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Coronavirus disease 2019 (COVID-19) is an infection characterized by the dysregulation of systemic cytokine levels. The measurement of serum levels of inflammatory cyto-/chemokines has been suggested as a tool in the management of COVID-19. The aim of this study is to highlight the significance of measured levels of interleukin (IL)-1α, IL-1ß, IL-6, IL-8, IL-10, IL-12(p70), IL-27, interferon (IFN)γ, interferon gamma-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α in serum samples from infected and recovered subjects, possibly predictive of severity and/or duration of the disease. Serum samples from healthy (HD), positive at hospital admittance (T0), and recovered subjects (T1, 31-60, or 70-200 days post-negativization) were collected and tested through a bead-based cytometric assay and confirmed through ELISA. IL-10 levels were increased in the T0 group compared to both HD and T1. IL-27 significantly decreased in the 31-60 group. IL-1ß significantly increased in the 70-200 day group. TNF-α significantly decreased in T0 compared to HD and in the 31-60 group versus HD. IP-10 significantly increased in T0 compared to HD. These results suggest that IP-10 could represent an early marker of clinical worsening, whereas IL-10 might be indicative of the possible onset of post-COVID-19 long syndrome.
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A new generation of an FFP2 (Filtering Face Piece of type 2) smart face mask is achieved by integrating broadband hybrid nanomaterials and a self-assembled optical metasurface. The multifunctional FFP2 face mask shows simultaneously white light-assisted on-demand disinfection properties and versatile biosensing capabilities. These properties are achieved by a powerful combination of white light thermoplasmonic responsive hybrid nanomaterials, which provide excellent photo-thermal disinfection properties, and optical metasurface-based colorimetric biosensors, with a very low limit of pathogens detection. The realized system is studied in optical, morphological, spectroscopic, and cell viability assay experiments and environmental monitoring of harmful pathogens, thus highlighting the extraordinary properties in reusability and pathogens detection of the innovative face mask.
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BACKGROUND: CD20+ T cells represent up to 5% of circulating T lymphocytes. These cells have been shown to produce higher levels of IL-17A and IFN-γ than those of CD20- T lymphocytes. Some reports described the role of CD20+ T cells in autoimmune disorders such as multiple sclerosis and rheumatoid arthritis possibly due to their ability to produce these inflammatory cytokines. This study is aimed at describing the behavior of CD20+ T lymphocytes in the most frequent autoimmune disorder, i.e., Hashimoto's thyroiditis (HT), presenting isolated or associated to further autoaggressive disorders in a frame of poly-autoimmunity. METHODS: The study group encompasses 65 HT patients: 23 presenting in isolated form (IT) and 42 with an associated non-endocrine autoimmune disorder [16 with chronic atrophic gastritis (CAG), 15 with nonsegmental vitiligo (VIT), and 11 with celiac disease (CD)]. Twenty healthy donors act as control group (HD). Chronic use of interfering drugs, severe or chronic disorders, and pregnancy and lactation were used as exclusion criteria. Whole blood samples (100 µl) were stained with fluorescent-labeled antibodies (anti-CD45, anti-CD3, anti-CD19, anti-CD16, anti-CD56, anti-CD4, anti-CD8, anti-CD20). Red blood cells were then lysed by adding 1 ml of hypotonic buffer, and samples were acquired on a Flow Cytometer. RESULTS: CD3+CD8+CD20+ T lymphocytes' percentages, were significantly higher in the whole group of autoimmune patients compared to healthy donors (p = 0.0145). Dividing HT patients based on the type of presentation of autoimmune thyroiditis, CAG group showed the highest percentage of these cells as compared to HD and CD (p = 0.0058). IT patients showed higher percentages of CD3+ CD8+CD20+ cells than those of HD patients although not reaching statistical significance. However, dividing IT group based on thyroid function, hypothyroid patients showed higher CD8+CD20+ cell percentages than those of HD and euthyroid patients (p = 0.0111). Moreover, in IT patients, these cells were negatively correlated with FT4 levels (p = 0.0171; r = -0.4921). CONCLUSIONS: These preliminary findings indicate that CD8+CD20+ T cells are activated in patients with autoimmune thyroiditis and may behave differently according to the presence of poly-autoimmunity and hypothyroidism.
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OBJECTIVE: To assess the role of CD3+ CD20+ CD4- CD8- double-negative (DN) or CD3+CD20+ CD4/CD8+ T cells and the related pro-inflammatory cytokines in the humor aqueous, in mediating retinal microvascular changes in patients with chronic plaque-type moderate to severe psoriasis. DESIGN: A total of 76 patients (57.6 ± 11.7 years) with chronic plaque-type psoriasis were initially evaluated. Nineteen patients (19 eyes) and 19 healthy volunteers (19 eyes) were subjected to dermatological evaluation with Psoriasis Area Severity Index (PASI) and the Dermatology life quality index (DLQI). Retinal images were processed using an automatized software. On the same day, a venous sample was collected and analyzed using multiparametric flow cytometry. Three out of 6 patients who presented cataract, consented to perform surgery with humor aqueous collection. The samples were analyzed using a Multi-Analyte ELISA kit for the simultaneous quantification of IL1α, IL1ß, IL2, IL4, IL6, IL8, IL10, IL12, IL17A, IFNγ, TNF-α, GMCSF. RESULTS: The CD3+CD4+/CD8+CD20+CD56- T cells expression was greater in the psoriatic patients (+73.9%, P < 0.001) compared to controls, but not the DN T cells (-8.2%, P = 0.30). Ocular complications were diagnosed in 61.1% of patients, microvascular parameters including artero-venous ratio (P = 0.04), subfoveal choriocapillaris/Sattler's layer, and choroidal thickness (CT, both P < 0.001) were significantly altered in psoriasis subgroup. The increased circulating levels of the CD3+CD4+/CD8+CD20+CD56- T cells were associated with thinning of subfoveal CT (P = 0.03) and Haller's layer (P = 0.01). Instead, the DN T cells presented an inverse relationship with disease duration (P = 0.02), DLQI score (P = 0.02), and the use of biological therapy (P = 0.05). The related cytokine patterns possibly modified in this cellular context have been investigated. No significant differences were observed in cytokines levels between psoriasis and controls, the most significant difference was detected on IL-6, without reaching statistical significance (fold change of 1.4, P = 0.13). CONCLUSION: Our findings demonstrated that CD20+ T cell subpopulation is highly represented in psoriasis regardless of the use of immunomodulatory therapies, and the diffuse microvascular alterations suggested possible endothelial damage as mainstream for the genesis of psoriatic-mediated complications as further supported by the comparable concentrations of cytokines, at least as humor aqueous content, with respect to healthy eyes.
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Antígenos CD20/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Oftalmopatias/imunologia , Psoríase/imunologia , Vasos Retinianos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Doença Crônica , Oftalmopatias/etiologia , Oftalmopatias/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Psoríase/complicações , Psoríase/patologia , Vasos Retinianos/patologiaRESUMO
Psoriasis is a chronic inflammatory systemic disease caused by deregulation of the interleukin-23/-17 axis that allows the activation of Th17 lymphocytes and the reprogramming of keratinocytes proliferative response, thereby inducing the secretion of cyto-/chemokines and antimicrobial peptides. Beside cell-to-cell contacts and release of cytokines, hormones and second messengers, cells communicate each other through the release of extracellular vesicles containing DNA, RNA, microRNAs and proteins. It has been reported the alteration of extracellular vesicles trafficking in several diseases, but there is scarce evidence of the involvement of extracellular vesicles trafficking in the pathogenesis of psoriasis. The main goal of the study was to characterize the release, the cargo content and the capacity to transfer bioactive molecules of extracellular vesicles produced by keratinocytes following recombinant IL-17A treatment if compared to untreated keratinocytes. A combined approach of standard ultracentrifugation, RNA isolation and real-time RT-PCR techniques was used to characterize extracellular vesicles cargo. Flow cytometry was used to quantitatively and qualitatively analyse extracellular vesicles and to evaluate cell-to-cell extracellular vesicles transfer. We report that the treatment of human keratinocytes with IL-17A significantly modifies the extracellular vesicles cargo and release. Vesicles from IL-17A-treated cells display a specific pattern of mRNA which is undid by IL-17A neutralization. Extracellular vesicles are taken up by acceptor cells irrespective of their content but only those derived from IL-17A-treated cells enable recipient cells to express psoriasis-associated mRNA. The results imply a role of extracellular vesicles in amplifying the pro-inflammatory cascade induced in keratinocyte by pro-psoriatic cytokines.
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Vesículas Extracelulares/efeitos dos fármacos , Interleucina-17/farmacologia , Queratinócitos/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Transformada , Quimiocina CCL20/biossíntese , Quimiocina CCL20/genética , Quimiocinas CXC/biossíntese , Quimiocinas CXC/genética , Endocitose , Vesículas Extracelulares/metabolismo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/biossíntese , Interleucina-6/genética , Queratinócitos/metabolismo , Tamanho da Partícula , Psoríase/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia , Succinimidas/metabolismo , beta-Defensinas/biossíntese , beta-Defensinas/genéticaRESUMO
The connection between chronic inflammation and risk of cancer has been supported by several studies. The development of cancer might be a process driven by the presence of a specific combination of inflammatory mediators, including cytokines, chemokines and enzymes, in the tumor microenvironment. Virus-induced tumors, like HPV-induced Squamous Cell Carcinomas, represent a paradigmatic example of the interplay between inflammation, as integral part of the innate antiviral response, and malignant transformation. Here, the role of inflammatory microenvironment in the HPV-induced carcinogenesis is addressed, with a specific focus on the involvement of the immune molecules as well as their delivery through the microvesicle cargo possibly correlated to the different HPV genotype. The expression of the inflammatory mediators in HPV positive cells has been analyzed in primary human foreskin keratinocytes and keratinocytes transduced by E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 genotypes. HPV E6 and E7 proteins can modulate the expression of immune mediators in HPV-infected cells and can affect the levels of immune molecules, mainly chemokines, in the extracellular milieu. HPV-16 E6 and E7 oncoproteins have been silenced to confirm the specificity of the modulation of the inflammatory microenvironment. Our results suggest that the expression of HPV oncoproteins allows the modification of the tumor milieu through the synthesis and release of specific pro-inflammatory cytokines and chemokines, affecting the efficacy of the immune response. The microenvironment can also be conditioned by an altered mRNA cargo delivered by extracellular vesicles, thereby efficiently affecting the surrounding cells with possible implication for tumorigenesis and tumor diagnosis.
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Microambiente Celular , Vesículas Extracelulares/metabolismo , Mediadores da Inflamação/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular , Inativação Gênica , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Hashimoto thyroiditis (HT) may occur isolated or associated with other non-endocrine autoimmune disorders (NEAD). No data are available about Breg cells in these disorders and this represented the aim of the study. Th17 and Breg cells subset were characterized on peripheral blood mononuclear cells isolated from 18 healthy donors (HD), 19 patients with isolated HT and 26 patients with HT+NEAD. Th17 were higher in patients with isolated HT than in HD but no further changes were seen in patients with HT+NEAD. CD24hiCD38hi unstimulated Breg cells were similar in HT patients and in HD, but significantly higher in patients with HT+NEAD than in both HT and in HD. CD19+CD24hiCD27+ Breg memory phenotype was similar in HD and in HT patients, but decreased in patients with HT+NEAD (23.4%vs38.5%). Upon CpG-stimulation, CD24hiCD38hi IL-10+ Breg cells were higher in HT patients than in HD (3.9%vs1.8%) but similar in patients with HT+NEAD (2.4%).
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Linfócitos B Reguladores/imunologia , Doença Celíaca/imunologia , Gastrite Atrófica/imunologia , Doença de Hashimoto/imunologia , Células Th17/imunologia , Vitiligo/imunologia , ADP-Ribosil Ciclase 1/imunologia , Adulto , Antígenos CD19/imunologia , Doenças Autoimunes/complicações , Doenças Autoimunes/imunologia , Antígeno CD24/imunologia , Estudos de Casos e Controles , Doença Celíaca/complicações , Feminino , Gastrite Atrófica/complicações , Doença de Hashimoto/complicações , Humanos , Interleucina-10/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , Pessoa de Meia-Idade , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Vitiligo/complicaçõesRESUMO
Human Papilloma Viruses (HPVs) are the causative agents of cervical cancer although other types of cancers are associated with HPV infection. Type I Interferons can interfere with HPV E6- and/or E7-dependent transformation and can affect microRNA (miRNA) expression. Cancer cells show a specific pattern of miRNA expression and HPVs are able to modulate miRNAs expressed in infected cells. Keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38) were studied to analyze the involvement of HPV oncoproteins in the anti-proliferative activity of IFN-ß. In view of our previous data showing senescence induction by the cytokine in K38 cells, we observe that IFN-ß treatment leads to p53-indipendent apoptosis in K16 cells whereas induces senescence in K16 cells if E6 is silenced and p53 expression is restored. The levels of selected miRNAs, deregulated in K16 and K38 cells, can be modulated by IFN-ß when E6 and E7 proteins of HPV-16, but not HPV-38, are expressed.
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Apoptose/efeitos dos fármacos , Papillomavirus Humano 16/metabolismo , Interferon beta/farmacologia , Queratinócitos/metabolismo , MicroRNAs/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/metabolismo , Apoptose/genética , Linhagem Celular Transformada , Papillomavirus Humano 16/genética , Humanos , Queratinócitos/patologia , Queratinócitos/virologia , MicroRNAs/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Oxidative stress represents a hallmark for many degenerative pathologies of the Central Nervous System. Throughout life, the constant pressure of noxious stimuli and/or episodes of traumatic events may expose the brain to a microenvironment where the non-balanced reactive oxygen species inevitably lead to neuronal loss and cognitive decline. HO-1, a 32 kDa heat-shock protein catalyzing the degradation of heme into carbon monoxide (CO), iron and biliverdin/bilirubin is considered one of the main antioxidant defense mechanisms playing pivotal roles in neuroprotection. Restoring the redox homeostasis is the goal of many natural or synthetic antioxidant molecules pursuing beneficial effects on brain functions. Here, we investigated the antioxidant capacity of four selected benzofuran-2-one derivatives in a cellular model of neurodegeneration represented by differentiated SH-SY5Y cells exposed to catechol-induced oxidative stress. Our main results highlight how all the molecules have antioxidant properties, especially compound 9, showing great abilities in reducing intracellular ROS levels and protecting differentiated SH-SY5Y cells from catechol-induced death. This compound above all seems to boost HO-1 mRNA and perinuclear HO-1 protein isoform expression when cells are exposed to the oxidative insult. Our findings open the way to consider benzofuran-2-ones as a novel and promising adjuvant antioxidant strategy for many neurodegenerative disorders.
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Psoriasis is a chronic inflammatory disease affecting skin and joints characterized by a chronically altered immune and inflammatory response. Several factors occur from the onset to the development of this disease due to different types of cells spatially and temporally localized in the affected area, such as, keratinocytes, macrophages, neutrophils and T helper lymphocytes. This scenario leads to the chronic release of high levels of inflammatory mediators (i.e., IL-17, IL-23, IL-22, TNF-α, S100 proteins, Defensins) and lastly parakeratosis and thickening of the stratum spinosum. Extracellular vesicles (EVs) are small double membraned biological nanoparticles that are secreted by all cell types and classified, based on dimension and biogenesis, into exosomes, microvesicles and apoptotic bodies. Their role as vessels for long range molecular signals renders them key elements in the pathogenesis of psoriasis, as well as innovative platforms for potential biomarker discovery and delivery of fine-tuned anti-inflammatory therapies. In this review, the role of EVs in the pathogenesis of psoriasis and the modulation of cellular microenvironment has been summarized. The biotechnological implementation of EVs for therapy and research for new biomarkers has been also discussed.
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Biomarcadores , Vesículas Extracelulares , Psoríase , Humanos , Psoríase/imunologia , Psoríase/metabolismo , Psoríase/etiologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Animais , Pele/patologia , Pele/imunologia , Pele/metabolismo , Microambiente Celular/imunologiaRESUMO
BACKGROUND: Low-dose-medicine is based on the administration of low doses of biological regulators to restore the immunologic balance altered in the disease. Cytokines are pivotal regulators of cellular and tissue functions and impaired crosstalk, due to an imbalance between specific cytokines, it is fundamental in acute inflammation and diseases correlated to low-grade chronic inflammation. Osteoarthritis is the most prevalent arthritic disease and a leading cause of disability. In the treatment of muscle-skeletal pathologies, the therapeutic integration of conventional medicine with homotoxicology, or low-dose-medicine appears to be beneficial. OBJECTIVE: This study aims to get more insights into the role of inflammatory cytokines and chemokines during the development of osteoarthritis and to evaluate a possible blocking strategy using anti-inflammatory molecules, we resort to an in vitro experimental model using an established human chondrosarcoma cell line that underwent to a well known pro-inflammatory stimulus as bacterial lipopolysaccharide. METHOD: We tested the production of inflammatory-related cytokines and chemokines, and the efficacy of low-dose (LD) administration of anti-inflammatory compounds, namely IL-10 and anti-IL-1, to block inflammatory cellular pathways. RESULTS: Following an inflammatory insult, chondrocytes upregulated the expression of several pro-inflammatory cyto-/chemokines and this induction could be counteracted by LD IL-10 and anti-IL-1. We reported that these effects could be ascribed to an interfering effect of LD drugs with the NF-κB signaling. CONCLUSION: Our results provided a good indication that LD drugs can be effective in inhibiting the inflammatory response in chondrocytes opening the way to new therapies for the treatment of diseases such as osteoarthritis.
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Actinic keratosis (AK) is a carcinoma in situ precursor of cutaneous squamous cell carcinoma (cSCC), the second most common cancer affecting the Caucasian population. AK is frequently present in the sun-exposed skin of the elderly population, UV radiation being the main cause of this cancer, and other risk factors contributing to AK incidence. The dysregulation of microRNAs (miRNAs) observed in different cancers leads to an improper expression of miRNA targets involved in several cellular pathways. The TaqMan Array Human MicroRNA Card assay for miRNA expression profiling was performed in pooled AK compared to healthy skin scraping samples from the same patients. Forty-three miRNAs were modulated in the AK samples. The expression of miR-19b (p < 0.05), -31, -34a (p < 0.001), -126, -146a (p < 0.01), -193b, and -222 (p < 0.05) was validated by RT-qPCR. The MirPath tool was used for MiRNA target prediction and enriched pathways. The top DIANA-mirPath pathways regulated by the targets of the 43 miRNAs are TGF-beta signaling, Proteoglycans in cancer, Pathways in cancer, and Adherens junction (7.30 × 10-10 < p < 1.84 × 10-8). Selected genes regulating the KEGG pathways, i.e., TP53, MDM2, CDKN1A, CDK6, and CCND1, were analyzed. MiRNAs modulated in AK regulate different pathways involved in tumorigenesis, indicating miRNA regulation as a critical step in keratinocyte cancer.
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Purpose: Extracellular Vesicles (EVs) are a heterogeneous group of cell-derived membranous nanoparticles involved in several physiopathological processes. EVs play a crucial role in the definition of the extracellular microenvironment through the transfer of their cargo. Psoriasis is a prototypical chronic inflammatory disease characterized by several secreted mediators, among which antimicrobial peptides (AMPs) are considered pivotal in the development of the psoriatic inflammatory microenvironment. The role of EVs in the pathogenesis of psoriasis has not been elucidated yet, even if emerging evidence demonstrated that interleukin-17A (IL-17A), the psoriasis-related principal cytokine, modifies EVs release and cargo content. The aim of this work was to analyze whether, besides IL-17A, other psoriasis-related cytokines (ie, IFN-γ, TNF-α, IL-22 and IL-23) could affect EVs release and their AMPs mRNAs cargo as well as to analyze the potential biological effect due to EVs internalization by different acceptor cells. Methods: Nanoparticle tracking analysis (NTA) was performed on supernatants of HaCaT cells stimulated with IL-17A, IFN-γ, TNF-α, IL-22 or IL-23 to enumerate EVs. Real-Time RT-PCR was used for gene expression analysis in cells and EVs. Confocal microscopy and Flow cytometry were used to, respectively, study Netosis and EVs internalization. Results: IL-17A and IFN-γ increased EVs release by HaCaT cells. All the tested cytokines modulated AMPs mRNA expression in parental cells and in their respective EVs. S100A12 and hBD2 mRNAs were upregulated following IL-17A and IL-22 treatments. Interestingly, EVs derived from cytokine treated HaCaT cells induced Netosis in freshly isolated neutrophils. Upregulation of S100A12 and hBD2 mRNA was also detectable in acceptor cells incubated with EVs derived from cells treated with psoriasis-related cytokines. Conclusion: The obtained results highlighted the role of EVs in the composition of psoriasis-associated secretome and microenvironment also suggesting the EV involvement in the spreading of the disease mediators and in the possible associated comorbidities.
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Systemic sclerosis (SSc) is a systemic autoimmune disease in which gastrointestinal disorders represent a complication in up to 90% of patients. SSc may associate with thyroid autoimmune disorders, with Hashimoto's thyroiditis (HT) being the more prevalent worldwide. Previous studies have examined the behavior of Th17 lymphocytes and Breg cells in patients with HT and concomitant autoimmune organ-specific disorders. These immune phenotypes seem to play a significant role in the pathogenesis of both these autoimmune processes, but their behavior when these two disorders coexist has not been described. We analyzed Th17 and Breg (CD24hiCD38hi) cell subsets in 50 subjects (45F/5M; median age = 49 years): 18 were healthy donors (HD), 20 had isolated HT, and 12 had SSc, seven of whom had both HT and SSc. Breg cells' function was also evaluated by measuring their IL-10 production when stimulated by specific activators. An increased percentage of Th17 lymphocytes characterized HT patients as compared to both HD and the whole group of SSc patients (p = 0.0018). On the contrary, the percentage of unstimulated Breg cells in SSc patients was higher (p = 0.0260), either associated or not with HT, as compared to both HT patients and HD, which, instead, showed a similar percentage of Breg cells. Following a specific stimulation with CpG, the percentages of Breg cells were increased in the whole sample of SSc patients (p < 0.001) as well as in isolated SSc and in SSc+HT ones as compared to isolated HT. However, qualitative analysis, obtained through the detection of the IL-10-producing phenotype, revealed that the percentage of CpG-stimulated CD24hiCD38hi-IL10+cells was significantly decreased in SSc patients (p < 0.0001) with no difference between isolated SSc and SSc+HT patients. The IL-10-producing phenotype was instead slightly increased in HT patients as compared to HD (4.1% vs. 2.8%). The presence of SSc seems to be characterized by an enrichment of total Breg cells but by a reduced Breg IL-10-producing phenotype, representing functional Bregs. This last finding was entirely due to the presence of SSc independently from the association with HT. This behavior is different from the ones described about the association of HT with organ-specific autoimmune disorders.
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Doenças Autoimunes , Linfócitos B Reguladores , Doença de Hashimoto , Escleroderma Sistêmico , Doenças Autoimunes/patologia , Humanos , Interleucina-10RESUMO
BACKGROUND: The ß3 human papillomavirus (HPV)49 induces immortalization of primary keratinocytes through the action of E6 and E7 oncoproteins with an efficiency similar to alpha high risk (HR)-HPV16. Since HR-HPV oncoproteins are known to alter microRNA (miRNA) expression and extracellular vesicle (EV) production, we investigated the impact of HPV49 E6 and E7 proteins on miRNA profile and EV expression, and their involvement in the control of cell proliferation. METHODS: The miRNA expression was evaluated by a miRNA array and validated by RT-qPCR in primary human keratinocytes immortalized by ß3 HPV49 (K49) or α9 HR-HPV16 (K16), and in EVs from K49 and K16. The modulation of miRNA target proteins was investigated by immunoblotting analyses. RESULTS: By comparing miRNA expression in K49 and K16 and the derived EVs, six miRNAs involved in HPV tumorigenesis were selected and validated. MiR-19a and -99a were found to be upregulated and miR-34a downregulated in both cell lines; miR-17 and -590-5p were upregulated in K49 and downmodulated in K16; miR-21 was downregulated only in K16. As for EV-carried miRNAs, the expression of miR-17, -19a, -21 and -99a was decreased and miR-34a was increased in K49 EVs. In K16 EVs, we revealed the same modulation of miR-19a, -34a, and -99a observed in producing cells, while miR-21 was upregulated. Cyclin D1, a common target of the selected miRNAs, was downmodulated in both cell lines, whereas cyclin-dependent kinase 4 was down-modulated in K49 but upregulated in K16. CONCLUSION: These data suggest that E6 and E7 proteins of ß3 HPV49 and α9 HR-HPV16 affect key factors of cell cycle control by indirect mechanisms based on miRNA modulation.
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BACKGROUND: Actinic keratosis (AK) is a precursor of cutaneous squamous cell carcinoma (cSCC). UV radiation is the major risk factor for AK, but certain human papillomaviruses (HPVs) of the beta genus are also involved in its development. Differently, the role of polyomaviruses (PyVs) in skin carcinogenesis is still debated. Fiftheen PyVs have been isolated from human tissues so far, including Merkel cell polyomavirus (MCPyV), the aetiological agent of Merkel cell carcinoma. METHODS: The presence of 13 PyVs was assessed in skin samples from AK patients (n = 342). Matched fresh-frozen scrapings from healthy skin (HS) and AK lesions from 242 patients, and formalin-fixed paraffin-embedded AK biopsies from a different cohort of 100 patients were analyzed by multiplex PyVs genotyping assay. RESULTS: The most frequent lesion site was the scalp in men (27.3%), and the cheek area in women (29.0%). Differences between men and women were significant for the scalp, the cheek area and the lips. Almost all the scrapings were PyV-positive (HS: 89.7%, AK: 94.6%; p = 0.04). The three most frequent PyVs were MCPyV, HPyV6 and JCPyV (HS: 87.2%, 58.7%, 6.6%, respectively; AK: 88.8%, 51.2%, 9.9%, respectively). HPyV9, TSPyV, BKPyV, HPyV7, LIPyV and SV40 were detected in < 2% of the scrapings. In most cases, matched HS and AK scrapings were both positive (MCPyV: 78.1%, HPyV6: 41.7%), or both negative for the individual genotypes (for the remaining PyVs). PyV prevalence in AK biopsies was 22.0%. Only MCPyV (21.0%) and HPyV6 (3.0%) were detected in these samples. CONCLUSIONS: PyV prevalence in HS and AK scrapings was high, but detection of PyVs exclusively in AK scrapings was rare. PyV positivity rate in AK biopsies was modest. Further research is need to reach firm conclusions regarding the role of these viruses in AK development.
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Mycobacterium bovis (M. bovis) is the causative agent of animal tuberculosis (bTB), infecting and causing disease in several animal species. In areas where there are complex interactions between reservoir hosts and susceptible species, the control of this pathogen is a challenge. The authors report two outbreaks of goat tuberculosis caused by M. bovis in multi-host ecosystems within two protected natural areas of Sicily, where TB is historically endemic. The first outbreak (Farm A) was identified after the incidental detection at the slaughterhouse of TB-like lesions in goat viscera ready to be disposed. Single intradermal cervical tuberculin test (SICT) was performed in Farm A on 205 goats, resulting positive in 10 (4.9%). After slaughtering, six out of ten animals showed TB-like lesions, from which M. bovis spoligotype SB0841 was isolated. The typing did not reveal any epidemiological connection with the neighboring cattle, suggesting that free-ranging type of management exposed the affected goat livestock or wildlife infected with other strains. The second outbreak (Farm B) was detected in a mixed farm (bovine, caprine, and ovine), where relapsing outbreaks of TB in cattle were registered in the previous years after performing the SICT in cohabiting goats. SICT resulted positive in 6/153 (3.9%), and two animals showed bTB-like lesions. No mycobacteria were cultured, and the final diagnosis of TB was achieved by histopathology and immunohistochemistry. The reported outbreaks highlight the importance of assessing the epidemiological, diagnostic, and regulatory critical issue, which is fundamental to optimizing the strategies of eradicating TB in the endemic multi-host ecosystem described.
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OBJECTIVES: Extracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here, we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL. METHODS: We tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL-derived EVs. RESULTS: A subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in human umbilical vein endothelial cells (HUVECs) and increasing the angiogenic tubularly-like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched with small RNAs and a high amount of miR-126, the most abundant angiogenic miRNA in platelets. The transfer of miR-126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as a downstream target in this system. CONCLUSION: PL is a biological source of available EVs with angiogenic effects involving a miRNAs-based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs.