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Purpose To assess the clinical and retinal imaging features of patients in whom retinal vascular occlusion (VO) had developed in temporal associations with COVID-19 vaccination. Methods In this retrospective case series, all consecutive adult patients with new onset VO within 6 weeks of vaccination against COVID-19 were included in the study between May 1 and October 31, 2021. All patients had a systemic medical health assessment, full ophthalmic evaluation, and complete fundus imaging. Results Fifteen eyes of VO (14 patients) after COVID-19 vaccinations were identified. The median time between vaccination and symptoms onset was 14 days (range 7–42 days). The mean best-corrected visual acuity (BCVA) was 20/55 with a range of 20/20 to 20/200. Eleven of 15 eyes (73.3%) had visual acuity improvement after intravitreal treatment at 60–90 days (range, 45–105 days) from the presentation. Four of 5 cases without systemic risk factors for VO had a mean BCVA > 20/32 at presentation and > 20/25 at the latest evaluation. Between May 1 and October 31, 2021, a temporal association was found between the 15 reported cases and COVID-19 vaccination out of a total of 29 VO (p = 0.05). The incidence of VO was higher in the considered period compared to the equivalent 6-month period in 2019 (1.17% vs 0.52%, respectively; p = 0.0134). Conclusions Retinal vascular occlusion with different grades of severity are reported in temporal association with COVID-19 vaccination. The exact pathogenic mechanism needs to be further studied. No certain causal relationship can be established from this case series.

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.

Glioblastoma (GB) is the most frequent and aggressive primary brain tumor. It is a highly infiltrating tumor. Complete eradication by surgery is difficult. The probability of survival beyond two years is very low. Magnetic resonance imaging (MRI) is a routine procedure for diagnosing gliomas. However, morphological MRI often fails to identify and quantify the presence of infiltration in peritumoral non-enhanced areas, resulting in inaccuracy in the assessment of invasive margins. Up to 50% of the patients show pseudo-progression. Morphological MRI fails to distinguish the marked enhancement in the tumor bed caused by radionecrosis from actual tumor progression. Diagnosis of GB recurrence through MR morphological sequences has a sensitivity and specificity of about 68% and 77%. Such percentages rise thanks to the use of non-morphological sequences (PWI, DWI, MRS), which are used in routine tumor protocols only in large centers and interpreted by expert neuroradiologists. Biomarkers in conjunction with MRI may help improve the classification, prognosis, and treatment choice. GB is highly infiltrated with monocytic cells, tumor-associated macrophages (TAMs), that invade the peripheral blood. Our group has recently identified a splice isoform of FK506-binding protein 51 (FKBP51) as a reliable TAM marker. We found an expansion in circulating CD163/FKBP51s monocytes in a cohort of GB patients. In patients with an MRI diagnosis of complete surgical tumor removal, these monocytes dramatically decreased. In patients with an MRI diagnosis of residual tumor, most of the peripheral CD163 monocytes that in the preoperative stage were FKBP51s- had turned into FKBP51s+. CD163/FKBP51s+ monocytes helped to distinguish between radionecrosis and actual tumor progression. These results suggest that circulating CD163/FKBP51s+ monocytes are associated with GB and can be helpful in monitoring GB patients. Citation Format: Carolina Giordano, Giuseppe Maria Della Pepa, Simona Romano, Laura Marrone, Carlo Maria Donzelli, Maria Fiammetta Romano, Simona Gaudino. Combined use of MRI and TAM analysis in GB monitoring. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5474.

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.

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.

Purpose We developed and applied a universal strategy for preimplantation genetic testing for all cystic fibrosis gene mutations (PGT-CF) based on next-generation sequencing (NGS). Methods A molecular protocol was designed to diagnose all CF mutations at preimplantation stage. The detection of CF mutations was performed by direct gene sequencing and linkage strategy testing 38 specific SNPs located upstream and inside the gene for PGT-CF. Seventeen couples at risk of CF transmission decided to undergo PGT-CF. Trophectoderm cell biopsies were performed on day 5–6 blastocysts. PGT for aneuploidy (PGT-A) was performed from the same samples. Tested embryos were transferred on further natural cycles. Results PGT was performed on 109 embryos. Fifteen CF mutations were tested. PGT-CF and PGT-A were conclusive for respectively 92.7% and 95.3% of the samples. A mean of 24.1 SNPs was informative per couple. After a single embryo transfer on natural cycle, 81.3% of the transferred tested embryos were implanted. Conclusions The present protocol based on the entire CFTR gene together with informative SNPs outside and inside the gene can be applied to diagnose all CF mutations at preimplantation stage.

We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL.

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.

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.

Purpose To present two consecutive cases of Central Retinal Artery Occlusion (CRAO) with unusual Optical Coherence Tomography (OCT) presentation of Hyperreflective Spots (HRS) in the posterior vitreous. Case Report The first patient was a 59 years-old male who developed CRAO in the post-operative period after aortic valve replacement. OCT scans revealed the presence of many HRS in the posterior vitreous, remarkably decreased in number at two months follow-up. The second patient was a 74-year-old male who developed CRAO after arterial chemoembolization for recurrent hepatocellular carcinoma. OCT scans showed again the presence of hyperreflective spots in the posterior vitreous and their reduction at two months follow-up with inner retinal atrophy as the final outcome in both cases. Conclusions HRS in the posterior vitreous have been described in inflammatory eye conditions such as uveitis, diabetic macular edema, post cataract surgery, and considered a clinical sign of inflammation. To our knowledge, the combination of CRAO and HRS has not been previously reported. The number of HRS seems to decrease over time, suggesting a role for inflammatory response in the acute stage of CRAO. A similar pathogenic process is known to occur during cerebral ischemia, where the inflammatory response may exacerbate brain injury and post-ischemic damage.

Background Which fertilization method, between ICSI and IVF in split insemination treatments, has the highest clinical efficiency in producing clinically usable blastocyst? Methods 211 infertile couples underwent split insemination treatments for a non-severe male factor. 1300 metaphase II (MII) oocytes were inseminated by conventional IVF and 1302 MII oocytes were micro-injected with the same partner’s semen. Embryo development until blastocyst stage on day V and clinical outcomes were valuated trough conventional key performance indicators (KPI), and new KPIs such as blastocyst rate per used MII oocytes and the number of MII oocytes to produce one clinically usable blastocyst from ICSI and IVF procedures. Results The results were globally analyzed and according to ovarian stimulation protocol, infertility indication, and female age. The conventional KPI were online with the expected values from consensus references. From global results, 2.3 MII oocyte was needed to produce one clinically usable blastocyst after ICSI compared to 2.9 MII oocytes in IVF. On the same way, more blastocysts for clinical use were produced from fewer MII oocytes in ICSI compared to IVF in all sub-groups. Conclusions In split insemination treatments, the yield of clinically usable blastocysts was always superior in ICSI compared to IVF. The new KPI "number of needed oocytes to produce one clinically usable embryo" tests the clinical efficiency of the IVF laboratory.

Purpose To report the unique case of a pair of phenotypically discordant monozygotic twins, with one of them affected by unilateral Coats disease. Case report Both patients underwent a complete ophthalmologic evaluation and were genetically tested with whole-exome sequencing (WES). Any known or unknown potential genetic determinant of Coats disease wasn't found. Conclusion It may suggest a non-genetic etiology for this disorder. This represents, to the best of our knowledge, the first case of genetic analysis of monozygotic twins, one of whom is affected by Coats disease. Further studies are warranted, including performing genetic analysis directly on retinal biopsy tissue.

TGFβ is essential for the generation of anti-tumor Th9 cells, while it causes resistance against anti-tumor immunity. Pei et al. identify BFAR as an E3 ligase of TGFβR1, through which it coordinates TGFβ signaling to modulate Th9-mediated cancer immunotherapy., TGFβ is essential for the generation of anti-tumor Th9 cells; on the other hand, it causes resistance against anti-tumor immunity. Despite recent progress, the underlying mechanism reconciling the double-edged effect of TGFβ signaling in Th9-mediated cancer immunotherapy remains elusive. Here, we find that TGFβ-induced down-regulation of bifunctional apoptosis regulator (BFAR) represents the key mechanism preventing the sustained activation of TGFβ signaling and thus impairing Th9 inducibility. Mechanistically, BFAR mediates K63-linked ubiquitination of TGFβR1 at K268, which is critical to activate TGFβ signaling. Thus, BFAR deficiency or K268R knock-in mutation suppresses TGFβR1 ubiquitination and Th9 differentiation, thereby inhibiting Th9-mediated cancer immunotherapy. More interestingly, BFAR-overexpressed Th9 cells exhibit promising therapeutic efficacy to curtail tumor growth and metastasis and promote the sensitivity of anti–PD-1–mediated checkpoint immunotherapy. Thus, our findings establish BFAR as a key TGFβ-regulated gene to fine-tune TGFβ signaling that causes Th9 induction insensitivity, and they highlight the translational potential of BFAR in promoting Th9-mediated cancer immunotherapy.

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 Supplementary Information The online version contains supplementary material available at 10.1007/s12265-021-10117-9.

We developed and applied a universal strategy for preimplantation genetic testing for all cystic fibrosis gene mutations (PGT-CF) based on next-generation sequencing (NGS).A molecular protocol was designed to diagnose all CF mutations at preimplantation stage. The detection of CF mutations was performed by direct gene sequencing and linkage strategy testing 38 specific SNPs located upstream and inside the gene for PGT-CF. Seventeen couples at risk of CF transmission decided to undergo PGT-CF. Trophectoderm cell biopsies were performed on days 5-6 blastocysts. PGT for aneuploidy (PGT-A) was performed from the same samples. Tested embryos were transferred on further natural cycles.PGT was performed on 109 embryos. Fifteen CF mutations were tested. PGT-CF and PGT-A were conclusive for, respectively, 92.7% and 95.3% of the samples. A mean of 24.1 SNPs was informative per couple. After single embryo transfer on natural cycle, 81.3% of the transferred tested embryos implanted.The present protocol based on the entire CFTR gene sequencing together with informative SNPs outside and inside the gene can be applied to diagnose all CF mutations at preimplantation stage.

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.

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.

Stat6 is known to drive macrophage M2 polarization. However, how macrophage polarization is fine-tuned by Stat6 is poorly understood. Here, we find that Lys383 of Stat6 is acetylated by the acetyltransferase CREB-binding protein (CBP) during macrophage activation to suppress macrophage M2 polarization. Mechanistically, Trim24, a CBP-associated E3 ligase, promotes Stat6 acetylation by catalyzing CBP ubiquitination at Lys119 to facilitate the recruitment of CBP to Stat6. Loss of Trim24 inhibits Stat6 acetylation and thus promotes M2 polarization in both mouse and human macrophages, potentially compromising antitumor immune responses. By contrast, Stat6 mediates the suppression of TRIM24 expression in M2 macrophages to contribute to the induction of an immunosuppressive tumor niche. Taken together, our findings establish Stat6 acetylation as an essential negative regulatory mechanism that curtails macrophage M2 polarization., Stat6 promotes M2 macrophage polarization. Here the authors characterize Trim24-CBP-Stat6 circuit regulating M2 macrophage polarization via Stat6 acetylation, and show it contributes to pro-tumorigenic macrophage activity in mice.

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.

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.

SummaryBackground 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 MF patients. 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. This article is protected by copyright. All rights reserved.

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults, and despite advances in neuro-oncology, the prognosis for patients remains dismal. The signal transducer and activator of transcription-3 (STAT3) has been reported as a key regulator of the highly aggressive mesenchymal GBM subtype, and its direct silencing (by RNAi oligonucleotides) has revealed a great potential as an anti-cancer therapy. However, clinical use of oligonucleotide-based therapies is dependent on safer ways for tissue-specific targeting and increased membrane penetration. The objective of this study is to explore the use of nucleic acid aptamers as carriers to specifically drive a STAT3 siRNA to GBM cells in a receptor-dependent manner. Using an aptamer that binds to and antagonizes the oncogenic receptor tyrosine kinase PDGFRβ (Gint4.T), here we describe the design of a novel aptamer-siRNA chimera (Gint4.T-STAT3) to target STAT3. We demonstrate the efficient delivery and silencing of STAT3 in PDGFRβ+GBM cells. Importantly, the conjugate reduces cell viability and migration in vitro and inhibits tumor growth and angiogenesis in vivo in a subcutaneous xenograft mouse model. Our data reveals Gint4.T-STAT3 conjugate as a novel molecule with great translational potential for GBM therapy. Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults, and despite advances in neuro-oncology, the prognosis for patients remains dismal. The signal transducer and activator of transcription-3 (STAT3) has been reported as a key regulator of the highly aggressive mesenchymal GBM subtype, and its direct silencing (by RNAi oligonucleotides) has revealed a great potential as an anti-cancer therapy. However, clinical use of oligonucleotide-based therapies is dependent on safer ways for tissue-specific targeting and increased membrane penetration. The objective of this study is to explore the use of nucleic acid aptamers as carriers to specifically drive a STAT3 siRNA to GBM cells in a receptor-dependent manner. Using an aptamer that binds to and antagonizes the oncogenic receptor tyrosine kinase PDGFRβ (Gint4.T), here we describe the design of a novel aptamer-siRNA chimera (Gint4.T-STAT3) to target STAT3. We demonstrate the efficient delivery and silencing of STAT3 in PDGFRβ+ GBM cells. Importantly, the conjugate reduces cell viability and migration in vitro and inhibits tumor growth and angiogenesis in vivo in a subcutaneous xenograft mouse model. Our data reveals Gint4.T-STAT3 conjugate as a novel molecule with great translational potential for GBM therapy.

// Alessandra Capasso 1, * , Carmen Cerchia 2, * , Carmen Di Giovanni 2 , Giuseppina Granato 1 , Francesco Albano 1 , Simona Romano 1 , Emmanuele De Vendittis 1 , Maria Rosaria Ruocco 1 , Antonio Lavecchia 2 1 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, 80131 Naples, Italy 2 Department of Pharmacy, “Drug Discovery” Laboratory, University of Naples Federico II, 80131 Naples, Italy * These authors have contributed equally to this work Correspondence to: Maria Rosaria Ruocco, e-mail: mariarosaria.ruocco2@unina.it Antonio Lavecchia, e-mail: antonio.lavecchia@unina.it Keywords: cancer, CDC25 phosphatases, drug discovery, cell cycle, melanoma cells Received: April 26, 2015 Accepted: October 02, 2015 Published: October 13, 2015 ABSTRACT CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5–9, 21, 24, and 25) were identified with K i values for CDC25A, -B and -C ranging from 0.01 to 4.4 μM. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds.

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.

Goodson, William H. et al., © The Author 2015. 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., We gratefully acknowledge the support of the National Institute of Health-National Institute of Environmental Health Sciences (NIEHS) conference grant travel support (R13ES023276); Glenn Rice, Office of Research and Development, United States Environmental Protection Agency, Cincinnati, OH, USA also deserves thanks for his thoughtful feedback and inputs on the manuscript; William H.Goodson III was supported by the California Breast Cancer Research Program, Clarence Heller Foundation and California Pacific Medical Center Foundation; Abdul M.Ali would like to acknowledge the financial support of the University of Sultan Zainal Abidin, Malaysia; Ahmed Lasfar was supported by an award from the Rutgers Cancer Institute of New Jersey; Ann-Karin Olsen and Gunnar Brunborg were supported by the Research Council of Norway (RCN) through its Centres of Excellence funding scheme (223268/F50), Amancio Carnero’s lab was supported by grants from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0306-2012); Matilde E. Lleonart was supported by a trienal project grant PI12/01104 and by project CP03/00101 for personal support. Amaya Azqueta would like to thank the Ministerio de Educacion y Ciencia (‘Juande la Cierva’ programme, 2009) of the Spanish Government for personal support; Amedeo Amedei was supported by the Italian Ministry of University and Research (2009FZZ4XM_002), and the University of Florence (ex60%2012); Andrew R.Collins was supported by the University of Oslo; Annamaria Colacci was supported by the Emilia-Romagna Region - Project ‘Supersite’ in Italy; Carolyn Baglole was supported by a salary award from the Fonds de recherche du Quebec-Sante (FRQ-S); Chiara Mondello’s laboratory is supported by Fondazione Cariplo in Milan, Italy (grant n. 2011-0370); Christian C.Naus holds a Canada Research Chair; Clement Yedjou was supported by a grant from the National Institutes of Health (NIH-NIMHD grant no. G12MD007581); Daniel C.Koch is supported by the Burroughs Wellcome Fund Postdoctoral Enrichment Award and the Tumor Biology Training grant: NIH T32CA09151; Dean W. Felsher would like to acknowledge the support of United States Department of Health and Human Services, NIH grants (R01 CA170378 PQ22, R01 CA184384, U54 CA149145, U54 CA151459, P50 CA114747 and R21 CA169964); Emilio Rojas would like to thank CONACyT support 152473, Ezio Laconi was supported by AIRC (Italian Association for Cancer Research, grant no. IG 14640) and by the Sardinian Regional Government (RAS); Eun-Yi Moon was supported by grants from the Public Problem-Solving Program (NRF-015M3C8A6A06014500) and Nuclear R&D Program (#2013M2B2A9A03051296 and 2010-0018545) through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science and Technology (MEST) in Korea; Fahd Al-Mulla was supported by the Kuwait Foundation for the Advancement of Sciences (2011-1302-06); Ferdinando Chiaradonna is supported by SysBioNet, a grant for the Italian Roadmap of European Strategy Forum on Research Infrastructures (ESFRI) and by AIRC (Associazione Italiana Ricerca sul Cancro; IG 15364); Francis L.Martin acknowledges funding from Rosemere Cancer Foundation; he also thanks Lancashire Teaching Hospitals NHS trust and the patients who have facilitated the studies he has undertaken over the course of the last 10 years; Gary S.Goldberg would like to acknowledge the support of the New Jersey Health Foundation; Gloria M.Calaf was supported by Fondo Nacional de Ciencia y Tecnología (FONDECYT), Ministerio de Educación de Chile (MINEDUC), Universidad de Tarapacá (UTA); Gudrun Koppen was supported by the Flemish Institute for Technological Research (VITO), Belgium; Hemad Yasaei was supported from a triennial project grant (Strategic Award) from the National Centre for the Replacement, Refinement and Reduction (NC3Rs) of animals in research (NC.K500045.1 and G0800697); Hiroshi Kondoh was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Science and Technology Agency and by JST, CREST; Hsue-Yin Hsu was supported by the Ministry of Science and Technology of Taiwan (NSC93-2314-B-320-006 and NSC94-2314-B-320-002); Hyun Ho Park was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) of the Ministry of Education, Science and Technology (2012R1A2A2A01010870) and a grant from the Korea Healthcare Technology R&D project, Ministry of Health and Welfare, Republic of Korea (HI13C1449); Igor Koturbash is supported by the UAMS/NIH Clinical and Translational Science Award (UL1TR000039 and KL2TR000063) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000; Jan Vondráček acknowledges funding from the Czech Science Foundation (13-07711S); Jesse Roman thanks the NIH for their support (CA116812), John Pierce Wise Sr. and Sandra S.Wise were supported by National Institute of Environmental Health Sciences (ES016893 to J.P.W.) and the Maine Center for Toxicology and Environmental Health; Jonathan Whitfield acknowledges support from the FERO Foundation in Barcelona, Spain; Joseph Christopher is funded by Cancer Research UK and the International Journal of Experimental Pathology; Julia Kravchenko is supported by a philanthropic donation by Fred and Alice Stanback; Jun Sun is supported by a Swim Across America Cancer Research Award; Karine A.Cohen-Solal is supported by a research scholar grant from the American Cancer Society (116683-RSG-09-087-01-TBE); Laetitia Gonzalez received a postdoctoral fellowship from the Fund for Scientific Research–Flanders (FWO-Vlaanderen) and support by an InterUniversity Attraction Pole grant (IAP-P7-07); Laura Soucek is supported by grant #CP10/00656 from the Miguel Servet Research Contract Program and acknowledges support from the FERO Foundation in Barcelona, Spain; Liang-Tzung Lin was supported by funding from the Taipei Medical University (TMU101-AE3-Y19); Linda Gulliver is supported by a Genesis Oncology Trust (NZ) Professional Development Grant, and the Faculty of Medicine, University of Otago, Dunedin, New Zealand; Louis Vermeulen is supported by a Fellowship of the Dutch Cancer Society (KWF, UVA2011-4969) and a grant from the AICR (14–1164); Mahara Valverde would like to thank CONACyT support 153781; Masoud H. Manjili was supported by the office of the Assistant Secretary of Defense for Health Affairs (USA) through the Breast Cancer Research Program under Award No. W81XWH-14-1-0087 Neetu Singh was supported by grant #SR/FT/LS-063/2008 from the Department of Science and Technology, Government of India; Nicole Kleinstreuer is supported by NIEHS contracts (N01-ES 35504 and HHSN27320140003C); P.K. Krishnakumar is supported by the Funding (No. T.K. 11-0629) of King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia; Paola A.Marignani is supported by the Dalhousie Medical Research Foundation, The Beatrice Hunter Cancer Institute and CIHR and the Nova Scotia Lung Association; Paul Dent is the holder of the Universal Inc.Chair in Signal Transduction Research and is supported with funds from PHS grants from the NIH (R01-CA141704, R01-CA150214, R01-DK52825 and R01-CA61774); Petr Heneberg was supported by the Charles University in Prague projects UNCE 204015 and PRVOUK P31/2012, and by the Czech Science Foundation projects P301/12/1686 and 15-03834Y; Po Sing Leung was supported by the Health and Medical Research Fund of Food and Health Bureau, Hong Kong Special Administrative Region, Ref. No: 10110021; Qiang Cheng was supported in part by grant NSF IIS-1218712; R. Brooks Robey is supported by the United States Department of Veterans Affairs; Rabindra Roy was supported by United States Public Health Service Grants (RO1 CA92306, RO1 CA92306-S1 and RO1 CA113447); Rafaela Andrade-Vieira is supported by the Beatrice Hunter Cancer Research Institute and the Nova Scotia Health Research Foundation, Renza Vento was partially funded by European Regional Development Fund, European Territorial Cooperation 2007–13 (CCI 2007 CB 163 PO 037, OP Italia-Malta 2007–13) and grants from the Italian Ministry of Education, University and Research (MIUR) ex-60%, 2007; Riccardo Di Fiore was a recipient of fellowship granted by European Regional Development Fund, European Territorial Cooperation 2007–2013 (CCI 2007 CB 163 PO 037, OP Italia-Malta 2007–2013); Rita Dornetshuber-Fleiss was supported by the Austrian Science Fund (FWF, project number T 451-B18) and the Johanna Mahlke, geb.-Obermann-Stiftung; Roberta Palorini is supported by a SysBioNet fellowship; Roslida Abd Hamid is supported by the Ministry of Education, Malaysia-Exploratory Research Grant Scheme-Project no: ERGS/1-2013/5527165; Sabine A.S.Langie is the beneficiary of a postdoctoral grant from the AXA Research Fund and the Cefic-LRI Innovative Science Award 2013; Sakina Eltom is supported by NIH grant SC1CA153326; Samira A.Brooks was supported by National Research Service Award (T32 ES007126) from the National Institute of Environmental Health Sciences and the HHMI Translational Medicine Fellowship; Sandra Ryeom was supported by The Garrett B. Smith Foundation and the TedDriven Foundation; Thierry Massfelder was supported by the Institut National de la Santé et de la Recherche Médicale INSERM and Université de Strasbourg; Thomas Sanderson is supported by the Canadian Institutes of Health Research (CIHR; MOP-115019), the Natural Sciences and Engineering Council of Canada (NSERC; 313313) and the California Breast Cancer Research Program (CBCRP; 17UB-8703); Tiziana Guarnieri is supported by a grant from Fundamental Oriented Research (RFO) to the Alma Mater Studiorum University of Bologna, Bologna, Italy and thanks the Fondazione Cassa di Risparmio di Bologna and the Fondazione Banca del Monte di Bologna e Ravenna for supporting the Center for Applied Biomedical Research, S.Orsola-Malpighi University Hospital, Bologna, Italy; W.Kimryn Rathmell is supported by the V Foundation for Cancer Research and the American Cancer Society; William K.Decker was supported in part by grant RP110545 from the Cancer Prevention Research Institute of Texas; William H.Bisson was supported with funding from the NIH P30 ES000210; Yon Rojanasakul was supported with NIH grant R01-ES022968; Zhenbang Chen is supported by NIH grants (MD004038, CA163069 and MD007593); Zhiwei Hu is grateful for the grant support from an institutional start-up fund from The Ohio State University College of Medicine and The OSU James Comprehensive Cancer Center (OSUCCC) and a Seed Award from the OSUCCC Translational Therapeutics Program.

The transcription factor ZNF224 plays a key proapoptotic role in chronic myelogenous leukemia (CML), by modulating Wilms Tumor protein 1 (WT1) dependent apoptotic genes transcription. Recently, we demonstrated that Bcr-Abl signaling represses ZNF224 expression in Bcr-Abl positive CML cell lines and in CML patients. Interestingly, Imatinib and second-generation tyrosine kinase inhibitors specifically increase ZNF224 expression. On the other hand, Bcr-Abl positively modulates, via JAK2 activation, the expression of the c-Myc oncogene, which is required for Bcr-Abl oncogenic transformation in CML. Consequently, JAK2 inhibitors represent promising molecular therapeutic tools in CML. In this work, we demonstrate that ZNF224 is a novel transcriptional repressor of c-Myc in CML. We also show that ZNF224 induction by Imatinib and AG490, a specific JAK2 inhibitor, is responsible for the transcriptional repression of c-MYC, thus highlighting the crucial role of the ZNF224/c-Myc axis in Imatinib responsiveness. Interestingly, we also report that ZNF224 is induced by AG490 in Imatinib-resistant CML cells, leading to c-Myc repression and apoptosis induction. These findings suggest that the development of molecular tools able to induce ZNF224 expression could provide promising means to bypass Imatinib resistance in CML.

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.

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.

To report a novel spectral-domain optical coherence tomography (SD-OCT) finding in children affected by tilted disc syndrome (TDS), and to correlate it with early visual field defects. Patients between 5 and 17 years old with TDS were enrolled in this study. The diagnosis of TDS was made by stereoscopic fundus photography, when the upper edge of the optic disc protruded anteriorly relative to its lower edge. All eyes were examined with 12 radial SD-OCT B-scans of 12 mm centered on the optic disc; the fundus area encompassing the optic nerve was additionally scanned using several vertical and horizontal scans.. C-scan SD-OCT were acquired using the Macular Cube 512 x128 to create the en face image. Standard automated perimetry 24–2 tests were performed on all patients. Thirty-eight eyes of 20 pediatric patients with TDS syndrome were enrolled during this 24-months clinical trial. Their mean age was 10.9 ± 2.7 years (range 7–15 years), 12 (60%) were male and eight (40%) were female. The OCT images of the optic discs showed a protrusion of the upper edge of Bruch’s membrane and choroid at the nasal edge of the optic disc in 39.5% of the eyes. The retinal nerve fiber tissue appeared to be herniated into this protrusion and bent superiorly in 15 eyes. This severe bending corresponded to early visual field anomalies that were not reduced by corrective lenses in 46.7% of the eyes. Visual field defects that do not improve by increased myopic correction in TDS may be due to the severe bending of the retinal nerve fiber tissue, which would impair axonal flow.

Tirofiban is a fibrinogen receptor antagonist, generated using the tripeptide Arg-Gly-Asp (RGD) as a template. RGD activates integrin receptors and integrin-mediated signals are necessary for normal cells to promote survival and stimulate cell cycle progression. We investigated whether tirofiban activated growth-stimulatory signals in endothelium. For this study human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) were used. Analysis of cell proliferation, by cell counts, showed that the number of endothelial cells doubled after 72 h of culture in the absence of tirofiban, while they were tripled and even quadrupled, in the presence of increasing doses of the drug. Moreover, tirofiban-stimulated cells had a greater ability to migrate through the transwell filters of Boyden chamber, compared to unstimulated cells. The scratch assay, which mimics cell migration during wound healing, showed that tirofiban stimulated HUVECs to migrate into the leading hedge of the scratch. Western blot showed that tirofiban increased the expression levels of VEGF and the downstream effectors Erk and cyclin D. An inhibitor of VEGFR2 counteracted tirofiban-induced-proliferation, suggesting a role for VEGF in such effect. Our study shows that tirofiban stimulates the migration and proliferation of endothelial cells suggesting that it can promote endothelial repair. Ex vivo cultures of arterial rings confirmed the growth stimulatory effect of tirofiban on endothelium. Thus, the benefits of tirofiban in those with acute coronary syndromes undergoing PTCA may be due to rapid endothelization of damaged vessel, besides antiplatelet effects.

Metastasis, also called secondary neoplastic disease, is a tumour newly formed in a site different from that of origin, as a consequence of cancer progression and dissemination largely through blood and lymphatic vessels. The ability to form metastases is the main property that distinguishes malignant from benign tumours. Treatments for metastatic cancer are similar in practice to those for primary tumours, but such treatments are mostly palliative; indeed, almost all deaths caused by solid tumours occur in the metastatic phase. Increasing evidence supports the concept that therapies for primary tumours are inadequate to treat metastasis and can even promote formation of metastases, while exerting local growth control. Furthermore, recurrent tumours, which are denoted by increased aggressiveness and therapy resistance in comparison with the primary tumour, have an increased metastatic potential. Genetic modifications occurring during tumour progression lead to substantial differences between the primary and metastatic tumours. This emphasises the importance of designing novel therapies for metastasis. In the last decade, a number of studies have contributed to the understanding of the genetic rearrangements underlying the conversion of cancer cells into the metastasis founder cells. The present article aims at reviewing recent advances in metastasis research and attempts to discuss the reasons for which the therapeutic strategies against primary tumours may not satisfactorily address their metastatic counterparts.