Particle Accelerators and Detectors for medical Diagnostics and Therapy

This Habilitationsschrift (Habilitation thesis) is focused on my research activities on medical applications of particle physics and was written in 2013 to obtain the Venia Docendi (Habilitation) in experimental physics at the University of Bern. It is based on selected publications, which represented at that time my major scientific contributions as an experimental physicist to the field of particle accelerators and detectors applied to medical diagnostics and therapy. The thesis is structured in two parts. In Part I, Chapter 1 presents an introduction to accelerators and detectors applied to medicine, with particular focus on cancer hadrontherapy and on the production of radioactive isotopes. In Chapter 2, my publications on medical particle accelerators are introduced and put into their perspective. In particular, high frequency linear accelerators for hadrontherapy are discussed together with the new Bern cyclotron laboratory. Chapter 3 is dedicated to particle detectors with particular emphasis on three instruments I contributed to propose and develop: segmented ionization chambers for hadrontherapy, a proton radiography apparatus with nuclear emulsion films, and a beam monitor detector for ion beams based on doped silica fibres. Selected research and review papers are contained in Part II. For copyright reasons, they are only listed and not reprinted in this on-line version. They are available on the websites of the journals.

Long-term ticagrelor monotherapy versus standard dual antiplatelet therapy followed by aspirin monotherapy in patients undergoing biolimus-eluting stent implantation: rationale and design of the GLOBAL LEADERS trial

AIMS The GLOBAL LEADERS trial is a superiority study in patients undergoing percutaneous coronary intervention, with a uniform use of Biolimus A9-eluting stents (BES) and bivalirudin. GLOBAL LEADERS was designed to assess whether a 24-month antithrombotic regimen with ticagrelor and one month of acetylsalicylic acid (ASA), compared to conventional dual antiplatelet therapy (DAPT), improves outcomes. METHODS AND RESULTS Patients (n >16,000) are randomised (1:1 ratio) to ticagrelor 90 mg twice daily for 24 months plus ASA ≤100 mg for one month versus DAPT with either ticagrelor (acute coronary syndrome) or clopidogrel (stable coronary artery disease) for 12 months plus ASA ≤100 mg for 24 months. The primary outcome is a composite of all-cause mortality or non-fatal, new Q-wave myocardial infarction at 24 months. The key safety endpoint is investigator-reported class 3 or 5 bleeding according to the Bleeding Academic Research Consortium (BARC) definitions. Sensitivity analysis will be carried out to explore potential differences in outcome across geographic regions and according to specific angiographic and clinical risk estimates. CONCLUSIONS The GLOBAL LEADERS trial aims to assess the role of ticagrelor as a single antiplatelet agent after a short course of DAPT for the long-term prevention of cardiac adverse events, across a wide spectrum of patients, following BES implantation.

Encouragement to Increase the Use of Psychosocial Skills in the Diagnosis and Therapy of Patients With Functional Dysphonia.

Clinicians believe that psychosocial factors play a causal role in the etiology of many forms of functional dysphonia (FD). But for decades, all attempts to confirm such causation have failed. This paper aims to show the logic of this failure, to discuss the possibilities of employing psychology in therapy nonetheless, and to encourage clinicians to use their psychosocial knowledge and skills. The failure to confirm psychic and social factors as causal in the etiology of FD is basically a consequence of a principal shortcoming of evidence-based medicine (EBM). As the gold standard for validity, reliability, and objectivity in medical research, EBM is based on calculability and hence the processing of quantitative data. But life paths and life situations are best or sometimes only expressible in qualitative, experiential, and idiographic terms. Thus EBM-guided evaluation undervalues most psychosocial studies. This report of an experienced multidisciplinary voice team proposes alternative pathways for integrating psychosocial knowledge into the diagnosis and the treatment of FD. The difference between the fields of activity of psychotherapists and speech-language pathologists is discussed, and the latter group is shown the potential benefits of using more of their psychosocial knowledge and skills.

Protrusio acetabuli: joint loading with severe pincer impingement and its theoretical implications for surgical therapy

Severe pincer impingement (acetabular protrusio) is an established cause of hip pain and osteoarthritis. The proposed underlying pathomechanism is a dynamic pathological contact of the prominent acetabular rim with the femoral head-neck junction. However, this cannot explain the classically described medial osteoarthritis in these hips. We therefore asked: (1) Does an overload exist in the medial aspect of the protrusio joint? and (2) What is the influence of three contemporary joint-preserving procedures on load distribution in protrusio hips? In vivo force and motion data for walking and standing to sitting were applied to six 3D finite element models (normal, dysplasia, protrusio, acetabular rim trimming, acetabular reorientation, and combined reorientation/rim trimming). Compared with dysplasia, the protrusio joint resulted in opposite patterns of von Mises stress and contact pressure during walking. In protrusio hips, we found an overload at the medial margin of the lunate surface (54% higher than normal). Isolated rim trimming further increased the medial overload (up to 28% higher than protrusio), whereas acetabular reorientation with/without rim trimming reduced stresses by up to 25%. Our results can be used as an adjunct for surgical decision making in the treatment of acetabular protrusio.

Implementation and Operational Research: Risk Charts to Guide Targeted HIV-1 Viral Load Monitoring of ART: Development and Validation in Patients From Resource-Limited Settings.

BACKGROUND HIV-1 RNA viral load (VL) testing is recommended to monitor antiretroviral therapy (ART) but not available in many resource-limited settings. We developed and validated CD4-based risk charts to guide targeted VL testing. METHODS We modeled the probability of virologic failure up to 5 years of ART based on current and baseline CD4 counts, developed decision rules for targeted VL testing of 10%, 20%, or 40% of patients in 7 cohorts of patients starting ART in South Africa, and plotted cutoffs for VL testing on colour-coded risk charts. We assessed the accuracy of risk chart-guided VL testing to detect virologic failure in validation cohorts from South Africa, Zambia, and the Asia-Pacific. RESULTS In total, 31,450 adult patients were included in the derivation and 25,294 patients in the validation cohorts. Positive predictive values increased with the percentage of patients tested: from 79% (10% tested) to 98% (40% tested) in the South African cohort, from 64% to 93% in the Zambian cohort, and from 73% to 96% in the Asia-Pacific cohort. Corresponding increases in sensitivity were from 35% to 68% in South Africa, from 55% to 82% in Zambia, and from 37% to 71% in Asia-Pacific. The area under the receiver operating curve increased from 0.75 to 0.91 in South Africa, from 0.76 to 0.91 in Zambia, and from 0.77 to 0.92 in Asia-Pacific. CONCLUSIONS CD4-based risk charts with optimal cutoffs for targeted VL testing maybe useful to monitor ART in settings where VL capacity is limited.

Association of hormone therapy and incident gout: population-based case-control study

OBJECTIVE This study aims to assess the odds of developing incident gout in association with the use of postmenopausal estrogen-progestogen therapy, according to type, timing, duration, and route of administration of estrogen-progestogen therapy. METHODS We conducted a retrospective population-based case-control analysis using the United Kingdom-based Clinical Practice Research Datalink. We identified women (aged 45 y or older) who had a first-time diagnosis of gout recorded between 1990 and 2010. We matched one female control with each case on age, general practice, calendar time, and years of active history in the database. We used multivariate conditional logistic regression to calculate odds ratios (ORs) with 95% CIs (adjusted for confounders). RESULTS The adjusted OR for gout with current use of oral formulations of opposed estrogens (estrogen-progestogen) was 0.69 (95% CI, 0.56-0.86) compared with never use. Current use was associated with a decreased OR for gout in women without renal failure (adjusted OR, 0.71; 95% CI, 0.57-0.87) and hypertension (adjusted OR, 0.62; 95% CI, 0.44-0.87) compared with never use. Tibolone was associated with a decreased OR for gout (adjusted OR, 0.77; 95% CI, 0.63-0.95) compared with never use. Estrogens alone did not alter the OR for gout. CONCLUSIONS Current use of oral opposed estrogens, but not unopposed estrogens, is associated with a decreased OR for incident gout in women without renal failure and is more pronounced in women with hypertension. Use of tibolone is associated with a decreased OR for incident gout. The decreased OR for gout may be related to the progestogen component rather than the estrogen component.

Everolimus-Eluting Bioresorbable Vascular Scaffold System in the Treatment of Cardiac Allograft Vasculopathy: the CART (Cardiac Allograft Reparative Therapy) Prospective Multicenter Pilot Study.

Cardiac allograft vasculopathy (CAV) is a form of accelerated atherosclerosis, which represents the leading cause of late morbidity and mortality after heart transplantation. The recent bioresorbable vascular scaffold (BVS) technology represents a potential novel therapeutic tool, in the context of CAV, by allowing transient scaffolding and concomitant vessel healing. Eligible subjects will be treated by using the Absorb Everolimus-Eluting BVS (Abbott Vascular, Santa Clara, CA, USA), and evaluated at pre-determined time points, up to 3 years since the index procedure. Both clinical and imaging data will be collected in dedicated case report forms (CRF). All imaging data will be analyzed in an independent core laboratory. The primary aim of the study is to evaluate the angiographic performance at 1 year of second-generation Absorb BVS, in heart transplant recipients affected by CAV.

Interventions for hirsutism (excluding laser and photoepilation therapy alone)

BACKGROUND Hirsutism occurs in 5% to 10% of women of reproductive age when there is excessive terminal hair growth in androgen-sensitive areas (male pattern). It is a distressing disorder with a major impact on quality of life. The most common cause is polycystic ovary syndrome. There are many treatment options, but it is not clear which are most effective. OBJECTIVES To assess the effects of interventions (except laser and light-based therapies alone) for hirsutism. SEARCH METHODS We searched the Cochrane Skin Group Specialised Register, CENTRAL (2014, Issue 6), MEDLINE (from 1946), EMBASE (from 1974), and five trials registers, and checked reference lists of included studies for additional trials. The last search was in June 2014. SELECTION CRITERIA Randomised controlled trials (RCTs) in hirsute women with polycystic ovary syndrome, idiopathic hirsutism, or idiopathic hyperandrogenism. DATA COLLECTION AND ANALYSIS Two independent authors carried out study selection, data extraction, 'Risk of bias' assessment, and analyses. MAIN RESULTS We included 157 studies (sample size 30 to 80) comprising 10,550 women (mean age 25 years). The majority of studies (123/157) were 'high', 30 'unclear', and four 'low' risk of bias. Lack of blinding was the most frequent source of bias. Treatment duration was six to 12 months. Forty-eight studies provided no usable or retrievable data, i.e. lack of separate data for hirsute women, conference proceedings, and losses to follow-up above 40%.Primary outcomes, 'participant-reported improvement of hirsutism' and 'change in health-related quality of life', were addressed in few studies, and adverse events in only half. In most comparisons there was insufficient evidence to determine if the number of reported adverse events differed. These included known adverse events: gastrointestinal discomfort, breast tenderness, reduced libido, dry skin (flutamide and finasteride); irregular bleeding (spironolactone); nausea, diarrhoea, bloating (metformin); hot flushes, decreased libido, vaginal dryness, headaches (gonadotropin-releasing hormone (GnRH) analogues)).Clinician's evaluation of hirsutism and change in androgen levels were addressed in most comparisons, change in body mass index (BMI) and improvement of other clinical signs of hyperandrogenism in one-third of studies.The quality of evidence was moderate to very low for most outcomes.There was low quality evidence for the effect of two oral contraceptive pills (OCPs) (ethinyl estradiol + cyproterone acetate versus ethinyl estradiol + desogestrel) on change from baseline of Ferriman-Gallwey scores. The mean difference (MD) was -1.84 (95% confidence interval (CI) -3.86 to 0.18).There was very low quality evidence that flutamide 250 mg, twice daily, reduced Ferriman-Gallwey scores more effectively than placebo (MD -7.60, 95% CI -10.53 to -4.67 and MD -7.20, 95% CI -10.15 to -4.25). Participants' evaluations in one study with 20 participants confirmed these results (risk ratio (RR) 17.00, 95% CI 1.11 to 259.87).Spironolactone 100 mg daily was more effective than placebo in reducing Ferriman-Gallwey scores (MD -7.69, 95% CI -10.12 to -5.26) (low quality evidence). It showed similar effectiveness to flutamide in two studies (MD -1.90, 95% CI -5.01 to 1.21 and MD 0.49, 95% CI -1.99 to 2.97) (very low quality evidence), as well as to finasteride in two studies (MD 1.49, 95% CI -0.58 to 3.56 and MD 0.40, 95% CI -1.18 to 1.98) (low quality evidence).Although there was very low quality evidence of a difference in reduction of Ferriman-Gallwey scores for finasteride 5 mg to 7.5 mg daily versus placebo (MD -5.73, 95% CI -6.87 to -4.58), it was unlikely it was clinically meaningful. These results were reinforced by participants' assessments (RR 2.06, 95% CI 0.99 to 4.29 and RR 11.00, 95% CI 0.69 to 175.86). However, finasteride showed inconsistent results in comparisons with other treatments, and no firm conclusions could be reached.Metformin demonstrated no benefit over placebo in reduction of Ferriman-Gallwey scores (MD 0.05, 95% CI -1.02 to 1.12), but the quality of evidence was low. Results regarding the effectiveness of GnRH analogues were inconsistent, varying from minimal to important improvements.We were unable to pool data for OCPs with cyproterone acetate 20 mg to 100 mg due to clinical and methodological heterogeneity between studies. However, addition of cyproterone acetate to OCPs provided greater reductions in Ferriman-Gallwey scores.Two studies, comparing finasteride 5 mg and spironolactone 100 mg, did not show differences in participant assessments and reduction of Ferriman-Gallwey scores (low quality evidence). Ferriman-Gallwey scores from three studies comparing flutamide versus metformin could not be pooled (I² = 62%). One study comparing flutamide 250 mg twice daily with metformin 850 mg twice daily for 12 months, which reached a higher cumulative dosage than two other studies evaluating this comparison, showed flutamide to be more effective (MD -6.30, 95% CI -9.83 to -2.77) (very low quality evidence). Data showing reductions in Ferriman-Gallwey scores could not be pooled for four studies comparing finasteride with flutamide as the results were inconsistent (I² = 67%).Studies examining effects of hypocaloric diets reported reductions in BMI, but which did not result in reductions in Ferriman-Gallwey scores. Although certain cosmetic measures are commonly used, we did not identify any relevant RCTs. AUTHORS' CONCLUSIONS Treatments may need to incorporate pharmacological therapies, cosmetic procedures, and psychological support. For mild hirsutism there is evidence of limited quality that OCPs are effective. Flutamide 250 mg twice daily and spironolactone 100 mg daily appeared to be effective and safe, albeit the evidence was low to very low quality. Finasteride 5 mg daily showed inconsistent results in different comparisons, therefore no firm conclusions can be made. As the side effects of antiandrogens and finasteride are well known, these should be accounted for in any clinical decision-making. There was low quality evidence that metformin was ineffective for hirsutism and although GnRH analogues showed inconsistent results in reducing hirsutism they do have significant side effects.Further research should consist of well-designed, rigorously reported, head-to-head trials examining OCPs combined with antiandrogens or 5α-reductase inhibitor against OCP monotherapy, as well as the different antiandrogens and 5α-reductase inhibitors against each other. Outcomes should be based on standardised scales of participants' assessment of treatment efficacy, with a greater emphasis on change in quality of life as a result of treatment.

Retention in care during the first 3 years of antiretroviral therapy for women in Malawi's option B+ programme: an observational cohort study

Background Studies of Malawi's option B+ programme for HIV-positive pregnant and breastfeeding women have reported high loss to follow-up during pregnancy and at the start of antiretroviral therapy (ART), but few data exist about retention during breastfeeding and after weaning. We examined loss to follow-up and retention in care in patients in the option B+ programme during their first 3 years on ART. Methods We analysed two data sources: aggregated facility-level data about patients in option B+ who started ART between Oct 1, 2011, and June 30, 2012, at 546 health facilities; and patient-level data from 20 large facilities with electronic medical record system for HIV-positive women who started ART between Sept 1, 2011, and Dec 31, 2013, under option B+ or because they had WHO clinical stages 3 or 4 disease or had CD4 counts of less than 350 cells per μL. We used facility-level data to calculate representative estimates of retention and loss to follow-up. We used patient-level data to study temporal trends in retention, timing of loss to follow-up, and predictors of no follow-up and loss to follow-up. We defined patients who were more than 60 days late for their first follow-up visit as having no follow-up and patients who were more than 60 days late for a subsequent visit as being lost to follow-up. We calculated proportions and cumulative probabilities of patients who had died, stopped ART, had no follow-up, were lost to follow-up, or were retained alive on ART for 36 months. We calculated odds ratios and hazard ratios to examine predictors of no follow-up and loss to follow-up. Findings Analysis of facility-level data about patients in option B+ who had not transferred to a different facility showed retention in care to be 76·8% (20 475 of 26 658 patients) after 12 months, 70·8% (18 306 of 25 849 patients) after 24 months, and 69·7% (17 787 of 25 535 patients) after 36 months. Patient-level data included 29 145 patients. 14 630 (50·2%) began treatment under option B+. Patients in option B+ had a higher risk of having no follow-up and, for the first 2 years of ART, higher risk of loss to follow-up than did patients who started ART because they had CD4 counts less than 350 cells per μL or WHO clinical stage 3 or 4 disease. Risk of loss to follow-up during the third year was low and similar for patients retained for 2 years. Retention rates did not change as the option B+ programme matured. Interpretation Our data suggest that pregnant and breastfeeding women who start ART immediately after they are diagnosed with HIV can be retained on ART through the option B+ programme, even after many have stopped breastfeeding. Interventions might be needed to improve retention in the first year on ART in option B+. Funding Bill & Melinda Gates Foundation, Partnerships for Enhanced Engagement in Research Health, and National Institute of Allergy and Infectious Diseases.

A sorrow shared is a sorrow halved? Internet-based group therapy versus individual therapy for social anxiety disorder: A randomized controlled trial

Purpose: Social anxiety disorder is one of the most researched conditions in the field of Internet-based self-help. Various studies have shown that cognitive-behavioral treatments can be efficacious to reduce social phobic symptoms. Most of the interventions tested include some form of support, whereas the efficacy of a web-based group format has yet to be investigated. The present study aims at investigating the possible added value of therapist-guided group support in an Internet-based guided self-help treatment for SAD. Methods: A total of 150 adults with a diagnosis of SAD are randomly assigned to either a wait-list control group or one of two active treatment conditions. Participants in the two active conditions use the same Internet-based self-help program, either with individual guidance by a therapist or with the support of a therapist-guided group of 6 individuals. In the group condition, participants communicate with each other via an integrated, protected discussion forum. The primary outcome variables are symptoms of SAD and diagnostic status immediately after the intervention (12 weeks) and at 6-month follow-up. Secondary endpoints are general symptomatology, depression, quality of life and adherence to treatment. Furthermore, process variables such as group processes and the working alliance are studied. Results: Results are currently being analyzed. Results at post-treatment will be presented and discussed. Potential moderating and mediating variables of treatment success will be addressed. Conclusion: The results of this study should indicate whether therapist-guided group support could enhance the efficacy of an internet based self-help treatment for SAD. This novel treatment format, if shown efficacious, could represent a cost-effective option and could be further modified to treat other conditions.

Clinical effectiveness of cognitive behavioral therapy for depression in routine care: A propensity score based comparison between randomized controlled trials and clinical practice

Background: The efficacy of cognitive behavioral therapy (CBT) for the treatment of depressive disorders has been demonstrated in many randomized controlled trials (RCTs). This study investigated whether for CBT similar effects can be expected under routine care conditions when the patients are comparable to those examined in RCTs. Method: N=574 CBT patients from an outpatient clinic were stepwise matched to the patients undergoing CBT in the National Institute of Mental Health Treatment of Depression Collaborative Research Program (TDCRP). First, the exclusion criteria of the RCT were applied to the naturalistic sample of the outpatient clinic. Second, propensity score matching (PSM) was used to adjust the remaining naturalistic sample on the basis of baseline covariate distributions. Matched samples were then compared regarding treatment effects using effect sizes, average treatment effect on the treated (ATT) and recovery rates. Results: CBT in the adjusted naturalistic subsample was as effective as in the RCT. However, treatments lasted significantly longer under routine care conditions. Limitations: The samples included only a limited amount of common predictor variables and stemmed from different countries. There might be additional covariates, which could potentially further improve the matching between the samples. Conclusions: CBT for depression in clinical practice might be equally effective as manual-based treatments in RCTs when they are applied to comparable patients. The fact that similar effects under routine conditions were reached with more sessions, however, points to the potential to optimize treatments in clinical practice with respect to their efficiency.

Feasibility study of a single-blind randomised controlled trial of an occupational therapy intervention.

BACKGROUND Several factors facilitate or hinder efficacy research in occupational therapy. Strategies are needed, therefore, to support the successful implementation of trials. AIM To assess the feasibility of conducting a randomised controlled trial (RCT). The main feasibility objectives of this study were to assess the process, resources, management, and scientific basis of a trial RCT. MATERIAL AND METHODS A total of 10 occupational therapists, between the ages of 30 and 55 (M 43.4; SD 8.3) with seven to 26 years' (M 14.3; SD 6.1) experience, participated in this study. Qualitative data collected included minutes of meetings, reports, and field notes. The data were analysed based on the principles of content analysis, using feasibility objectives as the main categories. RESULTS Data analysis revealed strengths in relation to retention and inclusion criteria of participants, the study protocol, study organisation, and the competence of researchers. Weaknesses were found related to recruitment, randomisation, data collection, time for training and communication, commitment, and design. CONCLUSION The findings indicated that there are several factors which had a considerable impact on the implementation of an RCT in practice. However, it was useful to assess methods and procedures of the trial RCT as a basis to refine research plans.

When to Monitor CD4 Cell Count and HIV RNA to Reduce Mortality and AIDS-Defining Illness in Virologically Suppressed HIV-Positive Persons on Antiretroviral Therapy in High-Income Countries: A Prospective Observational Study.

OBJECTIVE To illustrate an approach to compare CD4 cell count and HIV-RNA monitoring strategies in HIV-positive individuals on antiretroviral therapy (ART). DESIGN Prospective studies of HIV-positive individuals in Europe and the USA in the HIV-CAUSAL Collaboration and The Center for AIDS Research Network of Integrated Clinical Systems. METHODS Antiretroviral-naive individuals who initiated ART and became virologically suppressed within 12 months were followed from the date of suppression. We compared 3 CD4 cell count and HIV-RNA monitoring strategies: once every (1) 3 ± 1 months, (2) 6 ± 1 months, and (3) 9-12 ± 1 months. We used inverse-probability weighted models to compare these strategies with respect to clinical, immunologic, and virologic outcomes. RESULTS In 39,029 eligible individuals, there were 265 deaths and 690 AIDS-defining illnesses or deaths. Compared with the 3-month strategy, the mortality hazard ratios (95% CIs) were 0.86 (0.42 to 1.78) for the 6 months and 0.82 (0.46 to 1.47) for the 9-12 month strategy. The respective 18-month risk ratios (95% CIs) of virologic failure (RNA >200) were 0.74 (0.46 to 1.19) and 2.35 (1.56 to 3.54) and 18-month mean CD4 differences (95% CIs) were -5.3 (-18.6 to 7.9) and -31.7 (-52.0 to -11.3). The estimates for the 2-year risk of AIDS-defining illness or death were similar across strategies. CONCLUSIONS Our findings suggest that monitoring frequency of virologically suppressed individuals can be decreased from every 3 months to every 6, 9, or 12 months with respect to clinical outcomes. Because effects of different monitoring strategies could take years to materialize, longer follow-up is needed to fully evaluate this question.

Non-Viral Gene Therapy to Cells of the bovine Intervertebral Disc

Background. Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and degeneration. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM).1 Further studies showed that growth factors from transforming growth factor β (TGFβ) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC).2 Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods. Bovine nucleus pulposus (bNPC) and annulus fibrosus cells (bAFC) were harvested from bovine coccygeal IVD. Primary cells were then electroporized with plasmid GDF6 (Origene, vector RG211366) by optimizing parameters using the Neon Transfection system (Life Technologies, Basel). After transfection, cells were cultured in 2D monolayer or 3D alginate beads for 7, 14 or 21 days. Transfection efficiency of pGDF6 was analyzed by immunohistochemistry and fluorescent microscopy. Cell phenotype was quantified by real-time RT-PCR. To test a non-viral gene therapy applied directly to 3D whole organ culture, coccygeal bovine IVDs were harvested as previously described. Bovine IVDs were transfected by injection of plasmid GDF6 into the center. Electroporation was performed with ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) using 2-needle array electrode or tweezertrodes. 72 h after tranfection discs were fixed and cryosectioned and analyzed by immunofluorescence against GDF6. Results. RT-PCR and immunohistochemistry confirmed up-regulation of GFP and GDF6 in the primary bNPC/bAFC culture. The GFP-tagged GDF6 protein, however, was not visible, possibly due to failure of dimer formation as a result of fusion structure. Organ IVD culture transfection revealed GDF6 positive staining in the center of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GDF6 positive cells. Conclusion. Non-viral transfection is an appealing approach for gene therapy as it fulfills the translational safety aspects of transiency and lacks the toxic effects of viral transduction. We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgements. This project was funded by the Lindenhof Foundation (Funds “Research & Teaching”) Project no. 13-02-F. The imaging part of this study was performed with the facility of the Microscopy Imaging Center (MIC), University of Bern. References. Roughly PJ (2004): Spine (Phila), 29:2691-2699 Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014), Arthritis Research & Therapy, 16:R67