The anti-arrhythmia properties of bonnecor metabolites

The experiments on the model of aconitine-induced arrhythmia in anesthetized rats and awake dogs with cardiac rhythm disorders caused by the coronary artery occlusion showed that all four metabolites of bonnecor possessed the antiarrhythmic activity. Metabolite 4 proved to be the most active on both models of cardiac rhythm disorders.     

Therapeutic endoscopy in the complex treatment of patients with chronic gastroduodenal ulcer

Under the ambulatory treatment there were 300 patients: 120 of them had gastric ulcers and 180 had ulcers of the duodenum. The immediate and long-term results of the treatment were followed-up in 243 patients. The analysis has shown that Solcoseryl and film-forming glue "Statizol" included in the complex of general therapy of chronic ulcers of the stomach and duodenum by means of a local application (through the endoscope) made the average terms of healing 2 times shorter as compared with the routine treatment of ulcers and considerably reduced recidivation of the disease (in the main group recurrences took place in 19-10%, in the control group--in 36-42.4% of the cases). Most effective was the complex treatment including the application of Solcoseryl and film-forming glue "Statizol" simultaneously.     

Use of Statizol film-forming glue for treating gastric and duodenal ulcers

Patients with chronic gastroduodenal ulcers (210) and gastric ulcers (256) were treated in the out-patient clinic. The ulcerous defect of the mucous membrane was covered by the aerosolic film-making glue "Statizol" through a fibroendoscope (about 10 applications of the drug). The method was used in 110 patients after polypectomy in addition to the routine therapeutic treatment given to all the patients. The application of the film-making glue "Statizol" in the complex therapy of peptic ulcers was shown to shorten the period of healing the ulcerous defects, to give less amount of recurrent ulcer diseases (not more than 14.5%) and to be a prophylactic measure against bleedings after polypectomy.     

Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation

BACKGROUND: Liver function after transplantation is determined by the quality of the donor organ and the influences of preservation, flush, and reperfusion injury. In this regard, cell death (apoptosis) plays an important role in organ preservation and rejection. Therefore, we examined the possibility of genetic modification of the liver graft with a recombinant adenovirus vector encoding the Bcl-2 gene to reduce apoptosis during the preservation time. METHODS: Liver grafts from C57B1/6 mice were procured and preserved using standard techniques. A replication defective adenovirus vector (deltaE1) containing the human Bcl-2 gene (AdCMVhBcl-2) was developed in our laboratory. An adenovirus vector encoding an irrelevant gene (Escherichia coli beta-galactosidase) was used as a control. Each mouse received 1 x 10(9) plaque forming units administered i.v. 48 hr before the liver procurement. Analyses of liver enzyme activities were determined in the preservation solution. Apoptosis in liver biopsies was determined by DNA fragmentation with an in situ histochemical assay. RESULTS: Immunohistochemical analysis and RT-PCR confirmed the expression of hBcl-2 in the grafts. Grafts from livers expressing hBcl-2 showed significant reduction of the aspartame amino transferase (AST) and lactate dehydrogenase (LDH) release compared with grafts from the control groups. After rewarming, significant cytoprotection was also observed in grafts from animals treated with AdCMVhBcl-2. Histological analysis correlated with the hepatocellular injury determined with transaminases and LDH in the preservation solution. Significant reduction in the number of apoptotic cells was observed in grafts expressing hBcl-2. CONCLUSIONS: We have demonstrated a novel approach to reducing the preservation injury to liver grafts with the human Bcl-2 gene. This approach may allow a longer preservation time, potentially reduce the incidence of primary nonfunction, decrease the immunogenicity of the cold injured organ, and increase the safer use of "marginal" liver grafts.     

Gamma camera dual imaging with a somatostatin receptor and thymidine kinase after gene transfer with a bicistronic adenovirus in mice

PURPOSE: To compare two systems for assessing gene transfer to cancer cells and xenograft tumors with noninvasive gamma camera imaging. MATERIALS AND METHODS: A replication-incompetent adenovirus encoding the human type 2 somatostatin receptor (hSSTr2) and the herpes simplex virus thymidine kinase (TK) enzyme (Ad-hSSTr2-TK) was constructed. A-427 human lung cancer cells were infected in vitro and mixed with uninfected cells at different ratios. A-427 tumors in nude mice (n = 23) were injected with 1 x 10(6) to 5 x 10(8) plaque-forming units (pfu) of Ad-hSSTr2-TK. The expressed hSSTr2 and TK proteins were imaged owing to internally bound, or trapped, technetium 99m ((99m)Tc)-labeled hSSTr2-binding peptide (P2045) and radioiodinated 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (FIAU), respectively. Iodine 125 ((125)I)-labeled FIAU was used in vitro and iodine 131 ((131)I)-labeled FIAU, in vivo. The (99m)Tc-labeled P2045 and (125)I- or (131)I-labeled FIAU were imaged simultaneously with different window settings with an Anger gamma camera. Treatment effects were tested with analysis of variance. RESULTS: Infected cells in culture trapped (125)I-labeled FIAU and (99m)Tc-labeled P2045; uptake correlated with the percentage of Ad-hSSTr2-TK-positive cells. For 100% of infected cells, 24% +/- 0.4 (mean +/- SD) of the added (99m)Tc-labeled P2045 was trapped, which is significantly lower (P <.05) than the 40% +/- 2 of (125)I-labeled FIAU that was trapped. For the highest Ad-hSSTr2-TK tumor dose (5 x 10(8) pfu), the uptake of (99m)Tc-labeled P2045 was 11.1% +/- 2.9 of injected dose per gram of tumor (thereafter, dose per gram), significantly higher (P <.05) than the uptake of (131)I-labeled FIAU at 1.6% +/- 0.4 dose per gram. (99m)Tc-labeled P2045 imaging consistently depicted hSSTr2 gene transfer in tumors at all adenovirus doses. Tumor uptake of (99m)Tc-labeled P2045 positively correlated with Ad-hSSTr2-TK dose; (131)I-labeled FIAU tumor uptake did not correlate with vector dose. CONCLUSION: The hSSTr2 and TK proteins were simultaneously imaged following dual gene transfer with an adenovirus vector.     

Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells.

Gene delivery efficiency in clinical cancer gene therapy trials with recombinant adenoviruses (Ads) based on serotype 5 (Ad5) has been limited partly because of variable expression of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human primary cancer cells. As a means of circumventing CAR deficiency, Ad vectors have been retargeted by creating chimeric fibers possessing knob domains of alternate Ad serotypes. In this study, we have constructed an Ad5-based vector, Ad5/3luc1, with a chimeric fiber protein featuring a knob domain derived from Ad3. This virus is retargeted to the Ad3 receptor and, therefore, has different tissue tropism. A novel knob binding assay was used to measure expression of CAR and the Ad3 receptor. Further, to evaluate the correlation of receptor expression and infectivity by Ad, a panel of ovarian cancer cell lines and purified primary cancer cells were infected with Ad5luc1 and Ad5/3luc1 at 50, 200, and 1000 viral particles/cell. Our results confirm that Ad5/3luc1 is retargeted to the Ad3 receptor. Furthermore, the Ad3 receptor is present at higher levels than CAR on ovarian adenocarcinoma cells. Also, the amount of binding to primary receptor appears to be the major factor determining the efficiency of transgene expression. The Ad5/3 chimera displays enhanced infectivity for ovarian cancer cell lines and purified primary tumor cells, which could translate into increased efficacy in clinical trials.     

Roentgenological and pathomorphological changes in the canine heart during 6-months hypokinesia

The effect of 6-month hypokinesia on the cardiac function and pathomorphological changes in 8 dogs was investigated. The heart size during systolic and diastolic contractions, stroke volume and contractile function were measured once a month, using an X-ray unit and a kymograph. After the hypokinetic exposure 6 dogs were sacrificed and their hearts were examined morphologically and histochemically. The recovery processes were investigated on 2 other dogs that were allowed to survive for 30 days after exposure. The 6-month hypokinesia led to a significant decrease in the heart size, stroke volume, cardiac index, and the contractile function. Post-mortem morphological examinations revealed atrophic changes in the myocardium. Electron-microscopy investigations demonstrated focal destructive changes in myofibers and in mitochondria: some of them were denser while others had a more transparent matrix and degraded crystae. Histochemical data (increased acid and alkaline phosphatase) also suggested atrophic and destructive changes in the myocardium. The above changes did not return to normal within 30 days of the recovery period.     

Blood-based screening and light based imaging for the early detection and monitoring of ovarian cancer xenografts

We report a novel technology for in vivo early detection, identification, and monitoring of ovarian cancer in live mice leading to better treatment outcome. A genetic dualistic reporter system that uses an adenoviral (Ad) vector to transfer the genetic reporters to the ovarian cancer is described. Infection of the cancer cells leads to expression of one reporter that is detected in blood, namely, secreted human placental alkaline phosphatase (SEAP). A second reporter, namely, enhanced green fluorescent protein (GFP) is also delivered by the Ad, leading to expression at the site of ovarian cancer. The SEAP gene under control of the cytomegalovirus (CMV) promoter element is linked to the GFP gene with an IRES element. A diagnostic adenoviral vector (Ad) encoding the SEAP and GFP (Ad5-SEAP-GFP) is produced. Efficacy of newly developed diagnostic vector is tested in cell culture and animal models. SKOV3ip.1 cells are infected with Ad5-SEAP-GFP. Over time the cells are monitored for fluorescence and SEAP is also measured in the growth media supernatant. For animal experiments, SKOV3ip.1 cells are implanted first in nude mice either subcutaneously (SC) or intraperitoneally (IP) separately. After 4-7 days, the Ad5-SEAP-GFP is administered. Control mice do not receive any Ad vector. All mice are imaged with a fluorescent stereomicroscope after 24 h, and blood is collected for SEAP analyses. Increasing green fluorescence is detected in all SKOV3ip.1 cells infected with Ad5-SEAP-GFP, while SEAP levels in growth media increase over monitoring period. Expression of GFP in both SC and IP tumors is detected by 24 h in the live mice. At this time, the SEAP blood levels are more than 2-3 fold greater than blood levels of control group. GFP fluorescence and SEAP levels continue to increase in all mice that are injected with Ad5-SEAP-GFP until termination. Control mice (both SC and IP) do not express GFP or SEAP throughout the experiment. GFP contrast is necessary to differentiate between micro-sized early stage non-palpable ovarian tumor nodules and surrounding normal tissue. While the studies are conducted in mice, it is envisioned that the dual-based approach will eventually be translated into human applications for routine diagnosis and monitoring of ovarian cancer when an ovarian cancer specific promoter will be available. Due to the thickness of the abdominal wall in human laparoscopy or laparotomy will be necessary. This system will provide gynecologic oncologists with a more effective tool for treating patients. The blood-based screening assay provides a quick test to determine the presence of the ovarian cancer at its earliest stage. The location of the ovarian cancer is afforded by the light-based imaging component, which represents a new and improving technology with tremendous advantages of sensitivity and spatial resolution to localize micro-sized tumor nodules. The novelty of the dualistic system is the linkage of blood-based reporter screening as a selection criteria for subsequent light-based imaging procedures. This combination will lead to an accurate and widely applicable method for the early detection and monitoring of ovarian cancer, especially in high-risk women     

Infectivity enhanced, cyclooxygenase-2 promoter-based conditionally replicative adenovirus for pancreatic cancer

BACKGROUND AND AIMS: Pancreatic cancer is one of the most aggressive human malignancies. Conditionally replicative adenoviruses (CRAds) have shown some promise in the treatment of cancers. However, to date, their application for pancreatic cancer has met several obstacles: one is lack of a good control element to regulate replication, and the other is relatively low adenoviral infectivity. Thus, we constructed infectivity enhanced cyclooxygenase (COX)-2 promoter-based CRAds to develop a safe and effective therapeutic modality. METHODS: The CRAds were designed to achieve COX-2 promoter-controlled E1 expression for regulated replication (COX-2 CRAds). The infectivity-enhanced CRAds also have an RGD-4C motif in the adenoviral fiber-knob region. The selectivity and efficacy of these constructs were analyzed with cell lines in vitro. The in vivo therapeutic effect and viral replication were analyzed with a xenograft model. Pathology of the major organs and E1 RNA levels in the liver were also studied after systemic administration. RESULTS: The COX-2 CRAds showed a selective cytocidal effect in vitro in COX-2-positive cells and killed most of the pancreatic cancer cells. In vivo, intratumoral administration of the infectivity-enhanced COX-2 CRAds (10(9) particles) showed a strong antitumor effect comparable to wild-type virus, whereas the COX-2 CRAds without infectivity enhancement showed a limited effect. Viral replication was confirmed in the xenograft tumors. Systemic administration did not cause any detectable toxicity; the E1 RNA level in the liver after COX-2 CRAd administration was minimal. CONCLUSIONS: Infectivity-enhanced COX-2 CRAd is a promising agent for the treatment of pancreatic cancer.     

Bioluminescence imaging reveals a significant role for complement in liver transduction following intravenous delivery of adenovirus

The effect of complement on transgene expression was evaluated in vivo and in vitro using mice lacking complement components. Complement component 3 (C3) deficient mice (C3-/-) and appropriate wild-type controls were intravenously injected with a replication incompetent, luciferase-expressing normal Ad5 (Ad5Luc1), or fibritin-fiber Ad5 (Ad5FFLuc1). Repeated, noninvasive bioluminescence imaging was conducted over 35 days. Our data show for the first time that C3 facilitates both short- and long-term hepatic expression of luciferase following systemic delivery. C3-/- mice showed significantly less (P < 0.05) luciferase expression in their liver than treatment-matched wild-type mice when 2.3 x 10(9) (Ad5Luc1) and 4.0 x 10(9) (Ad5Luc1 or Ad5FFLuc1) viral particles (v.p.) were infused. The maximal difference in luciferase activity between C3-/- and wild-type mice was 99-fold difference at 3 days for the 2.3 x 10(9) v.p. dose (Ad5Luc1), 35-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5Luc1), and 22-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5FFLuc1). Preincubation of Ad5Luc1 with wild-type, C1q-/-, or factor B (FB) deficient mouse sera for 5 min significantly (P < 0.05) increased transduction of mouse liver cells, as compared to preincubation with C3-/- sera or PBS. These results suggest the classical or alternate complement pathway enhances Ad5-mediated liver transduction.