S100A9 is indispensable for survival of pneumococcal pneumonia in mice.

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn2+ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620).

Immunosuppressant Monitoring-Performance of the First Mass Spectrometry-Based Automated Clinical Analyzer Cascadion.

BACKGROUND: Automatic analyzers simplify processes and may help improve standardization. The first automated analyzer based on mass spectrometry is available and offers a panel for monitoring cyclosporin A, tacrolimus, sirolimus, and everolimus. Method comparisons and evaluation tests are presented to verify the capability of the Cascadion system for use in a clinical laboratory. METHODS: Sample preparation and measurements were performed using the Cascadion clinical analyzer. More than 1000 measurement values of patient samples were compared with an in vitro diagnostic-certified assay run on a liquid chromatography tandem mass spectrometry instrument. Precision and accuracy were determined using commercial quality control and external quality assessment (EQA) samples. RESULTS: A good correlation between the 2 instruments was observed (Pearson correlation r = 0.956-0.996). Deming regression revealed 95% confidence intervals of slopes and intercepts covering the values 1 and 0, for sirolimus and everolimus, respectively, indicating equivalence of both measuring systems. However, for cyclosporin A, a bias was observed and confirmed using a Bland-Altman plot (-9.1%). Measurement repeatability and intermediate measurement precision were appropriate showing coefficients of variation of 0.9%-6.1% and 2.0%-5.3%, respectively. Accuracy according to internal quality controls was 85%-111% and 81%-100% in the EQA samples of Reference Institute of Bioanalytics and Laboratory of the Government Chemist, respectively. High robustness was found with regard to the linearity of the calibration lines (linear regression coefficient r2 > 0.99). Carryover was negligible (0.1%). CONCLUSIONS: The Cascadion automatic analyzer produced convincing results in the measurement of patient, control, and EQA samples. The throughput was sufficient for routine use. Overall, it can be used as an alternative to open liquid chromatography tandem mass spectrometry instruments for immunosuppressant monitoring, simplifying processes without the need for specially trained personnel.

The urinary proteomics classifier chronic kidney disease 273 predicts cardiovascular outcome in patients with chronic kidney disease.

BACKGROUND: The urinary proteomic classifier chronic kidney disease 273 (CKD273) is predictive for the development and progression of chronic kidney disease (CKD) and/or albuminuria in type 2 diabetes. This study evaluates its role in the prediction of cardiovascular (CV) events in patients with CKD Stages G1-G5. METHODS: We applied the CKD273 classifier in a cohort of 451 patients with CKD Stages G1-G5 followed prospectively for a median of 5.5 years. Primary endpoints were all-cause mortality, CV mortality and the composite of non-fatal and fatal CV events (CVEs). RESULTS: In multivariate Cox regression models adjusting for age, sex, prevalent diabetes and CV history, the CKD273 classifier at baseline was significantly associated with total mortality and time to fatal or non-fatal CVE, but not CV mortality. Because of a significant interaction between CKD273 and CV history (P = 0.018) and CKD stages (P = 0.002), a stratified analysis was performed. In the fully adjusted models, CKD273 classifier was a strong and independent predictor of fatal or non-fatal CVE only in the subgroup of patients with CKD Stages G1-G3b and without a history of CV disease. In those patients, the highest tertile of CKD273 was associated with a >10-fold increased risk as compared with the lowest tertile. CONCLUSIONS: The urinary CKD273 classifier provides additional independent information regarding the CV risk in patients with early CKD stage and a blank CV history. Determination of CKD273 scores on a random urine sample may improve the efficacy of intensified surveillance and preventive strategies by selecting patients who potentially will benefit most from early risk management.

Adherence in pediatric renal recipients and its effect on graft outcome, a single-center, retrospective study.

BACKGROUND: In recent years, treatment-adherence gained increasing attention in nearly every area of medicine including transplant medicine. Medication adherence following solid organ transplantation is known to be indispensable for a satisfactory allograft survival. METHODS: We examined 60 patients between the ages of four months and 20 years who underwent kidney transplantation at Hannover Medical School between January 2011 and August 2017. Age at transplantation varied from 4 months to 20 years. 12 patients (20%) already underwent their second solid organ transplantation. 5 patients (8.3%) had a combined kidney-liver-transplantation. We used two different methods for rating adherence: An objective one based on the coefficient of variation (CoV%) of immunosuppressant trough levels, and a subjective questionnaire answered by the patients themselves, their parents or legal custodians, the treating pediatrician, as well as by the attending psychologist. RESULTS: The CoV% in our study was by-trend higher in those patients who suffered from a biopsy-proven rejection (x̅CoV% = 35.7, σ CoV% = 30.1 in patients with rejection vs. x̅ CoV% = 26.0, σ CoV% = 10.5 in patients without rejection). Furthermore, the psychologist's assessment correlated significantly both with rejections as well as with the formation of de novo donor-specific antibodies (dnDSA) while the pediatrician's rating showed no correlation (Prejections  = 0.005 and PdnDSA  = 0.03 for psychologist's rating vs. Prejections  = 0.50 and PdnDSA  = 0.50 for pediatrician). CONCLUSIONS: Apart from underlining the importance of medication adherence, the present research stresses the role of a multi-disciplinary treatment approach to support pediatric renal transplant recipients and their families.

Effect of Therapeutic Plasma Exchange on Immunoglobulin Deficiency in Early and Severe Septic Shock.

BACKGROUND: Deficiency of immunoglobulins of the classes IgG, IgG1, IgA and IgM is associated with severity of disease and mortality in sepsis and septic shock. Therapeutic plasma exchange (TPE) with fresh frozen plasma (FFP) has recently gained attention as an adjunctive therapeutic option in early septic shock. We hypothesized that TPE might modulate immunoglobulin deficiencies besides sole elimination of circulating injurious molecules. METHODS: We conducted a prospective single center study with TPE in 33 patients with early septic shock (onset < 12 h) requiring high doses of norepinephrine (NE > 0.4µg/kg/min). Clinical and biochemical data, including measurement of immunoglobulin subgroups IgG, IgG1, IgM and IgA were obtained before and after TPE. The following immunoglobulin cut-off values were used to analyze subgroups with low immunoglobulin concentrations at baseline (IgG ≤ 6.5, IgG1 ≤ 3, IgM ≤ 1.5 and IgA ≤ 0.35 g/L). RESULTS: At inclusion, median (IQR) SOFA score was 18 (15-20) and NE dose was 0.8 (0.6-1.2) µg/kg/min. The majority of patients demonstrated profound reductions in immunoglobulins levels of all classes. Globally, immunoglobulin levels were not significantly changed after a single TPE session. However, in patients with low baseline immunoglobulin levels a significant increase in all classes was observed (IgG 1.92 (0.96-3) g/L (+41%), IgG1 2.1 (1.46-2.32) g/L (+96%), IgA 0.44 (0.12-0.62) g/L (59%) and IgM 0.18 (0.14-0.34) g/L (+55%), p < 0.001 for comparison to patients above cut-off). CONCLUSIONS: The majority of early and severe septic shock patients had reduced immunoglobulin levels and a single TPE could attenuate immunoglobulin deficiencies of all classes. The clinical relevance of this observation has to be investigated in a proper designed trial.

A systematic review of subgroup analyses in randomised clinical trials in cardiovascular disease.

BACKGROUND: Subgroup analyses are frequently used to assess heterogeneity of treatment effects in randomised clinical trials. Inconsistent, improper and incomplete implementation, reporting and interpretation have been identified as ongoing challenges. Further, subgroup analyses were frequently criticised because of unreliable or potentially misleading results. More recently, recommendations and guidelines have been provided to improve the reporting of data in this regard. METHODS: This systematic review was based on a literature search within the digital archives of three selected medical journals, The New England Journal of Medicine, The Lancet and Circulation. We reviewed articles of randomised clinical trials in the domain of cardiovascular disease which were published in 2015 and 2016. We screened and evaluated the selected articles for the mode of implementation and reporting of subgroup analyses. RESULTS: We were able to identify a total of 130 eligible publications of randomised clinical trials. In 89/130 (68%) articles, results of at least one subgroup analysis were presented. This was dependent on the considered journal (p < 0.001), the number of included patients (p < 0.001) and the lack of statistical significance of a trial's primary analysis (p < 0.001). The number of reported subgroup analyses ranged from 1 to 101 (median = 13). We were able to comprehend the specification time of reported subgroup analyses for 71/89 (80%) articles, with 55/89 (62%) articles presenting exclusively pre-specified analyses. This information was not always traceable on the basis of provided trial protocols and often did not include the pre-definition of cut-off values for the categorization of subgroups. The use of interaction tests was reported in 84/89 (94%) articles, with 36/89 (40%) articles reporting heterogeneity of the treatment effect for at least one primary or secondary trial outcome. Subgroup analyses were reported more frequently for larger randomised clinical trials, and if primary analyses did not reach statistical significance. Information about the implementation of subgroup analyses was reported most consistently for articles from The New England Journal of Medicine, since it was also traceable on the basis of provided trial protocols. We were able to comprehend whether subgroup analyses were pre-specified in a majority of the reviewed publications. Even though results of multiple subgroup analyses were reported for most published trials, a corresponding adjustment for multiple testing was rarely considered. CONCLUSION: Compared to previous reviews in this context, we observed improvements in the reporting of subgroup analyses of cardiovascular randomised clinical trials. Nonetheless, critical shortcomings, such as inconsistent reporting of the implementation and insufficient pre-specification, persist.

Total haemoglobin - a reference measuring system for improvement of standardisation.

Background Total haemoglobin (Hb) concentration in blood belongs to the most requested measurands, and the HiCN method (hemiglobincyanide) is accepted as a reference. Although the reaction principle is clearly characterised, measurement conditions and settings are not consistently defined, some of them influencing the results. An improvement of standardisation is the object. Methods After method optimization, measurement results between different calibration laboratories (CL) were compared with each other and also with results of the National Metrology Institute of Germany (PTB), with target values of certified reference material, within the RELA scheme, and to >1500 results from routine laboratories. Results Overall deviations between three CLs were ≤0.5% (n = 24 samples) in a measurement range of 20 g/L to 300 g/L. A CV of 0.4% was determined in pooled blood (1 year long-term imprecision, 99.0%-101.1% recovery of the mean). For selected measurements (n = 4 samples) the PTB participated without significant differences to three CLs, and no significant differences were observed comparing CLs to certified values of reference materials. The expanded measurement uncertainty (probability 95%) was estimated as 1.1%. Conclusions A reference measuring system, comprising measuring instruments and other devices, including reagents and supply, to generate reference measurement values for total Hb concentration of high accuracy and low measurement uncertainty is presented. Measurement parameters are investigated and defined. The reference measuring system is ready to offer service to EQA providers and to the IVD industry for certifying control materials or calibrators.

Using ISO/TS 20914:2019 to calculate the measurement uncertainty of immunosuppressive drugs in a clinical laboratory.

According to the standard ISO 15189 clinical routine laboratories shall estimate measurement uncertainty (MU) of patient results of their provided measurands. Up to now there was no accepted description on how to perform. Recently, the ISO technical standard ISO/TS 20914 was published giving a practical guide for uncertainty estimation. The immunosuppressive drugs Everolimus, Ciclosporin, Sirolimus and Tacrolimus have narrow therapeutic windows. Hence, their MU should be considered for deducing clinical decisions. Here, a pathway is presented in detail on how to estimate MU measuring immunosuppressants using a widespread CE certified assay via LC-MS/MS technology. Namely, the expanded measurement uncertainties are from 13% to 27% depending on analyte and concentration. The calculation based on n > 2000 measurements each of four control levels within one year. Lower uncertainties were observed if the material was native pooled blood (13% to 17%, n > 300 measurements, one year).

Development and evaluation of point-of-care testing recertification with e-learning.

One of the main requirements in point-of-care testing (POCT) is efficient operator training to avoid diagnostic errors. Considering a variety of users and time-independent learning, e-learning is preferred. However, in our experience, e-learning is not always accepted by employees. After using a commercial e-learning program with little success, we developed a specific e-learning offer to achieve a better acceptance of online-based training. Herein our goal was to identify the most relevant aspects for better acceptance. The new e-learning modules were implemented with the learning management system ILIAS and dealt with typical sources of error. The implementation was accompanied by an anonymous online questionnaire within the POCT operators examining differences between the acceptance of the commercial e-learning and the hospital-specific. The results show higher acceptance for clinic-specific e-learning whereby online training of the POCT operators could successfully established. Most relevant aspects are the relevance of contents for the personal work and the working processes within the clinic as well as processing time. Thereby, the recertification of the POCT operators based on the successful completion of the learning modules was fully integrated in the POCT process. In respect to the need for regular recertification of POCT operators, our study shows that the acceptance of e-learning could be improved by adapting e-learning modules to the specific workflows in the hospital.

Lipid nanoparticle-mediated siRNA delivery for safe targeting of human CML in vivo.

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.

Association of Human FOS Promoter Variants with the Occurrence of Knee-Osteoarthritis in a Case Control Association Study.

Our aim was to analyse (i) the presence of single nucleotide polymorphisms (SNPs) in the JUN and FOS core promoters in patients with rheumatoid arthritis (RA), knee-osteoarthritis (OA), and normal controls (NC); (ii) their functional influence on JUN/FOS transcription levels; and (iii) their associations with the occurrence of RA or knee-OA. JUN and FOS promoter SNPs were identified in an initial screening population using the Non-Isotopic RNase Cleavage Assay (NIRCA); their functional influence was analysed using reporter gene assays. Genotyping was done in RA (n = 298), knee-OA (n = 277), and NC (n = 484) samples. For replication, significant associations were validated in a Finnish cohort (OA: n = 72, NC: n = 548). Initially, two SNPs were detected in the JUN promoter and two additional SNPs in the FOS promoter in perfect linkage disequilibrium (LD). JUN promoter SNP rs4647009 caused significant downregulation of reporter gene expression, whereas reporter gene expression was significantly upregulated in the presence of the FOS promoter SNPs. The homozygous genotype of FOS promoter SNPs showed an association with the susceptibility for knee-OA (odds ratio (OR) 2.12, 95% confidence interval (CI) 1.2⁻3.7, p = 0.0086). This association was successfully replicated in the Finnish Health 2000 study cohort (allelic OR 1.72, 95% CI 1.2⁻2.5, p = 0.006). FOS Promoter variants may represent relevant susceptibility markers for knee-OA.

Cellular and Molecular Effects of High-Molecular-Weight Heparin on Matrix Metalloproteinase 9 Expression.

Blood sampling with different anticoagulants alters matrix metalloproteinase (MMP-) 9 expression, thus influencing its concentration and diagnostic validity. Here, we aimed to evaluate the effects of different anticoagulants on MMP-9 regulation. MMP-9 expression was assessed in response to ethylenediaminetetraacetic acid, citrate, and high-/low-molecular-weight heparin (HMWH, LMWH) in co-culture experiments using THP-1, Jurkat, and HT cells (representing monocytes, T, and B cells). Triple and double cell line co-culture experiments revealed that HMWH treatment of THP-1 and Jurkat led to a significant MMP-9 induction, whereas other anticoagulants and cell type combinations had no effect. Supernatant of HMWH-treated Jurkat cells also induced MMP-9 in THP-1 suggesting monocytes as MMP-9 producers. HMWH-induced cytokine/chemokine secretion was assessed in co-culture supernatant, and the influence of cytokines/chemokines on MMP-9 production was analyzed. These experiments revealed that Jurkat-derived IL-16 and soluble intercellular adhesion molecule (sICAM-) 1 are able to induce MMP-9 and IL-8 production by THP-1. As a consequence, the increased MMP-9 expression found in HMWH blood samples may be influenced by HMWH-dependent secretion of IL-16 and sICAM-1 by T cells resulting in an increased production of MMP-9 and IL-8 by monocytes. IL-8, in turn, may support MMP-9 and its own expression in a positive autocrine feedback loop.

Proteome and Phosphoproteome Analysis in TNF Long Term-Exposed Primary Human Monocytes.

To better understand the inflammation-associated mechanisms modulating and terminating tumor necrosis factor (TNF-)induced signal transduction and the development of TNF tolerance, we analyzed both the proteome and the phosphoproteome in TNF long term-incubated (i.e., 48 h) primary human monocytes using liquid chromatography-mass spectrometry. Our analyses revealed the presence of a defined set of proteins characterized by reproducible changes in expression and phosphorylation patterns in long term TNF-treated samples. In total, 148 proteins and 569 phosphopeptides were significantly regulated (103 proteins increased, 45 proteins decreased; 377 peptides with increased and 192 peptides with decreased phosphorylation). A variety of these proteins are associated with the non-canonical nuclear factor κB (NF-κB) pathway (nuclear factor κB (NFKB) 2, v-rel reticuloendotheliosis viral oncogene homolog (REL) B, indolamin-2,3-dioxygenase (IDO), kynureninase (KYNU)) or involved in the negative regulation of the canonical NF-κB system. Within the phosphopeptides, binding motifs for specific kinases were identified. Glycogen synthase kinase (GSK) 3 proved to be a promising candidate, since it targets NF-κB inhibiting factors, such as CCAAT/enhancer binding protein (C/EBP) ß. Our experiments demonstrate that both proteome and phosphoproteome analysis can be effectively applied to study protein/phosphorylation patterns of primary monocytes. These results provide new regulatory candidates and evidence for a complex network of specific but synergistically acting/cooperating mechanisms enabling the affected cells to resist sustained TNF exposure and resulting in the resolution of inflammation.

Identification of two forms of TNF tolerance in human monocytes: differential inhibition of NF-κB/AP-1- and PP1-associated signaling.

The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we observed blockade of IκBα phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IκBα levels were found, accompanied by increased IκBα degradation, suggesting an increased IκBα turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser(536), p38, and c-jun was inhibited, and basal inhibitory p65-Ser(468) phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and protein phosphatase 1-dependent mechanisms. To our knowledge, we present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-κB/AP-1-associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inflammation and may be involved in immune paralysis.

Lipopolysaccharide binding protein, interleukin-10, interleukin-6 and C-reactive protein blood levels in acute ischemic stroke patients with post-stroke infection.

BACKGROUND: Ischemic stroke patients are prone to infection by stroke-induced immunodepression. We hypothesized that levels of lipopolysaccharide binding protein (LBP), interleukin-10 (IL-10), IL-6 and C-reactive protein (CRP) are early predictors for the development of stroke-associated infection. METHODS: Fifty-six patients with ischemic stroke (n = 51) and transient ischemic attack (TIA) (n = 5) who presented within 6 hours after symptom onset and who were free of detectable infection on admission were included in the study. Of these, 20 developed early infections during the first week. Blood samples were taken at 6, 12, and 24 hours and at 3 and 7 days after stroke onset. Levels of LBP, Il-10, IL-6 and CRP, as well as S100B, were measured as markers of inflammation and brain damage by commercially available immunometric tests. RESULTS: In the univariate analysis, levels of LBP, IL-10, IL-6 and CRP significantly differed between patients who developed an infection and those who did not. In the binary logistic regression analysis, which was adjusted for National Institutes of Health Stroke Scale (NIHSS) on admission, stroke subtype and S100B peak levels, as indicator of the extent of brain damage, IL-10 at 6 hours, CRP at 6 hours and NIHSS on admission were identified as independent predictors of infection (IL-10: P = 0.009; CRP: P = 0.018; NIHSS: P = 0.041). The area under the curve (AUC) of the receiver operating characteristic (ROC) curves in relation to the dichotomized status of the infection (infection versus no infection) was 0.74 (95% confidence interval: 0.59 to 0.88) for CRP at 6 hours, 0.76 (0.61 to 0.9) for IL-10 at 6 hours, 0.83 (0.71 to 0.94) for NIHSS on admission and 0.94 (0.88 to 1) for the combination of CRP, IL-10 and NIHSS. In a subanalysis, 16 patients with early infections were matched with 16 patients without infection according to S100B peak levels. Here, the temporal pattern of LBP, IL-10, IL-6 and CRP significantly differed between the patient groups. CONCLUSIONS: Our data show that blood levels of inflammation markers may be used as early predictors of stroke-associated infection. We propose prospective studies to investigate if the calculated cut-offs of CRP, IL-10 and NIHSS might help to identify patients who should receive early preventive antibiotic treatment.

Performance of the New RapidFire System for Therapeutic Monitoring of Immunosuppressants.

BACKGROUND: Typical runtimes for quantitation of immunosuppressants in whole blood based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) are in the order of a few minutes per sample. The Agilent RapidFire-MS/MS system uses solid phase extraction cartridges instead of chromatography columns and benefits from runtimes below 15 seconds. The purpose of this study was to figure out if this high-throughput instrument could be capable for application in the clinical laboratory to quantify cyclosporine A, everolimus, sirolimus, and tacrolimus. METHODS: 1172 measurement results from patient samples were compared between our routinely used LC-MS/MS system and the RapidFire 360 coupled to a tandem mass spectrometer. Sample preparation and routine measurement were performed using a certified kit. Imprecision and accuracy were investigated analyzing 3 commercial quality controls of different concentrations. RESULTS: Both measurement procedures showed excellent agreement (Pearson correlation coefficients r = 0.964-0.995). Deming regression revealed confidence intervals (95%) for slopes and intercepts of the linear equation, which covered the values 1 and 0, respectively, in almost all cases. The relative differences between both methods were marginal (1.7%-2.9%). Good intraday precision (coefficients of variation, 0.7%-9.0%) and interday precision (coefficients of variation, 2.8%-8.4%) for all immunosuppressants were achieved. The recovery of target concentrations was 81%-116%. High robustness was found with regard to linearity of the calibration lines (linear regression coefficients r ≥ 0.99). There was only negligible carry-over (cyclosporine A, 0.07%) and high endurance of the solid phase extraction cartridge (>3000 injections). CONCLUSIONS: The RapidFire-MS/MS system provided convincing results measuring patient samples and quality control materials. Together with a high throughput and a high robustness, it could represent an alternative to LC-MS/MS instruments in therapeutic drug monitoring.

Regulation of monocyte differentiation by specific signaling modules and associated transcription factor networks.

Monocyte/macrophages are important players in orchestrating the immune response as well as connecting innate and adaptive immunity. Myelopoiesis and monopoiesis are characterized by the interplay between expansion of stem/progenitor cells and progression towards further developed (myelo)monocytic phenotypes. In response to a variety of differentiation-inducing stimuli, various prominent signaling pathways are activated. Subsequently, specific transcription factors are induced, regulating cell proliferation and maturation. This review article focuses on the integration of signaling modules and transcriptional networks involved in the determination of monocytic differentiation.

The urinary proteome as correlate and predictor of renal function in a population study.

BACKGROUND: We investigate whether the urinary proteome refines the diagnosis of renal dysfunction, which affects over 10% of the adult population. METHODS: We measured serum creatinine, estimated glomerular filtration rate (eGFR) and 24-h albuminuria in 797 people randomly recruited from a population. We applied capillary electrophoresis coupled with mass spectrometry to measure multi-dimensional urinary proteomic classifiers developed for renal dysfunction (CKD273) or left ventricular dysfunction (HF1 and HF2). Renal function was followed up in 621 participants and the incidence of cardiovascular events in the whole study population. RESULTS: In multivariable-adjusted cross-sectional analyses, higher biomarker levels analysed separately or combined by principal component analysis into a single factor (SF), correlated (P ≤ 0.010) with worse renal function. Over 4.8 years, higher HF1 and SF predicted (P ≤ 0.014) lowering of eGFR; higher HF2 predicted (P ≤ 0.049) increase in serum creatinine and decrease eGFR. HF1, HF2 and SF predicted progression from CKD Stages 2 or ≤2 to Stage ≥3, with risk estimates for a 1-SD increment in the urinary biomarkers ranging from 38 to 71% (P ≤ 0.039). HF1, HF2 and SF yielded a net reclassification improvement of 31-51% (P ≤ 0.029). Over 6.1 years, 47 cardiovascular events occurred. HF2 and SF, independent of baseline eGFR, 24-h albuminuria and other covariables were significant predictors of cardiovascular complications with risk estimates for 1-SD increases ranging from 32 to 41% (P ≤ 0.047). CONCLUSIONS: The urinary proteome refines the diagnosis of existing or progressing renal dysfunction and predicts cardiovascular complications.

Multicentre prospective validation of a urinary peptidome-based classifier for the diagnosis of type 2 diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is one of the major late complications of diabetes. Treatment aimed at slowing down the progression of DN is available but methods for early and definitive detection of DN progression are currently lacking. The 'Proteomic prediction and Renin angiotensin aldosterone system Inhibition prevention Of early diabetic nephRopathy In TYpe 2 diabetic patients with normoalbuminuria trial' (PRIORITY) aims to evaluate the early detection of DN in patients with type 2 diabetes (T2D) using a urinary proteome-based classifier (CKD273). METHODS: In this ancillary study of the recently initiated PRIORITY trial we aimed to validate for the first time the CKD273 classifier in a multicentre (9 different institutions providing samples from 165 T2D patients) prospective setting. In addition we also investigated the influence of sample containers, age and gender on the CKD273 classifier. RESULTS: We observed a high consistency of the CKD273 classification scores across the different centres with areas under the curves ranging from 0.95 to 1.00. The classifier was independent of age (range tested 16-89 years) and gender. Furthermore, the use of different urine storage containers did not affect the classification scores. Analysis of the distribution of the individual peptides of the classifier over the nine different centres showed that fragments of blood-derived and extracellular matrix proteins were the most consistently found. CONCLUSION: We provide for the first time validation of this urinary proteome-based classifier in a multicentre prospective setting and show the suitability of the CKD273 classifier to be used in the PRIORITY trial.

Factors influencing voriconazole plasma level in intensive care patients.

Background: In clinical routine, voriconazole plasma trough levels (Cmin) out of target range are often observed with little knowledge about predisposing influences. Objectives: To determine the distribution and influencing factors on voriconazole blood levels of patients treated on intensive- or intermediate care units (ICU/IMC). Patients and methods: Data were collected retrospectively from patients with at least one voriconazole trough plasma level on ICU/IMC (n = 153) to determine the proportion of sub-, supra- or therapeutic plasma levels. Ordinal logistic regression analysis was used to assess factors hindering patients to reach voriconazole target range. Results: Of 153 patients, only 71 (46%) reached the target range at the first therapeutic drug monitoring, whereas 66 (43%) patients experienced too-low and 16 (10%) too-high plasma levels. Ordinal logistic regression analysis identified the use of extra corporeal membrane oxygenation (ECMO), low international normalized ratio (INR) and aspartate-aminotransferase (AST) serum levels as predictors for too-low plasma levels. Conclusion: Our data highlight an association of ECMO, INR and AST levels with voriconazole plasma levels, which should be considered in the care of critically ill patients to optimize antifungal therapy with voriconazole.