Biotin Homeostasis and Human Disorders: Recent Findings and Perspectives

Biotin (vitamin B7, or vitamin H) is a water-soluble B-vitamin that functions as a cofactor for carboxylases, i.e., enzymes involved in the cellular metabolism of fatty acids and amino acids and in gluconeogenesis; moreover, as reported, biotin may be involved in gene regulation. Biotin is not synthesized by human cells, but it is found in food and is also produced by intestinal bacteria. Biotin status/homeostasis in human individuals depends on several factors, including efficiency/deficiency of the enzymes involved in biotin recycling within the human organism (biotinidase, holocarboxylase synthetase), and/or effectiveness of intestinal uptake, which is mainly accomplished through the sodium-dependent multivitamin transporter. In the last years, administration of biotin at high/“pharmacological” doses has been proposed to treat specific defects/deficiencies and human disorders, exhibiting mainly neurological and/or dermatological symptoms and including biotinidase deficiency, holocarboxylase synthetase deficiency, and biotin–thiamine-responsive basal ganglia disease. On the other hand, according to warnings of the Food and Drug Administration, USA, high biotin levels can affect clinical biotin-(strept)avidin assays and thus lead to false results during quantification of critical biomarkers. In this review article, recent findings/advancements that may offer new insight in the abovementioned research fields concerning biotin will be presented and briefly discussed

Study of prothymosin α using specific radiolabeled derivatives of the polypeptide in in vitro and in vivo assays

Prothymosin alpha (ProTα) is a conserved mammalian polypeptide (human sequence 109amino acid-long) exerting a pleiotropic immunoenhancing activity. The N-terminal fragment[1-28] of the molecule, which is identical with the immunostimulating peptide thymosin α1(Tα1), was earlier considered as the immunoactive region of the polypeptide; however,recent data suggest that ProTα may exert a discrete immunomodulating action through itsC-terminal decapeptide [100-109] or its central region [51-89], via targeting Toll-likereceptor- 4 (TLR4). In the frame of this work, a derivative of the C-terminal fragmentProTα[100-109] (ProTα-D1) that can be radiolabeled with 99mTc was developed. Thebiological activity of the non-radioactive 185/187rhenium-complex of this derivative([Re]ProTα-D1, structurally similar with [99mTc]ProTα-D1) was evaluated in suitable in vitrobioassays and in assays correlated with TLR4 surface expression using humanneutrophils, in comparison with intact ProTα, various synthetic ProTα fragments/derivatives and negative control peptides. Subsequent cell-binding studies revealed specific, time-dependent and saturable binding of [99mTc]ProTα-D1 on neutrophils, which was inhibited by intact ProTα and the synthetic ProTα[100-109], as well as by a “prototype” TLR4-ligand (lipopolysaccharide from Escherichia coli, LPS), but not by synthetic Τα1, the central ProTα region and negative control peptides. Our results support the existence of ProTα-binding sites on human neutrophils, recognizing the C-terminal region [100-109], which might involve TLR4. Due to the properties of the radioisotope used for the labeling, [99mTc]ProTα-D1 was further evaluated in in vivo biodistribution and whole body imaging studies in mice bearing an inflammation model. These studies provided important information related to the pharmacokinetic profile of [99mTc]ProTα-D1 but above all revealed the capacity of [99mTc]ProTα-D1 to selectively target inflammatory loci in vivo.The accumulation of [99mTc]ProTα-D1 in inflammatory sites is specific and due to thepresence of the C-terminal decapeptide ProTα[100-109]. Overall, the present thesissignificantly contributed to the enrichment of our basic knowledge on the extracellularmode of action of ProTα, both in vitro and in vivo. Additionally, such data will facilitatefuture exploitation of the polypeptide or a smaller active fragment thereof, such as ProTα[100-109], in the clinical setting.Η Προθυμοσίνη α (ΠροΤα) αποτελεί ένα εξελικτικά συντηρημένο πολυπεπτίδιο των θηλαστικών (μήκους 109 αμινοξέων στον άνθρωπο), το οποίο εμφανίζει πλειοτροπική ανοσοενισχυτική δράση. Το αμινο(Ν)-τελικό θραύσμα [1-28] του μορίου, το οποίο ταυτίζεται με το ανοσοενισχυτικό πεπτίδιο Θυμοσίνη α1 (Τα1) θεωρήθηκε αρχικά ως η ανοσοδραστική περιοχή του πολυπεπτιδίου. Πρόσφατα όμως δεδομένα υποστηρίζουν ότι η ΠροΤα ασκεί μια διακριτή ανοσοεπαγωγική δράση μέσω του καρβοξυ(C)-τελικού της θραύσματος [100-109] ή της κεντρικής περιοχής [51-89], στοχεύοντας τον υποδοχέα τύπου Toll 4 (Toll Like Receptor 4, TLR4). Στα πλαίσια της παρούσας διατριβής,αναπτύχθηκε ένα εξειδικευμένο παράγωγο της C-τελικής περιοχής ΠροΤα [100-109](ΠροΤα-D1), ικανό να ραδιοεπισημανθεί με τεχνήτιο-99m (99m Tc). Η ανοσοδραστικότητα του συμπλόκου του νέου παραγώγου με μη ραδιενεργό ρήνιο (185/187Re) ([185/187Re]ΠροΤαD1),δομικά όμοιο με το [99m Tc]ΠροΤα-D1, αξιολογήθηκε in vitro σε κατάλληλη ανοσοδοκιμασία και σε δοκιμασία κινητικής μελέτης της επιφανειακής έκφρασης του υποδοχέα TLR4 σε ανθρώπινα ουδετερόφιλα, συγκριτικά πάντα με την αντίστοιχη δραστικότητα του ακέραιου μορίου της ΠροΤα, διαφόρων πεπτιδικών ΠροΤα-παραγώγων αλλά και κατάλληλων αρνητικών μαρτύρων. Μελέτες κυτταρικής δέσμευσης που ακολούθησαν αποκάλυψαν εξειδικευμένη πρόσδεση του παραγώγου [99m Tc]ΠροΤα-D1 σε ανθρώπινα ουδετερόφιλα, η οποία είναι χρονο-εξαρτώμενη, υπόκειται σε κορεσμό και αναστέλλεται από την παρουσία του ακέραιου μορίου της ΠροΤα, του θραύσματος ΠροΤα[100-109] και του λιποπολυσακχαρίτη LPS, αλλά όχι από την Τα1, το κεντρικό θραύσμα ΠροΤα[51-89] και τους αρνητικούς μάρτυρες. Τα αποτελέσματα αυτά υποστηρίζουν την ύπαρξη ειδικών θέσεων πρόσδεσης της ΠροΤα σε ανθρώπινα ουδετερόφιλα, οι οποίες αναγνωρίζουν την C-τελική περιοχή ΠροΤα[100-109] και πιθανότατα εμπλέκουν τον υποδοχέα TLR4. Χάρη στις ιδιότητες του ραδιοϊσοτόπου που χρησιμοποιήθηκε για την επισήμανση του παραγώγου, το σύμπλοκο [99m Tc]ΠροΤα-D1 αξιολογήθηκε περαιτέρω σε in vivo μελέτες βιοκατανομής και απεικόνισης σε μοντέλο φλεγμονής σε ποντίκια. Οι μελέτες αυτές έδωσαν σημαντικές πληροφορίες φαρμακοκινητικής φύσεως αλλά κυρίως κατέδειξαν την ικανότητα του [99m Tc]ΠροΤα-D1 για εκλεκτική στόχευση εστιών φλεγμονής. Η στρατολόγηση του [99m Tc]ΠροΤα-D1 σε τόπους φλεγμονής, με βάση και τα αποτελέσματα κατάλληλου αρνητικού μάρτυρα, είναι ειδική και οφείλεται στην παρουσία της C-τελικής περιοχής ΠροΤα[100-109]. Συνολικά, η παρούσα διατριβή συνέβαλλε στη συλλογή σημαντικών πληροφοριών για την αποσαφήνιση του εξωκυτταρικού ρόλου της ΠροΤα, οι οποίες θα διευκολύνουν την πιθανή μελλοντική αξιοποίηση του πολυπεπτιδίου ή και του μικρότερου C-τελικού δεκαπεπτιδίου ΠροΤα[100-109] στην κλινική πράξη

Peptide-Based Vaccines for Neurodegenerative Diseases: Recent Endeavors and Future Perspectives

The development of peptide-based vaccines for treating human neurodegenerative diseases has been the eventual aim of many research endeavors, although no active immunotherapies have been approved for clinical use till now. A typical example of such endeavors is the effort to develop vaccines for Alzheimer’s disease based on the beta-amyloid peptide, which continues to be intensively investigated despite previous setbacks. In this paper, recent developments in peptide-based vaccines which target beta-amyloid as well as tau protein and α-synuclein are presented. Particular focus has been directed toward peptide epitopes and formulation systems selected/developed and employed to enhance vaccine efficacy and safety. Results from both, human clinical trials and animal preclinical studies conducted mainly in transgenic mice have been included. Future perspectives on the topic are also briefly discussed

Fast and Accurate Determination of Minute Ochratoxin A Levels in Cereal Flours and Wine with the Label-Free White Light Reflectance Spectroscopy Biosensing Platform

Ochratoxin A (OTA) is one of the most toxic naturally encountered contaminants and is found in a variety of foods and beverages, including cereals and wine. Driven by the strict regulations regarding the maximum allowable OTA concentration in foodstuff and the necessity for on-site determination, the development of fast and sensitive methods for the OTA determination in cereal flours and wine samples, based on white light reflectance spectroscopy, is presented. The method relied on appropriately engineered silicon chips, on top of which an OTA-protein conjugate was immobilized. A polyclonal antibody against OTA was then employed to detect the analyte in the framework of a competitive immunoassay; followed by the subsequent addition of a biotinylated secondary antibody and streptavidin for signal enhancement. A small size instrument performed all assay steps automatically and the bioreactions were monitored in real time as the software converted the spectral shifts into effective biomolecular adlayer thickness increase. The assay developed had a detection limit of 0.03 ng/mL and a working range up to 200 ng/mL. The assay lasted 25 min (less than 1h, including calibrators/antibody pre-incubation) and was accomplished following a simple sample preparation protocol. The method was applied to corn and wheat flour samples and white and red wines with recovery values ranging from 87.2 to 111%. The simplicity of the overall assay protocol and convenient instrumentation demonstrates the potential of the immunosensor developed for OTA detection at the point of need

Antitumor reactive T-cell responses are enhanced in vivo by DAMP prothymosin alpha and Its C-terminal decapeptide

Prothymosin α (proTα) and its C-terminal decapeptide proTα(100–109) were shown to pleiotropically enhance innate and adaptive immune responses. Their activities have been broadly studied in vitro, focusing primarily on the restoration of the deficient immunoreactivity of cancer patients’ leukocytes. Previously, we showed that proTα and proTα(100–109) act as danger-associated molecular patterns (DAMPs), ligate Toll-like receptor-4, signal through TRIF-and MyD88-dependent pathways, promote the maturation of dendritic cells and elicit T-helper type 1 (Th1) immune responses in vitro, leading to the optimal priming of tumor antigen-reactive T-cell functions. Herein, we assessed their activity in a preclinical melanoma model. Immunocompetent mice bearing B16.F1 tumors were treated with two cycles of proTα or proTα(100–109) together with a B16.F1-derived peptide vaccine. Coadministration of proTα or proTα(100–109) and the peptide vaccine suppressed melanoma-cell proliferation, as evidenced by reduced tumor-growth rates. Higher melanoma infiltration by CD3+ T cells was observed, whereas ex vivo analysis of mouse total spleen cells verified the in vivo induction of melanoma-reactive cytotoxic responses. Additionally, increased levels of proinflammatory and Th1-type cytokines were detected in mouse serum. We propose that, in the presence of tumor antigens, DAMPs proTα and proTα(100–109) induce Th1-biased immune responses in vivo. Their adjuvant ability to orchestrate antitumor immunoreactivities can eventually be exploited therapeutically in humans. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Antitumor reactive T-cell responses are enhanced in vivo by DAMP prothymosin alpha and its C-terminal decapeptide

Prothymosin α (proTα) and its C-terminal decapeptide proTα(100–109) were shown to pleiotropically enhance innate and adaptive immune responses. Their activities have been broadly studied in vitro, focusing primarily on the restoration of the deficient immunoreactivity of cancer patients’ leukocytes. Previously, we showed that proTα and proTα(100–109) act as danger-associated molecular patterns (DAMPs), ligate Toll-like receptor-4, signal through TRIF- and MyD88-dependent pathways, promote the maturation of dendritic cells and elicit T-helper type 1 (Th1) immune responses in vitro, leading to the optimal priming of tumor antigen-reactive T-cell functions. Herein, we assessed their activity in a preclinical melanoma model. Immunocompetent mice bearing B16.F1 tumors were treated with two cycles of proTα or proTα(100–109) together with a B16.F1-derived peptide vaccine. Coadministration of proTα or proTα(100–109) and the peptide vaccine suppressed melanoma-cell proliferation, as evidenced by reduced tumor-growth rates. Higher melanoma infiltration by CD3+ T cells was observed, whereas ex vivo analysis of mouse total spleen cells verified the in vivo induction of melanoma-reactive cytotoxic responses. Additionally, increased levels of proinflammatory and Th1-type cytokines were detected in mouse serum. We propose that, in the presence of tumor antigens, DAMPs proTα and proTα(100–109) induce Th1-biased immune responses in vivo. Their adjuvant ability to orchestrate antitumor immunoreactivities can eventually be exploited therapeutically in humans

Prothymosin α and Its C-Terminal Immunoreactive Decapeptide Show no Evidence of Acute Toxicity: A Preliminary in Silico, in Vitro and in Vivo Investigation.

BACKGROUND: Members of the α-thymosin family have long been studied for their immunostimulating properties. Among them, the danger-associated molecular patterns (DAMPs) prothymosin α (proTα) and its C-terminal decapeptide proTα(100-109) have been shown to act as immunomodulators in vitro, due to their ability to promote T helper type 1 (Th1) responses. Recently, we verified these findings in vivo, showing that both proTα and proTα(100-109) enhance antitumor-reactive T cell-mediated responses. METHODS: In view of the eventual use of proTα and proTα(100-109) in humans, we investigated their safety profile in silico, in human leukocytes and cancer cells lines in vitro, and in immunocompetent mice in vivo, in comparison to the proTα derivative thymosin alpha 1 (Τα1), a 28-mer peptide extensively studied for its safety in clinical trials. RESULTS: In silico prediction via computational tools showed that all three peptide sequences likely are non-toxic or do not induce allergic regions. In vitro, proTα, proTα(100-109) and Tα1 did not affect the viability of human cancer cell lines and healthy donor-derived leukocytes, did not promote apoptosis or alter cell cycle distribution. Furthermore, mice injected with proTα, proTα(100-109) and Tα1 at doses equivalent to the suggested dose regimen of Tα1 in humans, did not show signs of acute toxicity, whereas proTα and proTα(100-109) increased the levels of proinflammatory and Th1-type cytokines in their peripheral blood. CONCLUSION: Our preliminary findings suggest that proTα and proTα(100-109), even at high concentrations, are non-toxic in vitro and in an acute toxicity model in vivo; moreover, we show that the two peptides retain their immunomodulatory properties in vivo and, eventually, could be considered for therapeutic use in humans