A multicenter study of asymmetric and symmetric dimethylarginine as predictors of mortality risk in hospitalized COVID-19 patients.

Mortality of patients hospitalized with COVID-19 has remained high during the consecutive SARS-CoV-2 pandemic waves. Early discrimination of patients at high mortality risk is crucial for optimal patient care. Symmetric (SDMA) and asymmetric dimethylarginine (ADMA) have been proposed as possible biomarkers to improve risk prediction of COVID-19 patients. We measured SDMA, ADMA, and other L-arginine-related metabolites in 180 patients admitted with COVID-19 in four German university hospitals as compared to 127 healthy controls. Patients were treated according to accepted clinical guidelines and followed-up until death or hospital discharge. Classical inflammatory markers (leukocytes, CRP, PCT), renal function (eGFR), and clinical scores (SOFA) were taken from hospital records. In a small subgroup of 23 COVID-19 patients, sequential blood samples were available and analyzed for biomarker trends over time until 14 days after admission. Patients had significantly elevated SDMA, ADMA, and L-ornithine and lower L-citrulline concentrations than controls. Within COVID-19 patients, SDMA and ADMA were significantly higher in non-survivors (n = 41, 22.8%) than in survivors. In ROC analysis, the optimal cut-off to discriminate non-survivors from survivors was 0.579 µmol/L for SDMA and 0.599 µmol/L for ADMA (both p < 0.001). High SDMA and ADMA were associated with odds ratios for death of 11.45 (3.37-38.87) and 5.95 (2.63-13.45), respectively. Analysis of SDMA and ADMA allowed discrimination of a high-risk (mortality, 43.7%), medium-risk (15.1%), and low-risk group (3.6%); risk prediction was significantly improved over classical laboratory markers. We conclude that analysis of ADMA and SDMA after hospital admission significantly improves risk prediction in COVID-19.

Study design for development of novel safety biomarkers of drug-induced liver injury by the translational safety biomarker pipeline (TransBioLine) consortium: a study protocol for a nested case-control study.

A lack of biomarkers that detect drug-induced liver injury (DILI) accurately continues to hinder early- and late-stage drug development and remains a challenge in clinical practice. The Innovative Medicines Initiative's TransBioLine consortium comprising academic and industry partners is developing a prospective repository of deeply phenotyped cases and controls with biological samples during liver injury progression to facilitate biomarker discovery, evaluation, validation and qualification.In a nested case-control design, patients who meet one of these criteria, alanine transaminase (ALT) ≥ 5 × the upper limit of normal (ULN), alkaline phosphatase ≥ 2 × ULN or ALT ≥ 3 ULN with total bilirubin > 2 × ULN, are enrolled. After completed clinical investigations, Roussel Uclaf Causality Assessment and expert panel review are used to adjudicate episodes as DILI or alternative liver diseases (acute non-DILI controls). Two blood samples are taken: at recruitment and follow-up. Sample size is as follows: 300 cases of DILI and 130 acute non-DILI controls. Additional cross-sectional cohorts (1 visit) are as follows: Healthy volunteers (n = 120), controls with chronic alcohol-related or non-alcoholic fatty liver disease (n = 100 each) and patients with psoriasis or rheumatoid arthritis (n = 100, 50 treated with methotrexate) are enrolled. Candidate biomarkers prioritised for evaluation include osteopontin, glutamate dehydrogenase, cytokeratin-18 (full length and caspase cleaved), macrophage-colony-stimulating factor 1 receptor and high mobility group protein B1 as well as bile acids, sphingolipids and microRNAs. The TransBioLine project is enabling biomarker discovery and validation that could improve detection, diagnostic accuracy and prognostication of DILI in premarketing clinical trials and for clinical healthcare application.

Unification of Treatments and Interventions for Tinnitus Patients (UNITI): a study protocol for a multi-center randomized clinical trial.

BACKGROUND: Tinnitus represents a relatively common condition in the global population accompanied by various comorbidities and severe burden in many cases. Nevertheless, there is currently no general treatment or cure, presumable due to the heterogeneity of tinnitus with its wide variety of etiologies and tinnitus phenotypes. Hence, most treatment studies merely demonstrated improvement in a subgroup of tinnitus patients. The majority of studies are characterized by small sample sizes, unstandardized treatments and assessments, or applications of interventions targeting only a single organ level. Combinatory treatment approaches, potentially targeting multiple systems as well as treatment personalization, might provide remedy and enhance treatment responses. The aim of the present study is to systematically examine established tinnitus therapies both alone and in combination in a large sample of tinnitus patients. Further, it wants to provide the basis for personalized treatment approaches by evaluating a specific decision support system developed as part of an EU-funded collaborative project (Unification of treatments and interventions for tinnitus patients; UNITI project). METHODS/STUDY DESIGN: This is a multi-center parallel-arm randomized clinical trial conducted at five different clinical sites over the EU. The effect of four different tinnitus therapy approaches (sound therapy, structured counseling, hearing aids, cognitive behavioral therapy) applied over a time period of 12 weeks as a single or rather a combinatory treatment in a total number of 500 chronic tinnitus patients will be investigated. Assessments and interventions are harmonized over the involved clinical sites. The primary outcome measure focuses on the domain tinnitus distress assessed via the Tinnitus Handicap Inventory. DISCUSSION: Results and conclusions from the current study might not only provide an essential contribution to combinatory and personalized treatment approaches in tinnitus but could also provide more profound insights in the heterogeneity of tinnitus, representing an important step towards a cure for tinnitus. TRIAL REGISTRATION: ClinicalTrials.gov NCT04663828 . Registered on 11 December 2020.

Towards a unification of treatments and interventions for tinnitus patients: The EU research and innovation action UNITI.

Tinnitus is the perception of a phantom sound and the patient's reaction to it. Although much progress has been made, tinnitus remains a scientific and clinical enigma of high prevalence and high economic burden, with an estimated prevalence of 10%-20% among the adult population. The EU is funding a new collaborative project entitled "Unification of Treatments and Interventions for Tinnitus Patients" (UNITI, grant no. 848261) under its Horizon 2020 framework. The main goal of the UNITI project is to set the ground for a predictive computational model based on existing and longitudinal data attempting to address the question of which treatment or combination of treatments is optimal for a specific patient group based on certain parameters. Clinical, epidemiological, genetic and audiological data, including signals reflecting ear-brain communication, as well as patients' medical history, will be analyzed making use of existing databases. Predictive factors for different patient groups will be extracted and their prognostic relevance validated through a Randomized Clinical Trial (RCT) in which different patient groups will undergo a combination of tinnitus therapies targeting both auditory and central nervous systems. From a scientific point of view, the UNITI project can be summarized into the following research goals: (1) Analysis of existing data: Results of existing clinical studies will be analyzed to identify subgroups of patients with specific treatment responses and to identify systematic differences between the patient groups at the participating clinical centers. (2) Genetic and blood biomarker analysis: High throughput Whole Exome Sequencing (WES) will be performed in well-characterized chronic tinnitus cases, together with Proximity Extension Assays (PEA) for the identification of blood biomarkers for tinnitus. (3) RCT: A total of 500 patients will be recruited at five clinical centers across Europe comparing single treatments against combinational treatments. The four main treatments are Cognitive Behavioral Therapy (CBT), hearing aids, sound stimulation, and structured counseling. The consortium will also make use of e/m-health applications for the treatment and assessment of tinnitus. (4) Decision Support System: An innovative Decision Support System will be implemented, integrating all available parameters (epidemiological, clinical, audiometry, genetics, socioeconomic and medical history) to suggest specific examinations and the optimal intervention strategy based on the collected data. (5) Financial estimation analysis: A cost-effectiveness analysis for the respective interventions will be calculated to investigate the economic effects of the interventions based on quality-adjusted life years. In this paper, we will present the UNITI project, the scientific questions that it aims to address, the research consortium, and the organizational structure.

Overcoming multidrug resistance by RNA interference.

The ATP-binding cassette (ABC)-transporter P-glycoprotein (Pgp, also known as ABCB1) is the best characterized factor involved in multidrug resistance (MDR) of cancer cells. Pgp, which is encoded by the MDR1 gene, acts as a membrane-embedded drug extrusion pump for multiple structurally unrelated cytotoxic drugs. Inhibition of the pump activity of Pgp by low-molecular weight pharmacologically active compounds as a method to reverse MDR in cancer patients has been studied extensively, but so far clinical trials have generally been disappointing. Thus, experimental strategies for overcoming MDR are under investigation. These approaches include the application of the RNA interference (RNAi) technology. RNAi is a physiological mechanism triggered by small double-stranded RNA molecules resulting in a sequence-specific gene-silencing. Besides its potential for development of novel therapeutics, RNAi also offers the possibility for specific inhibition of cellular targets in functional investigations. For specific inhibition of Pgp by triggering the RNAi pathway, transient gene-silencing by application of small interfering RNA (siRNA), and stable inhibition by transfection of MDR cancer cells with short hairpin RNA (shRNA) encoding expression cassettes encoded on plasmid DNA are described. Efficacy of RNAi on MDR1 mRNA expression level is determined by quantitative real-time RT-PCR and Northern blot. The consequences of RNAi on protein expression level are measured by Western blot and immunohistochemistry. The effects on the drug extrusion activity are measured by a drug accumulation assay based on flow cytometry, and reversal of the drug-resistant phenotype by assessment of drug-specific IC(50)-values by a cell proliferation assay based on colorimetry.

Complete in vivo reversal of the multidrug resistance phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.

Triggering the RNA interference (RNAi) pathway by inducing the expression of short hairpin RNA (shRNA) molecules has become a promising tool for efficient silencing of a given gene in gene therapy applications. In this study, shRNA encoding DNA was utilized to reverse the classical MDR1/P-glycoprotein (MDR1/P-gp)-mediated multidrug resistance (MDR) phenotype in vivo. For the first time, the nonviral jet-injection technology was applied for delivering naked shRNA-vector constructs for direct intratumoral in vivo transfer. The highly efficient anti-MDR1 shRNA expression vectors were applied twice in the human MDR1/P-gp overexpressing MaTu/ADR cancer xenograft-bearing mice, and twice in the corresponding drug-sensitive parental MaTu tumor xenograft bearing mice as well. Two days after anti-MDR1 shRNA vector injection, the expression level of the MDR1 messenger RNA (mRNA) was decreased by more than 90% and the corresponding MDR1/P-gp protein was no longer detectable in the tumors. Two jet-injections of anti-MDR1 shRNA vectors into the tumors, combined with two intravenous (IV) administrations of doxorubicin, were sufficient to achieve complete reversal of the drug-resistant phenotype. The data show that jet-injection delivery of shRNA-expressing vectors is effective in reversing MDR1/P-gp-mediated MDR in vivo, and is therefore a promising strategy for making tumors with an MDR1/Pgp-dependent MDR phenotype revert to a drug-sensitive state.

Complete reversal of ABCG2-depending atypical multidrug resistance by RNA interference in human carcinoma cells.

In the chemotherapeutic treatment of patients with disseminated neoplasms, multidrug resistance (MDR) is a major obstacle. ABCG2 (BCRP/MXR), a member of the superfamily of adenosine triphosphate-binding cassette (ABC) transporters, was demonstrated to be associated with "atypical" forms of multidrug-resistant phenotypes of cancer cells. To overcome the ABCG2-depending MDR, two specific anti-ABCG2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the human gastric carcinoma cell line EPG85-257RNOV, exhibiting an atypical MDR phenotype. Because both siRNAs showed biological activity, for stable inhibition of ABCG2 corresponding short hairpin RNA (shRNA) expression vectors were constructed. By treatment of EPG85-257RNOV cells with these constructs, expression of the targeted ABCG2-encoding mRNA and transport protein was inhibited completely. Furthermore, anti-ABCG2 shRNA-treated cells increased cellular drug accumulation to the same level measured in drug-sensitive parental cells. These effects were accompanied by complete reversal of the drug-resistant phenotype. Thus, the data indicate that siRNA- and shRNA-mediated RNAi-based gene therapy may be applicable in preventing and reversing ABCG2-depending atypical MDR.

RNA interference-triggered reversal of ABCC2-dependent cisplatin resistance in human cancer cells.

The adenosine triphosphate binding cassette (ABC)-transporter ABCC2 (MRP2/cMOAT) can mediate resistance against the commonly used anticancer drugs cisplatin and paclitaxel. To overcome the ABCC2-depending drug resistance, two specific anti-ABCC2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS. Since both siRNAs showed biological activity, for stable inhibition of ABCC2 a corresponding short hairpin RNA (shRNA)-encoding expression vector was designed. By treatment of A2780RCIS cells with this construct, the expressions of the targeted ABCC2 encoding mRNA and transport protein were inhibited. These effects were accompanied by reversal of resistance against cisplatin and paclitaxel. Thus, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing and reversing ABCC2-depending drug resistance.

Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.

Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp). For stable reversal of "classical" MDR by RNA interference (RNAi) technology, an H1-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed. By introduction of anti-MDR1/P-gp shRNA expression vectors into the extremely high drug-resistant human gastric carcinoma cell line EPG85-257RDB, the MDR phenotype was completely reversed. The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells. The data indicate that stable shRNA-mediated RNAi can be tremendously effective in reversing MDR1/P-gp-mediated MDR and is therefore a promising strategy for overcoming MDR by gene therapeutic applications.

Glypican-3 is involved in cellular protection against mitoxantrone in gastric carcinoma cells.

Elevated expression of the heparan sulphate proteoglycan glypican-3 (GPC3) was found on mRNA and protein levels in the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV, which was established by in vitro selection against mitoxantrone. In order to elucidate a putative role of GPC3 in the drug-resistant phenotype, the mitoxantrone-resistant cell line EPG85-257RNOV was transfected with an expression vector construct carrying an anti-GPC3 hammerhead ribozyme. It could be demonstrated that in anti-GPC3 ribozyme-transfected cell clones, the GPC3-specific mRNA and corresponding protein expression levels were decreased to levels that are similar to those observed in nonresistant, parental cells. The anti-GPC3 ribozyme-containing clones reduced the mitoxantrone resistance level up to 21% of the original resistance and the crossresistance against etoposide to 33% of the original value. This reversal of drug resistance was accompanied by an increased cellular mitoxantrone accumulation in the anti-GPC3 ribozyme-expressing cells. In conclusion, it was verified that GPC3 is involved in the cellular protection against mitoxantrone in the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV.

Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi).

For reversal of MDR1 gene-dependent multidrug resistance (MDR), two small interfering RNA (siRNA) constructs were designed to inhibit MDR1 expression by RNA interference. SiRNA duplexes were used to treat human pancreatic carcinoma (EPP85-181RDB) and gastric carcinoma (EPG85-257RDB) cells. In both cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels. Resistance against daunorubicin was decreased to 89% (EPP85-181RDB) or 58% (EPG85-257RDB). The data indicate that this approach may be applicable to cancer patients as a specific means to reverse tumors with a P-glycoprotein-dependent MDR phenotype back to a drug-sensitive one.