Tumor-specific suicide gene therapy for hepatocellular carcinoma by transcriptionally targeted retroviral replicating vectors.

Replicating virus vectors are attractive tools for anticancer gene therapy, but the potential for adverse events due to uncontrolled spread of the vectors has been a major concern. To design a tumor-specific retroviral replicating vector (RRV), we replaced the U3 region of the RRV ACE-GFP with a regulatory sequence consisting of the hepatitis B virus enhancer II (EII) and human α-fetoprotein (AFP) core promoter to produce ACE-GFP-EIIAFP, a hepatocellular carcinoma (HCC)-targeting RRV. Similar to ACE-GFP, ACE-GFP-EIIAFP exhibited robust green fluorescent protein (GFP) expression in HCC cells and, most importantly, it exhibited HCC-specific replication and did not replicate in non-HCC tumor cells or normal liver cells. We sequenced the promoter region of ACE-GFP-EIIAFP collected from serial infection cycles to examine the genomic stability of the vector during its replicative spread, and found that the vector could retain the hybrid promoter in the genome for at least six infection cycles. In vitro studies revealed that ACE-CD-EIIAFP and ACE-PNP-EIIAFP, which express the yeast cytosine deaminase and Escherichia coli purine nucleoside phosphorylase, respectively, exert a highly potent cytotoxic effect on HCC cells in the presence of their respective prodrugs. In vivo, ACE-CD-EIIAFP-mediated suicide gene therapy efficiently suppressed HCC tumor growth and no detectable RRV signal was observed in extratumoral tissues. These results suggest that the tumor-specific, suicide-gene-encoding RRV may fulfill the promise of retroviral gene therapy for cancer.

Severe acute pyelonephritis: a review of clinical outcome and risk factors for mortality.

OBJECTIVE. To review demographics of patients with acute pyelonephritis, their outcomes of severe upper urinary tract infection, and to identify risk factors for long hospital stay and mortality. DESIGN. Case series. SETTING. A regional hospital in Hong Kong. PATIENTS. Patients admitted between June 2007 and June 2012 for acute pyelonephritis were identified. Those with the most severe outcomes were analysed of their mortality, need for care in the intensive care unit, or necessitation of urological intervention. RESULTS. Overall, 68 patients fulfilled our criteria for severe acute pyelonephritis. The female-to-male ratio was 7:3. Their mean age was 58 years. Overall, 57% of the patients had impaired renal function and 37% were diabetic; 47% developed shock after admission and 56% required further intensive care unit care; 75% of the patients demonstrated radiological evidence of urinary tract obstruction and required subsequent drainage procedures. Five patients died due to severe acute pyelonephritis. The prevalence of bacteraemia and bacteriuria was 57% and 74%, respectively. Escherichia coli accounted for the majority of causative organisms. Four risk factors-bacteraemia, shock, need for intensive care, and suppurative pyelonephritis-were associated with hospital stay of longer than 14 days. Old age (≥65 years), male sex, deranged renal function, and presence of disseminated intravascular coagulation were associated with mortality. CONCLUSION. There was high prevalence of bacteraemia and septic shock in patients with severe acute pyelonephritis. The factors of old age (≥65 years), male sex, deranged renal function, and presence of disseminated intravascular coagulation were associated with mortality. With the support of intensive care, early recognition of urinary tract obstruction and timely drainage, patients with severe acute pyelonephritis generally carry a good prognosis.

Nurse-initiated defibrillation: are nurses confident enough?

OBJECTIVES: To determine the capability of nurses to identify ventricular fibrillation (VF) and ventricular tachycardia (VT) rhythms on an ECG and carry out subsequent defibrillation on their own as soon as they identify and confirm cardiac arrest. METHODS: This was a prospective cohort study to determine the capability of emergency department (ED) nurses to recognise VF or pulseless VT correctly and their willingness to perform defibrillation immediately in an ED of a teaching hospital in Hong Kong. A questionnaire was completed before and after a teaching session focusing on the identification of rhythms in cardiac arrest and defibrillation skills. Correct answers for both ECG interpretation and defibrillation decisions scored one point for each question. The differences in mean scores between the pre-teaching and post-teaching questionnaires of all nurses were calculated. RESULTS: 51 pre-teaching and 43 post-teaching questionnaires were collected. There were no statistically significant changes in ECG scores after teaching. For defibrillation scores, there was an overall improvement in the defibrillation decision (absolute mean difference 0.42, p=0.014). Performance was also improved by the teaching (absolute mean difference 0.465, p=0.046), reflected by the combination of both scores. Two-thirds (67%) of nurses became more confident in managing patients with shockable rhythms. CONCLUSION: Nurses improve in defibrillation decision-making skills and confidence after appropriate brief, focused in-house training.

Enhanced efficiency of prodrug activation therapy by tumor-selective replicating retrovirus vectors armed with the Escherichia coli purine nucleoside phosphorylase gene.

Gene transfer of the Escherichia coli purine nucleoside phosphorylase (PNP) results in potent cytotoxicity after administration of the prodrug fludarabine phosphate (F-araAMP). Here, we have tested whether application of this strategy in the context of replication-competent retrovirus (RCR) vectors, which can achieve highly efficient tumor-restricted transduction as well as persistent expression of transgenes, would result in effective tumor inhibition, or, alternatively, would adversely affect viral replication. We found that RCR vectors could achieve high levels of PNP expression concomitant with the efficiency of their replicative spread, with significant cell killing activity in vitro and potent therapeutic effects in vivo. In U-87 xenograft models, replicative spread of the vector resulted in progressive transmission of the PNP transgene, as evidenced by increasing PNP enzyme activity with time after vector inoculation. On F-araAMP administration, high efficiency gene transfer of PNP by the RCR vector resulted in significant suppression of tumor growth and extended survival time. As the RCR mediates stable integration of the PNP gene and continuous expression, an additional round of F-araAMP administration resulted in further survival benefit. RCR-mediated PNP suicide gene therapy thus represents a highly efficient form of intracellular chemotherapy, and may achieve effective antitumor activity with less systemic toxicity.

Laparoscopic biliary bypass--a single centre experience.

BACKGROUND/AIMS: To review the results of laparoscopic biliary bypass for both benign and malignant pathologies in a minimal access surgery training center. METHODOLOGY: Retrospective review of a prospectively maintained database of laparoscopic biliary bypass during the period 1995-2004. RESULTS: During the review period 1995-2004, there were 26 laparoscopic biliary bypasses performed in our center which included 23 laparoscopic choledochoduodenostomy (LCD), 2 laparoscopic roux-en-Y choledochojejunostomy (LCJ) and 1 laparoscopic cholecystojejunostomy (LCCJ). Of the 23 LCD, all except 1 patient were operated for recurrent pyogenic cholangitis (RPC). The 2 LCJ and 1 LCCJ were performed for patients with advanced carcinoma in the periampullary region and simultaneous laparoscopic gastrojejunostomy (LGJ) was also performed to relieve the gastric outflow obstruction. Among the 23 LCD, there were 2 open conversions (7.7%) for lost broken tip of ultrasonic dissector and significant bleeding during choledochotomy respectively. Major complications occurred in 6 patients (23%), which included 3 bile leaks (11.5%), 1 intraabdominal collection (3.8%). 1 wound infection (3.8%) and 1 gastric stasis (3.8%). The only mortality in our series was a patient with carcinoma of head of pancreas undergoing simultaneous roux-en-Y LCCJ and LGJ. He had persistent gastric stasis after operation and required revision surgery for the kinked cholecystojejunostomy anastomosis. He finally died of myocardial ischemia after the second operation. As for the postoperative pain control, the mean pethidine consumption was 243.4 +/- 254.7 mg (range 0-1200 mg) and mean dologesic usage was 16.2 +/- 20.4 tablets (range 0-94 tablets). The average postoperative hospital stay was 12.6 +/- 11.5 days (range 5-60 days). The long-term functional results were satisfactory and only 1 patient had recurrent stone upon a mean follow-up of 32.3 months. Among the patients with malignant biliary obstruction, the only mishap was as previously mentioned and the remaining 2 patients could enjoy satisfactory palliation for more than a year before death. CONCLUSIONS: Laparoscopic bypass is not only feasible but also highly effective in relieving biliary obstruction with good postoperative results in both benign and malignant conditions.

Antegrade biliary stenting versus T-tube drainage after laparoscopic choledochotomy--a comparative cohort study.

BACKGROUND/AIMS: Laparoscopic exploration of the common bile duct (LECBD) has been proven to be an effective and preferred treatment approach for uncomplicated common bile duct stones. However there is still controversy regarding the choice of biliary decompression after laparoscopic choledochotomy. METHODOLOGY: This is a retrospective comparison between the use of antegrade biliary stenting and T-tube drainage following successful laparoscopic choledochotomy. During the period between January 1995 and July 2003, biliary decompression was achieved by either antegrade biliary stenting or T-tube drainage based on the discretion of the operating surgeon. For antegrade biliary stenting, a 10-Fr Cotton-Leung biliary stent was inserted through the choledochotomy and passed down across the papilla. The stent position was confirmed by on-table choledochoscopy before interrupted single-layered closure of the common bile duct. Endoscopic retrograde cholangiopancreatography (ERCP) was performed to remove the stent 4 weeks after operation and at the same time to check for any residual stones or other complications like stricture or leak. In the T-tube group, a 16-Fr latex T-tube was used and the long limb was brought out through the subcostal trocar port followed by the same method of bile duct closure. Cholangiogram through the T-tube was performed on day 7 and the tube would be taken off 1 week later (about 2 weeks after operation) if the cholangiogram did not reveal any abnormality. The two groups were compared according to the demographic data, operation time, length of hospital stay and complication rates. RESULTS: During the study period, 108 laparoscopic explorations of the common bile duct were performed in our centre of which 95 were attempted laparoscopic choledochotomies and 13 were transcystic duct explorations. Of the 95 patients with attempted laparoscopic choledochotomy, there were 9 open conversions, 17 laparoscopic bilioenteric bypasses and 6 primary closures of the common bile duct. All of these patients together with those receiving transcystic duct explorations were excluded and the remaining 63 patients having postoperative bile diversion by either antegrade biliary stenting or T-tube drainage were included in this study. Bile diversion was achieved by antegrade biliary stenting in 35 patients whereas 28 patients had T-tube drainage. There was no difference between the two groups in terms of age, clinical presentation, bilirubin level, length of hospital stay, follow-up duration, common bile duct size, size of common bile duct stones, incidence of residual/recurrent stone and complication rate. It was observed that more patients in the stenting group developed bile leak (14.2% vs. 3.5%) and required more intramuscular pethidine injections (182.86 +/- 139.30 vs. 92.81+/-81.15mg, P=0.000). On the other hand, the T-tube group had longer operation time (141.4+/-45.1 vs. 11 1.1+/-33.9 minutes, P=0.006) and had a longer postoperative hospital stay (10.0+/-7.4 vs. 8.8+/-9.3 days, P=0.020) reaching statistical significance. CONCLUSIONS: Postoperative bile diversion by antegrade biliary stenting after laparoscopic choledochotomy is shown to shorten operation duration and postoperative stay as compared to T-tube drainage, but the problem of bile leak needs further refinement of insertion technique.

Laparoscopy versus open left lateral segmentectomy for recurrent pyogenic cholangitis.

BACKGROUND: Recurrent pyogenic cholangitis (RPC) is a common disease in Southeast Asia. Its classical presentation is repeated attacks of cholangitis with multiple recurrences of bile duct stones. The stones are commonly located in the left lateral segments (2 and 3) and therefore complete clearance is difficult to achieve by either endoscopic retrograde cholangiopancreatography or surgical exploration of the common bile duct. The definitive treatment usually involves resection of the stone-harboring segments. The recent advent in laparoscopic surgery has shown that hand-assisted laparoscopic segmentectomy is a safe and feasible, alternative. This study aimed to compare hand-assisted laparoscopic segmentectomy with open segmentectomy in patients with recurrent, RPC. METHODS: This study retrospectively reviewed a prospectively maintained database of both open and laparoscopic treatments for RPC in a single center between 1994 and 2004. During this period, patients with RPC and left intrahepatic (segments 2 and 3) ductal stones not amendable to endoscopic treatment were recruited for analysis. Patients with concomitant gallbladder stones and common bile duct stones were offered left lateral segmentectomy with cholecystectomy and exploration of the common bile duct. Selected patients would have choledochoduodenostomy drainage during the same operation. The operations were performed via either the hand-assisted laparoscopic approach or the open approach using an ultrasonic surgical aspirator. The two cohorts were compared with respect to perioperative parameters to determine whether there would be any advantage in attempting hand-assisted laparoscopic segmentectomy. RESULTS: During the study period from 1994 to 2004, 17 patients underwent left lateral segmentectomy for RPC. Of the 17 patients, 10 had hand-assisted laparoscopic resections, and 7 underwent open resections. All open resections were performed before 1999. Despite the small number of patients and potential type 2 error, there were no differences in age, sex distribution, number of cholangitic attacks, sessions of endoscopic retrograde cholangiopancreatography before surgery, or number of previous operation between the two groups. The median operating time was shorter in the open group (232.5 vs 150 min; p = 0.007), whereas the median blood loss was similar (350 vs 400 ml; p = 0.551). The median postoperative stay was 8 days for hand-assisted laparoscopic group versus 14 days for the open group. This difference was statistically significant (p = 0.019). There was one open conversion in the hand-assisted laparoscopic group because of intraoperative bleeding from the left hepatic vein. Postoperative complication rates were lower in hand-assisted laparoscopic group, but the difference was not statistically significant (20% vs 57%; p = 126). The intramuscular pethidine requirement again was less in hand-assisted laparoscopic group (0 vs 600 mg; p = 0.002). There was no operative mortality in either group of patients. No recurrent cholangitis was noted in either groups during the median follow-up period of more than 3 years. CONCLUSION: This study not only confirmed the feasibility of hand-assisted laparoscopic segmentectomy for recurrent pyogenic cholangitis, but also showed that this treatment approach is associated with less pain and shorter hospital stay. However, hand-assisted laparoscopic segmentectomy is a lengthier operation and technically more challenging. Nevertheless, the authors believe that with more experience and further improvement of ancillary technology, this procedure can become a standard treatment for recurrent pyogenic cholangitis in selected cases.

Laparoscopic exploration of common bile duct in difficult choledocholithiasis.

BACKGROUND: This review investigated the role played by laparoscopic exploration of the common bile duct (LECBD) in the management of difficult choledocholithiasis. METHODS: This retrospective study reviewed a prospective database of LECBD for difficult choledocholithiasis during the period 1995 to 2003. RESULTS: Of the 97 LECBDs performed in the authors' center from 1995 to 2003, 25 were performed for difficult choledocholithiasis. Difficult choledocholithiasis was defined as failure of endoscopic stone retrieval for the following reasons: access and cannulation difficulty, the difficult nature of common bile duct (CBD) stones, and the presence of endoscopic retrograde cholangiopancreatography (ERCP)-related complications. There were seven unsuccessful cannulations because of previous gastrectomy (n = 5) and periampullary diverticulum (n = 2). Among the 18 patients with failed endoscopic extraction, there were 10 impacted stones, 2 incomplete stone clearances after multiple attempts, 2 type 2 Mirizzi syndromes, 1 proximal stent migration, 1 repeated post-ERCP pancreatitis, 1 situs inversus, and 1 stricture at the distal common bile duct. There were 14 male and 11 female patients with a mean age of 67.8 +/- 15 years. Initial presentations included cholangitis (n = 14, 56%), biliary colic (n = 3, 12%), jaundice/deranged liver function ( n = 5, 20%), cholecystitis (n = 2, 8%), and pancreatitis (n = 1, 4%). Regarding the approach for LECBD, there were 2 transcystic duct explorations and 23 choledochotomies. The mean operative time was 149.4 +/- 49.3 min, and there were three conversions (12%). The stone clearance rate was 100%, and no recurrence was detected during a mean follow-up period of 16.8 months. Five complications were encountered, which included bile leak (3 patients) and wound infection (2 patients). When the results were compared with the remaining 72 LECBDs for nondifficult stones during the same period, the complication rate, conversion rate, and rate of residual stones were similar despite a longer operation time (149.4 +/- 49.4 min vs 121.6 +/- 50.5 min). CONCLUSION: When ERCP is impossible or stone retrieval is incomplete, LECBD is the solution to difficult CBD stones.

Genomic stability of murine leukemia viruses containing insertions at the Env-3' untranslated region boundary.

Retroviruses containing inserts of exogenous sequences frequently eliminate the inserted sequences upon spread in susceptible cells. We have constructed replication-competent murine leukemia virus (MLV) vectors containing internal ribosome entry site (IRES)-transgene cassettes at the env-3' untranslated region boundary in order to examine the effects of insert sequence and size on the loss of inserts during viral replication. A virus containing an insertion of 1.6 kb replicated with greatly attenuated kinetics relative to wild-type virus and lost the inserted sequences in a single infection cycle. In contrast, MLVs containing inserts of 1.15 to 1.30 kb replicated with kinetics only slightly attenuated compared to wild-type MLV and exhibited much greater stability, maintaining their genomic integrity over multiple serial infection cycles. Eventually, multiple species of deletion mutants were detected simultaneously in later infection cycles; once detected, these variants rapidly dominated the population and thereafter appeared to be maintained at a relative equilibrium. Sequence analysis of these variants identified preferred sites of recombination in the parental viruses, including both short direct repeats and inverted repeats. One instance of insert deletion through recombination with an endogenous retrovirus was also observed. When specific sequences involved in these recombination events were eliminated, deletion variants still arose with the same kinetics upon virus passage and by apparently similar mechanisms, although at different locations in the vectors. Our results suggest that while lengthened, insert-containing genomes can be maintained over multiple replication cycles, preferential deletions resulting in loss of the inserted sequences confer a strong selective advantage.

A uniquely stable replication-competent retrovirus vector achieves efficient gene delivery in vitro and in solid tumors.

A major obstacle in cancer gene therapy is the limited efficiency of in vivo gene transfer by replication-defective retrovirus vectors in current use. One strategy for circumventing this difficulty would be to use vectors capable of replication within tumor tissues. We have developed a replication-competent retrovirus (RCR) vector derived from murine leukemia virus (MuLV). This vector utilizes a unique design strategy in which an internal ribosome entry site-transgene cassette is positioned between the env gene and the 3' long terminal repeat (LTR). The ability of this vector to replicate and transmit a transgene was examined in culture and in a solid tumor model in vivo. The RCR vector exhibited replication kinetics similar to those of wildtype MuLV and mediated efficient delivery of the transgene throughout an entire population of cells in culture after an initial inoculation with 1 plaque-forming unit (PFU) of vector per 2000 cells. After injection of 6 x 10(3) PFU of vector into established subcutaneous tumors, highly efficient spread of the transgene was observed over a period of 7 weeks, in some cases resulting in spread of the transgene throughout the entire tumor. MuLV-based RCR vectors show significant advantages over standard replication-defective vectors in efficiency of gene delivery both in culture and in vivo. This represents the first example of the use of an RCR vector in an adult mammalian host, and their first application to transduction of solid tumors.