Plant-Made Bet v 1 for Molecular Diagnosis

Allergic disease diagnosis is currently experiencing a breakthrough due to the use of allergenic molecules in serum-based assays rather than allergen extracts in skin tests. The former methodology is considered a very innovative technology compared with the latter, since it is characterized by flexibility and adaptability to the patient’s clinical history and to microtechnology, allowing multiplex analysis. Molecular-based analysis requires pure allergens to detect IgE sensitization, and a major goal, to maintain the diagnosis cost-effective, is to limit their production costs. In addition, for the production of recombinant eukaryotic proteins similar to natural ones, plant-based protein production is preferred to bacterial-based systems due to its ability to perform most of the post-translational modifications of eukaryotic molecules. In this framework, Plant Molecular Farming (PMF) may be useful, being a production platform able to produce complex recombinant proteins in short time-frames at low cost. As a proof of concept, PMF has been exploited for the production of Bet v 1a, a major allergen associated with birch (Betula verrucosa) pollen allergy. Bet v 1a has been produced using two different transient expression systems in Nicotiana benthamiana plants, purified and used in a new generation multiplex allergy diagnosis system, the patient-Friendly Allergen nano-BEad Array (FABER). Plant-made Bet v 1a is immunoreactive, binding IgE and inhibiting IgE-binding to the Escherichia coli expressed allergen currently available in the FABER test, thus suggesting an overall similar though non-overlapping immune activity compared with the E. coli expressed form

Nutritional status and the risk of malnutrition in older adults with chronic kidney disease – implications for low protein intake and nutritional care: A critical review endorsed by ERN-ERA and ESPEN

Increased life expectancy is posing unprecedented challenges to healthcare systems worldwide. These include a sharp increase in the prevalence of chronic kidney disease (CKD) and of impaired nutritional status with malnutrition-protein-energy wasting (PEW) that portends worse clinical outcomes, including reduced survival. In older adults with CKD, a nutritional dilemma occurs when indications from geriatric nutritional guidelines to maintain the protein intake above 1.0 g/kg/day to prevent malnutrition need to be adapted to the indications from nephrology guidelines, to reduce protein intake in order to prevent or slow CKD progression and improve metabolic abnormalities. To address these issues, the European Society for Clinical Nutrition and Metabolism (ESPEN) and the European Renal Nutrition group of the European Renal Association (ERN-ERA) have prepared this conjoint critical review paper, whose objective is to summarize key concepts related to prevention and treatment of both CKD progression and impaired nutritional status using dietary approaches, and to provide guidance on how to define optimal protein and energy intake in older adults with differing severity of CKD. Overall, the authors support careful assessment to identify the most urgent clinical challenge and the consequent treatment priority. The presence of malnutrition-protein-energy wasting (PEW) suggests the need to avoid or postpone protein restriction, particularly in the presence of stable kidney function and considering the patient's preferences and quality of life. CKD progression and advanced CKD stage support prioritization of protein restriction in the presence of a good nutritional status. Individual risk-benefit assessment and appropriate nutritional monitoring should guide the decision-making process. Higher awareness of the challenges of nutritional care in older adult patients with CKD is needed to improve care and outcomes. Research is advocated to support evidence-based recommendations, which we still lack for this increasingly large patient subgroup

Biometal Dyshomeostasis in Olfactory Mucosa of Alzheimer's Disease Patients

Olfactory function, orchestrated by the cells of the olfactory mucosa at the rooftop of the nasal cavity, is disturbed early in the pathogenesis of Alzheimer's disease (AD). Biometals including zinc and calcium are known to be important for sense of smell and to be altered in the brains of AD patients. Little is known about elemental homeostasis in the AD patient olfactory mucosa. Here we aimed to assess whether the disease-related alterations to biometal homeostasis observed in the brain are also reflected in the olfactory mucosa. We applied RNA sequencing to discover gene expression changes related to metals in olfactory mucosal cells of cognitively healthy controls, individuals with mild cognitive impairment and AD patients, and performed analysis of the elemental content to determine metal levels. Results demonstrate that the levels of zinc, calcium and sodium are increased in the AD olfactory mucosa concomitantly with alterations to 17 genes related to metal-ion binding or metal-related function of the protein product. A significant elevation in alpha-2-macroglobulin, a known metal-binding biomarker correlated with brain disease burden, was observed on the gene and protein levels in the olfactory mucosa cells of AD patients. These data demonstrate that the olfactory mucosa cells derived from AD patients recapitulate certain impairments of biometal homeostasis observed in the brains of patients.Peer reviewe

Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco

Background: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10. Results: Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T1 plants were 10.8 and 37.0 \u3bcg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells. Conclusion: Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein

Single-Cell RNA-Seq Analysis of Olfactory Mucosal Cells of Alzheimer's Disease Patients

Olfaction is orchestrated by olfactory mucosal cells located in the upper nasal cavity. Olfactory dysfunction manifests early in several neurodegenerative disorders including Alzheimer's disease, however, disease-related alterations to the olfactory mucosal cells remain poorly described. The aim of this study was to evaluate the olfactory mucosa differences between cognitively healthy individuals and Alzheimer's disease patients. We report increased amyloid-beta secretion in Alzheimer's disease olfactory mucosal cells and detail cell-type-specific gene expression patterns, unveiling 240 differentially expressed disease-associated genes compared to the cognitively healthy controls, and five distinct cell populations. Overall, alterations of RNA and protein metabolism, inflammatory processes, and signal transduction were observed in multiple cell populations, suggesting their role in Alzheimer's disease-related olfactory mucosa pathophysiology. Furthermore, the single-cell RNA-sequencing proposed alterations in gene expression of mitochondrially located genes in AD OM cells, which were verified by functional assays, demonstrating altered mitochondrial respiration and a reduction of ATP production. Our results reveal disease-related changes of olfactory mucosal cells in Alzheimer's disease and demonstrate the utility of single-cell RNA sequencing data for investigating molecular and cellular mechanisms associated with the disease.Peer reviewe

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs

Effects of chronic inflammatory bowel diseases on left ventricular structure and function: a study protocol

BACKGROUND: Experimental evidences suggest an increased collagen deposition in inflammatory bowel diseases (IBD). In particular, large amounts of collagen type I, III and V have been described and correlated to the development of intestinal fibrotic lesions. No information has been available until now about the possible increased collagen deposition far from the main target organ. In the hypothesis that chronic inflammation and increased collagen metabolism are reflected also in the systemic circulation, we aimed this study to evaluate the effects on left ventricular wall structure by assessing splancnic and systemic collagen metabolism (procollagen III assay), deposition (ultrasonic tissue characterization), and cardiac function (echocardiography) in patients with different long standing history of IBD, before and after surgery. METHODS: Thirty patients affected by active IBD, 15 with Crohn and 15 with Ulcerative Colitis, submitted to surgery will be enrolled in the study in a double blind fashion. They will be studied before the surgical operation and 6, 12 months after surgery. A control group of 15 healthy age and gender-matched subjects will also be studied. At each interval blood samples will be collected in order to assess the collagen metabolism; a transthoracic echocardiogram will be recorded for the subsequent determination of cardiac function and collagen deposition. DISCUSSION: From this study protocol we expect additional information about the association between IBD and cardiovascular disorders; in particular to address the question if chronic inflammation, through the altered collagen metabolism, could affect left ventricular structure and function in a manner directly related to the estimated duration of the disease

Elevated serum procollagen type III peptide in splanchnic and peripheral circulation of patients with inflammatory bowel disease submitted to surgery

BACKGROUND: In the hypothesis that the increased collagen metabolism in the intestinal wall of patients affected by inflammatory bowel disease (IBD) is reflected in the systemic circulation, we aimed the study to evaluate serum level of procollagen III peptide (PIIIP) in peripheral and splanchnic circulation by a commercial radioimmunoassay of patients with different histories of disease. METHODS: Twenty-seven patients, 17 with Crohn and 10 with ulcerative colitis submitted to surgery were studied. Blood samples were obtained before surgery from a peripheral vein and during surgery from the mesenteric vein draining the affected intestinal segment. Fifteen healthy age and sex matched subjects were studied to determine normal range for peripheral PIIIP. RESULTS: In IBD patients peripheral PIIIP level was significantly higher if compared with controls (5.0 ± 1.9 vs 2.7 ± 0.7 μg/l; p = 0.0001); splanchnic PIIIP level was 5.5 ± 2.6 μg/l showing a positive gradient between splanchnic and peripheral concentrations of PIIIP. No significant differences between groups nor correlations with patients' age and duration of disease were found. CONCLUSIONS: We provide evidence that the increased local collagen metabolism in active IBD is reflected also in the systemic circulation irrespective of the history of the disease, suggesting that PIIIP should be considered more appropiately as a marker of the activity phases of IBD