Quantification of sirolimus by liquid chromatography-tandem mass spectrometry using on-line solid-phase extraction

Quantification of the new immunosuppressant sirolimus (syn. rapamycin; Rapamune((R))) in whole blood by chromatography is essential for its clinical use since no immunoassay is available although monitoring is mandatory. Here we report on a rapid and convenient liquid chromatography (LC)-tandem mass spectrometry method and describe our practical experience with its routine use. Whole blood samples were hemolyzed and deproteinized using an equal volume (150 mul) of a mixture of methanol/zinc sulfate solution containing the internal standard desmethoxy-rapamycin. After centrifugation, the clear supernatants were submitted to an on-line solid-phase extraction procedure using the polymeric Waters Oasis HLB(R) material, with elution of the extracts onto the analytical column in the back-flush mode by column switching. For analytical chromatography a RP-C18 column was used with 90/10 methanol/2 mM ammonium acetate as the mobile phase. A 1:10 split was used for the transfer to the mass spectrometer, a Micromass Quattro LC-tandem mass spectrometry system equipped with a Z-spray((R)) source and used in the positive electrospray ionization mode. The following transitions were recorded: sirolimus, 931>864 m/z, and desmethoxy-rapamycin (I.S.), 901>834 m/z. The analytical running time was 5 min, including on-line extraction. The method has a linear calibration curve (r>0.99; range 1.6-50 mug/l) and is rugged and precise with monthly CVs <7% at a sirolimus concentration of 13.1 mug/l in routine use; the instrumentation proved to be reliable and required minimal maintenance

Ten-Year Results of a Randomized Trial Comparing Tacrolimus Versus Cyclosporine A in Combination With Mycophenolate Mofetil After Heart Transplantation

Background. Long-term results of prospective randomized trials comparing triple immunosuppressive strategies combining tacrolimus (TAC) or cyclosporine A (CsA) with mycophenolate mofetil (MMF) and steroids after heart transplantation (HTX) are rarely published. Therefore, we collected long-term follow-up data of an intervention cohort 10 years after randomization. Methods. Ten-year follow-up data of 60 patients included in a prospective, randomized trial between 1998 and 2000 were analyzed as intention-to-treat (TAC-MMF n=30; CsA-MMF n=30). Baseline characteristics were well balanced. Cardiac allograft vasculopathy (CAV) was graduated in accordance with the new ISHLT classification. Results. Survival at 1, 5, and 10 years was 96.7%, 80.0%, and 66.7% for TAC-MMF and 90.0%, 83.3%, and 80.0% for CsA-MMF (P=ns). Freedom from acute rejection (AR) was significantly higher in TAC-MMF versus CsA-MMF (65.5% vs. 21.7%, log-rank 8.3, P=0.004). Freedom from ISHLT >= CAV(1) after 5 and 10 years was in TAC-MMF 64.0% and 45.8%, and in CsA-MMF 36.0% (log-rank 3.0, P=0.085) and 8.0% (log-rank 9.0, P=0.003). No difference in long-term results for freedom from coronary angioplasty or stenting, renal dysfunction, diabetes mellitus, CMV infection, or malignancy was detected. Conclusion. Cross-over effects because of treatment switch may result in impairment of significance between the groups. The long-term analysis resulted in a significant difference in manifestation of CAV between the groups after 10 years. Less rejection in the TAC-group might have contributed to the lower incidence of CAV. Superior freedom from AR and CAV in the TAC-MMF group did not result in better long-term survival

Pineapple Lectin AcmJRL Binds SARS-CoV-2 Spike Protein in a Carbohydrate-Dependent Fashion

The highly glycosylated spike protein of SARS-CoV-2 is essential for infection and constitutes a prime target for antiviral agents and vaccines. The pineapple-derived jacalin-related lectin AcmJRL is present in the medication bromelain in significant quantities and has previously been described to bind mannosides. Here, we performed a large ligand screening of AcmJRL by glycan array analysis, quantified the interaction with carbohydrates and validated high-mannose glycans as preferred ligands. Because the SARS-CoV-2 spike protein was previously reported to carry a high proportion of high-mannose N-glycans, we tested the binding of AcmJRL to the recombinantly produced extraviral domain of spike protein. We could demonstrate that AcmJRL binds the spike protein with a low-micromolar KD in a carbohydrate-dependent fashion

Isoflurane vs. propofol for sedation in invasively ventilated patients with acute hypoxemic respiratory failure: an a priori hypothesis substudy of a randomized controlled trial

Background Acute hypoxemic respiratory failure (AHRF) is a leading concern in critically ill patients. Experimental and clinical data suggest that early sedation with volatile anesthestics may improve arterial oxygenation and reduce the plasma and alveolar levels of markers of alveolar epithelial injury and of proinflammatory cytokines. Conclusions In patients with AHRF, inhaled sedation with isoflurane for a duration of up to 48 h did not lead to improved oxygenation in comparison to intravenous sedation with propofol. Trial registration The main study was registered in the European Medicines Agency's EU Clinical Trial register (EudraCT), 2016-004551-67, before including the first patient. The present substudy was registered at German Clinical Trials Register (DRKS, ID: DRKS00018959) on January 7th, 2020, before opening the main study data base and obtaining access to study results

Experimental Realisation of High-sensitivity Laboratory X-ray Grating-based Phase-contrast Computed Tomography

The possibility to perform high-sensitivity X-ray phase-contrast imaging with laboratory grating-based phase-contrast computed tomography (gbPC-CT) setups is of great interest for a broad range of high-resolution biomedical applications. However, achieving high sensitivity with laboratory gbPC-CT setups still poses a challenge because several factors such as the reduced flux, the polychromaticity of the spectrum, and the limited coherence of the X-ray source reduce the performance of laboratory gbPC-CT in comparison to gbPC-CT at synchrotron facilities. In this work, we present our laboratory X-ray Talbot-Lau interferometry setup operating at 40 kVp and describe how we achieve the high sensitivity yet unrivalled by any other laboratory X-ray phase-contrast technique. We provide the angular sensitivity expressed via the minimum resolvable refraction angle both in theory and experiment, and compare our data with other differential phase-contrast setups. Furthermore, we show that the good stability of our high-sensitivity setup allows for tomographic scans, by which even the electron density can be retrieved quantitatively as has been demonstrated in several preclinical studies

Prognostic Factors for Distress After Genetic Testing for Hereditary Cancer

The psychological impact of an unfavorable genetic test result for counselees at risk for hereditary cancer seems to be limited: only 10-20 % of counselees have psychological problems after testing positive for a known familial mutation. The objective of this study was to find prognostic factors that can predict which counselees are most likely to develop psychological problems after presymptomatic genetic testing. Counselees with a 50 % risk of BRCA1/2 or Lynch syndrome completed questionnaires at three time-points: after receiving a written invitation for a genetic counseling intake (T1), 2-3 days after receiving their DNA test result (T2), and 4-6 weeks later (T3). The psychological impact of the genetic test result was examined shortly and 4-6 weeks after learning their test result. Subsequently, the influence of various potentially prognostic factors on psychological impact were examined in the whole group. Data from 165 counselees were analyzed. Counselees with an unfavorable outcome did not have more emotional distress, but showed significantly more cancer worries 4-6 weeks after learning their test result. Prognostic factors for cancer worries after genetic testing were pre-existing cancer worries, being single, a high risk perception of getting cancer, and an unfavorable test result. Emotional distress was best predicted by pre-existing cancer worries and pre-existing emotional distress. The psychological impact of an unfavorable genetic test result appears considerable if it is measured as "worries about cancer." Genetic counselors should provide additional guidance to counselees with many cancer worries, emotional distress, a high risk perception or a weak social network

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