Neuroprotective Action of Humanin and Humanin Analogues: Research Findings and Perspectives.

Humanin is a 24-mer peptide first reported in the early 2000s as a new neuroprotective/cytoprotective factor rescuing neuronal cells from death induced by various Alzheimer's disease-associated insults. Nowadays it is known that humanin belongs to the novel class of the so-called mitochondrial-derived peptides (which are encoded by mitochondrial DNA) and has been shown to exert beneficial cytoprotective effects in a series of in vitro and/or in vivo experimental models of human diseases, including not only neurodegenerative disorders but other human diseases as well (e.g., age-related macular degeneration, cardiovascular diseases, or diabetes mellitus). This review article is focused on the presentation of recent in vitro and in vivo research results associated with the neuroprotective action of humanin as well as of various, mainly synthetic, analogues of the peptide; moreover, the main mode(s)/mechanism(s) through which humanin and humanin analogues may exert in vitro and in vivo regarding neuroprotection have been reported. The prospects of humanin and humanin analogues to be further investigated in the frame of future research endeavors against neurodegenerative/neural diseases have also been briefly discussed.

Immunosensors for Autoimmune-Disease-Related Biomarkers: A Literature Review.

Immunosensors are a special class of biosensors that employ specific antibodies for biorecognition of the target analyte. Immunosensors that target disease biomarkers may be exploited as tools for disease diagnosis and/or follow-up, offering several advantages over conventional analytical techniques, such as rapid and easy analysis of patients' samples at the point-of-care. Autoimmune diseases have been increasingly prevalent worldwide in recent years, while the COVID-19 pandemic has also been associated with autoimmunity. Consequently, demand for tools enabling the early and reliable diagnosis of autoimmune diseases is expected to increase in the near future. To this end, interest in immunosensors targeting autoimmune disease biomarkers, mainly, various autoantibodies and specific pro-inflammatory proteins (e.g., specific cytokines), has been rekindled. This review article presents most of the immunosensors proposed to date as potential tools for the diagnosis of various autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis. The signal transduction and the immunoassay principles of each immunosensor have been suitably classified and are briefly presented along with certain sensor elements, e.g., special nano-sized materials used in the construction of the immunosensing surface. The main concluding remarks are presented and future perspectives of the field are also briefly discussed.

Cortisol Immunosensors: A Literature Review.

Cortisol is a steroid hormone that is involved in a broad range of physiological processes in human/animal organisms. Cortisol levels in biological samples are a valuable biomarker, e.g., of stress and stress-related diseases; thus, cortisol determination in biological fluids, such as serum, saliva and urine, is of great clinical value. Although cortisol analysis can be performed with chromatography-based analytical techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), conventional immunoassays (radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), etc.) are considered the "gold standard" analytical methodology for cortisol, due to their high sensitivity along with a series of practical advantages, such as low-cost instrumentation, an assay protocol that is fast and easy to perform, and high sample throughput. Especially in recent decades, research efforts have focused on the replacement of conventional immunoassays by cortisol immunosensors, which may offer further improvements in the field, such as real-time analysis at the point of care (e.g., continuous cortisol monitoring in sweat through wearable electrochemical sensors). In this review, most of the reported cortisol immunosensors, mainly electrochemical and also optical ones, are presented, focusing on their immunosensing/detection principles. Future prospects 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.

Recent Developments in the Field of Optical Immunosensors Focusing on a Label-Free, White Light Reflectance Spectroscopy-Based Immunosensing Platform.

Optical immunosensors represent a research field of continuously increasing interest due to their unique features, which can mainly be attributed to the high-affinity and specific antibodies they use as biorecognition elements, combined with the advantageous characteristics of the optical transducing systems these sensors employ. The present work describes new developments in the field, focusing on recent bioanalytical applications (2021-2022) of labeled and label-free optical immunosensors. Special attention is paid to a specific immunosensing platform based on White Light Reflectance Spectroscopy, in which our labs have gained specific expertise; this platform is presented in detail so as to include developments, improvements, and bioanalytical applications since the mid-2000s. Perspectives on the field are been briefly discussed as well, highlighting the potential of optical immunosensors to eventually reach the state of a reliable, highly versatile, and widely applicable analytical tool suitable for use at the Point-of-Care.

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 cell 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, pro- Tα, 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.

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.

IgY technology: Methods for developing and evaluating avian immunoglobulins for the in vitro detection of biomolecules.

The term "IgY technology" was introduced in the literature in the mid 1990s to describe a procedure involving immunization of avian species, mainly laying hens and consequent isolation of the polyclonal IgYs from the "immune" egg yolk (thus avoiding bleeding and animal stress). IgYs have been applied to various fields of medicine and biotechnology. The present article will deal with specific aspects of IgY technology, focusing on the currently reported methods for developing, isolating, evaluating and storing polyclonal IgYs. Other topics such as current information on isolation protocols or evaluation of IgYs from different avian species are also discussed. Specific advantages of IgY technology (e.g., novel antibody specificities that may emerge via the avian immune system) will also be discussed. Recent in vitro applications of polyclonal egg yolk-derived IgYs to the field of disease diagnosis in human and veterinary medicine through in vitro immunodetection of target biomolecules will be presented. Moreover, ethical aspects associated with animal well-being as well as new promising approaches that are relevant to the original IgY technology (e.g., development of monoclonal IgYs and IgY-like antibodies through the phage display technique or in transgenic chickens) and future prospects in the area will also be mentioned.

Fast and Sensitive Determination of the Fungicide Carbendazim in Fruit Juices with an Immunosensor Based on White Light Reflectance Spectroscopy.

Carbendazim is a systemic benzimidazole-type fungicide with broad-spectrum activity against fungi that undermine food products safety and quality. Despite its effectiveness, carbendazim constitutes a major environmental pollutant, being hazardous to both humans and animals. Therefore, fast and reliable determination of carbendazim levels in water, soil, and food samples is of high importance for both food industry and public health. Herein, an optical biosensor based on white light reflectance spectroscopy (WLRS) for fast and sensitive determination of carbendazim in fruit juices is presented. The transducer is a Si/SiO2 chip functionalized with a benzimidazole conjugate, and determination is based on a competitive immunoassay format. Thus, for the assay, a mixture of an in-house developed rabbit polyclonal anti-carbendazim antibody with the standards or samples is pumped over the chip, followed by biotinylated secondary antibody and streptavidin. The WLRS platform allows for real-time monitoring of biomolecular interactions carried out onto the Si/SiO2 chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. The sensor is able to detect 20 ng/mL of carbendazim in fruit juices with high accuracy and precision (intra- and inter-assay CVs ≤ 6.9% and ≤9.4%, respectively) in less than 30 min, applying a simple sample treatment that alleviates any "matrix-effect" on the assay results and a 60 min preincubation step for improving assay sensitivity. Excellent analytical characteristics and short analysis time along with its small size render the proposed WLRS immunosensor ideal for future on-the-spot determination of carbendazim in food and environmental samples.

In Vitro Immunodetection of Prothymosin Alpha in Normal and Pathological Conditions.

Prothymosin alpha (ProTα) is a highly acidic polypeptide, ubiquitously expressed in almost all mammalian cells and tissues and consisting of 109 amino acids in humans. ProTα is known to act both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similar to molecules termed as "alarmins". Antibodies and immunochemical techniques for ProTα have played a leading role in the investigation of the biological role of ProTα, several aspects of which still remain unknown and contributed to unraveling the diagnostic and therapeutic potential of the polypeptide. This review deals with the so far reported antibodies along with the related immunodetection methodology for ProTα (immunoassays as well as immunohistochemical, immunocytological, immunoblotting, and immunoprecipitation techniques) and its application to biological samples of interest (tissue extracts and sections, cells, cell lysates and cell culture supernatants, body fluids), in health and disease states. In this context, literature information is critically discussed, and some concluding remarks are presented.

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.

Development of a specific IgY-based ELISA for prothymosin alpha, a bioactive polypeptide with diagnostic and therapeutic potential.

Prothymosin alpha (ProTα) is a highly conserved polypeptide (109 amino acids in humans) with diagnostic and therapeutic potential; ProTα exerts intra- and extra-cellular biological functions associated with cell proliferation, apoptosis and immune regulation, while it has been suggested to act as a damage-associated molecular pattern (DAMP) or alarmin. In this work, chicken polyclonal anti-ProTα antibodies that had been developed several years ago were immunochemically evaluated and proven to retain immunoreactivity for ProTα, with remarkable thermal and pH stability. Moreover, the antibodies showed practically no cross-reactivity with a series of ProTα-fragments, eventually intracellularly produced -such as ProTα[1-28] (also known as Tα1) and ProTα[100-109], which exert per se biological activity and might be present in biological samples along with the intact molecule, being therefore highly specific for whole-length ProTα. Based on the above antibodies (IgYs-3e), a highly specific competitive ProTα-ELISA with well-studied analytical characteristics (intra- and inter-assay CVs: ≤5% and ≤12%, respectively, limit of detection: 2.1 ng/mL, recovery: 88-104%) was developed. The new ProTα-ELISA was applied to the analysis of supernatants of HeLa cells driven to necrosis; intact ProTα was measured in cell culture supernatants, at levels that seemed to depend on % cell necrosis.

White light reflectance spectroscopy biosensing system for fast quantitative prostate specific antigen determination in forensic samples.

A label-free biosensor based on white light reflectance spectroscopy for the determination of PSA as semen indicator in forensic samples is presented. The sensor is based on a two-step immunoassay which employs the same polyclonal anti-PSA antibody as capture and detection antibody followed by reaction with streptavidin as a signal enhancement step. The whole assay time was set to 10min; 5min reaction of immobilized antibody with the PSA calibrators or the samples, 3min reaction with the biotinylated anti-PSA antibody and 2min reaction with streptavidin. Following this protocol, a detection limit of 0.5ng/mL was achieved and the assay's linear response range extended up to 500ng/mL. Thus, taking into account the quantification limit of 1.0ng/mL and the average PSA concentration in semen (0.2-5.5mg/mL), semen quantities of a few nanoliters could be detected. The accuracy of the sensor developed was demonstrated through recovery (% recovery ranged from 89.6 to 106) and semen dilution experiments. A linear correlation was found for semen dilutions ranging from 5000 to 360,000. The lack of interference by other bodily fluids was confirmed by analysing stains of blood, urine and saliva prior to and after the addition of semen. Finally, the sensor was evaluated by analysing 51 forensic casework samples which were also analysed with a semi-quantitative membrane strip test (Seratec® PSA), through microscopic detection of spermatozoa, and male DNA identification through detection of Y chromosome. The results obtained with the sensor were in excellent agreement with those provided by an immunoradiometric assay kit (PSA-RIACT) and in complete agreement with the findings using the membrane strip assay, spermatozoa and Y chromosome detection. The excellent analytical performance and small size of the instrument make the sensor developed an attractive tool for use in forensic evidence screening for semen detection.

Prothymosin Alpha: An Alarmin and More...

BACKGROUND/OBJECTIVE: Prothymosin alpha (proTα) is a ubiquitous polypeptide first isolated by Haritos in 1984, whose role still remains partly elusive. We know that proTα acts both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similarly to molecules termed as "alarmins". Our research team pioneered the elucidation of the mechanisms underlying the observed activities of proTα. RESULTS: We were the first to demonstrate that proTα levels increase during normal and abnormal cell proliferation. We showed that proTα acts pleiotropically, inducing immunomodulatory effects on immune cell populations. We revealed that the immunoreactive region of proTα is the carboxyterminal decapeptide proTα(100-109) and both molecules stimulate innate immune responses, signaling through Toll-like receptors (TLRs), specifically TLR-4. We reported that proTα and proTα(100-109) bind on the surface of human neutrophils on sites involving TLR-4, and cell activation is complemented by cytoplasmic calcium ion influx. Further, we showed that proTα and proTα(100-109) act as adjuvants upstream of lymphocyte stimulation and, in the presence of antigen, promote the expansion of antigen-reactive effectors. Most recently, we reported that proTα(100-109) may accumulate in experimentally inflamed sites and can serve as a surrogate biomarker in severe bacterial infections, proposing that extracellular release of proTα or proTα(100- 109) alerts the immune system during conditions of danger. CONCLUSION: We, therefore, suggest that proTα, and likely proTα(100-109), act as alarmins, being important immune mediators as well as biomarkers, and could eventually become targets for new therapeutic/diagnostic approaches in immune-related diseases like cancer, inflammation, and sepsis.

In vivo biodistribution and imaging studies with a 99mTc-radiolabeled derivative of the C-terminus of prothymosin alpha in mice bearing experimentally-induced inflammation.

Prothymosin alpha (ProTα) is a highly conserved mammalian polypeptide (109 amino acids in man) exerting in vitro and in vivo immunoenhancing activities. Recently, our team has developed a 99mTc-radiolabeled derivative of the C-terminal bioactive decapeptide of ProTα ([99mTc]C1) and employed it in in vitro studies, the results of which support the existence of binding sites on human neutrophils that recognize [99mTc]C1, intact ProTα as well as the C-terminal decapeptide of ProTα and presumably involve Toll-like receptor 4. In the present work, [99mTc]C1 was administered to Swiss albino mice with experimentally-induced inflammation for in vivo biodistribution and imaging studies, in parallel with a suitable negative control, which differs from [99mTc]C1 only in bearing a scrambled version of the ProTα decapeptide. The biodistribution data obtained with [99mTc]C1 demonstrated fast clearance of radioactivity from blood, heart, lungs, normal muscle, and predominantly urinary excretion. Most importantly, slow clearance of radioactivity from the inflammation focus was observed, resulting in a high ratio of inflamed/normal muscle tissue (9.15 at 30min post injection, which remained practically stable up to 2h). The inflammation-targeting capacity of [99mTc]C1 was confirmed by imaging studies and might be attributed to neutrophils, which are recruited at the inflamed areas and bear binding sites for [99mTc]C1. In this respect, apart from being a valuable tool for further studies on ProTα in in vitro and in vivo systems, [99mTc]C1 merits further evaluation as a radiopharmaceutical for specific imaging of inflammation foci.

Specific in vitro binding of a new (99m)Tc-radiolabeled derivative of the C-terminal decapeptide of prothymosin alpha on human neutrophils.

Prothymosin alpha (ProTα) is a conserved mammalian polypeptide with intracellular functions associated with cell proliferation and apoptosis and an extracellular role associated with immunopotentiation. The N-terminal fragment [1-28], 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 its central or C-terminal region, via targeting Toll-like receptor- 4 (TLR4). In this work, a derivative of the C-terminal fragment ProTα[100-109] (ProTα-D1) that can be radiolabeled with (99m)Tc was developed. The biological activity of the non-radioactive (185/187)rhenium-complex of this derivative ([(185/187)Re]ProTα-D1, structurally similar with [(99m)Tc]ProTα-D1) was verified through suitable in vitro bioassays on human neutrophils. Subsequent cell-binding studies revealed specific, time-dependent and saturable binding of [(99m)Tc]ProTα-D1 on neutrophils, which was inhibited by intact ProTα and ProTα[100-109], as well as by a "prototype" TLR4-ligand (lipopolysaccharide from Escherichia coli). Overall, our results support the existence of ProTα-binding sites on human neutrophils, recognizing [(99m)Tc]ProTα-D1, which might involve TLR4. [(99m)Tc]ProTα-D1 may be a useful tool for conducting further in vitro and in vivo studies, aiming to elucidate the extracellular mode of action of ProTα and, eventually, develop ProTα-based immunotherapeutics.

Commercially available chemicals as immunizing haptens for the development of a polyclonal antibody recognizing carbendazim and other benzimidazole-type fungicides.

Carbendazim is a fungicide widely used for controlling fungi affecting fruits, vegetables, field crops etc. Determination of carbendazim in water, soil and various crops is frequently required to assure compliance with national/European regulations. A polyclonal antibody recognizing carbendazim was developed by using commercially available 2-(2-aminoethyl) benzimidazole, 2-benzimidazole propionic acid and 2-mercaptobenzimidazole as immunizing haptens; each of the above derivatives was directly conjugated to the carrier protein keyhole limpet hemocyanin and a mixture of the conjugates was administered to New Zealand white rabbits. Immunochemical functionality of the antisera and the corresponding isolated antibody (whole IgG fraction) was evaluated through titer and displacement curves in an in-house developed ELISA, which employed a 2-mercaptobenzimidazole - functionalized lysine-dendrimer as the immobilized hapten. As shown with ELISA-displacement curves, the above antibody could recognize carbendazim as well as other benzimidazole-type fungicides, i.e. benomyl and thiabendazole, and also intact benzimidazole, while it did not cross-react with the structurally different pesticides carbaryl and imazalil. Considering the rather simple approach which has led to its development and its highly promising immunochemical profile, the new antibody may be exploited in immunoanalytical systems for detecting benzimidazole-type pesticides e.g. in samples of environmental interest. The above antibody is being currently tested as a biorecognition element in the novel FOODSCAN cell biosensor platform for pesticide residue detection based on the Bioelectric Recognition Assay technology.

Complexes of an alpha thymosin derivative with ¹85/¹87Re and (99m)Tc: structural analysis and initial biological evaluation.

Alpha thymosins are a family of immunostimulating peptides first isolated from thymus gland. In the present work, the structure of the ReO(V)³âº complex of an alpha 1 thymosin derivative containing the metal-chelating N,N-dimethylglycyl-L-seryl-L-cysteinyl group was studied with NMR, CD, and ESI. The analysis indicated the existence of two interconverting diastereomers depending on the orientation of the side chain of the chelated Ser syn- or anti- to the oxygen of the ReO(V)³âº core. The two diastereomers could be separated on HPLC under a slow gradient showing the ratio of syn/anti to be 3:2, in agreement with the NMR data. The conversion process was shown to involve the coordination of a water molecule to the ReO(V)³âº core through the incubation of the complex in ¹8O-enriched water and subsequent ESI analysis. HPLC analysis of the analogous radioactive (99m)TcO(V)³âº complex showed the formation of two isomers in the same syn/anti 3:2 ratio. Biodistribution studies of the (99m)TcO(V)³âº complex in Swiss albino mice with experimentally induced inflammation showed higher accumulated radioactivity in inflamed tissue compared to normal (ratio of inflamed/control tissue 3.9). (99m)Tc-labeled complexes of alpha thymosin derivatives are expected to facilitate research on alpha thymosins and accelerate exploitation of these peptides in immunotherapy protocols.