Jens Eriksen

Efficiency of High- and Low-Voltage Pulse Combinations for Gene Electrotransfer in Muscle, Liver, Tumor, and Skin

Gene electrotransfer is gaining momentum as an efficient methodology for nonviral gene transfer. In skeletal muscle, data suggest that electric pulses play two roles: structurally permeabilizing the muscle fibers and electrophoretically supporting the migration of DNA toward or across the permeabilized membrane. To investigate this further, combinations of permeabilizing short high-voltage pulses (HV; hundreds of V/cm) and mainly electrophoretic long low-voltage pulses (LV; tens of V/cm) were investigated in muscle, liver, tumor, and skin in rodent models. The following observations were made: (1) Striking differences between the various tissues were found, likely related to cell size and tissue organization; (2) gene expression is increased, if there was a time interval between the HV pulse and the LV pulse; (3) the HV pulse was required for high electrotransfer to muscle, tumor, and skin, but not to liver; and (4) efficient gene electrotransfer was achieved with HV field strengths below the detectability thresholds for permeabilization; and (5) the lag time interval between the HV and LV pulses decreased sensitivity to the HV pulses, enabling a wider HV amplitude range. In conclusion, HV plus LV pulses represent an efficient and safe option for future clinical trials and we suggest recommendations for gene transfer to various types of tissues.

Gene expression profiles in skeletal muscle after gene electrotransfer

BackgroundGene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have therefore investigated transcriptional changes through gene expression profile analyses, morphological changes by histological analysis, and physiological changes by force generation measurements. DNA electrotransfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 μs) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment.ResultsDifferentially expressed genes were investigated by microarray analysis, and descriptive statistics were performed to evaluate the effects of 1) electroporation, 2) DNA injection, and 3) time after treatment. The biological significance of the results was assessed by gene annotation and supervised cluster analysis.Generally, electroporation caused down-regulation of structural proteins e.g. sarcospan and catalytic enzymes. Injection of DNA induced down-regulation of intracellular transport proteins e.g. sentrin. The effects on muscle fibres were transient as the expression profiles 3 weeks after treatment were closely related with the control muscles. Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration. Histological analysis revealed structural changes with loss of cell integrity and striation pattern in some fibres after DNA+HV+LV treatment, while HV+LV pulses alone showed preservation of cell integrity. No difference in the force generation capacity was observed in the muscles 2 weeks after DNA electrotransfer.ConclusionThe small and transient changes found in the gene expression profiles are of great importance, as this demonstrates that DNA electrotransfer is safe with minor effects on the muscle host cells. These findings are essential for introducing the DNA electrotransfer to muscle for clinical use. Indeed the HV+LV pulse combination used has been optimised to ensure highly efficient and safe DNA electrotransfer.

Duration and level of transgene expression after gene electrotransfer to skin in mice

In development of novel vaccines, attention is drawn to DNA vaccinations. They are heat stable and can be easily produced. Gene electrotransfer is a simple and nonviral means of transferring DNA to cells and tissues and is attracting increasing interest. One very interesting perspective with gene electrotransfer is that choice of tissue can determine the duration of transgene expression. With gene electrotransfer to muscle, long-term expression, that is beyond 1 year, can be obtained, whereas gene electrotransfer to skin gives short-term expression, which is desirable in, for example, DNA vaccinations. Level and duration of transgene expression after gene electrotransfer to skin is essential and here we present data from two independent quantitative studies. Using in vivo bioimaging of a far-red fluorescent molecule, Katushka, allowing for continuous monitoring of local gene expression, compared with measurements of a systemic transgene, that is, serum erythropoietin (EPO) after gene electrotransfer with EPO to skin, we found a significant increase in transgene expression (P< 0.01) with a peak 9 days (Katushka) and 14 days (EPO) after transfection. Duration of expression could be 3–4 weeks, which is a suitable time frame for vaccinations and is applicable, for example, in gene therapy for wound healing or treatment of cancer.

Calcium electroporation in three cell lines: a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions.

BACKGROUNDnElectroporation with calcium (calcium electroporation) can induce ATP depletion-associated cellular death. In the clinical setting, the cytotoxic drug bleomycin is currently used with electroporation (electrochemotherapy) for palliative treatment of tumors. Calcium electroporation offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions, as is the case with cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing calcium electroporation and electrochemotherapy.nnnMETHODSnThe effects of calcium electroporation and bleomycin electroporation (alone or in combination) were compared in three different cell lines (DC-3F, transformed Chinese hamster lung fibroblast; K-562, human leukemia; and murine Lewis Lung Carcinoma). Furthermore, the effects of electrical pulsing parameters and calcium compound on treatment efficacy were determined.nnnRESULTSnElectroporation with either calcium or bleomycin significantly reduced cell survival (p<0.0001), without evidence of a synergistic effect. Cellular death following calcium or bleomycin treatment occurred at similar applied voltages, suggesting that similar parameters should be applied. At equimolar concentrations, calcium chloride and calcium glubionate resulted in comparable decreases in cell viability.nnnCONCLUSIONSnCalcium electroporation and bleomycin electroporation significantly reduce cell survival at similar applied voltage parameters. The effect of calcium electroporation is independent of calcium compound.nnnGENERAL SIGNIFICANCEnThis study strongly supports the use of calcium electroporation as a potential cancer therapy and the results may aid in future clinical trials.

No differences in short-term morbidity and mortality after robot-assisted laparoscopic versus laparoscopic resection for colonic cancer: a case–control study of 263 patients

BackgroundRobot-assisted laparoscopy has been reported to be a safe and feasible alternative to traditional laparoscopy. The aim of this study was to compare short-term results in patients with colonic cancer who underwent robot-assisted laparoscopic colonic resection (RC) or laparoscopic colonic resection (LC).MethodsThe study was a retrospective case control study of all patients with colonic cancer who underwent RC from March 2010 to March 2012 or LC from January 2009 to December 2011 at a tertiary-care university hospital. Data were retrieved from the national chart database and patient journals. Biochemical markers [C-reactive protein (CRP), hemoglobin, white blood cell count, and thrombocyte count] were recorded before surgery and for the first 3xa0days after surgery.ResultsA total of 101 patients underwent RC and 162 patients underwent LC. There were no significant differences in the rate of conversion to open surgery, number of permanent enterostomies, number of intraoperative complications, level of postoperative cellular stress response, number of postoperative complications, length of postoperative hospital stay, or 30-day mortality between the two groups. There was a significantly longer setup time for RC (77.1 vs. 69.7xa0min, Pxa0=xa00.000), but surgical time was significantly shorter for RC (165.8 vs. 183.4xa0min, Pxa0=xa00.006) and there was no difference in the overall procedure time (254.0 vs. 243.6xa0min, Pxa0=xa00.086).ConclusionWe found RC to be a safe and feasible alternative to LC for colonic cancer. We found that for RC surgical time was shorter and overall procedure time was comparable to that for LC; however, these results should be confirmed in future randomized clinical trials.

Tet-On Induction with Doxycycline after Gene Transfer in Mice: Sweetening of Drinking Water is not a Good Idea

Gene transfer to skeletal muscle leads to long-term, stable expression of transferred genes. An exiting development is the use of inducible expression systems. Using the inducible Tet-On system, it has been customary to administer doxycycline in drinking water with added sucrose to ameliorate the bitter taste. During a study aiming at regulating electrotransferred genes through the Tet-On system, we observed excessive drinking behavior among mice. Removal of sugar from the drinking water led to normal drinking behavior and most importantly did not affect the level of gene expression. Based on this study, the practice of adding sucrose to drinking water in doxycycline induction studies should be abandoned.

In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.

Reply to: Re: No differences in short-term morbidity and mortality after robot-assisted laparoscopic versus laparoscopic resection for colonic cancer: A case-control study of 263 patients (Surg Endosc 2013)

We thank Dr. Colantonio for his comments. We agree that there is an overlap in the literature where ‘‘case-control’’ is inappropriately used in studies where the more correct term would be ‘‘retrospective cohort’’ study. We thank Dr. Colantonio for bringing this to attention. We also agree with Dr. Colantonio that an obvious limitation of retrospective study designs such as ours is the difference in risk factors between the observed groups. However, when looking at relatively new techniques (such as robot-assisted colectomy for colon cancer), the small number of surgical cases can become an obstacle for the use of analytic methods, such as stratification and matching. We agree that restriction with regard to surgeon experience would have been preferable, but because our hospital is a teaching hospital, a large part of the laparoscopic colectomies are performed by junior surgeons under supervision. Restriction would have entailed a significant risk of selection bias in favor of the robotic colectomies, because one can assume that the patients selected for laparoscopic operation by a senior surgeon would have more advanced cancers, older age, and poorer health status with regard to comorbidity. In contrast, all patients were eligible for robotic surgery and were selected for robotic surgery on the basis of robot and surgeon availability. We addressed these limitations in our discussion, and we emphasized that our outcomes, including the shorter surgical time in the robot-assisted laparoscopic colectomy group, could indeed be affected by them. Our study concluded that ‘‘robot-assisted laparoscopic colonic resection is a safe and feasible alternative to traditional laparoscopic resection for colonic cancer.’’ We do not dissuade surgeons from using robot-assisted laparoscopy for colonic resection because we believe that randomized clinical trials of robotassisted laparoscopic colectomy versus laparoscopic colectomy, larger than those currently available, could yet prove that the superior technical possibilities of robotassisted surgery will translate into improved functional and oncological outcomes in patients with colon cancer.

The anaesthesiologist and chronic pain

THE 3RD International Copenhagen Symposium on Chronic Pain was held in January 2001 with the title ‘‘Pain in cancer and non-cancer conditions – similarities and differences’’. The symposium attempted to look at the controversies and dilemmas anaesthesiologists and others working with chronic pain patients have to face in the forthcoming years. In keeping with tradition, each of the distinguished speakers of the symposium has submitted an article, which is published in the present issue of Acta Anaesthesiologica Scandinavica. This collection of well-written papers from leading pain physicians and scientists depicts the multitudinous complexity of chronic pain and emphasizes the challenges and demands pain physicians have to meet. If chronic pain is to be managed successfully, knowledge not only about the physical components of the pain condition but also about the psychological/ psychiatric, socio-economic and existential/spiritual aspects is necessary. In recent years comprehensive activities to create research and treatment facilities for pain management have taken place. However, due to this very development, a subdivision of the area of pain management is rapidly emerging. The most conspicuous development is the increasing tendency to separate acute and chronic pain. Acute pain (postoperative pain) in the hospital setting is in more advanced units handled as a part of a multidisciplinary perioperative subspeciality comprising not only pain but also mobilization, nutrition etc. A subspeciality dealing with perioperative medicine may readily be established in units in which a multidisciplinary staff consisting of anaesthesiologists, surgeons, physiotherapists and others are working together. Patients with chronic pain of non-malignant origin have to be treated and cared for in specialized, multidisciplinary staffed departments, realizing that such settings are mandatory in order to obtain a reasonable treatment outcome in these often heavily stigmatized

Short- and long-term outcomes after colorectal anastomotic leakage is affected by surgical approach at reoperation

BackgroundAnastomotic leakage is the most serious surgical complication following colorectal resection, and surgical intervention is often required. The purpose of the study was to investigate short- and long-term outcomes after reoperation for anastomotic leakage.MethodPatients with a symptomatic anastomotic leakage following a laparoscopic colorectal cancer resection from January 2009 to December 2014 were identified from our local prospective database. Patients were grouped according to the management of anastomotic leaks: local, lap, or open approach. Primary outcomes were length of stay, chance of bowel continuity, and overall mortality.ResultsA total of 113 patients were included. The median follow-up time was 40xa0months (0–82xa0months). Overall mortality was significantly associated with UICC stage III–VI disease (vs. UICC stage I–II disease) [adj. HR 5.35 (CI 2.32–12.4), pu2009=u20090.0001] and minimal invasive reoperation compared with open approach [local: adj. HR 0.12 (CI 0.03–0.52), pu2009=u20090.004; lap: adj. HR 0.32 (CI 0.12–0.86), pu2009=u20090.024]. Chance of bowel continuity was significantly increased in younger patients below 67xa0years [adj. OR 6.15 (1.76–21.5), pu2009=u20090.004] and following a local procedure [adj. OR 7.45 (1.07–51.8), pu2009=u20090.043]. Patients in the open group had significantly longer length of stay and time to initiation of adjuvant chemotherapy compared with those in the lap group.ConclusionOur data confirms that minimal invasive reoperation for anastomotic leakage is a safe and feasible approach associated with short- and long-term advantages and can be chosen in selected cases.