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Mpox (monkeypox) is a zoonotic viral disease caused by the mpox virus (MPXV). Recently in 2022, a multi-country Mpox outbreak has determined great concern as the disease rapidly spreads. The majority of cases are being noticed in European regions and are unrelated to endemic travel or known contact with infected individuals. In this outbreak, close sexual contact appears to be important for MPXV transmission, and an increasing prevalence in people with multiple sexual partners and in men who have sex with men has been observed. Although Vaccinia virus (VACV)-based vaccines have been shown to induce a cross-reactive and protective immune response against MPXV, limited data support their efficacy against the 2022 Mpox outbreak. Furthermore, there are no specific antiviral drugs for Mpox. Host-cell lipid rafts are small, highly dynamic plasma-membrane microdomains enriched in cholesterol, glycosphingolipids and phospholipids that have emerged as crucial surface-entry platforms for several viruses. We previously demonstrated that the antifungal drug Amphotericin B (AmphB) inhibits fungal, bacterial and viral infection of host cells through its capacity to sequester host-cell cholesterol and disrupt lipid raft architecture. In this context, we discuss the hypothesis that AmphB could inhibit MPXV infection of host cells through disruption of lipid rafts and eventually through redistribution of receptors/co-receptors mediating virus entry, thus representing an alternative or additional therapeutic tool for human Mpox.

Several flaviviruses such as Hepatitis C virus, West Nile virus, Dengue virus and Japanese Encephalitis virus exploit the raft platform to enter host cells whereas the involvement of lipid rafts in Zika virus–host cell interaction has not yet been demonstrated. Zika virus disease is caused by a flavivirus transmitted by Aedes spp. Mosquitoes, although other mechanisms such as blood transfusion, sexual and maternal–fetal transmission have been demonstrated. Symptoms are generally mild, such as fever, rash, joint pain and conjunctivitis, but neurological complications, including Guillain-Barré syndrome, have been associated to this viral infection. During pregnancy, it can cause microcephaly and other congenital abnormalities in the fetus, as well as pregnancy complications, representing a serious health threat. In this study, we show for the first time that Zika virus employs cell membrane lipid rafts as a portal of entry into Vero cells. We previously demonstrated that the antifungal drug Amphotericin B (AmphB) hampers a microbe–host cell interaction through the disruption of lipid raft architecture. Here, we found that Amphotericin B by the same mechanism of action inhibits both Zika virus cell entry and replication. These data encourage further studies on the off-label use of Amphotericin B in Zika virus infections as a new and alternate antiviral therapy.

Silibinin, a natural polyphenolic flavonoid, is known to possess anti-inflammatory, anticancer, antioxidant, and immunomodulatory properties. However, the effects of Silibinin on the maturation and immunostimulatory functions of human dendritic cells (DC) remain to be elucidated. In this study, we have attempted to ascertain whether Silibinin influences the maturation, cytokine production, and antigen-presenting capacity of human monocyte-derived DC. We show that Silibinin significantly suppresses the upregulation of costimulatory and MHC molecules in LPS-stimulated mature DC and inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-12, IL-23, and TNF-α production. Furthermore, Silibinin impairs the proliferation response of the allogenic memory CD4 T lymphocytes elicited by LPS-matured DC and their Th1/Th17 profile. These findings demonstrate that Silibinin displays immunosuppressive activity by inhibiting the maturation and activation of human DC and support its potential application of adjuvant therapy in the treatment of autoimmune diseases.

Hypovitaminosis D is involved in various inflammatory, infectious and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Moreover, the active form of vitamin D, calcitriol, has been shown to modulate the immune response, playing an anti-inflammatory effect. However little is known about the mechanisms underlying this anti-inflammatory effect and the potential sex differences of calcitriol immune regulation. Hence, the aim of this study was to investigate whether calcitriol could act differently in modulating T cell immunity of age-matched male and female healthy donors. We analyzed the effects of calcitriol in T lymphocytes from healthy women and men on the expression levels of the vitamin D receptor (VDR) and pro- and anti-inflammatory cytokine production. We showed that a treatment with calcitriol induced a significant increase in the VDR expression levels of activated T lymphocytes from male and female healthy subjects. Moreover, we found that calcitriol significantly reduced the expression level of pro-inflammatory cytokines IL-17, INF-γ and TNF-α in the T lymphocytes of both sexes. Notably, we observed that calcitriol induced a significant increase in the expression level of anti-inflammatory cytokine IL-10 only in the T lymphocytes from female healthy donors. In conclusion, our study provides new insights regarding the sex-specific anti-inflammatory role of calcitriol in T cell immunity.

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention.

Background Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0–6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions A crucial step of melanoma progression does occur at melanoma intermediate –stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement. Electronic supplementary material The online version of this article (10.1186/s13046-018-0915-z) contains supplementary material, which is available to authorized users.

Caveolae have been indicated as a center of cytoskeleton regulation for Src kinase/Rho GTPase signaling. In addition, Src recruitment on intact cortical actin cytoskeleton appears to be required for bFGF/FGFR signal activation. Recently, we established a relationship between caveolin-1 (Cav-1) expression and cell migration in human malignant melanoma, constitutively activated by a bFGF autoregulatory loop. This work intends to investigate whether caveolae's asset, through bFGF/FGFR/c-Src/Rho signaling, could be related to melanoma cell anchorage. Accordingly, we revealed the existence of a FGFR/Src kinase pathway in Cav-1 enriched detergent-resistant membranes (DRMs) of Me665/1 metastatic melanoma cells, as confirmed by FGFR silencing. Moreover, we determined the expression and phosphorylation levels of Cav-1/Src/Erk signal pathway as a function of FGFR activation and cell density. A sucrose density gradient ultracentrifugation was employed to monitor Cav-1 membrane association and buoyancy in Me665/1 cells treated for actin fragmentation or for altered phosphorylation signals. As a result, melanoma cells show remarkable resistance to Cav-1 disassembly, together with persisting cell signal activity, being Src and Cav-1 crucial modulators of Rho GTPases. In conclusion, our study primarily highlights, in a metastatic melanoma cell line expressing caveolin, the circumstances whereby caveola structural and functional endurance enables the FGFR/Src/Rho GTPases pathway to keep on cell progression.

Caveolin-1 (Cav-1), a member of the caveolin family, regulates caveolae-associated signaling proteins, which are involved in many biological processes, including cancer development. Cav-1 was found to exert a complex and ambiguous role as oncogene or tumor suppressor depending on the cellular microenvironment. Here we investigated Cav-1 expression and function in a panel of melanomas, finding its expression in all the cell lines. The exception was the primary vertical melanoma cell line, WM983A, characterized by the lack of Cav-1, and then utilized as a recipient for Cav-1 gene transduction to address a series of functional studies. The alleged yet controversial role of phospho (Ph)-Cav-1 on cell regulation was also tested by transducing the nonphosphorylatable Cav-1Y14A mutant. Wild-type Cav-1, but not mutated Cav-1Y14A, increased tumorigenicity as indicated by enhanced proliferation, migration, invasion and capacity of forming foci in semisolid medium. Accordingly, Cav-1 silencing inhibited melanoma cell growth reducing some of the typical traits of malignancy. Finally, we detected a secreted fraction of Cav-1 associated with cell released microvesicular particles able to stimulate in vitro anchorage independence, migration and invasion in a paracrine/autocrine fashion and, more important, competent to convey metastatic asset from the donor melanoma to the less aggressive recipient cell line. A direct correlation between Cav-1 levels, the amount of microvesicles released in the culture medium and MMP-9 expression was also observed.

Recent research has revealed that propranolol, a beta-adrenoceptor antagonist, causes extracellular signal-regulated kinase (ERK) cascade activation, nuclear translocation of phospho-ERK and increased transcriptional activity in cultured cell lines. Given the importance of beta-adrenoceptor antagonists in the treatment of heart failure, we evaluated the capability of propranolol of promoting the ERK-dependent gene expression at the cardiomyocyte level. To this end, the gene expression of the early growth response factor 1 (Egr1), a well-recognized indicator of nuclear extracellular signal-regulated kinase 1/2 (ERK1/2) activation, was assessed by quantitative real-time RT-PCR in vivo as well as in vitro experiments. Propranolol, administered at the dose of 10 mg/kg/day in C57BL/6 mice, caused a approximately 19-fold increase of Egr1 mRNA expression in left ventricular myocardium along with a approximately 2.1-fold increase of Egr1 protein expression. Isoproterenol, a nonselective beta-adrenoceptor agonist, also increased Egr1 mRNA and protein expression but to a lesser degree. Remarkably, isoproterenol administration was associated with the development of cardiac hypertrophy, whereas propranolol-treated mice showed a completely normal cardiac morphology. The effect of propranolol on Egr1 mRNA expression was abrogated in mice lacking beta(1)- and beta(2)-adrenoceptors indicating that propranolol increases Egr1 mRNA expression in a beta-adrenoceptor-dependent manner. The role of beta-adrenoceptors was further confirmed by showing that propranolol was able to increase Egr1 mRNA and protein levels in cultured neonatal cardiomyocytes. Collectively, these results indicate that propranolol promotes Egr1 gene expression in cardiomyocytes via beta-adrenoceptors with a mechanism which is independent of its ability to antagonize the effects of catecholamines. It is also suggested that cardiomyocyte growth and Egr1 gene overexpression are not obligate processes.

Transferrin receptor 2 (TfR2) possesses a YQRV motif similar to the YTRF motif of transferrin receptor 1 (TfR1) responsible for the internalization and secretion through the endosomal pathway. Raft biochemical dissection showed that TfR2 is a component of the low-density Triton-insoluble (LDTI) plasma membrane domain, able to co-immunoprecipitate with caveolin-1 and CD81, two structural raft proteins. In addition, subcellular fractionation experiments showed that TfR1, which spontaneously undergoes endocytosis and recycling, largely distributed to intracellular organelles, whereas TfR2 was mainly associated with the plasma membrane. Given the TfR2 localization in lipid rafts, we tested its capability to activate cell signalling. Interaction with an anti-TfR2 antibody or with human or bovine holotransferrin showed that it activated ERK1/ERK2 and p38 MAP kinases. Integrity of lipid rafts was required for MAPK activation. Co-localization of TfR2 with CD81, a raft tetraspanin exported through exosomes, prompted us to investigate exosomes released by HepG2 and K562 cells into culture medium. TfR2, CD81 and to a lesser extent caveolin-1, were found to be part of the exosomal budding vesicles. In conclusion, the present study indicates that TfR2 localizes in LDTI microdomains, where it promotes cell signalling, and is exported out of the cells through the exosome pathway, where it acts as an intercellular messenger.

Although HIV uses CD4 and coreceptors (CCR5 and CXCR4) for productive infection of T cells, glycosphingolipids (GSL) may play ancillary roles in lymphoid and non-lymphoid cells. Interactions of the HIV Envelope Glycoprotein (Env) with GSL may help HIV in various steps of its pathogenesis. Physical-chemical aspects of the interactions between HIV Env and GSL leading to CD4-dependent entry into lymphocytes, the role of GSL in HIV transcytosis, and CD4-independent entry into non-lymphoid cells are reviewed. An overview of signaling properties of HIV receptors is provided with some speculation on how GSL may play a role in these events by virtue of being in membrane rafts. Finally, we summarize how interactions between HIV and coreceptors leading to signaling and/or fusion can be analyzed by the use of various tyrosine kinase and cytoskeletal inhibitors. Published in 2004.

Skeletal muscle tissue is one of the main sites where glucose uptake occurs in response to insulin. The glucose transporter type-4 (GLUT4) is primarily responsible for the insulin-stimulated increase in glucose uptake. Upon insulin stimulation, GLUT4 is recruited from intracellular reserves to the plasma membrane. The molecular mechanisms that regulate the translocation of GLUT4 to the sarcolemma remain to be fully identified. Here, we demonstrate that GLUT4 is localized to perinuclear stores that contain flotillin-1, a marker of lipid rafts, in skeletal muscle cells. Stimulation with insulin for 10 min results in the translocation of flotillin-1/GLUT4-containing domains to the plasma membrane in a PI3K- and PKCzeta-dependent manner. We also demonstrate that caveolin-3, a marker of caveolae, is required for the insulin receptor-mediated activation of the PI3K-dependent pathway, which occurs 2 min after insulin stimulation. In fact, we demonstrate that lack of caveolin-3 significantly reduces insulin-stimulated glucose uptake in caveolin-3 null myotubes by inhibiting both PI3K and Akt, as well as the movement of GLUT4 to the plasma membrane. Interestingly, caveolin-3 moves away from the plasma membrane toward the cytoplasm 5 min after insulin stimulation and temporarily interacts with flotillin-1/GLUT4-containing domains before they reach the sarcolemma, with the consequent movement of the insulin receptor from caveolin-3-containing domains to flotillin-1-containing domains. Such translocation temporally matches the insulin-stimulated movement of Cbl and CrkII in flotillin-1/GLUT4-containing domains, as well as the activation of the GDP-GTP exchange factor C3G. Disruption of flotillin-1-based domains prevents the activation of C3G, movement of GLUT4 to the sarcolemma, and glucose uptake in response to insulin. Thus, the activation of the Cbl/C3G/TC10-dependent pathway, which occurs before flotillin-1/GLUT4-containing domains reach the plasma membrane, is flotillin-1 mediated and follows the activation of the PI3K-mediated signaling. Taken together, these results indicate that flotillin-1 and caveolin-3 may regulate muscle energy metabolism through the spatial and temporal segregation of key components of the insulin signaling.

One of the key oncogenic pathways involved in melanoma aggressiveness, development and progression is the RAS/BRAF/MEK pathway, whose alterations are found in most patients. These molecular anomalies are promising targets for more effective anti-cancer therapies. Some Mek inhibitors showed promising antitumor activity, although schedules and doses associated with low systemic toxicity need to be defined. In addition, it is now accepted that cancers can arise from and be maintained by the cancer stem cells (CSC) or tumor-initiating cells (TIC), commonly expanded in vitro as tumorspheres from several solid tumors, including melanoma (melanospheres). Here, we investigated the potential targeting of MEK pathway by exploiting highly reliable in vitro and in vivo pre-clinical models of melanomas based on melanospheres, as melanoma initiating cells (MIC) surrogates. MEK inhibition, through PD0325901, provided a successful strategy to affect survival of mutated-BRAF melanospheres and growth of wild type-BRAF melanospheres. A marked citotoxicity was observed in differentated melanoma cells regardless BRAF mutational status. PD0325901 treatment, dramatically inhibited growth of melanosphere-generated xenografts and determined impaired tumor vascularization of both mutated- and wild type-BRAF tumors, in the absence of mice toxicity. These results suggest that MEK inhibition might represent a valid treatment option for patients with both mutated- or wild type-BRAF melanomas, affecting tumor growth through multiple targets.

The treatment with beta-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated beta-adrenergic signaling antagonizes alpha(1)-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the beta-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an alpha(1)-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of beta-ARs with isoproterenol, a beta-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to alpha(1)-ARs, cardiomyocytes were treated with prostaglandin F(2)alpha, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of beta-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using beta1- and beta2-AR double knockout mice, in which the predominant cardiac beta-AR subtypes are lacking, or by administering isoproterenol (ISO), a beta-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in beta(1)- and beta(2)-AR gene deficient mice. Conversely, we found that ANP, beta-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated beta-adrenergic signaling negatively regulates Gq cascade activation-induced fetal gene expression in cultured cardiomyocytes and that this inhibitory regulation is already operative in the mouse heart under physiological conditions.