Natural killer (NK) cell-based therapies are emerging as highly promising candidates for cancer treatment, but their development and quality control depend on robust assessment of key critical quality attributes, particularly their cytotoxicity. Despite the availability of various approaches for assessing cytotoxicity, existing techniques often suffer from high data variability and show limited reproducibility. We compared commonly used approaches for NK-cell cytotoxicity assessment, including calcein release, lactate dehydrogenase release, and flow cytometry (FCM)-based analysis, using NK-92 effector and GFP-labelled K562 target cells. Our results provide insight into the shortcomings of these methods, as well as problems resulting from unharmonized evaluation criteria and limitations of endpoint measurements, which are commonly applied in literature. We selected FCM as the most suitable platform for standardized evaluation and developed an automated gating workflow for cytotoxicity analysis. The automated workflow was benchmarked against three independent manual evaluations to assess agreement, bias, and performance. The optimized workflow showed agreement with manual analysis while remaining essentially unbiased. In addition, automated analysis reduced evaluator dependence by producing deterministic outputs from identical input data. Further comparison revealed directional bias in manual gating, indicating that a relevant portion of measurement variability arose from manual evaluation, rather than biology alone. We present autogating as a fit-for-purpose, automated FCM-based strategy for NK-cell cytotoxicity evaluation that preserves agreement with manual analysis while improving standardization and reproducibility, thereby providing a practical route toward more harmonized cytotoxicity testing in cell therapy workflows.

Abstract Background Autoimmune connective tissue diseases (ACTD), including systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjögren’s disease (SD), and idiopathic inflammatory myopathies (IIM), are associated with markedly increased cardiovascular risk (CVR) that is insufficiently captured by conventional risk scores. Reliable biomarkers for CVR stratification in ACTD are lacking. This exploratory, hypothesis-generatig study aimed to investigate whether metabolomic alterations reflect CVR across ACTD subtypes. Methods In this cross-sectional, exploratory study, patients with SLE (n = 33), SSc (n = 18), SD (n = 16), and IIM (n = 9) were recruited from a tertiary rheumatology center. Serum metabolomic profiling was performed using ¹H-NMR spectroscopy. Associations between 113 quantified metabolites and clinical CVR parameters (including age, sex, body mass index, glucocorticoid use, Framingham score, hypertension, diabetes, lipid parameters, and lifestyle factors) were analyzed using non-parametric statistics with false discovery rate correction. Correlation analyses were conducted using Spearman coefficients. Results Metabolomic alterations varied substantially across ACTD subtypes and CVR factors. Lipid metabolites showed the strongest and most consistent associations with CVR parameters. The Framingham score correlated with 11 metabolites in SLE, 1 in SSc, 2 in SD, and 93 in IIM, predominantly involving lipid components. Diabetes mellitus was associated with extensive metabolomic changes, particularly in SSc (n = 62 metabolites), followed by SD (n = 20) and SLE (n = 5). In contrast, arterial hypertension showed minimal metabolomic differentiation across most ACTD, except in IIM. Body mass index correlated mainly with lipid metabolites in SSc, but not in SLE. Glucocorticoid therapy and dosage were strongly associated with alterations in lipid metabolism, especially in SLE and SSc. Across all entities, correlations with total cholesterol, LDL, and HDL were dominated by lipid metabolites. Associations with renal markers (UACR) were limited. Conclusions ACTD are characterized by heterogeneous metabolomic signatures associated with cardiovascular risk factors. Larger, longitudinal studies are required to validate these findings and to determine their clinical utility in cardiovascular risk stratification in ACTD.

BACKGROUND:Microvascular damage by oxidative stress is central in the pathogenesis of generalized COVID-19 disease. Hence, a disbalance of endothelial-derived oxidative and anti-oxidative factors in COVID-19 patients can be expected, and the extent of this disbalance might correlate with disease severity. METHODS:We measured xanthine oxidase (XO), superoxide dismutase (SOD), catalase (CAT), and ischemia modified albumin (IMA) in serum samples of 166 COVID-19 patients and 238 controls. We then cathegorized the COVID-19 group further into mild, moderate, severe and lethal courses and tested these for correlation with each parameter alone, and with multi-parametric logistic regression analysis. RESULTS:Compared to controls, XO was significantly lower in COVID-19 patients, SOD and CAT were significantly higher. Difference in IMA was insignificant. In the single parameter analysis, only CAT concentration was significantly correlated to disease severity. In the logistic regression analysis, XO and SOD were negatively correlated with disease severity. CONCLUSIONS:Oxidative stress in COVID-19 does derive from other sources than endothelial XO. The rise in protective enzymes like SOD and CAT may be the result of enzyme induction. Since the correlation of CAT with disease severity was highest, we propose this parameter as a possible predictor for a severe clinical course.

Steatotic liver disease (SLD) is characterised by profound metabolic reprogramming, yet no single biomarker reliably distinguishes disease entities, stages or sex-specific risk profiles. By integrating serum metabolomic signatures as a liquid biopsy with tumour-associated CSC marker profiles in a sex-stratified analytical framework, we aimed to identify biologically meaningful differences and improve strategies for early, presymptomatic detection of SLD progression and HCC. The present study focuses on a targeted panel of 12 strongly dysregulated serum metabolites as candidate biomarkers of disease progression, quantified by NMR-based metabolomics and ELISA and complemented by CSC marker staining. We combined these NMR-based metabolomic ‘liquid biopsy’ data with circulating tumour-associated biomarkers, MELD-based risk assessment and tissue-level CSC marker expression across MetALD, MASLD, immune-mediated and cancerogenic liver disease, HCC and healthy controls. Female MetALD patients showed the second highest mortality after HCC, with lower survival than male cancer patients, despite MELD 3.0 assigning ~50% higher scores in women. MetALD mortality clustered with GP73, CD44, metabolomics and AA/3HB ratio, indicating a distinct, high-risk female phenotype. Integrating liquid-based metabolomic profiling, AA/3HB redox assessment, CSC markers and MELD 3.0 into sex-sensitive diagnostic pathways may improve early detection and risk stratification of alcohol-associated SLD, especially in women.

Natural killer cells are emerging as promising “off-the-shelf” effectors for cancer immunotherapy, yet expansion of the NK-92 cell line in batch cultivation leads to rapid loss of cytotoxicity concomitant with lactate accumulation. In this study we developed and validated a two-phase manufacturing strategy that decouples cell proliferation from functional recovery in order to obtain an improved final product potency. Our 8-day kinetic survey determined declines in viability, metabolite profiles and cytotoxicity during static batch expansion. Guided by these data, in a 32-run full-factorial design-of-experiments approach we varied fresh cultivation medium proportion, temperature, dissolved oxygen, and recovery duration; partial least squares modeling identified fresh-medium ratio and recovery time as the primary drivers of cytotoxicity restoration. Identified optimal conditions (90% fresh medium, 37.2 °C, 3.7 days recovery) recovered cytotoxicity and maximized cytotoxic capacity. These setpoints were then translated to a 2 L stirred-tank bioreactor, where a fed-batch expansion under controlled pH and lactate levels produced 2.0 × 109 cells, followed by recovery that achieved 43% ± 8% cytotoxicity. This scalable, two-phase paradigm minimizes medium usage and obviates continuous perfusion, offering a potential workflow to increase NK-92 potency and a base for manufacturing high-quality advanced therapy medicinal products.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide despite recent advances in systemic therapy. The introduction of immune checkpoint inhibitors has substantially expanded treatment options for advanced disease; however, durable responses are observed only in a subset of patients. Increasing evidence indicates that this limited efficacy is largely driven by the complex immune landscape of HCC, which is shaped by disease aetiology, tumour microenvironment-mediated immune suppression, and impaired innate immune surveillance. Chronic viral hepatitis and metabolically driven liver diseases represent the principal drivers of HCC and profoundly influence immune regulation within the liver. Persistent antigen exposure, metabolic reprogramming, and stromal remodelling contribute to dysfunctional T- and natural killer (NK) cell responses, while immunosuppressive components of the tumour microenvironment, including tumour-associated macrophages, myeloid-derived suppressor cells, regulatory T cells, and inhibitory ligands such as HLA-G, facilitate immune escape and therapeutic resistance. In this review, we discuss recent advances in HCC immunotherapy, focusing on emerging checkpoint pathways such as TIGIT and Tim-3, glypican-3 targeted cellular therapies and bispecific antibodies, and NK cell-based therapeutic strategies. We further highlight the role of liquid biopsy approaches for treatment monitoring and biomarker development. Together, these insights emphasize the need for biomarker-guided patient stratification and integrated therapeutic strategies to improve the clinical efficacy of immunotherapy in HCC.

Background: Patients with rheumatoid arthritis (RA) have a disproportionately increased cardiovascular risk (CVR). Serological CVR predictors that capture this disease-associated risk increase are currently not available. Elevated levels of oxidized high-density lipoprotein (oxHDL) have been associated with coronary artery disease and atrial fibrillation. The aim of this study was to evaluate the suitability of oxHDL as a CVR predictor in RA. Methods: An observational cross-sectional study was conducted in patients with confirmed RA undergoing variable disease-modifying anti-rheumatic drug (DMARD) therapy. Anamnestic, clinical, and laboratory surrogate markers of cardiovascular morbidity and disease activity were collected. Th quantification of oxHDL was conducted using a fluorometric biochemical cell-free assay. Results: A total of 200 RA patients were included in the study. ox-HDL showed significant correlations with the following variables: body mass index (BMI) (positive), total cholesterol, and average daily prednisolone dose (both negative). Overweight and hypertensive individuals exhibited higher levels of oxHDL. Additionally, oxHDL correlated positively with the number of tender small joints. Conclusion: oxHDL is associated with distinct CVR indicators in RA. Both prospective and follow-up data are needed to definitively evaluate the role of oxHDL in CVR assessment in RA.

Background Natural killer cells play a critical role in innate immunity by targeting and eliminating cancerous and infected cells.The cell line NK-92, currently used in several clinical trials, has shown great potential in cancer immunotherapy due to strong cytotoxic capabilities and suitability as an off-the-shelf therapy. However, effective clinical application of NK-92 therapies requires optimised cell culture conditions, including cultivation medium composition and expansion methods. This study aimed at identifying optimal cultivation medium formulations and intensifying NK-92 cell expansion by systematically evaluating critical quality attributes and key performance indicators. Results We screened various cultivation media formulations, including alpha minimum essential medium, Roswell Park Memorial Institute medium, and stem cell growth medium, supplemented with either human serum, fetal bovine serum, chemically defined serum replacement, and a combination of fetal bovine serum and horse serum. Cells cultivated in alpha minimum essential medium supplemented with human serum significantly outperformed the other formulations, achieving the highest growth rate, maximum viable cell count, and superior cytotoxicity at high cost-efficiency. Media containing chemically defined serum replacement showed reduced cell proliferation despite high cytotoxicity per cell, indicating a complex balance between cell quantity and functionality. Following cultivation medium selection, cultivation strategies including batch, fed-batch, and repetitive batch were compared. The repetitive batch method, where cultivation medium is replenished repeatedly throughout the cultivation, demonstrated superior expansion capability, long-term cell viability, significantly improved cytotoxicity, although associated with higher medium consumption and thus costs. Conclusions This study identified alpha minimum essential medium supplemented with human serum in repetitive batch cultivation as the optimal approach for NK-92 cell expansion.This combination enhances cell growth, viability, and cytotoxicity, meeting essential clinical quality attributes while maintaining cost-effectiveness.These findings provide valuable guidance and should act as a baseline for consistent, scalable, and commercially viable production of NK cell-based cancer therapies.

Human natural killer (NK) cells represent a promising leukocyte population for use in immune cell therapies. Hematologic as well as solid tumors have been shown to respond to treatment with autologous or allogeneic NK cells. Providing NK cells in sufficient quantity and quality (i.e., efficacy, safety, identity, purity, and yield), remains a challenge. A deeper insight into the critical process parameters (CPP) and critical quality attributes (CQA) of primary NK cell culture is needed for the understanding and control of NK cell manufacturing. Due to the large number of potential CPP’s, a design of experiments (DoE) approach was used for systematic planning and reduction of the number of experiments. Primary NK cells from two donors were cultivated under batch conditions and varying CPP’s. A detailed analysis of the impact of the investigated CPP’s was conducted, and the DoE based experimental setup allowed the interpretation of the influence of each selected process parameter as well as their combination on the relevant CQAs. Based on this data, a statistical model was formulated, which allows predictions of the maximal viable cell number. This approach allows for detailed insight into the CPP’s and possible optimizations in the production of cell therapy products.

Abstract Background Mycobacterium tuberculosis (MT) infections represent a global health problem and latent tuberculosis infection (LTBI) affects an estimated 25% of the world population. 10.6 million people fell ill with tuberculosis (TB) worldwide in 2021 and a total of 1.6 million TB-associated deaths were reported. Thus, reliable diagnosis of LTBI is crucial to ensure adequate treatment. We tested three novel MT antigens of the dormancy survival regulator (DosR) complex, ACR, Rv1733, Rv2626, for improvement of MT specific interferon gamma release assays (IGRA) for diagnosing TB. Furthermore, we specifically investigated the potential of the complement factor C5a and the toll like receptor (TLR) agonists CpG ODN as well as Poly(I: C) as costimulators in order to increase diagnostic quality of MT IGRAs. Three MT IGRAs were evaluated, i.e. our in-house IGRA, a prototypic EUROIMMUN Quan-T-Cell TB assay and the gold standard QuantiFERON Tb-Gold Plus assay. Methods In this single-center, prospective trial, whole blood from 71 patients with tuberculosis disease was stimulated using our in-house IGRA with ACR, Rv1733, Rv2626 compared to the current gold standard MT antigen formulation encompassing MT antigens ESAT-6, CFP-10 and TB10.4. Further, C5a, CpG ODN and Poly(I: C) were tested as co-stimulators. IFN-γ levels in plasma were quantified using ELISA. Results The three novel antigens ACR, Rv1733 and Rv2626 failed to elicit equal or stronger IFN-γ-responses compared to the gold standard antigen formulation with ESAT-6, CFP-10 and TB10.4. The TLR9 agonist CpG ODN increased IFN-γ responses in whole blood of tuberculosis patients using our in-house assays (6,768 ± 21,097 mlU/ml vs. 2,971 ± 4,780 mlU/ml, p = 0.31), yet not significantly. The same trend was found for the prototypic EUROIMMUN Quan-T-Cell TB assay (3,355 ± 5,425 mlU/ml vs. 2,548 ± 4,145 mlU/ml, p = 0.1) and the QuantiFERON Tb-Gold Plus assay (3,627 ± 5,992 mlU/ml vs. 2,635 m ± 4,475 mlU/ml, p = 0.08, for tube 1; 3,257 ± 5,349 vs. 2,759 ± 4,446 mIU/ml, p = 0.25, for tube 2). No increase of IFN-γ release was seen using Poly(I: C) or C5a in all three assays. Conclusions ACR, Rv1733 and Rv2626 failed to elicit equal or even better IFN-γ responses in our in-house IGRA compared to ESAT-6, CFP-10 and TB10.4 in patients with MT infection. The TLR9 agonist CpG ODN might be useful as co-stimulator in MT IGRAs.

Background/Objectives: Hepatitis E virus (HEV) is one of the leading causes of acute hepatitis, with immunosuppressed individuals, such as oncology patients, being particularly vulnerable to chronic infections that may progress to liver disease or fatal outcomes. Assay variability complicates HEV prevalence assessment in at-risk groups. This study aimed to compare the reliability and concordance of three HEV antibody assays—Wantai, Euroimmun, and Elecsys®—in immunosuppressed oncology patients. Methods: In this prospective pilot study, serum samples were obtained from oncology patients between September 2020 and October 2021. Samples were collected both at baseline (treatment-naive) and during ongoing treatment. A healthy control group was retrospectively included for comparative analysis. Anti-HEV IgM and IgG antibodies were tested in all samples using enzyme-linked immunosorbent assays (Wantai, Euroimmun) and an electrochemiluminescence immunoassay (Elecsys®). Demographic and clinical data, along with information on HEV risk factors, were extracted from medical records and patient questionnaires. Results: HEV IgM prevalence ranged from 0% (Wantai) to 6% (Elecsys®), while IgG prevalence was 12% (Euroimmun), 38% (Wantai), and 53% (Elecsys®). Concordance was poor, with Cohen’s Kappa values indicating slight to moderate agreement (κ = 0.000–0.553). Patients with hematological malignancies exhibited the highest IgG seroprevalence. Risk factor analysis revealed the highest association between HEV exposure and the consumption of undercooked pork or crop-based agriculture. Conclusions: Significant variability among HEV serological assays highlights the challenges of reliable HEV diagnostics in immunosuppressed oncology patients. Assay selection and improved testing strategies are critical for this high-risk group.

Background:Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease, and it significantly increases the risk of cardiovascular disease and death. The evaluation of cardiovascular risk (CVR) is crucial in these patients, but it may be underestimated using the current criteria, as they do not include nontraditional CVR factors. Soluble ST-2, which is the circulating form of the IL-33 receptor, has been identified as a biomarker for cardiovascular and rheumatic diseases. In this study, we examined the role of sST-2 in assessing CVR in RA. Methods:Monocentric, retrospective, observational trial. Inclusion of RA patients on variable DMARD therapy. Analysis of RA disease using established scores (DAS 28, VAS, HFQ), clinical findings (number of swollen and painful joints), and laboratory investigation. Documentation of numerous CVR variables. Quantification of soluble sST-2 by ELISA. Results:In total, 129 individuals were included. Soluble sST-2 did neither correlate nor was associated with any variable of RA disease activity. In contrast, significant associations were identified between sST-2 and a number of established CVR markers. Conclusions:The data indicates a novel role for sST-2 in CVR prediction in RA.

Background:Rheumatoid arthritis (RA) significantly increases the overall risk of cardiovascular disease (CVD). In addition to conventional risk factors, the inflammatory activity of the disease itself and medications that promote atherosclerosis contribute to an even greater risk. In this study, we performed metabolomic analysis in RA patients, both on and off disease-modifying anti-rheumatic drug (DMARD) therapy, with the aim of identifying new candidates for more sophisticated cardiovascular risk (CVR) assessment. Methods:This is an observational, cross-sectional investigation that included patients with established RA. DMARD therapy, if prescribed, consisted of methotrexate (MTX) alone or in combination with other conventional disease-modifying anti-rheumatic drugs (cDMARDs) or biologic disease-modifying anti-rheumatic drugs (bDMARDs), or other cDMARDs or bDMARDs without MTX, respectively. Metabolomic profiling was conducted using a Bruker AVANCE NEO 600 MHz nuclear magnetic resonance (NMR) spectrometer. The spectra obtained were Fourier transformed using TopSpin software (version 4.0, Bruker Biospin, Germany). All spectra were automatically phased and subjected to baseline correction. Subsequently, the spectra were analyzed using the proprietary Profiler software (version 1.4_Blood, lifespin GmbH, Germany), and a quantitative metabolite list was generated. Results:In total, 200 patients were included in the study, 54 subjects were not receiving any DMARDs (n = 47 untreated at the time of inclusion, n = 7 with established disease but not receiving DMARD therapy), and 146 were receiving DMARD treatment. No metabolic differences were found in relation to drug therapy or RA activity. The following CVR factors were associated with significant metabolic abnormalities: distress, arterial hypertension, diabetes mellitus and an average higher Framingham score. Distressed individuals showed abnormalities in histidine metabolism. Conclusions:Our findings have aided in the identification of potential surrogate markers for assessing the burden of CVD in individuals with RA. Histidine may be of particular diagnostic importance in CVR assessment in RA.

Objective To conduct a metabolomics analysis in patients with spondyloarthritis (SpA) and compare results with those from healthy controls. The overall goal was to identify small-molecule substances that may have potential pathogenic and diagnostic significance in SpA. Methods This was an observational, cross-sectional, single-centre study that included patients with axial (ankylosing spondylitis [AS]), peripheral (psoriatic arthritis [PsA]) and healthy controls. Results The study included 50 patients with AS, 50 patients with PsA, and 164 controls. When compared with healthy controls, patients with SpA showed significant differences in 35 metabolites, primarily associated with amino acid and lipid metabolism. However, only two differences were found between the AS and PsA cohorts (glucose and glycerol). Conclusions Our data suggest that patients with SpA exhibit significant disruptions in amino acid and lipid metabolism. The large number of identified metabolites offers promising opportunities, both for discovering new SpA biomarkers and for gaining a deeper understanding of the pathophysiology of these chronic inflammatory diseases.

Background/Objectives: Pneumocystis jirovecii pneumonia (PJP) is the most frequently diagnosed AIDS-defining illness in Europe, with especially high mortality in HIV-negative patients caused by delayed diagnosis and low awareness. This study aims to evaluate cytokine release assays (CRA) to facilitate a less invasive and resource-efficient PJP specific diagnostic test. We focus on the P. jirovecii antigens Kexin 1 (KEX1), MSG1, and MSG2, which were identified in prior studies as immunologically relevant. Methods: Whole blood samples from 50 participants—22 healthy individuals and 28 immunocompromised individuals, including 8 with proven PJP—were stimulated in vitro with full-length and partial KEX1, MSG1, MSG2, and a combination of all three antigens (PJ-MIX). Following 24 h incubation at 37 °C, cytokine levels of IL-2, IFN-γ, IL-17A, and IL-17F were measured. Results: Stimulation with full-length KEX1, MSG1, MSG2, and PJ-MIX antigens induced higher IL-2 concentrations in the healthy control group compared to the groups IL-2 baseline levels and to the group of proven PJP cases. Similarly, stimulation with full-length KEX1, MSG1, and PJ-MIX elevated IFN-γ levels in the healthy control group compared to baseline IFN-γ levels. Conclusions: Our findings highlight the potential of IL-2 and IFN-γ release following stimulation with PJ antigens, with PJ-MIX eliciting the strongest and most significant responses, suggesting a cumulative antigen effect. This pilot study establishes a foundation for a PJP-specific CRA, deepening our knowledge of T-cell immunity against PJP. Clinically, such a test could, among other applications, evaluate at-risk patients who should receive prophylaxis and may consequently reduce PJP-related morbidity and mortality.

Background: Rheumatoid arthritis (RA) significantly increases the overall risk of cardiovascular disease (CVD). In addition to conventional risk factors, the inflammatory activity of the disease itself and medications that promote atherosclerosis contribute to an even greater risk. However, there is a lack of additional biomarkers for CVD risk assessment. In recent years, metabolomics has emerged as a valuable tool for identifying potential biomarkers in various diseases. Objectives: In this study, we conducted metabolomics analysis in RA patients, both with and without disease-modifying antirheumatic drug initiation (DMARD) therapy. Methods: Observational, cross-sectional investigation that included patients with established RA. DMARD therapy, if prescribed, consisted of methotrexate alone or in combination with other cDMARDs or bDMARDs or other cDMARDs or bDMARDs without MTX, respectively. Metabolomic profiling was conducted using a Bruker AVANCE NEO 600 MHz NMR spectrometer. The spectra obtained were Fourier transformed using TopSpin software (version 4.0, Bruker Biospin, Germany). All spectra were automatically phased and subjected to baseline correc-tion. Subsequently, the spectra were analyzed using the proprietary Profiler software (version 1.4_Blood, lifespin GmbH, Germany) and a quantitative metabolite list was generated. Results: In total, 47 patients with untreated and 153 individuals with DMARD-treated RA were included. In general, RA patients with arterial hypertension showed higher glucose and mannose levels than non-hypertensive subjects. Also, diabetics displayed significant differences between several intermediates (n=15) of the amino acid and lipid metabolism. Patients with a Framingham score of 20+ showed 19 distinct metabolic aberrations from individuals with a score of below 10. Finally, the following metabolites significantly differed between DMARD- and DMARD+ RA: 1,2-propanediol, glycerol, high density lipoprotein, and leucine. Conclusion: Our findings have aided in the identification of potential surrogate markers for assessing the burden of cardiovascular disease in individuals with RA. Selected candidates may have the potential to enhance the accuracy of cardiovascular risk assessment in RA thus allowing for a more comprehensive evaluation. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Susann Patschan: None declared, Inga Claus: None declared, Meike Hoffmeister: None declared, Selina Strathmeyer Lifespin GmbH, Steffen Heelemann Lifespin GmbH, Constantin Remus: None declared, Werner Dammermann: None declared, Oliver Ritter: None declared, Daniel Patschan: None declared.

Background: Improving precision medicine in chemotherapy requires highly sensitive and easily applicable diagnostic tools. In addition, non-invasive molecular real-time monitoring of cytotoxic response is highly desirable. Here, we employed the kinetics of DNA double-strand breaks (DSB) and cell-free DNA (cfDNA) in a cell model of topoisomerase II-inhibitors in T cell leukemia (Jurkat cells) compared to normal cells (peripheral blood mononuclear cells, PBMCs). Methods: We applied automated microscopy to quantify immuno-stained phosphorylated H2AX (γH2AX) as a marker for either DNA damage response (DDR) or cell death and quantitative PCR-based analysis of nuclear and mitochondrial cfDNA concentrations. Results: Jurkat cells displayed a DDR to cytotoxic drug treatment significantly earlier than PBMCs, and etoposide (ETP) induced DSB formation faster than doxorubicin (DOX) in both Jurkat and PBMCs. Jurkat cells exhibited an earlier cytotoxic response compared to PBMC, with a significantly increased mitochondrial cfDNA formation after 2 h of DOX application. In PBMCs, increased cell death was detected after 4 h of incubation with ETP, whereas DOX treatment was less effective. Conclusions: Both automated microscopy and mitochondrial cfDNA quantification analysis indicate that (malignant) Jurkat cells are more sensitive to DOX than (healthy) PBMC. Our real-time approach can improve DDR inducing drug selection and adaptation in cancer therapy and aids in decisions for optimal patient biosampling.

BACKGROUND:Atrial fibrillation (AF) is the most common heart arrhythmia and considered to be a progressive chronic disease associated with increased morbidity and mortality. Recent data suggest a link between inflammation, oxidative stress, and AF, although the underlying mechanisms are not fully understood. Because oxidized lipoproteins cause structural damage and electrophysiologic changes in cardiomyocytes, it is feasible that the transformation of atheroprotective high-density lipoprotein (HDL) into dysfunctional HDL contributes to the development of AF. OBJECTIVE:The purpose of this study was to determine whether a reduced antioxidant function of HDL is associated with the presence of AF. METHODS:In this multicenter cross-sectional cohort study, we assessed HDL function in sera of 1206 participants. Patients were divided into groups according to the presence of AF (n = 233) or no AF (n = 973). A validated cell-free biochemical assay was used to determine reduced HDL antioxidant function as assessed by increased normalized HDL lipid peroxide content (nHDLox). RESULTS:Participants with AF had a 9% higher mean relative nHDLox compared to persons without AF (P = .025). nHDLox was strongly associated with AF in all models of logistic regression, including the analysis adjusted for age, sex, and risk factors for AF (all P ≤.01). CONCLUSION:Reduced antioxidant HDL function is associated with the presence of AF, which supports growing evidence that impaired lipoprotein function is linked to electrophysiological changes in cardiomyocytes. nHDLox is one of several contributors to the initiation and perpetuation of AF.

Background: Unhealthy habits, such as overeating processed and high-calorie foods, alcohol abuse, and smoking, negatively impact human health. It has been suggested that the inflammatory process and the resulting growth of nerve fibers within the intervertebral disc (IVD) fissures is the main reason for the pain accompanying IVD degeneration (IVDD). Objectives: The aim of this study was to determine whether smoking, alcohol consumption, overweight/obesity, or diabetes comorbidity contribute to the development of IVDD and how the aforementioned factors affect the levels of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and growth associated protein 43 (GAP-43) in the study and control groups (intervertebral discs, IVDs from cadavers, and serum samples from voluntary blood donors). Methods: The study group comprised 113 patients diagnosed with IVDD who qualified for microdiscectomy. Two control groups (I and II) were used in this study. The first included 81 IVDs obtained from Caucasian human cadavers. Control group II, on the other hand, included serum samples obtained from 113 voluntary blood donors. The expression profiles of BDNF, GDNF, and GAP-43 were determined by enzyme-linked immunosorbent assay (ELISA). Results: Our statistical analysis confirmed that patients who were overweight/obese, smoked tobacco, consumed alcohol, or had diabetes had a higher risk of IVDD (OR > 1). Statistical analysis showed that BDNF, GAP-43, and GDNF concentrations were significantly higher in the IVDs and serum samples obtained from the study group compared to the control group (p < 0.05). In addition, higher levels of BDNF, GDNF, and GAP-43 were noted in IVDD patients who consumed alcohol, smoked tobacco, were overweight/obese, or had comorbid diabetes compared to patients without these risk factors (p < 0.05). Conclusion: We showed that changes in energy metabolism, habits, and lifestyle, as well as the degenerative process of IVD in the lumbosacral spine contribute to changing the concentration profile of the analyzed neurotrophic factors.

OBJECTIVES:Due to disease- or therapy-associated immunosuppression, oncological patients suffer from significantly higher morbidity and mortality due to infections transmitted by respiratory pathogens such as Streptococcus pneumoniae and influenza virus. Although the German Standing Committee on Vaccination (STIKO) provides specific recommendations for vaccination against these pathogens, there is no data on vaccination rates in this high-risk population. METHODS:Data from the interventional EVO study were analyzed to provide information on vaccination rates against Streptococcus pneumoniae and influenza virus in oncological patients. Numbers presented in this publication summarize baseline and follow-up data of the control group; thus, data were not influenced by the intervention. RESULTS:Data of 370 patients were analyzed; 20.5% of patients were treated for hematological malignancies and 79.5% for solid cancer. 28.1% of patients had received vaccination against influenza and 32.2% against Streptococcus pneumoniae; for the latter only 7.3% according recommendations. While vaccination rates where even lower for patients with thoracic carcinoma (influenza 26.7% and Streptococcus pneumoniae 6.0% according to STIKO recommendations), rates in patients with multiple myeloma were remarkably higher (39.0% and 14.6%). CONCLUSIONS:Despite strong recommendations to vaccinate and the clear clinical need to prevent infections in the vulnerable group of oncological patients, only the minority was vaccinated against Streptococcus pneumoniae or influenza, underlining the urgent need for better vaccination strategies in this high-risk population.