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Infection with Hepatitis E Virus (HEV) is often asymptomatic but can also lead to chronic infection in immunosuppressed individuals. Although fecal–oral transmission of HEV is well established, transmission by blood transfusion has also been reported. Here, we studied HEV seroprevalence in a cohort of 1000 blood donors (50% male, age 18–73 years, mean 35 years) at the University Medical Center Hamburg–Eppendorf in Germany. We found a seroprevalence of anti-HEV IgG of 16.6%. Interestingly, 1.3% of the blood donors had positive IgM serology despite testing negative for HEV by polymerase chain reaction (PCR). Analysis of preceding and follow-up samples showed persistence of IgM antibodies for up to seven months in asymptomatic individuals. In eight individuals, anti-HEV IgM positivity persisted for 0 to 7 months (median 2 months), as confirmed by testing stored samples. This study demonstrates that anti-HEV IgM positivity can persist for more than six months in individuals who had neither clinically overt hepatitis E nor a viremia duration that would allow PCR positivity to be detected.

Assessment of a low skeletal muscle mass (SM) is important for diagnosis of ageing and disease-associated sarcopenia and is hindered by heterogeneous methods and terminologies that lead to differences in diagnostic criteria among studies and even among consensus definitions. The aim of this review was to analyze and summarize previously published cut-offs for SM applied in clinical and research settings and to facilitate comparison of results between studies. Multiple published reference values for discrepant parameters of SM were identified from 64 studies and the underlying methodological assumptions and limitations are compared including different concepts for normalization of SM for body size and fat mass (FM). Single computed tomography or magnetic resonance imaging images and appendicular lean soft tissue by dual X-ray absorptiometry (DXA) or bioelectrical impedance analysis (BIA) are taken as a valid substitute of total SM because they show a high correlation with results from whole body imaging in cross-sectional and longitudinal analyses. However, the random error of these methods limits the applicability of these substitutes in the assessment of individual cases and together with the systematic error limits the accurate detection of changes in SM. Adverse effects of obesity on muscle quality and function may lead to an underestimation of sarcopenia in obesity and may justify normalization of SM for FM. In conclusion, results for SM can only be compared with reference values using the same method, BIA- or DXA-device and an appropriate reference population. Limitations of proxies for total SM as well as normalization of SM for FM are important content-related issues that need to be considered in longitudinal studies, populations with obesity or older subjects.

Although clinically relevant, the detection rates of EpCAM positive CTCs in non-small cell lung cancer (NSCLC) are surprisingly low. To find new clinically informative markers for CTC detection in NSCLC, the expression of EGFR and HER3 was first analyzed in NSCLC tissue (n = 148). A positive EGFR and HER3 staining was observed in 52.3% and 82.7% of the primary tumors, and in 62.7% and 91.2% of brain metastases, respectively. Only 3.0% of the brain metastases samples were negative for both HER3 and EGFR proteins, indicating that the majority of metastases express these ERBB proteins, which were therefore chosen for CTC enrichment using magnetic cell-separation. Enrichment based on either EGFR or HER3 detected CTCs in 37.8% of the patients, while the combination of EGFR/HER3 enrichment with the EpCAM-based CellSearch technique detected a significantly higher number of 66.7% CTC-positive patients (Cohen’s kappa = −0.280) which underlines the existence of different CTC subpopulations in NSCLC. The malignant origin of keratin-positive/CD45-negative CTC clusters and single CTCs detected after EGFR/HER3 based enrichment was documented by the detection of NSCLC-associated mutations. In conclusion, EGFR and HER3 expression in metastasized NSCLC patients have considerable value for CTC isolation plus multiple markers can provide a novel liquid biopsy approach.

Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions. CTCs were enriched based on their EpCAM expression (CellSearch ) or by size and deformability (Parsortix ), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling. Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8). Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality. HCC is characterized by high heterogeneity and, subsequently, adaptation by developing resistance to current treatments. Applying individualized models is crucial to understanding the potential of approved therapies. Therefore, we classify a primary-like cell line derived from the core region of an HCC with underlying HIV-HCV co-infection employing deep analysis on the pathway regulation level. Methods: We employed DEG analysis, followed by pathway analysis, to characterize the preservation level of the LC4 cells and the level of adoption. Next, we classify the new model for HCC research by employing healthy donor samples, commonly used HCC cell lines, and global RNAseq datasets. Results: LC4 cells reflect the characteristics of the parental cancer region, including immunosuppression and metabolic reprogramming, characterized by the downregulation of drug-metabolizing enzymes compared to healthy individuals, indicating a transition to alternate metabolic pathways. Moreover, we identified specific biomarkers equally regulated in the parental tissue, in global datasets of the same entities as well as in LC4 cells. Conclusions: We classified LC4 cells as an individual immunosuppressive and highly progressive primary-like HCC cell line. LC4 cells are applicable as a model for preclinical drug testing, minimizing the lack of preclinical models in HCV-HIV-induced HCC research.

Introduction Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates pathophysiological processes involved in sepsis progression, including endothelial permeability, cytokine release, and vascular tone. The aim of this study was to investigate whether serum-S1P concentrations are associated with disease severity in patients with sepsis. Methods This single-center prospective-observational study includes 100 patients with systemic inflammatory response syndrome (SIRS) plus infection (n = 40), severe sepsis (n = 30), or septic shock (n = 30) and 214 healthy blood donors as controls. Serum-S1P was measured by mass spectrometry. Blood parameters, including C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), lactate, and white blood cells (WBCs), were determined by routine assays. The Sequential Organ Failure Assessment (SOFA) score was generated and used to evaluate disease severity. Results Serum-S1P concentrations were lower in patients than in controls ( P  < 0.01), and the greatest difference was between the control and the septic shock groups ( P  < 0.01). Serum-S1P levels were inversely correlated with disease severity as determined by the SOFA score ( P  < 0.01) as well as with IL-6, PCT, CRP, creatinine, lactate, and fluid balance. A receiver operating characteristic analysis for the presence or absence of septic shock revealed equally high sensitivity and specificity for S1P compared with the SOFA score. In a multivariate logistic regression model calculated for prediction of septic shock, S1P emerged as the strongest predictor ( P  < 0.001). Conclusions In patients with sepsis, serum-S1P levels are dramatically decreased and are inversely associated with disease severity. Since S1P is a potent regulator of endothelial integrity, low S1P levels may contribute to capillary leakage, impaired tissue perfusion, and organ failure in sepsis.

The relevance of blood-based assays to monitor minimal residual disease (MRD) in non-metastatic prostate cancer (PCa) remains unclear. Proving that clinically relevant circulating tumor cells (CTCs) can be detected with available technologies could address this. This study aimed to improve CTC detection in non-metastatic PCa patients by combining three independent CTC assays: the CellSearch system, an in vivo CellCollector and the EPISPOT. Peripheral blood samples from high-risk PCa patients were screened for CTCs before and three months after radical prostatectomy (RP). Combining the results of both time points, CTCs were detected in 37%, 54.9% and 58.7% of patients using CellSearch, CellCollector and EPISPOT, respectively. The cumulative positivity rate of the three CTC assays was 81.3% (87/107) with 21.5% (23/107) of patients harboring ≥5 CTCs/7.5 ml blood. Matched pair analysis of 30 blood samples taken before and after surgery indicated a significant decrease in CTCs captured by the CellCollector from 66% before RP to 34% after therapy (p = 0.031). CTC detection by EPISPOT before RP significantly correlated with PSA serum values (p < 0.0001) and clinical tumor stage (p = 0.04), while the other assays showed no significant correlations. In conclusion, CTC-based liquid biopsies have the potential to monitor MRD in patients with non-metastatic prostate cancer.

The aim of this study was to define the breadth and specificity of dominant SARS-CoV-2-specific T cell epitopes using a comprehensive set of 135 overlapping 15-mer peptides covering the SARS-CoV-2 envelope (E), membrane (M) and nucleoprotein (N) in a cohort of 34 individuals with acute (n = 10) and resolved (n = 24) COVID-19. Following short-term virus-specific in vitro cultivation, the single peptide-specific CD4+ T cell response of each patient was screened using enzyme linked immuno spot assay (ELISpot) and confirmed by single-peptide intracellular cytokine staining (ICS) for interferon-γ (IFN-γ) production. 97% (n = 33) of patients elicited one or more N, M or E-specific CD4+ T cell responses and each patient targeted on average 21.7 (range 0–79) peptide specificities. Overall, we identified 10 N, M or E-specific peptides that showed a response frequency of more than 36% and five of them showed high binding affinity to multiple HLA class II binders in subsequent in vitro HLA binding assays. Three peptides elicited CD4+ T cell responses in more than 55% of all patients, namely Mem_P30 (aa146-160), Mem_P36 (aa176-190), both located within the M protein, and Ncl_P18 (aa86-100) located within the N protein. These peptides were further defined in terms of length and HLA restriction. Based on this epitope and restriction data we developed a novel DRB*11 tetramer (Mem_aa145-164) and examined the ex vivo phenotype of SARS-CoV-2-specific CD4+ T cells in one patient. This detailed characterization of single T cell peptide responses demonstrates that SARS-CoV-2 infection universally primes a broad T cell response directed against multiple specificities located within the N, M and E structural protein.

Purpose New biomarkers for the detection and monitoring of aggressive variant prostate cancer (AVPC) including therapy-induced neuroendocrine prostate cancer (NEPC) are urgently needed, as measuring prostate-specific antigen (PSA) is not reliable in androgen-indifferent diseases. Molecular analysis of circulating tumor cells (CTC) enables repeated analysis for monitoring and allows to capture the heterogeneity of the disease. Experimental design 102 blood samples from 76 metastatic prostate cancer (mPC) patients, including 37 samples from histologically proven NEPC, were collected and CTCs were enriched using label-dependent and label-independent methods. Relevant transcripts were selected for CTC profiling using semi-quantitative RT-PCR analysis and validated in published datasets and cell lines. Transcriptional profiles in patient samples were analyzed using supervised and unsupervised methods. Results CTC counts were increased in AVPC and NEPC as compared to metastatic hormone-sensitive prostate cancer (mHSPC). Gene expression profiles of CTCs showed a high degree of inter-patient heterogeneity, but NEPC-specific transcripts were significantly increased in patients with proven NEPC, while adenocarcinoma markers were decreased. Unsupervised analysis identified four distinct clusters of CTC low , AR high , amphicrine and pure NEPC gene expression profiles that reflected the clinical groups. Based on the transcript panel, NEPC could be distinguished from mHSPC or AVPC patients with a specificity of 95.5% and 88.2%, respectively. Conclusion Molecular subtypes of mPC can be distinguished by transcriptional profiling of CTCs. In the future, our convenient PCR-based analysis may complement the monitoring of advanced PCa patients and allow timely detection of resistance to androgen receptor pathway inhibitors.

Host iron deficiency is protective against severe malaria as the human malaria parasite Plasmodium falciparum depends on bioavailable iron from its host to proliferate. The essential pathways of iron acquisition, storage, export, and detoxification in the parasite differ from those in humans, as orthologs of the mammalian transferrin receptor, ferritin, or ferroportin, and a functional heme oxygenase are absent in P. falciparum . Thus, the proteins involved in these processes may be excellent targets for therapeutic development, yet remain largely unknown. Here, we show that parasites cultured in erythrocytes from an iron-deficient donor displayed significantly reduced growth rates compared to those grown in red blood cells from healthy controls. Sequencing of parasite RNA revealed diminished expression of genes involved in overall metabolism, hemoglobin digestion, and metabolite transport under low-iron versus control conditions. Supplementation with hepcidin, a specific ferroportin inhibitor, resulted in increased labile iron levels in erythrocytes, enhanced parasite replication, and transcriptional upregulation of genes responsible for merozoite motility and host cell invasion. Through endogenous GFP tagging of differentially expressed putative transporter genes followed by confocal live-cell imaging, proliferation assays with knockout and knockdown lines, and protein structure predictions, we identified six proteins that are likely required for ferrous iron transport in P. falciparum . Of these, we localized Pf VIT and Pf ZIPCO to cytoplasmic vesicles, Pf MRS3 to the mitochondrion, and the novel putative iron transporter Pf E140 to the plasma membrane for the first time in P. falciparum . Pf NRAMP/ Pf DMT1 and Pf CRT were previously reported to efflux Fe 2+ from the digestive vacuole. Our data support a new model for parasite iron homeostasis, in which Pf E140 is involved in iron uptake across the plasma membrane, Pf MRS3 ensures non-redundant Fe 2+ supply to the mitochondrion as the main site of iron utilization, Pf VIT transports excess iron into cytoplasmic vesicles, and Pf ZIPCO exports Fe 2+ from these organelles in case of iron scarcity. These results provide new insights into the parasite’s response to differential iron availability in its environment and into the mechanisms of iron transport in P. falciparum as promising candidate targets for future antimalarial drugs.