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Background: Mesenchymal stromal cells (MSCs) exert immunomodulatory effects, primarily through released extracellular vesicles (EVs). For the clinical-grade manufacturing of MSC-EV products culture conditions need to support MSC expansion and allow the manufacturing of potent MSC-EV products. Traditionally, MSCs are expanded in fetal bovine serum-supplemented media. However, according to good manufacturing practice (GMP) guidelines the use of animal sera should be avoided. To this end, human platelet lysate (hPL) has been qualified as an animal serum replacement. Although hPL outcompetes animal sera in promoting MSC expansion, hPL typically contains components of the coagulation system that need to be inhibited or removed to avoid coagulation reactions in the cell culture. Commonly, heparin is utilized as an anticoagulant; however, higher concentrations of heparin can negatively impact MSC viability, and conventional concentrations alone do not sufficiently prevent clot formation in prepared media., Methods: To circumvent unwanted coagulation processes, this study compared various clotting prevention strategies, including different anticoagulants and calcium chloride (CaCl 2 )-mediated declotting methods, which in combination with heparin addition was found effective. We evaluated the influence of the differently treated hPLs on the proliferation and phenotype of primary bone marrow-derived MSCs and identified the CaCl 2 -mediated declotting method as the most effective option. To determine whether CaCl 2 declotted hPL allows the manufacturing of immunomodulatory MSC-EV products, EVs were prepared from conditioned media of MSCs expanded with either conventional or CaCl 2 declotted hPL. In addition to metric analyses, the immunomodulatory potential of resulting MSC-EV products was assessed in a recently established multi-donor mixed lymphocyte reaction assay., Results and Conclusions: Our findings conclusively show that CaCl 2 -declotted hPLs support the production of immunomodulatory-active MSC-EV products., Competing Interests: Declaration of Competing Interest BG is a scientific advisory board member of Innovex Therapeutics SL, Mursla Ltd, ReNeuron Ltd and PL BioScience. He is a founding director of Exosla Ltd. HH is founder and executive director at PL Bioscience a company, selling hPLs as cell culture supplements. All other authors report no conflicts of interest., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: The use of cell salvage and autologous blood transfusion is an important and widespread method of blood conservation during surgeries with expected high blood loss. The continuous autotransfusion device CATSmart ® (Fresenius Kabi, Germany) contains two new washing programs on the device called Flex wash 3 and Flex wash 5. To the best of our knowledge, there are no published clinical data regarding the performance of the two new washing programs., Methods: In total, 69 patients undergoing cardiac or orthopedic surgery were included in this randomized, controlled, bicentric trial to validate the red cell separation process and washout quality of Flex wash 3 compared to Flex wash 5. After washing, the primary quality target was to determine hematocrit value, recovery rate, albumin, and total protein elimination rate in the packed red cells (PRCs). The secondary objective was to assess the elimination of heparin by measuring the factor anti-Xa activity by a 1- and 2-stage assay in PRC after washing., Results: In the whole cohort of patients, hematocrit was 16.00% [9.15%; 21.30%] (median [Q1; Q3]) in the wound blood and 69.90% [51.10%; 80.90%] in the PRC resulting in a recovery rate of 63.92% [47.06%; 88.13%]. The albumin elimination rate was 98.77% [97.94%; 99.27%], and the total protein elimination rate was 98.85% [97.76%; 99.42%]. The heparin elimination rate was 99.95% [99.90%; 99.97%] in the 1-stage assay and 99.70% [99.41%; 99.87%] in the 2-stage assay. There was no difference between Flex wash 3 and Flex wash 5 washing procedure regarding the recovery rate 63.75% [46.64%; 78.65%] versus 67.89% [47.20%; 92.69%] ( p = 0.85), albumin elimination rate 98.74% [97.67%; 99.27%] versus 98.78% [98.10%; 99.28%] ( p = 0.97), protein elimination rate 98.79% [97.94%; 99.47%] versus 98.92% [97.58%; 99.42%] ( p = 0.88), and anti-Xa elimination rate in the 1-stage assay 99.94% [99.79%; 99.97%] versus 99.95% [99.92%; 99.97%] ( p = 0.24) and in 2-stage assay 99.66% [99.20%; 99.86%] versus 99.77% [99.47%; 99.90%] ( p = 0.23)., Conclusions: The two new washing procedures, Flex wash 3 and Flex wash 5, enable sufficient and comparable red cell separation and washout quality of albumin, total protein, as well as heparin., Competing Interests: The authors have no conflicts of interest to declare., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Background: Post-acute sequelae of COVID-19 (PASC) comprise a broad spectrum of symptoms such as fatigue, general weakness, compromised attention and sleep or anxiety disorders. PASC represents a medical and socio-economic challenge., Objectives: Our study evaluated cytokines (IL-1β, IL-6 and TNFα) and cortisol levels in a cohort of typical patients with PASC, suffering concentration problems, fatigue and difficulties finding words., Design: This was a prospective cohort study. Four groups were analysed and compared: those who had never contracted SARS-CoV-2 ( n  = 13), infected but had no PASC ( n  = 34), infected with former PASC that resolved ( n  = 40) and patients with ongoing PASC after infection ( n  = 91)., Methods: Cytokine and cortisol serum levels were determined in patients' blood samples., Results: Cytokine levels of IL-1β, IL-6, TNFα and cortisol levels did not differ between groups analysed., Conclusion: This may indicate a non-organic/psychosomatic genesis of PASC; further studies are needed to elucidate the underlying causes of PACS, and non-organic causes should not be overlooked., Competing Interests: MF, LA, EU, AKM, FS, LV, DZ, CK and MS declare that they have no competing interest in connection with the submitted material., (© The Author(s), 2024.)

Background: Malignant ascites commonly occurs in advanced or recurrent stages of epithelial ovarian cancer during peritoneal carcinomatosis and is correlated with poor prognosis. Due to its complex composition of cellular and acellular components malignant ascites creates a unique tumor microenvironment, which mediates immunosuppression and promotes progression of disease. However, the immunosuppressive mechanisms remain poorly understood., Methods: In the present study, we explored the antitumor activity of healthy donor NK and T cells directed against ovarian cancer cells in presence of malignant ascites derived from patients with advanced or recurrent peritoneal carcinomatosis. A wide range of methods was used to study the effect of ascites on NK and T cells (FACS, ELISA, EliSpot, qPCR, Live-cell and confocal microscopy, Western blot and electrolyte flux assays). The ascites components were assessed using quantitative analysis (nephelometry, potentiometry and clinical chemistry) and separation methods (dialysis, ultracentrifugal filtration and lipid depletion)., Results: Ascites rapidly inhibited NK cell degranulation, tumor lysis, cytokine secretion and calcium signaling. Similarly, target independent NK and T cell activation was impaired in ascites environment. We identified imbalanced electrolytes in ascites as crucial factors causing extensive immunosuppression of NK and T cells. Specifically, high sodium, low chloride and low potassium content significantly suppressed NK-mediated cytotoxicity. Electrolyte imbalance led to changes in transcription and protein expression of electrolyte channels and impaired NK and T cell activation. Selected inhibitors of sodium electrolyte channels restored intracellular calcium flux, conjugation, degranulation and transcript expression of signaling molecules. The levels of ascites-mediated immunosuppression and sodium/chloride/potassium imbalance correlated with poor patient outcome and selected molecular alterations were confirmed in immune cells from ovarian cancer patients., Conclusion: Our data suggest a novel electrolyte-based mechanism of immunosuppression in malignant ascites of patients with peritoneal carcinomatosis. We show for the first time that the immunosuppression of NK cytotoxicity in coculture assays is correlated to patient poor survival. Therapeutic application of sodium channel inhibitors may provide new means for restoring immune cell activity in ascites or similar electrolyte imbalanced environments., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

Context: The bone-derived adipokine lipocalin-2 is relevant for body weight regulation by stimulating the leptin-melanocortin pathway., Objective: We aimed to (i) detect variants in the lipocalin-2 gene ( LCN2 ) which are relevant for body weight regulation and/or anorexia nervosa (AN); (ii) describe and characterize the impact of LCN2 and MC4R variants on circulating lipocalin-2 level., Methods: Sanger sequencing of the coding region of LCN2 in 284 children and adolescents with severe obesity or 287 patients with anorexia nervosa. In-silico analyses to evaluate functional implications of detected LCN2 variants. TaqMan assays for rare non-synonymous variants (NSVs) in additional independent study groups. Serum levels of lipocalin-2 were measured by ELISA in 35 females with NSVs in either LCN2 or MC4R , and 33 matched controls without NSVs in the two genes., Results: Fourteen LCN2 -variants (five NSVs) were detected. LCN2 -p.Leu6Pro and p.Gly9Val located in the highly conserved signal peptide region may induce functional consequences. The secondary structure change of lipocalin-2 due to LCN 2-p.Val89Ile may decrease solubility and results in a low lipocalin-2 level in a heterozygotes carrier (female recovered from AN). Lean individuals had lower lipocalin-2 levels compared to patients with obesity ( p = 0.033)., Conclusion: Lipocalin-2 levels are positively associated with body mass index (BMI). Single LCN2 -variants might have a profound effect on lipocalin-2 levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zheng, Rajcsanyi, Kowalczyk, Giuranna, Herpertz-Dahlmann, Seitz, de Zwaan, Herzog, Ehrlich, Zipfel, Giel, Egberts, Burghardt, Föcker, Al-Lahham, Hebebrand, Fuhrer, Tan, Zwanziger, Peters and Hinney.)

Aim of the Study: The aim of the project was to investigate regional differences in thyroid stimulating hormone (TSH), and free thyroxine (fT4) concentrations and iodine status in comparable German and European cohort studies., Methods: Sex- and age-stratified TSH, fT4, and urine iodine concentrations of thyroid-healthy participants (age group 45-75 years) of the HNR (Heinz Nixdorf Recall) Study in the Ruhr region of Germany, the southern German KORA (Cooperative Health Research in the Augsburg Region) and northeastern German SHIP (Study of Health in Pomerania) studies, as well as the Norwegian HUNT (Nord-Trøndelag Health) study (age group 40-79 years), the English EPIC (European Prospective Investigation of Cancer)-Norfolk study, and the Dutch Rotterdam study were compared. The TSH reference range for the HNR study population was calculated and compared to the KORA and SHIP studies., Results: Regional differences showed a stronger influence on TSH and fT4 concentrations than sex and age of the subjects in the 45- to 75-year age group. The estimated difference in medians, as measured by the HNR study, was lowest in the SHIP study, -0.47 (95% CI: -0.53; -0.41) for men and -0.41 (-0.53; -0.41) for women. The Rotterdam study had the highest difference in medians for both men and women (men: 0.56 with 0.44; 0.68 and women: 0.62 with 0.46; 0.78). The lowest median TSH concentrations, across all age categories considered, were seen in the German cohorts., Conclusions: Comparison of thyroid function parameters and iodine in elderly subjects between six comparable cohort studies from Germany and Europe showed a significant influence of region, which exceeded the sex and age dependence of the parameters., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Background and Aims: Non-thyroidal illness syndrome (NTIS) is frequent in critically ill patients and associated with adverse outcomes. We aimed to characterize the evolution of NTIS in patients with acute decompensation (AD) of cirrhosis and acute-on-chronic liver failure (ACLF), since NTIS is not well described in these newly defined syndromes., Methods: Thyroid hormones (TH) were quantified at baseline in consecutive patients with cirrhosis. In addition, 76 inflammatory mediators were quantified by proximity extension analysis assay in a subgroup of patients. Associations between TH, cirrhosis stage, mortality and inflammation were assessed., Results: Overall, 437 patients were included, of whom 165 (37.8%), 211 (48.3%), and 61 (14%) had compensated cirrhosis (CC), AD, and ACLF. FT 3 concentrations were lower in AD versus CC, and further decreased in ACLF. Importantly, NTIS was present in 83 (39.3%) patients with AD and in 44 (72.1%) patients with ACLF (P<0.001). Yet, TSH and TSH-based indexes (TSH/FT 3 -ratio, thyroid index) showed an U-shaped evolution during progression of cirrhosis, suggesting a partially preserved responsiveness of the hypothalamus and pituitary in AD. Infections were associated with lower FT 3 concentrations in AD, but not in ACLF. Low FT 3 concentrations correlated significantly with 90-day mortality. Both, AD/ACLF and NTIS, were associated with signatures of inflammatory mediators, which were partially non-overlapping., Conclusion: NTIS is frequent already in AD and therefore precedes critically illness in a subgroup of patients with decompensated cirrhosis. This might constitute a new paradigm of TH signaling in cirrhosis, offering opportunities to explore preventive effects of TH in AD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Langer, Bauschen, Guckenbiehl, Klauss, Lutz, Denk, Zwanziger, Moeller and Lange.)

Women are more prone to develop either hypothyroidism or cholesterol gallstones than men. However, a male predominance in cholesterol gallstones under hypothyroidism was reported. Recently, a novel pathogenic link between thyroid hormone (TH) deficiency and cholesterol gallstones has been described in male mice. Here, we investigate if TH deficiency impacts cholesterol gallstone formation in females by the same mechanism. Three-month-old C57BL/6J mice were randomly divided into a control, a TH deficient, a lithogenic, and a lithogenic + TH deficient group and diet-treated for two, four, and six weeks. Gallstone prevalence, liver function tests, bile composition, hepatic gene expression, and gallbladder aquaporin expression and localization were investigated. Cholesterol gallstones were observed in lithogenic + TH deficient but not lithogenic only female mice. Diminished hydrophilicity of primary bile acids due to decreased gene expression of hepatic detoxification phase II enzymes was observed. A sex-specific expression and localization of hepatobiliary aquaporins involved in transcellular water and glycerol permeability was observed under TH deficient and lithogenic conditions. TH deficiency promotes cholesterol gallstone formation in female C57BL/6J mice by the same mechanism as observed in males. However, cholesterol gallstone prevalence was lower in female than male C57BL/6J mice. Interestingly, the sex-specific expression and localization of hepatobiliary aquaporins could protect female C57BL/6J mice to cholestasis and could reduce biliary water transport in male C57BL/6J mice possibly contributing to the sex-dependent cholesterol gallstone prevalence under TH deficiency.

Background: Chronic kidney disease (CKD) is responsible for large personal health and societal burdens. Screening populations at higher risk for CKD is effective to initiate earlier treatment and decelerate disease progress. We externally validated clinical prediction models for unknown CKD that might be used in population screening., Methods: We validated six risk models for prediction of CKD using only non-invasive parameters. Validation data came from 4,185 participants of the German Heinz-Nixdorf-Recall study (HNR), drawn in 2000 from a general population aged 45-75 years. We estimated discrimination and calibration using the full model information, and calculated the diagnostic properties applying the published scoring algorithms of the models using various thresholds for the sum of scores., Results: The risk models used four to nine parameters. Age and hypertension were included in all models. Five out of six c-values ranged from 0.71 to 0.73, indicating fair discrimination. Positive predictive values ranged from 15 to 19%, negative predictive values were > 93% using score thresholds that resulted in values for sensitivity and specificity above 60%., Conclusions: Most of the selected CKD prediction models show fair discrimination in a German general population. The estimated diagnostic properties indicate that the models are suitable for identifying persons at higher risk for unknown CKD without invasive procedures., (© 2022. The Author(s).)

Background: Despite the high level of protection against severe COVID-19 provided by the currently available vaccines some breakthrough infections occur. Until now, there is no information whether a potential risk of a breakthrough infection can be inferred from the level of antibodies after booster vaccination., Methods: Levels of binding antibodies and neutralization capacity after the first, one and six month after the second, and one month after the third (booster) vaccination against COVID-19 were measured in serum samples from 1391 healthcare workers at the University Hospital Essen. Demographics, vaccination scheme, pre-infection antibody titers and neutralization capacity were compared between individuals with and without breakthrough infections., Results: The risk of developing an Omicron breakthrough infection was independent of vaccination scheme, sex, body mass index, smoking status or pre-existing conditions. In participants with low pre-infection anti-spike antibodies (≤ 2641.0 BAU/ml) and weaker neutralization capacity (≤ 65.9%) against Omicron one month after the booster vaccination the risk for developing an Omicron infection was 10-fold increased ( P = 0.001; 95% confidence interval, 2.36 - 47.55)., Conclusion: Routine testing of anti-SARS-CoV-2 IgG antibodies and surrogate virus neutralization can quantify vaccine-induced humoral immune response and may help to identify subjects who are at risk for a breakthrough infection. The establishment of thresholds for SARS-CoV-2 IgG antibody levels identifying "non"-, "low" and "high"-responders may be used as an indication for re-vaccination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Möhlendick, Čiučiulkaitė, Elsner, Anastasiou, Trilling, Wagner, Zwanziger, Jöckel, Dittmer and Siffert.)

Context: 3,5,3'-L-triiodothyronine (T3) is a potent inducer of hepatocyte proliferation via the Wnt/β-catenin signaling pathway. Previous studies suggested the involvement of rapid noncanonical thyroid hormone receptor (TR) β signaling, directly activating hepatic Wnt/β-catenin signaling independent from TRβ DNA binding. However, the mechanism by which T3 increases Wnt/β-catenin signaling in hepatocytes has not yet been determined., Objective: We aimed to determine whether DNA binding of TRβ is required for stimulation of hepatocyte proliferation by T3., Methods: Wild-type (WT) mice, TRβ knockout mice (TRβ KO), and TRβ mutant mice with either specifically abrogated DNA binding (TRβ GS) or abrogated direct phosphatidylinositol 3 kinase activation (TRβ 147F) were treated with T3 for 6 hours or 7 days. Hepatocyte proliferation was assessed by Kiel-67 (Ki67) staining and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Activation of β-catenin signaling was measured in primary murine hepatocytes. Gene expression was analyzed by microarray, gene set enrichment analysis (GSEA), and quantitative reverse transcription polymerase chain reaction., Results: T3 induced hepatocyte proliferation with an increased number of Ki67-positive cells in WT and TRβ 147F mice (9.2% ± 6.5% and 10.1% ± 2.9%, respectively) compared to TRβ KO and TRβ GS mice (1.2% ± 1.1% and 1.5% ± 0.9%, respectively). Microarray analysis and GSEA showed that genes of the Wnt/β-catenin pathway-among them, Fzd8 (frizzled receptor 8) and Ctnnb1 (β-catenin)-were positively enriched only in T3-treated WT and TRβ 147F mice while B-cell translocation gene anti-proliferation factor 2 was repressed. Consequently, expression of Ccnd1 (CyclinD1) was induced., Conclusions: Instead of directly activating Wnt signaling, T3 and TRβ induce key genes of the Wnt/β-catenin pathway, ultimately stimulating hepatocyte proliferation via CyclinD1. Thus, canonical transcriptional TRβ action is necessary for T3-mediated stimulation of hepatocyte proliferation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)