Quantification of Bile Acids in Cerebrospinal Fluid: Results of an Observational Trial.

(1) Background: Bile acids, known as aids in intestinal fat digestion and as messenger molecules in serum, can be detected in cerebrospinal fluid (CSF), although the blood-brain barrier is generally an insurmountable obstacle for bile acids. The exact mechanisms of the occurrence, as well as possible functions of bile acids in the central nervous system, are not precisely understood. (2) Methods: We conducted a single-center observational trial. The concentrations of 15 individual bile acids were determined using an in-house LC-MS/MS method in 54 patients with various acute and severe disorders of the central nervous system. We analyzed CSF from ventricular drainage taken within 24 h after placement, and blood samples were drawn at the same time for the presence and quantifiability of 15 individual bile acids. (3) Results: At a median time of 19.75 h after a cerebral insult, the concentration of bile acids in the CSF was minute and almost negligible. The CSF concentrations of total bile acids (TBAs) were significantly lower compared to the serum concentrations (serum 0.37 µmol/L [0.24, 0.89] vs. 0.14 µmol/L [0.05, 0.43]; p = 0.033). The ratio of serum-to-CSF bile acid levels calculated from the respective total concentrations were 3.10 [0.94, 14.64] for total bile acids, 3.05 for taurocholic acid, 14.30 [1.11, 27.13] for glycocholic acid, 0.0 for chenodeoxycholic acid, 2.19 for taurochenodeoxycholic acid, 1.91 [0.68, 8.64] for glycochenodeoxycholic acid and 0.77 [0.0, 13.79] for deoxycholic acid; other bile acids were not detected in the CSF. The ratio of CSF-to-serum S100 concentration was 0.01 [0.0, 0.02]. Serum total and conjugated (but not unconjugated) bilirubin levels and serum TBA levels were significantly correlated (total bilirubin p = 0.031 [0.023, 0.579]; conjugated bilirubin p = 0.001 [0.193, 0.683]; unconjugated p = 0.387 [-0.181, 0.426]). No correlations were found between bile acid concentrations and age, delirium, intraventricular blood volume, or outcome measured on a modified Rankin scale. (4) Conclusions: The determination of individual bile acids is feasible using the current LC-MS/MS method. The results suggest an intact blood-brain barrier in the patients studied. However, bile acids were detected in the CSF, which could have been achieved by active transport across the blood-brain barrier.

Humoral and cellular immune responses in fully vaccinated individuals with or without SARS-CoV-2 breakthrough infection: Results from the CoV-ADAPT cohort.

Despite recent advances in prophylactic vaccination, SARS-CoV-2 infections continue to cause significant morbidity. A better understanding of immune response differences between vaccinated individuals with and without later SARS-CoV-2 breakthrough infection is urgently needed. CoV-ADAPT is a prospective long-term study comparing humoral (anti-spike-RBD-IgG, neutralization capacity, avidity) and cellular (spike-induced T-cell interferon-γ [IFN-γ] release) immune responses in individuals vaccinated against SARS-CoV-2 at four different time points (three before and one after third vaccination). In this cohort study, 62 fully vaccinated individuals presented with SARS-CoV-2 breakthrough infections vs 151 without infection 3-7 months following third vaccination. Breakthrough infections significantly increased anti-spike-RBD-IgG (p < 0.01), but not spike-directed T-cell IFN-γ release (TC) or antibody avidity. Despite comparable surrogate neutralization indices, the functional neutralization capacity against SARS-CoV-2-assessed via a tissue culture-based assay-was significantly higher following breakthrough vs no breakthrough infection. Anti-spike-RBD-IgG and antibody avidity decreased with age (p < 0.01) and females showed higher anti-spike-RBD-IgG (p < 0.01), and a tendency towards higher antibody avidity (p = 0.051). The association between humoral and cellular immune responses previously reported at various time points was lost in subjects after breakthrough infections (p = 0.807). Finally, a machine-learning approach based on our large immunological dataset (a total of 49 variables) from different time points was unable to predict breakthrough infections (area under the curve: 0.55). In conclusion, distinct differences in humoral vs cellular immune responses in fully vaccinated individuals with or without breakthrough infection could be demonstrated. Breakthrough infections predominantly drive the humoral response without boosting the cellular component. Breakthrough infections could not be predicted based on immunological data, which indicates a superior role of environmental factors (e.g., virus exposure) in individualized risk assessment.

Biomarker profiles in heart failure with preserved vs. reduced ejection fraction: results from the DIAST-CHF study.

AIMS: Chronic heart failure (HF) is a common disease and one of the leading causes of death worldwide. Heart failure with preserved ejection fraction (HFpEF) and with reduced ejection fraction (HFrEF) are different diseases with distinct as well as comparable pathophysiologies and diverse responses to therapeutic agents. We aimed to identify possible pathobiochemical signalling pathways and biomarkers in HFpEF and HFrEF by using a broad proteomic approach. METHODS AND RESULTS: A total of 180 biomarkers in the plasma of a representative subgroup (71 years old) of HFpEF (70% female) with a left ventricular ejection fraction (LVEF) ≥ 50% and HFrEF (18% female) with an LVEF ≤ 40% patients (n = 127) from the Prevalence and Clinical Course of Diastolic Dysfunction and Diastolic Heart Failure (DIAST-CHF) trial were examined and compared with a healthy control group (n = 40; 48% female). We were able to identify 35 proteins that were expressed significantly different in both HF groups compared with the control group. We determine 29 unique proteins expressed in HFpEF and 33 unique proteins in HFrEF. Significantly up-regulated trefoil factor 3 (TFF3) and down-regulated contactin-1 could be identified as previously unknown biomarkers for HF. However, TFF3 is also a predictive factor for the occurrence of a cardiovascular event in HFpEF patients. In HFpEF, serine protease 27 was found at reduced levels for the first time, which could offer a new therapeutic target. Additionally, network analyses showed a special role of platelet-derived growth factor subunit A, Dickkopf-related protein 1, and tumour necrosis factor receptor superfamily member 6 in HFpEF patients, whereas perlecan and junctional adhesion molecule A stood out in the HFrEF group. Overall, signalling pathways of metabolic processes, cellular stress, and iron metabolism seemed to be important for HFrEF, whereas for HFpEF, oxygen stress, haemostasis, cell renewal, cell migration, and cell proliferation are in the foreground. CONCLUSIONS: The identified proteins and signalling pathways offer new therapeutic and diagnostic approaches for patients with chronic HF.

Influence of Shear Stress, Inflammation and BRD4 Inhibition on Human Endothelial Cells: A Holistic Proteomic Approach.

Atherosclerosis is an important risk factor in the development of cardiovascular diseases. In addition to increased plasma lipid concentrations, irregular/oscillatory shear stress and inflammatory processes trigger atherosclerosis. Inhibitors of the transcription modulatory bromo- and extra-terminal domain (BET) protein family (BETi) could offer a possible therapeutic approach due to their epigenetic mechanism and anti-inflammatory properties. In this study, the influence of laminar shear stress, inflammation and BETi treatment on human endothelial cells was investigated using global protein expression profiling by ion mobility separation-enhanced data independent acquisition mass spectrometry (IMS-DIA-MS). For this purpose, primary human umbilical cord derived vascular endothelial cells were treated with TNFα to mimic inflammation and exposed to laminar shear stress in the presence or absence of the BRD4 inhibitor JQ1. IMS-DIA-MS detected over 4037 proteins expressed in endothelial cells. Inflammation, shear stress and BETi led to pronounced changes in protein expression patterns with JQ1 having the greatest effect. To our knowledge, this is the first proteomics study on primary endothelial cells, which provides an extensive database for the effects of shear stress, inflammation and BETi on the endothelial proteome.

Adverse drug reactions following lymphocyte immunotherapy for the treatment of infertility: A retrospective study.

AIM: Unexplained infertility is a major burden for couples who want to have children. Lymphocyte immunotherapy (LIT) could be a therapeutic help for these couples. Although LIT has been carried out for decades, the data on the success of therapy are still controversial and there is hardly information on possible adverse drug reactions. METHODS: In this study, we used a questionnaire to determine the frequency of local and systemic adverse drug reactions in our patients who were treated with LIT between 2017 and 2020 (n = 302). In addition, we asked about pregnancies and/or live births after LIT in a 2-year follow-up (n = 140). RESULTS: Most of the patients reported the occurrence of mild local adverse drug reactions in a period of less than 4 weeks: Over 75% reported moderate erythema, itching or swelling, over 10% erythema, itching or swelling as more pronounced adverse drug reaction. Blistering was specified in 10% of the cases. Serious adverse drug reactions or adverse events were not described. In the follow-up, 69% of our patients stated a pregnancy after LIT, and 50% a life birth. CONCLUSIONS: Overall, LIT represents a well-tolerated therapy for couples with unexplained infertility, however, more evidence is needed on the benefits.

Comparison of a novel automated DiaSys procalcitonin immunoassay with four different BRAHMS-partnered immunoassays.

Objectives: Procalcitonin (PCT) is an important biomarker of sepsis and respiratory infections. Various automated immunoassays for measuring PCT in patient plasma are available in medical laboratories. However, due to a lack of international reference material for PCT, the assays are not always comparable. Design and methods: In this study, we compared a new turbidimetric immunoassay from DiaSys, measured on the Abbott Architect c16000 and Alinity c, with four BRAHMS-associated chemiluminescence immunoassays (Abbott Architect i2000SR, Alinity i, Roche Cobas e411 and DiaSorin Liaison XL) using 120 random patient plasma samples from the clinical laboratory routine at the University Medical Center Goettingen. Results: The DiaSys assay showed clear differences as compared to the BRAHMS-associated assays when measured on Architect c: i.e. 58% positive mean bias vs. Architect i, 67% vs. Cobas and 23% vs. Liaison. As a result, additional 19% our patients would have a suspected bacterial infection, when using PCT values from the DiaSys assay and commonly accepted decision limits. A crosscheck of the DiaSys calibrator on the BRAHMS-associated systems showed a low recovery of the calibrator material (approx. 50%). Conclusions: Overall, this study shows significant differences between the DiaSys and BRAHMS-associated assays. This could be attributed to a potential DiaSys calibrator problem. This highlights the need for an international reference material for harmonization of the PCT assays.

Analytical interference of 33 different hemoglobin variants on HbA1c measurements comparing high-performance liquid chromatography with whole blood enzymatic assay: A multi-center study.

BACKGROUND AND AIMS: The concentration of glycated hemoglobin (HbA1c) is an essential diagnostic and therapeutic biomarker in diabetes mellitus. However, it is known that Hb structural variants and synthesis disorders, can affect the HbA1c measurement in different assays. Although the analytical interference of various hemoglobinopathies on the chromatographic measurement of HbA1c using HPLC has been well studied, data on the interference on the enzymatic assay are few. MATERIALS AND METHODS: In this multi-center study, a large number (n = 104) of 33 different hemoglobin variants were collected over a period of one year and compared between an HPLC (Tosoh G8 and G11) and an enzymatic assay (Abbott Alinity c). RESULTS: A good comparability between ion-exchange HPLC and the Alinity assay for most Hb variants was found. However, we were able to determine for the first time that certain Hb variants (Hb Okayama, HbAE, Hb Lepore) can lead to clinically relevant discordant results. HbF (>5%) can already cause a relevant aberration. CONCLUSIONS: Overall, using the Abbott HbA1c assay in the presence of certain hemoglobin variants can induce clinically relevant interference that can affect diagnosis and therapy monitoring decisions, mainly because the enzymatic assay cannot provide any information about Hb variants.

Interdependencies of cellular and humoral immune responses in heterologous and homologous SARS-CoV-2 vaccination.

BACKGROUND: Homologous and heterologous SARS-CoV-2 vaccinations yield different spike protein-directed humoral and cellular immune responses. This study aimed to explore their currently unknown interdependencies. METHODS: COV-ADAPT is a prospective, observational cohort study of 417 healthcare workers who received vaccination with homologous ChAdOx1 nCoV-19, homologous BNT162b2 or with heterologous ChAdOx1 nCoV-19/BNT162b2. We assessed humoral (anti-spike-RBD-IgG, neutralizing antibodies, and avidity) and cellular (spike-induced T-cell interferon-γ release) immune responses in blood samples up to 2 weeks before (T1) and 2-12 weeks following secondary immunization (T2). RESULTS: Initial vaccination with ChAdOx1 nCoV-19 resulted in lower anti-spike-RBD-IgG compared with BNT162b2 (70 ± 114 vs. 226 ± 279 BAU/ml, p < .01) at T1. Booster vaccination with BNT162b2 proved superior to ChAdOx1 nCoV-19 at T2 (anti-spike-RBD-IgG: ChAdOx1 nCoV-19/BNT162b2 2387 ± 1627 and homologous BNT162b2 3202 ± 2184 vs. homologous ChAdOx1 nCoV-19 413 ± 461 BAU/ml, both p < .001; spike-induced T-cell interferon-γ release: ChAdOx1 nCoV-19/BNT162b2 5069 ± 6733 and homologous BNT162b2 4880 ± 7570 vs. homologous ChAdOx1 nCoV-19 1152 ± 2243 mIU/ml, both p < .001). No significant differences were detected between BNT162b2-boostered groups at T2. For ChAdOx1 nCoV-19, no booster effect on T-cell activation could be observed. We found associations between anti-spike-RBD-IgG levels (ChAdOx1 nCoV-19/BNT162b2 and homologous BNT162b2) and T-cell responses (homologous ChAdOx1 nCoV-19 and ChAdOx1 nCoV-19/BNT162b2) from T1 to T2. Additionally, anti-spike-RBD-IgG and T-cell response were linked at both time points (all groups combined). All regimes yielded neutralizing antibodies and increased antibody avidity at T2. CONCLUSIONS: Interdependencies between humoral and cellular immune responses differ between common SARS-CoV-2 vaccination regimes. T-cell activation is unlikely to compensate for poor humoral responses.

National external quality assessment and direct method comparison reflect crucial deviations of Procalcitonin measurements in Germany.

BACKGROUND AND AIMS: Sepsis is a major concern worldwide, affecting 49 million individuals and being related to 11 million deaths. Its fast diagnosis is the key factor to guarantee a positive prognosis. Procalcitonin (PCT) has emerged as one powerful biomarker to early diagnose sepsis and for monitoring of antibiotic treatment. However, its clinical utility is jeopardized by missing standardisation. MATERIALS AND METHODS: Here we present a 1-year follow-up of the External Quality Assessment (EQA) in Germany, depicting substantial discrepancies among manufacturers and the used assay technology of current PCT measurements. A direct method comparison on two immunoassays (Abbott vs. DiaSys) on a set of 135 routine samples was used to analyse the causes of observed deviations. RESULTS: All BRAHMS-licensed manufacturers (Thermo, Roche, Abbott, Siemens, Biomérieux), the Beckman and DiaSys immunoassays as well as all assay types (fluorescence, luminescence, PETIA) reveal substantial recovery differences between each other. However, upon a non-linear re-standardization of calibrators, the two directly compared methods (Abbott, DiaSys) are well interchangeable. CONCLUSION: This work demonstrates the heterogenic situation of PCT measurements in Germany among manufacturers and all methods. By introducing dedicated correction factors, comparable results of PCT can be achieved. This work also strengthens the inevitability of calibrator traceability and higher metrological reference materials on PCT.

Plasma Biomarker Profiling in Heart Failure Patients with Preserved Ejection Fraction before and after Spironolactone Treatment: Results from the Aldo-DHF Trial.

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) is poorly understood and therapeutic strategies are lacking. This study aimed to identify plasma proteins with pathophysiological relevance in HFpEF and with respect to spironolactone-induced effects. We assessed 92 biomarkers in plasma samples from 386 HFpEF patients-belonging to the Aldo-DHF trial-before (baseline, BL) and after one-year treatment (follow up, FU) with spironolactone (verum) or a placebo. At BL, various biomarkers showed significant associations with the two Aldo-DHF primary end point parameters: 33 with E/e' and 20 with peak VO2. Ten proteins including adrenomedullin, FGF23 and inflammatory peptides (e.g., TNFRSF11A, TRAILR2) were significantly associated with both parameters, suggesting a role in the clinical HFpEF presentation. For 13 proteins, expression changes from BL to FU were significantly different between verum and placebo. Among them were renin, growth hormone, adrenomedullin and inflammatory proteins (e.g., TNFRSF11A, IL18 and IL4RA), indicating distinct spironolactone-mediated effects. BL levels of five proteins, e.g., inflammatory markers such as CCL17, IL4RA and IL1ra, showed significantly different effects on the instantaneous risk for hospitalization between verum and placebo. This study identified plasma proteins with different implications in HFpEF and following spironolactone treatment. Future studies need to define their precise mechanistic involvement.

Inconsistent Findings of Cardiac Troponin T and I in Clinical Routine Diagnostics: Factors of Influence.

Cardiac troponins are crucial for the diagnosis of acute myocardial infarction. Despite known differences in their diagnostic implication, there are no recommendations for only one of the two troponins, cardiac troponin I (cTnI) and troponin T (cTnT) so far. In an everyday routine diagnostic, cTnT (Roche) as well as cTnI (Abbott) were measured in 5667 samples from 3264 patient cases. We investigated the number of identical or discrepant troponin findings. Regarding cTnI, we considered both, sex-dependent and unisex cutoffs. In particular, the number of cTnT positive and cTnI negative results was strikingly high in 14.0% of cTnT positive samples and increases to 23.8% by using sex-specific cTnI cutoffs. This group was considerably greater than the group of cTnI positive and cTnT negative results, also after elimination of patients with an eGFR < 60 mL/min/1.73 m2. Comparing the troponin cases with a dynamic increase or decrease between two measurements, we saw a balanced number of discrepant cases (between cTnT+/cTnI- and cTnT-/cTnI+), which was, however, still present. Using ROC analysis, sex-dependent cutoffs improved sensitivity and specificity of cTnI. This study shows in a large cohort that comparing the two cardiac troponins does not amount to identical analytical results. Consideration of sex-dependent cutoffs may improve sensitivity and specificity.

Intoxication cases associated with the novel designer drug 3',4'-methylenedioxy-α-pyrrolidinohexanophenone and studies on its human metabolism using high-resolution mass spectrometry.

Among the increasing number of new psychoactive substances, 3',4'-methylenedioxy-α-pyrrolidinohexanophenone (MDPHP) belongs to the group of synthetic cathinones, which are the derivatives of the naturally occurring compound cathinone, the main psychoactive ingredient in the khat plant. Currently, only limited data are available for MDPHP, and no information is available on its human metabolism. We describe the toxicological investigation of nine cases associated with the use of MDPHP during the period February-June 2019. Serum MDPHP concentrations showed a high variability ranging from 3.3 to 140 ng/mL (mean 30.3 ng/mL and median 16 ng/mL). Intoxication symptoms of the described cases could not be explained by the abuse of MDPHP alone because in all cases the co-consumption of other psychotropic drugs with frequent occurrence of opiates and benzodiazepines could be verified. Therefore, the patients showed different clinical symptoms, including aggressive behaviour, delayed physical response, loss of consciousness and coma. Liquid chromatography-high-resolution mass spectrometry was successfully used to investigate the human in vivo metabolism of MDPHP using authentic human urine samples. The metabolism data for MDPHP were further substantiated by the analysis of human urine using gas chromatography-mass spectrometry (GC-MS, a widely used systematic toxicological analysis method appropriate for the toxicological detection of MDPHP intake), which revealed the presence of seven phase I metabolites and three phase II metabolites as glucuronides. GC-MS spectral data for MDPHP and metabolites are provided. The identified metabolite pattern corroborates the principal metabolic pathways of α-pyrrolidinophenones in humans.

Macro-CK type 2 in metastatic prostate cancer.

The isoenzyme creatine kinase muscle/brain (CK-MB) still plays an important role for the differential diagnosis of CK elevations and the clarification of their origin from heart or skeletal muscle. Therefore, it is necessary to know the diagnostic pitfalls in interpreting CK-MB results. We demonstrate a case of macro-CK type 2 in a 75-year-old patient with metastatic castration-resistant prostate cancer and its identification by isoenzyme electrophoresis, which can be typical for cancer diseases.

Assessment of the Therapeutic Potential of Metallothionein-II Application in Focal Cerebral Ischemia In Vitro and In Vivo.

Metallothionein-II (MT-II) is an ubiquitously expressed small-molecular-weight protein and highly induced in various species and tissues upon stress, inflammation, and ischemia. MT-deficiency exacerbates ischemic injury in rodent stroke models in vitro and in vivo. However, there is conflicting data on the potential neuroprotective effect of exogenously applied metallothionein. Thus, we applied MT-II in an in vitro stroke model and intraperitoneally (i.p.) in two in vivo standard models of transient middle cerebral artery occlusion (MCAO) (a 'stringent' one [60 min MCAO/48 h reperfusion] and a 'mild' one [30 min MCAO/72 h reperfusion]), as well as i.v. together with recombinant tissue plasminogen activator (rtPA) to evaluate if exogenous MT-II-application protects against ischemic stroke. Whereas MT-II did not protect against 60 min MCAO, there was a significant reduction of direct and indirect infarct volumes and neurological deficit in the MT-II (i.p.) treated animals in the 'mild' model at 3d after MCAO. Furthermore, MT-II also improved survival of the mice after MCAO, suppressed TNF-α mRNA induction in ischemic brain tissue, and protected primary neuronal cells against oxygen-glucose-deprivation in vitro. Thus, exogenous application of MT-II protects against ischemic injury in vitro and in vivo. However, long-term studies with different species and larger sampling sizes are required before a clinical use can be envisaged.