Profile Of Cells Research Articles

Anti-inflammatory potential of Piper betleoides C. DC., a promising Piper species of Northeast India: in vitro and in vivo evidence and mechanistic insight.

The present study aims to investigate the anti-inflammatory potential of the leaf hydroalcoholic extract of Piper betleoides C. DC., also known as "Jangli Paan" in Northeast India, using lipopolysaccharide (LPS)-treated both cell culture (RAW264.7, macrophage cells) and animal (albino rat) model of inflammation. Treatment with leaf hydroalcoholic extract of Piper betleoides (PBtE) dose-dependently (5, 10, and 20µg/mL) decreased the secretion of pro-inflammatory (TNF-α, IL-6, and MCP-1) and increased anti-inflammatory (IL-4 and IL-10) cytokines in LPS-treated macrophages. Similarly, treatment with PBtE also prevented the alternation in mRNA expression of inflammatory markers (TNF-α, CCL-2, IL-6, and IL-10) in LPS-treated macrophages. Dose-dependent supplementation with PBtE further reduced the production of intracellular ROS and increased the phagocytosis efficacies in LPS-treated cells. Further in vivo studies demonstrated that treatment with PBtE dose-dependently (50, 100, and 200mg/kg body weight) prevented the dysregulation of the secretion of inflammatory cytokines (TNF-α, IL-4, IL-6, and IL-10) and reduced the circulatory levels of prostaglandin (PGE2) and nitric oxide products (nitrite) in LPS-treated animals. In addition, alternation of blood cell profiling and the liver as well as kidney dysfunctions were also prevented by the treatment with PBtE in LPS-treated rats. The anti-inflammatory potential of PBtE was comparable to those seen in sodium diclofenac (positive control) treated group. LC-MS analyses showed piperine, piperlongumine, piperolactam-A, and dehydropipernonaline and GC-MS analyses demonstrated phytol, caryophyllene, and falcarinol as the phytochemicals present in Piper betleoides, which might play an important role in preventing inflammation and associated pathophysiology. Different treatments didn't cause any toxicity in cell culture and animal models. This study for the first time demonstrated the promising anti-inflammatory potential of the leaf hydroalcoholic extract of Piper betleoides.

Modulation of brain energy metabolism in hepatic encephalopathy: impact of glucose metabolic dysfunction.

Cerebral function is linked to a high level of metabolic activity and relies on glucose as its primary energy source. Glucose aids in the maintenance of physiological brain activities; as a result, a disruption in metabolism has a significant impact on brain function, launching a chain of events that leads to neuronal death. This metabolic insufficiency has been observed in a variety of brain diseases and neuroexcitotoxicity disorders, including hepatic encephalopathy. It is a significant neurological complication that develops in people with liver disease, ranging from asymptomatic abnormalities to coma. Hyperammonemia is the main neurotoxic villain in the development of hepatic encephalopathy and induces a wide range of complications in the brain. The neurotoxic effects of ammonia on brain function are thought to be mediated by impaired glucose metabolism. Accordingly, in this review, we provide an understanding of deranged brain energy metabolism, emphasizing the role of glucose metabolic dysfunction in the pathogenesis of hepatic encephalopathy. We also highlighted the differential metabolic profiles of brain cells and the status of metabolic cooperation between them. The major metabolic pathways that have been explored are glycolysis, glycogen metabolism, lactate metabolism, the pentose phosphate pathway, and the Krebs cycle. Furthermore, the lack of efficacy in current hepatic encephalopathy treatment methods highlights the need to investigate potential therapeutic targets for hepatic encephalopathy, with regulating deficient bioenergetics being a viable alternative in this case. This review also demonstrates the importance of the development of glucose metabolism-focused disease diagnostics and treatments, which are now being pursued for many ailments.

KCNE4 is a crucial host factor for Orf virus infection by mediating viral entry

The orf virus (ORFV) poses a serious threat to the health of domestic small ruminants (i.e., sheep and goats) and humans on a global scale, causing around $150 million in annual losses to livestock industry. However, the host factors involved in ORFV infection and replication are still elusive. In this study, we compared the RNA-seq profiles of ORFV-infected or non-infected sheep testicular interstitial cells (STICs) and identified a novel host gene, potassium voltage-gated channel subfamily E member 4 (KCNE4), as a key host factor involved in the ORFV infection. Both RNA-seq data and RT-qPCR assay revealed a significant increase in the expression of KCNE4 in the infected STICs from 9 to 48 h post infection (hpi). On the other hand, the RT-qPCR assay detected a decrease in ORFV copy number in both the STICs transfected by KCNE4 siRNA and the KCNE4 knockout (KO) HeLa cells after the ORFV infection, together with a reduced fluorescence ratio of ORFV-GFP in the KO HeLa cells at 24 hpi, indicating KCNE4 to be critical for the ORFV infection. Furthermore, the attachment and internalization assays showed decreased ORFV attachment, internalization, replication, and release by the KO HeLa cells, demonstrating a potential inhibition of ORFV entry into the cells by KCNE4. Pretreatment with the KCNE4 inhibitors such as quinidine and fluoxetine significantly repressed the ORFV infection. All our findings reveal KCNE4 as a novel host regulator of the ORFV entry and replication, shedding new insight into the interactive mechanism of ORFV infection. The study also highlights the K+ channels as possible druggable targets to impede viral infection and disease.

Unravelling tRNA fragments in DENV pathogenesis: Insights from RNA sequencing

Small non-coding RNAs (sncRNAs) derived from tRNAs are known as tRNA-derived small RNAs (tsRNAs). These tsRNAs are further categorized into tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs), which play significant roles in the various molecular mechanisms underlying certain human diseases. However, the generation of tsRNAs and their potential roles during Dengue virus (DENV) infection is not yet known. Here, we performed small RNA sequencing to identify the generation and alterations in tsRNAs expression profiles of DENV-infected Huh7 cells. Upon DENV infection, tRNA fragmentation was found to be increased. We identified a significant number of differentially expressed tsRNAs during DENV infection. Interestingly, the 3′tRF population showed upregulation, while the i-tRF population exhibited downregulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to analyze the impact of differentially expressed tsRNAs on DENV pathogenesis. Our results suggest that differentially expressed tsRNAs are involved in transcriptional regulation via RNA polymerase II promoter and metabolic pathways. Overall, our study contributes significantly to our understanding of the roles played by tsRNAs in the complex dynamics of DENV infection.

Deciphering single-cell transcriptomic landscape of tumors in colorectal cancer treated with photothermal semiconducting polymers to design the combination therapy with checkpoint blockades for inhibition of tumor progression and metastasis

Immunotherapy is often less effective for tumors with “immune-excluded,” or “immune-desert” microenvironment. To circumvent this hurdle, new therapeutic strategies need to be designed to reprogram the tumor microenvironment (TME). It is well known that photothermal therapy (PTT) can prime the TME and provoke the systemic anti-tumor immunity. But few studies perform extensive exploration in depth to decipher the changes in TME after PTT with cutting-edge technology besides conventional flow cytometry analysis. In this study, we designed a nanosystem consisting of organic semiconductor polymer liposome nanoparticles (Y8 NPs) as a multimodal imaging and photothermal agent to image and treat tumors. Upon laser irradiation, Y8 NPs in tumors could rapidly increase the temperature, induce massive cancer cell apoptosis and also promote the immunogenic cell death (ICD), ultimately remodulating the TME. We employed single cell RNA sequencing (scRNA-seq) technology to comprehensively explore transcriptomic profiling of individual cells constituting TME, demonstrating heterogenous alterations in the subtypes of immune cells, even tumor cells. Considering the findings by scRNA-seq and TME remodulation, the combinational strategy with checkpoint blockade PD-1 antibody was designed to treat the tumors, demonstrating that the combination not only impedes primary tumor progression but also produce profound abscopal anti-tumor effect. In conclusion, we provide a simple strategy for the remodulation of TME with a photothermal nanosystem to achieve activation of anti-tumor immunity, and suppress primary and metastatic tumor progression.

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states.

Schlemm's canal is a critical ocular drainage structure that maintains intraocular pressure with important consequences for glaucoma. Despite this, Schlemm's canal has been relatively understudied with the molecular process that mediate its function, maintain its health, or that are compromised in glaucoma needing much deeper characterization. This study is important as it provides the most comprehensive molecular characterization of Schlemm's canal cells to date providing a wealth of molecular details. In addition to highlighting important biological pathways, this information will guide studies to develop new treatments against glaucoma.

Genomic and transcriptomic profiles associated with response to eribulin and nivolumab combination in HER-2-negative metastatic breast cancer

BackgroundBiomarkers for predicting response to the immunotherapy and chemotherapy combination in breast cancer patients are not established. In this study, we report exploratory genomic and transcriptomic analyses of pretreatment tumor tissues from patients enrolled in phase II clinical trial of a combination of eribulin and nivolumab for HER-2-negative metastatic breast cancer (MBC) (KORNELIA trial, NCT04061863).MethodsWe analyzed associations between tumor molecular profiles based on genomic (n = 76) and transcriptomic data (n = 58) and therapeutic efficacy. Patients who achieved progression-free survival (PFS) ≥ 6 months were defined as PFS6-responders and PFS6-nonresponders otherwise.FindingsAnalyses on tumor mutation burden (TMB) showed a tendency toward a favorable effect on efficacy, while several analyses related to homologous recombination deficiency (HRD) indicated a potentially negative impact on efficacy. Patients harboring TP53 mutations showed significantly poor PFS6 rate and PFS, which correlated with the enrichment of cell cycle-related signatures in PFS6-nonresponders. High antigen presentation gene set enrichment scores (≥ median) were significantly associated with longer PFS. Naïve B-cell and plasma cell proportions were considerably higher in long responders (≥ 18 months).InterpretationGenomic features including TMB, HRD, and TP53 mutations and transcriptomic features related to immune cell profiles and cell cycle may distinguish responders. Our findings provide insights for further exploring the combination regimen and its biomarkers in these tumors.

Impact of different formulations of platelet lysate on proliferative and immune profile of equine mesenchymal stromal cells.

Platelet lysate (PL) is investigated as a potential replacement for fetal bovine serum (FBS) in cell culture. However, there is limited research on its impact on the immune profile of equine mesenchymal stromal cells (eMSCs). This study aimed to evaluate the effects of different PL formulations on the proliferative capacity, multipotentiality, and immune profile of equine adipose tissue-derived MSCs (eAD-MSCs). In vitro growth kinetics and trilineage differentiation of eAD-MSCs (n = 7) were assessed under three culture conditions: medium-concentration PL (MPL), high-concentration PL (HPL), and FBS as a control. The immune profile was evaluated by studying the expression of immunogenic receptors such as MHC I, MHC II, and immunomodulatory molecules IL-6, IL-10, and TNF-α, determined by gene expression, surface marker expression, and cytokine quantification. Both PL formulations, pooled from 5 donors, exhibited 3.3 and 6.5-fold higher platelet counts than baseline plasma for MPL and HPL, respectively. Higher concentrations of TGF-β and PDGF were found in both PL formulations compared to baseline. Furthermore, MPL and HPL subcultures demonstrated proliferative, clonogenic, and multipotent capacities similar to FBS. The immune profile of PL-cultured cells exhibited gene expression levels related to immunogenicity and immunomodulation similar to the reference condition, and the surface antigen presence of MHC II was also similar. However, HPL media exhibited higher IL-6, IL-10, and TNF-α concentrations in the culture supernatant. In conclusion, both PL media contained higher concentrations of growth factors compared to FBS, supporting the in vitro culture of eAD-MSCs with proliferative, clonogenic, and multipotent capacity similar to the reference medium. Nonetheless, PL usage led to a variation in the immunomodulatory cytokine microenvironment, with higher concentrations of IL-6, IL-10, and TNF-α in HPL media compared to MPL and FBS.

Lifestyle Intervention Modulates the CD4+ T Cell Profile in the Blood of Crohn's Disease Patients.

Crohn's disease (CD) significantly affects patients' well-being and is influenced by stress and lifestyle factors, highlighting the importance of improving quality of life in CD management. An imbalance between pro- and anti-inflammatory CD4+ T cell responses is a key factor in CD, and stress has been shown to alter the function of CD4+ T cells. Therefore, this study aimed to evaluate the effect of a mind-body medicine stress management and lifestyle modification (MBM) program on the CD4+ T cell profile in CD patients. Circulating CD4+ T cells from CD patients were analyzed by flow cytometry following the MBM program. Patients were randomly assigned to either a guided intervention group (IG) or a self-guided waitlist control group (CG) over a 9-month trial and compared with healthy blood donors. Lifestyle intervention reduced regulatory T cell (Treg) frequencies in the blood of CD patients. Notably, we observed a significant correlation between the quality of life improvement and Treg frequencies in the IG but not in the CG. Furthermore, differential activation and expression of the gut-homing molecules G protein-coupled receptor 15 and CCR9 on circulating Tregs and CD4+ effector T cells were detected in both the IG and CG. The MBM program, whether guided or self-directed, has the potential to restore the CD4+ T cell profile of CD patients to levels comparable to healthy blood donors. Lifestyle interventions may benefit CD progression, symptoms, and immunological status, but further analysis is needed to substantiate these findings and to fully understand their clinical implications. (ClinicalTrials.gov: NCT05182645).

Myeloid Cells in Myocardial Ischemic Injury: The Role of the Macrophage Migration Inhibitory Factor.

Ischemic heart disease, manifesting as myocardial infarction (MI), remains the leading cause of death in the western world. Both ischemia and reperfusion (I/R) cause myocardial injury and result in cardiac inflammatory responses. This sterile inflammation in the myocardium consists of multiple phases, involving cell death, tissue remodeling, healing, and scar formation, modulated by various cytokines, including the macrophage migration inhibitory factor (MIF). Meanwhile, different immune cells participate in these phases, with myeloid cells acting as first responders. They migrate to the injured myocardium and regulate the initial phase of inflammation. The MIF modulates the acute inflammatory response by affecting the metabolic profile and activity of myeloid cells. This review summarizes the role of the MIF in regulating myeloid cell subsets in MI and I/R injury and discusses emerging evidence of metabolism-directed cellular inflammatory responses. Based on the multifaceted role of the MIF affecting myeloid cells in MI or I/R, the MIF can be a therapeutic target to achieve metabolic balance under pathology and alleviate inflammation in the heart.

Testing of NMC and LFP Li-ION cells for surface temperature at various conditions

Battery electric vehicles (BEVs) are being considered as a replacement technology for diesel vehicles in underground mining of mineral resources. However, many issues related to BEVs in industrial applications still require more detailed research. One significant topic is the heat load—BEVs' heat emission into the working environment during regular operation. This paper provides an overview of the thermal behavior of NMC and LFP Li-ion cells tested under various conditions, which is an important step in determining the overall heat load of BEVs. The surface temperature of three NMC and one LFP 18650 Li-ion cells was monitored during charging and discharging at four different rates, at constant ambient temperatures of 30 °C and 40 °C. The main results pertain to the cell's thermal behavior during charging and discharging, highlighting the distinctive temperature profiles of NMC and LFP cells.

49 Inhibition of AXL along with c-Met potentially halts resistance development in renal cell carcinoma

Abstract Background c-Met, a receptor tyrosine kinase (RTK), is overexpressed in renal cell carcinoma (RCC) and correlates with a decreased survival rate. Upon binding to its specific ligand, Hepatocyte Growth Factor (HGF), c-Met activates pro-tumorigenic signaling pathways. Cabozantinib (Cabo) inhibits c-Met and a few other RTKs, including AXL, and is approved for the treatment of patients with advanced-stage RCC. AXL and its ligand, GAS6, are overexpressed in RCC and are also markers of poor prognosis. The pro-tumorigenic role of AXL and its potential crosstalk with c-Met in renal cancer need to be thoroughly investigated. Methods We generated a Cabo-resistant (CaboR) cell line from wild-type Cabo-sensitive (CaboS) Caki-1 clear-cell RCC cells, and CRISPR/Cas9-mediated AXL knock-out cells (AXL-KO) from 786-O clear-cell RCC cells. Chromatin immunoprecipitation and sequencing (ChIP-seq) was performed to profile H3K27ac, a histone post-translational modification associated with active promoters and enhancers, in CaboR and CaboS cell lines. We also performed RNA sequencing (RNA-seq) on CaboR and CaboS and evaluated differentially expressed genes and enriched pathways using gene set enrichment analysis (GSEA). Transcriptional profiles of control clones and AXL-KO cells were also compared to identify the effect of AXL-KO on pro-tumorigenic signaling. We utilized an AXL-specific small molecule inhibitor, TP-0903, in combination with Cabo to validate our data. Finally, we studied how AXL silencing or inhibition may affect resistance to Cabo. Results Our findings revealed that AXL forms a complex with c-Met and may have a significant crosstalk which can be involved in therapeutic resistance. We found that prolonged treatment with c-Met inhibitors Cabo, Crizotinib, and PF-4217903, induced c-Met and AXL overexpression in RCC cells. Interestingly, c-Met inhibitor(s)-induced overexpressed c-Met can also be increasingly phosphorylated (at low concentrations) in the presence of HGF, which may cause enhanced downstream tumor-promoting signaling. We found that silencing AXL can prevent this c-Met-inhibitors-mediated c-Met overexpression. Moreover, we found marked activation of the promoters and enhancers of several transcription factors, including ETV1, HOXA9, and FOXK1, in CaboR cells compared to CaboS cells. In the CaboR cells, genes involved in oxidative phosphorylation, progression through the division cycle, DNA replication, the NRF2 pathway, and DNA repair were upregulated, whereas genes associated with glycolysis, angiogenesis, hypoxia, and the Met pathway were downregulated (Figure 1). Finally, silencing AXL (using siRNA) or inhibiting AXL (using TP0903) in CaboR cells, induced significant apoptosis. Conclusions Together, our data suggest that CaboR cells have a distinct epigenomic and transcriptomic profile, and that targeting AXL along with c-Met inhibition can be beneficial in preventing acquired therapeutic resistance in RCC.

Sphingosine-1 phosphate receptor 1 (S1PR1) expression maintains stemness of acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) arises from leukemia stem cells (LSCs) and is maintained by cells which have acquired features of stemness. We compared transcription profiles of AML cells with/without stem cell features defined as in vitro clonogenicity and serial engraftment in immune-deficient mice xenograft model. We used multi-parameter flow cytometry (MPFC) to separate CD34+ bone marrow-derived leukemia cells into sphingosine-1 phosphate receptor 1 (S1PR1)+ and S1PR1- fractions. Cells in the S1PR1+ fraction demonstrated significantly higher clonogenicity and higher engraftment potential compared with those in the S1PR1- fraction. In contrast, CD34+ bone marrow cells from normal samples showed reduced clonogenicity in the S1PR1+ fraction compared with the S1PR1- fraction. Inhibition of S1PR1 expression in an AML cell line reduced the colony-forming potential of KG1 cells. Transcriptomic analyses and rescue experiments indicated PI3K/AKT pathway and MYBL2 are downstream mediators of S1PR1-associated stemness. These findings implicate S1PR1 as a functional biomarker of LSCs and suggest its potential as a therapeutic target in AML treatment.

A targeted CRISPR screen identifies ETS1 as a regulator of HIV latency.

HIV latency is a major obstacle for the eradication of HIV. However, molecular mechanisms that restrict proviral expression during therapy are not well understood. Identification of host cell factors that silence HIV would create opportunities for targeting these factors to reverse latency and eliminate infected cells. Our study aimed to explore mechanisms of latency in infected cells by employing a lentiviral CRISPR screen and CRISPR/Cas9 knockout in primary CD4 T cells. These experiments revealed that ETS1 is essential for maintaining HIV latency in primary CD4 T cells and we further confirmed ETS1's role in maintaining HIV latency through CRISPR/Cas9 knockout in CD4 T cells from antiretroviral therapy (ART)-suppressed individuals with HIV. Transcriptomic profiling of ETS1-depleted cells from these individuals identified several host cell pathways involved in viral transcription regulated by ETS1, including the long non-coding RNA MALAT1. Overall, our study demonstrates that ETS1 is a critical regulator of HIV latency, affecting the expression of several cellular genes that directly or indirectly influence HIV expression.

Lack of pathogenic involvement of CCL4 and its receptor CCR5 in arthritogenic alphavirus disease.

Arthritogenic alphaviruses, including chikungunya virus (CHIKV), Mayaro virus (MAYV), Ross River virus (RRV), and O'nyong nyong virus (ONNV) are emerging and reemerging viruses that cause disease characterized by fever, rash, and incapacitating joint swelling. Alphavirus infection induces robust immune responses in infected hosts, leading to the upregulation of several cytokines and chemokines, including chemokine C ligand 4 (CCL4). CCL4 is a chemoattractant for immune cells such as T cells, natural killer cells, monocytes/macrophages, and dendritic cells, recruiting these cells to the site of infection, stimulating the release of proinflammatory mediators, and inducing T cell differentiation. CCL4 has been found at high levels in both the acute and chronic phases of chikungunya disease; however, the role of CCL4 in arthritogenic alphavirus disease development remains unexplored. Here, we tested the effect of CCL4 on MAYV infection in mice through antibody depletion and treatment with recombinant mouse CCL4. We observed no differences in mice depleted of CCL4 or treated with recombinant CCL4 in terms of disease progression such as weight loss and footpad swelling or the development of viremia. CCL4 uses the G protein-coupled receptor C-C chemokine receptor type 5 (CCR5). To determine whether CCR5 deficiency would alter disease outcomes or virus replication in mice, we inoculated CCR5 knockout (CCR5-/-) mice with MAYV and observed no effect on disease development and immune cell profile of blood and footpads between CCR5-/- and wild type mice. These studies failed to identify a clear role for CCL4 or its receptor CCR5 in MAYV infection.

Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia.

Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm (NPM1c+). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a proapoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm and DNA damage-induced apoptosis is caspase-2 dependent in NPM1c+ but not in NPM1wt AML cells. Strikingly, in NPM1c+ cells, caspase-2 loss results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment of the AKT/mTORC1 pathways, and inhibition of Rictor cleavage. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells lacking caspase-2. Our results show that caspase-2 is essential for proliferation and self-renewal of AML cells expressing mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function.

Evaluating batch correction methods for image-based cell profiling

High-throughput image-based profiling platforms are powerful technologies capable of collecting data from billions of cells exposed to thousands of perturbations in a time- and cost-effective manner. Therefore, image-based profiling data has been increasingly used for diverse biological applications, such as predicting drug mechanism of action or gene function. However, batch effects severely limit community-wide efforts to integrate and interpret image-based profiling data collected across different laboratories and equipment. To address this problem, we benchmark ten high-performing single-cell RNA sequencing (scRNA-seq) batch correction techniques, representing diverse approaches, using a newly released Cell Painting dataset, JUMP. We focus on five scenarios with varying complexity, ranging from batches prepared in a single lab over time to batches imaged using different microscopes in multiple labs. We find that Harmony and Seurat RPCA are noteworthy, consistently ranking among the top three methods for all tested scenarios while maintaining computational efficiency. Our proposed framework, benchmark, and metrics can be used to assess new batch correction methods in the future. This work paves the way for improvements that enable the community to make the best use of public Cell Painting data for scientific discovery.

Integrated multiplex analysis of cell death regulators in stage II colorectal cancer suggests patients with ‘persister’ cell profiles fail to benefit from adjuvant chemotherapy

ObjectiveInducing tumour cell apoptosis is a primary objective of chemotherapy but, to date, there are no validated biomarkers of apoptosis sensitivity or resistance. Our objective was to image multiple apoptosis...

Inter-tissue differences in oxidative stress susceptibility reveal a less stable endothelial barrier in the brain than in the retina

Oxidative stress can impair the endothelial barrier and thereby enable autoantibody migration in Neuromyelitis optica spectrum disorder (NMOSD). Tissue-specific vulnerability to autoantibody-mediated damage could be explained by a differential, tissue-dependent endothelial susceptibility to oxidative stress. In this study, we aim to investigate the barrier integrity and complement profiles of brain and retinal endothelial cells under oxygen-induced oxidative stress to address the question of whether the pathomechanism of NMOSD preferentially affects the brain or the retina.Primary human brain microvascular endothelial cells (HBMEC) and primary human retinal endothelial cells (HREC) were cultivated at different cell densities (2.5*104 to 2*105 cells/cm2) for real-time cell analysis. Both cell types were exposed to 100, 500 and 2500 μM H2O2. Immunostaining (CD31, VE-cadherin, ZO-1) and Western blot, as well as complement protein secretion using multiplex ELISA were performed.HBMEC and HREC cell growth phases were cell type-specific. While HBMEC cell growth could be categorized into an initial peak, proliferation phase, plateau phase, and barrier breakdown phase, HREC showed no proliferation phase, but entered the plateau phase immediately after an initial peak. The plateau phase was 7 h shorter in HREC. Both cell types displayed a short-term, dose-dependent adaptive response to H2O2. Remarkably, at 100 μM H2O2, the transcellular resistance of HBMEC exceeded that of untreated cells. 500 μM H2O2 exerted a more disruptive effect on the HBMEC transcellular resistance than on HREC. Both cell types secreted complement factors H (FH) and I (FI), with FH secretion remaining stable after 2 h, but FI secretion decreasing at higher H2O2 concentrations.The observed differences in resistance to oxidative stress between primary brain and retinal endothelial cells may have implications for further studies of NMOSD and other autoimmune diseases affecting the eye and brain. These findings may open novel perspectives for the understanding and treatment of such diseases.

Rapid and robust isolation of microglia and vascular cells from brain subregions for integrative single-cell analyses

Cell isolation protocols from brain tissue include prolonged ex vivo processing durations, rendering them suboptimal for transcriptomic studies. Particularly for microglia and vascular cells, current isolation methods produce lower yields, necessitating addition of an enrichment step, and use of large tissue volumes - in most cases whole brain tissue - to obtain sufficient yields. Here, we developed a simple, rapid, and reproducible cell isolation method for generating single-cell suspensions from micro-dissected brain regions, enriched for microglia and vascular cells, without an enrichment step. Cells isolated using this method are suitable for molecular profiling studies using 10 × Genomics Chromium single-cell RNA sequencing with high reproducibility. Our method is valuable for longitudinal unbiased molecular profiling of microglia and vascular cells within different brain regions, spanning multiple time points across physiological development or disease progression.