Paracelsus Medical University Salzburg Research Articles

Cardiovascular risk markers in patients with spinal cord injury: the Austrian wheelchair dancer study

Journal Article Accepted manuscript Cardiovascular Risk Markers in Patients with Spinal Cord Injury: The Austrian Wheelchair Dancer Study Get access Barbara Mayr, Barbara Mayr Institute of Sports Medicine, Prevention and Rehabilitation and Research Institute of Molecular Sports Medicine and Rehabilitation, Paracelsus Medical University, Salzburg, Lindhofstraße 20, 5020 Salzburg, Austria https://orcid.org/0000-0002-2835-3938 Search for other works by this author on: Oxford Academic Google Scholar Michelle Felber, Michelle Felber Institute of Sports Medicine, Prevention and Rehabilitation and Research Institute of Molecular Sports Medicine and Rehabilitation, Paracelsus Medical University, Salzburg, Lindhofstraße 20, 5020 Salzburg, Austria Search for other works by this author on: Oxford Academic Google Scholar Vanessa Frey, Vanessa Frey Department of Neurology, Christian Doppler University Hospital, Paracelsus Medical University and Centre for Cognitive Neuroscience Salzburg, Austria. Member of the European Reference Network EpiCARE https://orcid.org/0000-0002-5337-1273 Search for other works by this author on: Oxford Academic Google Scholar Nora Renz, Nora Renz Department of Neurology, Christian Doppler University Hospital, Paracelsus Medical University and Centre for Cognitive Neuroscience Salzburg, Austria. Member of the European Reference Network EpiCARE https://orcid.org/0000-0001-8011-6684 Search for other works by this author on: Oxford Academic Google Scholar Kerstin Schwenker, Kerstin Schwenker Department of Neurology, Christian Doppler University Hospital, Paracelsus Medical University and Centre for Cognitive Neuroscience Salzburg, Austria. Member of the European Reference Network EpiCAREKarl Landsteiner Institute for Neurorehabilitation and Space Neurology, Salzburg, AustriaNeuroscience Institute, Christian Doppler University Hospital, Center for Cognitive Neuroscience Salzburg, Paracelsus Medical University, Salzburg, AustriaSpinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University Salzburg, Austria https://orcid.org/0000-0002-0316-6108 Search for other works by this author on: Oxford Academic Google Scholar Eugen Trinka, Eugen Trinka Department of Neurology, Christian Doppler University Hospital, Paracelsus Medical University and Centre for Cognitive Neuroscience Salzburg, Austria. Member of the European Reference Network EpiCAREKarl Landsteiner Institute for Neurorehabilitation and Space Neurology, Salzburg, AustriaNeuroscience Institute, Christian Doppler University Hospital, Center for Cognitive Neuroscience Salzburg, Paracelsus Medical University, Salzburg, AustriaSpinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University Salzburg, Austria https://orcid.org/0000-0002-5950-2692 Search for other works by this author on: Oxford Academic Google Scholar Josef Niebauer Josef Niebauer Institute of Sports Medicine, Prevention and Rehabilitation and Research Institute of Molecular Sports Medicine and Rehabilitation, Paracelsus Medical University, Salzburg, Lindhofstraße 20, 5020 Salzburg, Austria Corresponding author: Prof. Josef Niebauer, MD, PhD, MBA, University Institute of Sports Medicine, Prevention and Rehabilitation, Paracelsus Medical University, Lindhofstraße 20, 5020 Salzburg, Austria. ph.:+43(0)5 7255-23200, fax: +43(0)5 7255-23299. E-mail: j.niebauer@salk.at https://orcid.org/0000-0002-2811-9041 Search for other works by this author on: Oxford Academic Google Scholar European Journal of Preventive Cardiology, zwad128, https://doi.org/10.1093/eurjpc/zwad128 Published: 24 April 2023 Article history Received: 06 March 2023 Revision received: 18 April 2023 Accepted: 20 April 2023 Published: 24 April 2023

"Investigation of the Antimicrobial Activity of Hospital Furniture Coated with Melamine Resin Endowed with Catalysts as In Situ Generated Biocides"

Guggenbichler S1, M Hell2 and Guggenbichler JP3* Author Affiliations 1Division for Vascular Surgery and Endovascular Surgery of the University Clinic for Cardiac Surgery, Vascular Surgery and Endovascular Surgery, Paracelsus Medical University Salzburg, Austria 2MEDILAB, Teaching Laboratory of the Paracelsus Medical University Salzburg, Department of Clinical Microbiology and Infection Control, Salzburg, Austria 3Department or Pediatrics, AmiSTec GMbH und Co KG, Kössen, Austria Received: May 03, 2021 | Published: May 10, 2021 Corresponding author: Guggenbichler JP, Department or Pediatrics, Univ. Erlangen, AmiSTec GMbH und Co KG, Kössen, Austria DOI: 10.26717/BJSTR.2021.35.005737

Results of a hospitalization policy of asymptomatic and pre-symptomatic COVID-19-positive long-term care facility residents in the province of Salzburg\u2014a report from the AGMT COVID-19 Registry

COVID-19-associated case fatality rates up to 48% were reported among nursing facility residents. During the first wave of the COVID-19 pandemic, routine SARS-CoV-2 testing in long-term care facilities in the Province of Salzburg and centralized hospitalization in the COVID-19 unit of the Paracelsus Medical University Salzburg (Austria) irrespective of symptoms was implemented. Baseline characteristics and the course of COVID-19 disease were assessed among hospitalized long-term care facility residents within the COVID-19 Registry of the Austrian Group Medical Tumor Therapy (AGMT; NCT04351529). Between the 24th of March and the 20th of April 2020, 50 COVID-19-positive residents were hospitalized. The median age was 84.5 years (range: 79–88) and the median number of comorbidities and baseline medication classes was 6 (IQR: 4–7) and 5 (IQR: 3–6), respectively. At admission, 31 residents (62%) were symptomatic, nine residents (18%) pre-symptomatic whereas ten residents (20%) remained asymptomatic. The 30-day mortality rate from hospitalization was 32% and significantly higher in symptomatic residents at admission when compared to asymptomatic residents including pre-symptomatic residents (48% [95% CI: 27–63%] versus 5% [95% CI: 0–15%], p=0.006). The Early Warning Score (EWS) at admission was associated with 30-day mortality: high risk: 100%, intermediate risk: 50% (95% CI: 0–78%), and low risk: 21% (95% CI: 7-32%) (p<0.001). In light of comparably low mortality rates between asymptomatic and pre-symptomatic hospitalized COVID-19-positive residents, we suggest the supply of comparable intensity and quality of monitoring and care in long-term care facilities as an alternative to immediate hospitalization upon a positive COVID-19 test in asymptomatic residents.

Planning and Design of a Prospective Randomised Multi-Centre Trial on the Repair of Large Hiatal Hernias with Sutures vs. Pledgeted Sutures vs. Absorbable Mesh

The hernia recurrence rate after surgical treatment of large hiatal hernias is still very high. The optimal technique to reduce the recurrence rate is still under debate. The aim of this work is to clarify whether pledgeted reinforced sutures or a resorbable mesh can reduce the recurrence rate compared to hiatus closure with only sutures. An Austria-wide, multi-centre, prospective, randomised study was planned. The study protocol was prepared by the main test centre (University Clinic for General, Visceral and Thoracic Surgery, Paracelsus Medical University Salzburg). The study includes patients who are scheduled to undergo laparoscopic or robot-assisted surgery for a large symptomatic hiatal hernia. A large hiatal hernia is defined as > 5 cm in manometry or gastroscopy or at least ⅓ of the stomach lying intrathoracically. The primary study endpoint is defined as the hernia recurrence rate, objectively assessed by gastroscopy. After inclusion in the study, patients will be followed up for 6 months, 1 year, 3 years and 5 years after the operation, using standardised questionnaires and gastroscopy. The power calculation showed a requirement of 55 patients per group. Preoperative randomisation and data management are software-based. The study approval by the leading ethics committee is currently pending and the study itself has been registered on ClinicalTrials.gov since October 2020. The Clinical Trials Registration Number is NCT04591860. Five clinics are participating in the study at the moment and all centres are actively enrolling patients. The duration of the study is set until January 2027. This study is the world's first prospective randomised study that examines the value of pledgets and resorbable mesh to reduce the recurrence rate after treatment of large hiatal hernias. The results will help to find the optimal technique to close the hiatus of large hiatal hernias.

Impact of PD-L1 Scores and Changes on Clinical Outcome in Rectal Cancer Patients Undergoing Neoadjuvant Chemoradiotherapy.

Reports on the prognostic role of programmed death-ligand 1 (PD-L1) expression in rectal cancer are controversial. We investigated expression patterns and changes of PD-L1 in rectal cancer patients undergoing neoadjuvant chemoradiotherapy (CRT). Seventy-two patients diagnosed with rectal cancer and/or treated with fluorouracil-based neoadjuvant CRT at the Department of Internal Medicine III of the Paracelsus Medical University Salzburg (Austria) between January 2003 and October 2012 were included. PD-L1 scoring was performed according to the tumor proportion score (TPS), combined positive score (CPS), and immune cell score (IC). PD-L1 TPS prior to neoadjuvant CRT had a statistically significant impact on survival (median: ≤1%: 95.4 months (95% CI: 51.8—not reached) vs. >1%: not reached, p = 0.03, log-rank). Patients with a PD-L1 TPS ≤1% prior to and after CRT showed an inferior survival compared to all other patients (median: 56.7 months (95% CI: 51.4—not reached) vs. not reached, p = 0.005, log-rank). In multivariate analysis, PD-L1 TPS prior to neoadjuvant CRT (>1% vs. ≤1%, hazard ratio: 0.29 (95% CI: 0.11–0.76), p = 0.01) remained independently associated with survival. In conclusion, low PD-L1 TPS was associated with inferior survival in rectal cancer patients undergoing neoadjuvant CRT. A prospective validation of the prognostic value of PD-L1 expression in rectal cancer patients within a clinical trial is necessitated.

Evaluation of circulating cell-free DNA as a molecular monitoring tool in patients with metastatic cancer.

The clinical decisions made when treating patients with metastatic cancer require knowledge of the current tumor extent and response to therapy. For the majority of solid tumors, a response assessment, which is based on imaging, is used to guide these decisions. However, measuring serum protein biomarkers (i.e. tumor markers) may be of additional use. Furthermore, tumor markers exhibit variable specificity and sensitivity and cannot therefore be solely relied upon when making decisions regarding cancer treatment. Therefore, there is a clinical requirement for the identification of specific, sensitive and quantitative biomarkers. In recent years, circulating cell-free DNA (cfDNA) and mutation-specific circulating cell-free tumor DNA (cftDNA) have been identified as novel potential biomarkers. In the current study, cfDNA and cftDNA were compared using imaging-based staging and current tumor markers in 15 patients with metastatic colorectal, pancreatic or breast cancer. These patients were treated at the Third Medical Department of Paracelsus Medical University Salzburg (Austria). The results of the current study demonstrated a statistically significant correlation between the concentration changes of cfDNA and cftDNA and response to treatment, which was assessed by imaging. A correlation was not indicated with current clinically used tumor markers, including carcinoembryonic antigen, carcinoma antigen 15-3 and carcinoma antigen 19-9. The present study also indicated a correlation between cfDNA and cftDNA and the tumor volume of metastatic lesions, which was not observed with the current clinically used tumor markers. In conclusion, cfDNA and cftDNA exhibit the potential to become novel biomarkers for the response assessment following cancer treatment, and may serve as a tool for the estimation of tumor volume. The current study further supports the increasingly important role of cfDNA and cftDNA as new monitoring tools for use during cancer therapy.

Randomised controlled trials evaluating endometrial scratching: assessment of methodological issues.

Do randomised controlled trials (RCTs) evaluating endometrial scratching suffer from methodological issues including insufficient trial registration, statistical errors or irreproducibility, randomisation errors or miscellaneous issues? The majority of RCTs investigating endometrial scratching have methodological issues. A large number of small RCTs investigating the effectiveness of endometrial scratching prior to in vitro fertilisation (IVF) and intrauterine insemination (IUI)/intercourse have reported favourable findings. Subsequently, systematic reviews incorporating these RCTs yielded meta-analyses in favour of endometrial scratching. Endometrial scratching has been widely adopted by infertility specialists around the world. Recently, an international RCT including 1364 women reported no benefit from endometrial scratching before IVF. We evaluated several methodological issues of RCTs investigating the effectiveness of endometrial scratching prior to IVF and IUI/intercourse. We identified 25 RCTs for IVF and 12 RCTs for IUI/intercourse with full-text publication. We assessed the RCTs on the following criteria: adequacy of trial registration, statistical issues (description of statistical methods and reproducibility of univariable statistical analysis), excessive similarity or difference in baseline characteristics that is not compatible with chance (Monte Carlo simulations and Kolmogorov-Smirnov test) and miscellaneous methodological issues. Of 25 RCTs evaluating endometrial scratching prior to IVF, only eight (32%) had adequate trial registration. In total, 10 (40%) RCTs had issues regarding statistical methods. Nine (69%, 13 applicable) RCTs had at least one inconsistency between reported and reproduced univariable statistical analysis for categorical baseline/intermediate characteristics. Statistical results of at least one outcome were not reproducible in 14 (74%, 19 applicable) RCTs. Only two (8%) RCTs had none of the above issues. Suggested by the simulations, these RCTs did not significantly violate the null hypothesis that the baseline characteristics were the results of a properly conducted randomisation process (P= 0.4395).Of 12 IUI/intercourse RCTs, only 2 (17%) had adequate trial registration. In total, five (42%) studies had issues of statistical methods. Inconsistency between reported and reproduced univariable analysis for baseline/intermediate categorical variable(s) was found in four (57%, 7 applicable) RCTs. Statistical analysis was not reproducible for at least one outcome in eight (80%, 10 applicable) studies. All RCTs had at least one of the above issues. These RCTs were inconsistent with the null hypothesis that their baseline characteristics were the results of proper randomised allocation (P= 1.659*10-7). We were unable to assess RCTs which were not published as full-text papers. We could not analyse individual participant data to investigate possible reasons for statistical inconsistencies. The method to infer the likelihood of proper random sampling rests on assumptions including independent baseline characteristics, simple randomisation and no publication bias. The methodological issues common to RCTs evaluating endometrial scratching may have biased the results of the trials. Further development and validation of these novel methods may be helpful for the critical appraisal of RCTs. No external funding was sought to support this work. B.W.M. is supported by a National Health Medical Research Council (NHMRC) Practitioner Fellowship (GNT1082548). B.W.M. reports consultancy for ObsEva, Merck and Guerbet. D.W. is supported by a grant from the Paracelsus Medical University Salzburg, Austria (PMU Research Fund-PMU FFF Number: L-18/02/006-WET) and by Drs Haackert Foundation, Germany. S.L. is an author of a trial included in this study, an author of an included systematic review and a Cochrane editor. All other authors have no conflicts of interest. N/A.

The Austrian Spinal Cord Injury Study: a registry for patients living with a traumatic spinal cord injury

Study designEstablishing the structure of a prospective spinal cord injury (SCI) patient registry.ObjectivesTo develop a registry for patients with traumatic spinal cord injury (tSCI) in Austria as a base for addressing research questions, improving patient outcomes, and establishing a platform for future clinical trials.SettingsCoordinating institution: Paracelsus Medical University Salzburg, Austria; participating partners are located in nine states in Austria.MethodsThe Austrian Spinal Cord Injury Study (ASCIS) collects longitudinal data on simple forms within a 7-stage follow-up examination timeline.ResultsThe implementation of the ASCIS in 2012 created the first nationwide SCI patient registry in Austria. ASCIS is currently implemented in 17 trauma hospitals in 9 Austrian states, and over 150 individuals with acute tSCI have been registered to date. As in Austria, the structure of the health-care system does not involve a specialized SCI center covering the primary health care and the rehabilitation care, major challenges have to be overcome to involve all participating primary centers and rehabilitation centers, which perform tSCI patient care, for ASCIS. Through implementing ASCIS, a network of SCI clinicians and researchers, which is now beginning to support translational research and to initiate clinical trials for patients with tSCI, has formed.ConclusionsASCIS is uniquely positioned in Austria to capture detailed information from the early acute to the chronic phases of tSCI, to provide this information also to bigger and translational settings, and to connect researchers and clinicians to facilitate clinical research on tSCI.

Red blood cell alloimmunization in 184 patients with myeloid neoplasms treated with azacitidine – A retrospective single center experience

Alloimmunization to Red Blood Cell (RBC) antigens frequently occurs in patients with myeloid neoplasms (AML, MDS and CMML) and potentially poses the patient at risk for delayed hemolytic transfusion reactions and limited supply of compatible RBC-units. However, there is comparatively little data on transfusion associated characteristics in this patient cohort. We therefore retrospectively analyzed transfusion requirements and clinical outcomes of 184 patients with myloid neoplasms treated with azacitidine at the Paracelsus Medical University Salzburg, which were included in the Austrian Registry of Hypomethylating Agents.The mean blood component requirements for AML, MDS and CMML were 39.8, 67.4 and 31.4 RBC units and 31.7, 27.6 and 19.1 platelet (PLT) units respectively. In spite of an extended and stringent RBC unit matching policy (ABO, RhD, RhCcEe and K antigens), 20 (11%) patients formed at least one alloantibody (“allo-group”), whereas 164 patients (89%) did not (“non-allo-group”). The most frequent antibody specificity was anti-E, followed by anti-Wra −Lua, −D, −C and −Jka. Alloimmunization was associated with higher numbers of transfused RBC units (68 vs. 38; p=0.001), as well as with longer time under transfusion (16.7 vs. 9.4 months; p=0.014). Median overall survival (OS) did not differ significantly between the “allo”- and “non-allo-group”.

Status epilepticus in the elderly—A retrospective study on 120 patients

PurposeStatus epilepticus (SE) is one of the most common neurological emergencies with a high incidence in the elderly. Major determinants of prognosis are patients’ age, duration of SE and underlying etiology. We aimed identifying differences in clinical presentation of SE, etiologies and outcome between patients (pts.) sixty years or older (≥60) and younger than sixty (<60) years (yrs). MethodsWe retrospectively analyzed 120 patients (48 women) with SE admitted to the Neurological Intensive Care Unit (NICU), Department of Neurology, Paracelsus Medical University Salzburg, Austria between 1/2011 and 01/2013. Key findingsMedian age was 69 years (range 14–90) (63% ≥60yrs). Generalized tonic clonic SE was the most common SE type, whereas non convulsive SE with and without coma tended to occur more frequently in the elderly (33% ≥60 yrs. vs. 20%<60 yrs, Chi2=3.511, p=0.061). Preexisting history of epilepsy was more common in the younger age group (64% vs 41% p=0.014). An acute symptomatic cause of SE was identified in 25% (31/120), with cerebrovascular diseases being more frequent in the elderly (47% vs. 11%; p<0.01). Duration of SE did not differ between the age groups (p=0.63). Mortality was higher in elderly patients (31% vs. 7%, p=0.028, Chi Square=5.18) and moderate disability in younger patients (42% vs 17%; p=0.005, Chi Square=7.83). After Bonferroni correction only the higher rate of cerebrovascular etiologies in the elderly was statistically significant. SignificanceIn the elder population, SE occurs more often in patients without preexisting epilepsy and is most frequently caused by cerebrovascular diseases. NCSE tends to be more frequent in the elderly and diagnosis is complicated by subtle clinical presentation. Even though comorbidities represent treatment limitations, in our sample no differences in choice of AED as well as dosage were observed between the age groups, reflecting a trend toward AEDs with more favorable adverse event profile in all patients. SE in older patients is associated with poorer outcome and higher mortality.

167 Long-term in vivo correction of a recessive dystrophic epidermolysis bullosa phenotype using RNA trans-splicing repair

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

169 Regeneration of a functional epidermis at a large, long-standing wound by gene-corrected autologous epidermal stem cells

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

Randomized controlled clinical trial to evaluate different intraocular lenses for the surgical compensation of low to moderate-to-high regular corneal astigmatism during cataract surgery

To evaluate vector analysis, rotational stability, and visual outcomes after implantation of toric IOLs with low (<2.25 diopter [D]) toric values (Acrysof IQ SN6AT3 or SN6AT4) and moderate-to-high toric (3.00 D to 6.00 D) values (Acrysof IQ SN6AT5, SN6AT6, SN6AT7, SN6AT8, or SN6AT9) versus after implantation of a nontoric IOL (Acrysof SN60WF) in eyes with regular topographic corneal astigmatism. Department of Ophthalmology, Paracelsus Medical University Salzburg, Salzburg, Austria. Randomized controlled clinical trial. The study included eyes of patients undergoing phacoemulsification and IOL implantation. Patients were randomized into 3 groups to bilaterally receive the nontoric IOL, the low toric IOL, or the moderate-to-high toric IOL. Over a 6-week follow-up period, rotational stability and astigmatic changes were evaluated in the 2 toric IOL groups using the Alpins vector method. The study included 78 eyes of 39 patients. Postoperatively mean refractive astigmatism decreased significantly from 1.45 D ± 1.18 (SD) to 0.36 ± 0.44 D in the low toric group and from 1.92 ± 1.09 D to 0.31 ± 0.46 D in the moderate-to-high toric group. There was no significant difference between mean difference vectors in the low toric IOL group and the moderate-to-high toric IOL group. A significant difference in the correction index was detected, revealing overcorrection with low toric IOLs (1.03 ± 0.12 D) and undercorrection with moderate-to-high toric IOLs (0.95 ± 0.12 D). Implantation of low toric IOLs and medium-to-high toric IOLs in patients with low to moderate-to-high corneal astigmatism was shown to be effective in correcting regular corneal astigmatism. Undercorrection in the moderate-to-high group could be related to misalignment. This research project was financially supported by the Fuchs Foundation for the Promotion of Research in Ophthalmology, Salzburg, Austria. Alcon Inc. financially supports the Fuchs-Foundation as the clinical research center of the Department of Ophthalmology of the Paracelsus Medical University Salzburg, Salzburg, Austria (Grant Number 2010-37). No author has a financial or proprietary interest in any material or method mentioned.

Intraindividual aqueous flare comparison after implantation of hydrophobic intraocular lenses with or without a heparin-coated surface

To assess the efficacy of a heparin-surface-modified (HSM) hydrophobic acrylic intraocular lens (IOL) (EC-1YH PAL) and the same IOL without heparin coating (EC-1Y-PAL) by the flare and cell intensity in the anterior chamber after uneventful cataract surgery. Department of Ophthalmology, Paracelsus Medical University Salzburg, Austria. Comparative case series. Routine phacoemulsification with randomized implantation of an HSM IOL in 1 eye (HSM IOL group) and an uncoated IOL (uncoated IOL group) in the fellow eye was performed. Postoperative inflammation was assessed objectively using a laser flare-cell meter (FM-600) preoperatively as well as 1 day and 1 and 3 months postoperatively. Aqueous cells in the anterior chamber, distance visual acuities, and subjective manifest refraction were also evaluated at each visit. One hundred eyes (50 patients) were enrolled. In both groups, the mean flare values increased significantly from preoperatively to 1 day postoperatively (P<.001) and nearly reached preoperative values by 3 months postoperatively. One day postoperatively, the mean flare value was statistically significantly lower in the HSM IOL group (14.92 photons per millisecond [ph/ms] ± 7.47 [SD]) than in the uncoated IOL group (mean 16.73 ± 7.81 ph/ms) (P=.04); there was no statistically significant difference between groups 1 and 3 months postoperatively (both P>.58). The HSM IOL group had a greater and quicker decrease in aqueous cells, reaching statistical significance 1 month postoperatively (P=.01). The HSM IOL showed a significant lower inflammatory reaction in the early postoperative stage with a faster disappearance of inflammatory signs. No author has a financial or proprietary interest in any material or method mentioned.

Results of dacryocystorhinostomy according to Toti from 2000 to 2012

The aim of this study was to describe the efficiency, long-term success, patient satisfaction and recurrence rate after dacryocystorhinostomy according to Toti in a retrospective analysis. At the Eye Clinic of the Paracelsus Medical University Salzburg 188 eyes of 176 patients with stenosis/obstruction of lacrimal duct were surgically treated between January 2000 and December 2012. Of these patients 173 eyes had no surgery before DCR according to Toti and 15 eyes had already been operated with diverse dacryocystorhinostomies. All patients underwent dacryocystorhinostomy according to Toti. Pre- and postoperative clinical finings were analysed retrospectively. Postoperative long-term results regarding patient satisfaction, absence of inflammation, epiphora and annoyance through local scar building had been evaluated with a questionnaire in a cross-sectional analysis. The primary success rate of DCR according to Toti was 91,9 % and the secondary success rate 98,3 % in patients without surgery preoperatively. Classifying by localisation of stenosis, better results could be reached in patients with postsaccal stenosis/obstructions (94,3 %) than in those with presaccal obstructions (77,8 %) or with mixed pre- and postsaccal stenosis/obstructions (86,7 %). Also in the patient group with previous surgery primary and secondary success rates of 93,3 % could be achieved after DCR according to Toti. This study confirms that DCR according to Toti is a highly successful technique minimising epiphora and discomfort. The best success rate was found in patients with postsaccal stenosis/obstructions. The local scar was not found to be disturbing.

Insights From Major Themes at EuroPRevent 2013, Rome, Italy

39 ISSN 1479-6678 10.2217/FCA.13.46 © 2014 Future Medicine Ltd Future Cardiol. (2014) 10(1), 39–41 1University Institute of Sports Medicine, Prevention & Rehabilitation, Salzburg, Austria 2Research Institute of Molecular Sports Medicine & Rehabilitation, Paracelsus Medical University Salzburg, Salzburg, Austria 3Institute of Sports Medicine of the State of Salzburg, Sports Medicine of the Olympic Center Salzburg-Rif, Lindhofstr. 20, 5020 Salzburg, Austria 4The School of Physical Therapy & Rehabilitation Science, The University of Montana, Missoula, MT, USA *Author for correspondence: Tel.: +43 662 4482 4270; Fax: +43 662 4482 4274; j.niebauer@salk.at EuroPRevent, the annual meeting of the European Association of Cardiovascular Prevention and Rehabilitation (EACPR), was held in Rome, Italy in April 2013. While a broad range of sessions covered many topics related to exercise, from biomarkers to public health initiatives, several relevant themes emerged including the primary and secondary prevention of cardiovascular disease (CVD), and the continued evolving use of cardiopulmonary exercise testing to accurately assess the performance of patients with chronic heart failure and those receiving novel therapies, such as ventricular-assist devices. Stemming the cardiovascular event rate in those with and without CVD, and more effectively managing patients with chronic heart failure, is vital to reduce the social and economic burden associated with the rising prevalence of these patient populations. EuroPRevent, the annual meeting of the EACPR, was held in Rome, Italy between 18 and 20 April 2013, with nearly 1600 clinicians and scientists in attendance. This years overall theme was the ‘Universal Approach to Cardiovascular Preventive Cardiology’, which was a very well-chosen topic since the UN target to achieve a reduction in cardiovascular mortality of 25% by 2025 is ambitious but realistic, provided all partners in the medical profession work together. The notion that only a team effort will lead to success was substantiated by the conduction of the first global forum on CVD prevention in clinical practice, but also underscored by an unprecedented number of high-quality sessions that were co-organized with partner societies, as well as Working Groups on Atherosclerosis & Vascular Biology, Hypertension and the Heart, Peripheral Circulation and Thrombosis. The main program of the EACPR meeting consisted of sessions on prevention and rehabilitation, epidemiology, preventive and rehabilitative implementation, sports cardiology, translational science, clinical cases and exercise. A large number of poster presentations provided studies in Insights from major themes at EuroPRevent 2013, Rome, Italy

An Easy and Novel Method of Preoperative Laser-Assisted Marking for Body Lift Procedures

Sir: We read with great interest the article “Simplifying Circumferential Body Contouring: The Central Body Lift Evolution” by Rohrich et al.1 The authors present the evolution of their technique and experience with the central body lift procedure in a greater than 12-year period and emphasize the key operative elements and strict safety guidelines that afford optimal results with minimal morbidity. We would hereby like to introduce a novel technique to facilitate the preoperative planning and marking concerning the body lift procedure in massive weight loss patients. Preoperative markings are set in standing position,2,3 and this sometimes implicates difficulties for the surgeon, especially in drawing straight vertical and horizontal lines, which later on will be the position of the horizontal scar on the typical position of the abdomen and the back. Scar placement is crucial and the result of a precise marking technique. In the massive weight loss patient, excess skin may make identification of the anatomical landmarks (which are often used for orientation to achieve symmetry) difficult.4 Kalbermatten et al.5 in 2009 suggested body taping to visualize the optimal position for skin incision. This technique makes preoperative marking objectively reproducible. The authors also postulated demands for a sufficient marking technique, which are all fulfilled by our new method too: minimal expenditure of time, congruence of marked and later resected area, and finally cost efficiency. In this Viewpoint, we present a new simple method of reproducibly defining the incision lines for body lift procedures with an available laser marking device (Fig. 1).Fig. 1: Laser device placed opposite the standing patient (Stanley XP180 Cross Line and Laser Plumb; Stanley Deutschland–HDL Industrievertretung GmbH).The patient is placed standing with hanging arms to avoid a tightening effect on the lateral trunk. The laser marking light lines (horizontal and vertical light) are turned on and the vertical light line is placed exactly on the anal cleft. The designated scar line is generally defined by placing the horizontal laser light approximately 2 cm below the upper impression of the underwear’s elastic strap. After marking of a median line, further vertical lines are drawn at a distance of 5 cm (2 inches) as far as the spina iliaca anterior superior by turning the patient. For defining the resection margins, skin is moved upward and downward using the tip of the index finger at each vertical line. Immediately before skin slips of the fingertip, a skin mark is set at the place where the vertical line and laser light intersect. This maneuver is started medially and continued to the lateral side as far as resection is planned. To complete the drawing of the inferior and superior incision line, these points are connected (Fig. 2).Fig. 2: Laser marking light lines (horizontal and vertical light) placed on the back of the patient.The use of this simple instrument allows placement of reproducible skin markings, pays respect to varying tissue mobility, and reduces time needed for preoperative skin marking. As a sighting device, the laser marking device gives the surgeon a secure instrument with which to draw precise lines that finally will result in a more precise positioning of the scar and thus a better postoperative outcome for the massive weight loss patient. DISCLOSURE The authors have no financial interest to declare in relation to the content of this communication. No funding was received for this work. Lorenz Larcher, M.D. Hospital of St. John of God (Barmherzige Brüder) Salzburg (A) Department of Plastic, Aesthetic, and Reconstructive Surgery Academic Teaching Hospital Paracelsus Medical University Salzburg Karl Schwaiger Paracelsus Medical University Salzburg Florian Ensat, M.D. Gottfried Wechselberger, M.D., M.Sc. Heinrich M. Schubert, M.D. Hospital of St. John of God (Barmherzige Brüder) Salzburg (A) Department of Plastic, Aesthetic, and Reconstructive Surgery Academic Teaching Hospital Paracelsus Medical University Salzburg Salzburg, Austria

Rotational stability and visual outcome after implantation of a new toric intraocular lens for the correction of corneal astigmatism during cataract surgery

To evaluate rotational stability and the refractive and visual outcomes of a new aspheric toric intraocular lens (IOL) for correction of preexisting corneal astigmatism during routine cataract surgery. Department of Ophthalmology, Paracelsus Medical University Salzburg, Salzburg, Austria. Case series. Aspheric Bi-Flex T toric IOLs were monolaterally or bilaterally implanted after phacoemulsification in patients with topographic corneal astigmatism between 1.5 diopters (D) and 4.0 D. Preoperative IOL calculations were performed by laser interference biometry (Haigis formula). Appropriate IOL-torus alignment was facilitated by combined imaging and eye-tracking technology. Refraction and uncorrected distance (UDVA) and corrected distance visual acuities were measured 1 day, 1 week, and 1 and 3 months postoperatively. At each visit, photodocumentation in retroillumination was performed to evaluate torus position and potential toric IOL rotation. The mean refractive astigmatism decreased from 1.93 D ± 0.90 (SD) (range 0.5 to 4.0 D) to 0.30 ± 0.54 D (range 0.0 to 1.5 D) at 3 months. Patients achieved a mean UDVA of 0.05 ± 0.12 logMAR (range -0.18 to 0.30 logMAR [∼20/20]). Intraoperative to 3-month postoperative comparison of IOL axis alignment showed low levels of rotation (mean 2.12 ± 3.45 degrees; range -2 to +5 degrees). Implantation of the new aspheric toric IOL was effective, safe, and stable in correcting preexisting regular corneal astigmatism during cataract surgery. Combined imaging and eye tracking seems to be a promising technology to evaluate the correct axis for IOL torus alignment.

Discontinuation of combined resistance–endurance training increases cardiovascular risk factors

risk factors Robert Berent ⁎, Serge P. von Duvillard , Stephen F. Crouse , Johann Auer , John S. Green , Josef Niebauer , Helmut Sinzinger , Peter Schmid a a Center for Cardiovascular Rehabilitation, Bad Schallerbach, Austria b Department of Physical Performance, Norwegian School of Sport Sciences, Oslo, Norway c Texas A&M University, College Station, TX, USA d Department of Cardiology, General Hospital Braunau, Austria e Institute of Sports Medicine, Prevention and Rehabilitation, Paracelsus Medical University Salzburg, Austria f ATHOS, Institute for Diagnosis and Treatment of Atherosclerosis and Lipid Disorders, Vienna, Austria