Abstract Background Vδ2 T cells are promising candidates for approaches of immunotherapy due to their unique pleiotropic functions; they were recently shown to enhance antiviral protection in hematopoietic stem cell transplantation (HSCT) recipients via innate effector activity and modulation of virus-specific adaptive T-cell response. Extracellular Vesicles (EVs) are key carriers of immunomodulatory signals and Vδ2-derived EVs (Vδ2-EVs) exhibit antitumor activity but their role in viral infection remain unclear. The aim of this study was to investigate the direct and immunomodulatory antiviral functions of Vδ2-EVs in healthy subjects and HSCT patients. Methods The direct antiviral activity of Vδ2-EVs were tested in vitro using a model of Cytomegalovirus (CMV) replication. The immunomodulatory antiviral activities of Vδ2-EVs were evaluated in both healthy donors and HSCT recipients by functional immunological assays (cytokine release and proliferation capability of virus-specific T cells). Finally, their molecular cargo was characterized through miRNA sequencing. Results Our findings reveal that Vδ2-EVs efficiently inhibit CMV replication, reducing the frequency of CMV-infected fibroblast cells. Moreover, Vδ2-EVs are taken up by myeloid cells and were able to activate antigen-presenting cells, leading to an increased frequency of CMV-specific T cells, as measured by IFN-γ production. Accordingly, Vδ2-EVs enhanced the proliferation of CMV-specific T-cell clones in HSCT pediatric recipients. Finally, the analysis of miRNA content in Vδ2-EVs highlighted the enrichment of miRNAs that target genes regulating critical antiviral response processes such as SOCS1. Conclusions Altogether, this study provides new insights into the antiviral functions of Vδ2-EVs and underscores their translational therapeutic potential as modulators of antiviral immunity in immunocompromised settings.

Introduction:Elevated inflammation and immune dysregulation are the main consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The dysregulated inflammatory state persists after coronavirus disease 2019 (COVID-19), establishing the post-acute sequelae of SARS-CoV-2 infection in individuals with long COVID (LC). The role of CD169+ monocytes in the early diagnosis of SARS-CoV-2 infection and their association with severe outcomes were demonstrated in COVID-19 patients (COV). We aimed to delineate specific myeloid activation that characterizes the acute and post-acute phases of SARS-CoV-2 infection, evaluating the correlation between cellular and extracellular vesicles (EVs). Methods:Blood samples from COV, LC, and healthy donors (HD) were collected at Tor Vergata University Hospital in Rome. Plasmatic EVs were isolated by differential centrifugation and evaluated by flow cytometry and atomic force microscopy (AFM). Leukocyte subpopulations and different sizes of circulating EVs (100-200, 240-500, >500 nm) were characterized for HLA-DR and CD169 expression in COV, LC, and HD through flow cytometry. Serum inflammatory markers were assessed by the ELLA immunoassay system. The analyzed markers were associated with clinical and biochemical parameters in COV and LC. Results:The analysis of HLA-DR+, CD169+, and HLA-DR+CD169+ leukocytes confirmed our previous results in which the activated monocytes CD169+HLA-DR+ were found significantly high in COV, persisting in LC, and correlated differently with coagulation markers and inflammatory cytokines. Similar to cellular levels, the percentage and number of HLA-DR+CD169+ EVs were significantly elevated in COV and persisted in LC compared to HD. Different HLA-DR and CD169 expressions were found according to EV size in COV, LC, and HD, and correlations with biochemical parameters and circulating inflammatory markers were found. A positive correlation of HLA-DR and CD169 expression among monocytes and circulating EVs was found, supporting a possible connection between the two compartments and circulating inflammatory mediators. Moreover, the characterization by flow cytometry of EV cell derivation and cytokine cargo revealed EVs as sensitive indicators of both acute and persistent immune perturbations, bridging viral antigen persistence with inflammatory signaling in long COVID. Conclusion:Myeloid activation markers and inflammatory cytokines are dynamically expressed between blood cells and circulating extracellular vesicles, underlining multilevel cell-to-cell communications, opening new possibilities to monitor COVID-19 and long COVID-associated sequelae.

Human T-Lymphotropic virus type 1 (HTLV-1) lifelong infects at least 5-10 million people worldwide, a minority of whom develop severe lethal diseases including adult T-cell Leukemia/lymphoma and HTLV-1-associated myelopathy or tropical spastic paraparesis. Currently, no vaccines or curative therapies to fight HTLV-1 infection or diseases are available. Recently we found that a tributyltin molecule, Bu3SnOCOCF3 (TBT), which is more potent than cisplatin in inducing cytotoxic effects towards a panel of cell lines including high-tumorigenic cells, also exerted potent cytotoxic effects even towards HTLV-1-infected cell lines, mimicking different states of virus-driven transformation. The type of cell death involved was elusive. In the present study, the effects of TBT on virological and cell death parameters were investigated in HTLV-1-infected immortalized lymphocytes generated by in vitro infection and rendered with or without progressive independence from interleukin-2 as a growth factor. Molecular studies demonstrated that TBT affected HTLV-1 viral gene expression, especially HBZ. TBT confirmed its high cytotoxic potential on the HTLV-1-infected cell lines assayed, especially towards the IL-2-independent HTLV-1-infected cells. Investigation of mechanisms involved in cell death induced by TBT in HTLV-1-infected cells confirmed that caspase 3 and 8 activation, as well as apoptotic response, were relevant. In addition, pyroptosis as well as other unspecifed forms of lytic death presumably contribute to cell death induced by TBT in HTLV-1-infected cells, while a concomitant activation of an autophagic response by this compound seems to mitigate it. Overall, these experimental results outline a particular profile of TBT-induced cell death in HTLV-1-infected cells that is useful for future studies aimed at verifying the real potential of tin-based compounds to contrast diseases caused by HTLV-1.

Extracellular vesicles (EVs) have emerged as pivotal structural mediators of immune dysregulation and viral antigen persistence in long COVID. Rather than passive biological carriers, EVs released during SARS-CoV-2 infection function as highly organized lipid nanostructures that preserve the native conformational integrity of viral proteins, including the spike glycoprotein, thereby facilitating chronic signaling and systemic inflammation. Recent advancements in single-particle analytical techniques, such as cryo-electron tomography and atomic force microscopy, are now overcoming the limitations of bulk analysis, enabling the visualization of EV heterogeneity and the quantitative profiling of their nanomechanical properties. This review synthesizes current insights into how EV-associated viral remnants and host-derived inflammatory cargo propagate neuro-immune crosstalk and vascular injury. We conclude by evaluating the potential of EVs as precision biomarkers and bioengineered therapeutic platforms, emphasizing the need for standardized structural characterization to transition these nanovesicles from physiological mediators to clinical diagnostic and regenerative tools.

Perinatal depression (PD) actually affects 10-15% of pregnant women and represents one of the most debated topics as potentially implicated in offspring neurodevelopmental disorders etiology, particularly Autism Spectrum Disorder (ASD). Scientific evidence supports the role of Human Endogenous Retroviruses (HERVs) in ASD, as a link among environmental stimuli, epigenetic remodeling and biological processes. The aim of the present study was to characterize the expression profile of different HERVs and selected cytokines in peripheral blood mononuclear cells from women who have experienced PD in comparison to women without history of PD and their respective children stratified according to ASD diagnosis, by RT Real-Time PCR. We showed that ASD children and their PD mothers share abnormal expression of pHERV-W, syncytin-2 and IL-6 likely influenced by the common environment, maternal status, genetic predisposition, or postnatal factors. Of note, mothers with a history of PD were also evaluated at the time of blood sampling using the Hamilton Depression Rating Scale, and a positive correlation with pHERV-W levels was observed. Together with previous results in preclinical models and human studies, our results support the role of HERVs in autism as a contributing factor in creating an adverse environment for normal neurodevelopment, strengthening the view of a mother-child association in the context of autism. PD being associated with the altered activity of HERVs could be considered to be an additional risk factor in the pathogenesis of autism.

IntroductionTo evaluate the efficacy of a novel eye drop formulation containing hyaluronic acid (0.2%) and butyroyl-glutathione (GSHC4, 0.4%) in glaucoma-associated ocular surface disease (G-OSD), and to explore putative mechanisms of action through in vitro assays of corneal epithelial wound healing and cytokine modulation under basal, inflammatory, and oxidative stress conditions.MethodsIn this preliminary, hypothesis-generating, prospective, double-blind, cross-over study, 16 patients with glaucoma or ocular hypertension and coexisting dry eye symptoms were randomized to receive either HA alone or HA + GSH-C4 for 4 weeks, separated by a 1-week washout. Clinical endpoints included the Ocular Surface Disease Index (OSDI), FACES scale, tear film break-up time (TFBUT), Schirmer test, and NEI fluorescein staining. Tear cytokines were quantified at multiple timepoints. In vitro, human corneal epithelial cells underwent scratch-wound assays and cytokine profiling.ResultsFifteen of sixteen patients completed the study. HA + GSH-C4 significantly improved OSDI (p < 0.001), FACES scores (p = 0.012), TFBUT (p < 0.001), and NEI-SS (p < 0.001) compared with HA alone. No treatment-related adverse events were observed. Tear cytokine analysis revealed a reversible treatment-specific elevation of selected mediators (e.g., IFN-γ, IL-12p70) consistent with a controlled pro-repair response. The HA + GSH-C4 accelerated epithelial wound closure and selectively suppressed MCP-1 across all tested conditions.DiscussionThe HA + GSH-C4 formulation improved G-OSD symptoms and ocular surface stability, accompanied by a dynamic cytokine response in tears. These findings support its clinical utility and suggest a dual mechanism involving barrier restoration and modulation of ocular surface immune responses.

Among the metal-derived complexes, recently, tin derivatives have been investigated as promising anti-cancer drug candidates. Our previous study showed that the tin-based compound Bu3SnOCOCF3 (TBT) exerts cytotoxic activity on solid tumor cell lines. In the present study, the effects of TBT were evaluated in vitro on HTLV-1-infected human lymphocytic cell lines at different stages of viral transformation, consisting of IL-2-dependent (PB2/IL-2) and IL-2-independent (PB2/NO-IL-2) cells, generated in our laboratory by HTLV-1 in vitro infection of lymphocytes from the same donor, and the C91/PL cell line established by co-cultivation with T cells from a patient with HTLV-1-positive leukemia. TBT induced a reliable and reproducible dose-dependent inhibition of metabolic activity and viability in the HTLV-1-infected cells. The effect was cell-type-dependent, with C91/PL cells being quite resistant. An investigation into the cytotoxic effects induced by TBT in HTLV-1-infected cells and data on caspase inhibitors/caspase activation indicated that apoptotic cell death was involved, but also that the possible involvement of other forms of cell death could not be excluded. Taken together, the results show for the first time that the tin-based compound, although not devoid of a certain cytotoxicity toward uninfected cells, can induce typical and potent effects on HTLV-1-infected cells.

Urinary tract infections (UTIs) are among the most common bacterial infections worldwide. They occur in the urinary system when a microorganism, commonly present on the perineal skin or rectum, reaches the bladder through the urethra, and adheres to the luminal surface of uroepithelial cells, forming biofilms. The treatment of UTIs includes antibiotics, but their indiscriminate use has favored the development of multidrug-resistant bacteria strains, which represent a serious challenge to today's microbiology. The pathogenesis of the infection and antibiotic resistance synergistically contribute to hindering the eradication of the disease while favoring the establishment of persistent infections. The repeated requirement for antibiotic treatment and the limited therapeutic options have further contributed to the increase in antibiotic resistance and the occurrence of potential relapses by therapeutic failure. To limit antimicrobial resistance and broaden the choice of non-antibiotic preventive approaches, this review reports studies focused on the bacteriostatic/bactericidal activity, inhibition of bacterial adhesion and quorum sensing, restoration of uroepithelial integrity and immune response of molecules, vitamins, and compounds obtained from plants. To date, different supplementations are recommended by the European Association of Urology for the management of UTIs as an alternative approach to antibiotic treatment, while a variety of bioactive compounds are under investigation, mostly at the level of in vitro and preclinical studies. Although the evidence is promising, they are far from being included in the clinical practice of UTIs.

In the context of long-term therapy in virologically suppressed people living with HIV-1 (PLWH), the identification of new biomarkers associated with immuno-virological discordance, and the risk of disease progression is needed. Herein we investigated HERVs expression in association with immuno-virological discordance parameters for the identification of novel markers for the clinical monitoring of virologically suppressed PLWH. It is known the human endogenous retroviruses (HERVs), relics of ancestral exogenous retroviral infections comprising 8% of human genome, could be reactivated by exogenous viruses including HIV-1. The study included 31 virologically suppressed PLWH and 10 healthy donors; blood HIV-DNA levels and residual plasma viremia were quantified by droplet digital-PCR, the expression of HERVs by RT-Real time PCR, and immunophenotyping by flow cytometry. The results revealed a dynamic association of HERVs with several virological and immunological parameters such as the HIV-1 reservoir, CD4 cell count, CD4 nadir and with CD8+ and CD19 lymphocyte activation. In an era of searching innovative biomarkers for people living with HIV-1, the interconnection of HERVs with the HIV-1 reservoir and lymphocyte activation opens to further investigation on HERVs role in persistent immune activation in virologically suppressed PLWH, proposing them as potential new markers for clinical monitoring.

ABSTRACT:Dysregulated expression of human endogenous retrovirus K (HERV-K) has been found in many types of tumors. Previously, we demonstrated the concomitant expression of HERVs and embryonic genes in cancer cells with aggressive and stemness features. In the field of onco-hematology, some studies have described alterations of HERV expression in chronic lymphocytic leukemia (CLL), the most common adult leukemia in the Western world. Despite numerous achievements in CLL clinical research, given the heterogeneity of the disease and the different treatment choices, identification of new biomarkers for patient management is needed. On this basis, this work aimed to evaluate the expression of HERVs and embryonic genes as novel combined biomarkers in CLL and their potential association with clinical features and therapy regimens. Peripheral blood mononuclear cells were isolated from 49 healthy donors (HDs) and 74 patients with CLL, evaluating their treatment regimen. The expression of different HERVs and embryonic genes was analyzed by real-time polymerase chain reaction. Molecular analysis showed higher expression of HERVs and embryonic genes in patients than HDs, differently expressed according to treatment status. Using principal component analysis, we found complex expression profiles of HERVs and embryonic genes associated with CLL and different treatment regimens. In ibrutinib-treated patients, HERVs were found to be associated with unfavorable prognostic factors of CLL. These findings, although requiring confirmation in larger patient cohorts, highlight the interconnection between HERVs and embryonic genes in CLL, suggesting their use as potential new biomarkers in monitoring innovative treatments.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections of human germ-line cells, which are mostly silenced during evolution, but could be de-repressed and play a pathological role. Infection with some exogenous viruses, including herpesviruses, HIV-1 and SARS-CoV-2, was demonstrated to induce the expression of HERV RNAs and proteins.

Endogenous retroviruses (ERVs) are genetic elements derived from a process of germline infection by exogenous retroviruses. Some ERVs have been co-opted for physiological functions, and their activation has been associated with complex diseases, including Autism Spectrum Disorder (ASD). We have already demonstrated an abnormal expression of ERVs in the BTBR T + tf/J (BTBR) mouse model of ASD during intrauterine life till adulthood. Thus, starting from the assumptions that ERVs may contribute to the derailment of neurodevelopment and that ASD has fetal origins as a consequence of adverse intrauterine conditions, the present study aims to characterize the transcriptional activity of selected ERVs (MusD, IAP, Syn-A, Syn-B, ARC and GLN), LINE-1, inflammatory mediators (IL-6, IL-10, IL-11 CXCL-1) at the maternal–fetal interface and in dissected embryos from BTBR mice. Our results highlight the deregulation of ERVs and inflammatory mediators at the maternal–fetal interface, and in cephalic and non-cephalic embryonic tissues from BTBR compared to C57BL/6 J. Several correlations among ERV expression levels emerged in different tissues from C57BL/6 J mice while, in BTBR mice, no correlations were found, suggesting that in this model, the acquisition of autistic-like traits might be linked to the dysregulation of ERV activity occurring during intra-uterine life.

Infection with SARS-CoV-2, the virus responsible for COVID-19 diseases, can impact different tissues and induce significant cellular alterations. The production of extracellular vesicles (EVs), which are physiologically involved in cell communication, is also altered during COVID-19, along with the dysfunction of cytoplasmic organelles. Since circulating EVs reflect the state of their cells of origin, they represent valuable tools for monitoring pathological conditions. Despite challenges in detecting EVs due to their size and specific cellular compartment origin using different methodologies, flow cytometry has proven to be an effective method for assessing the role of EVs in COVID-19. This review summarizes the involvement of plasmatic EVs in COVID-19 patients and individuals with Long COVID (LC) affected by post-acute sequelae of SARS-CoV-2 infection (PASC), highlighting their dual role in exerting both pro- and antiviral effects. We also emphasize how flow cytometry, with its multiparametric approach, can be employed to characterize circulating EVs, particularly in infectious diseases such as COVID-19, and suggest their potential role in chronic impairments during post-infection.

Increasing evidence indicates that human endogenous retroviruses (HERVs) are important to human health and are an underexplored component of many diseases. Certain HERV families show unique expression patterns and immune responses in autism spectrum disorder (ASD) patients compared to healthy controls, suggesting their potential as biomarkers. Despite these interesting findings, the role of HERVs in ASD needs to be further investigated. In this review, we discuss recent advances in genetic research on ASD, with a particular emphasis on the implications of HERVs on neurodevelopment and future genomic initiatives aimed at discovering ASD-related genes through Artificial Intelligence. Given their pro-inflammatory and autoimmune characteristics, the existing literature suggests that HERVs may contribute to the onset or worsening of ASD in individuals with a genetic predisposition. Therefore, we propose that investigating their fundamental properties could not only improve existing therapies but also pave the way for new therapeutic strategies.

Phenotypic drug discovery (PDD) involves screening compounds for their effects on cells, tissues, or whole organisms without necessarily understanding the underlying molecular targets. PDD differs from target-based strategies as it does not require knowledge of a specific drug target or its role in the disease. This approach can lead to the discovery of drugs with unexpected therapeutic effects or applications and allows for the identification of drugs based on their functional effects, rather than through a predefined target-based approach. Ultimately, disease definitions are mostly symptom-based rather than mechanism-based, and the therapeutics should be likewise. In recent years, there has been a renewed interest in PDD due to its potential to address the complexity of human diseases, including the holistic picture of multiple metabolites engaging with multiple targets constituting the central hub of the metabolic host–microbe interactions. Although PDD presents challenges such as hit validation and target deconvolution, significant achievements have been reached in the era of big data. This article explores the experiences of researchers testing the effect of a thymic peptide hormone, thymosin alpha-1, in preclinical and clinical settings and discuss how its therapeutic utility in the precision medicine era can be accommodated within the PDD framework.

The use of CD169 as a marker of viral infection has been widely discussed in the context of COVID-19, and in particular, its crucial role in the early detection of SARS-CoV-2 infection and its association with the severity and clinical outcome of COVID-19 were demonstrated. COVID-19 patients show relevant systemic alteration and immunological dysfunction that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC). It is critical to implement the characterization of the disease, focusing also on the possible impact of the different COVID-19 waves and the consequent effects found after infection. On this basis, we evaluated by flow cytometry the expression of CD169 and HLA-DR on monocytes from COVID-19 patients and PASC individuals to better elucidate their involvement in immunological dysfunction, also evaluating the possible impact of different pandemic waves. The results confirm CD169 RMFI is a good marker of viral infection. Moreover, COVID-19 patients and PASC individuals showed high percentage of CD169+ monocytes, but low percentage of HLA-DR+ monocytes and the alteration of systemic inflammatory indices. We have also observed alterations of CD169 and HLA-DR expression and indices of inflammation upon different COVID-19 waves. The persistence of specific myeloid subpopulations suggests a role of CD169+ monocytes and HLA-DR in COVID-19 disease and chronic post-infection inflammation, opening new opportunities to evaluate the impact of specific pandemic waves on the immune response impairment and systemic alterations with the perspective to provide new tools to monitoring new variants and diseases associated to emerging respiratory viruses.

The complex alterations of the immune system and the immune-mediated multiorgan injury plays a key role in host response to SARS-CoV-2 infection and in the pathogenesis of COVID-19, being also associated with adverse outcomes. Thymosin alpha 1 (Tα1) is one of the molecules used in the treatment of COVID-19, as it is known to restore the homeostasis of the immune system during infections and cancer. The use of Tα1 in COVID-19 patients had been widely used in China and in COVID-19 patients, it has been shown to decrease hospitalization rate, especially in those with greater disease severity, and reduce mortality by restoring lymphocytopenia and more specifically, depleted T cells. Persistent dysregulation with depletion of naive B and T cell subpopulations and expansion of memory T cells suggest a chronic stimulation of the immune response in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC). Our data obtained from an ex vivo study, showed that in PASC individuals with a chronically altered immune response, Tα1 improve the restoration of an appropriate response, most evident in those with more severe illness and who need respiratory support during acute phase, and in those with specific systemic and psychiatric symptoms of PASC, confirming Tα1 treatment being more effective in compromised patients. The results obtained, along with promising reports on recent trials on Tα1 administration in patients with COVID-19, offer new insights into intervention also for those patients with long-lasting inflammation with post-infectious symptoms, some of which have a delayed onset.