Gianpaolo Vidili

Functionalized multiwalled carbon nanotubes as ultrasound contrast agents

Ultrasonography is a fundamental diagnostic imaging tool in everyday clinical practice. Here, we are unique in describing the use of functionalized multiwalled carbon nanotubes (MWCNTs) as hyperechogenic material, suggesting their potential application as ultrasound contrast agents. Initially, we carried out a thorough investigation to assess the echogenic property of the nanotubes in vitro. We demonstrated their long-lasting ultrasound contrast properties. We also showed that ultrasound signal of functionalized MWCNTs is higher than graphene oxide, pristine MWCNTs, and functionalized single-walled CNTs. Qualitatively, the ultrasound signal of CNTs was equal to that of sulfur hexafluoride (SonoVue), a commercially available contrast agent. Then, we found that MWCNTs were highly echogenic in liver and heart through ex vivo experiments using pig as an animal model. In contrast to the majority of ultrasound contrast agents, we observed in a phantom bladder that the tubes can be visualized within a wide variety of frequencies (i.e., 5.5–10 MHz) and 12.5 MHz using tissue harmonic imaging modality. Finally, we demonstrated in vivo in the pig bladder that MWCNTs can be observed at low frequencies, which are appropriate for abdominal organs. Importantly, we did not report any toxicity of CNTs after 7 d from the injection by animal autopsy, organ histology and immunostaining, blood count, and chemical profile. Our results reveal the enormous potential of CNTs as ultrasound contrast agents, giving support for their future applications as theranostic nanoparticles, combining diagnostic and therapeutic modalities.

Long-term Clinical Outcomes of Splanchnic Vein Thrombosis: Results of an International Registry

IMPORTANCE Little information is available on the long-term clinical outcome of patients with splanchnic vein thrombosis (SVT). OBJECTIVE To assess the incidence rates of bleeding, thrombotic events, and mortality in a large international cohort of patients with SVT. DESIGN, SETTING, AND PARTICIPANTS A prospective cohort study was conducted beginning May 2, 2008, and completed January 30, 2014, at hospital-based centers specialized in the management of thromboembolic disorders; a 2-year follow-up period was completed January 30, 2014, and data analysis was conducted from July 1, 2014, to February 28, 2015. Participants included 604 consecutive patients with objectively diagnosed SVT; there were no exclusion critieria. Information was gathered on baseline characteristics, risk factors, and antithrombotic treatment. Clinical outcomes during the follow-up period were documented and reviewed by a central adjudication committee. MAIN OUTCOMES AND MEASURES Major bleeding, defined according to the International Society on Thrombosis and Hemostasis; bleeding requiring hospitalization; thrombotic events, including venous and arterial thrombosis; and all-cause mortality. RESULTS Of the 604 patients (median age, 54 years; 62.6% males), 21 (3.5%) did not complete follow-up. The most common risk factors for SVT were liver cirrhosis (167 of 600 patients [27.8%]) and solid cancer (136 of 600 [22.7%]); the most common sites of thrombosis were the portal vein (465 of 604 [77.0%]) and the mesenteric veins (266 of 604 [44.0%]). Anticoagulation was administered to 465 patients in the entire cohort (77.0%) with a mean duration of 13.9 months; 175 of the anticoagulant group (37.6%) received parenteral treatment only, and 290 patients (62.4%) were receiving vitamin K antagonists. The incidence rates (reported with 95% CIs) were 3.8 per 100 patient-years (2.7-5.2) for major bleeding, 7.3 per 100 patient-years (5.8-9.3) for thrombotic events, and 10.3 per 100 patient-years (8.5-12.5) for all-cause mortality. During anticoagulant treatment, these rates were 3.9 per 100 patient-years (2.6-6.0) for major bleeding and 5.6 per 100 patient-years (3.9-8.0) for thrombotic events. After treatment discontinuation, rates were 1.0 per 100 patient-years (0.3-4.2) and 10.5 per 100 patient-years (6.8-16.3), respectively. The highest rates of major bleeding and thrombotic events during the whole study period were observed in patients with cirrhosis (10.0 per 100 patient-years [6.6-15.1] and 11.3 per 100 patient-years [7.7-16.8], respectively); the lowest rates were in patients with SVT secondary to transient risk factors (0.5 per 100 patient-years [0.1-3.7] and 3.2 per 100 patient-years [1.4-7.0], respectively). CONCLUSIONS AND RELEVANCE Most patients with SVT have a substantial long-term risk of thrombotic events. In patients with cirrhosis, this risk must be balanced against a similarly high risk of major bleeding. Anticoagulant treatment appears to be safe and effective in most patients with SVT.

Renal Hemodynamics and Renoprotection

Background: Angiotensin II (AII) is the well-known determinant of kidney damage increasing intraglomerular pressure, matrix expansion and fibroblast proliferation. Renin-angiotensin system (RAS) inhibition, limiting the hemodynamic effects of AII, reduces proteinuria and is renoprotective in the long term. Methods: We studied 15 chronic proteinuric patients by Doppler ultrasonography to clarify the intrarenal hemodynamic changes during RAS blockade by Benazepril (10–20 mg/day) alone and combined with Valsartan (80–160 mg/day). We also investigated the correlation between hemodynamic indices, RAS components and antiproteinuric effect. Results: After 1 month of Benazepril proteinuria, resistive index (RI) and pulsatility index (PI) significantly decreased and proteinuria reduction was directly correlated to decrease in RI (r = 0.55, p = 0.03). Contrarily, after 1 month of combined therapy, RI and PI restored to baseline and progressively increased in the following 3 months, while proteinuria decreased. Increase in RI was directly correlated to concomitant increase in plasma renin activity (r = 0.65, p = 0.01) suggesting a direct role of renin in restoring intrarenal resistances. Conclusion: The hemodynamic changes caused by RAS inhibitors partially contribute to the antiproteinuric effect. Other RAS components, such as renin, may contribute to renal vasoconstriction and could be a further determinant of kidney damage besides a promising target for renoprotection.

Both de novo synthetized and exogenous fatty acids support the growth of hepatocellular carcinoma cells

Although it is well established that fatty acids (FA) are indispensable for the proliferation and survival of cancer cells in hepatocellular carcinoma (HCC), inhibition of Fatty Acid Synthase (FASN) cannot completely repress HCC cell growth in culture. Thus, we hypothesized that uptake of exogenous FA by cancer cells might play an important role in the development and progression of HCC. Lipoprotein lipase (LPL) is the enzyme that catalyses the hydrolysis of triglycerides into free fatty acids (FFA) and increases the cellular uptake of FA.

A functional mammalian target of rapamycin complex 1 signaling is indispensable for c‐Myc‐driven hepatocarcinogenesis

Amplification and/or activation of the c‐Myc proto‐oncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c‐Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c‐Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c‐Myc‐dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of mTORC1, strongly inhibits c‐Myc liver tumor formation. Also, the p70 ribosomal S6 kinase/ribosomal protein S6 and eukaryotic translation initiation factor 4E‐binding protein 1/eukaryotic translation initiation factor 4E signaling cascades downstream of mTORC1 are required for c‐Myc‐driven tumorigenesis. Intriguingly, microarray expression analysis revealed up‐regulation of multiple amino acid transporters, including solute carrier family 1 member A5 (SLC1A5) and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c‐Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1 as their inhibition suppressed mTORC1 in c‐Myc tumor cells. In human hepatocellular carcinoma specimens, levels of c‐Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6. Conclusion: Our current study indicates that an intact mTORC1 axis is required for c‐Myc‐driven hepatocarcinogenesis; thus, targeting the mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of hepatocellular carcinoma with activated c‐Myc signaling. (Hepatology 2017;66:167–181).

Immune compatible cystine-functionalized superparamagnetic iron oxide nanoparticles as vascular contrast agents in ultrasonography

Superparamagnetic iron oxide nanoparticles (SPIONs) have been extensively investigated for many biomedical applications. A good quality functionalization that combines imaging goals with a high-level of biocompatibility remains one of the challenges for particle translation into medical practice. Here, we focus on a new functionalization of SPIONs with cystine (Cy-SPIONs). Cystine is able to make SPIONs stable and dispersible in water and in culture cell media. New insights are provided into the biological and immune effects of Cy-SPIONs with a wide variety of standard and molecular assays to evaluate cytotoxicity, cell activation, cytokine release and the expression of 84 genes related to immune responses. A good immune biocompatibility of Cy-SPIONs on primary immune cells was found. The great potential of Cy-SPIONs for further in vivo studies and as contrast agents for magnetic resonance imaging (MRI) is highlighted. In addition, we also exploited ultrasonography, since it is a safer, less expensive and common imaging technology. The good echogenic properties of Cy-SPIONs in water and in whole blood are shown, both in vitro and in a phantom vein for bloodstream simulations. Our results open up a new scenario for future applications of cystine-functionalized SPIONs as immune-compatible ultrasound and MRI contrast agents.

Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria phenotype in patients with splanchnic vein thrombosis

INTRODUCTION Splanchnic vein thrombosis (SVT) is a serious complication in patients with paroxysmal nocturnal hemoglobinuria (PNH). Mutant PNH clones can be associated with an increased risk of SVT even in the absence of overt disease, but their prevalence in non-selected SVT patients remains unknown. MATERIALS AND METHODS Patients with objective diagnosis of SVT and without known PNH were tested for the presence of PNH clone using high-sensitivity flow cytometric analysis. RESULTS A total of 202 SVT patients were eligible, 58.4% were males, mean age was 54.6years (range 17-94), site of thrombosis was portal in 103 patients, mesenteric in 67, splenic in 37, and supra-hepatic in 10. SVT was associated with JAK2 V6167F in 28 of 126 (22.2%) screened patients, liver cirrhosis in 15.3% patients, recent surgery in 10.9%, and myeloproliferative neoplasm in 10.6%, whereas in 34.6% of patients neither permanent nor transient risk factors were detected. None of the patients had a clearly demonstrable PNH clone, but in two patients (0.99%, 95% CI 0.17-3.91) we observed very small PNH clones (size 0.014% and 0.16%) confirmed in two independent samples. One patient had portal vein thrombosis and no associated risk factors, the second had superior mesenteric vein thrombosis and inflammatory bowel disease. CONCLUSIONS Very small PNH clones can be detected in patients with SVT and no clinical manifestations of disease. Future studies are needed to explore the potential role of this finding in the pathogenesis of SVT.

Pan-mTOR inhibitor MLN0128 is effective against intrahepatic cholangiocarcinoma in mice.

BACKGROUND & AIMS Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. We evaluated the therapeutic potential of MLN0128 vs. gemcitabine/oxaliplatin in a novel ICC mouse model. METHODS We established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their anti-tumor efficacy in vivo. Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for the characterization and mechanistic study. RESULTS Co-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro, inducing strong ICC cell apoptosis and only marginally affecting proliferation. CONCLUSIONS This study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics, such as gemcitabine/oxaliplatin-based regimens, especially in the subset of tumors exhibiting activated AKT/mTOR cascade. Lay summary: We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this new preclinical model, we evaluated the therapeutic potential of mTOR inhibitor MLN0128 vs. gemcitabine/oxaliplatin (the standard chemotherapy for ICC treatment). Our study shows the anti-neoplastic potential of MLN0128, suggesting that it may be superior to gemcitabine/oxaliplatin-based chemotherapy for the treatment of ICC, especially in the tumors exhibiting activated AKT/mTOR cascade.

A case of thyroid metastasis from pancreatic cancer: case report and literature review

BackgroundThyroid metastases are clinically rare, and usually occur in patients with a history of prior malignancy and when there are metastases elsewhere. Metastases of pancreatic carcinoma to the thyroid are extremely rare, with only three cases reported in the literature.Case presentationWe report a patient who had a pancreatic carcinoma with metastasis to the thyroid as initial clinical presentation of the disease. A 63-year-old man with a history of weight loss and fatigue presented with cervical lymphadenopathies and a large nodule in the right lobe of the thyroid. A fine needle aspiration of the nodule gave inconclusive cytological results for the origin of the neoplastic cells. An ultrasound-guided core biopsy revealed the presence of a poorly differentiated adenocarcinoma infiltrating the thyroid with atrophic thyroid follicles. Immunohistochemical staining of the lesion was strongly positive for Cytokeratin 19 suggesting a pancreatic origin of the metastasis. A contrast CT scan demonstrated an enlargement of the pancreatic body, dilatation of the pancreatic duct, diffuse retroperitoneal, paraaortic and cervical lymphadenopathy and secondary lesions in the liver.ConclusionMetastases to the thyroid from pancreatic carcinoma are extremely rare. A core biopsy of the lesion excluded a thyroid carcinoma and permitted the diagnosis of the primary neoplasm.

Jagged 1 is a major Notch ligand along cholangiocarcinoma development in mice and humans

Intrahepatic cholangiocarcinoma (ICC) is a rare yet deadly malignancy with limited treatment options. Activation of the Notch signalling cascade has been implicated in cholangiocarcinogenesis. However, while several studies focused on the Notch receptors required for ICC development, little is known about the upstream inducers responsible for their activation. Here, we show that the Jagged 1 (Jag1) ligand is almost ubiquitously upregulated in human ICC samples when compared with corresponding non-tumorous counterparts. Furthermore, we found that while overexpression of Jag1 alone does not lead to liver tumour development, overexpression of Jag1 synergizes with activated AKT signalling to promote liver carcinogenesis in AKT/Jag1 mice. Histologically, tumours consisted exclusively of ICC, with hepatocellular tumours not occurring in AKT/Jag1 mice. Furthermore, tumours from AKT/Jag1 mice exhibited extensive desmoplastic reaction, an important feature of human ICC. At the molecular level, we found that both AKT/mTOR and Notch cascades are activated in AKT/Jag1 ICC tissues, and that the Notch signalling is necessary for ICC development in AKT/Jag1 mice. In human ICC cell lines, silencing of Jag1 via specific small interfering RNA reduces proliferation and increases apoptosis. Finally, combined inhibition of AKT and Notch pathways is highly detrimental for the in vitro growth of ICC cell lines. In summary, our study demonstrates that Jag1 is an important upstream inducer of the Notch signalling in human and mouse ICC. Targeting Jag1 might represent a novel therapeutic strategy for the treatment of this deadly disease.