Standard versus Endocuff versus cap-assisted colonoscopy for adenoma detection: A randomised controlled clinical trial.

BACKGROUND AND AIMS: Adenoma detection rate (ADR) in colon cancer screening is most important for cancer prophylaxis. This work is the first three-armed randomised controlled clinical trial aimed at comparing a head-to-head setting standard colonoscopy (SC) with Endocuff-assisted colonoscopy (EC) and cap-assisted colonoscopy (CAC) for improvement of ADR. METHODS: Patients from Poland and Germany with independent indication for colonoscopy were randomised into three arms of this trial: EC, CAC and SC. Exclusion criteria were age <18 years, active Crohn's disease or ulcerative colitis, known stenosis and post-colonic resection status. RESULTS: A total of 585 patients (195 SC, 189 EC and 186 CAC) were enrolled in this study. Indications were not different between the groups (colorectal cancer screening 51%, diagnostic colonoscopy in 31% and post-polypectomy follow-up in 18%; p = 0.94). Withdrawal time was a mean of 7 min in all groups (p = 0.658), and bowel preparation did not differ between the groups. The time to reach the caecum was significantly reduced when using the cap (a mean of 6 min for CAC vs. 7 min for SC; p = 0.0001). There was no significant difference in the primary outcome of the ADR between the groups (EC 32%, CAC 30%, SC 30%; p = 0.815). EC proved to be superior (EC vs. SC) in the sigmoid colon and transverse colon for polyp detection. CONCLUSION: The use of EC increased the total number of polyps seen during colonoscopy. In contrast to recent studies, no significant improvement of the ADR was detected.

Higher adenoma detection rates with endocuff-assisted colonoscopy - a randomized controlled multicenter trial.

OBJECTIVES: The Endocuff is a device mounted on the tip of the colonoscope to help flatten the colonic folds during withdrawal. This study aimed to compare the adenoma detection rates between Endocuff-assisted (EC) colonoscopy and standard colonoscopy (SC). METHODS: This randomized prospective multicenter trial was conducted at four academic endoscopy units in Germany. PARTICIPANTS: 500 patients (235 males, median age 64[IQR 54-73]) for colon adenoma detection purposes were included in the study. All patients were either allocated to EC or SC. The primary outcome measure was the determination of the adenoma detection rates (ADR). RESULTS: The ADR significantly increased with the use of the Endocuff compared to standard colonoscopy (35.4%[95% confidence interval{CI} 29-41%] vs. 20.7%[95%CI 15-26%], p<0.0001). Significantly more sessile polyps were detected by EC. Overall procedure time and withdrawal time did not differ. Caecal and ileum intubation rates were similar. No major adverse events occurred in both groups. In multivariate analysis, age (odds ratio [OR] 1.03; 95%[CI] 1.01-1.05), male sex (OR 1.74; 95%CI 1.10-2.73), withdrawal time (OR 1.16; 95%CI 1.05-1.30), procedure time (OR 1.07; 95%CI 1.04-1.10), colon cleanliness (OR 0.60; 95%CI 0.39-0.94) and use of Endocuff (OR 2.09; 95%CI 1.34-3.27) were independent predictors of adenoma detection rates. CONCLUSIONS: EC increases the adenoma detection rate by 14.7%(95%CI 6.9-22.5%). EC is safe, effective, easy to handle and might reduce colorectal interval carcinomas. TRIAL REGISTRATION: ClinicalTrials.gov NCT02034929.

Overexpression of lysosomal acid lipase and other proteins in atherosclerosis.

Atherosclerosis is one of the major causes of morbidity and mortality in the western world. The existing data of elevated expression levels of proteins like DNA damage and DNA repair enzymes in human atherosclerotic plaques are reviewed. From the literature, the effect of overexpression of different proteins using adenoviral vectors or the model of transgenic mice on the development of atherosclerosis will be discussed. Special focus is placed on the lysosomal acid lipase (LAL), because LAL connects extra-cellular with intra-cellular lipid metabolism and is the only hydrolase for cleavage of cholesteryl esters delivered to the lysosomes. Patients with a deficiency of LAL show an accumulation of lipids in the cells and develop pre-mature atherosclerosis. To answer the question of the influence of LAL in atherosclerosis if overexpressed, we show for the first time data of transgenic mice overexpressing LAL and the effect on the lipid level.

Systematic mutagenesis of potential glycosylation sites of lysosomal acid lipase.

Lysosomal acid lipase (LAL; EC 3.1.1.13) is a key enzyme in the intracellular lipid metabolism. It hydrolyzes exogenous triglycerides and cholesterol esters taken up by various cell types. LAL has six potential N-glycosylation sites and one potential O-glycosylation site. Elimination of each of the six Asn-(X)-Ser/Thr sites by site-directed mutagenesis and expression in baculovirus-infected Spodoptera frugiperda cells resulted in two single-mutant enzymes without lipolytic activities (N134Q and N246Q) and four mutants with preserved activities. The two inactive mutants were not detectable on immunoblot analysis, indicating that they were not secreted. Six double mutants in all possible combinations except for the two inactive single mutants were produced and expressed. Double mutants in combination with the N9 glycosylation site showed reduced activities as compared to the other mutants or the wild-type enzyme. Kinetic data of LAL glycosylation mutants indicate that substrate affinity of N9Q was not changed, but k (cat) of N9 mutants was reduced distinctly compared to the wild-type enzyme. Peanut agglutinin lectin did not recognize LAL, demonstrating that the protein has no core1 structure (Galbeta 1-3 GalNAc) of O-glycosylation. These data indicate that at least two of the six N-glycosylation sites are used in native lipase. N134 and N246 were found to be essential for LAL activity. We conclude that glycosylation plays an important role in the formation of functional LAL.

Low-dose adenoviral immunotherapy of rat hepatocellular carcinoma using single-chain interleukin-12.

Generation of antitumor immunity by adenoviral gene transfer of interleukin-12 (IL-12) is a very promising concept in cancer gene therapy. Systemically, IL-12 has provoked toxic side effects at therapeutically relevant doses. Native IL-12 lacks effectiveness in clinical trials even when expressed intratumorally from adenoviral vectors. Our strategy was to increase the therapeutic efficacy of IL-12 by expressing a fusion protein of its two subunits (scIL-12) in an adenoviral vector and to evaluate the effects after intratumoral administration. In a rat model of hepatocellular carcinoma, this vector revealed antitumor effects even at a low dosage of 4.6 x 10(5) i.u. in a dose-dependent manner. Long-term antitumor effects were determined at 2.3 x 10(6) and 2.3 x 10(7) i.u. per animal, resulting in 82% and 90% surviving animals, respectively. Magnetic resonance imaging (MRI) enabled individual tumor size follow-up and revealed the scIL-12 effects on large tumors. Treating one hepatic lesion also led to tumor elimination in a second non-treated hepatic lesion. Animals rechallenged with tumor cells remained tumor-free. Compared to studies applying native IL-12, our data show that the fusion of IL-12 subunits provides approximately 1000-fold higher biological activity. As a consequence of the observed gain in activity, scIL12 promises a substantially improved antitumor efficacy and safety profile of intratumoral adenoviral IL-12 immunotherapy, supporting its clinical use.

Lysosomal acid lipase as a preproprotein.

Lysosomal acid lipase (LAL; EC 3.1.1.13) hydrolyzes intracellular triglycerides and cholesterol esters taken up by various cell-types. Previously, LAL purified from human liver tissue was described as a preproprotein with a 27 amino acid signal peptide and a 49 amino acid propeptide. Three mutants of the putative proregion of LAL were produced and expressed in Spodoptera frugiperda insect cells. Pulse-chase experiments demonstrated that LAL undergoes proteolytical processing. The deletion of the 49 amino acids led to a complete loss of the LAL activity. The two other mutants were produced at the C-terminus of the pro-region, at positions 49 and 50, by site-directed mutagenesis. Mutant K49R showed wild-type LAL activity, but mutant G50A showed significantly reduced enzyme activity compared to wild-type LAL and a greater reduction in culture medium than in detergent cell extracts. Kinetic data suggest that mutant G50A is less stable than wild-type LAL and mutant K49R. In contrast to K49, the highly conserved amino acid residue G50 seems to be in a very important position and its mutation influences both secretion and enzyme activity of LAL. A three-dimensional model of LAL shows that K49 and G50 are localized in the loop-region between two beta-sheets, highly accessible for proteolytic enzymes. These data together indicate that LAL is indeed a preproprotein, in which the pro-region is essential for its folding and stability, secretion, and enzyme activity.

Highly suppressible expression of single-chain interleukin-12 by doxycycline following adenoviral infection with a single-vector Tet-regulatory system.

BACKGROUND: Adenoviral vectors have been shown to efficiently transfer DNA into a wide variety of eukaryotic cells in vitro and in vivo. However, the therapeutic benefit of this approach is limited by severe side effects as a result of uncontrolled transgene expression. METHODS: A bi-directional promoter that controls the desired transgene as well as a tetracycline-suppressible transactivator (tTA) was cloned into the E1-region of E1-deleted recombinant adenoviral vectors. Autoregulation within this construct was obtained by tTA expression under control of the operator, to which tTA binds in the absence of tetracycline. Consequently, binding of tetracycline to tTA results in downregulation of tTA as well as the co-expressed transgene in the infected cell. RESULTS: We were able to suppress luciferase-reporter gene expression by up to 16 000-fold in the presence of doxycycline (dox, 2 micro g/ml). Under control of this tetracycline-regulated system, single-chain interleukin-12 (scIL12) was expressed. Adenovirally mediated expression of this potentially lethal cytokine with strong activation of antitumoral immune response was downregulated by up to 6000-fold in the presence of dox. Subsequently, this downregulation also resulted in a highly significant reduction of interferon-gamma secretion by stimulated splenocytes. These mainly contribute to the toxicity of this immunotherapeutic approach. CONCLUSIONS: With expression levels exceeding those of the cytomegalovirus (CMV) promoter in almost all cell lines tested, these new vectors will also contribute to the safety of adenoviral approaches by controlled expression without compromising on maximum expression levels.

Lysosomal acid lipase deficiency: correction of lipid storage by adenovirus-mediated gene transfer in mice.

Lysosomal acid lipase (LAL) is the essential enzyme for hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. Its deficiency produces two human phenotypes: Wolman disease (WD) and cholesteryl ester storage disease (CESD). The LAL null (lal(-/-)) mouse mimicks aspects of human WD and CESD. The potential for gene therapy of LAL deficiency was tested with first-generation adenoviral vectors containing human LAL cDNA (Ad-hLAL) by intravenous injection into lal(-/-) mice. Compared with phosphate-buffered saline-injected controls, the mice receiving Ad-hLAL had increased hepatic LAL activity, decreased hepatomegaly, and normalization of histopathology. hLAL protein and mRNA were detected by immunohistochemical staining and in situ hybridization in hepatic parenchymal and sinusoid lining cells, splenic sinusoidal cells, lung macrophages, and adrenal cortical cells. Mice showed TG reductions in liver, spleen, and small intestine of 68, 54, and 50%, respectively, and cholesterol reductions of 55, 52, and 34%, respectively, at 20 days postinjection. These studies provide the basis for the use of gene therapy, in the form of gene transfer via intravenously administered adenovirus, to correct deficiency states, such as WD and CESD, and histopathology of a variety of tissues.