Lipopolysaccharide activates calcineurin in ventricular myocytes.

OBJECTIVES: We investigated whether lipopolysaccharide (LPS), a proximate cause of inflammation, activates calcineurin in cardiac myocytes and if calcineurin regulates apoptosis in this setting. BACKGROUND: Calcineurin regulates myocardial growth and hypertrophy, but its role in inflammation is unknown. Calcineurin has proapoptotic or antiapoptotic effects depending on the stimuli. METHODS: Calcineurin activity was measured in left ventricular myocytes from adult Sprague Dawley rats. Cardiac apoptosis was measured by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling staining and caspase-3 activity after in vitro and in vivo exposure to LPS. RESULTS: Lipopolysaccharide increased calcineurin activity in myocytes over 1 to 24 h (t 1/2 = 4.8 h) with an EC(50) of 0.80 ng/ml LPS (p < 0.05, n = 4). The LPS (10 ng/ml) effects were mimicked by angiotensin II (Ang II) (100 nmol/l); both increased calcineurin activity and induced apoptosis without additive effects (p < 0.05, n = 5 to 9). Lipopolysaccharide and/or Ang II effects were prevented by 1 h pre-treatment with an Ang II type 1 receptor blocker (losartan, 1 micromol/l), calcineurin inhibitor (cyclosporin A, 0.5 micromol/l), calcium chelator (1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester, 0.1 micromol/l), or by inhibiting sarcoplasmic reticulum (SR) calcium (Ca)-ATPase (thapsigargin, 1 micromol/l) or SR calcium release channel (ryanodine, 1 micromol/l). Left ventricular apoptosis increased from 4 to 24 h after LPS (1 mg/kg intravenously) in vivo, but not in rats pre-treated with cyclosporin A (20 mg/kg/day subcutaneously) for 3 days (p < 0.05, n = 5). CONCLUSIONS: In cardiac myocytes, LPS activates calcineurin in association with apoptosis by Ang II and SR calcium-dependent mechanisms. This expands the paradigm for cardiac calcineurin to be activated by low levels of LPS in inflammation and chronic conditions (e.g., infections, smoking, and heart failure).     

Microsatellite instability in in vitro ageing of T lymphocyte clones

Repair of mismatches in mammalian cell DNA is mediated by a complex of proteins that constitute the so-called mismatch repair system (MMR), the main post-replicative pathway for the correction of replication errors. Loss of MMR (as exemplified by germline mutations in some MMR genes, leading to hereditary non-polyposis colorectal cancer) results in increased mutation rates at both coding sequences and in non-coding regions such as microsatellites. In order to evaluate possible functional alterations of this repair system during ageing that could affect immune system efficiency, we studied microsatellite instability at five different loci interspersed in the genome (CD4, VWA31, Tpox, Fes/FPS and p53) in total DNA from T lymphocyte clones derived from hematopoietic stem cells, or peripheral T cells of young or elderly subjects. In addition, these clones had been maintained for different periods in vitro to represent a culture model of ageing. We observed increasing instability accumulating with increasing passages in culture, particularly in CD34+cell-derived clones, but no clear donor age relationship.     

Role of endoscopic retrograde cholangiopancreatography in diagnosis and management of congenital choledochal cysts: 28 pediatric cases

Background/purposeManagement of choledochal cysts consists of surgical excision and hepaticojejunal anastomosis. Endoscopic retrograde cholangiopancreatography (ERCP) can be used to resolve complications and to evaluate the biliary tract and pancreatobiliary duct junction. Our aim was to underline the importance of ERCP for optimal management.MethodsFrom 2005 to 2011, 28 patients were reviewed (21 female, 7 male; mean age, 5.71 years; range, 2-16 years). After imaging, all patients underwent elective ERCP and were referred for surgery.ResultsCholedochal cyst was diagnosed at ultrasound and magnetic resonance cholangiopancreatography in all examined patients; common biliopancreatic duct was diagnosed in 3 (20%) of 15 patients at magnetic resonance cholangiopancreatography and in none at ultrasound. Endoscopic retrograde cholangiopancreatography showed choledochal cyst in all patients and common biliopancreatic duct in 19 (68%) of 28 patients. Twelve patients underwent sphincterotomy. All patients underwent surgical extrahepatic biliary tree resection and hepaticojejunal anastomosis. Mean period of hospitalization was 9.5 days (range, 6-13 days). No major complications related to ERCP were observed. Two patients needed postoperative ERCP for complications (pancreatitis during follow-up).ConclusionsIn our pediatric experience, ERCP is feasible and safe. It can rule out other possible biliary tract anomalies and help plan the timing and choice of the appropriate surgical procedure.     

Strictureplasty and intestinal resection: different options in complicated pediatric-onset Crohn disease

Background/PurposeSurgical resection or strictureplasty (SP) are different options for intestinal Crohn disease (CD) strictures. The aim of this article is evaluation of long-term outcome of SP and resection.MethodsFrom 1996 to 2011, 39 patients (23 male, 16 female) with symptomatic ileal and ileocolonic CD strictures resistant to medical/nutritional therapy and treated with surgery in 2 different surgical units were reviewed. The mean age at diagnosis was 11.82 years (range, 4-17 years). Mean age at surgery was 15.94 years (range, 4-24 years). Mean follow-up was 6.88 years (range, 0.5-15 years). Patients underwent resection (group A) or different SP techniques (group B).ResultsTwenty patients underwent intestinal resection (ileal or ileocolonic resection), and 19 patients underwent SP (jejunal, ileal, or ileocolic). Early postsurgical complications were observed in 2 patients of group A. Follow-up of group A patients revealed that 1 patient needed emergency treatment after 8 months surgery because of adhesions and 1 patient developed recurrence treated with medical therapy. In the follow-up group B, 3 patients experienced disease recurrence, 2 of them at the site of previous surgery.ConclusionsAt long-term follow-up, no significant difference in relapsing rate was observed between the 2 groups. Strictureplasty and resection represent an effective treatment of pediatric CD strictures. Strictureplasty could represent the first option for intestinal preservation.     

Long-term results of survival in patients with thalassemia major treated with bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) is the only available curative approach for thalassemia major, although long-term morbidity and mortality are not established. The aim of this study was to assess the long-term clinical and hematological results in children and adults with thalassemia major treated with BMT. We analyzed the outcome of 115 patients (median age 9 years, range 11 months to 28 years) with thalassemia major undergoing BMT from a related donor between 1983 and 2006. All patients received the same protocol, consisting of busulfan and cyclophoshamide as conditioning therapy and cyclosporin (CSA) alone or CSA and methotrexate for graft-versus-host disease (GvHD) prophylaxis. The cumulative probability of graft rejection was 6.7%. The transplant-related mortality at 1 year was 8.7%. The 20-year Kaplan-Meier estimate of overall survival and disease-free survival was 89.2% and 85.7%, respectively. Ninety-nine patients out of 103 survivors were in excellent clinical and hematological conditions at last visit following a median follow-up of 15 years (range, 1-24 years) with the exception of two patients who had invalidating chronic GvHD. This study conducted with a large cohort of patients and covering a long period of observation time, showed BMT to be curative for the majority of patients with thalassemia major. The impact of long-term transplant-related sequelae was very limited.     

Atypical Lipomatous Tumors: Should They be Treated Like Other Sarcoma or Not? Surgical Consideration from a Bi-Institutional Experience

Background

To identify the best surgical approach to atypical lipomatous tumors we reviewed 171 patients who underwent surgery at two sarcoma referral centers with different surgical policies.

Methods

Of the 151 patients (88 %) with primary tumors, 95 were treated at Institution A and 76 were treated at Institution B. At Institution A, a wide surgical resection, including a slight cuff of soft tissue around the mass, was adopted, which was defined as marginal resection (MR) according to the Enneking classification. At Institution B, a simple tumor resection (SR), according to the Enneking classification, was employed. En bloc surgical resection was the goal in both centers. The primary outcomes of the study were local recurrence-free survival (LRFS), incidence of secondary dedifferentiation at recurrence, and presence of residual tumor after re-excision.

Results

Sixteen patients (9 %) had local recurrence. The 10-year LRFS was 82 %. No cases of secondary dedifferentiation were observed. Residual tumor after re-excision was found in 46 % of cases. In univariate analysis, sclerosing subtype, tumor rupture, and SR were unfavorable prognostic factors for LRFS. Sclerosing subtype and tumor rupture were independent prognostic factors for LRFS in multivariate analysis. SR was significantly associated with tumor rupture.

Conclusions

Sclerosing subtype and tumor rupture are unfavorable prognostic factors for local recurrence. MR is associated with a lower risk of tumor rupture than SR. Neurovascular and major muscle resections are not necessary in principle. Re-excision after unplanned surgery is not always mandatory. A preoperative core needle biopsy could be useful in identifying the sclerosing subtype.     

Clinical outcomes of compensated and decompensated cirrhosis: A long term study

AIM: To study these characteristics and prognostic patterns in a Greek patient population.METHODS: We analyzed a large cohort of cirrhotic patients referred to the department of Gastroenterology and Hepatology and the outpatient clinics of this tertiary hospital, between 1991 and 2008. We included patients with established cirrhosis, either compensated or decompensated, and further decompensation episodes were registered. A data base was maintained and updated prospectively throughout the study period.We analyzed differences in cirrhosis aetiology, time to and mode of decompensation, hepatocellular carcinoma(HCC) occurrence and ultimately patient survival.RESULTS: Five hundreds and twenty-two patients with median age 67(range, 29-91) years and average follow up 9 years-10 mo(range, 1-206 mo) were studied. Commonest aetiology was hepatitis C virus(HCV, 41%) followed by alcohol(31%). The median survival time in compensated cirrhotics was 115 mo(95%CI: 95-133), whereas in decompensated patients was 55 mo(95%CI: 36-75). HCV patients survived longer while HBV patients had over twice the risk of death of HCV patients. The median time to decompensation was 65 mo(95%CI: 51-79), with alcoholics having the highest risk(RR = 2.1 vs HCV patients). Hepatitis B virus(HBV) patients had the highest risk of HCC, alcoholics the lowest. Leading causes of death: liver failure, hepatorenal syndrome, sepsis and HCC progression. CONCLUSION: Cirrhosis aetiology and decompensation at presentation were predictors of survival. Alcoholics had the highest decompensation risk, HBV cirrhotics the highest risk of HCC and HCV cirrhotics the highest decompensation-free time.     

Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyses on Recovered Effector Cells and Supernatants

Cytotoxicity assays provide an in vitro evaluation of the lytic activity of NK and T cells against tumors or transformed cells. However, none of these methods allow the recovery of cells or supernatants after the assay. We standardized a microcytotoxicity test using calcein-acetoxymethyl (calcein-AM) dye that requires very small quantities of cells while maintaining the same sensitivity as the traditional ⁵¹Cr assay. The assay is applicable to resting as well as activated human effector cells and uses different targets such as human cell lines that are adherent or growing in suspension and resistant or sensitive. The most important feature of the method is the possibility of recovering cells and supernatants for additional analyses such as phenotyping and evaluation of soluble factors.     

Diagnostic yield of endoscopic ultrasound-guided tissue acquisition of solid pancreatic lesions after inconclusive percutaneous ultrasound-guided tissue acquisition

Abstract

Introduction

After a failed percutaneous ultrasound (US)-guided sampling, it is recommended that endoscopic ultrasound (EUS)-guided tissue acquisition (TA) be performed for non-resectable solid pancreatic lesions according to the European Federation of Societies for Ultrasound in Medicine and Biology. However, the diagnostic performance of EUS-guided TA in this setting is unknown.