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SARS-CoV-2 viral-derived particles have been proposed to have a causal role in tissue inflammation. Macrophage is the culprit cell in the pathogenesis of destructive inflammatory response to the SARS-CoV-2 virus. We investigated whether the spike protein might play a role in perturbing the physiological process of resolution of inflammation. Using an in vitro model of M2 polarized macrophages, we found that recombinant spike protein produced typical M1 morphological features in these alternative differentiated cells. In the presence of spike, M2-macrophages lose their elongated morphology, become rounded and acquire a strong capability to stimulate lymphocyte activation and proliferation. Moreover, in M2 macrophages, spike activated the signal transducer and activator-1 (STAT1) the pivotal mediator of pro-inflammatory macrophages. We observed STAT1 activation also in endothelial cells cultured with recombinant spike, accompanied by Bax upregulation and cell death. Blockade of beta3 integrin with the RGD mimetic tirofiban reverted the spike-induced costimulatory effects on M2 macrophages. Also, tirofiban counteracted STAT1 and Bax activation in endothelial cells cultured with spike and reduced endothelial cell death. In conclusion, we found that some proinflammatory effects of the spike protein can involve the integrin pathway and provide elements supporting use of RGD mimetics against SARS-Cov-2., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The primary treatment for glioblastoma (GBM) is removing the tumor mass as defined by MRI. However, MRI has limited diagnostic and predictive value. Tumor-associated macrophages (TAM) are abundant in GBM tumor microenvironment (TME) and are found in peripheral blood (PB). FKBP51 expression, with its canonical and spliced isoforms, is constitutive in immune cells and aberrant in GBM. Spliced FKBP51s supports M2 polarization. To find an immunologic signature that combined with MRI could advance in diagnosis, we immunophenotyped the macrophages of TME and PB from 37 patients with GBM using FKBP51s and classical M1-M2 markers. We also determined the tumor levels of FKBP51s, PD-L1, and HLA-DR. Tumors expressing FKBP51s showed an increase in various M2 phenotypes and regulatory T cells in PB, indicating immunosuppression. Tumors expressing FKBP51s also activated STAT3 and were associated with reduced survival. Correlative studies with MRI and tumor/macrophages cocultures allowed to interpret TAMs. Tumor volume correlated with M1 infiltration of TME. Cocultures with spheroids produced M1 polarization, suggesting that M1 macrophages may infiltrate alongside cancer stem cells. Cocultures of adherent cells developed the M2 phenotype CD163/FKBP51s expressing pSTAT6, a transcription factor enabling migration and invasion. In patients with recurrences, increased counts of CD163/FKBP51s monocyte/macrophages in PB correlated with callosal infiltration and were accompanied by a concomitant decrease in TME-infiltrating M1 macrophages. PB PD-L1/FKBP51s connoted necrotic tumors. In conclusion, FKBP51s identifies a GBM subtype that significantly impairs the immune system. Moreover, FKBP51s marks PB macrophages associated with MRI features of glioma malignancy that can aid in patient monitoring., Significance: Our research suggests that by combining imaging with analysis of monocyte/macrophage subsets in patients with GBM, we can enhance our understanding of the disease and assist in its treatment. We discovered a similarity in the macrophage composition between the TME and PB, and through association with imaging, we could interpret macrophages. In addition, we identified a predictive biomarker that drew more attention to immune suppression of patients with GBM., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Cancer stem cells (CSC) are the leading cause of the failure of anti-tumor treatments. These aggressive cancer cells are preserved and sustained by adjacent cells forming a specialized microenvironment, termed niche, among which tumor-associated macrophages (TAMs) are critical players. The cycle of tricarboxylic acids, fatty acid oxidation path, and electron transport chain have been proven to play central roles in the development and maintenance of CSCs and TAMs. By improving their oxidative metabolism, cancer cells are able to extract more energy from nutrients, which allows them to survive in nutritionally defective environments. Because mitochondria are crucial bioenergetic hubs and sites of these metabolic pathways, major hopes are posed for drugs targeting mitochondria. A wide range of medications targeting mitochondria, electron transport chain complexes, or oxidative enzymes are currently investigated in phase 1 and phase 2 clinical trials against hard-to-treat tumors. This review article aims to highlight recent literature on the metabolic adaptations of CSCs and their supporting macrophages. A focus is provided on the resistance and dormancy behaviors that give CSCs a selection advantage and quiescence capacity in particularly hostile microenvironments and the role of TAMs in supporting these attitudes. The article also describes medicaments that have demonstrated a robust ability to disrupt core oxidative metabolism in preclinical cancer studies and are currently being tested in clinical trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Marrone, Romano, Malasomma, Di Giacomo, Cerullo, Abate, Vecchione, Fratantonio and Romano.)

Game theory-inspired deep learning using a generative adversarial network provides an environment to competitively interact and accomplish a goal. In the context of medical imaging, most work has focused on achieving single tasks such as improving image resolution, segmenting images, and correcting motion artifacts. We developed a dual-objective adversarial learning framework that simultaneously 1) reconstructs higher quality brain magnetic resonance images (MRIs) that 2) retain disease-specific imaging features critical for predicting progression from mild cognitive impairment (MCI) to Alzheimer's disease (AD). We obtained 3-Tesla, T1-weighted brain MRIs of participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI, N=342) and the National Alzheimer's Coordinating Center (NACC, N = 190) datasets. We simulated MRIs with missing data by removing 50% of sagittal slices from the original scans (i.e., diced scans). The generator was trained to reconstruct brain MRIs using the diced scans as input. We introduced a classifier into the GAN architecture to discriminate between stable (i.e., sMCI) and progressive MCI (i.e., pMCI) based on the generated images to facilitate encoding of disease-related information during reconstruction. The framework was trained using ADNI data and externally validated on NACC data. In the NACC cohort, generated images had better image quality than the diced scans (Structural similarity (SSIM) index: 0.553 ± 0.116 versus 0.348 ± 0.108). Furthermore, a classifier utilizing the generated images distinguished pMCI from sMCI more accurately than with the diced scans (F1-score: 0.634 ± 0.019 versus 0.573 ± 0.028). Competitive deep learning has potential to facilitate disease-oriented image reconstruction in those at risk of developing Alzheimer's disease.

Recent advancements in generative artificial intelligence, particularly using large language models (LLMs), are gaining increased public attention. We provide a perspective on the potential of LLMs to analyze enormous amounts of data from medical records and gain insights on specific topics in neurology. In addition, we explore use cases for LLMs, such as early diagnosis, supporting patient and caregivers, and acting as an assistant for clinicians. We point to the potential ethical and technical challenges raised by LLMs, such as concerns about privacy and data security, potential biases in the data for model training, and the need for careful validation of results. Researchers must consider these challenges and take steps to address them to ensure that their work is conducted in a safe and responsible manner. Despite these challenges, LLMs offer promising opportunities for improving care and treatment of various neurologic disorders ., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Quantifying the risk of progression to Alzheimer's disease (AD) could help identify persons who could benefit from early interventions. We used data from the Alzheimer's Disease Neuroimaging Initiative (ADNI, n = 544, discovery cohort) and the National Alzheimer's Coordinating Center (NACC, n = 508, validation cohort), subdividing individuals with mild cognitive impairment (MCI) into risk groups based on cerebrospinal fluid amyloid-β levels and identifying differential gray matter patterns. We then created models that fused neural networks with survival analysis, trained using non-parcellated T1-weighted brain MRIs from ADNI data, to predict the trajectories of MCI to AD conversion within the NACC cohort (integrated Brier score: 0.192 [discovery], and 0.108 [validation]). Using modern interpretability techniques, we verified that regions important for model prediction are classically associated with AD. We confirmed AD diagnosis labels using postmortem data. We conclude that our framework provides a strategy for risk-based stratification of individuals with MCI and for identifying regions key for disease prognosis., Competing Interests: V.B.K. reports honoraria from invited scientific presentations not exceeding $5000/year. He also serves as a consultant to Davos Alzheimer’s Collaborative and AstraZeneca. R.A. is a scientific advisor to Signant Health and consultant to Biogen. The remaining authors declare no competing interests., (© 2023 The Author(s).)

Scaffold proteins are crucial regulators of signaling networks, and their abnormal expression may favor the development of tumors. Among the scaffold proteins, immunophilin covers a unique role as 'protein-philin' (Greek 'philin' = friend) that interacts with proteins to guide their proper assembly. The growing list of human syndromes associated with the immunophilin defect underscores the biological relevance of these proteins that are largely opportunistically exploited by cancer cells to support and enable the tumor's intrinsic properties. Among the members of the immunophilin family, the FKBP5 gene was the only one identified to have a splicing variant. Cancer cells impose unique demands on the splicing machinery, thus acquiring a particular susceptibility to splicing inhibitors. This review article aims to overview the current knowledge of the FKBP5 gene functions in human cancer, illustrating how cancer cells exploit the scaffolding function of canonical FKBP51 to foster signaling networks that support their intrinsic tumor properties and the spliced FKBP51s to gain the capacity to evade the immune system., Competing Interests: The authors declare that they have no conflicts of interest to report regarding the present study., (© 2023 Marrone et al.)

FKBP51 plays a relevant role in sustaining cancer cells, particularly melanoma. This cochaperone participates in several signaling pathways. FKBP51 forms a complex with Akt and PHLPP, which is reported to dephosphorylate Akt. Given the recent discovery of a spliced FKBP51 isoform, in this paper, we interrogate the canonical and spliced isoforms in regulation of Akt activation. We show that the TPR domain of FKBP51 mediates Akt ubiquitination at K63, which is an essential step for Akt activation. The spliced FKBP51, lacking such domain, cannot link K63-Ub residues to Akt. Unexpectedly, PHLPP silencing does not foster phosphorylation of Akt, and its overexpression even induces phosphorylation of Akt. PHLPP stabilizes levels of E3-ubiquitin ligase TRAF6 and supports K63-ubiquitination of Akt. The interactome profile of FKBP51 from melanoma cells highlights a relevant role for PHLPP in improving oncogenic hallmarks, particularly, cell proliferation., (© 2023. The Author(s).)

FKBP51 is constitutively expressed by immune cells. As other FKBP family members, FKBP51 acts as a coreceptor for the natural products FK506 and rapamycin, which exhibit immunosuppressive effects. However, little is known about the intrinsic role of this large FKBP in the primary function of lymphocytes, that is, the adaptive immune response against foreign antigens, for example, pathogens. This paper aimed to investigate whether FKBP51 expression was modulated during lymphocyte activation. Moreover, as we recently identified a splicing isoform of FKBP51, namely FKBP51s, we also measured this splice protein, along with the canonical one, at different times of a peripheral blood mononuclear cell culture stimulated via T cell receptor. Our results show that the two FKBP51 isoforms were alternatively induced during the proliferative burst. Canonical FKBP51 increased in the time window between 48 and 96 h and its expression levels correlated with cyclin D levels. FKBP51s transiently increased earlier, at 24-36 h to reappearing later, at 120 h, when cyclin D expression returned at resting levels and proliferation ceased. Interestingly, within these two specific timeframes, FKBP51s accumulated in the nucleus. Here FKBP51s colocalized with the Foxp3 transcription factor at 36 h. Regulatory T cell (Treg) counts significantly decreased when FKBP51s was downmodulated. The coculture suppression assay suggested that FKBP51s supports the suppressive capability of Tregs. At 120 h, chromatin immunoprecipitation experiments found FKBP51s linked to CCND1 gene, suggesting a possible effect on gene transcription regulation, as previously demonstrated in melanoma. In conclusion, our study shows that FKBP5 isoforms are upregulated during lymphocyte activation, albeit on different timeframes. The activation of canonical FKBP51 coincides with proliferation hallmarks; FKBP5 splicing occurs early to sustain Treg development and late when proliferation ceases., (© 2023 Wiley Periodicals LLC.)

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, whose presentation and clinical course are highly variable. Identification of novel prognostic factors may contribute to improving the CLL classification and providing indications for treatment options. The zinc finger protein ZNF224 plays a key role in cell transformation, through the control of apoptotic and survival pathways. In this study, we evaluated the potential application of ZNF224 as a novel marker of CLL progression and therapy responsiveness. To this aim, we analyzed ZNF224 expression levels in B lymphocytes from CLL patients at different stages of the disease and in patients showing different treatment outcomes. The expression of ZNF224 was significantly increased in disease progression and dramatically decreased in patients in complete remission after chemotherapy. Gene expression correlation analysis performed on datasets of CLL patients revealed that ZNF224 expression was well correlated with that of some prognostic and predictive markers. Moreover, bioinformatic analysis coupled ZNF224 to NF-κB pathway, and experimental data demonstrated that RNA interference of ZNF224 reduced the activity of the NF-κB survival pathway in CLL cells. Consistently with a pro-survival role, ZNF224 knockdown raised spontaneous and drug-induced apoptosis and inhibited the proliferation of peripheral blood mononuclear cells from CLL patients. Our findings provide evidence for the involvement of ZNF224 in the survival of CLL cells via NF-κB pathway modulation, and also suggest ZNF224 as a prognostic and predictive molecular marker of CLL disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Catapano, Sepe, Toscano, Paolella, Chiurazzi, Barbato, Bruzzese, Arianna, Grosso, Romano, Romano, Costanzo and Cesaro.)

In a recent article from Cell Reports Medicine, Kwak et al. generate novel insights about subtyping cognitively impaired individuals based on structural imaging. Quantifying heterogeneity in Alzheimer's disease via subtyping could help us harness new disease-modifying therapies and improve patient care by providing a more targeted approach., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Thrombocytopenia after TAVI is common and clinically detrimental. Retrospectively, we observed Portico recipients had a more profound platelet drop than Evolut recipients. We thus investigated periprocedural platelet damage and/orpro-inflammatory state in 64 TAVI recipients at baseline and after implantation. Platelet damage was assessed by annexin V staining and monocyte-phagocytic phenotype was assessed according to CD14/CD36 expression. Serum cytokines were measured in 20 patients. The formaldehyde-based storage solution altered platelets. When, before being loaded onto the delivery system, Portico underwent one additional flushing to those recommended, the receiving patients showed thrombocytopenia, platelet damage, and CD36-monocyte count were mitigated. A general increase in IL-6 was recorded in overall TAVI recipients, but a high serum level of IL-8, a potent thrombocytopenia inducer, was measured in Portico recipients only, including those with extra-rinsed valve. Our study suggests a platelet-injury effect by storage-solution and generates the hypothesis of a role for the biomaterial in stimulating innate-immunity. Larger prospective studies are needed. Graphical Abstract., (© 2021. The Author(s).)

Melanoma is one of the most immunogenic tumors and has the highest potential to elicit specific adaptive antitumor immune responses. Immune cells induce apoptosis of cancer cells either by soluble factors or by triggering cell-death pathways. Melanoma cells exploit multiple mechanisms to escape immune system tumoricidal control. FKBP51 is a relevant pro-oncogenic factor of melanoma cells supporting NF-κB-mediated resistance and cancer stemness/invasion epigenetic programs. Herein, we show that FKBP51-silencing increases TNF-related apoptosis-inducing ligand (TRAIL)-R2 (DR5) expression and sensitizes melanoma cells to TRAIL-induced apoptosis. Consistent with the general increase in histone deacetylases, as by the proteomic profile, the immune precipitation assay showed decreased acetyl-Yin Yang 1 (YY1) after FKBP51 depletion, suggesting an impaired repressor activity of this transcription factor. ChIP assay supported this hypothesis. Compared with non-silenced cells, a reduced acetyl-YY1 was found on the DR5 promoter, resulting in increased DR5 transcript levels. Using Crispr/Cas9 knockout (KO) melanoma cells, we confirmed the negative regulation of DR5 by FKBP51. We also show that KO cells displayed reduced levels of acetyl-EP300 responsible for YY1 acetylation, along with reduced acetyl-YY1. Reconstituting FKBP51 levels contrasted the effects of KO on DR5, acetyl-YY1, and acetyl-EP300 levels. In conclusion, our finding shows that FKBP51 reduces DR5 expression at the transcriptional level by promoting YY1 repressor activity. Our study supports the conclusion that targeting FKBP51 increases the expression level of DR5 and sensitivity to TRAIL-induced cell death, which can improve the tumoricidal action of immune cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tufano, Cesaro, Martinelli, Pacelli, Romano and Romano.)

Despite Glioblastoma (GBM) frequently expressing programmed cell death ligand-1 (PD-L1), treatment with anti-programmed cell death-1 (PD1) has not yielded brilliant results. Intratumor variability of PD-L1 can impact determination accuracy. A previous study on mouse embryonic fibroblasts (MEFs) reported a role for cyclin-D in control of PD-L1 expression. Because tumor-cell growth within a cancer is highly heterogeneous, we looked at whether PD-L1 and its cochaperone FKBP51s were influenced by cell proliferation, using U251 and SF767 GBM-cell-lines. PD-L1 was measured by Western blot, flow cytometry, confocal-microscopy, quantitative PCR (qPCR), CCND1 by qPCR, FKBP51s by Western blot and confocal-microscopy. Chromatin-Immunoprecipitation assay (xChIp) served to assess the DNA-binding of FKBP51 isoforms. In the course of cell culture, PD-L1 appeared to increase concomitantly to cyclin-D on G1/S transition, to decrease during exponential cell growth progressively. We calculated a correlation between CCND1 and PD-L1 gene expression levels. In the temporal window of PD-L1 and CCND1 peak, FKBP51s localized in ER. When cyclin-D declined, FKBP51s went nuclear. XChIp showed that FKBP51s binds CCND1 gene in a closed-chromatin configuration. Our finding suggests that the dynamism of PD-L1 expression in GBM follows cyclin-D fluctuation and raises the hypothesis that FKBP51s might participate in the events that govern cyclin-D oscillation.

Comparatively little is known about how new instrumental actions are encoded in the brain. Using whole-brain c-Fos mapping, we show that neural activity is increased in the anterior dorsolateral striatum (aDLS) of mice that successfully learn a new lever-press response to earn food rewards. Post-learning chemogenetic inhibition of aDLS disrupts consolidation of the new instrumental response. Similarly, post-learning infusion of the protein synthesis inhibitor anisomycin into the aDLS disrupts consolidation of the new response. Activity of D1 receptor-expressing medium spiny neurons (D1-MSNs) increases and D2-MSNs activity decreases in the aDLS during consolidation. Chemogenetic inhibition of D1-MSNs in aDLS disrupts the consolidation process whereas D2-MSN inhibition strengthens consolidation but blocks the expression of previously learned habit-like responses. These findings suggest that D1-MSNs in the aDLS encode new instrumental actions whereas D2-MSNs oppose this new learning and instead promote expression of habitual actions., (© 2021. The Author(s).)

Magnetic resonance imaging (MRI) is the gold standard for glioblastoma (GBM) patient evaluation. Additional non-invasive diagnostic modalities are needed. GBM is heavily infiltrated with tumor-associated macrophages (TAMs) that can be found in peripheral blood. FKBP51s supports alternative-macrophage polarization. Herein, we assessed FKBP51s expression in circulating monocytes from 14 GBM patients. The M2 monocyte phenotype was investigated by qPCR and flow cytometry using antibodies against PD-L1, CD163, FKBP51s, and CD14. MRI assessed morphologic features of the tumors that were aligned to flow cytometry data. PD-L1 expression on circulating monocytes correlated with MRI tumor necrosis score. A wider expansion in circulating CD163/monocytes was measured. These monocytes resulted in a dramatic decrease in patients with an MRI diagnosis of complete but not partial surgical removal of the tumor. Importantly, in patients with residual tumor, most of the peripheral monocytes that in the preoperative stage were CD163/FKBP51s- had turned into CD163/FKBP51s+. After Stupp therapy, CD163/FKBP51s+ monocytes were almost absent in a case of pseudoprogression, while two patients with stable or true disease progression showed sustained levels in such circulating monocytes. Our work provides preliminary but meaningful and novel results that deserve to be confirmed in a larger patient cohort, in support of potential usefulness in GBM monitoring of CD163/FKBP51s/CD14 immunophenotype in adjunct to MRI.

Testicular torsion is a known cause of morbidity in pediatric patients, but the burden in the adult population is poorly understood. We sought to determine the incidence of testicular torsion and risk factors for orchiectomy in a population encompassing all ages. A cohort analysis of 1625 males undergoing surgery for torsion was performed using the 2011 and 2012 Healthcare Cost and Utilization Project Nationwide Emergency Departments Sample. Patient and hospital factors were examined for association with orchiectomy vs. testicular salvage. The estimated yearly incidence of testicular torsion was 5.9 per 100,000 males ages 1-17 years and 1.3 per 100,000 males ≥18 years. Among those undergoing surgical intervention, orchiectomy was performed in 33.6%. The risk of orchiectomy was highest in patients 1-11 years of age and patients over 50 years of age (46.0% and 69.7% of patients, respectively). Orchiectomy was also associated with public insurance (Medicaid/Medicare) or self-pay as primary payer. While testicular torsion is less common in the adult population, the rate of orchiectomy is high. Those with disadvantaged payer status are also at increased risk for testicular loss.

Objective  This study was aimed to develop a method combining computed tomography (CT) and fluorescence imaging, allowing identification of microvasculature in anatomical donors and facilitating translational research and education. Methods  We investigated homogeneity and radiopacity of 30 different mixtures including radiopaque substances povidone-iodine (Betadine), barium sulfate (BaSO 4 ), and bismuth subsalicylate (Pepto-Bismol) varying in suspension and dilution with agar, latex, or gelatin. Three candidate mixtures were selected for testing the extent of perfusion in renal vasculature to establish methodology. From these candidate mixtures, two were selected for mixture with fluorescein and infusion into cadavers based on their ability to perfuse renal vasculature. The extent to which these two candidate mixtures combined with fluorescein were able to perfuse vasculature in a cadaver head was used to determine which mixture was superior. Results  BaSO 4 and bismuth subsalicylate-based mixtures demonstrated superior opacity in vials. In terms of solidifying agents, gelatin-based mixtures demonstrated increased friability and lower melting points compared with the other agents, so only latex and agar-based mixtures were used moving forward past the vial stage. Combinations of BaSO 4 and latex and BaSO 4 and 3% agar were found to perfuse kidneys superiorly to the mixture containing bismuth subsalicylate. Finally, in cadaver heads, the mixture containing BaSO 4 , agar, and fluorescein was found to perfuse the smallest vasculature. Conclusion  A final combination of BaSO 4 , 3% agar, and fluorescein proves to be a powerful and novel combination enabling CT imaging, fluorescence imaging, and dissection of vasculature. This paves the way for future translational research and education., Competing Interests: Conflict of Interest A.K.D. reports in addition, A.K.D. has a patent retractable endoscopic suction pending. All the other authors report no conflict of interest., (Thieme. All rights reserved.)

Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 μm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics., Competing Interests: Declaration of Interests O.A.S., C.L., K.D.P., W.M.P., and E.S.B. declare that they applied for a U.S. patent based on the work presented in this paper, Application No.: PCT/US2019/065773., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: FKBP51 immunophilin is abundantly expressed by immune cells. Co-inhibitory immune receptor signalling generates the splicing isoform FKBP51s. Tregs stained by FKBP51s are increased in melanoma patients and their counts are associated with anti-CTLA-4 response. An expansion of FKBP51s + PD-L1 + monocytes was measured in a group of non-responding patients to anti-CTLA-4. The aim of this work was to confirm the predictive value of response of FKBP51s + Tregs in a cohort of patients undergoing anti-PD1 treatment and shed light on a monocyte subset co-expressing PD-L1/FKBP51s., Methods: Co-cultures of organoids and autologous lymphocytes were used to confirm that tumour T-cell interaction can induce FKBP51s. PBMC immunophenotype and flow cytometry served to assess and monitor FKBP51s + Treg and FKBP51s + PD-L1 + monocytes in 22 advanced melanoma patients treated with anti-PD1. Silencing and overexpression of FKBP51s in human macrophages served to address the protein role in the tolerant macrophages' behaviour., Results: FKBP51s + Tregs count was increased in responders and had a prognostic value. Non-responders showed an early increase in FKBP51s + PD-L1 + monocytes during anti-PD1 treatment. Manipulation of FKBP51s modulated the macrophage-phenotype, with forced protein expression promoting aspects associated with tolerance., Conclusions: FKBP51s may guide in the selection and monitoring of melanoma patient candidates to immune-checkpoint-targeted therapy. Manipulation of FKBP51s may overcome resistance.

Neuroblastoma (NB) and malignant cutaneous melanoma (CMM) are neural crest cells (NCC)-derived tumors and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association studies (GWAS). We took a three-staged approach to conduct cross-disease meta-analysis of GWAS for NB and CMM (2101 NB cases and 4202 controls; 12 874 CMM cases and 23 203 controls) to identify shared loci. Findings were replicated in 1403 NB cases and 1403 controls of European ancestry and in 636 NB, 508 CMM cases and 2066 controls of Italian origin. We found a cross-association at locus 1p13.2 (rs2153977, odds ratio = 0.91, P = 5.36 × 10-8). We also detected a suggestive (P < 10-7) NB-CMM cross-association at 2q37.1 with opposite effect on cancer risk. Pathway analysis of 110 NB-CMM risk loci with P < 10-4 demonstrated enrichment of biological processes such as cell migration, cell cycle, metabolism and immune response, which are essential of human NCC development, underlying both tumors. In vitro and in silico analyses indicated that the rs2153977-T protective allele, located in an NB and CMM enhancer, decreased expression of SLC16A1 via long-range loop formation and altered a T-box protein binding site. Upon depletion of SLC16A1, we observed a decrease of cellular proliferation and invasion in both NB and CMM cell lines, suggesting its role as oncogene. This is the largest study to date examining pleiotropy across two NC cell-derived tumors identifying 1p13.2 as common susceptibility locus for NB and CMM risk. We demonstrate that combining genome-wide association studies results across cancers with same origins can identify new loci common to neuroblastoma and melanoma arising from tissues which originate from neural crest cells. Our results also show 1p13.2 confer risk to neuroblastoma and melanoma by regulating SLC16A1., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Recent advances in tumor immunology, fostered by dramatic outcomes with cancer immunotherapy, have opened new scenarios in cancer metastasis. The cancer stemness/mesenchymal phenotype and an excess of immune suppressive signals are emerging as Intertwined aspects of human tumors. This review examines recent studies that explored the mechanistic links between cancer cell stemness and immunoevasion, and the evidence points to these key events in cancer metastasis as two sides of the same coin. This review also covers the mechanisms involved in tumor expression of programmed cell death ligand 1 (PD-L1), a major factor exploited by human neoplasias to suppress immune control. We highlight the convergence of mesenchymal traits and PD-L1 expression and examine the functions of this immune inhibitory molecule, which confers cancer cell resistance and aggressiveness., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2019. Published by Elsevier Ltd.)

Selective inhibition of FKBP51 has emerged as possible novel treatment for diseases like major depressive disorder, obesity, chronic pain, and certain cancers. The current FKBP51 inhibitors are rather large, flexible, and have to be further optimized. By using a structure-based rigidification strategy, we hereby report the design and synthesis of a novel promising bicyclic scaffold for FKBP51 ligands. The structure-activity analysis revealed the decalin scaffold as the best moiety for the selectivity-enabling subpocket of FBKP51. The resulting compounds retain high potency for FKBP51 and excellent selectivity over the close homologue FKBP52. With the cocrystal structure of an advanced ligand in this novel series, we show how the decalin locks the key selectivity-inducing cyclohexyl moiety of the ligand in a conformation typical for FKBP51-selective binding. The best compound 29 produces cell death in a HeLa-derived KB cell line, a cellular model of cervical adenocarcinoma, where FKBP51 is highly overexpressed. Our results show how FKBP51 inhibitors can be rigidified and extended while preserving FKBP51 selectivity. Such inhibitors might be novel tools in the treatment of human cancers with deregulated FKBP51.

The immune system actively counteracts the tumorigenesis process; a breakout of the immune system function, or its ability to recognize transformed cells, can favor cancer development. Cancer becomes able to escape from immune system control by using multiple mechanisms, which are only in part known at a cellular and molecular level. Among these mechanisms, in the last decade, the role played by the so-called "inhibitory immune checkpoints" is emerging as pivotal in preventing the tumor attack by the immune system. Physiologically, the inhibitory immune checkpoints work to maintain the self-tolerance and attenuate the tissue injury caused by pathogenic infections. Cancer cell exploits such immune-inhibitory molecules to contrast the immune intervention and induce tumor tolerance. Molecular agents that target these checkpoints represent the new frontier for cancer treatment. Despite the heterogeneity and multiplicity of molecular alterations among the tumors, the immune checkpoint targeted therapy has been shown to be helpful in selected and even histologically different types of cancer, and are currently being adopted against an increasing variety of tumors. The most frequently used is the moAb-based immunotherapy that targets the Programmed Cell Death 1 protein (PD-1), the PD-1 Ligand (PD-L1) or the cytotoxic T lymphocyte antigen-4 (CTLA4). However, new therapeutic approaches are currently in development, along with the discovery of new immune checkpoints exploited by the cancer cell. This article aims to review the inhibitory checkpoints, which are known up to now, along with the mechanisms of cancer immunoediting. An outline of the immune checkpoint targeting approaches, also including combined immunotherapies and the existing trials, is also provided. Notwithstanding the great efforts devoted by researchers in the field of biomarkers of response, to date, no validated FDA-approved immunological biomarkers exist for cancer patients. We highlight relevant studies on predictive biomarkers and attempt to discuss the challenges in this field, due to the complex and largely unknown dynamic mechanisms that drive the tumor immune tolerance., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here we describe a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and is compatible with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, and number of neurons observable several-fold over previously published fully genetically encoded reagents 1-8 . Under conventional one-photon microscopy, SomArchon enables the routine population analysis of around 13 neurons at once, in multiple brain regions (cortex, hippocampus, and striatum) of head-fixed, awake, behaving mice. Using SomArchon, we detected both positive and negative responses of striatal neurons during movement, as previously reported by electrophysiology but not easily detected using modern calcium imaging techniques 9-11 , highlighting the power of voltage imaging to reveal bidirectional modulation. We also examined how spikes relate to the subthreshold theta oscillations of individual hippocampal neurons, with SomArchon showing that the spikes of individual neurons are more phase-locked to their own subthreshold theta oscillations than to local field potential theta oscillations. Thus, SomArchon reports both spikes and subthreshold voltage dynamics in awake, behaving mice.

Gliomas aberrantly express programmed cell death ligand-1 (PD-L1), which has a pivotal role in immunoevasion. The splicing isoform of FKBP5 , termed FKBP51s, is a PD-L1 foldase, assisting the immune checkpoint molecule in maturation and expression on the plasma membrane. The concept that PD-L1 supports tumor-intrinsic properties is increasingly emerging. The aim of the present work was to confirm the pro-tumoral effect of PD-L1 on human glioma cell survival, stemness capacity and resistance, and to address the issue of whether, by targeting its foldase either chemically or by silencing, the aggressive tumor features could be attenuated. PD-L1-depleted glioma cells have a reduced threshold for apoptosis, while PD-L1 forced expression increases resistance. Similar results were obtained with FKBP51s modulation. The ability of PD-L1 to counteract cell death was hampered by FKBP51s silencing. PD-L1 expression was particularly high in glioma cells with a cancer-stem-cell profile. Moreover, PD-L1 sustained the spheroid formation capability of glioma cells. Targeting of FKBP51s by small-interfering RNA (siRNA) or the specific inhibitor SAFit2, reduced the number of formed spheroids, along with PD-L1 expression. Finally, in an orthotopic mouse model of glioblastoma, daily treatment with SAFit2 significantly reduced tumor PD-L1 expression, and tumor growth. In treated mice, caspase-3 activation and reduced vimentin expression were observed in excised tumors. In conclusion, targeting of FKBP51s hampers PD-L1 and its pro-tumoral properties, thereby affecting the self-renewal and growth capacities of glioblastoma cells in vitro and in vivo., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2019.)

Striatal parvalbumin (PV) and cholinergic interneurons (CHIs) are poised to play major roles in behavior by coordinating the networks of medium spiny cells that relay motor output. However, the small numbers and scattered distribution of these cells have hindered direct assessment of their contribution to activity in networks of medium spiny neurons (MSNs) during behavior. Here, we build on recent improvements in single-cell calcium imaging combined with optogenetics to test the capacity of PVs and CHIs to affect MSN activity and behavior in mice engaged in voluntary locomotion. We find that PVs and CHIs have unique effects on MSN activity and dissociable roles in supporting movement. PV cells facilitate movement by refining the activation of MSN networks responsible for movement execution. CHIs, in contrast, synchronize activity within MSN networks to signal the end of a movement bout. These results provide new insights into the striatal network activity that supports movement.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, whose role in melanoma is controversial. Although high-dose TNF-α is approved for the treatment of patients with in transit-metastatic melanoma confined to the limb, diverse preclinical models of melanoma have shown that TNF-α can induce cell invasion. Biomarkers that can differentiate between the dual role of TNF-α are needed. TRAF2 is critical to TNF receptor-induced activation of nuclear factor-κB (NF-κB), allowing shifting from death to survival-signaling cascades. The large immunophilin FKBP51 acts as a scaffold and catalyst in the IκB kinase complex assembly and activation. Here, using microscopy and an electrophoretic mobility-shift assay, we provide further evidence in support of the essential role of FKBP51 in sustaining the TNF-α NF-κB signaling in melanoma. Through the cross-linking reaction with the chemical linker disuccinimidyl glutarate, we show that a direct interaction occurs between FKBP51 and TRAF2 in melanoma cells. Immunohistochemistry of tumor samples from 24 patients with cutaneous melanomas showed a correlation between the expressions of the two proteins. Given the association of FKBP51 and TRAF2 with TNF-α-induced NF-κB signaling and their correlation in tumor samples, we propose that the two proteins can be exploited as useful markers for the identification of those melanoma tumors that can benefit from TNF-α inhibition. Future studies will address this hypothesis.

An unexpectedly high incidence of thrombosis in patients that received the polylactic acid bioresorbable vascular scaffold (BVS) suggests a delayed/incomplete endothelial repair with this stent. The anti-platelet agent tirofiban stimulates endothelial cell migration and proliferation, mediated by VEGF production. We investigated the tirofiban effect on the migration and adhesion of endothelial cells to BVS, in vitro. We performed human umbilical endothelial cell (HUVEC) cultures in the presence of BVS. Tirofiban, similarly to VEGF, increased the ability of HUVEC to grow on the vascular scaffold, compared to unstimulated or abciximab-treated cells. Tirofiban increased HUVEC expression of β1 and β3 integrins along with collagen and fibronectin. A role for β1 integrin in the "pro-adhesive and -migratory" signals elicited by tirofiban was suggested by use of an anti-β1-blocking antibody that prevented poly-levo-lactic acid vascular scaffold colonization. Our study suggests that tirofiban may improve the outcomes of patients receiving BVS by accelerating stent endothelization.

Background: Mycosis fungoides (MF) is the major subtype of cutaneous T-cell lymphomas (CTCL). It usually has a prolonged indolent clinical course with a minority of cases acquiring a more aggressive biological profile and resistance to conventional therapies, partially attributed to the persistent activation of nuclear factor-kappa B (NF-κB) pathway. In the last decade, several papers suggested an important role for the FK506-binding protein 51 (FKBP51), an immunophilin initially cloned in lymphocytes, in the control of NF-κB pathway in different types of human malignancies., Objectives: We aimed to investigate the possible value of FKBP51 expression as a new reliable marker of outcome in patients with MF., Methods: We assessed by immunohistochemistry (IHC) FKBP51 expression in 44 patients with MF, representative of different stages of the disease. Immunohistochemical results were subsequently confirmed at mRNA level with quantitative PCR (qPCR) in a subset of enrolled patients. In addition, IHC and qPCR served to study the expression of some NF-κB-target genes, including the tumour necrosis factor receptor-associated factor 2 (TRAF2)., Results: Our results show that FKBP51 was expressed in all evaluated cases, with the highest level of expression characterizing MFs with the worst prognosis. Moreover, a significant correlation subsisted between FKBP51 and TRAF2 IHC expression scores., Conclusions: We hypothesize a role for FKBP51 as a prognostic marker for MF and suggest an involvement of this immunophilin in deregulated NF-κB pathway of this CTCL., (© 2017 European Academy of Dermatology and Venereology.)

Introduction: Doppler echocardiography with early diastolic transmitral velocity (E)/early mitral annular diastolic velocity (E') ratio has been proposed as the best predictor for evaluating left ventricle (LV) filling pressure. A dimensionless index E/(E' × S') ratio (S' = systolic mitral annulus velocity) resulted in readily, reproducible, and reliable predictor of LV filling pressure. We assessed the prognostic impact of E/E' × S') in patients with asymptomatic heart failure (HF)., Materials and Methods: We calculated E/(E' × S') in 337 patients (179 male, 53%; age 54.7 ± 13.7 years) using the average of septal and lateral mitral annular velocities. We considered a composite endpoint as follows: all-cause death, acute myocardial infarction, stroke, and HF exacerbation., Results: Baseline ejection fraction resulted 60.2 ± 11.8%; E/E' × S') was 1.45 ± 0.8, with S' 7.4 ± 2.4 cm/s and E/E' 9.5 ± 5.4. After a 22-month median follow-up, there were 42 events: 5 deaths (12%), 3 acute myocardial infarctions (7%), 1 stroke (2%), and 33 HF hospitalizations (79%). In patients reaching the composite endpoint, E/(E' × S') resulted 2.07 ± 1.1 versus 1.3 ± 0.7 in event-free population ( P < 0.001). In a Cox-regression analysis, adjusted for confounding clinical factors and conventional echo parameters, E/(E' × S') ( P < 0.001), age ( P < 0.001), and male gender ( P = 0.03) resulted independent predictors of the composite endpoint., Conclusions: E/(E' × S') was an independent predictor for the future cardiac events in asymptomatic HF., Competing Interests: There are no conflicts of interest.

The inhibitory immune checkpoint PD-L1/PD1 promotes the alternative splicing of the FKBP5 gene, resulting in increased expression of its variant 4 in the peripheral blood mononuclear cells of melanoma patients. The variant 4 transcript is translated into the truncated FKBP51s protein. Given the importance of co-inhibitory signalling in tumour immune escape, here we tested the potential for using FKBP51s expression to predict immunotherapy outcomes. To do this, we immunophenotyped PBMCs from 118 melanoma patients and 77 age- and sex-matched healthy controls. Blood samples were collected before patients underwent ipilimumab treatment. In 64 of the 118 patients, FKBP51s expression was also assessed in regulatory T cells (Tregs). We found that each PBMC subset analysed contained an FKBP51s pos fraction, and that this fraction was greater in the melanoma patients than healthy controls. In CD4 T lymphocytes, the FKBP51s neg fraction was significantly impaired. Tregs count was increased in melanoma patients, which is in line with previous studies. Also, by analyses of FKBP51s in Tregs, we identified a subgroup of ipilimumab nonresponder patients (p = 0.002). In conclusion, FKBP51s-based immunophenotyping of melanoma patients revealed several profiles related to a negative immune regulatory control and identified an unknown Treg subset. These findings are likely to be useful in the selection of the patients that are candidate for immunotherapy.

Background: FKBP51 is a co-chaperone with isomerase activity, abundantly expressed in glioma. We previously identified a spliced isoform (FKBP51s) and highlighted a role for this protein in the upregulation of Programmed Death Ligand 1 (PD-L1) expression in melanoma. Because gliomas can express PD-L1 causing a defective host anti-tumoral immunity, we investigated whether FKBP51s was expressed in glioma and played a role in PD-L1 regulation in this tumour., Methods: We used D54 and U251 glioblastoma cell lines that constitutively expressed PD-L1. FKBP51s was measured by immunoblot, flow cytometry and microscopy. In patient tumours, IHC and qPCR were used to measure protein and mRNA levels respectively. FKBP51s depletion was achieved by siRNAs, and its enzymatic function was inhibited using selective inhibitors (SAFit). We investigated protein maturation using N-glycosidase and cell fractionation approaches., Results: FKBP51s was expressed at high levels in glioma cells. Glycosylated-PD-L1 was increased and reduced by FKBP51s overexpression or silencing, respectively. Naïve PD-L1 was found in the endoplasmic reticulum (ER) of glioma cells complexed with FKBP51s, whereas the glycosylated form was measured in the Golgi apparatus. SAFit reduced PD-L1 levels (constitutively expressed and ionizing radiation-induced). SAFit reduced cell death of PBMC co-cultured with glioma., Conclusions: Here we addressed the mechanism of post-translational regulation of PD-L1 protein in glioma. FKBP51s upregulated PD-L1 expression on the plasma membrane by catalysing the protein folding required for subsequent glycosylation. Inhibition of FKBP51s isomerase activity by SAFit decreased PD-L1 levels. These findings suggest that FKBP51s is a potential target of immunomodulatory strategies for glioblastoma treatment., Competing Interests: CONFLICTS OF INTEREST No conflicts of interest is declared.

We evaluated the prognostic impact of a complex remodeling classification (CRC) in asymptomatic patients with arterial hypertension (AH). We retrospectively included 749 hypertensive patients (female 325, 43.4% age 62 ± 11.3 years) in Stages A and B of heart failure. CRC was evaluated including indexed left ventricular mass, end-diastolic volume, and relative wall thickness. After 45-month follow-up, we considered a composite endpoint: total mortality, myocardial infarction, myocardial revascularization, cerebrovascular events, and acute pulmonary edema. Blood pressure was controlled in 265 patients (35.4%), 317 (42.3%) were in Grade 1 of AH, 123 (16.4%) in Grade 2, and 44 (5.9%) in Grade 3. Considering CRC, 292 patients (38%) presented normal/physiological hypertrophy, 102 (13.6%) concentric remodeling, 29 (3.9%) eccentric remodeling, 157 (21%) concentric hypertrophy, 11 (1.5%) mixed hypertrophy, 52 (6.9%) dilated hypertrophy, and 36 (4.8%) eccentric hypertrophy. We observed a total of 73 events (9.7%). Kaplan-Meier method demonstrated a significant different survival in CRC-derived classes (P < .001). Cox regression demonstrated CRC as independent predictor (P = .01), after adjusting for age, gender, diabetes mellitus, grade of hypertension, antihypertensive therapy, stable ischemic heart disease, obesity, systolic and diastolic dysfunction, and classic remodeling classification. In asymptomatic patients with AH, CRC is an independent predictor of poor outcome., (Copyright © 2017 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.)

Up-to-date, several molecular markers of prognosis have been studied in Oral Squamous Cell Carcinoma (OSCC), but none entered in the clinical setting. Therapy of OSCC tumors mainly relies on surgery, radiotherapy and partially on chemotherapy; there is an urgent need for biomarkers able to better stratify OSCC patients' risk to address targeted therapeutic strategies. The role of immune response in the pathogenesis and biological behavior of OSCC has been investigated by several authors, and promising results have been obtained with immune checkpoint inhibitors. We already investigated the role of the immune modulator FK506-binding protein 51 (FKBP51), a FK506-binding immunophilin, in cutaneous melanoma biology, and its expression in several human solid tumors. In the present study, we aimed to assess the value of FKBP51 expression in OSCC tumor cells as a marker of outcome. We collected clinical data from 72 patients who underwent surgery for Squamous Cell Carcinoma (SCC) of the tongue, floor, lips and palate. FKBP51 expression was assessed by immunohistochemistry on paraffin-embedded tumor tissues. In addition, we evaluated the human papillomavirus (HPV) status of primary tumors by immunohistochemistry, viral subtyping and In Situ Hybridization (ISH) assay. We found that high FKBP51-expressing tumors characterized the OSCCs with the worst prognosis: the high immunohistochemical expression of FKBP51 associated with death occurring within five years from the diagnosis with a sensitivity of 88.46% and a specificity of 91.67%. The estimated positive predictive value of the test was 88.45% and negative predictive value 91.67%. We tested FKBP51 mRNA presence, by RT-PCR assay, in a selected series of OSCC tumors, and we found that mRNA correlated well to the protein expression and to the clinical outcome. Applying the Bayes formula, we estimated an 88% probability of dying within five years from the diagnosis of OSCC patients with a high FKBP51 immunohistochemical (IHC) test result (>51% of FKBP51 positive tumor cells). On the basis of our analysis, we propose tumor tissue expression of FKBP51 protein as a reliable prognostic marker for OSCC tumors.

Patients with asymptomatic heart failure (HF; stage A and B) are characterized by maladaptive left ventricular (LV) remodeling. Classic 4-group classification of remodeling considers only LV mass index and relative wall thickness as variables. Complex remodeling classification (CRC) includes also LV end-diastolic volume index. Main aim was to assess the prognostic impact of CRC in stage A and B HF. A total of 1,750 asymptomatic subjects underwent echocardiographic examination as a screening evaluation in the presence of cardiovascular risk factors. LV dysfunction, both systolic (ejection fraction) and diastolic (transmitral flow velocity pattern), was evaluated, together with LV remodeling. We considered a composite end point: all-cause death, myocardial infarction, coronary revascularizations, cerebrovascular events, and acute pulmonary edema. CRC was suitable for 1,729 patients (men 53.6%; age 58.3 ± 13 years). Two hundred thirty-eight patients presented systolic dysfunction (ejection fraction <50%) and 483 diastolic dysfunction. According to the CRC, 891 patients were normals or presented with physiologic hypertrophy, 273 concentric remodeling, 47 eccentric remodeling, 350 concentric hypertrophy, 29 mixed hypertrophy, 86 dilated hypertrophy, and 53 eccentric hypertrophy. Age and gender distribution was noticed (p <0.001). After a median follow-up of 21 months, Kaplan-Meier analysis showed different survival distribution (p <0.001) of the CRC patterns. In multivariate Cox regression (adjusted for age, gender, history of stable ischemic heart disease, classic remodeling classification, systolic, and diastolic dysfunction), CRC was independent predictor of primary end point (p = 0.044, hazard ratio 1.101, 95% CI 1.003 to 1.21), confirmed in a logistic regression (p <0.03). In conclusion, CRC could help physicians in prognostic stratification of patients in stage A and B HF., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Drug-eluting stents (DES) are now considered the most promising device to treat peripheral artery disease (PAD) and minimize restenosis. There is uncertainty however on the best antirestenotic drug for such devices. In particular, biolimus (i.e. umirolimus) and everolimus are two of the most promising agents, given the extensive data in support of their coronary safety and efficacy, but their comparative effectiveness for peripheral interventions is not established., Methods: Building upon our extensive experience in the percutaneous treatment of infra-inguinal artery disease with DES, we compared the acute and longterm outlook of patients treated with biolimus-eluting stents (BES) and everolimus-eluting stents (EES). We collected baseline, procedural and outcome details on all patients undergoing infra-inguinal BES or EES implantation. The endpoints of interest were death, amputation, revascularization, their composite, and change in Fontaine class. A total of 80 patients were included (20 treated with BES and 60 with EES). Most features were similar in the two groups, despite longer lesions in the EES group. Unadjusted analysis showed similar results irrespective of the drug used, with composite endpoint occurring, respectively, in 4 (20.0%) and 10 (16.7%) (p=0.741)., Results and Conclusion: However, analysis with inverse probability of treatment weighting showed significant differences in the risk of revascularization (hazard ratio of BES vs EES=9.55 [95% confidence interval 2.16-42.23], p=0.003) and composite endpoint (hazard ratio=5.11 [1.33-19.62], p=0.018). In conclusion, EES appear superior to BES for endovascular therapy of infrainguinal artery disease. Dedicated randomized trials are required to definitely confirm or disprove these findings., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Aging of the European population and interest in a healthy population in western countries have contributed to an increase in the number of health surveys, where the role of survey design, data collection, and data analysis methodology is clear and recognized by the whole scientific community. Survey methodology has had to couple with the challenges deriving from data collection through information and communications technology (ICT). Telemedicine systems have not used patients as a source of information, often limiting them to collecting only biometric data. A more effective telemonitoring system would be able to collect objective and subjective data (biometric parameters and symptoms reported by the patients themselves), and to control the quality of subjective data collected: this goal be achieved only by using and merging competencies from both survey methodology and health research., Objective: The objective of our study was to propose new metrics to control the quality of data, along with the well-known indicators of survey methodology. Web questionnaires administered daily to a group of patients for an extended length of time are a Web health monitoring survey (WHMS) in a telemedicine system., Methods: We calculated indicators based on paradata collected during a WHMS study involving 12 patients, who signed in to the website daily for 2 months., Results: The patients' involvement was very high: the patients' response rate ranged between 1.00 and 0.82, with an outlier of 0.65. Item nonresponse rate was very low, ranging between 0.0% and 7.4%. We propose adherence to the chosen time to connect to the website as a measure of involvement and cooperation by the patients: the difference from the median time ranged between 11 and 24 minutes, demonstrating very good cooperation and involvement from all patients. To measure habituation to the questionnaire, we also compared nonresponse rates to the items between the first and the second month of the study, and found no significant difference. We computed the time to complete the questionnaire both as a measure of possible burden for patient, and to detect the risk of automatic responses. Neither of these hypothesis was confirmed, and differences in time to completion seemed to depend on health conditions. Focus groups with patients confirmed their appreciation for this "new" active role in a telemonitoring system., Conclusions: The main and innovative aspect of our proposal is the use of a Web questionnaire to virtually recreate a checkup visit, integrating subjective (patient's information) with objective data (biometric information). Our results, although preliminary and if need of further study, appear promising in proposing more effective telemedicine systems. Survey methodology could have an effective role in this growing field of research and applications.

Tirofiban is used in the treatment of patients with acute coronary syndrome submitted to percutaneous coronary intervention (PCI). We have, previously, shown that tirofiban stimulates VEGF expression and promotes proliferation of endothelial cells. VEGF is a well known inhibitor of endothelial cell apoptosis. TNF-α is a pro-apoptotic cytokine released in the site of a vascular injury, including balloon angioplasty. We thought to investigate whether tirofiban was able to protect endothelial cells from cell death induced by TNF-α. For this study, we used human umbilical vein endothelial cells (HUVEC). Analysis of apoptosis was performed by propidium iodide incorporation, annexin V staining and measure of active caspase 3 levels. Western blot served for a semiquantitative measure of Akt activation, VEGF, and the pro-apoptotic Bim and Bak. Our results show that TNF-α was unable to activate caspase 3 and produce cell death in the presence of tirofiban. Activation of apoptosis was preceded by upregulation of Bim and Bak that resulted decreased after addition of tirofiban. The anti-apoptosis effect of tirofiban was reproduced by VEGF and counteracted by VEGFR2 blockade and the cation chelating agent ethylene glycol tetraacetic acid (EGTA). The use of p-Akt inhibitor, BEZ235,and Akt knockdown, suggested that pAkt mediated the prosurvival effect of tirofiban. In conclusion, tirofiban protects endothelial cells from apoptosis stimulated by TNF-α, due to its ability to stimulate VEGF production., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The PID1/NYGGF4/PCLI1 gene encodes for a protein with a phosphotyrosine-binding domain, which interacts with the lipoprotein receptor-related protein 1. Previous work by us and others suggested a function of the gene in cell proliferation of NIH3T3 fibroblasts and 3T3-L1 pre-adipocytes. The molecular characterization of PCLI1 protein, ectopically expressed in NIH3T3 fibroblasts, revealed two phosphorylation sites at Ser154 and Ser165. In order to clarify the functions of this gene, we analyzed the effects of its downregulation on cellular proliferation and cell cycle progression in NIH3T3 cell cultures. Downregulation of PID1/NYGGF4/PCLI1 mRNA levels by short hairpin RNAs (shRNAs) elicited decreased proliferation rate in mammalian cell lines; cell cycle analysis of serum-starved, synchronized NIH3T3 fibroblasts showed an increased accumulation of shRNA-interfered cells in the G1 phase. Decreased levels of FOS and MYC mRNAs were accordingly associated with these events. The molecular scenario emerging from our data suggests that PID1/NYGGF4/PCLI1 controls cellular proliferation and cell cycle progression in NIH3T3 cells.

Background: The use of inhibitors of glycoprotein IIb/IIIa (GPIIb/IIIa) has provided dramatic results in terms of the prevention of acute stent thrombosis and a reduction in major adverse coronary events in patients subjected to percutaneous coronary intervention. GPIIb/IIIa or αIIbβ3 is a member of the β3 subfamily of integrins, which also includes αVβ3. GPIIb/IIIa functions as a receptor for fibrinogen and several adhesion proteins sharing an arginine-glycine-aspartic acid (RGD) sequence. GPIIb/IIIa antagonists, through blockade of the receptor, prevent platelet aggregation. Among the three GPIIb/IIIa antagonists used in therapy, abciximab is an anti-β3 monoclonal antibody, while tirofiban and eptifibatide mimic the binding sequence of the fibrinogen ligand. Although antiplatelet aggregation represents the central function of GPIIb/IIIa inhibitors, further actions have been documented for these compounds., Objective: The aim of the present article is to review the structures and functions of GPIIb/IIIa antagonists and to highlight the clinical outcomes and results of randomized trials with these compounds. Hypotheses on the unexplored potential of GPIIb/IIIa antagonists will be put forward., Conclusion: GPIIb/IIIa inhibitors were developed to prevent platelet aggregation, however, these compounds can exert further biological functions, both platelet- and non-platelet-related. Large-scale studies comparing the efficacy and safety of GPIIb/IIIa antagonists are lacking. More insights into the functions of these compounds may lead to generation of novel small molecules able to antagonize platelet aggregation while promoting vascular repair.

Background: FK506 binding proteins (FKBP) are multifunctional proteins highly conserved across the species and abundantly expressed in the cell. In addition to a well-established role in immunosuppression, FKBPs modulate several signal transduction pathways in the cell, due to their isomerase activity and the capability to interact with other proteins, inducing changes in conformation and function of protein partners. Increasing literature data support the concept that FKBPs control cancer related pathways., Scope of the Review: The aim of the present article is to review current knowledge on FKBPs roles in regulation of key signaling pathways associated with cancer., Major Conclusions: Some family members appear to promote disease while others are protective against tumorigenesis., General Significance: FKBPs family proteins are expected to provide new biomarkers and small molecular targets, in the near future, increasing diagnostic and therapeutic opportunities in the cancer field. This article is part of a Special Issue entitled Proline-Directed Foldases: Cell Signaling Catalysts and Drug Targets., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Melanoma is the most aggressive skin cancer; its prognosis, particularly in advanced stages, is disappointing largely due to the resistance to conventional anticancer treatments and high metastatic potential. NF-κB constitutive activation is a major factor for the apoptosis resistance of melanoma. Several studies suggest a role for the immunophilin FKBP51 in NF-κB activation, but the underlying mechanism is still unknown. In the present study, we demonstrate that FKBP51 physically interacts with IKK subunits, and facilitates IKK complex assembly. FKBP51-knockdown inhibits the binding of IKKγ to the IKK catalytic subunits, IKK-α and -β, and attenuates the IKK catalytic activity. Using FK506, an inhibitor of the FKBP51 isomerase activity, we found that the IKK-regulatory role of FKBP51 involves both its scaffold function and its isomerase activity. Moreover, FKBP51 also interacts with TRAF2, an upstream mediator of IKK activation. Interestingly, both FKBP51 TPR and PPIase domains are required for its interaction with TRAF2 and IKKγ, whereas only the TPR domain is involved in interactions with IKKα and β. Collectively, these results suggest that FKBP51 promotes NF-κB activation by serving as an IKK scaffold as well as an isomerase. Our findings have profound implications for designing novel melanoma therapies based on modulation of FKBP51., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

FKBP51 (gene FKBP5) is an immunophilin capable of immunosuppression expressed in melanoma and lymphocytes. We found increased levels of a spliced FKBP5 variant in the PBMCs of 124 patients with melanoma. This variant encodes for an unknown isoform (FKBP51s). We hypothesized that FKBP51s resulted from tumour interaction with immune cells, through PDL-1/PD-1. To address this issue, we performed melanoma/PBMC cocultures. Furthermore, the immunohistochemistry of 76 melanoma specimens served to investigate whether FKBP51s stained tumour infiltrating lymphocytes. Our results showed that PBMCs expressed FKBP51s when cocultured with melanoma. Tumour PDL-1 knockdown or anti-PD-1 reduced FKBP51s expression in cocultured PBMCs. IHC showed a strong FKBP51s signal in tumour infiltrating lymphocytes, and lymphocytes of the invasion front of the tumour, along with melanoma PDL-1 expression. When overexpressed in melanoma, FKBP51s facilitated PDL-1 expression on the cell surface. In conclusion, our study shows that FKBP51s marks the PBMCs of patients with melanoma and is exploited by the tumour to immunomodulate through PDL-1., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Numerous studies have indicated that BMP4 signaling is involved in the regulation of the early steps of development. In mouse embryonic stem cells (ESCs), BMP4 is crucial to sustain pluripotency and blocks differentiation towards neural fate. Here, through a systematic analysis of miRNAs in ESCs, we establish that BMP4 signaling regulates miR-23a, 27a and 24-2, through the recruitment of phospho-Smads at the promoter of the gene encoding this miRNA cluster. Suppression of miR-23a/b, 27a/b and 24 does not affect self-renewal or pluripotency, but induces an evident change of ESC differentiation, with a significant increase of the cells undergoing apoptosis after the transition from ESCs to epiblast stem cells (EpiSCs). BMP4 has been previously reported to cause apoptosis during ESC differentiation. By blocking BMP4 signaling, we completely prevent the apoptosis induced by suppression of the miRs. This suggests that the effects of miR suppression are the result of enhanced BMP4 signaling. This hypothesis is further supported by the observation that Smad5, the transcription factor downstream of the BMP4 receptor, is targeted by the miRNAs of the 23a and 23b clusters. Altogether, our results highlight the existence of a regulatory loop, involving Smad5 and the miR-23a clusters, that modulates the apoptotic response of ESCs to BMP4.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology., (© The Author 2015. Published by Oxford University Press.)

Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)