Diagnostic leukapheresis reveals distinct phenotypes of NSCLC circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) hold immense promise for unraveling tumor heterogeneity and understanding treatment resistance. However, conventional methods, especially in cancers like non-small cell lung cancer (NSCLC), often yield low CTC numbers, hindering comprehensive analyses. This study addresses this limitation by employing diagnostic leukapheresis (DLA) to cancer patients, enabling the screening of larger blood volumes. To leverage DLA's full potential, this study introduces a novel approach for CTC enrichment from DLAs. METHODS: DLA was applied to six advanced stage NSCLC patients. For an unbiased CTC enrichment, a two-step approach based on negative depletion of hematopoietic cells was used. Single-cell (sc) whole-transcriptome sequencing was performed, and CTCs were identified based on gene signatures and inferred copy number variations. RESULTS: Remarkably, this innovative approach led to the identification of unprecedented 3,363 CTC transcriptomes. The extensive heterogeneity among CTCs was unveiled, highlighting distinct phenotypes related to the epithelial-mesenchymal transition (EMT) axis, stemness, immune responsiveness, and metabolism. Comparison with sc transcriptomes from primary NSCLC cells revealed that CTCs encapsulate the heterogeneity of their primary counterparts while maintaining unique CTC-specific phenotypes. CONCLUSIONS: In conclusion, this study pioneers a transformative method for enriching CTCs from DLA, resulting in a substantial increase in CTC numbers. This allowed the creation of the first-ever single-cell whole transcriptome in-depth characterization of the heterogeneity of over 3,300 NSCLC-CTCs. The findings not only confirm the diagnostic value of CTCs in monitoring tumor heterogeneity but also propose a CTC-specific signature that can be exploited for targeted CTC-directed therapies in the future. This comprehensive approach signifies a major leap forward, positioning CTCs as a key player in advancing our understanding of cancer dynamics and paving the way for tailored therapeutic interventions.

Correction: Broadly directed SARS-CoV-2-specific CD4+ T cell response includes frequently detected peptide specificities within the membrane and nucleoprotein in patients with acute and resolved COVID-19.

[This corrects the article DOI: 10.1371/journal.ppat.1009842.].

Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells.

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus-infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV-1-infected cells. By combining an unbiased large-scale screening approach with a functional assay, we identify TRAIL to be associated with NK cell degranulation against HIV-1-infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor-mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFNγ production. Moreover, TRAIL-mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL-mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti-HIV-1 activity of NK cells but also possesses a multifunctional role beyond receptor-mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.

Impact of the COVID-19 outbreak on blood supply in two large university hospitals.

OBJECTIVE: This study aimed to examine the relationship between the decrease in elective procedures and the need for blood donation during the novel coronavirus disease (COVID-19) pandemic at university hospitals. BACKGROUND: The COVID-19 pandemic has immensely impacted transfusion medicine. By cancelling elective surgery, the German government hoped to increase the available resources for patients infected with COVID-19, especially in intensive care units, and prevent the shortage of blood products. METHODS/MATERIALS: Over 26 weeks, from the 3rd of February 2020 to the 2nd of August 2020, during the first phase of the pandemic, we assessed the number of crossmatches, blood group typing, use of donated blood, and case mix indices by retrospectively analysing data from two major university hospitals' information systems in Essen and Hamburg, Germany. Data were pooled, analysed, and compared with that of the same period in the previous year. RESULTS: Following the cessation of elective procedures, the number of requests for crossmatches and blood group typing significantly decreased in 2020 compared to that in 2019. However, the number of blood transfusions required was reduced to a lesser extent. The number of outpatient and inpatient cases significantly decreased, whereas the cases requiring transfusion decreased only. CONCLUSION: During the initial phase of the pandemic, transfusion medicine, especially in large institutions, faced an almost unchanged high demand for donated blood. This should be considered regarding personnel and blood donation allocations. Therefore, we developed a monitoring system to display the availability of blood products in real-time. The quick and easy display of in-stock and expiring blood products can optimise the use of this valuable resource.

Pathogenicity of Human Anti-PLA 2 R1 Antibodies in Minipigs: A Pilot Study.

SIGNIFICANCE STATEMENT: Membranous nephropathy (MN) is an autoimmune kidney disease characterized by immune deposits in the glomerular basement membrane. Circulating anti-phospholipase A 2 receptor 1 (PLA 2 R1) antibodies are detectable in 70%-80% of patients with MN, but experimental evidence of pathogenicity has been lacking. This study demonstrates the pathogenicity of human anti-PLA 2 R1 antibodies in minipigs, a model for MN that intrinsically expresses PLA 2 R1 on podocytes. After passive transfer of human anti-PLA 2 R1 antibody-containing plasma from patients with PLA 2 R1-associated MN to minipigs, antibodies were detected in the minipig glomeruli, but not in response to plasma from healthy controls. The minipigs developed histomorphological characteristics of MN, local complement activation in the glomeruli, and low-level proteinuria within 7 days, showing that human anti-PLA 2 R1 antibodies are pathogenic. BACKGROUND: Primary membranous nephropathy (MN) is an autoimmune kidney disease in which immune complexes are deposited beneath the epithelium in the glomeruli. The condition introduces a high risk for end-stage kidney disease. Seventy percent to 80% of patients with MN have circulating antibodies against phospholipase A 2 receptor 1 (PLA 2 R1), and levels correlate with treatment response and prognosis. However, experimental evidence that human anti-PLA 2 R1 antibodies induce MN has been elusive. METHODS: In passive transfer experiments, minipigs received plasma or purified IgG from patients with PLA 2 R1-associated MN or from healthy controls. Anti-PLA 2 R1 antibodies and proteinuria were monitored using Western blot, ELISA, and Coomassie staining. Kidney tissues were analyzed using immunohistochemistry, immunofluorescence, electron microscopy, and proteomic analyses. RESULTS: Minipigs, like humans, express PLA 2 R1 on podocytes. Human anti-PLA 2 R1 antibodies bound to minipig PLA 2 R1 in vitro and in vivo . Passive transfer of human anti-PLA 2 R1 antibodies from patients with PLA 2 R1-associated MN to minipigs led to histological characteristics of human early-stage MN, activation of components of the complement cascade, and low levels of proteinuria. We observed development of an autologous, later phase of disease. CONCLUSIONS: A translational approach from humans to minipigs showed that human anti-PLA 2 R1 antibodies are pathogenic in MN, although in the heterologous phase of disease only low-level proteinuria developed.

Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1.

In cancer metastasis, single circulating tumor cells (CTCs) in the blood and disseminated tumor cells (DTCs) in the bone marrow mediate cancer metastasis. Because suitable biomarker proteins are lacking, CTCs and DTCs with mesenchymal attributes are difficult to isolate from the bulk of normal blood cells. To establish a procedure allowing the isolation of such cells, we analyzed the cell line BC-M1 established from DTCs in the bone marrow of a breast cancer patient by stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry. We found high levels of the transmembrane protein CUB domain-containing protein 1 (CDCP1) in breast cancer cell lines with mesenchymal attributes. Peripheral blood mononuclear cells were virtually negative for CDCP1. Confirmation in vivo by CellSearch revealed CDCP1-positive CTCs in 8 of 30 analyzed breast cancer patients. Only EpCam-positive CTCs were enriched by CellSearch. Using the extracellular domain of CDCP1, we established a magnetic-activated cell sorting (MACS) approach enabling also the enrichment of EpCam-negative CTCs. Thus, our approach is particularly suited for the isolation of mesenchymal CTCs with downregulated epithelial cancer that occur, for example, in triple-negative breast cancer patients who are prone to therapy failure.

PSMA-heterogeneity in metastatic castration-resistant prostate cancer: Circulating tumor cells, metastatic tumor burden, and response to targeted radioligand therapy.

BACKGROUND: We explored the interrelation between prostate-specific membrane antigen (PSMA) expression on circulating tumor cells (CTCs) and that of solid metastatic lesions as determined by whole-body PSMA-targeted positron emission tomography (PET) to refine the prediction of response to subsequent PSMA-targeted radioligand therapy (RLT). METHODS: A prospective study was performed in 20 patients with advanced mCRPC. Of these, 16 underwent subsequent RLT with [177 Lu]Lu-PSMA-617 at a dose of 7.4 GBq every 6-8 weeks. PSMA expression on CTCs using the CellSearch system was compared to clinical and serological results, and to marker expression in targeted imaging and available histological sections of prostatectomy specimens (19% of RLT patients). Clinical outcome was obtained after two cycles of RLT. RESULTS: Marked heterogeneity of PSMA expression was observed already at first diagnosis in available histological specimens. Targeted whole-body imaging also showed heterogeneous inter- and intra-patient PSMA expression between metastases. Heterogeneity of CTC PSMA expression was partially paralleled by heterogeneity of whole-body tumor burden PSMA expression. Twenty percent of CTC samples showed no PSMA expression, despite unequivocal PSMA expression of solid metastases at PET. A high fraction of PSMA-negative CTCs emerged as the sole predictor of poor RLT response (odds ratio [OR]: 0.9379 [95% confidence interval, CI, 0.8558-0.9902]; p = 0.0160), and was prognostic for both shorter progression-free survival (OR: 1.236 [95% CI, 1.035-2.587]; p = 0.0043) and overall survival (OR: 1.056 [95% CI, 1.008-1.141]; p = 0.0182). CONCLUSION: This proof-of-principle study suggests that liquid biopsy for CTC PSMA expression is complementary to PET for individual PSMA phenotyping of mCRPC.

Natural killer cell-mediated ADCC in SARS-CoV-2-infected individuals and vaccine recipients.

COVID-19, caused by SARS-CoV-2, has emerged as a global pandemic. While immune responses of the adaptive immune system have been in the focus of research, the role of NK cells in COVID-19 remains less well understood. Here, we characterized NK cell-mediated SARS-CoV-2 antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2 spike-1 (S1) and nucleocapsid (NC) protein. Serum samples from SARS-CoV-2 resolvers induced significant CD107a-expression by NK cells in response to S1 and NC, while serum samples from SARS-CoV-2-negative individuals did not. Furthermore, serum samples from individuals that received the BNT162b2 vaccine induced strong CD107a expression by NK cells that increased with the second vaccination and was significantly higher than observed in infected individuals. As expected, vaccine-induced responses were only directed against S1 and not against NC protein. S1-specific CD107a responses by NK cells were significantly correlated to NK cell-mediated killing of S1-expressing cells. Interestingly, screening of serum samples collected prior to the COVID-19 pandemic identified two individuals with cross-reactive antibodies against SARS-CoV-2 S1, which also induced degranulation of NK cells. Taken together, these data demonstrate that antibodies induced by SARS-CoV-2 infection and anti-SARS-CoV-2 vaccines can trigger significant NK cell-mediated ADCC activity, and identify some cross-reactive ADCC-activity against SARS-CoV-2 by endemic coronavirus-specific antibodies.

Broadly directed SARS-CoV-2-specific CD4+ T cell response includes frequently detected peptide specificities within the membrane and nucleoprotein in patients with acute and resolved COVID-19.

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.

Deciphering the Plasmodium falciparum malaria-specific CD4+ T-cell response: ex vivo detection of high frequencies of PD-1+TIGIT+ EXP1-specific CD4+ T cells using a novel HLA-DR11-restricted MHC class II tetramer.

Relatively little is known about the ex vivo frequency and phenotype of the Plasmodium falciparum-specific CD4+ T-cell response in humans. The exported protein 1 (EXP1) is expressed by plasmodia at both, the liver stage and blood stage, of infection making it a potential target for CD4+ and CD8+ effector T cells. Here, a fluorochrome-labelled HLA-DRB1∗11:01-restriced MHC class II tetramer derived from the P. falciparum EXP1 (aa62-74) was established for ex vivo tetramer analysis and magnetic bead enrichment in 10 patients with acute malaria. EXP1-specific CD4+ T cells were detectable in 9 out of 10 (90%) malaria patients expressing the HLA-DRB1∗11 molecule with an average ex vivo frequency of 0.11% (0-0.22%) of total CD4+ T cells. The phenotype of EXP1-specific CD4+ T cells was further assessed using co-staining with activation (CD38, HLA-DR, CD26), differentiation (CD45RO, CCR7, KLRG1, CD127), senescence (CD57), and co-inhibitory (PD-1, TIGIT, LAG-3, TIM-3) markers as well as the ectonucleotidases CD39 and CD73. EXP1-specific tetramer+ CD4+ T cells had a distinct phenotype compared to bulk CD4+ T cells and displayed a highly activated effector memory phenotype with elevated levels of co-inhibitory receptors and activation markers: EXP1-specific CD4+ T cells universally expressed the co-inhibitory receptors PD-1 and TIGIT as well as the activation marker CD38 and showed elevated frequencies of CD39. These results demonstrate that MHC class II tetramer enrichment is a sensitive approach to investigate ex vivo antigen-specific CD4+ T cells in malaria patients that will aid further analysis of the role of CD4+ T cells during malaria.

Expression Patterns and Corepressor Function of Retinoic Acid-induced 2 in Prostate Cancer.

BACKGROUND: Revealing molecular mechanisms linked to androgen receptor activity can help to improve diagnosis and treatment of prostate cancer. Retinoic acid-induced 2 (RAI2) protein is thought to act as a transcriptional coregulator involved in hormonal responses and epithelial differentiation. We evaluated the clinical relevance and biological function of the RAI2 protein in prostate cancer. METHODS: We assessed RAI2 gene expression in the Cancer Genome Atlas prostate adenocarcinoma PanCancer cohort and protein expression in primary tumors (n = 199) by immunohistochemistry. We studied RAI2 gene expression as part of a multimarker panel in an enriched circulating tumor cell population isolated from blood samples (n = 38) of patients with metastatic prostate cancer. In prostate cancer cell lines, we analyzed the consequences of androgen receptor inhibition on RAI2 protein expression and the consequences of RAI2 depletion on the expression of the androgen receptor and selected target genes. RESULTS: Abundance of the RAI2 protein in adenocarcinomas correlated with the androgen receptor; keratins 8, 18, and 19; and E-cadherin as well as with an early biochemical recurrence. In circulating tumor cells, detection of RAI2 mRNA significantly correlated with gene expression of FOLH1, KLK3, RAI2, AR, and AR-V7. In VCaP and LNCaP cell lines, sustained inhibition of hormone receptor activity induced the RAI2 protein, whereas RAI2 depletion augmented the expression of MME, STEAP4, and WIPI1. CONCLUSIONS: The RAI2 protein functions as a transcriptional coregulator of the androgen response in prostate cancer cells. Detection of RAI2 gene expression in blood samples from patients with metastatic prostate cancer indicated the presence of circulating tumor cells.

Circulating Cellular Communication Network Factor 1 Protein as a Sensitive Liquid Biopsy Marker for Early Detection of Breast Cancer.

BACKGROUND: Despite recent progress in liquid biopsy technologies, early blood-based detection of breast cancer is still a challenge. METHODS: We analyzed secretion of the protein cellular communication network factor 1 (CCN1, formerly cysteine-rich angiogenic inducer 61) in breast cancer cell lines by an enzyme-linked immunosorbent assay (ELISA). Soluble CCN1 in the plasma (2.5 µL) of 544 patients with breast cancer and 427 healthy controls was analyzed by ELISA. The breast cancer samples were acquired at the time of primary diagnosis prior to neoadjuvant therapy or surgery. A classifier was established on a training cohort of patients with breast cancer and age-adapted healthy controls and further validated on an independent cohort comprising breast cancer patients and healthy controls. Samples from patients with benign breast diseases were investigated as additional controls. Samples from patients with acute heart diseases (n = 127) were investigated as noncancer controls. The diagnostic accuracy was determined by receiver operating characteristic using the parameters area under the curve, sensitivity, and specificity. RESULTS: CCN1 was frequently secreted by breast cancer cell lines into the extracellular space. Subsequent analysis of clinical blood samples from patients with breast cancer and age-adjusted healthy controls revealed an overall specificity of 99.0% and sensitivity of 80.0% for cancer detection. Remarkably, 81.5% of small T1 cancers were already CCN1-positive, while CCN1 concentrations in patients with benign breast lesions were below the threshold for breast cancer detection. CONCLUSIONS: Circulating CCN1 is a potentially novel blood biomarker for the detection of breast cancer at the earliest invasive stage.

Sustained Response After Remdesivir and Convalescent Plasma Therapy in a B-Cell-Depleted Patient With Protracted Coronavirus Disease 2019 (COVID-19).

We provide detailed clinical, virological, and immunological data of a B-cell-depleted patient treated with obinutuzumab for follicular lymphoma with protracted coronavirus disease 2019 (COVID-19) and viremia. A sustained response was achieved after 2 courses of remdesivir and subsequent convalescent plasma therapy. Immunocompromised patients might require combined and prolonged antiviral treatment regimens.

Sensitive Blood-Based Detection of Asbestos-Associated Diseases Using Cysteine-Rich Angiogenic Inducer 61 as Circulating Protein Biomarker.

BACKGROUND: Detection of asbestos-associated diseases like asbestosis or mesothelioma is still challenging. We sought to improve the diagnosis of benign asbestos-associated disease (BAAD) by detection of the protein cysteine-rich angiogenic inducer 61 (Cyr61) in human plasma. METHODS: Plasma Cyr61 was quantified using an enzyme-linked immunosorbent assay. Plasma samples from males diagnosed with BAAD, but without a malignant disease (n = 101), and malignant mesothelioma (n = 21; 15 males, 6 females), as well as nonasbestos-exposed healthy control participants (n = 150; 58 males, 92 females) were analyzed. Clinical sensitivity and specificity of Cyr61 were determined by receiver operating characteristic analysis. RESULTS: The median plasma Cyr61 concentration for healthy control participants was 0.27 ng/mL. Cytoplasmic Cyr61 in peripheral blood mononuclear cells from healthy control participants was evenly distributed, as detected by immunofluorescent staining. The increase in plasma Cyr61 concentrations in the BAAD study group was statistically significant compared to the healthy control participants (P < 0.0001). For the detection of BAAD vs male healthy control participants, clinical sensitivity was 88% and clinical specificity 95% with an area under the curve of 0.924 at maximal Youden Index. For a predefined clinical specificity of 100%, the clinical sensitivity was 76%. For male mesothelioma patients vs male healthy control participants, the clinical sensitivity at maximal Youden Index was 95% with a clinical specificity of 100% (area under the curve, 0.997) and for a predefined clinical specificity of 100%, the clinical sensitivity was 93%. CONCLUSIONS: In our study, plasma Cyr61 protein concentrations showed to be a new biomarker for asbestos-associated diseases like BAAD and mesothelioma in men, which deserves further investigation in large-scale cohort studies.

Sphingosine-1-Phosphate, Motor Severity, and Progression in Parkinson's Disease (MARK-PD).

BACKGROUND: Treatment with sphingosine-1-phosphate (S1P) agonists confers neuroprotective effects in animal models of Parkinson's disease (PD). OBJECTIVES: We assessed the association of serum S1P levels with motor and cognitive symptoms in patients with PD. METHODS: S1P concentrations were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in serum of 196 PD patients and in 196 age- and sex-matched controls. Motor (Unified Parkinson's disease rating scale III [UPDRS III], Hoehn and Yahr) and cognitive (Montreal Cognitive Assessment [MoCA]) function were assessed at baseline. Follow-up data was available from 64 patients (median [interquartile range], 513 [381-677] days). RESULTS: S1P levels were lower in PD patients compared with controls, that is 1.75 (1.38-2.07) and 1.90 (1.59-2.18) µmol/L, respectively (P = 0.001). In PD patients, lower S1P concentrations were associated with higher UPDRS III scores and Hoehn and Yahr stage. In the follow-up cohort, S1P concentrations below the median were associated with faster motor decline (hazard ratio: 4.78 [95% CI, 1.98, 11.50]), but not with cognitive worsening. CONCLUSIONS: Our observations reveal an association of S1P with PD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Trimethyllysine, vascular risk factors and outcome in acute ischemic stroke (MARK-STROKE).

Trimethyllysine (TML) is involved in the generation of the pro-atherogenic metabolite trimethylamine-N-oxide (TMAO) by gut microbiota. In clinical studies, elevated TML levels predicted major adverse cardiovascular events (MACE) in patients with acute or stable coronary artery disease (CAD). In contrast to cardiovascular patients, the role of TML in patients with acute cerebral ischemia is unknown. Here, we evaluated circulating TML levels in 374 stroke patients from the prospective biomarkers in stroke (MARK-STROKE) study. Compared with 167 matched healthy controls, acute ischemic stroke patients had lower median TML plasma concentrations, i.e. 0.71 vs. 0.47 µmol/L (p < 0.001) and this difference persisted after adjusting for age and sex. TML plasma concentrations were associated with age, serum creatinine, glucose, cholesterol and lysine. Patients with prevalent arterial hypertension, atrial fibrillation or a history of myocardial infarction had increased TML levels, but this observation was not independent of age, sex and GFR. In 274 patients, follow-up data were available. During a median follow-up of 284 [25th-75th percentile: 198, 431] days, TML was not associated with incident MACE (stroke, myocardial infarction, death). In summary, our data suggests a different role of TML in acute ischemic stroke compared with CAD patients.

The effect of moderate intraoperative blood loss and norepinephrine therapy on sublingual microcirculatory perfusion in patients having open radical prostatectomy: An observational study.

BACKGROUND: It is not clear whether moderate intraoperative blood loss and norepinephrine used to restore the macrocirculation impair the microcirculation and affect microcirculation/macrocirculation coherence. OBJECTIVE: We sought to investigate the effect of moderate intraoperative blood loss and norepinephrine therapy administered to treat intraoperative hypotension on the sublingual microcirculation. DESIGN: Prospective observational study. SETTING: University Medical Center Hamburg-Eppendorf, Hamburg, Germany, from November 2018 to March 2019. PATIENTS: Thirty patients scheduled for open radical prostatectomy and 29 healthy volunteer blood donors. INTERVENTION: Simultaneous assessment of the macrocirculation using a noninvasive finger-cuff method and the sublingual microcirculation using vital microscopy. MAIN OUTCOME MEASURES: The main outcome measures were changes in the sublingual microcirculation caused by moderate intraoperative blood loss and norepinephrine therapy. RESULTS: General anaesthesia decreased median [IQR] mean arterial pressure from 100 [90 to 104] to 79 [69 to 87] mmHg (P < 0.001), median heart rate from 69 [63 to 79] to 53 [44 to 62] beats per minute (P < 0.001), median cardiac index from 2.67 [2.42 to 3.17] to 2.09 [1.74 to 2.49] l min-1 m-2 (P < 0.001), and median microvascular flow index from 2.75 [2.66 to 2.85] to 2.50 [2.35 to 2.63] (P = 0.001). A median blood loss of 600 [438 to 913] ml until the time of prostate removal and norepinephrine therapy to treat intraoperative hypotension had no detrimental effect on the sublingual microcirculation: There were no clinically important changes in the microvascular flow index, the proportion of perfused vessels, the total vessel density, and the perfused vessel density. Blood donation resulted in no clinically important changes in any of the macrocirculatory or microcirculatory variables. CONCLUSION: Moderate intraoperative blood loss and norepinephrine therapy administered to treat intraoperative hypotension have no detrimental effect on the sublingual microcirculation and the coherence between the macrocirculation and microcirculation in patients having open radical prostatectomy.

High-Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies.

Extracellular vesicles (EVs) are released from basically all cells. Over the last decade, small EVs (sEVs; 50-150 nm) have gained enormous attention in diagnostics and therapy. However, methodological limitations coupled to the lack of EV standards leave many questions in this quickly evolving field unresolved. Recently, by using enhanced green fluorescent protein (eGFP)-labeled sEVs as biological reference material, we systematically optimized imaging flow cytometry for single sEV analysis. Furthermore, we showed that sEVs stained with different fluorescent antibodies can be analyzed in a multiparametric manner. However, many parameters potentially affecting the sEV staining procedure still require further evaluation and optimization. Here, we present a concise, systematic evaluation of the impact of the incubation temperature (4°C, room temperature and 37°C) during sEV antibody staining on the outcome of experiments involving the staining of EVs with fluorescence-conjugated antibodies. We provide evidence that both the staining intensity and the sample recovery can vary depending on the incubation temperature applied, and that observed differences are less pronounced following prolonged incubation times. In addition, this study can serve as an application-specific example of parameter evaluation in EV flow cytometry. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Blood donation and donors: insights from a large German teaching hospital (2008-2017).

BACKGROUND AND OBJECTIVES: The availability of blood and blood products is crucial for the provision of high-quality hospital services. We analyse changes in whole blood donations, donors and their behaviour over 9 years at a large German teaching hospital. MATERIALS AND METHODS: A descriptive analysis using data from over 34 000 donors and 265 000 donations from a large university hospital's blood centre was conducted using data from July 2008 to December 2017. The analysis focussed on (a) whole blood donations and (b) donor characteristics and how they changed over time. We categorized donors into four categories according to their donation activity (First-Time, Highly Active, Active and Reactivated). RESULTS: We observed falling donations over time and that donors donated less frequently. Consequently, we show a downward trend in the number of Highly Active donors, whilst First-Time donors remained stable. We also provide evidence that donors donated well below their capacity and that the blood type of donors appeared to be in line with the wider German donor population. Lastly, we show a sharp drop in the return rates of First-Time donors over time. CONCLUSION: We recommend that Highly Active donors and former Highly Active donors are more carefully considered when planning donor engagement strategies and effort made in (at the very least) maintaining their donation activity. Our results in the context of the literature highlight the need for further research into the changing attitudes towards blood donation and prosocial activities.

FASN Is a Biomarker Enriched in Malignant Glioma-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are known for their important role in cancer progression and hold considerable potential as a source for tumor biomarkers. However, purification of tumor-specific EVs from patient plasma is still an urgent unmet need due to contamination by normal host cell-derived EVs, that results in compromised analytical sensitivity. Here we identified fatty acid synthase (FASN), a key lipogenic enzyme which is highly expressed in malignant glioma cells, to be elevated in CD63- and CD81-positive EVs in glioma patient plasma samples, opening vital opportunities to sort brain tumor-specific EVs.