SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition.

T cell immunity is central for the control of viral infections. To characterize T cell immunity, but also for the development of vaccines, identification of exact viral T cell epitopes is fundamental. Here we identify and characterize multiple dominant and subdominant SARS-CoV-2 HLA class I and HLA-DR peptides as potential T cell epitopes in COVID-19 convalescent and unexposed individuals. SARS-CoV-2-specific peptides enabled detection of post-infectious T cell immunity, even in seronegative convalescent individuals. Cross-reactive SARS-CoV-2 peptides revealed pre-existing T cell responses in 81% of unexposed individuals and validated similarity with common cold coronaviruses, providing a functional basis for heterologous immunity in SARS-CoV-2 infection. Diversity of SARS-CoV-2 T cell responses was associated with mild symptoms of COVID-19, providing evidence that immunity requires recognition of multiple epitopes. Together, the proposed SARS-CoV-2 T cell epitopes enable identification of heterologous and post-infectious T cell immunity and facilitate development of diagnostic, preventive and therapeutic measures for COVID-19.

A COVID-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity.

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins1,2, combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18-80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4+ and CD8+ T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency.

Deletion of IFT20 exclusively in the RPE ablates primary cilia and leads to retinal degeneration.

Vision impairment places a serious burden on the aging society, affecting the lives of millions of people. Many retinal diseases are of genetic origin, of which over 50% are due to mutations in cilia-associated genes. Most research on retinal degeneration has focused on the ciliated photoreceptor cells of the retina. However, the contribution of primary cilia in other ocular cell types has largely been ignored. The retinal pigment epithelium (RPE) is a monolayer epithelium at the back of the eye intricately associated with photoreceptors and essential for visual function. It is already known that primary cilia in the RPE are critical for its development and maturation; however, it remains unclear whether this affects RPE function and retinal tissue homeostasis. We generated a conditional knockout mouse model, in which IFT20 is exclusively deleted in the RPE, ablating primary cilia. This leads to defective RPE function, followed by photoreceptor degeneration and, ultimately, vision impairment. Transcriptomic analysis offers insights into mechanisms underlying pathogenic changes, which include transcripts related to epithelial homeostasis, the visual cycle, and phagocytosis. Due to the loss of cilia exclusively in the RPE, this mouse model enables us to tease out the functional role of RPE cilia and their contribution to retinal degeneration, providing a powerful tool for basic and translational research in syndromic and non-syndromic retinal degeneration. Non-ciliary mechanisms of IFT20 in the RPE may also contribute to pathogenesis and cannot be excluded, especially considering the increasing evidence of non-ciliary functions of ciliary proteins.

IGF1 promotes human myotube differentiation toward a mature metabolic and contractile phenotype.

Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal muscle models in vitro are incapable of fully recapitulating its physiological functions especially muscle contractility. By supplementation of insulin-like growth factor 1 (IGF1), a growth factor secreted by myofibers in vivo, we aimed to overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium in daily collected samples for 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical pulse stimulation (EPS) following day 6. Myotubes without IGF1 were almost incapable of contraction. IGF1 treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7, and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of proteins of the respiratory chain and elevated mitochondrial respiration. IGF1-treatment also upregulated glucose transporter (GLUT)4 and increased insulin-dependent glucose uptake compared with myotubes differentiated without IGF1. To conclude, addition of IGF1 to serum-free medium significantly improves the differentiation of human myotubes that showed enhanced myofibril formation, response to electrical pulse stimulation, oxidative respiratory capacity, and glucose metabolism overcoming limitations of previous standards. This novel protocol enables investigation of muscular exercise on a molecular level.NEW & NOTEWORTHY Human skeletal muscle models are highly valuable to study how exercise prevents type 2 diabetes without invasive biopsies. Current models did not fully recapitulate the function of skeletal muscle especially during exercise. By supplementing insulin-like growth factor 1 (IGF1), the authors developed a functional human skeletal muscle model characterized by inducible contractility and increased oxidative and insulin-sensitive metabolism. The novel protocol overcomes the limitations of previous standards and enables investigation of exercise on a molecular level.

STE20-type kinases MST3 and MST4 promote the progression of hepatocellular carcinoma: Evidence from human cell culture and expression profiling of liver biopsies.

Hepatocellular carcinoma (HCC) is one of the most fatal and fastest growing malignancies. Recently, nonalcoholic steatohepatitis (NASH), characterized by liver steatosis, inflammation, cell injury (hepatocyte ballooning), and different stages of fibrosis, has emerged as a major catalyst for HCC. Because the STE20-type kinases, MST3 and MST4, have been described as critical molecular regulators of NASH pathophysiology, we here focused on determining the relevance of these proteins in human HCC. By analyzing public datasets and in-house cohorts, we found that hepatic MST3 and MST4 expression was positively correlated with the incidence and severity of HCC. We also found that the silencing of both MST3 and MST4, but also either of them individually, markedly suppressed the tumorigenesis of human HCC cells including attenuated proliferation, migration, invasion, and epithelial-mesenchymal transition. Mechanistic investigations revealed lower activation of STAT3 signaling in MST3/MST4-deficient hepatocytes and identified GOLGA2 and STRIPAK complex as the binding partners of both MST3 and MST4. These findings reveal that MST3 and MST4 play a critical role in promoting the progression of HCC and suggest that targeting these kinases may provide a novel strategy for the treatment of liver cancer.

Redox state and altered pyruvate metabolism contribute to a dose-dependent metformin-induced lactate production of human myotubes.

Metformin-induced glycolysis and lactate production can lead to acidosis as a life-threatening side effect, but slight increases in blood lactate levels in a physiological range were also reported in metformin-treated patients. However, how metformin increases systemic lactate concentrations is only partly understood. Because human skeletal muscle has a high capacity to produce lactate, the aim was to elucidate the dose-dependent regulation of metformin-induced lactate production and the potential contribution of skeletal muscle to blood lactate levels under metformin treatment. This was examined by using metformin treatment (16-776 µM) of primary human myotubes and by 17 days of metformin treatment in humans. As from 78 µM, metformin induced lactate production and secretion and glucose consumption. Investigating the cellular redox state by mitochondrial respirometry, we found metformin to inhibit the respiratory chain complex I (776 µM, P < 0.01) along with decreasing the [NAD+]:[NADH] ratio (776 µM, P < 0.001). RNA sequencing and phospho-immunoblot data indicate inhibition of pyruvate oxidation mediated through phosphorylation of the pyruvate dehydrogenase (PDH) complex (39 µM, P < 0.01). On the other hand, in human skeletal muscle, phosphorylation of PDH was not altered by metformin. Nonetheless, blood lactate levels were increased under metformin treatment (P < 0.05). In conclusion, the findings suggest that metformin-induced inhibition of pyruvate oxidation combined with altered cellular redox state shifts the equilibrium of the lactate dehydrogenase (LDH) reaction leading to a dose-dependent lactate production in primary human myotubes.NEW & NOTEWORTHY Metformin shifts the equilibrium of lactate dehydrogenase (LDH) reaction by low dose-induced phosphorylation of pyruvate dehydrogenase (PDH) resulting in inhibition of pyruvate oxidation and high dose-induced increase in NADH, which explains the dose-dependent lactate production of differentiated human skeletal muscle cells.

Ex vivo instability of lipids in whole blood: preanalytical recommendations for clinical lipidomics studies.

Reliability, robustness, and interlaboratory comparability of quantitative measurements is critical for clinical lipidomics studies. Lipids' different ex vivo stability in blood bears the risk of misinterpretation of data. Clear recommendations for the process of blood sample collection are required. We studied by UHPLC-high resolution mass spectrometry, as part of the "Preanalytics interest group" of the International Lipidomics Society, the stability of 417 lipid species in EDTA whole blood after exposure to either 4°C, 21°C, or 30°C at six different time points (0.5 h-24 h) to cover common daily routine conditions in clinical settings. In total, >800 samples were analyzed. 325 and 288 robust lipid species resisted 24 h exposure of EDTA whole blood to 21°C or 30°C, respectively. Most significant instabilities were detected for FA, LPE, and LPC. Based on our data, we recommend cooling whole blood at once and permanent. Plasma should be separated within 4 h, unless the focus is solely on robust lipids. Lists are provided to check the ex vivo (in)stability of distinct lipids and potential biomarkers of interest in whole blood. To conclude, our results contribute to the international efforts towards reliable and comparable clinical lipidomics data paving the way to the proper diagnostic application of distinct lipid patterns or lipid profiles in the future.

Acylated- and unacylated ghrelin during an oral glucose tolerance test in humans at risk for type 2 diabetes mellitus.

BACKGROUND/OBJECTIVES: The orexigenic peptide hormone ghrelin has been implicated in the pathophysiology of obesity and type 2 diabetes mellitus through its effects on nutrient homeostasis. Ghrelin is subject to a unique post-translational acyl modification regulating its biochemical activity. SUBJECTS/METHODS: In this study we aimed to investigate the relation of acylated (AcG) as well as unacylated ghrelin (UnG) with body weight and insulin resistance in the fasting (n = 545) and post-oral glucose tolerance test (oGTT) state (n = 245) in a metabolically well characterized cohort covering a broad range of BMI (17.95 kg/m²-76.25 kg/m²). RESULTS: Fasting AcG (median 94.2 pg/ml) and UnG (median 175.3 pg/ml) were negatively and the AcG/UnG ratio was positively correlated with BMI (all p < 0.0001). Insulin sensitivity (ISI) correlated positively with AcG (p = 0.0014) and UnG (p = 0.0004) but not with the AcG/UnG ratio. In a multivariate analysis, including ISI and BMI, only BMI, but not ISI was independently associated with AcG and UnG concentrations. Significant changes of AcG and UnG concentrations were detectable after oGTT stimulation, with slight decreases after 30 min and increases after 90-120 min. Subject stratification into BMI-divergent groups revealed more pronounced AcG increases in the two groups with BMI < 40 kg/m². CONCLUSION: Our data demonstrate lower concentrations for both AcG and UnG with increasing BMI as well as an increased proportion of the biologically active, acylated form of ghrelin giving point to pharmacologic intervention in ghrelin acylation and/or increase in UnG for treatment of obesity despite decreased absolute AcG levels.

Acute and long-term exercise adaptation of adipose tissue and skeletal muscle in humans: a matched transcriptomics approach after 8-week training-intervention.

BACKGROUND: Exercise exerts many health benefits by directly inducing molecular alterations in physically utilized skeletal muscle. Molecular adaptations of subcutaneous adipose tissue (SCAT) might also contribute to the prevention of metabolic diseases. AIM: To characterize the response of human SCAT based on changes in transcripts and mitochondrial respiration to acute and repeated bouts of exercise in comparison to skeletal muscle. METHODS: Sedentary participants (27 ± 4 yrs) with overweight or obesity underwent 8-week supervised endurance exercise 3×1h/week at 80% VO2peak. Before, 60 min after the first and last exercise bout and 5 days post intervention, biopsies were taken for transcriptomic analyses and high-resolution respirometry (n = 14, 8 female/6 male). RESULTS: In SCAT, we found 37 acutely regulated transcripts (FC > 1.2, FDR < 10%) after the first exercise bout compared to 394, respectively, in skeletal muscle. Regulation of only 5 transcripts overlapped between tissues highlighting their differential response. Upstream and enrichment analyses revealed reduced transcripts of lipid uptake, storage and lipogenesis directly after exercise in SCAT and point to ß-adrenergic regulation as potential major driver. The data also suggest an exercise-induced modulation of the circadian clock in SCAT. Neither term was associated with transcriptomic changes in skeletal muscle. No evidence for beigeing/browning was found in SCAT along with unchanged respiration. CONCLUSIONS: Adipose tissue responds completely distinct from adaptations of skeletal muscle to exercise. The acute and repeated reduction in transcripts of lipid storage and lipogenesis, interconnected with a modulated circadian rhythm, can counteract metabolic syndrome progression toward diabetes.

Arginine-stimulated copeptin in children and adolescents.

OBJECTIVE: Copeptin is secreted in isomolar amounts along with arginine vasopressin peptide (AVP) from the neurohypophysis. Its stability makes it a perfect candidate for the endocrine approach in the diagnosis of AVP deficiency (AVPD; cranial diabetes insipidus; CDI). However, pediatric reference values are lacking. DESIGN AND PATIENTS: This is a monocentric retrospective analysis of donated residual serum samples from 72 children and adolescents who underwent arginine or growth hormone-releasing hormone-arginine stimulation to test GH secretory capacity from 2018 to 2022. MEASUREMENTS: Copeptin was measured in baseline, 30-, and 60-min samples by BRAHMS Copeptin proAVP Kryptor immunofluorescence assay. RESULTS: Of the 72 patients, 4 suffered from complete AVPD (CDI). The baseline level of copeptin in the 68 non-AVPD (non-CDI) patients was highly variable (range: 1.3-44.4 pmol/L). The increase after arginine was moderate (30 min range: 1.6-40.4 pmol/L). The median baseline and peak copeptin levels were 5.6 and 8.0 pmol/L, respectively. The 2.5th percentile of the baseline and peak values of copeptin were 2.1 and 3.3 pmol/L, respectively. The increase and peak value of copeptin were inversely related to age (R = -.405; p = .011, and R = -.335; p = .0072, respectively) but not to gender, body mass index (standard deviation score) or GH secretion. In the four patients with AVPD (CDI), baseline or stimulated copeptin was below the 2.5th percentile of non-AVPD (non-CDI) patients. CONCLUSIONS: Stimulated copeptin is a promising parameter for the differential diagnosis of polyuria-polydipsia syndrome. However, the low copeptin increase after arginine and the high limit of quantification of the assay are problematic for use in paediatrics.

SARS-CoV-2-reactive T-cell receptors isolated from convalescent COVID-19 patients confer potent T-cell effector function.

Both B cells and T cells are involved in an effective immune response to SARS-CoV-2, the disease-causing virus of COVID-19. While B cells-with the indispensable help of CD4+ T cells-are essential to generate neutralizing antibodies, T cells on their own have been recognized as another major player in effective anti-SARS-CoV-2 immunity. In this report, we provide insights into the characteristics of individual HLA-A*02:01- and HLA-A*24:02-restricted SARS-CoV-2-reactive TCRs, isolated from convalescent COVID-19 patients. We observed that SARS-CoV-2-reactive T-cell populations were clearly detectable in convalescent samples and that TCRs isolated from these T cell clones were highly functional upon ectopic re-expression. The SARS-CoV-2-reactive TCRs described in this report mediated potent TCR signaling in reporter assays with low nanomolar EC50 values. We further demonstrate that these SARS-CoV-2-reactive TCRs conferred powerful T-cell effector function to primary CD8+ T cells as evident by a robust anti-SARS-CoV-2 IFN-γ response and in vitro cytotoxicity. We also provide an example of a long-lasting anti-SARS-CoV-2 memory response by reisolation of one of the retrieved TCRs 5 months after initial sampling. Taken together, these findings contribute to a better understanding of anti-SARS-CoV-2 T-cell immunity and may contribute to paving the way toward immunotherapeutics approaches targeting SARS-CoV-2.

Evaluation of a laboratory-based high-throughput SARS-CoV-2 antigen assay.

OBJECTIVES: Antigen tests are an essential part of SARS-CoV-2 testing strategies. Rapid antigen tests are easy to use but less sensitive compared to nucleic acid amplification tests (NAT) and less suitable for large-scale testing. In contrast, laboratory-based antigen tests are suitable for high-throughput immunoanalyzers. Here we evaluated the diagnostic performance of the laboratory-based Siemens Healthineers SARS-CoV-2 Antigen (CoV2Ag) assay. METHODS: In a public test center, from 447 individuals anterior nasal swab specimens as well as nasopharyngeal swab specimens were collected. The nasal swab specimens were collected in sample inactivation medium and measured using the CoV2Ag assay. The nasopharyngeal swab specimens were measured by RT-PCR. Additionally, 9,046 swab specimens obtained for screening purposes in a tertiary care hospital were analyzed and positive CoV2Ag results confirmed by NAT. RESULTS: In total, 234/447 (52.3%) participants of the public test center were positive for SARS-CoV-2-RNA. Viral lineage B1.1.529 was dominant during the study. Sensitivity and specificity of the CoV2Ag assay were 88.5% (95%CI: 83.7-91.9%) and 99.5% (97.4-99.9%), respectively. Sensitivity increased to 93.7% (97.4-99.9%) and 98.7% (97.4-99.9%) for swab specimens with cycle threshold values <30 and <25, respectively. Out of 9,046 CoV2Ag screening tests from hospitalized patients, 21 (0.2%) swab specimens were determined as false-positive by confirmatory NAT. CONCLUSIONS: Using sample tubes containing inactivation medium the laboratory-based high-throughput CoV2Ag assay is a very specific and highly sensitive assay for detection of SARS-CoV-2 antigen in nasal swab specimens including the B1.1.529 variant. In low prevalence settings confirmation of positive CoV2Ag results by SARS-CoV-2-RNA testing is recommended.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in Households with Children, Southwest Germany, May-August 2020.

Resolving the role of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in households with members from different generations is crucial for containing the current pandemic. We conducted a large-scale, multicenter, cross-sectional seroepidemiologic household transmission study in southwest Germany during May 11-August 1, 2020. We included 1,625 study participants from 405 households that each had ≥1 child and 1 reverse transcription PCR-confirmed SARS-CoV-2-infected index case-patient. The overall secondary attack rate was 31.6% and was significantly higher in exposed adults (37.5%) than in children (24.6%-29.2%; p = <0.015); the rate was also significantly higher when the index case-patient was >60 years of age (72.9%; p = 0.039). Other risk factors for infectiousness of the index case-patient were SARS-CoV-2-seropositivity (odds ratio [OR] 27.8, 95% CI 8.26-93.5), fever (OR 1.93, 95% CI 1.14-3.31), and cough (OR 2.07, 95% CI 1.21-3.53). Secondary infections in household contacts generate a substantial disease burden.

Systematic, Modifying Group-Assisted Strategy Expanding Coverage of Metabolite Annotation in Liquid Chromatography-Mass Spectrometry-Based Nontargeted Metabolomics Studies.

From microbes to human beings, nontargeted metabolic profiling by liquid chromatography (LC)-mass spectrometry (MS) has been commonly used to investigate metabolic alterations. Still, a major challenge is the annotation of metabolites from thousands of detected features. The aim of our research was to go beyond coverage of metabolite annotation in common nontargeted metabolomics studies by an integrated multistep strategy applying data-dependent acquisition (DDA)-based ultrahigh-performance liquid chromatography (UHPLC)-high-resolution mass spectrometry (HRMS) analysis followed by comprehensive neutral loss matches for characteristic metabolite modifications and database searches in a successive manner. Using pooled human urine as a model sample for method establishment, we found 22% of the detected compounds having modifying structures. Major types of metabolite modifications in urine were glucuronidation (33%), sulfation (20%), and acetylation (6%). Among the 383 annotated metabolites, 100 were confirmed by standard compounds and 50 modified metabolites not present in common databases such as human metabolite database (HMDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were structurally elucidated. Practicability was tested by the investigation of urines from pregnant women diagnosed with gestational diabetes mellitus vs healthy controls. Overall, 83 differential metabolites were annotated and 67% of them were modified metabolites including five previously unreported compounds. To conclude, the systematic modifying group-assisted strategy can be taken as a useful tool to extend the number of annotated metabolites in biological and biomedical nontargeted studies.

Noninvasive, longitudinal imaging-based analysis of body adipose tissue and water composition in a melanoma mouse model and in immune checkpoint inhibitor-treated metastatic melanoma patients.

BACKGROUND: As cancer cachexia (CC) is associated with cancer progression, early identification would be beneficial. The aim of this study was to establish a workflow for automated MRI-based segmentation of visceral (VAT) and subcutaneous adipose tissue (SCAT) and lean tissue water (LTW) in a B16 melanoma animal model, monitor diseases progression and transfer the protocol to human melanoma patients for therapy assessment. METHODS: For in vivo monitoring of CC B16 melanoma-bearing and healthy mice underwent longitudinal three-point DIXON MRI (days 3, 12, 17 after subcutaneous tumor inoculation). In a prospective clinical study, 18 metastatic melanoma patients underwent MRI before, 2 and 12 weeks after onset of checkpoint inhibitor therapy (CIT; n = 16). We employed an in-house MATLAB script for automated whole-body segmentation for detection of VAT, SCAT and LTW. RESULTS: B16 mice exhibited a CC phenotype and developed a reduced VAT volume compared to baseline (B16 - 249.8 µl, - 25%; controls + 85.3 µl, + 10%, p = 0.003) and to healthy controls. LTW was increased in controls compared to melanoma mice. Five melanoma patients responded to CIT, 7 progressed, and 6 displayed a mixed response. Responding patients exhibited a very limited variability in VAT and SCAT in contrast to others. Interestingly, the LTW was decreased in CIT responding patients (- 3.02% ± 2.67%; p = 0.0034) but increased in patients with progressive disease (+ 1.97% ± 2.19%) and mixed response (+ 4.59% ± 3.71%). CONCLUSION: MRI-based segmentation of fat and water contents adds essential additional information for monitoring the development of CC in mice and metastatic melanoma patients during CIT or other treatment approaches.

Extracellular vesicles released by myeloid-derived suppressor cells from pregnant women modulate adaptive immune responses.

Immunological pregnancy complications are a main challenge in reproductive medicine. Mechanisms regulating the adaptation of the maternal immune system to pregnancy are incompletely understood and therapeutic options limited. Myeloid derived suppressor cells (MDSC) are immune-modulatory cells expanding during healthy pregnancy and seem to play a crucial role for maternal-fetal tolerance. Recent studies showed that exosomes produced by MDSC have immune-modulatory effects corresponding to their parental cells under different pathological conditions. Here, we investigated immunological effects of exosomes of GR-MDSC during pregnancy. Isolated GR-MDSC exosomes from peripheral blood of pregnant women were tested for functionality in different in vitro assays. We show that GR-MDSC exosomes exhibited profound immune-modulatory effects such as suppression of T-cell proliferation, T helper 2 (Th2)-cell polarization, induction of regulatory T-cells and inhibition of lymphocyte cytotoxicity. Our results confirm that MDSC-derived exosomes functionally correspond to their parental cells and identify them as an interesting therapeutic target for immunological pregnancy complications.

Evaluation of the first immunosuppressive drug assay available on a fully automated LC-MS/MS-based clinical analyzer suggests a new era in laboratory medicine.

OBJECTIVES: Due to its high specificity, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the gold standard in diagnostic areas such as therapeutic monitoring of immunosuppressive drugs (ISDs). However, many laboratories still rely on immunoassays for ISD quantification in a tradeoff between analytical performance and the advantages of fully automated analyzers - shorter turnaround times, greater ease of use, and 24/7 availability. METHODS: The LC-MS/MS-based Thermo Scientific™ Cascadion™ SM Immunosuppressant Panel was evaluated for >6 months in the routine laboratory of a university hospital. We assessed the analytical performance of the panel and compared it to conventional LC-MS/MS as well as to immunoassays (cyclosporine A, sirolimus, tacrolimus (Siemens) and everolimus (Thermo Fisher)). In addition, both ISD panel and Cascadion analyzer were scrutinized with regards to, e.g., turnaround time, usability, and robustness. RESULTS: All ISDs showed high linearity and precision (CV≤6%) and a good correlation with conventional LC-MS/MS. The mean deviation to the immunoassays was 17-19% and negative for all ISDs except everolimus with a positive 19% bias. No weak points were revealed when challenging assay and system with, e.g., high haematocrit, sedimented whole blood or priority samples. The Cascadion integrated well into our 24/7 routine and could easily be operated simultaneously with several other analyzers by technical staff without LC-MS experience. CONCLUSIONS: The ISD panel showed excellent analytical performance and demonstrated that a fully automated LC-MS-based analysis starting from primary samples is feasible, suggesting that LC-MS could become an integral part of 24/7 diagnostics in the near future.

Consolidation of Reward Memory during Sleep Does Not Require Dopaminergic Activation.

Sleep enhances memories, especially if they are related to future rewards. Although dopamine has been shown to be a key determinant during reward learning, the role of dopaminergic neurotransmission for amplifying reward-related memories during sleep remains unclear. In this study, we scrutinize the idea that dopamine is needed for the preferential consolidation of rewarded information. We impaired dopaminergic neurotransmission, thereby aiming to wipe out preferential sleep-dependent consolidation of high- over low-rewarded memories during sleep. Following a double-blind, balanced, crossover design, 17 young healthy men received the dopamine d2-like receptor blocker sulpiride (800 mg) or placebo, after learning a motivated learning task. The task required participants to memorize 80 highly and 80 lowly rewarded pictures. Half of them were presented for a short (750 msec) and a long (1500 msec) duration, respectively, which permitted dissociation of the effects of reward on sleep-associated consolidation from those of mere encoding depth. Retrieval was tested after a retention interval of approximately 22 hr that included 8 hr of nocturnal sleep. As expected, at retrieval, highly rewarded memories were remembered better than lowly rewarded memories, under placebo. However, there was no evidence for an effect of reducing dopaminergic neurotransmission with sulpiride during sleep on this differential retention of rewarded information. This result indicates that dopaminergic activation likely is not required for the preferential consolidation of reward-associated memory. Rather, it appears that dopaminergic activation only tags such memories at encoding for intensified reprocessing during sleep.

Feasibility of precise and reliable glucose quantification in human whole blood samples by 1 tesla benchtop NMR.

The standard procedure for blood glucose measurements is enzymatic testing. This method is cheap, but requires small samples of open blood with direct contact to the test medium. In principle, NMR provides non-contact analysis of body fluids, but high-field spectrometers are expensive and cannot be easily utilized under clinical conditions. Low-field NMR systems with permanent magnets are becoming increasingly smaller and more affordable. The studies presented here aim at exploring the capabilities of low-field NMR for measuring glucose concentrations in whole blood. For this purpose, a modern 1 T benchtop NMR spectrometer was used. Challenges arise from broad spectral lines, the glucose peak locations close to the water signal, low SNR and the interference with signals from other blood components. Whole blood as a sample comprises even more boundary conditions: crucial for reliable results are avoiding the separation of plasma and cells by gravitation and reliable reference values. First, the accuracy of glucose levels measured by NMR was tested using aqueous glucose solutions and commercially available bovine plasma. Then, 117 blood samples from oral glucose tolerance testing were measured with minimal preparation by simple pulse-acquire NMR experiments. The analysis itself is the key to achieve high precision, so several approaches were investigated: peak integration, orthogonal projection to latent structure analysis and support vector machine regression. Correlations between results from the NMR spectra and the routine laboratory automated analyzer revealed an RMSE of 7.90 mg/dL for the best model. 91.5% of the model output lies within the limits of the German Medical Association guidelines, which require the glucose measurement to be within 11% of the reference method. It is concluded that spectral quantification of glucose in whole blood samples by high-quality NMR spectrometers operating at 1 T is feasible with sufficient accuracy.

Insulin Action in the Hypothalamus Increases Second-Phase Insulin Secretion in Humans.

BACKGROUND: Animal studies and initial correlative data in humans indicate that insulin action in the brain may affect pancreatic insulin secretion. An important brain region for this process is the hypothalamus, an area that can develop insulin resistance. METHODS: Fifteen young, healthy men (27 ± 3 years) with a wide BMI spectrum (20-30 kg/m2) underwent 2 hyperglycemic clamps (target blood glucose: 10 mmol/L). In this double-blind study, subjects received 160 U of insulin or placebo as a nasal spray on 2 days in randomized order. On another day, insulin sensitivity of the hypothalamus was determined by functional magnetic resonance imaging. RESULTS: Glucose levels were comparable on both study days. In the whole group, C-peptide levels were not significantly different between conditions. Though, there was a significant interaction between insulin sensitivity of the hypothalamus × nasal spray × time on C-peptide levels (p = 10-6). The group was therefore divided according to median hypothalamic insulin sensitivity. C-peptide concentrations were higher after intranasal insulin compared to placebo spray in the group with a strong hypothalamic insulin response (p < 0.0001, ß = 6.00 ± 1.24) and lower in the brain insulin-resistant group (p = 0.005, ß = -2.68 ± 0.95). Neither somatostatin nor glucagon kinetics was altered by the nasal spray. CONCLUSIONS: In participants with high hypothalamic insulin sensitivity, insulin action in the brain enhanced second-phase insulin secretion from pancreatic beta cells. This reaction could, for example, contribute to late postprandial glucose regulation by suppressing hepatic glucose production by portal venous insulin.