Prothymosin a and a prothymosin α-derived peptide enhance TH1-type immune responses against defined HER-2/neu epitopes

Background: Active cancer immunotherapies are beginning to yield clinical benefit, especially those using peptide-pulsed dendritic cells (DCs). Different adjuvants, including Toll-like receptor (TLR) agonists, commonly co-administered to cancer patients as part of a DC-based vaccine, are being widely tested in the clinical setting. However, endogenous DCs in tumor-bearing individuals are often dysfunctional, suggesting that ex vivo educated DCs might be superior inducers of anti-tumor immune responses. We have previously shown that prothymosin alpha (proTα) and its immunoreactive decapeptide proTα(100–109) induce the maturation of human DCs in vitro. The aim of this study was to investigate whether proTα- or proTα(100–109)-matured DCs are functionally competent and to provide preliminary evidence for the mode of action of these agents. Results: Monocyte-derived DCs matured in vitro with proTα or proTα(100–109) express co-stimulatory molecules and secrete pro-inflammatory cytokines. ProTα- and proTα(100–109)-matured DCs pulsed with HER-2/neu peptides induce TH1-type immune responses, prime autologous naïve CD8-positive (+) T cells to lyse targets expressing the HER-2/neu epitopes and to express a polyfunctional profile, and stimulate CD4+ T cell proliferation in an HER-2/neu peptide-dependent manner. DC maturation induced by proTα and proTα(100–109) is likely mediated via TLR-4, as 25 shown by assessing TLR-4 surface expression and the levels of the intracellular adaptor molecules TIRAP, MyD88 and TRIF. Conclusions: Our results suggest that proTα and proTα(100–109) induce both the maturation and the T cell stimulatory capacity of DCs. Although further studies are needed, evidence for a possible proTα and proTα(100–109) interaction with TLR-4 is provided. The initial hypothesis that proTα and the proTα-derived immunoactive decapeptide act as “alarmins”, provides a rationale for their eventual use as adjuvants in DC-based anti-cancer immunotherapy

Prothymosin alpha: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy

The thymus is a central lymphoid organ with crucial role in generating T cells and maintaining homeostasis of the immune system. More than 30 peptides, initially referred to as “thymic hormones,” are produced by this gland. Although the majority of them have not been proven to be thymus-speciWc, thymic peptides comprise an eVective group of regulators, mediating important immune functions. Thymosin fraction Wve (TFV) was the Wrst thymic extract shown to stimulate lymphocyte proliferation and diVerentiation. Subsequent fractionation of TFV led to the isolation and characterization of a series of immunoactive peptides/polypeptides, members of the thymosin family. Extensive research on prothymosin (proT) and thymosin 1 (T1) showed that they are of clinical signiWcance and potential medical use. They may serve as molecular markers for cancer prognosis and/or as therapeutic agents for treating immunodeWciencies, autoimmune diseases and malignancies. Although the molecular mechanisms underlying their eVect are yet not fully elucidated proT and T1 could be considered as candidates for cancer immunotherapy. In this review, we will focus in principle on the eventual clinical utility of proT, both as a tumor biomarker and in triggering anticancer immune responses. Considering the experience acquired via the use of T1 to treat cancer patients, we will also discuss potential approaches for the future introduction of proT into the clinical setting

Mechanisms of leukocyte activation by immunoenhancing thymic peptides: development of an assay for the evaluation of the activity of the peptides

The aim of this thesis was to investigate the effect of proTα(100-109) and intact prothymosin (proTα) on basic cell functions of innate immunity cells expressing TLR-4 and to develop/evaluate an immunoassay to quantify proTα(100-109) extracellularly, in order to elucidate its mode of action. The activation of neutrophils and monocytes with proTα(100-109) or proTα enhanced their basic functions (phagocytosis, oxidative burst and cytotoxicity) and restored their suppressed functions in breast cancer patients to the levels of healthy individuals. Furthermore, using polyclonal antibodies, we developed a competitive ELISA for proTα(100-109) and determined its concentration in vitro, in supernatants of cells led to cell death, in vivo in the serum of mice infected with bacteria and ex vivo in human sera. Specifically, the levels of proTα(100-109) were higher in apoptotic supernatants compared to low concentrations in the corresponding controls. BALB/c and C57BL/6 mice infected in vivo with five bacterial strains showed differences in the concentration of proTα(100-109) depending on the strain, breeding conditions and the type of pathogen. In general, the levels of proTα(100-109) before infection were low and gradually increased during the progress of infection. Using MALDI-TOF mass spectrometry, we detected a peak corresponding to the molecular mass of the decapeptide only in sera of infected animals. In an aggressive model of lethal sepsis in ICR mice, using Klebsiella pneumoniae that is endocytosed, we observed a gradual increase in the concentration of proTα(100-109), with maximal levels determined at 48 hours postinfection. In animals infected with a standard strain of Klebsiella, we observed variations and activation mainly of necrotic death pathways, as the bacterium was not endocytosed. Finally, we determined the levels of proTα(100-109) in sera of healthy individuals, pediatric patients and patients hospitalized in ICU. ProTα(100-109) levels in healthy subjects were very low, whereas in infected children, we detected higher concentrations of the decapeptide compared to children with autoinflammatory diseases, where the concentration of proTα(100-109) was low. ICU patients’ samples serially collected from admission to exit, showed variable proTα(100-109) concentrations during the hospitalization of patients who developed bacterial infections, which were correlated with clinical symptoms and laboratory markers. In contrast, the concentration of proTα(100-109) was minimal in ICU patients who did not develop an infection. The results of this thesis suggest that proTα(100-109) acts as an adjuvant on neutrophils and monocytes and, eventually could be used as an immunotherapeutic molecule to enhance immune system functions in cancer patients. Moreover, the same molecule could be used as a diagnostic marker for the differential diagnosis between septic and aseptic inflammation, providing that analysis of a larger number of samples from infected patients confirms our results.Σκοπός της διατριβής ήταν η διερεύνηση της επίδρασης του προΤα(100-109) και της ακέραιης προθυμοσίνης α (προΤα) σε βασικές λειτουργίες κυττάρων της φυσικής ανοσίας που εκφράζουν TLR-4 και η ανάπτυξη/αξιολόγηση ανοσοδοκιμασίας, η οποία να ποσοτικοποιεί το προΤα(100-109) εξωκυτταρικά, ώστε να διαλευκανθεί ο τρόπος δράσης του. Η ενεργοποίηση ουδετερόφιλων και μονοκυττάρων με προΤα(100-109) ή προΤα ενίσχυσε τις βασικές τους λειτουργίες (φαγοκυττάρωση, οξειδωτική έκρηξη και κυτταροτοξική δράση) και επανέφερε τις κατασταλμένες λειτουργίες τους σε ασθενείς με καρκίνο του μαστού στα φυσιολογικά επίπεδα των υγιών. Παράλληλα, χρησιμοποιώντας πολυκλωνικά αντισώματα, αναπτύξαμε ανταγωνιστική ELISA για το προΤα(100-109), με την οποία προσδιορίστηκε η συγκέντρωση του in vitro, σε υπερκείμενα κυττάρων που οδηγήθηκαν σε κυτταρικό θάνατο, in vivo στον ορό ποντικών μολυσμένων με βακτήρια και ex vivo σε ανθρώπινους ορούς. Ειδικότερα, τα επίπεδα του προΤα(100-109) ήταν αυξημένα σε υπερκείμενα κυττάρων που οδηγήθηκαν in vitro σε απόπτωση, ενώ στα αντίστοιχα υπερκείμενα-μάρτυρες η συγκέντρωσή του ήταν χαμηλή. Σε BALB/c και C57BL/6 ποντίκια, τα οποία μολύνθηκαν in vivo με πέντε βακτηριακά στελέχη, παρατηρήσαμε διαφορές ως προς τη συγκέντρωση του προΤα(100-109) στον ορό ανάλογα με τη φυλή, τις συνθήκες διαβίωσης και το είδος του παθογόνου. Γενικά, τα επίπεδα του προΤα(100-109) πριν τη μόλυνση ήταν χαμηλά και αυξήθηκαν σταδιακά κατά την πρόοδο της λοίμωξης. Με MALDI-TOF φασματομετρία μάζας, ανιχνεύθηκε μικρή κορυφή που αντιστοιχούσε στη μοριακή μάζα του δεκαπεπτιδίου μόνο στους ορούς των μολυσμένων ζώων. Σε επιθετικό μοντέλο θανατηφόρου σηψαιμίας σε ICR ποντίκια με Klebsiella pneumoniae που ενδοκυτταρώνεται, παρατηρήθηκε σταδιακή αύξηση της συγκέντρωσης του προΤα(100-109), με τη μέγιστη ποσότητά του να ανιχνεύεται 48 ώρες μετά τη λοίμωξη. Σε ζώα που μολύνθηκαν με πρότυπο στέλεχος Klebsiella, παρατηρήσαμε αυξομειώσεις και ενεργοποίηση κυρίως νεκρωτικών μονοπατιών θανάτου, λόγω της μη ενδοκυττάρωσης του βακτηρίου. Τέλος, μετρήθηκαν τα επίπεδα του προΤα(100-109) σε ορούς υγιών ατόμων, παιδιατρικών ασθενών και ασθενών της ΜΕΘ. Τα επίπεδα του προΤα(100-109) σε υγιή άτομα ήταν ιδιαίτερα χαμηλά, ενώ στα παιδιά με λοιμώξεις ανιχνεύθηκαν υψηλότερες συγκεντρώσεις δεκαπεπτιδίου σε σχέση με τα παιδιά με αυτοφλεγμονώδη νοσήματα, όπου η συγκέντρωση ήταν χαμηλή. Στα δείγματα της ΜΕΘ, τα οποία ελήφθησαν σειριακά από τη στιγμή της εισαγωγής του ασθενούς μέχρι την έξοδό του, καταγράφηκαν διακυμάνσεις στη συγκέντρωση του προΤα(100-109) κατά την πορεία νοσηλείας ασθενών που εμφάνισαν βακτηριακές λοιμώξεις, οι οποίες συσχετίζονται με την αντίστοιχη κλινική εικόνα και τους εργαστηριακούς δείκτες. Αντίθετα, ελάχιστη ήταν η συγκέντρωσή του σε περιστατικά που νοσηλεύθηκαν στη ΜΕΘ χωρίς σημεία λοίμωξης. Όπως φαίνεται από τα αποτελέσματα της παρούσας διατριβής, το προΤα(100-109) δρα ανοσοενισχυτικά σε ουδετερόφιλα και μονοκύτταρα και, ίσως, μελλοντικά θα μπορούσε να χρησιμοποιηθεί ως ανοσοθεραπευτικό μόριο για την ενίσχυση των λειτουργιών του ανοσοποιητικού συστήματος καρκινοπαθών. Παράλληλα, το ίδιο μόριο θα μπορούσε να χρησιμοποιηθεί ως διαγνωστικό εργαλείο διαφοροδιάγνωσης μεταξύ σηπτικής και άσηπτης φλεγμονής, εφόσον αυτό επιβεβαιωθεί με ανάλυση μεγάλου αριθμού δειγμάτων ασθενών με λοιμώξεις

Decoding the impact of autoinflammatory/autoimmune diseases on inner ear harmony and hearing loss

Autoimmune and autoinflammatory diseases affecting the inner ear can cause symptoms such as hearing loss, imbalance, vertigo, and tinnitus, presenting demanding and often underdiagnosed conditions. Diagnostic challenges arise due to their diverse manifestations, potential long-term consequences, and the absence of specific serological markers, necessitating a multidisciplinary approach combining clinical evaluation, audiological assessments, and imaging techniques. Various autoimmune disorders, including systemic lupus erythematosus, rheumatoid arthritis, and Sjogren’s syndrome, have been implicated in immune-mediated damage to auditory structures, resulting in inner ear dysfunction. Inflammatory processes in autoinflammatory diseases like Cogan’s syndrome and relapsing polychondritis can also affect the inner ear. While the exact mechanisms of inner ear involvement in these conditions are still being studied, immune-mediated inflammation, damage to auditory structures, and vascular involvement play significant roles in auditory impairments. Treatment strategies primarily focus on immunomodulation and inflammation control using corticosteroids, immunosuppressants, and targeted biologic agents to ameliorate symptoms and preserve hearing function. Hearing aids and cochlear implants may be also considered for severe hearing loss. Individualized approaches are necessary due to patient response heterogeneity. This review provides a concise overview of key autoimmune and autoinflammatory diseases impacting the inner ear, highlighting clinical manifestations, diagnostics, pathophysiology, and treatment options. Early recognition and appropriate management are crucial for optimizing patient outcomes. Further research is needed to understand underlying mechanisms and identify novel therapeutic targets. Collaboration between otolaryngologists, rheumatologists, and immunologists is crucial for improving the quality of life in these complex conditions

Antiproliferative Activity of (-)-Rabdosiin Isolated from Ocimum sanctum L.

Background: Ocimum sanctum L. (holy basil; Tulsi in Hindi) is an important medicinal plant, traditionally used in India. Methods: The phytochemical study of the nonpolar (dichloromethane 100%) and polar (methanol:water; 7:3) extracts yielded fourteen compounds. Compounds 6, 7, 9, 11, 12, and 13, along with the methanol:water extract were evaluated for their cytotoxicity against the human cancer cell lines MCF-7, SKBR3, and HCT-116, and normal peripheral blood mononuclear cells (PBMCs). Results: Five terpenoids, namely, ursolic acid (1), oleanolic acid (2), betulinic acid (3), stigmasterol (4), and β-caryophyllene oxide (5); two lignans, i.e., (-)-rabdosiin (6) and shimobashiric acid C (7); three flavonoids, luteolin (8), its 7-O-β-D-glucuronide (9), apigenin 7-O-β-D-glucuronide (10); and four phenolics, (E)-p-coumaroyl 4-O-β-D-glucoside (11), 3-(3,4-dihydroxyphenyl) lactic acid (12), protocatechuic acid (13), and vanillic acid (14) were isolated. Compound 6 was the most cytotoxic against the human cancer lines assessed and showed very low cytotoxicity against PBMCs. Conclusions: Based on these results, the structure of compound 6 shows some promise as a selective anticancer drug scaffold