicenter prospective analysis.

Introduction: Poor graft function (PGF) is a rare but serious complication of allogeneic hematopoietic cell transplantation (alloHCT). Due to their hematopoietic supporting properties and immune regulatory effects, multipotent mesenchymal stromal cells (MSC) could be considered a good candidate to help to restore bone marrow (BM) niches homeostasis and facilitate hematopoiesis after alloHCT. Methods: We prospectively assessed the efficacy and safety of ex-vivo expanded BM-derived MSC from third-party donor in a series of 30 patients with prolonged severe cytopenia and PGF after alloHCT. This multicenter trial was registered at www.clinicaltrials.gov (#NTC00603330). Results: Within 90 days post-MSC infusion, 53% (95% CI, 35 - 71%) of patients improved at least one cytopenia (overall response, OR) and 37% (95% CI, 19 - 54%) achieved a complete hematological response (CR: absolute neutrophil count, ANC >0.5 x 109/L, Hb > 80g/L and platelet count > 20 x 109/L with transfusion independence). Corresponding response rates increased to 67% (95% CI, 50 - 84%) OR and 53% (95% CI, 35 - 71%) CR within 180 days after MSC infusion. A significant decrease in red blood cells and platelets transfusion requirement was observed after MSC (median of 30-days transfusion requirement of 0.5 and 0 from d90-120 post-MSC versus 5 and 6.5 before MSC, respectively, p ≤0.001). An increase in ANC was also noted by day +90 and +180, with 3/5 patients with severe neutropenia having recovered an ANC > 1 x 109/L within the 90-120 days after MSC infusion. Overall survival at 1 year post-MSC was 70% (95% CI, 55.4 - 88.5), with all but one of the patients who achieved CR being alive. A single infusion of third-party MSC appeared to be safe, with the exception of one deep vein thrombotic event possibly related to the intervention. Discussion: In conclusion, a single i.v. infusion of BM-derived MSC from third party donor seemed to improve hematological function after alloHCT, although spontaneous amelioration cannot be excluded. Comparative studies are warranted to confirm these encouraging results.

Genome skimming and microsatellite analysis reveal contrasting patterns of genetic diversity in a rare sandhill endemic (Erysimum teretifolium, Brassicaceae).

Barriers between islands often inhibit gene flow creating patterns of isolation by distance. In island species, the majority of genetic diversity should be distributed among isolated populations. However, a self-incompatible mating system leads to higher genetic variation within populations and very little between-population subdivision. We examine these two contrasting predictions in Erysimum teretifolium, a rare self-incompatible plant endemic to island-like sandhill habitats in Santa Cruz County, California. We used genome skimming and nuclear microsatellites to assess the distribution of genetic diversity within and among eight of the 13 remaining populations. Phylogenetic analyses of the chloroplast genomes revealed a deep separation of three of the eight populations. The nuclear ribosomal DNA cistron showed no genetic subdivision. Nuclear microsatellites suggest 83% of genetic variation resides within populations. Despite this, 18 of 28 between-population comparisons exhibited significant population structure (mean FST = 0.153). No isolation by distance existed among all populations, however when one outlier population was removed from the analysis due to uncertain provenance, significant isolation by distance emerged (r2 = 0.5611, p = 0.005). Population census size did not correlate with allelic richness as predicted on islands. Bayesian population assignment detected six genetic groupings with substantial admixture. Unique genetic clusters were concentrated at the periphery of the species' range. Since the overall distribution of nuclear genetic diversity reflects E. tereifolium's self-incompatible mating system, the vast majority of genetic variation could be sampled within any individual population. Yet, the chloroplast genome results suggest a deep split and some of the nuclear microsatellite analyses indicate some island-like patterns of genetic diversity. Restoration efforts intending to maximize genetic variation should include representatives from both lineages of the chloroplast genome and, for maximum nuclear genetic diversity, should include representatives of the smaller, peripheral populations.

Reproductive success through high pollinator visitation rates despite self incompatibility in an endangered wallflower.

PREMISE OF THE STUDY: Self incompatibility (SI) in rare plants presents a unique challenge-SI protects plants from inbreeding depression, but requires a sufficient number of mates and xenogamous pollination. Does SI persist in an endangered polyploid? Is pollinator visitation sufficient to ensure reproductive success? Is there evidence of inbreeding/outbreeding depression? We characterized the mating system, primary pollinators, pollen limitation, and inbreeding/outbreeding depression in Erysimum teretifolium to guide conservation efforts. METHODS: We compared seed production following self pollination and within- and between-population crosses. Pollen tubes were visualized after self pollinations and between-population pollinations. Pollen limitation was tested in the field. Pollinator observations were quantified using digital video. Inbreeding/outbreeding depression was assessed in progeny from self and outcross pollinations at early and later developmental stages. KEY RESULTS: Self-pollination reduced seed set by 6.5× and quadrupled reproductive failure compared with outcross pollination. Pollen tubes of some self pollinations were arrested at the stigmatic surface. Seed-set data indicated strong SI, and fruit-set data suggested partial SI. Pollinator diversity and visitation rates were high, and there was no evidence of pollen limitation. Inbreeding depression (δ) was weak for early developmental stages and strong for later developmental stages, with no evidence of outbreeding depression. CONCLUSIONS: The rare hexaploid E. teretifolium is largely self incompatible and suffers from late-acting inbreeding depression. Reproductive success in natural populations was accomplished through high pollinator visitation rates consistent with a lack of pollen limitation. Future reproductive health for this species will require large population sizes with sufficient mates and a robust pollinator community.

Orchestration of an uncommon maturation cascade of the house dust mite protease allergen quartet.

In more than 20% of the world population, sensitization to house dust mite allergens triggers typical allergic diseases such as allergic rhinitis and asthma. Amongst the 23 mite allergen groups hitherto identified, group 1 is cysteine proteases belonging to the papain-like family whereas groups 3, 6, and 9 are serine proteases displaying trypsin, chymotrypsin, and collagenolytic activities, respectively. While these proteases are more likely to be involved in the mite digestive system, they also play critical roles in the initiation and in the chronicity of the allergic response notably through the activation of innate immune pathways. All these allergenic proteases are expressed in mite as inactive precursor form. Until recently, the exact mechanisms of their maturation into active proteases remained to be fully elucidated. Recent breakthroughs in the understanding of the activation mechanisms of mite allergenic protease precursors have highlighted an uncommon and unique maturation pathway orchestrated by group 1 proteases that tightly regulates the proteolytic activities of groups 1, 3, 6, and 9 through complex intra- or inter-molecular mechanisms. This review presents and discusses the currently available knowledge of the activation mechanisms of group 1, 3, 6, and 9 allergens of Dermatophagoides pteronyssinus laying special emphasis on their localization, regulation, and interconnection.

Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus.

BACKGROUND: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade. METHODS: The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfer method. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanism by mite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus. RESULTS: All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite. CONCLUSIONS: Der p 1 in either its recombinant form or in the natural context of house dust mite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases. GENERAL SIGNIFICANCE: This finding suggests that Der p 1 may be valuable target against mites.

The proline-rich motif of the proDer p 3 allergen propeptide is crucial for protease-protease interaction.

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.