Intra-tumoral gene delivery of feIL-2, feIFN-gamma and feGM-CSF using magnetofection as a neoadjuvant treatment option for feline fibrosarcomas: a phase-I study.

Despite aggressive pre- or postoperative treatment, feline fibrosarcomas have a high relapse rate. In this study, a new treatment option based on immune stimulation by intra-tumoral delivery of three feline cytokine genes was performed. The objective of this phase-I dose-escalation study was to determine a safe dose for further evaluation in a subsequent phase-II trial. Twenty-five client-owned cats with clinical diagnosis of fibrosarcoma - primary tumours as well as recurrences - entered the study. Four increasing doses of plasmids coding for feIL-2, feIFN-gamma or feGM-CSF, respectively, were previously defined. In groups I, II, III and IV these doses were 15, 50, 150 and 450 microg per plasmid and a corresponding amount of magnetic nanoparticles. Two preoperative intra-tumoral injections of the magnetic DNA solution were followed by magnetofection. A group of four control cats received only surgical treatment. Side effects were registered and graded according to the VCOG-CTCAE scale and correlated to treatment. Statistical analyses included one-way anova, post hoc and Kruskal-Wallis tests. ELISA tests detecting plasma feIFN-gamma and plasma feGM-CSF were performed. One cat out of group IV (450 microg per plasmid) showed adverse events probably related to gene delivery. As these side effects were self-limiting and occurred only in one of eight cats in group IV, this dose was determined to be well tolerable. Altogether six cats developed local recurrences during a 1-year observation period. Four of these cats had been treated with dose IV. Regarding these observations, a subsequent phase-II trial including a representative amount of cats should be tested for the efficacy of dose IV as well as dose III.

Allogeneic retrovirally transduced, IL-2- and IFN-gamma-secreting cancer cell vaccine in patients with hormone refractory prostate cancer--a phase I clinical trial.

BACKGROUND: The purpose of this vaccine study was to determine the safety and feasibility of vaccination with an allogeneic prostate carcinoma cell line, LNCaP, expressing recombinant interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) and to evaluate the efficacy of inducing tumor-specific immune responses in HLA-A2-matched patients with hormone refractory prostate cancer (HRPC). METHODS: In a dose-escalating phase I study, HLA-A2-matched HRPC patients received four vaccinations of irradiated allogeneic LNCaP cells retrovirally transduced to secrete IL-2 and IFN-gamma at study day 1, 15, 29 and 92 and subsequently every 91 days unless tumor progression was evident. RESULTS: Three patients receiving the first dose level (7.5 million cells) showed no evidence of dose-limiting toxicity or vaccine-related adverse events including autoimmunity. One of three patients receiving the second dose level (15 million cells) developed a transient self-limiting grade 3 local injection site reaction (ulceration) after the eighth vaccination. Vaccine-induced immune responses against a broad array of prostate tumor associated antigens were detected in all six patients. Two of the three patients receiving the higher dose showed a decline in serum prostate-specific antigen (PSA) values of more than 50%, with one patient remaining on protocol for 3 years. CONCLUSIONS: Immunisation with the allogeneic LNCaP/IL-2/IFN-gamma vaccine is safe and feasible without any dose-limiting toxicity or autoimmunity. A 50% PSA decline was achieved in two of the six patients. This encouraging data provides the scientific rationale for further investigation of the vaccine in a phase II trial.

Prediction of flap necrosis with laser induced indocyanine green fluorescence in a rat model.

Prediction of necrosis has a clinical relevance in all fields of plastic surgery. The new application of indocyanine green (ICG) fluoroscopy in plastic surgery allows an objective quantification of skin perfusion and a high topographical resolution. The aim of the present study is to determine threshold values for flap perfusion under well-defined experimental conditions. Twenty random pattern flaps with a length to width ratio of 4:1 (8 x 2 cm(2)) were dissected on the anterior abdominal wall of 20 male Sprague-Dawley rats. ICG fluoroscopy was performed at the end of the operation. The animals were sacrificed at the seventh postoperative day with a reliable necrosis of the distal part of the flaps. Postoperative ICG fluoroscopy then was analysed both in regions that will survive and undergo necrosis. At day 7 a mean area of 5.5 cm(2) (57% of the total flap area) survived and a mean of 3.8 cm(2) (43%) became necrotic. The surviving part of the flap had a mean perfusion index of 62% compared to reference skin. The distal parts of the flap that necrotised showed an average perfusion index of only 19% postoperatively. Differences were statistically highly significant (p<0.001). Indocyanine green fluoroscopy is a useful tool to evaluate perfusion topographically and predict necrosis. From a statistical point of view a perfusion index of less than 25% of the reference skin can be considered as a sign of developing flap necrosis.

Nerve replacement strategies for cavernous nerves.

OBJECTIVE: This article reviews novel restorative therapies for cavernous nerves that may be used to replace resected cavernous nerves at the time of pelvic surgery. METHODS: A literature-based presentation (Medline search) on current nerve replacement strategies was conducted with emphasis on neurobiological factors contributing to the restoration of erectile function after cavernous nerve injuries. RESULTS: A promising alternative to autologous nerve grafts for extending the length of successful nerve regeneration are artificial nerve guides. The addition of neurotrophic factors, extracellular matrix components and Schwann cells has been shown to promote cavernous nerve regeneration. Neurotrophic factors can be incorporated in the scaffold or can be supplied by cells seeded into the stroma. The regenerative capacity of these cells can be further enhanced by genetic modification with neurotrophic factor encoding genes. CONCLUSIONS: Artificial nerve guides, especially biodegradable ones containing growth-promoting factors or cells, are a promising option for the repair of cavernous nerve lesions.

Uptake of radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil in cardiac cells after adenoviral transfer of the herpesvirus thymidine kinase gene: the cellular basis for cardiac gene imaging.

BACKGROUND: Gene therapy is a promising approach for the treatment of cardiac diseases. Coexpression of therapeutic genes with a suitable marker gene would allow for the noninvasive imaging of successful gene transfer and expression via radiolabeled marker substrates. In the present study, such an approach was first applied to cardiac tissue. METHODS AND RESULTS: The combination of the herpesvirus thymidine kinase reporter gene (HSV1-tk) and radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil (FIAU) was evaluated. H9c2 rat cardiomyoblasts were infected in vitro with a replication-defective HSV1-tk-containing adenovirus and a negative control virus. The intracellular uptake of [(14)C]FIAU increased with increasing multiplicity of infection and with time after infection. Uptake in negative controls remained <15% of positive controls. Additionally, vectors were applied intramyocardially in Wistar rats. The marker substrate [(125)I]FIAU was injected intravenously 3 days later, and animals were killed after 24 hours. Autoradiographically, regional transgene expression was clearly identified in animals receiving the adenovirus containing HSV1-tk (3. 4+/-2.2-fold increase of radioactivity at vector administration site compared with remote myocardium), whereas nonspecific uptake in negative controls was low (<10% of positive controls). CONCLUSIONS: Using an adenoviral vector, HSV1-tk can be successfully expressed in cardiac cells in vitro and in vivo, yielding high uptake of radiolabeled FIAU. The results suggest that imaging transgene expression in the heart is feasible and may be used to monitor gene therapy noninvasively.

Enhanced cardiac contractility after gene transfer of V2 vasopressin receptors In vivo by ultrasound-guided injection or transcoronary delivery.

BACKGROUND: Systemic levels of arginine vasopressin (AVP) are increased in congestive heart failure, resulting in vasoconstriction and reduced cardiac contractility via V(1) vasopressin receptors. V(2) vasopressin receptors (V2Rs), which promote activation of adenylyl cyclase, are physiologically expressed only in the kidney and are absent in the myocardium. Heterologous expression of V2Rs in the myocardium could result in a positive inotropic effect by using the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: We tested gene transfer with a recombinant adenovirus for the human V2R (Ad-V2R) to stimulate contractility of rat or rabbit myocardium in vivo. Ultrasound-guided direct injection or transcoronary delivery of adenovirus in vivo resulted in recombinant receptor expression in the myocardial target area, leading to a substantial increase in [(3)H]AVP binding. In 50% of the cardiomyocytes isolated from the directly injected area, single-cell shortening measurements detected a significant increase in contraction amplitude after exposure to AVP or the V2R-specific desmopressin (DDAVP). Echocardiography of the target myocardial area documented a marked increase in local fractional shortening after systemic administration of DDAVP in V2R-expressing animals but not in control virus-treated hearts. Simultaneous measurement of global contractility (dP/dt(max)) confirmed a positive inotropic effect of DDAVP on left ventricular function in the Ad-V2R-injected animals. CONCLUSIONS: Adenoviral gene transfer of the V2R into the myocardium increases cardiac contractility in vivo. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in the therapy of congestive heart failure by bypassing the desensitized beta-adrenergic receptor-signaling cascade.

[Isoflurane anesthesia in rabbits in a closed anesthetic system]. / Zur Isoflurannarkose beim Kaninchen im geschlossenen Narkosesystem.

Using the Stephens anaesthetic apparatus-which is a closed system with an in-circuit, nonprecision vaporizer-and isoflurane as anaesthetic gas, 18 rabbits were anaesthetized and showed sufficient hypnosis, analgesia, and muscle relaxation during bone surgery. Induction of anaesthesia was achieved with intravenous propofol and all rabbits were intubated afterwards. During the following isoflurane inhalation anaesthesia the mean arterial blood pressure decreased considerably (compared to control measures before induction), the heart rate remained the same or showed a slight increase, and the respiratory rate decreased. The arterial pO2 decreased corresponding to the respiratory depression after propofol induction and increased again during spontaneous ventilation with 100% oxygen. The changes in arterial pCO2 and pH were representative for a rise in the CO2-stimulation threshold. A moderate metabolic acidosis could be seen due to preanaesthetic excitement of the animals. Recovery time was short (between one and 11 minutes) and no signs of excitation could be detected. The consumed volume of isoflurane was 0.80 ml/kg BW/h.

[CO2/O2 anesthesia for the castration of male piglets (preliminary results)]. / Die CO2/O2-Anästhesie zur Kastration von männlichen Ferkeln (vorläufige Ergebnisse).

Introduction of anaesthesia with CO2/O2 (60% to 40%) is possible within 90 and 120 seconds. There are moderate to excessive excitations occurring as part of state II of anaesthesia. Anaesthesia (during castration) in CO2/O2-atmosphere produces excellent analgesia and relaxation. The duration of castration surgery is much shorter under CO2-anaesthesia than without anaesthesia. Blood cortisol levels are significantly higher after castration without CO2-anaesthesia. About 5 minutes after CO2/O2-anaesthesia and castration surgery, piglets are already awake and standing.