Persistence of SARS-CoV-2 infection and viral intra- and inter-host evolution in COVID-19 hospitalized patients.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) persistence in COVID-19 patients could play a key role in the emergence of variants of concern. The rapid intra-host evolution of SARS-CoV-2 may result in an increased transmissibility, immune and therapeutic escape which could be a direct consequence of COVID-19 epidemic currents. In this context, a longitudinal retrospective study on eight consecutive COVID-19 patients with persistent SARS-CoV-2 infection, from January 2022 to March 2023, was conducted. To characterize the intra- and inter-host viral evolution, whole genome sequencing and phylogenetic analysis were performed on nasopharyngeal samples collected at different time points. Phylogenetic reconstruction revealed an accelerated SARS-CoV-2 intra-host evolution and emergence of antigenically divergent variants. The Bayesian inference and principal coordinate analysis analysis showed a host-based genomic structuring among antigenically divergent variants, that might reflect the positive effect of containment practices, within the critical hospital area. All longitudinal antigenically divergent isolates shared a wide range of amino acidic (aa) changes, particularly in the Spike (S) glycoprotein, that increased viral transmissibility (K417N, S477N, N501Y and Q498R), enhanced infectivity (R346T, S373P, R408S, T478K, Q498R, Y505H, D614G, H655Y, N679K and P681H), caused host immune escape (S371L, S375F, T376A, K417N, and K444T/R) and displayed partial or complete resistance to treatments (G339D, R346K/T, S371F/L, S375F, T376A, D405N, N440K, G446S, N460K, E484A, F486V, Q493R, G496S and Q498R). These results suggest that multiple novel variants which emerge in the patient during persistent infection, might spread to another individual and continue to evolve. A pro-active genomic surveillance of persistent SARS-CoV-2 infected patients is recommended to identify genetically divergent lineages before their diffusion.

T-cell immunity against Severe Acute Respiratory Syndrome Coronavirus 2 proteins in patients with type 1 diabetes.

AIMS: Individuals with type 1 diabetes (T1D) do not appear to have an elevated risk of severe Coronavirus Disease 19 (COVID-19). Pre-existing immune reactivity to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in unexposed individuals may serve as a protective factor. Hence, our study was designed to evaluate the existence of T cells with reactivity against SARS-CoV-2 antigens in unexposed patients with T1D. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from SARS-CoV-2 unexposed patients with T1D and healthy control subjects. SARS-CoV-2 specific T cells were identified in PBMCs by ex-vivo interferon (IFN)γ-ELISpot and flow cytometric assays. The epitope specificity of T cells in T1D was inferred through T Cell Receptor sequencing and GLIPH2 clustering analysis. RESULTS: T1D patients unexposed to SARS-CoV-2 exhibited higher rates of virus-specific T cells than controls. The T cells primarily responded to peptides from the ORF7/8, ORF3a, and nucleocapsid proteins. Nucleocapsid peptides predominantly indicated a CD4+ response, whereas ORF3a and ORF7/8 peptides elicited both CD4+ and CD8+ responses. The GLIPH2 clustering analysis of TCRß sequences suggested that TCRß clusters, associated with the autoantigens proinsulin and Zinc transporter 8 (ZnT-8), might share specificity towards ORF7b and ORF3a viral epitopes. Notably, PBMCs from three T1D patients exhibited T cell reactivity against both ORF7b/ORF3a viral epitopes and proinsulin/ZnT-8 autoantigens. CONCLUSIONS: The increased frequency of SAR-CoV-2- reactive T cells in T1D patients might protect against severe COVID-19 and overt infections. These results emphasise the long-standing association between viral infections and T1D.

The biological variation of serum 1,25-dihydroxyvitamin D and parathyroid hormone, and plasma fibroblast growth factor 23 in healthy individuals.

BACKGROUND AND AIMS: Measuring 1,25-dihydroxyvitamin D (1,25(OH)2D), parathyroid hormone 1-84 (PTH 1-84) and intact FGF23 (iFGF23) is crucial for diagnosing a variety of diseases affecting bone and mineral homeostasis. Biological variability (BV) data are important for defining analytical quality specifications (APS), the usefulness of reference intervals, and the significance of variations in serial measurements in the same subject. The aim of this study was to pioneer the provision of BV estimates for 1,25(OH)2D and to improve existing BV estimates for iFGF23 and PTH 1-84. MATERIALS AND METHODS: Serum and plasma-EDTA samples of sixteen healthy subjects have been collected for seven weeks and measured in duplicate by chemiluminescent immunoassay on the DiaSorin Liaison platform. After variance verification, within-subject (CVI) and between-subject (CVG) BV estimates were assessed by either standard ANOVA, or CV-ANOVA. The APSs were calculated according to the EFLM-BV-model. RESULTS: We found the following CVI estimates with 95% confidence intervals:1,25(OH)2D, 22.2% (18.9-26.4); iFGF23, 16.1% (13.5-19.5); and PTH 1-84, 17.9% (14.8-21.8). The CVG were: 1,25(OH)2D, 21.2% (14.2-35.1); iFGF23, 21.1% (14.5-35.8); and PTH 1-84, 31.1% (22.1-50.8). CONCLUSIONS: We report for the first time BV estimates for 1,25(OH)2D and enhance existing data about iFGF23-BV and PTH 1-84-BV through cutting-edge immunometric methods.

Enhanced pro-apoptotic activity of rituximab through IBTK silencing in non-Hodgkin lymphoma B-cells.

Rituximab is a commonly used chemotherapeutic drug for patients with aggressive lymphomas, such as non-Hodgkin's lymphoma (NHL). Currently, the combination of Rituximab and chemotherapy (R-CHOP) stands as the most prevalent first-line therapy for NHL. Nevertheless, the development of new therapeutic approaches remains imperative. An increasing body of evidence highlights a novel role for IBTK in tumorigenesis and cancer growth. In this study, we aim to broaden our understanding of IBTK's function in B-lymphoma, with a particular focus on its impact on the expression of the oncogene MYC. Here, we assessed the effects of combining Rituximab with IBTK silencing on cell viability through cell cycle analysis and Annexin V assays in vitro. Furthermore, we leveraged the transplantability of Eµ-myc lymphomas to investigate whether the inhibition of IBTK could elicit anti-tumor effects in the treatment of lymphomas in vivo. Our data suggests that IBTK silencing may serve as an effective anti-tumor agent for aggressive B-Lymphomas, underscoring its role in promoting apoptosis when used in combination with Rituximab, both in in vitro and in vivo settings.

Humoral and T-cell response to SARS-CoV-2 mRNA vaccine in multiple sclerosis patients: Correlations with DMTs and clinical variables.

Disease-modifying therapies (DMTs) can affect vaccine responses in individuals with multiple sclerosis (MS). We assessed the humoral and T-cell responses following SARS-CoV-2 mRNA vaccination in MS patients receiving various DMTs. We prospectively enrolled 243 participants, including 113 healthy control subjects and 130 MS patients. Blood samples for detecting SARS-CoV-2 antibodies were collected at three time points: T0, before the first vaccine dose; T1, before the second dose; and T2, one month after the second dose. In a subgroup of 51 patients and 20 controls, samples were collected at T0 and T2 to assess the T-cell immune response to the Spike antigen of SARS-CoV-2 using ELISPOT-IFNγ. The IgG levels in patients treated with fingolimod and ocrelizumab (159.1 AU/ml and 467.1 AU/ml, respectively) were significantly lower than those in healthy controls and patients on other DMTs (P â€‹< â€‹0.0001). The mean Ig titers were higher in patients with an absolute lymphocyte count ≥1000 â€‹cells/mm3 compared to those with a count between 500 and 1000 and with a count <500 (mean â€‹± â€‹SD:7205.6 â€‹± â€‹7339.2, 2413.1 â€‹± â€‹4515.4 and 165.9 â€‹± â€‹152.2, respectively; p â€‹= â€‹0.008). We found correlations between antibody levels and age (r â€‹= â€‹0.233, p â€‹= â€‹0.008). A positive Spike-specific T-cell response was detectable in 100 â€‹% of vaccinated healthy controls and patients treated with teriflunomide, dimethyl-fumarate, and natalizumab, in 90.5 â€‹% of fingolimod patients, and in 63.8 â€‹% of ocrelizumab patients. There is a correlation between IgG-specific titer after SARS-CoV-2 vaccination and clinical variables (age, lymphocyte count). Notably, a T-cell-specific response to SARS-CoV-2 developed in patients treated with fingolimod and ocrelizumab, even with lower rates of humoral response.

The spike-specific TCRß repertoire shows distinct features in unvaccinated or vaccinated patients with SARS-CoV-2 infection.

BACKGROUND: The evolving variants of SARS-CoV-2 may escape immunity from prior infections or vaccinations. It's vital to understand how immunity adapts to these changes. Both infection and mRNA vaccination induce T cells that target the Spike protein. These T cells can recognize multiple variants, such as Delta and Omicron, even if neutralizing antibodies are weakened. However, the degree of recognition can vary among people, affecting vaccine efficacy. Previous studies demonstrated the capability of T-cell receptor (TCR) repertoire analysis to identify conserved and immunodominant peptides with cross-reactive potential among variant of concerns. However, there is a need to extend the analysis of the TCR repertoire to different clinical scenarios. The aim of this study was to examine the Spike-specific TCR repertoire profiles in natural infections and those with combined natural and vaccine immunity. METHODS: A T-cell enrichment approach and bioinformatic tools were used to investigate the Spike-specific TCRß repertoire in peripheral blood mononuclear cells of previously vaccinated (n = 8) or unvaccinated (n = 6) COVID-19 patients. RESULTS: Diversity and clonality of the TCRß repertoire showed no significant differences between vaccinated and unvaccinated groups. When comparing the TCRß data to public databases, 692 unique TCRß sequences linked to S epitopes were found in the vaccinated group and 670 in the unvaccinated group. TCRß clonotypes related to spike regions S135-177, S264-276, S319-350, and S448-472 appear notably more prevalent in the vaccinated group. In contrast, the S673-699 epitope, believed to have super antigenic properties, is observed more frequently in the unvaccinated group. In-silico analyses suggest that mutations in epitopes, relative to the main SARS-CoV-2 variants of concern, don't hinder their cross-reactive recognition by associated TCRß clonotypes. CONCLUSIONS: Our findings reveal distinct TCRß signatures in vaccinated and unvaccinated individuals with COVID-19. These differences might be associated with disease severity and could influence clinical outcomes. TRIAL REGISTRATION: FESR/FSE 2014-2020 DDRC n. 585, Action 10.5.12, noCOVID19@UMG.

Iron-mediated oxidative stress induces PD-L1 expression via activation of c-Myc in lung adenocarcinoma.

Introduction: The PD-1/PD-L1 axis is hijacked by lung adenocarcinoma (LUAD) cells to escape immune surveillance. PD-L1 expression in LUAD is affected, among others, by the metabolic trafficking between tumor cells and the tumor microenvironment (TME). Methods: Correlation between PD-L1 expression and iron content within the TME was established on FFPE LUAD tissue samples. The effects of an iron rich microenvironment on PD-L1 mRNA and protein levels were assessed in vitro in H460 and A549 LUAD by using qPCR, western blot and flow citometry. c-Myc knockdown was performed to validate the role of this transcription factor on PD-L1 expression. The effects of iron-induced PD-L1 on T cell immune function was assessed by quantifying IFN-γ release in a co-colture system. TCGA dataset was used to analyse the correlation between PD-L1 and CD71 mRNA expression in LUAD patients. Results: In this study, we highlight a significant correlation between iron density within the TME and PD-L1 expression in 16 LUAD tissue specimens. In agreement, we show that a more pronounced innate iron-addicted phenotype, indicated by a higher transferrin receptor CD71 levels, significantly correlates with higher PD-L1 mRNA expression levels in LUAD dataset obtained from TCGA database. In vitro, we demonstrate that the addition of Fe3+ within the culture media promotes the significant overexpression of PD-L1 in A549 and H460 LUAD cells, through the modulation of its gene transcription mediated by c-Myc. The effects of iron lean on its redox activity since PD-L1 up-regulation is counteracted by treatment with the antioxidant compound trolox. When LUAD cells are co-cultured with CD3/CD28-stimulated T cells in an iron-rich culture condition, PD-L1 up-regulation causes the inhibition of T-lymphocytes activity, as demonstrated by the significant reduction of IFN-γ release. Discussion: Overall, in this study we demonstrate that iron abundance within the TME may enhance PD-L1 expression in LUAD and, thus, open the way for the identification of possible combinatorial strategies that take into account the iron levels within the TME to improve the outcomes of LUAD patients treated with anti-PD-1/PD-L1-based therapies.

Severe and mild-moderate SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: An exploratory study.

BACKGROUND: Despite an apparent effective vaccination, some patients are admitted to the hospital after SARS-CoV-2 infection. The role of adaptive immunity in COVID-19 is growing; nonetheless, differences in the spike-specific immune responses between patients requiring or not hospitalization for SARS-CoV-2 infection remains to be evaluated. In this study, we aim to evaluate the spike-specific immune response in patients with mild-moderate or severeSARS-CoV-2 infection, after breakthrough infection following two doses of BNT162b2 mRNA vaccine. METHODS: We included three cohorts of 15 cases which received the two BNT162b2 vaccine doses in previous 4 to 7 months: 1) patients with severe COVID-19; 2) patients with mild-moderate COVID-19 and 3) vaccinated individuals with a negative SARS-CoV-2 molecular pharyngeal swab (healthy subjects). Anti-S1 and anti-S2 specific SARS-CoV-2 IgM and IgG titers were measured through a chemiluminescence immunoassay technology. In addition, the frequencies of IFNγ-releasing cells were measured by ELISpot. RESULTS: The spike-specific IFNγ-releasing cells were significantly lower in severe patients (8 [0; 26] s.f.c.×106), as compared to mild-moderate patients (135 [64; 159] s.f.c.×106; p<0.001) and healthy subjects (103 [50; 188] s.f.c.×106; p<0.001). The anti-Spike protein IgG levels were similar among the three cohorts of cases (p = 0.098). All cases had an IgM titer below the analytic sensitivity of the test. The Receiver Operating Curve analysis indicated the rate of spike-specific IFNγ-releasing cells can discriminate correctly severe COVID-19 and mild-moderate patients (AUC: 0.9289; 95%CI: 0.8376-1.000; p< 0.0001), with a diagnostic specificity of 100% for s.f.c. > 81.2 x 106. CONCLUSIONS: 2-doses vaccinated patients requiring hospitalization for severe COVID-19 show a cellular-mediated immune response lower than mild-moderate or healthy subjects, despite similar antibody titers.

Ferritin Heavy Chain Binds Peroxiredoxin 6 and Inhibits Cell Proliferation and Migration.

The H Ferritin subunit (FTH1), as well as regulating the homeostasis of intracellular iron, is involved in complex pathways that might promote or inhibit carcinogenesis. This function may be mediated by its ability to interact with different molecules. To gain insight into the FTH1 interacting molecules, we analyzed its interactome in HEK293T cells. Fifty-one proteins have been identified, and among them, we focused our attention on a member of the peroxiredoxin family (PRDX6), an antioxidant enzyme that plays an important role in cell proliferation and in malignancy development. The FTH1/PRDX6 interaction was further supported by co-immunoprecipitation, in HEK293T and H460 cell lines and by means of computational methods. Next, we demonstrated that FTH1 could inhibit PRDX6-mediated proliferation and migration. Then, the results so far obtained suggested that the interaction between FTH1/PRDX6 in cancer cells might alter cell proliferation and migration, leading to a less invasive phenotype.

Spike-specific T-cell responses in patients with COVID-19 successfully treated with neutralizing monoclonal antibodies against SARS-CoV-2.

OBJECTIVES: Neutralizing monoclonal antibodies (moAbs) improves clinical outcomes in patients with COVID-19 when administered during the initial days of infection. The action of moAbs may impair the generation or maintenance of effective immune memory, similar to that demonstrated in other viral diseases. We aimed to evaluate short-term memory T-cell responses in patients effectively treated with bamlanivimab/etesevimab, casirivimab/imdevimab, or sotrovimab (SOT). METHODS: Spike (S)-specific T-cell responses were analyzed in 23 patients with COVID-19 (vaccinated or unvaccinated) before and after a median of 50 (range: 28-93) days from moAb treatment, compared with 11 vaccinated healthy controls. T-cell responses were measured by interferon-γ-enzyme-linked immunospot and flow cytometric activation-induced marker assay. RESULTS: No statistically significant difference in S-specific T-cell responses was observed between patients treated with moAb and vaccinated healthy controls. Bamlanivimab/etesevimab and casirivimab/imdevimab groups showed significant increases in cellular responses in paired baseline/postrecovery series, as well as vaccinated patients receiving SOT. In contrast, unvaccinated patients prescribed SOT presented no statistically significant increases in T-cell-responses, suggesting diverse impacts of different moAbs on the evolution of S-specific T-cell responses in vaccinated and unvaccinated patients. CONCLUSION: The moAbs did not hinder short-term memory S-specific T-cell responses in the overall group of patients; however, differences among moAbs must be further investigated both in vaccinated and unvaccinated individuals.

Multiparametric Magnetic Resonance Imaging-Ultrasound Fusion Transperineal Prostate Biopsy: Diagnostic Accuracy from a Single Center Retrospective Study.

The management of prostate biopsy in men with clinical suspicion of prostate cancer has changed in the last few years, especially with the introduction of imaging techniques, to overcome the low efficacy of risk stratification based on PSA levels. Here, we aimed to compare the diagnostic accuracy of multiparametric MRI with fusion ultrasound-guided prostate biopsy and standard biopsy, both performed through the transperineal route. To this end, we retrospectively analyzed 272 patients who underwent combined transperineal targeted and standard biopsy during the same session. The primary outcome was to compare the cancer detection rate between targeted and standard biopsy. The secondary outcome was to evaluate the added value of combined targeted and standard biopsy approach as compared to only targeted or standard biopsy. Results showed that a rate of 16.7% clinically significant tumors (International Society of Urological Pathology (ISUP) grade ≥ 2) would have been lost if only the standard biopsy had been used. The combined targeted and standard biopsy showed an added value of 10.3% and 9.9% in reducing the risk of prostate cancer missing after targeted or standard biopsy alone, respectively. The combined targeted and standard biopsy pathway is recommended to reduce the risk of missing clinically significant prostate cancer.

FTH1 Pseudogenes in Cancer and Cell Metabolism.

Ferritin, the principal intracellular iron-storage protein localized in the cytoplasm, nucleus, and mitochondria, plays a major role in iron metabolism. The encoding ferritin genes are members of a multigene family that includes some pseudogenes. Even though pseudogenes have been initially considered as relics of ancient genes or junk DNA devoid of function, their role in controlling gene expression in normal and transformed cells has recently been re-evaluated. Numerous studies have revealed that some pseudogenes compete with their parental gene for binding to the microRNAs (miRNAs), while others generate small interference RNAs (siRNAs) to decrease functional gene expression, and still others encode functional mutated proteins. Consequently, pseudogenes can be considered as actual master regulators of numerous biological processes. Here, we provide a detailed classification and description of the structural features of the ferritin pseudogenes known to date and review the recent evidence on their mutual interrelation within the complex regulatory network of the ferritin gene family.

FtH-Mediated ROS Dysregulation Promotes CXCL12/CXCR4 Axis Activation and EMT-Like Trans-Differentiation in Erythroleukemia K562 Cells.

The cell-microenvironment communication is essential for homing of hematopoietic stem cells in stromal niches. Recent evidences support the involvement of epithelial-to-mesenchymal (EMT) process in hematopoietic stem cell homeostasis as well as in leukemia cells invasiveness and migration capability. Here, we demonstrate that the alteration of iron homeostasis and the consequent increase of redox metabolism, mediated by the stable knock down of ferritin heavy chain (FtH), enhances the expression of CXCR4 in K562 erythroleukemia cells, thus promoting CXCL12-mediated motility. Indeed, addition of the CXCR4 receptor antagonist AMD3100 reverts this effect. Upon FtH knock down K562 cells also acquire an "EMT-like" phenotype, characterized by the increase of Snail, Slug and Vimentin with the parallel loss of E-cadherin. By using fibronectin as substrate, the cell adhesion assay further shows a reduction of cell adhesion capability in FtH-silenced K562 cells. Accordingly, confocal microscopy shows that adherent K562 control cells display a variety of protrusions while FtH-silenced K562 cells remain roundish. These phenomena are largely due to the reactive oxygen species (ROS)-mediated up-regulation of HIF-1α/CXCR4 axis which, in turn, promotes the activation of NF-κB and the enhancement of EMT features. These data are confirmed by treatments with either N-acetylcysteine (NAC) or AMD3100 or NF-κB inhibitor IκB-alpha which revert the FtH-silenced K562 invasive phenotype. Overall, our findings demonstrate the existence of a direct relationship among iron metabolism, redox homeostasis and EMT in the hematological malignancies. The effects of FtH dysregulation on CXCR4/CXCL12-mediated K562 cell motility extend the meaning of iron homeostasis in the leukemia cell microenvironment.

Molecular T-Cell Repertoire Analysis as Source of Prognostic and Predictive Biomarkers for Checkpoint Blockade Immunotherapy.

The T cells are key players of the response to checkpoint blockade immunotherapy (CBI) and monitoring the strength and specificity of antitumor T-cell reactivity remains a crucial but elusive component of precision immunotherapy. The entire assembly of T-cell receptor (TCR) sequences accounts for antigen specificity and strength of the T-cell immune response. The TCR repertoire hence represents a "footprint" of the conditions faced by T cells that dynamically evolves according to the challenges that arise for the immune system, such as tumor neo-antigenic load. Hence, TCR repertoire analysis is becoming increasingly important to comprehensively understand the nature of a successful antitumor T-cell response, and to improve the success and safety of current CBI.

Insights into Thymus Development and Viral Thymic Infections.

T-cell development in the thymus is a complex and highly regulated process, involving a wide variety of cells and molecules which orchestrate thymocyte maturation into either CD4+ or CD8+ single-positive (SP) T cells. Here, we briefly review the process regulating T-cell differentiation, which includes the latest advances in this field. In particular, we highlight how, starting from a pool of hematopoietic stem cells in the bone marrow, the sequential action of transcriptional factors and cytokines dictates the proliferation, restriction of lineage potential, T-cell antigen receptors (TCR) gene rearrangements, and selection events on the T-cell progenitors, ultimately leading to the generation of mature T cells. Moreover, this review discusses paradigmatic examples of viral infections affecting the thymus that, by inducing functional changes within this lymphoid gland, consequently influence the behavior of peripheral mature T-lymphocytes.

Chemoresistance in H-Ferritin Silenced Cells: The Role of NF-κB.

Nuclear Factor-κB (NF-κB) is frequently activated in tumor cells contributing to aggressive tumor growth and resistance to chemotherapy. Here we demonstrate that Ferritin Heavy Chain (FHC) protein expression inversely correlates with NF-κB activation in cancer cell lines. In fact, FHC silencing in K562 and SKOV3 cancer cell lines induced p65 nuclear accumulation, whereas FHC overexpression correlated with p65 nuclear depletion in the same cell lines. In FHC-silenced cells, the p65 nuclear accumulation was reverted by treatment with the reactive oxygen species (ROS) scavenger, indicating that NF-κB activation was an indirect effect of FHC on redox metabolism. Finally, FHC knock-down in K562 and SKOV3 cancer cell lines resulted in an improved cell viability following doxorubicin or cisplatin treatment, being counteracted by the transient expression of inhibitory of NF-κB, IκBα. Our results provide an additional layer of information on the complex interplay of FHC with cellular metabolism, and highlight a novel scenario of NF-κB-mediated chemoresistance triggered by the downregulation of FHC with potential therapeutic implications.

Cross-talk among HMGA1 and FoxO1 in control of nuclear insulin signaling.

As a mediator of insulin-regulated gene expression, the FoxO1 transcription factor represents a master regulator of liver glucose metabolism. We previously reported that the high-mobility group AT-hook 1 (HMGA1) protein, a molecular switch for the insulin receptor gene, functions also as a downstream target of the insulin receptor signaling pathway, representing a critical nuclear mediator of insulin function. Here, we investigated whether a functional relationship existed between FoxO1 and HMGA1, which might help explain insulin-mediated gene transcription in the liver. To this end, as a model study, we investigated the canonical FoxO1-HMGA1-responsive IGFBP1 gene, whose hepatic expression is regulated by insulin. By using a conventional GST-pull down assay combined with co-immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) analyses, we provide evidence of a physical interaction between FoxO1 and HMGA1. Further investigation with chromatin immunoprecipitation, confocal microscopy, and Fluorescence Recovery After Photobleaching (FRAP) technology indicated a functional significance of this interaction, in both basal and insulin-stimulated states, providing evidence that, by modulating FoxO1 transactivation, HMGA1 is essential for FoxO1-induced IGFBP1 gene expression, and thereby a critical modulator of insulin-mediated FoxO1 regulation in the liver. Collectively, our findings highlight a novel FoxO1/HMGA1-mediated mechanism by which insulin may regulate gene expression and metabolism.

The receptor protein tyrosine phosphatase PTPRJ negatively modulates the CD98hc oncoprotein in lung cancer cells.

PTPRJ, a receptor protein tyrosine phosphatase strongly downregulated in human cancer, displays tumor suppressor activity by negatively modulating several proteins involved in proliferating signals. Here, through a proteomic-based approach, we identified a list of potential PTPRJ-interacting proteins and among them we focused on CD98hc, a type II glycosylated integral membrane protein encoded by SLC3A2, corresponding to the heavy chain of a heterodimeric transmembrane amino-acid transporter, including LAT1. CD98hc is widely overexpressed in several types of cancers and contributes to the process of tumorigenesis by interfering with cell proliferation, adhesion, and migration. We first validated PTPRJ-CD98hc interaction, then demonstrated that PTPRJ overexpression dramatically reduces CD98hc protein levels in A549 lung cancer cells. In addition, following to the treatment of PTPRJ-transduced cells with MG132, a proteasome inhibitor, CD98hc levels did not decrease compared to controls, indicating that PTPRJ is involved in the regulation of CD98hc proteasomal degradation. Moreover, PTPRJ overexpression combined with CD98hc silencing consistently reduced cell proliferation and triggered apoptosis of lung cancer cells. Interestingly, by interrogating the can Evolve database, we observed an inverse correlation between PTPRJ and SLC3A2 gene expression. Indeed, the non-small cell lung cancers (NSCLCs) of patients showing a short survival rate express the lowest and the highest levels of PTPRJ and SLC3A2, respectively. Therefore, the results reported here contribute to shed lights on PTPRJ signaling in cancer cells: moreover, our findings also support the development of a novel anticancer therapeutic approach by targeting the pathway of PTPRJ that is usually downregulated in highly malignant human neoplasias.

Multimodal imaging for a theranostic approach in a murine model of B-cell lymphoma with engineered nanoparticles.

Nanoparticles (NPs) are a promising tool for in vivo multimodality imaging and theranostic applications. Hyaluronic acid (HA)-based NPs have numerous active groups that make them ideal as tumor-targeted carriers. The B-lymphoma neoplastic cells express on their surfaces a clone-specific immunoglobulin receptor (Ig-BCR). The peptide A20-36 (pA20-36) selectively binds to the Ig-BCR of A20 lymphoma cells. In this work, we demonstrated the ability of core-shell chitosan-HA-NPs decorated with pA20-36 to specifically target A20 cells and reduce the tumor burden in a murine xenograft model. We monitored tumor growth using high-frequency ultrasonography and demonstrated targeting specificity and kinetics of the NPs via in vivo fluorescent reflectance imaging. This result was also confirmed by ex vivo magnetic resonance imaging and confocal microscopy. In conclusion, we demonstrated the ability of NPs loaded with fluorescent and paramagnetic tracers to act as multimodal imaging contrast agents and hence as a non-toxic, highly specific theranostic system.