Reflotron plus system

In the ILERVAS cohort, a fasting dried blood spot sample was obtained by a fingertip puncture according to standard protocols. Creatinine, uric acid, and total cholesterol levels were assessed with the REFLOTRON^® Plus system (Roche Diagnostics, Germany). It is a validated clinical chemistry system with highly correlated results to well-standardized laboratory methods (21 (link)–23 (link)). The glycosylated hemoglobin test was performed using a point-of-care instrument (Cobas B101^®, Roche Diagnostics, Germany) that meets the generally accepted performance criteria for its measurement (24 (link)). In the NEFRONA cohort, biochemical parameters were obtained from a routine fasting blood test taken no more than 3 months apart from vascular examination. GFR was estimated according to international guidelines using the CKD-EPI equation in both cohorts (25 (link)).

Plasma levels of TC, HDL, Triglycerides, and Glucose were measured with the Reflotron^® Plus system (Roche Diagnostics GmbH, Mannheim, Germany) using specific test strips. The atherogenic risk was evaluated by calculating the atherogenic index plasma (AIP) as the log (TAG/HDL-C) [53 (link)].

Naive mice were transferred with 7 × 10⁶ naive (CD44 negative) OT-I cells and infected with 1 × 10⁹ plaque-forming unit (PFU) adenovirus on day 0. Memory mice were infected with 1 × 10⁹ PFU adenovirus 30 days after OT-I cell transfer and/or immunization (see also “Generation of T cell memory”). For the induction of a cytokine-mediated hepatitis, mice were infected with 2 × 10⁹ PFU AdGOL i.v. and injected with 0.4 µg TNF i.v. on day 2 after infection. ALT levels in the serum were measured with the Reflotron®plus system (Roche).

Blood was collected in heparin tubes by direct puncture of the left heart ventricle. After centrifugation (2.000 × g for 10 min, 4°C), 32 μl blood plasma per measurement was analyzed using diagnostic test strips (Reflotron®, Roche, Germany, ALT and AST) and the Reflotron® Plus system, Roche, Germany.

Capillary blood samples from the right earlobe were taken for the analysis of lactate (Lactate Pro 2, Arkray KDK, Kyoto, Japan) and CK (Reflotron Plus System, Roche, Basel, Switzerland) in duplicate, with the mean being utilized for statistical analysis. Upon repeated measurement, the standard errors for lactate and CK were 0.1 mmol⋅l^–1 and 4.1 U⋅l^–1, respectively.

For the measurement of AST, ALT, bilirubin, and creatinine, ten male CB-17/IcrHanHsd-Prkdcscid mice (Harlan Laboratories, San Pietro al Natisone, Italy) that were 6–8 weeks old, were randomly assigned to three groups: two to the control (i.e., no treatment), four to the solvent control (10% DMSO), and four to the beauvericin treatment group (5 mg/kg bw/day beauvericin). Mice were treated in two cycles of five and four days, respectively. One day and two weeks after the last treatment, blood was collected from mice. Samples were allowed to coagulate at room temperature and serum was obtained by two centrifugation steps (1000 rpm, 10 min, 4 °C). Concentrations of AST, ALT, bilirubin, and serum creatinine were determined in serum samples by Reflotron^® Plus System (Roche, Basel, Switzerland) according to the manufacturer’s instructions.