Berger F.H.,VU University Amsterdam |
Nieboer K.H.,Vrije Universiteit Brussel |
Pinto A.,Cardarelli Hospital |
Scaglione M.,Pineta Grande Medical Center
Radiologia Medica | Year: 2014
To avoid detection at border crossings or airport customs, drug trafficking is increasingly performed by intra-corporeal concealment. Body packers may ingest packets of varying size and containing varying drugs (mostly cocaine, heroin and cannabis) mixed with other compounds, while body pushers will insert packets in the rectum or vaginal cavity. Body packing may lead to potential life-threatening complications with acute overdose syndromes after packet rupture and intestinal obstruction with possible ensuing bowel rupture being the most significant complications. Physicians including radiologists should be aware of the capabilities of imaging techniques to screen for presence of drug packets as well as the potential complications. Although conventional radiography has long been and still is the most important imaging modality for screening for presence of intestinal packets, the better test characteristics in conjunction with the decreasing radiation exposure, will likely render computed tomography (CT) more important in the future. For imaging of symptomatic patients, CT already is the modality of choice. Besides these modalities, ultrasound and magnetic resonance imaging will be discussed in this paper, together with more general background and clinical information. © 2014, Italian Society of Medical Radiology. Source
Masala S.,University of Rome Tor Vergata |
Anselmetti G.C.,Institute for Cancer Research and Treatment |
Muto M.,Cardarelli Hospital |
Mammucari M.,University of Rome Tor Vergata |
And 2 more authors.
Clinical Orthopaedics and Related Research | Year: 2011
Background: Percutaneous vertebroplasty is currently an alternative for treating vertebral fractures of the thoracic and lumbar spine, providing both pain control and vertebral stabilization. In the cervical spine, however, percutaneous vertebroplasty is technically challenging because of the complex anatomy of this region. Questions/purposes: We evaluated the technical feasibility, complication rate, and ability of percutaneous vertebroplasty to provide pain relief in patients with painful metastatic cervical fractures. Methods: We retrospectively reviewed 62 patients (24 men) who, between May 2005 and May 2009, underwent vertebroplasty to treat painful metastatic cervical fractures. Each patient was evaluated by a visual analog scale for pain, number of pain analgesics, and CT and MRI before, the day after, and at 3 months after the procedure. Results: Two of the 62 patients had asymptomatic cement leakage in the soft tissues. We observed no delayed complications. Mean pretreatment and 24-hour posttreatment visual analog scale pain scores were 7.9 ± 1.7 and 1.5 ± 2, respectively. Immediately after surgery, the pain completely disappeared in 25 (40%) patients. Administration of analgesics was suspended in 34 (55%) patients whereas in 27 (39%) patients the median analgesics use decreased from two pills per day (range, 0-3) to 0 (range, 0-3). In two (3%) patients, analgesics administration was continued due to the persistence of pain. At 3 months, the patients reported a mean visual analog scale pain score of 1.7 ± 2. Conclusions: Our data suggest, in selected patients, percutaneous vertebroplasty may be performed with a high technical success rate combined with a low complication rate, providing immediate pain relief lasting at least 3 months and a reduction in the use of analgesic drugs. Level of Evidence: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence. © The Association of Bone and Joint Surgeons® 2010. Source
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. Source
Pinto A.,Cardarelli Hospital |
Caranci F.,University of Naples Federico II |
Romano L.,Cardarelli Hospital |
Carrafiello G.,University of Insubria |
And 2 more authors.
Seminars in Ultrasound, CT and MRI | Year: 2012
An important goal of error analysis is to create processes aimed at reducing or preventing the occurrence of errors and minimizing the degree of harm. The discovery of any errors presents an opportunity to study the types that occur and to examine their sources and develop measures to prevent them from recurring. The development of an effective system for detecting and appropriately managing errors is essential to substantially attenuate their consequences. At this stage, the error analysis process identifies contributing factors to enable the implementation of concrete steps to prevent such errors from occurring in the future. Active and comprehensive management of errors and adverse events requires ongoing surveillance processes. Educational programs, morbidity and mortality meetings, and a comprehensive and respected root cause analysis process are also essential components of this comprehensive approach. To reduce the incidence of errors, health care providers must identify their causes, devise solutions, and measure the success of improvement efforts. Moreover, accurate measurements of the incidence of error, based on clear and consistent definitions, are essential prerequisites for effective action. © 2012 Elsevier Inc.. Source