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News Article | December 9, 2015
Site: www.nature.com

The Atg7fl/fl line was from T. Eissa; the GFP–LC3 mouse line was from N. Mizushima. The Prx1-Cre line was purchased from Jackson Laboratories (strain no. 005584). The Col2a1-Cre line was from B. Lee. The Fgf18- and Fgfr3-knockout mice were from D. Ornitz. The Fgfr4-knockout mice were from J. Seavitt. All mice used were maintained in a C57BL/6 strain background. The number of mice used in each experiment is specified in figure legends. Sex of the mice was not taken into account until P9. At later time points (P30 and P120) only male mice were analysed. Mice were randomly assigned to treatment groups. The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Cardarelli Hospital in Naples and authorized by the Italian Ministry of Health. Skeletons were fixed in 95% ethanol overnight and stained with alcian blue and alizarin red according to standardized protocols (http://empress.har.mrc.ac.uk/browser/). Measurement of bone length was performed using ImageJ software. Histology was performed according to standardized procedures (http://empress.har.mrc.ac.uk/browser/). Briefly, femurs were fixed overnight in 4% (wt/vol) paraformaldehyde (PFA) and then demineralized in 10% EDTA (pH 7.4) for 48 h (demineralization was performed only if specimens were isolated from mice older than P5). Specimens were then dehydrated, embedded in paraffin and sectioned at 7 μm, and stained with haematoxylin and eosin. For BrdU staining, mice were injected with 100 μl of 10 mM BrdU (Sigma) 4 h before being killed. BrdU incorporation was detected using a Zymed BrdU staining kit (Invitrogen). The TdT-mediated dUTP nick end labelling (TUNEL) assay was performed using the In situ Cell Death Detection kit (Roche). Counterstaining was performed using haematoxylin. For immunofluorescence, femurs were dissected from euthanized mice and fixed with buffered 4% PFA overnight at 4 °C, then washed with PBS and cryoprotected in successive sucrose solutions diluted with PBS (10% for 2 h, 20% for several hours and 30% overnight at 4 °C; all wt/vol), and finally embedded in OCT (Sakura). Cryostat sections were cut at 10 μm. Sections were blocked and permeabilized in 3% (wt/vol) BSA, 5% fetal bovine serum (FBS) in PBS plus 0.3% Triton X-100 for 3 h and then incubated with the primary antibody overnight. Sections were washed three times with 3% BSA in PBS plus 0.3% Triton X-100 and then incubated for 3 h with secondary antibodies conjugated with Alexa Fluor 488, or Alexa Fluor 568. The extracellular Col2 staining was performed by pre-treating sections with chondroitinase ABC (Sigma) at 0.2 U ml−1 for 1 h at 37 °C before the blocking step. Intracellular PC2 staining was performed without chondroitinase ABC pre-treatment to stain only the PC2 molecules that were not masked by proteoglycans. Primary antibodies used were: GFP, LAMP1 and HSP47 (Abcam), Col2a1 (1:30, Hybridoma Bank, II6B3), FGFR3 and FGFR4 (Cell Signaling), VapA, Sec31, giantin, GM130, P115 and calreticulin were previously described29. Nuclei were stained with DAPI and sections were mounted with vectashield (Vector laboratories). Images were captured using a Zeiss LSM700 confocal microscope. Co-localization analysis was performed calculating Mander’s coefficient using ImageJ (co-localization analysis plug-in). Semi-quantitative analyses were performed by an investigator blinded to the genotype of the mice. This was performed using the Sircol soluble collagen assay (Biocolor) following the manufacturer’s protocol. Briefly, femoral and tibia cartilages were microdissected and collagen was acid pepsin extracted and complexed with Sircol dye. Absorbance was measured at 555 nm and concentration was calculated using a standard curve. Values were normalized to DNA levels calculated measuring the absorbance at 260 nm. Femoral cartilages were isolated from three mice with the same genotype, pooled and homogenized in 0.5 ml of 1 mg ml−1 cold (4 °C) pepsin in 0.2 M NaCl, 0.5 M acetic acid to pH 2.1 with HCl and then digested at 4 °C for 24 h, twice. The pellet was discarded and an equal volume (1 ml) of 4 M NaCl in 1 M acetic acid was added to precipitate collagen. The pellet was then resuspended in 0.8 ml of 0.2 M NaCl in 0.5 M acetic acid and was precipitated again three times. After the last precipitation the pellet was washed twice with 70% EtOH to remove residual NaCl. The pellet was then dissolved in 0.8 ml 0.5 M acetic acid, and lyophilized. Subsequently it was resuspended in Laemmli buffer without EtSH at a concentration of 2 mg ml−1, denatured at 80 °C for 5 min and loaded on 6% SDS–PAGE. Gels were then stained with Coomassie blue R-250. GAG (glycosaminoglycan) quantification was performed using the Blyscan sulfated glycosaminoglycan assay (Biocolor) following the manufacturer’s protocol. Briefly, femoral and tibia cartilages were microdissected and GAGs were papain extracted at 65 °C overnight and complexed with Blyscan dye. Absorbance was measured at 656 nm and concentration was calculated using a standard curve. Values were normalized to DNA levels calculated measuring the absorbance at 260 nm. For EM analysis, growth plates were fixed in 1% glutaraldehyde in 0.2 M HEPES buffer. Small blocks of growth plates were then post-fixed in uranyl acetate and in OsO . After dehydration through a graded series of ethanol, tissue samples were cleared in propylene oxide, embedded in Epoxy resin (Epon 812) and polymerized at 60 °C for 72 h. From each sample, thin sections were cut with a Leica EM UC6 ultramicrotome and images were acquired using a FEI Tecnai −12 (FEI) electron microscope equipped with Veletta CCD camera for digital image acquisition. Newborn mice were intraperitoneally injected daily with Tat–beclin-1 peptide (Beclin-1 Activator II, retro-inverso Tat-beclin-1, Millipore) at 2 mg kg−1 resuspended in PBS. Control mice were injected with vehicle only. Mice were killed after 6 or 9 days for PC2 immunofluorescence experiments and total collagen quantification, respectively. Leupeptin (Sigma catalogue L2884) was resuspended in water at 10 mM. Mice were given an intraperitoneal injection at 40 mg kg−1. Six hours after injection tissues were harvested and processed for western blotting. Femoral and tibia cartilages were microdissected and lysed using a TissueLyser (Qiagen) in RIPA buffer supplemented with 0.5% SDS, PhosSTOP and EDTA-free protease inhibitor tablets (Roche). Samples were incubated for 30 min on ice, briefly sonicated on ice and the soluble fraction was isolated by centrifugation at 14,000 r.p.m. for 10 min at 4 °C. FGF18 (50 ng ml−1 unless otherwise indicated), PTHrP (10 μg ml−1) and BMP2 (500 ng ml−1) were from Peprotech, rhSHH (10 μg ml−1) was from R&D Systems. JNK inhibitor (SP600125, Sigma-Aldrich) was used at 50 μM. Tannic acid (Fluka Chemika) was used at 0.5% final concentration in the medium for 1 h at 37 °C. Bafilomycin A1 (Sigma) was used at 200 nM. Primary cultured chondrocytes were prepared from rib cartilage of P5 mice. Rib cages were first incubated in DMEM (Euroclone) using 0.2% collagenase D (Roche) and after adherent connective tissue had been removed (1.5 h) the specimens were washed and incubated in fresh collagenase D solution for a further 4.5 h. Isolated chondrocytes were maintained in DMEM supplemented with 10% FCS (Invitrogen) and ascorbic acid (Sigma Aldrich) (50 μg ml−1). Given the incomplete deletion of the Atg7 gene in Atg7fl/fl; Col2a1-Cre chondrocytes (Extended Data Fig. 1c) and the lack of Cre expression in chondrocostal chondrocytes of Prx1-Cre mice, collagen secretion experiments were performed in the RCS chondrocyte cell line30, in which autophagy was inhibited by Atg7 RNA interference and by Spautin-1 treatment. Cells were transfected with Lipofectamine LTX and Plus reagent (Invitrogen) following a reverse transfection protocol. For siRNA experiments, siGENOME SMARTpool siRNAs (Dharmacon Thermo Scientific) were transfected to the final concentration of 50 nM. Cells were harvested 72 h after transfection. Plasmids: GFP–LC3 and mRFP–GFP–LC3 were from T. Yoshimori, GFP–LAMP1 was from A. Fraldi mCherry–PC2 was previously described29; BCL2–haemagglutinin (HA) was from M. Renna, 2×FYYE–GFP was from S. Tooze. FGFR3 plasmid was from Addgene and FGFR4 from DNASU plasmid repositories. Chondrocytes were fixed for 10 min in 4% PFA in PBS and permeabilized for 30 min in 0.05% (w/v) saponin, 0.5% (w/v) BSA, 50 mM NH Cl and 0.02% NaN in PBS (blocking buffer). For the detection of endogenous LC3 cells were methanol-fixed. The cells were incubated for 1 h with the primary antibodies, washed three times in PBS, incubated for 1 h with the secondary (Alexa Fluor-labelled) antibody, washed three times in PBS, incubated for 20 min with 1 μg ml−1 Hoechst 33342 and finally mounted in Mowiol. All confocal experiments showing co-localization were acquired using slice thickness of 0.5 μm using the LSM 710 confocal microscope equipped with a 63 × 1.4 numerical aperture oil objective. To prevent fusion of exocytic organelles with PM, chondrocytes were incubated for 1 h at 37 °C in 20 mM HEPES-buffered DMEM supplemented with 0.5% tannic acid (TA). At the end of the incubation, TA-containing medium was removed and cells were fixed and processed for immunofluorescence. RCS chondrocytes were reversed transfected with mCherry–PC2 and GFP–LC3 plasmids and plated in Mattek glass bottomed dishes. Collagen synchronization was performed by incubating cells at 40 °C on the heated stage for 2.5 h. Collagen release was initiated by lowering the temperature of the stage to 32 °C and medium being supplemented with 50 μg ml−1 of ascorbate, in a humidified atmosphere with 5% CO . Time-lapse videos were acquired for 45 min, starting from collagen release, during temperature drop (4 min), and at 32 °C. One frame was acquired roughly every 20 s with lasers set at 30% power or below. RCS chondrocytes were reverse transfected and plated in Mattek glass-bottomed dishes. RCS cells were synchronized on the heated stage for 2.5 h at 40 °C and released at 32 °C, in medium supplemented with 50 μg ml−1 ascorbic acid in a humidified atmosphere with 5% CO . The critical angle used was 65°, giving an evanescent field of 137 nm. Appropriate filter sets were used for GFP and mCherry detection. Frames were acquired on loop with no time delay (one frame roughly every 3 s), for 15 min. All live-cell imaging experiments were performed with a ×60 Plan Apo oil immersion lens using a Nikon Eclipse Ti Spinning Disk microscope, and images and videos were annotated using the NIS Elements 4.20 software. Cells were washed twice with PBS and then scraped in lysis buffer (RIPA lysis buffer in the presence of PhosSTOP and EDTA-free protease inhibitor tablets; Roche). Cell lysates were incubated on ice for 20 min, then the soluble fraction was isolated by centrifugation at 14,000 r.p.m. for 10 min at 4 °C. Total protein concentration in cellular extracts was measured using the colorimetric BCA protein assay kit (Pierce Chemical). Protein extracts, separated by SDS–PAGE and transferred onto PVDF or nitrocellulose (for collagen) membranes, were probed with antibodies against FGFR3, FGFR4, phospho (p)-JNK, JNK, p-BCL2, p-c-JUN, PDI, p-P38, P38, beclin-1, p-ERK, ERK1/2, p-P70S6K, P70S6K, p-4EBP1, 4EBP1, ATG7, p-AKT, AKT, p-mTORC1, mTORC1 (Cell Signaling Technology), HA, H3 histone, VPS34 (Sigma-Aldrich), LC3B, β-actin (Novus Biologicals), SQSTM1 (BD Transduction Laboratories and Abnova), GOLPH3 (Abcam), GAPDH, p-AMPKa, AMPKa (Santa Cruz Biotecnology), type II collagen (CIIC1b; Hybridoma Bank). Proteins of interest were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:2,000, Vector Laboratories) and visualized with the Super Signal West Dura substrate (Thermo Scientific), according to the manufacturer’s protocol. The western blotting images were acquired using the Chemidoc-lt imaging system (UVP) and band intensity was calculated using ImageJ software using the ‘Gels and Plot lanes’ plug-in. Primary chondrocytes were plated in CellCarrier-96 Black plates (6005558, Perkin Elmer). After identifying the nuclei with Hoechst 33342 (405 nm) staining, a cytoplasmic mask was drawn using Col2 staining (568 nm). To carry out the analysis, the number of GFP–LC3 spots in the cytoplasm of Col2-positive cells were counted, and expressed per cell. Levels of co-localization between GFP–LC3 and Col2a1 were assessed and expressed as a percentage using the parameters: area of co-localization of red spots with area of green spots normalized to total area of green spots. Image acquisition was performed using Opera High Content Screening System (PerkinElmer); image analysis was performed using Acapella High Content Imaging and Analysis Software (PerkinElmer). Repeated-measures ANOVA was performed with Tukey’s post-hoc test. RCS chondrocytes (100-mm dish) were grown in DMEM medium with 10% FBS and antibiotics. For FGF18 treatment, 70–80% confluent cells were cultured overnight in DMEM with 10% adult bovine serum (Sigma-Aldrich) and then treated with FGF18 (50 ng ml−1, 2 h) (Peprotech) or dimethylsulfoxide (DMSO) vehicle. RCS chondrocytes were rinsed off the plate with ice-cold PBS, washed, and then scraped in IP lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1% NP-40, with one PhosSTOP and one EDTA-free protease inhibitor tablet per 10 ml). Cell lysates were rotated at 4 °C for at least 30 min, and then the soluble fraction was isolated by centrifugation at 14,000 r.p.m. for 10 min at 4 °C. A fraction of the clarified lysate was used for western blot analysis. Primary antibody or rabbit pre-immune IgG were added to the lysates and rotated overnight at 4 °C, and then 25 μl of Protein A Sepharose beads (Sigma-Aldrich) were added and rotated for 2 h at 4 °C. Immunoprecipitates were washed three times with cold lysis buffer. Whole-cell lysates and immunoprecipitated proteins were boiled in 30 μl sample buffer, separated by SDS–PAGE on precast 4–15% gels (BioRad), transferred on PVDF membranes and probed with antibodies against beclin-1 (Santa Cruz Biotechnology), VPS34 (Sigma-Aldrich) and BCL2 (Cell Signaling Technology), FGFR3 (Santa Cruz), FGFR4 (Cell Signaling) and p-tyrosine (4G10, Millipore). PtdIns(3)K activity in the beclin-1 immunoprecipitates was determined using the Class III PI3K ELISA kit (Echelon Biosciences) according to the manufacturer’s instructions. Immunocomplexes were incubated with a reaction mixture containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P ) substrate and ATP for 3 h, and the amount of PtdIns(3,4,5)P generated from PtdIns(4,5)P by PtdIns(3)K was quantified using a competitive ELISA. Equal amounts of beclin-1 immunoprecipitate were evaluated by western blotting using beclin-1 antibody. To follow PC2 secretion in RCS chondrocytes, cells were pre-treated overnight with ascorbic acid (100 μg ml−1) in DMEM without FCS. Cells were then labelled with 37.5 μCi ml−1 2,3 3H-Proline (Perkin Elmer) for 4 h at 40 °C in DMEM, without FCS supplemented with ascorbic acid (100 μg ml−1), then shifted to 32 °C in DMEM without FCS containing cold proline (10 mM), 20 mM HEPES pH 7.2 and ascorbic acid (100 μg ml−1). After 0, 30 and 60 min the medium and cells were collected, lysed and proteins precipitated in saturated ammonium sulfate overnight, then resuspended in Laemmli buffer. Samples were run on 4–15% precast gels (Biorad), transferred onto nitrocellulose membrane (Whatman, Perkin Elmer) and developed by autoradiography using the BetaIMAGER-D system and analysed using M3 Vision software (Biospace Lab). Paired Student’s t-test was performed when comparing the same cell population with two different treatments. Unpaired Student’s t-test was performed when comparing two groups of mice or different primary chondrocyte preparations. One-way ANOVA and Tukey’s post-hoc tests were performed when comparing more than two groups relative to a single factor (time or treatment/genotype). Two-way ANOVA and Tukey’s post-hoc tests were performed when comparing more than two groups relative to two factors (time and treatment/genotype). No statistical methods were used to predetermine sample size.


Dellinger E.P.,University of Washington | Forsmark C.E.,Florida College | Layer P.,Israelitic Hospital | Levy P.,Pole des Maladies de lAppareil Digestif | And 7 more authors.
Annals of Surgery | Year: 2012

OBJECTIVE: To develop a new international classification of acute pancreatitis severity on the basis of a sound conceptual framework, comprehensive review of published evidence, and worldwide consultation. BACKGROUND: The Atlanta definitions of acute pancreatitis severity are ingrained in the lexicon of pancreatologists but suboptimal because these definitions are based on empiric description of occurrences that are merely associated with severity. METHODS: A personal invitation to contribute to the development of a new international classification of acute pancreatitis severity was sent to all surgeons, gastroenterologists, internists, intensivists, and radiologists who are currently active in clinical research on acute pancreatitis. The invitation was not limited to members of certain associations or residents of certain countries. A global Web-based survey was conducted and a dedicated international symposium was organized to bring contributors from different disciplines together and discuss the concept and definitions. RESULT: The new international classification is based on the actual local and systemic determinants of severity, rather than description of events that are correlated with severity. The local determinant relates to whether there is (peri)pancreatic necrosis or not, and if present, whether it is sterile or infected. The systemic determinant relates to whether there is organ failure or not, and if present, whether it is transient or persistent. The presence of one determinant can modify the effect of another such that the presence of both infected (peri)pancreatic necrosis and persistent organ failure have a greater effect on severity than either determinant alone. The derivation of a classification based on the above principles results in 4 categories of severity-mild, moderate, severe, and critical. CONCLUSIONS: This classification is the result of a consultative process amongst pancreatologists from 49 countries spanning North America, South America, Europe, Asia, Oceania, and Africa. It provides a set of concise up-to-date definitions of all the main entities pertinent to classifying the severity of acute pancreatitis in clinical practice and research. This ensures that the determinant-based classification can be used in a uniform manner throughout the world. Copyright © 2012 by Lippincott Williams & Wilkins.


Bucci C.,University of Salerno | Bucci C.,L Curto Hospital | Rotondano G.,Maresca Hospital | Hassan C.,Nuova Regina Margherita Hospital | And 5 more authors.
Gastrointestinal Endoscopy | Year: 2015

Background: Colonoscopy is considered the criterion standard for detecting colorectal cancer; adequate preparation is crucial for an effective colonoscopy, but definitive data on the optimal preparation are lacking. Objective: Our aim was to assess the efficacy of split-dose versus non-split-dose preparations, the rate of adequate preparation according to type and dose of laxatives, the role of "runway time" (the interval time between the last drink of purgative and the beginning of colonoscopy), and to evaluate compliance as an additive risk factor for colon cleansing. Design: A series of meta-analyses of controlled studies. Setting: Randomized clinical trial of split dose regimen versus entire dose taken on the day preceding colonoscopy. Patients: Published trials (1960-2013) comparing split-dose versus non-split-dose preparations in adults undergoing colonoscopy were selected by using MEDLINE, the Cochrane Central Register of Controlled Trials, clinicaltrial.gov, ISI Web of Science, and Scopus. Interventions: Colonoscopy. Main Outcome Measurements: Rate difference of the degree of colon cleansing between split dose and whole dose was the primary measure of treatment effect. Results: We included 29 studies. Overall, an adequate preparation was obtained in 85% of patients in the splitdose group and in 63% of the non-split-dose group (rate difference 22%). The heterogeneity was caused by 5 factors: the runway time (the longer, the worse the cleansing), type of diet, male sex, use of polyethylene glycol 4 L, and the Jadad score. Compliance was significantly higher in the split-dose group. Limitations: Average quality of the included studies and publication bias. Conclusion: We provided further evidence of the superiority of a split-dose regimen over a non-split-dose regimen and showed that, regardless of type and dose, the superiority of split-dose regimens remains valid if the "golden 5 hours" rule is preserved. Copyright © 2014 by the American Society for Gastrointestinal Endoscopy.


Ferrara F.,Cardarelli Hospital | Schiffer C.A.,Barbara Ann Karmanos Cancer Institute
The Lancet | Year: 2013

The outlook for patients with acute myeloid leukaemia has improved in the past 30 years. Unlike other cancers, much of this progress is attributable to refinement of supportive treatment, rather than the introduction of new drugs. New antibacterial and antifungal agents, antiemetics, and improved transfusion support have decreased the rate of early death, and morbidity and mortality from allogeneic stem cell transplantation has been substantially reduced. However, more than half of young adult patients and about 90% of older patients still die from their disease. Refractoriness to initial induction treatment and, more frequently, relapse after complete remission, are still the main obstacles to cure. Accordingly, new treatment approaches with mechanisms of action different from those of conventional chemotherapy are needed. Our knowledge of the various chromosomal and molecular abnormalities implicated in the pathogenesis of the many subtypes of the disease has greatly expanded; as a result, clinical research is moving towards the investigation of new non-cytotoxic agents in combination with chemotherapy. The goal is to target the molecular abnormalities identified at diagnosis; however, several aberrations can coexist in subclones of acute myeloid leukaemia, making the disease less likely to be inhibited by a single agent. © 2013 Elsevier Ltd.


Ferrara F.,Cardarelli Hospital
Expert Opinion on Investigational Drugs | Year: 2012

Introduction: The prognosis of acute myeloid leukemia (AML) is improved in the last two decades, even though induction and consolidation chemotherapy has not involved new drugs. The more effective use of well-known agents as well as refinement of supportive care during the inevitable phase of severe pancytopenia following intensive chemotherapy accounts for the reduction of treatment-related death rate. In addition, mortality due to allogeneic and autologous stem cell transplantation has also been reduced, due to adoption of more effective therapies for graft versus host disease and other transplant-related complications. Areas covered: The multitude of chromosomal and molecular abnormalities makes the treatment of AML a challenging prospect. In addition, genetic aberrations are not mutually exclusive and coexist in the leukemic cells. As a consequence, the clinical development of new biologic agents proceeds slowly. Data for this review were identified from PubMed and references from relevant articles published in English from 2000 to 2011. Expert opinion: In Phase II studies, different new agents have been found to be active in AML and are currently under investigation in Phase III trials also in combination with conventional chemotherapy. In the near future, we would have more information about the possibility of introducing new drugs into daily practice. © 2012 Informa UK, Ltd.


Ferrara F.,Cardarelli Hospital
Hematological Oncology | Year: 2014

Acute myeloid leukaemia (AML) is the second more frequent hematologic malignancy in developed countries and primarily affects older adults with a median age at diagnosis of 69years. Given the progressive ageing of the general population, the incidence of the disease in elderly people is expected to further increase in the years to come. Along with cytogenetics at diagnosis, age represents the most relevant prognostic factor in AML, in that the outcome steadily declines with increasing age. Reasons for poor prognosis include more frequent unfavourable karyotype and other adverse biologic characteristics, such as high rates of expression of genes drug resistance related and high prevalence of secondary AML. Noticeably, as compared with young adults, poorer results in elderly patients have been reported within any cytogenetic and molecular prognostic subgroup, because of frequent comorbid diseases, which render many patients ineligible to intensive chemotherapy. Therefore, predictive models have been developed with the aim of achieving best therapeutic results avoiding unnecessary toxicity. Following conventional induction therapy, older AML patients have complete remission rates in the range of 45-65%, and fewer than 10% of them survive for a minimum of 5years. On the other hand, hypomethylating agents, such as azacytidine and decitabine offer the possibility of long-term disease control without necessarily achieving complete remission and can represent a reasonable alternative to intensive chemotherapy. Either intensive chemotherapy or hypomethylating agents have lights and shadows, and the therapeutic selection is often influenced by physician's and patient's attitude rather than definite criteria. Research is progress in order to assess predictive biologic factors, which would help clinicians in the selection of patients who can take actual benefit from different therapeutic options. © 2013 John Wiley & Sons, Ltd.


di Costanzo G.G.,Cardarelli Hospital | Tortora R.,Cardarelli Hospital
World Journal of Hepatology | Year: 2015

Intermediate stage, or stage B according to Barcelona Clinic Liver Cancer classification, of hepatocellular carcinoma (HCC) comprises a heterogeneous population with different tumor burden and liver function. This heterogeneity is confirmed by the large variability of treatment choice and disease-relate survival. The aim of this review was to highlight the existing evidences regarding this specific topic. In a multidisciplinary evaluation, patients with large (> 5 cm) solitary HCC should be firstly considered for liver resection (LR). When LR is unfeasible, locoregional treatments are evaluable therapeutic options, being transarterial chemoembolization (TACE), the most used procedure. Percutaneous ablation can be an evaluable treatment for large HCC. However, the efficacy of all ablative procedures decrease as tumor size increases over 3 cm. In clinical practice, a combination treatment strategy [TACE or transarterial radioembolization (TARE)-plus percutaneous ablation] is "a priori" preferred in a relevant percentage of these patients. On the other hands, sorafenib is the treatment of choice in patients who are unsuitable to surgery and/or with a contraindication to locoregional treatments. In multifocal HCC, TACE is the first-line treatment. The role of TARE is still undefined. Surgery may have also a role in the treatment of multifocal HCC in selected cases (patients with up to three nodules, multifocal HCC involving 2-3 adjacent liver segments). In some patients with bilobar disease the combination of LR and ablative treatment may be a valuable option. The choice of the best treatment in the patient with intermediate stage HCC should be "patient-tailored" and made by a multidisciplinary team. © 2015 Baishideng Publishing Group Inc.


Ferrara F.,Cardarelli Hospital
Expert Opinion on Pharmacotherapy | Year: 2010

Importance of the field: Acute promyelocytic leukemia (APL) represents a paradigm of therapeutic success in clinical hematology. Since the introduction of all-trans-retinoic-acid in the early 1980s, complete remission rates exceed 90% and the cure rate is > 70%. Notwithstanding, various questions concerning the management of APL remain unanswered. Areas covered in this review: The aim of this article is to focus on still controversial issues in the management of APL, such as the role of arsenic trioxide as front-line therapy, the management of older unfit patients, the potential utility of gemtuzumab-ozogamycin and the effectiveness (if any) of maintenance therapy for patients in molecular remission. In addition, the possibility of reducing the intensity of post-remission therapy, which is associated with substantial morbidity in potentially cured patients, is discussed. What the reader will gain: Current and future therapeutic options for the treatment of newly diagnosed and relapsed APL. Take home message: To date, the therapy of APL is the most successful example of differentiation therapy and its scientific history can serve as a model for subsequent development of similar treatments in other leukemias and cancers. However, treatment strategies continue to evolve rapidly, with particular focus on minimizing the early and late effects of cytotoxic chemotherapy. © 2010 Informa UK Ltd.


Ronga I.,Cardarelli Hospital | Gallucci F.,Cardarelli Hospital | Riccardi F.,Cardarelli Hospital | Uomo G.,Cardarelli Hospital
Advances in Medical Sciences | Year: 2014

About 80% of all pancreatic ductal adenocarcinoma patients suffer from a wasting syndrome referred to as the "cancer anorexia-cachexia syndrome" (CACS) characterized by abnormally low weight, weakness and loss of skeletal muscle mass with or without loss of body fat, which directly impacts overall survival, quality of life, and physical activity. The aim of this review was to examine recent findings about CACS' pathophysiology and to describe the current pharmacological approaches. In recent years many efforts were made to improve our knowledge of CACS; currently we know that cachexia arises from a complex and multifactorial interaction between various mechanisms including inflammation, anorexia/malnutrition, alterations of protein and lipid metabolism; consequently its management requires multidisciplinary and multipharmacological approach that should address the different causes underlying this clinical event. On these premises, several drugs have been proposed starting from the first pharmacological treatment based on progestational agents or corticosteroids; most of them are in the preclinical phase, but some have already reached the clinical experimentation stage. In conclusion, to date, there is no standard effective treatment and further studies are needed to unravel the basic mechanisms underlying CACS and to develop newer therapeutic strategies with the hope to improve the quality of life of pancreatic cancer patients. © 2014 Medical University of Bialystok.


Guarnieri G.,Cardarelli Hospital | Masala S.,University of Rome Tor Vergata | Muto M.,Cardarelli Hospital
Interventional Neuroradiology | Year: 2015

Vertebroplasty (VP) is a percutaneous mini-invasive technique developed in the late 1980s as antalgic and stabilizing treatment in patients affected by symptomatic vertebral fracture due to porotic disease, traumatic injury and primary or secondary vertebral spine tumors. The technique consists of a simple metameric injection of an inert cement (poly-methylmethacrylate, PMMA), through a needle by trans-peduncular, parapeduncular or trans-somatic approach obtaining a vertebral augmentation and stabilization effect associated with pain relief. The technique is simple and fast, and should be performed under fluoroscopy or CT guidance in order to obtain a good result with low complication rate. The aim of this paper is to illustrate the utility of VP, the indications-contraindications criteria, how to technically perform the technique using imaging guidance, and the results and complications of this treatment in patients affected by symptomatic vertebral compression fracture. © The Author(s) 2015.

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