The apoptogenic response of human myeloid leukaemia cell lines and of normal and malignant haematopoietic progenitor cells to the proteasome inhibitor PSI

Degradation of several intracellular proteins involved in cell cycle control and tumour growth is regulated by the ubiquitin-dependent multicatalytic protease complex (proteasome). We report that proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leucinal (PSI) was cytotoxic on most human myeloid leukaemia cell lines at IC50 doses ranging from 5 to 25 nmol/l. Additionally, PSI pre-treatment enhanced cytotoxicity by taxol and cisplatinum. PSI was more active on leukaemic than on normal CD34(+) bone marrow progenitors because the 50% growth inhibition of colony-forming unit granulocyte macrophage (CFU-GM) from cases of chronic myelogenous leukaemia (CML) and normal subjects was achieved by 15 nmol/l and 50 nmol/l PSI respectively. PSI killed cells by apoptosis as revealed by ultrastructural changes, nuclear DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and of beta-catenin, and was antagonized by ectopic expression of Bcl-2 but not by inactivating mutations of p53. This event was associated with a slight accumulation of Bcl-2, a decrease of Bax but no changes in Bcl-X(L) protein expression at any time point. In Ph(+) cell lines BCR-ABL protein was only down-regulated after 48 h of treatment with 10 nmol/l PSI. Altogether, these results indicate that PSI, alone or in association with other cytotoxic agents, has anti-tumour activity against myeloid malignancies and is more effective on leukaemic than on normal haematopoietic progenitor cells.     

Incision and abdominal wall hernias in patients with aneurysm or occlusive aortic disease

INTRODUCTION: Patients undergoing midline incision for abdominal aortic reconstruction appear to be at greater risk for postoperative incision hernia compared with patients undergoing celiotomy for general surgical procedures. Controversy exists as to whether incidence of abdominal wall hernia and increased risk for incision hernia is higher in patients with abdominal aortic aneurysm (AAA) than in patients operated on because of aortoiliac occlusive disease (AOD). We conducted a prospective multi-institutional study to assess frequency of incision hernia after aortic surgery through a midline laparotomy and of previous abdominal wall hernia. METHODS: Patients with AAA (n = 177) or AOD (n = 82) from three major institutions were prospectively enrolled in the study and examined. Data collected included demographic data, cardiopulmonary risk factors, smoking status, history of previous or current abdominal wall hernia (incision, inguinal, umbilical, femoral), previous midline incision, suture type, and postoperative complications. At a minimum of 6 months after laparotomy, patients were evaluated clinically for a new incision hernia. Differences were tested with the unpaired t test, X(2) test, or Fisher exact test, and multiple logistic regression was used to control for confounding variables. RESULTS: Mean follow-up of the cohort was 32.8 +/- 2.3 months. Rate of abdominal wall hernia and inguinal hernia in patients with AAA versus AOD was 38.4% versus 11% (P =.001) and 23.7% versus 6.1% (P =.003), respectively. Rate of postoperative incision hernia in patients with AAA was 28.2%, and in patients with AOD was 11.0% (P =.002). Adjusting for age, smoking, chronic obstructive pulmonary disease, body mass index, diabetes, bowel obstruction, and suture type, patients with AAA had almost a ninefold risk for postoperative incision hernia formation (odds ratio [OR], 8.8; P =.0049). CONCLUSION: Compared with patients with AOD, patients with AAA have a higher frequency of abdominal wall hernia and inguinal hernia, and are at significant increased risk for development of incision hernia postoperatively. The higher frequency of hernia formation in patients with AAA suggests the presence of a structural defect within the fascia. Further studies are needed to delineate the molecular changes of the aorta and its relation to the abdominal wall fascia.     

Inhibition of CD40–CD154 costimulatory pathway by a cyclic peptide targeting CD154

Disruption of the CD40–CD154 interaction was found to be effective in the prevention and treatment of several immune-mediated diseases. The antibody-based strategy of inhibition was in humans limited by platelet activation leading to thrombotic effects. Other strategies different from antibody technology may be useful to create tools to interfere with CD40–CD154 pathway. In the present study, we selected and characterized from a phage display library, cyclic hepta-peptides specific for human CD154 through biopanning against plate-immobilized recombinant hCD154-muCD8. Nine phage clones were selected for the ability to bind CD154 expressed on the surface of J558L cells transfected with human CD154. From the nine selected phage clones, we obtained seven different amino acidic sequences, and the corresponding hepta-peptides rendered cyclic by two cysteines were synthesized. All the peptides specifically bound CD154 expressed on J558L. However, only the peptide 4.10 (CLPTRHMAC) was found to recognize the active binding site of CD154, as it competed with the blocking anti-CD154 antibody. When changes in the amino acid composition were introduced in the sequence of 4.10 peptide, the binding to CD154 was abrogated, suggesting that the amino acid sequence was critical for its specificity. This peptide was found to inhibit the CD40–CD154 interaction, preventing CD40-dependent activation of B lymphocytes in vitro as it was able, as the blocking anti-human CD154 mAb, to prevent the expression of CD80 and CD86 costimulatory molecules and switching of Ig isotype induced by CD154. Moreover, the peptide 4.10 inhibited the in vitro endothelial cell motility and organization into capillary-like structures, and the in vivo angiogenesis of human umbilical cord-derived endothelial cells implanted in Matrigel in severe combined immunodeficiency mice. In vitro studies on platelet activation demonstrated that the 4.10 peptide, at variance of the anti-CD154 mAb, was unable to prime human platelet activation and aggregation. In conclusion, we identify a cyclic hepta-peptide able to displace the binding of human CD154 to CD40 expressed on cell surface and to abrogate some biological effects related to the CD40 stimulation, such as B cell activation and endothelial triggered angiogenesis.     

Revisiting signs, strengths and weaknesses of Standard Chest Radiography in patients of Acute Dyspnea in the Emergency Department

Dyspnoea, defined as an uncomfortable awareness of breathing, together with thoracic pain are two of the most frequent symptoms of presentation of thoracic diseases in the Emergency Department (ED). Causes of dyspnoea are various and involve not only cardiovascular and respiratory systems. In the emergency setting, thoracic imaging by standard chest X-ray (CXR) plays a crucial role in the diagnostic process, because it is of fast execution and relatively not expensive. Although radiologists are responsible for the final reading of chest radiographs, very often the clinicians, and in particular the emergency physicians, are alone in the emergency room facing this task. In literature many studies have demonstrated how important and essential is an accurate direct interpretation by the clinician without the need of an immediate reading by the radiologist. Moreover, the sensitivity of CXR is much impaired when the study is performed at bedside by portable machines, particularly in the diagnosis of some important causes of acute dyspnoea, such as pulmonary embolism, pneumothorax, and pulmonary edema. In these cases, a high inter-observer variability of bedside CXR reading limits the diagnostic usefulness of the methodology and complicates the differential diagnosis. The aim of this review is to analyze the radiologic signs and the correct use of CXR in the most important conditions that cause cardiac and pulmonary dyspnoea, as acute exacerbation of chronic obstructive pulmonary disease, acute pulmonary oedema, acute pulmonary trombo-embolism, pneumothorax and pleural effusion, and to focus indications and limitations of this diagnostic tool.     

Effects of epidermal growth factor receptor blockade on ependymoma stem cells in vitro and in orthotopic mouse models

Some lines of evidence suggest that tumors, including ependymoma, might arise from a subpopulation of cells, termed cancer stem cells (CSCs), with self-renewal and tumor-initiation properties. Given the strict dependence of CSCs on epidermal growth factor (EGF) through EGF receptor (EGFR), we investigated the effects of EGFR inhibitors in ependymoma-stem cells (SCs) in vitro and in orthotopic mouse models. We established two ependymoma-SC lines from two recurrent pediatric ependymoma. Both lines expressed markers of radial glia--the candidate SCs of ependymoma--and showed renewal ability, multipotency, and tumorigenicity after orthotopic implantation, despite markedly different expression of CD133 (94 vs. 6%). High phosphorylated-EGFR/EGFR ratio was detected, which decreased after differentiation. EGFR inhibitors (gefitinib and AEE788) reduced clonogenicity, proliferation and survival of ependymoma-SC lines dose-dependently, and blocked EGF-induced activation of EGFR, Akt and extracellular signal-regulated kinase 1/2. Overall, AEE788 was more effective than gefitinib. EGFR blockade as well as differentiation strongly reduced CD133 expression. However, ex vivo treatment with AEE788 did not impair orthotopic tumor engraftment, whereas ex vivo differentiation did, suggesting that CD133 does not absolutely segregate for tumorigenicity in ependymoma-SCs. Orally administered AEE788 prolonged survival of mice bearing ependymoma-SC-driven orthotopic xenografts from 56 to 63 days, close to statistical significance (log-rank p=0.06). Our study describes for the first time EGFR signaling in ependymoma-SCs and the effects of EGFR blockade in complementary in vitro and in vivo systems. The experimental models we developed can be used to further investigate the activity of EGFR inhibitors or other antineoplastic agents in this tumor.