Altered Extracellular Vesicle-Derived Protein and microRNA Signatures in Bronchoalveolar Lavage Fluid from Patients with Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease for which there is no cure. Accumulating research results suggest a role for extracellular vesicles (EVs) in the pathogenesis of COPD. This study aimed to uncover the involvement of EVs and their molecular cargo in the progression of COPD by identification of EV-associated protein and microRNA (miRNA) profiles. We isolated EVs from the bronchial alveolar lavage fluid (BALF) of 18 patients with COPD and 11 healthy controls using size-exclusion chromatography. EV isolates were characterized using nanoparticle tracking analysis and protein content. Proteomic analysis revealed a higher abundance of 284 proteins (log2FC > 1) and a lower abundance of 3 proteins (log2FC < -1) in EVs derived from patients with COPD. Ingenuity pathway analysis showed that proteins enriched in COPD-associated EVs trigger inflammatory responses, including neutrophil degranulation. Variances in surface receptors and ligands associated with COPD EVs suggest a preferential interaction with alveolar cells. Small RNAseq analysis identified a higher abundance of ten miRNAs and a lower abundance of one miRNA in EVs from COPD versus controls (Basemean > 100, FDR < 0.05). Our data indicate that the molecular composition of EVs in the BALF of patients with COPD is altered compared to healthy control EVs. Several components in COPD EVs were identified that may perpetuate inflammation and alveolar tissue destruction.

Growth behavior and mRNA expression profiling during growth of IPEC-J2 cells.

OBJECTIVE: The IPEC-J2 cell line is used as an in vitro small intestine model for swine, but it is also used as a model for the human intestine, presenting a relatively unique setting. By combining electric cell-substrate impedance sensing, with next-generation-sequencing technology, we showed that mRNA gene expression profiles and related pathways can depend on the growth phase of IPEC-J2 cells. Our investigative approach welcomes scientists to reproduce or modify our protocols and endorses putting their gene expression data in the context of the respective growth phase of the cells. RESULTS: Three time points are presented: (TP1) 1 h after medium change (= 6 h after seeding of cells), (TP2) the time point of the first derivative maximum of the cell growth curve, and a third point at the beginning of the plateau phase (TP3). Significantly outstanding at TP1 compared to TP2 was upregulated PLEKHN1, further FOSB and DEGS2 were significantly downregulated at TP2 compared to TP3. Any provided data can be used to improve next-generation experiments with IPEC-J2 cells.

Identification of microRNA biomarkers simultaneously expressed in circulating extracellular vesicles and atherosclerotic plaques.

Background: Atherosclerosis is a widespread disorder of the cardiovascular system. The early detection of plaques by circulating biomarkers is highly clinically relevant to prevent the occurrence of major complications such as stroke or heart attacks. It is known that extracellular vesicles (EVs) are important in intercellular communication in atherosclerotic disorders and carry many components of their cells of origin, including microRNAs (miRNAs). In this study, we test the assumption that miRNAs present in material acquired from plaques in patients undergoing surgery for atherosclerotic carotid artery stenosis are also expressed in circulating EVs obtained from the identical patients. This would allow the adoption of a liquid biopsy approach for the detection of plaques. Methods: We studied 22 surgical patients with atherosclerotic carotid arterial stenosis and 28 healthy controls. EVs were isolated from serum by precipitation. miRNA expression profiles of serum-derived EVs were obtained by small RNA sequencing and in plaque material simultaneously acquired from patients. A comparative analysis was performed to identify circulating atherosclerosis-associated miRNAs that are also detectable in plaques. Results: Seven miRNAs were found to be differentially regulated in patient serum compared with the serum of healthy controls. Of these, miR-193b-5p, miR-193a-5p, and miR-125a-3p were significantly upregulated in patients compared with that in healthy controls and present in both, circulating EVs and plaque material. An overrepresentation analysis of experimentally validated mRNA targets revealed an increased regulation of inflammation and vascular growth factors, key players in atherosclerosis and plaque formation. Conclusion: Our findings suggest that circulating EVs reflect plaque development in patients with symptomatic carotid artery stenosis, which can serve as biomarker candidates for detecting the presence of atherosclerotic plaques.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Extracellular vesicle miRNAs drive aberrant macrophage responses in NSAID-exacerbated respiratory disease.

BACKGROUND: Extracellular vesicles (EVs) have been implicated in the pathogenesis of asthma, however, how EVs contribute to immune dysfunction and type 2 airway inflammation remains incompletely understood. We aimed to elucidate roles of airway EVs and their miRNA cargo in the pathogenesis of NSAID-exacerbated respiratory disease (N-ERD), a severe type 2 inflammatory condition. METHODS: EVs were isolated from induced sputum or supernatants of cultured nasal polyp or turbinate tissues of N-ERD patients or healthy controls by size-exclusion chromatography and characterized by particle tracking, electron microscopy and miRNA sequencing. Functional effects of EV miRNAs on gene expression and mediator release by human macrophages or normal human bronchial epithelial cells (NHBEs) were studied by RNA sequencing, LC-MS/MS and multiplex cytokine assays. RESULTS: EVs were highly abundant in secretions from the upper and lower airways of N-ERD patients. N-ERD airway EVs displayed profoundly altered immunostimulatory capacities and miRNA profiles compared to airway EVs of healthy individuals. Airway EVs of N-ERD patients, but not of healthy individuals induced inflammatory cytokine (GM-CSF and IL-8) production by NHBEs. In macrophages, N-ERD airway EVs exhibited an impaired potential to induce cytokine and prostanoid production, while enhancing M2 macrophage activation. Let-7 family miRNAs were highly enriched in sputum EVs from N-ERD patients and mimicked suppressive effects of N-ERD EVs on macrophage activation. CONCLUSION: Aberrant airway EV miRNA profiles may contribute to immune dysfunction and chronic type 2 inflammation in N-ERD. Let-7 family miRNAs represent targets for correcting aberrant macrophage activation and mediator responses in N-ERD.

A pipeline for the development and analysis of extracellular vesicle-based transcriptomic biomarkers in molecular diagnostics.

Extracellular vesicles are shed by every cell type and can be found in any biofluid. They contain different molecules that can be utilized as biomarkers, including several RNA species which they protect from degradation. Here, we present a pipeline for the development and analysis of extracellular vesicle-associated transcriptomic biomarkers that our group has successfully applied multiple times. We highlight the key steps of the pipeline and give particular emphasis to the necessary quality control checkpoints, which are linked to numerous available guidelines that should be considered along the workflow. Our pipeline starts with patient recruitment and continues with blood sampling and processing. The purification and characterization of extracellular vesicles is explained in detail, as well as the isolation and quality control of extracellular vesicle-associated RNA. We point out the possible pitfalls during library preparation and RNA sequencing and present multiple bioinformatic tools to pinpoint biomarker signature candidates from the sequencing data. Finally, considerations and pitfalls during the validation of the biomarker signature using RT-qPCR will be elaborated.

The proteome of bacterial membrane vesicles in Escherichia coli-a time course comparison study in two different media.

Introduction: Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. Methods: To gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1ß and IL-8 in the human monocyte cell line THP-1 upon bMV treatment. Results: Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1ß and IL-8 expressions. Conclusion: Our study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

Extracellular Vesicles and Endocannabinoid Signaling in Patients with COVID-19.

Introduction: Endocannabinoids in COVID-19 have immunomodulatory and anti-inflammatory properties but the functional role and the regulation of endocannabinoid signaling in this pandemic disorder is controversial. To exercise their biologic function, endocannabinoids need to travel across the intercellular space and within the blood stream to reach their target cells. How the lipophilic endocannabinoids are transported in the vascular system and how these hydrophobic compounds cross cell membranes is still unclear. Extracellular vesicles (EVs) are released and incorporated by many cell types including immune cells. EVs are small lipid-membrane covered particles and contain RNA, lipids and proteins. They play an important role in intercellular communication by transporting these signaling molecules from their cells of origin to specific target cells. EVs may represent ideal transport vehicles for lipophilic signaling molecules like endocannabinoids and this effect could also be evident in COVID-19. Materials and Methods: We measured the endocannabinoids anandamide, 2-AG, SEA, PEA and OEA in patients with COVID-19 in EVs and plasma. RNA sequencing of microRNAs (miRNAs) derived from EVs (EV-miRNAs) and mRNA transcripts from blood cells was used for the construction of signaling networks reflecting endocannabinoid and miRNA communication by EVs to target immune cells. Results: With the exception of anandamide, endocannabinoid concentrations were significantly enriched in EVs in comparison to plasma and increased with disease severity. No enrichment in EVs was seen for the more hydrophilic steroid hormones cortisol and testosterone. High EV-endocannabinoid concentrations were associated with downregulation of CNR2 (CB2) by upregulated EV-miRNA miR-146a-5p and upregulation of MGLL by downregulated EV-miR-199a-5p and EV-miR-370-5p suggesting counterregulatory effects. In contrast, low EV-levels of anandamide were associated with upregulation of CNR1 by downregulation of EV-miR-30c-5p and miR-26a-5p along with inhibition of FAAH. Immunologically active molecules in immune cells regulated by endocannabinoid signaling included VEGFA, GNAI2, IGF1, BDNF, IGF1R and CREB1 and CCND1 among others. Discussion and Conclusions: EVs carry immunologically functional endocannabinoids in COVID-19 along with miRNAs which may regulate the expression of mRNA transcripts involved in the regulation of endocannabinoid signaling and metabolism. This mechanism could fine-tune and adapt endocannabinoid effects in recipient cells in relationship to the present biological context.

Uptake and effects of polystyrene nanoplastics in comparison to non-plastic silica nanoparticles on small intestine cells (IPEC-J2).

Nanoplastics smaller than 1 µm accumulate as anthropogenic material in the food chain, but only little is known about their uptake and possible effects on potentially strongly exposed cells of the small intestine. The aim of the study was to observe the uptake of 100 nm polystyrene nanoplastics into a non-tumorigenic small intestine cell culture model (IPEC-J2 cells) and to monitor the effects on cell growth and gene regulation, compared to a 100 nm non-plastic silica nanoparticle reference. The intracellular uptake of both types of nanoparticles was proven via (confocal) fluorescence microscopy and complemented with transmission electron microscopy. Fluorescence microscopy showed a growth phase-dependent uptake of nanoparticles into the cells, hence further experiments included different time points related to epithelial closure, determined via electric cell substrate impedance sensing. No retardations in epithelial closure of cells after treatment with polystyrene nanoparticles could be found. In contrast, epithelial cell closure was partly negatively influenced by silica nanoparticles. An increased production of organic nanoparticles, like extracellular vesicles, was not measurable via nanoparticle tracking analysis. An assessment of messenger RNA by next generation sequencing and subsequent pathway analysis revealed that the TP53 pathway was influenced significantly by the polystyrene nanoparticle treatment. In both treatments, dysregulated mRNAs were highly enriched in the NOTCH signaling pathway compared to the non-particle control.

Extensive blood transcriptome analysis reveals cellular signaling networks activated by circulating glycocalyx components reflecting vascular injury in COVID-19.

Background: Degradation of the endothelial protective glycocalyx layer during COVID-19 infection leads to shedding of major glycocalyx components. These circulating proteins and their degradation products may feedback on immune and endothelial cells and activate molecular signaling cascades in COVID-19 associated microvascular injury. To test this hypothesis, we measured plasma glycocalyx components in patients with SARS-CoV-2 infection of variable disease severity and identified molecular signaling networks activated by glycocalyx components in immune and endothelial cells. Methods: We studied patients with RT-PCR confirmed COVID-19 pneumonia, patients with COVID-19 Acute Respiratory Distress Syndrome (ARDS) and healthy controls (wildtype, n=20 in each group) and measured syndecan-1, heparan sulfate and hyaluronic acid. The in-silico construction of signaling networks was based on RNA sequencing (RNAseq) of mRNA transcripts derived from blood cells and of miRNAs isolated from extracellular vesicles from the identical cohort. Differentially regulated RNAs between groups were identified by gene expression analysis. Both RNAseq data sets were used for network construction of circulating glycosaminoglycans focusing on immune and endothelial cells. Results: Plasma concentrations of glycocalyx components were highest in COVID-19 ARDS. Hyaluronic acid plasma levels in patients admitted with COVID-19 pneumonia who later developed ARDS during hospital treatment (n=8) were significantly higher at hospital admission than in patients with an early recovery. RNAseq identified hyaluronic acid as an upregulator of TLR4 in pneumonia and ARDS. In COVID-19 ARDS, syndecan-1 increased IL-6, which was significantly higher than in pneumonia. In ARDS, hyaluronic acid activated NRP1, a co-receptor of activated VEGFA, which is associated with pulmonary vascular hyperpermeability and interacted with VCAN (upregulated), a proteoglycan important for chemokine communication. Conclusions: Circulating glycocalyx components in COVID-19 have distinct biologic feedback effects on immune and endothelial cells and result in upregulation of key regulatory transcripts leading to further immune activation and more severe systemic inflammation. These consequences are most pronounced during the early hospital phase of COVID-19 before pulmonary failure develops. Elevated levels of circulating glycocalyx components may early identify patients at risk for microvascular injury and ARDS. The timely inhibition of glycocalyx degradation could provide a novel therapeutic approach to prevent the development of ARDS in COVID-19.

Anesthetic­specific lncRNA and mRNA profile changes in blood during colorectal cancer resection: A prospective, matched­case pilot study.

Prometastatic and antitumor effects of different anesthetics have been previously analyzed in several studies with conflicting results. Thus, the underlying perioperative molecular mechanisms mediated by anesthetics potentially affecting tumor phenotype and metastasis remain unclear. It was hypothesized that anesthetic­specific long non­coding RNA (lncRNA) expression changes are induced in the blood circulation and play a crucial role in tumor outcome. In the present study, high­throughput sequencing and quantitative PCR were performed in order to identify lncRNA and mRNA expression changes affected by two therapeutic regimes, total intravenous anesthesia (TIVA) and volatile anesthetic gas (VAG) in patients undergoing colorectal cancer (CRC) resection. Total blood RNA was isolated prior to and following resection and characterized using RNA sequencing. mRNA­lncRNA interactions and their roles in cancer­related signaling of differentially expressed lncRNAs were identified using bioinformatics analyses. The comparison of these two time points revealed 35 differentially expressed lncRNAs in the TIVA­group, and 25 in the VAG­group, whereas eight were shared by both groups. Two lncRNAs in the TIVA­group, and 23 in the VAG­group of in silico identified target­mRNAs were confirmed as differentially regulated in the NGS dataset of the present study. Pathway analysis was performed and cancer relevant canonical pathways for TIVA were identified. Target­mRNA analysis of VAG revealed a markedly worsened immunological response against cancer. In this proof­of­concept study, anesthesic­specific expression changes in lncRNA and mRNA profiles in blood were successfully identified. Moreover, the data of the present study provide the first evidence that anesthesia­induced lncRNA pattern changes may contribute further in the observed differences in CRC outcome following tumor resection.

Using High-Resolution Differential Cell Counts (HRDCCs) in Bovine Milk and Blood to Monitor the Immune Status over the Entire Lactation Period.

Differential cell counts in milk offer a deeper insight into the immunology of the mammary gland and might even provide information about the systemic health status of a dairy cow. Consequently, their potential as a diagnostic method to identify biomarkers has been a subject of research for quite some time. The objective of our study was to closely monitor the immune status of eight healthy dairy cows throughout their whole lactation. For this, high-resolution differential cell counts in milk and blood were determined by means of flow cytometry, which included 10 subpopulations of the 3 main populations of immune cells and their viability. Milk and blood samples were taken twice a week in the first 100 days after calving and once a week during the remaining lactation period: in total, 55 (52-57) blood and 55 (52-57) milk samples per animal. In addition, six well-established routine laboratory biomarkers, i.e., haptoglobin, calcium, and different metabolic parameters (non-esterified fatty acids, ß-hydroxybutyric acid, bilirubin, and glutamate dehydrogenase), were analyzed in all blood samples. Furthermore, a standard differential blood cell count was performed on all blood samples. We found substantial differences between cell count progressions in the blood and milk. The distribution of cell populations in the blood remained mostly stable throughout the lactation, albeit at different individual levels. Several cell populations in the milk showed a noticeable dynamic over time, which caused a separation of different lactation stages in clustering analyses. Gamma delta T cells and CD4+ T cells in the milk stood out as they showed characteristic fluctuations during the course of lactation as well as minor changes in the case of inflammation. The determination of a differential cell count has the potential to be a sensitive diagnostic and prognostic tool in bovine milk. Further studies need to show to what extent the method is suitable for routine diagnostics and how to deal with animal-specific differences.

Obtaining Reliable RT-qPCR Results in Molecular Diagnostics-MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery.

In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of "key steps" in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine.

A panel of blood-derived miRNAs with a stable expression pattern as a potential pan-cancer detection signature.

Introduction: MicroRNAs have a significant role in the regulation of the transcriptome. Several miRNAs have been proposed as potential biomarkers in different malignancies. However, contradictory results have been reported on the capability of miRNA biomarkers in cancer detection. The human biological clock involves molecular mechanisms that regulate several genes over time. Therefore, the sampling time becomes one of the significant factors in gene expression studies. Method: In the present study, we have tried to find miRNAs with minimum fluctuation in expression levels at different time points that could be more accurate candidates as diagnostic biomarkers. The small RNA-seq raw data of ten healthy individuals across nine-time points were analyzed to identify miRNAs with stable expression. Results: We have found five oscillation patterns. The stable miRNAs were investigated in 779 small-RNA-seq datasets of eleven cancer types. All miRNAs with the highest differential expression were selected for further analysis. The selected miRNAs were explored for functional pathways. The predominantly enriched pathways were miRNA in cancer and the P53-signaling pathway. Finally, we have found seven miRNAs, including miR-142-3p, miR-199a-5p, miR-223-5p, let-7d-5p, miR-148b-3p, miR-340-5p, and miR-421. These miRNAs showed minimum fluctuation in healthy blood and were dysregulated in the blood of eleven cancer types. Conclusion: We have found a signature of seven stable miRNAs which dysregulate in several cancer types and may serve as potential pan-cancer biomarkers.

Extracellular Vesicle Associated miRNAs Regulate Signaling Pathways Involved in COVID-19 Pneumonia and the Progression to Severe Acute Respiratory Corona Virus-2 Syndrome.

Background: Extracellular vesicles (EVs) are mediators of cell-to-cell communication in inflammatory lung diseases. They function as carriers for miRNAs which regulate mRNA transcripts and signaling pathways after uptake into recipient cells. We investigated whether miRNAs associated with circulating EVs regulate immunologic processes in COVID-19. Methods: We prospectively studied 20 symptomatic patients with COVID-19 pneumonia, 20 mechanically ventilated patients with severe COVID-19 (severe acute respiratory corona virus-2 syndrome, ARDS) and 20 healthy controls. EVs were isolated by precipitation, total RNA was extracted, profiled by small RNA sequencing and evaluated by differential gene expression analysis (DGE). Differentially regulated miRNAs between groups were bioinformatically analyzed, mRNA target transcripts identified and signaling networks constructed, thereby comparing COVID-19 pneumonia to the healthy state and pneumonia to severe COVID-19 ARDS. Results: DGE revealed 43 significantly and differentially expressed miRNAs (25 downregulated) in COVID-19 pneumonia when compared to controls, and 20 miRNAs (15 downregulated) in COVID-19 ARDS patients in comparison to those with COVID-19 pneumonia. Network analysis for comparison of COVID-19 pneumonia to healthy controls showed upregulated miR-3168 (log2FC=2.28, padjusted<0.001), among others, targeting interleukin-6 (IL6) (25.1, 15.2 - 88.2 pg/ml in COVID-19 pneumonia) and OR52N2, an olfactory smell receptor in the nasal epithelium. In contrast, miR-3168 was significantly downregulated in COVID-19 ARDS (log2FC=-2.13, padjusted=0.003) and targeted interleukin-8 (CXCL8) in a completely activated network. Toll-like receptor 4 (TLR4) was inhibited in COVID-19 pneumonia by miR-146a-5p and upregulated in ARDS by let-7e-5p. Conclusion: EV-derived miRNAs might have important regulative functions in the pathophysiology of COVID-19: CXCL8 regulates neutrophil recruitment into the lung causing epithelial damage whereas activated TLR4, to which SARS-CoV-2 spike protein binds strongly, increases cell surface ACE2 expression and destroys type II alveolar cells that secrete pulmonary surfactants; both resulting in pulmonary-capillary leakage and ARDS. These miRNAs may serve as biomarkers or as possible therapeutic targets.

Development of an advanced flow cytometry based high-resolution immunophenotyping method to benchmark early immune response in dairy cows.

The determination of the somatic cell count of a milk sample is one of the most common methods to monitor udder health of a dairy cow. However, this procedure does not take into account the fact that cells in milk present a great variety of different cell types. The objective of our study was to establish a high-resolution differential cell count (HRDCC) by means of flow cytometry in blood and milk. We were able to detect ten subpopulations among the three main populations of immune cells and to determine their viability. Additionally, blood samples were analyzed for common laboratory biomarkers, i.e. differential blood counts, haptoglobin levels and several metabolic parameters. In this first feasibility study, we used three different vaccines to stimulate the immune system of five healthy cows each. Samples were collected shortly before, in between and after the vaccinations. Using multivariate statistical methods we saw a diagnostic benefit when HRDCCs were included compared to only the standard laboratory parameters. The impacts of all three vaccinations on the immune system were visible in blood HRDCCs as well as in milk HRDCCs. Cluster of Differentiation 8+ (CD8+) T cells, B cells and monocyte/macrophage subpopulations were among the most important and statistically relevant parameters for all treatments in both biofluids. Moreover, in one of the treatment groups intermediate monocytes showed a significant increase after both vaccinations. Although the use of HRDCC in blood or milk was shown to be highly relevant for early systemic diagnostic, to confirm these subpopulations further investigations in cows of different breed, lactation stage or health status are required.

Detection of Atherosclerosis by Small RNA-Sequencing Analysis of Extracellular Vesicle Enriched Serum Samples.

Atherosclerosis can occur throughout the arterial vascular system and lead to various diseases. Early diagnosis of atherosclerotic processes and of individual disease patterns would be more likely to be successful if targeted therapies were available. For this, it is important to find reliable biomarkers that are easily accessible and with little inconvenience for patients. There are many cell culture, animal model or tissue studies that found biomarkers at the microRNA (miRNA) and mRNA level describing atherosclerotic processes. However, little is known about their potential as circulating and liquid biopsy markers in patients. In this study, we examined serum-derived miRNA - profiles from 129 patients and 28 volunteers to identify potential biomarkers. The patients had four different atherosclerotic manifestations: abdominal aneurysm (n = 35), coronary heart disease (n = 34), carotid artery stenosis (n = 24) and peripheral arterial disease (n = 36). The samples were processed with an extracellular vesicle enrichment protocol, total-RNA extraction and small RNA-sequencing were performed. A differential expression analysis was performed bioinformatically to find potentially regulated miRNA biomarkers. Resulting miRNA candidates served as a starting point for an overrepresentation analysis in which relevant target mRNAs were identified. The Gene Ontology database revealed relevant biological functions in relation to atherosclerotic processes. In patients, expression of specific miRNAs changed significantly compared to healthy volunteers; 27 differentially expressed miRNAs were identified. We were able to detect a group-specific miRNA fingerprint: miR-122-5p, miR-2110 and miR-483-5p for abdominal aortic aneurysm, miR-370-3p and miR-409-3p for coronary heart disease, miR-335-3p, miR-381-3p, miR493-5p and miR654-3p for carotid artery stenosis, miR-199a-5p, miR-215-5p, miR-3168, miR-582-3p and miR-769-5p for peripheral arterial disease. The results of the study show that some of the identified miRNAs have already been associated with atherosclerosis in previous studies. Overrepresentation analysis on this data detected biological processes that are clearly relevant for atherosclerosis, its development and progression showing the potential of these miRNAs as biomarker candidates. In a next step, the relevance of these findings on the mRNA level is to be investigated and substantiated.

Progranulin signaling in sepsis, community-acquired bacterial pneumonia and COVID-19: a comparative, observational study.

BACKGROUND: Progranulin is a widely expressed pleiotropic growth factor with a central regulatory effect during the early immune response in sepsis. Progranulin signaling has not been systematically studied and compared between sepsis, community-acquired pneumonia (CAP), COVID-19 pneumonia and a sterile systemic inflammatory response (SIRS). We delineated molecular networks of progranulin signaling by next-generation sequencing (NGS), determined progranulin plasma concentrations and quantified the diagnostic performance of progranulin to differentiate between the above-mentioned disorders using the established biomarkers procalcitonin (PCT), interleukin-6 (IL-6) and C-reactive protein (CRP) for comparison. METHODS: The diagnostic performance of progranulin was operationalized by calculating AUC and ROC statistics for progranulin and established biomarkers in 241 patients with sepsis, 182 patients with SIRS, 53 patients with CAP, 22 patients with COVID-19 pneumonia and 53 healthy volunteers. miRNAs and mRNAs in blood cells from sepsis patients (n = 7) were characterized by NGS and validated by RT-qPCR in an independent cohort (n = 39) to identify canonical gene networks associated with upregulated progranulin at sepsis onset. RESULTS: Plasma concentrations of progranulin (ELISA) in patients with sepsis were 57.5 (42.8-84.9, Q25-Q75) ng/ml and significantly higher than in CAP (38.0, 33.5-41.0 ng/ml, p < 0.001), SIRS (29.0, 25.0-35.0 ng/ml, p < 0.001) and the healthy state (28.7, 25.5-31.7 ng/ml, p < 0.001). Patients with COVID-19 had significantly higher progranulin concentrations than patients with CAP (67.6, 56.6-96.0 vs. 38.0, 33.5-41.0 ng/ml, p < 0.001). The diagnostic performance of progranulin for the differentiation between sepsis vs. SIRS (n = 423) was comparable to that of procalcitonin. AUC was 0.90 (95% CI = 0.87-0.93) for progranulin and 0.92 (CI = 0.88-0.96, p = 0.323) for procalcitonin. Progranulin showed high discriminative power to differentiate bacterial CAP from COVID-19 (sensitivity 0.91, specificity 0.94, AUC 0.91 (CI = 0.8-1.0) and performed significantly better than PCT, IL-6 and CRP. NGS and partial RT-qPCR confirmation revealed a transcriptomic network of immune cells with upregulated progranulin and sortilin transcripts as well as toll-like-receptor 4 and tumor-protein 53, regulated by miR-16 and others. CONCLUSIONS: Progranulin signaling is elevated during the early antimicrobial response in sepsis and differs significantly between sepsis, CAP, COVID-19 and SIRS. This suggests that progranulin may serve as a novel indicator for the differentiation between these disorders. TRIAL REGISTRATION: Clinicaltrials.gov registration number NCT03280576 Registered November 19, 2015.

Molecular RNA Correlates of the SOFA Score in Patients with Sepsis.

The most common scoring system for critically ill patients is the Sequential Organ Failure Assessment (SOFA) score. Little is known about specific molecular signaling networks underlying the SOFA criteria. We characterized these networks and identified specific key regulatory molecules. We prospectively studied seven patients with sepsis and six controls with high-throughput RNA sequencing (RNAseq). Quantitative reverse transcription PCR (RT-qPCR) confirmation was performed in a second independent cohort. Differentially and significantly expressed miRNAs and their target mRNA transcripts were filtered for admission SOFA criteria and marker RNAs for the respective criteria identified. We bioinformatically constructed molecular signaling networks specifically reflecting these criteria followed by RT-qPCR confirmation of RNAs with important regulatory functions in the networks in the second cohort. RNAseq identified 82 miRNAs (45% upregulated) and 3254 mRNAs (50% upregulated) differentially expressed between sepsis patients and controls. Bioinformatic analysis characterized 6 miRNAs and 76 mRNA target transcripts specific for the SOFA criteria. RT-qPCR validated miRNA and mRNAs included IGFBP2 (respiratory system); MMP9 and PDE4B (nervous system); PPARG (cardiovascular system); AKR1B1, ANXA1, and LNC2/NGAL (acute kidney injury); GFER/ALR (liver); and miR-30c-3p (coagulopathy). There are specific canonical networks underlying the SOFA score. Key regulatory miRNA and mRNA transcripts support its biologic validity.

miREV: An Online Database and Tool to Uncover Potential Reference RNAs and Biomarkers in Small-RNA Sequencing Data Sets from Extracellular Vesicles Enriched Samples.

Extracellular vesicles (EVs) are nano-sized, membrane-enclosed vesicles released by cells for intercellular communication. EVs are involved in pathological processes and miRNAs in EVs have gained interest as easily accessible biomolecules in liquid biopsies for diagnostic purposes. To validate potential miRNA biomarker, transcriptome analyses must be carried out to detect suitable reference miRNAs. miREV is a database with over 400 miRNA sequencing data sets and helps the researcher to find suitable reference miRNAs for their individual experimental setup. The researcher can put together a specific sample set in miREV, which is similar to his own experimental concept in order to find the most suitable references. This allows to run validation experiments without having to carry out a complex and costly transcriptome analysis priorly. Additional read count tables of each generated sample set are downloadable for further analysis. miREV is freely available at https://www.physio.wzw.tum.de/mirev/.