Autoimmunity against Nucleus Ambiguous Is Putatively Possible in Both Long-COVID-19 and Vaccinated Subjects: Scientific Evidence and Working Hypothesis.

As reported by the World Health Organization (WHO), about 10-20% of people have experienced mid- to long-term effects following SARS-CoV-2 infection, collectively referred to as post-COVID-19 condition or long-COVID, including some neurovegetative symptoms. Numerous findings have suggested that the onset of these neurovegetative symptoms upon viral infection may be caused by the production of autoantibodies through molecular mimicry phenomena. Accordingly, we had previously demonstrated that 22 of the human proteins sharing putatively immunogenic peptides with SARS-CoV-2 proteins are expressed in the dorsal motor nucleus and nucleus ambiguous. Therefore, if molecular mimicry occurs following severe forms of COVID-19, there could be transitory or permanent damage in some vagal structures, resulting in a lower vagal tone and all the related clinical signs. We investigated the presence of autoantibodies against two proteins of vagal nuclei sharing a peptide with SARS-CoV-2 spike glycoprotein using an immunoassay test on blood obtained from patients with cardiorespiratory symptoms in patients affected by ongoing symptomatic COVID-19 (long-COVID), subjects vaccinated without a history of SARS-CoV-2 infection, and subjects not vaccinated without a history of SARS-CoV-2 infection. Interestingly, putative autoantibodies were present in both long-COVID-19 and vaccinated groups, opening interesting questions about pathogenic mechanisms of the disease.

Interplay between the Chaperone System and Gut Microbiota Dysbiosis in Systemic Lupus Erythematosus Pathogenesis: Is Molecular Mimicry the Missing Link between Those Two Factors?

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by self-immune tolerance breakdown and the production of autoantibodies, causing the deposition of immune complexes and triggering inflammation and immune-mediated damage. SLE pathogenesis involves genetic predisposition and a combination of environmental factors. Clinical manifestations are variable, making an early diagnosis challenging. Heat shock proteins (Hsps), belonging to the chaperone system, interact with the immune system, acting as pro-inflammatory factors, autoantigens, as well as immune tolerance promoters. Increased levels of some Hsps and the production of autoantibodies against them are correlated with SLE onset and progression. The production of these autoantibodies has been attributed to molecular mimicry, occurring upon viral and bacterial infections, since they are evolutionary highly conserved. Gut microbiota dysbiosis has been associated with the occurrence and severity of SLE. Numerous findings suggest that proteins and metabolites of commensal bacteria can mimic autoantigens, inducing autoimmunity, because of molecular mimicry. Here, we propose that shared epitopes between human Hsps and those of gut commensal bacteria cause the production of anti-Hsp autoantibodies that cross-react with human molecules, contributing to SLE pathogenesis. Thus, the involvement of the chaperone system, gut microbiota dysbiosis, and molecular mimicry in SLE ought to be coordinately studied.

Nanovesicular Mediation of the Gut-Brain Axis by Probiotics: Insights into Irritable Bowel Syndrome.

BACKGROUND: Dysbiosis, influenced by poor diet or stress, is associated with various systemic diseases. Probiotic supplements are recognized for stabilizing gut microbiota and alleviating gastrointestinal issues, like irritable bowel syndrome (IBS). This study focused on the tryptophan pathways, which are important for the regulation of serotonin levels, and on host physiology and behavior regulation. METHODS: Nanovesicles were isolated from the plasma of subjects with chronic diarrhea, both before and after 60 days of consuming a probiotic mix (Acronelle®, Bromatech S.r.l., Milan, Italy). These nanovesicles were assessed for the presence of Tryptophan 2,3-dioxygenase 2 (TDO 2). Furthermore, the probiotics mix, in combination with H2O2, was used to treat HT29 cells to explore its cytoprotective and anti-stress effect. RESULTS: In vivo, levels of TDO 2 in nanovesicles were enhanced in the blood after probiotic treatment, suggesting a role in the gut-brain axis. In the in vitro model, a typical H2O2-induced stress effect occurred, which the probiotics mix was able to recover, showing a cytoprotective effect. The probiotics mix treatment significantly reduced the heat shock protein 60 kDa levels and was able to preserve intestinal integrity and barrier function by restoring the expression and redistribution of tight junction proteins. Moreover, the probiotics mix increased the expression of TDO 2 and serotonin receptors. CONCLUSIONS: This study provides evidence for the gut-brain axis mediation by nanovesicles, influencing central nervous system function.

Speeding up Glioblastoma Cancer Research: Highlighting the Zebrafish Xenograft Model.

Glioblastoma multiforme (GBM) is a very aggressive and lethal primary brain cancer in adults. The multifaceted nature of GBM pathogenesis, rising from complex interactions between cells and the tumor microenvironment (TME), has posed great treatment challenges. Despite significant scientific efforts, the prognosis for GBM remains very poor, even after intensive treatment with surgery, radiation, and chemotherapy. Efficient GBM management still requires the invention of innovative treatment strategies. There is a strong necessity to complete cancer in vitro studies and in vivo studies to properly evaluate the mechanisms of tumor progression within the complex TME. In recent years, the animal models used to study GBM tumors have evolved, achieving highly invasive GBM models able to provide key information on the molecular mechanisms of GBM onset. At present, the most commonly used animal models in GBM research are represented by mammalian models, such as mouse and canine ones. However, the latter present several limitations, such as high cost and time-consuming management, making them inappropriate for large-scale anticancer drug evaluation. In recent years, the zebrafish (Danio rerio) model has emerged as a valuable tool for studying GBM. It has shown great promise in preclinical studies due to numerous advantages, such as its small size, its ability to generate a large cohort of genetically identical offspring, and its rapid development, permitting more time- and cost-effective management and high-throughput drug screening when compared to mammalian models. Moreover, due to its transparent nature in early developmental stages and genetic and anatomical similarities with humans, it allows for translatable brain cancer research and related genetic screening and drug discovery. For this reason, the aim of the present review is to highlight the potential of relevant transgenic and xenograft zebrafish models and to compare them to the traditionally used animal models in GBM research.

The Interplay between Glioblastoma Cells and Tumor Microenvironment: New Perspectives for Early Diagnosis and Targeted Cancer Therapy.

Glioblastoma multiforme (GBM) stands out as the most tremendous brain tumor, constituting 60% of primary brain cancers, accompanied by dismal survival rates. Despite advancements in research, therapeutic options remain limited to chemotherapy and surgery. GBM molecular heterogeneity, the intricate interaction with the tumor microenvironment (TME), and non-selective treatments contribute to the neoplastic relapse. Diagnostic challenges arise from GBM advanced-stage detection, necessitating the exploration of novel biomarkers for early diagnosis. Using data from the literature and a bioinformatic tool, the current manuscript delineates the molecular interplay between human GBM, astrocytes, and myeloid cells, underscoring selected protein pathways belonging to astroglia and myeloid lineage, which can be considered for targeted therapies. Moreover, the pivotal role of extracellular vesicles (EVs) in orchestrating a favorable microenvironment for cancer progression is highlighted, suggesting their utility in identifying biomarkers for GBM early diagnosis.

Hsp70 and Calcitonin Receptor Protein in Extracellular Vesicles from Glioblastoma Multiforme: Biomarkers with Putative Roles in Carcinogenesis and Potential for Differentiating Tumor Types.

Glioblastoma multiforme (GBM) is a malignancy of bad prognosis, and advances in early detection and treatment are needed. GBM is heterogenous, with varieties differing in malignancy within a tumor of a patient and between patients. Means are needed to distinguish these GMB forms, so that specific strategies can be deployed for patient management. We study the participation of the chaperone system (CS) in carcinogenesis. The CS is dynamic, with its members moving around the body in extracellular vesicles (EVs) and interacting with components of other physiological systems in health and disease, including GBM. Here, we describe the finding of high amounts of Hsp70 (HSPA1A) and the calcitonin receptor protein (CTR) in EVs in patients with GBM. We present a standardized protocol for collecting, purifying, and characterizing EVs carrying Hsp70 and CTR in plasma-derived EVs from patients with GBM. EVs from GBM patients were obtained just before tumor ablative surgery (T0) and 7 days afterwards (T1); Hsp70 was highly elevated at T0 and less so at T1, and CTR was greatly increased at T0 and reduced to below normal values at T1. Our results encourage further research to assess Hsp70 and CTR as biomarkers for differentiating tumor forms and to determine their roles in GBM carcinogenesis.

Putative Roles and Therapeutic Potential of the Chaperone System in Amyotrophic Lateral Sclerosis and Multiple Sclerosis.

The putative pathogenic roles and therapeutic potential of the chaperone system (CS) in amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) are reviewed to provide a bibliographic and conceptual platform for launching research on the diagnostic and therapeutic applications of CS components. Various studies suggest that dysfunction of the CS contributes to the pathogenesis of ALS and MS, and here, we identify some of the implicated CS members. The physiology and pathophysiology of the CS members can be properly understood if they are studied or experimentally or clinically manipulated for diagnostic or therapeutic purposes, bearing in mind that they belong to a physiological system with multiple interacting and dynamic components, widespread throughout the body, intra- and extracellularly. Molecular chaperones, some called heat shock protein (Hsp), are the chief components of the CS, whose canonical functions are cytoprotective. However, abnormal chaperones can be etiopathogenic factors in a wide range of disorders, chaperonopathies, including ALS and MS, according to the data reviewed. Chaperones typically form teams, and these build functional networks to maintain protein homeostasis, the canonical role of the CS. However, members of the CS also display non-canonical functions unrelated to protein homeostasis. Therefore, chaperones and other members of the CS, if abnormal, may disturb not only protein synthesis, maturation, and migration but also other physiological processes. Thus, in elucidating the role of CS components in ALS and MS, one must look at protein homeostasis abnormalities and beyond, following the clues emerging from the works discussed here.

Immunohistochemistry of Human Hsp60 in Health and Disease: Recent Advances in Immunomorphology and Methods for Assessing the Chaperonin in Extracellular Vesicles.

The subject matter of this chapter is defined by the title of its two previous editions, "Immunohistochemistry of human Hsp60 in health and disease: From autoimmunity to cancer," the latest of which appeared in 2018. Since then, considerable advances have been made in the fields of autoimmunity and cancer and some of them are closely linked to progress in the understanding of the chaperone system (CS). This is a physiological system composed of molecular chaperones, co-chaperones, chaperone cofactors, and chaperone interactors and receptors. The molecular chaperones are the chief members of the CS, and here we focus on one of them, Hsp60. Since extracellular vesicles (EVs) have also emerged as key factors in the functioning of the CS and in carcinogenesis, we have incorporated a detailed section about them. This chapter explains how to assess Hsp60 in tissues and in EVs for application in diagnosis, prognostication, and patient monitoring and, eventually, for developing methods using them as therapeutic targets and tools. We describe immunohistochemical techniques, immunofluorescence and double immunofluorescence-confocal microscopy, and methods for collecting and isolating EVs from blood plasma and for assessing their contents in Hsp60 and related microRNAs (miRNAs). All these procedures have proven to be reliable and useful in the study and management of various types of cancer and inflammatory and autoimmune conditions.

Identification of Molecular Targets and Anti-Cancer Agents in GBM: New Perspectives for Cancer Therapy.

The authors of the present literature piece were invited to participate in the present Special Issue at the beginning of 2022, and we were all very enthusiastic at the prospect of assembling a series of articles on new molecular targets and anti-cancer agents in glioblastoma multiforme (GBM) [...].

Air Pollution: Role of Extracellular Vesicles-Derived Non-Coding RNAs in Environmental Stress Response.

Air pollution has increased over the years, causing a negative impact on society due to the many health-related problems it can contribute to. Although the type and extent of air pollutants are known, the molecular mechanisms underlying the induction of negative effects on the human body remain unclear. Emerging evidence suggests the crucial involvement of different molecular mediators in inflammation and oxidative stress in air pollution-induced disorders. Among these, non-coding RNAs (ncRNAs) carried by extracellular vesicles (EVs) may play an essential role in gene regulation of the cell stress response in pollutant-induced multiorgan disorders. This review highlights EV-transported ncRNAs' roles in physiological and pathological conditions, such as the development of cancer and respiratory, neurodegenerative, and cardiovascular diseases following exposure to various environmental stressors.

Oligomeric State and Holding Activity of Hsp60.

Similar to its bacterial homolog GroEL, Hsp60 in oligomeric conformation is known to work as a folding machine, with the assistance of co-chaperonin Hsp10 and ATP. However, recent results have evidenced that Hsp60 can stabilize aggregation-prone molecules in the absence of Hsp10 and ATP by a different, "holding-like" mechanism. Here, we investigated the relationship between the oligomeric conformation of Hsp60 and its ability to inhibit fibrillization of the Ab40 peptide. The monomeric or tetradecameric form of the protein was isolated, and its effect on beta-amyloid aggregation was separately tested. The structural stability of the two forms of Hsp60 was also investigated using differential scanning calorimetry (DSC), light scattering, and circular dichroism. The results showed that the protein in monomeric form is less stable, but more effective against amyloid fibrillization. This greater functionality is attributed to the disordered nature of the domains involved in subunit contacts.

Molecular Pathways Implicated in Radioresistance of Glioblastoma Multiforme: What Is the Role of Extracellular Vesicles?

Glioblastoma multiforme (GBM) is a primary brain tumor that is very aggressive, resistant to treatment, and characterized by a high degree of anaplasia and proliferation. Routine treatment includes ablative surgery, chemotherapy, and radiotherapy. However, GMB rapidly relapses and develops radioresistance. Here, we briefly review the mechanisms underpinning radioresistance and discuss research to stop it and install anti-tumor defenses. Factors that participate in radioresistance are varied and include stem cells, tumor heterogeneity, tumor microenvironment, hypoxia, metabolic reprogramming, the chaperone system, non-coding RNAs, DNA repair, and extracellular vesicles (EVs). We direct our attention toward EVs because they are emerging as promising candidates as diagnostic and prognostication tools and as the basis for developing nanodevices for delivering anti-cancer agents directly into the tumor mass. EVs are relatively easy to obtain and manipulate to endow them with the desired anti-cancer properties and to administer them using minimally invasive procedures. Thus, isolating EVs from a GBM patient, supplying them with the necessary anti-cancer agent and the capability of recognizing a specified tissue-cell target, and reinjecting them into the original donor appears, at this time, as a reachable objective of personalized medicine.

Contribution of Extracellular Vesicles and Molecular Chaperones in Age-Related Neurodegenerative Disorders of the CNS.

Many neurodegenerative disorders are characterized by the abnormal aggregation of misfolded proteins that form amyloid deposits which possess prion-like behavior such as self-replication, intercellular transmission, and consequent induction of native forms of the same protein in surrounding cells. The distribution of the accumulated proteins and their correlated toxicity seem to be involved in the progression of nervous system degeneration. Molecular chaperones are known to maintain proteostasis, contribute to protein refolding to protect their function, and eliminate fatally misfolded proteins, prohibiting harmful effects. However, chaperone network efficiency declines during aging, prompting the onset and the development of neurological disorders. Extracellular vesicles (EVs) are tiny membranous structures produced by a wide range of cells under physiological and pathological conditions, suggesting their significant role in fundamental processes particularly in cellular communication. They modulate the behavior of nearby and distant cells through their biological cargo. In the pathological context, EVs transport disease-causing entities, including prions, α-syn, and tau, helping to spread damage to non-affected areas and accelerating the progression of neurodegeneration. However, EVs are considered effective for delivering therapeutic factors to the nervous system, since they are capable of crossing the blood-brain barrier (BBB) and are involved in the transportation of a variety of cellular entities. Here, we review the neurodegeneration process caused mainly by the inefficiency of chaperone systems as well as EV performance in neuropathies, their potential as diagnostic biomarkers and a promising EV-based therapeutic approach.

Circulating Molecular Chaperones in Subjects with Amnestic Mild Cognitive Impairment and Alzheimer's Disease: Data from the Zabùt Aging Project.

Molecular chaperones play essential roles in many processes such as cell differentiation, tissue homeostasis, and organ remodeling. Recent data indicate that chaperones can act as cytoprotectants for brain cells during the progression of neurodegenerative diseases, including Alzheimer's disease (AD). However, very few data on the levels of chaperones in dementia, including its prodromal phases, have been reported. In this study, we used biological samples and epidemiological data collected during the Zabùt Aging Project (a prospective, community-based, cohort study of normal/pathological aging conducted in Sicily, Italy, with a follow-up of ten years) to determine if there is an association between plasma levels of the chaperones Hsp60, Hsp70, and Hsp90 with amnestic mild cognitive impairment (aMCI) and AD. Twenty-six aMCI individuals, 26 AD and 26 controls, matched for age and sex, were enrolled. After adjustment for education, subjects with AD showed significantly higher levels of Hsp60 than aMCI (OR = 1.16, 95% CI 1.04-1.30) and controls (OR = 1.12, 95% CI 1.03-1.22), while Hsp70 was significantly higher only in AD (OR = 1.84, 95% CI 1.09-3.10) than controls. In contrast, circulating levels of Hsp90 were significantly diminished in aMCI (OR = 0.69, 95% CI 0.52-0.91) and AD (OR = 0.51, 95% CI 0.35-0.75) compared to controls. However, these results were no longer significant after adjustment for multiple comparisons. Although the results lost significance after adjustment for multiple comparisons, they are encouraging despite the smallness of the sample and new studies should be carried out with larger populations to determine to what extent sequential measurement of serum chaperones in aMCI and AD can be trusted as indicators of disease status and progression.

Extracellular heat shock proteins in cancer: From early diagnosis to new therapeutic approach.

In cancer, human cells lose the ability to properly control the series of events that occur constantly during cell growth and division, including protein expression, stability, and dynamics. Heat shock proteins (Hsps) are key molecules in these events, constitutively expressed at high levels and could furthermore be induced by the response to cancer-induced stress. In tumor cells, Hsps have been shown to be implicated in the regulation of apoptosis, immune responses, angiogenesis and metastasis; in some cases, they can be overexpressed and dysregulated, representing important cancer hallmarks. In the past few years, it has been demonstrated that Hsps can be released by tumor cells through several secreting pathways, including the extracellular vesicles (EVs), thus modulating the tumor microenvironment as well as long-distance intercellular communication and metastatization. In this review, we discuss the role of extracellular Hsps in cancer, with a particular interest in Hsps in EVs. We would also like to highlight the importance of fully understanding of the role of extracellular Hsps released by EVs and encourage further research in this field the use of Hsps as early cancer biomarkers and therapeutic targets.

Molecular Chaperones and miRNAs in Epilepsy: Pathogenic Implications and Therapeutic Prospects.

Epilepsy is a pathologic condition with high prevalence and devastating consequences for the patient and its entourage. Means for accurate diagnosis of type, patient monitoring for predicting seizures and follow up, and efficacious treatment are desperately needed. To improve this adverse outcome, miRNAs and the chaperone system (CS) are promising targets to understand pathogenic mechanisms and for developing theranostics applications. miRNAs implicated in conditions known or suspected to favor seizures such as neuroinflammation, to promote epileptic tolerance and neuronal survival, to regulate seizures, and others showing variations in expression levels related to seizures are promising candidates as useful biomarkers for diagnosis and patient monitoring, and as targets for developing novel therapies. Components of the CS are also promising as biomarkers and as therapeutic targets, since they participate in epileptogenic pathways and in cytoprotective mechanisms in various epileptogenic brain areas, even if what they do and how is not yet clear. The data in this review should help in the identification of molecular targets among the discussed miRNAs and CS components for research aiming at understanding epileptogenic mechanisms and, subsequently, develop means for predicting/preventing seizures and treating the disease.

JNK pathway and heat shock response mediate the survival of C26 colon carcinoma bearing mice fed with the mushroom Pleurotus eryngii var. eryngii without affecting tumor growth or cachexia.

In the last few years, there has been emerging interest in developing treatments against human diseases using natural bioactive content. Here, the powder of the edible mushroom Pleurotus eryngii var. eryngii was mixed with the normal diet of mice bearing C26 colon carcinoma. Interestingly, it was evidenced by a significant increase in the survival rate of C26 tumor-bearing mice accompanied by a significant increase in Hsp90 and Hsp27 protein levels in the tumors. These data were paralleled by a decrease in Hsp60 levels. The mushroom introduced in the diet induced the inhibition of the transcription of the pro-inflammatory cytokines IL-6 and IL-1 exerting an anti-inflammatory action. The effects of the mushroom were mediated by the activation of c-Jun NH2-terminal kinases as a result of metabolic stress induced by the micronutrients introduced in the diet. In the tumors of C26 bearing mice fed with Pleurotus eryngii there was also a decreased expression of the mitotic regulator survivin and the anti-apoptotic factor Bcl-xL as well as an increase in the expression levels of Atg7, a protein that drives autophagy. In our hypothesis the interplay of these molecules favored the survival of the mice fed with the mushroom. These data are promising for the introduction of Pleurotus eryngii as a dietary supplement or as an adjuvant in anti-cancer therapy.

The Role of Molecular Chaperones in Virus Infection and Implications for Understanding and Treating COVID-19.

The COVID-19 pandemic made imperative the search for means to end it, which requires a knowledge of the mechanisms underpinning the multiplication and spread of its cause, the coronavirus SARS-CoV-2. Many viruses use members of the hosts' chaperoning system to infect the target cells, replicate, and spread, and here we present illustrative examples. Unfortunately, the role of chaperones in the SARS-CoV-2 cycle is still poorly understood. In this review, we examine the interactions of various coronaviruses during their infectious cycle with chaperones in search of information useful for future research on SARS-CoV-2. We also call attention to the possible role of molecular mimicry in the development of autoimmunity and its widespread pathogenic impact in COVID-19 patients. Viral proteins share highly antigenic epitopes with human chaperones, eliciting anti-viral antibodies that crossreact with the chaperones. Both, the critical functions of chaperones in the infectious cycle of viruses and the possible role of these molecules in COVID-19 autoimmune phenomena, make clear that molecular chaperones are promising candidates for the development of antiviral strategies. These could consist of inhibiting-blocking those chaperones that are necessary for the infectious viral cycle, or those that act as autoantigens in the autoimmune reactions causing generalized destructive effects on human tissues.

Extracellular Vesicles-Based Drug Delivery Systems: A New Challenge and the Exemplum of Malignant Pleural Mesothelioma.

Research for the most selective drug delivery to tumors represents a fascinating key target in science. Alongside the artificial delivery systems identified in the last decades (e.g., liposomes), a family of natural extracellular vesicles (EVs) has gained increasing focus for their potential use in delivering anticancer compounds. EVs are released by all cell types to mediate cell-to-cell communication both at the paracrine and the systemic levels, suggesting a role for them as an ideal nano-delivery system. Malignant pleural mesothelioma (MPM) stands out among currently untreatable tumors, also due to the difficulties in achieving an early diagnosis. Thus, early diagnosis and treatment of MPM are both unmet clinical needs. This review looks at indirect and direct evidence that EVs may represent both a new tool for allowing an early diagnosis of MPM and a potential new delivery system for more efficient therapeutic strategies. Since MPM is a relatively rare malignant tumor and preclinical MPM models developed to date are very few and not reliable, this review will report data obtained in other tumor types, suggesting the potential use of EVs in mesothelioma patients as well.