Upregulation Of Adhesion Molecules Research Articles

Microvascular C5b-9 deposition in non-lesional skin in patients with SLE and its correlation with active lupus nephritis: a prospective observational study

ObjectiveTissue damage in lupus nephritis (LN) is mediated by activation of the classical complement pathway. Complement-mediated upregulation of endothelial cell adhesion molecules is seen in dermal blood vessels of non-lesional...

Epigenetic Mechanisms in Vascular Inflammation: Modulation of Endothelial Adhesion Molecules and Endothelium-Leukocyte Adhesion.

The endothelium, an essential component of the vascular system, plays a critical role in the inflammatory response. Under pro-inflammatory stimuli, endothelial cells undergo activation and dysfunction, leading to the release of inflammatory mediators and upregulation of cell adhesion molecules. These changes facilitate the adhesion, rolling, and transmigration of leukocytes into the subendothelial space. Emerging evidence suggests that epigenetic mechanisms, including nucleic acid methylation, post-translational histone modifications, and non-coding RNA, contribute significantly to the regulation of vascular inflammation and expression of cell adhesion molecules. Understanding the epigenetic molecular signatures that govern these processes may provide new insights into the development of therapeutic strategies to combat vascular inflammation and associated diseases. This review aims to summarize the current knowledge on the epigenetic mechanisms involved in modulating the intricate processes underlying vascular inflammation, with a specific focus on the expression of endothelial adhesion molecules and endothelium-leukocyte adhesion.

Role of toll-like receptors and nod-like receptors in acute lung infection.

The respiratory system exposed to microorganisms continuously, and the pathogenicity of these microbes not only contingent on their virulence factors, but also the host's immunity. A multifaceted innate immune mechanism exists in the respiratory tract to cope with microbial infections and to decrease tissue damage. The key cell types of the innate immune response are macrophages, neutrophils, dendritic cells, epithelial cells, and endothelial cells. Both the myeloid and structural cells of the respiratory system sense invading microorganisms through binding or activation of pathogen-associated molecular patterns (PAMPs) to pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and NOD-like receptors (NLRs). The recognition of microbes and subsequent activation of PRRs triggers a signaling cascade that leads to the activation of transcription factors, induction of cytokines/5chemokines, upregulation of cell adhesion molecules, recruitment of immune cells, and subsequent microbe clearance. Since numerous microbes resist antimicrobial agents and escape innate immune defenses, in the future, a comprehensive strategy consisting of newer vaccines and novel antimicrobials will be required to control microbial infections. This review summarizes key findings in the area of innate immune defense in response to acute microbial infections in the lung. Understanding the innate immune mechanisms is critical to design host-targeted immunotherapies to mitigate excessive inflammation while controlling microbial burden in tissues following lung infection.

Neflamapimod inhibits endothelial cell activation, adhesion molecule expression, leukocyte attachment and vascular inflammation by inhibiting p38 MAPKα and NF-κB signaling

Neflamapimod, a selective inhibitor of the alpha isoform of p38 mitogen-activated protein kinase (MAPKα), was investigated for its potential to inhibit lipopolysaccharide (LPS)-induced activation of endothelial cells (ECs), adhesion molecule induction, and subsequent leukocyte attachment to EC monolayers. These events are known to contribute to vascular inflammation and cardiovascular dysfunction. Our results demonstrate that LPS treatment of cultured ECs and rats leads to significant upregulation of adhesion molecules, both in vitro and in vivo, which can be effectively inhibited by neflamapimod treatment. Western blotting data further reveals that neflamapimod inhibits LPS-induced phosphorylation of p38 MAPKα and the activation of NF-κB signaling in ECs. Additionally, leukocyte adhesion assays demonstrate a substantial reduction in leukocyte attachment to cultured ECs and the aorta lumen of rats treated with neflamapimod. Consistent with vascular inflammation, LPS-treated rat arteries exhibit significantly diminished vasodilation response to acetylcholine, however, arteries from rats treated with neflamapimod maintain their vasodilation capacity, demonstrating its ability to limit LPS-induced vascular inflammation. Overall, our data demonstrate that neflamapimod effectively inhibits endothelium activation, adhesion molecule expression, and leukocyte attachment, thereby reducing vascular inflammation.

Uptake of tumor-derived microparticles induces metabolic reprogramming of macrophages in the early metastatic lung

Pre-metastatic niche formation is a critical step during the metastatic spread of cancer. One way by which primary tumors prime host cells at future metastatic sites is through the shedding of tumor-derived microparticles as a consequence of vascular sheer flow. However, it remains unclear how the uptake of such particles by resident immune cells affects their phenotype and function. Here, we show that ingestion of tumor-derived microparticles by macrophages induces a rapid metabolic and phenotypic switch that is characterized by enhanced mitochondrial mass and function, increased oxidative phosphorylation, and upregulation of adhesion molecules, resulting in reduced motility in the early metastatic lung. This reprogramming event is dependent on signaling through the mTORC1, but not the mTORC2, pathway and is induced by uptake of tumor-derived microparticles. Together, these data support a mechanism by which uptake of tumor-derived microparticles induces reprogramming of macrophages to shape their fate and function in the early metastatic lung.

Upregulation of CD226 on subsets of T cells and NK cells is associated with upregulated adhesion molecules and cytotoxic factors in patients with tuberculosis

Human T cells and natural killer (NK) cells are major effector cells of innate immunity exerting potential immune surveillance against tuberculosis infection. CD226 is an activating receptor playing vital roles in the functions of T cells and NK cells during HIV infection and tumorigenesis. However, CD226 is a less-studied activating receptor during Mycobacterium tuberculosis (Mtb) infection. In this study, we used peripheral blood from tuberculosis patients and healthy donors to evaluate CD226 immunoregulation functions from two independent cohorts using Flow cytometry. Here, we found that a subset of T cells and NK cells that constitutively express CD226 exhibit a distinct phenotype in TB patients. In fact, the proportions of CD226+ and CD226- cell subsets differ between healthy people and tuberculosis patients, and the expression of immune checkpoint molecules (TIGIT, NKG2A) and adhesion molecules (CD2, CD11a) in CD226+ and CD226- subsets of T cells and NK cells exhibits special regulatory roles. Furthermore, CD226+ subsets produced more IFN-γ and CD107a than CD226- subsets in tuberculosis patients. Our results imply that CD226 may be a potential predictor of disease progression and clinical efficacy in tuberculosis by mediating the cytotoxic capacity of T cells and NK cells.

High levels of endothelial ICAM-1 prohibit natalizumab mediated abrogation of CD4+ T cell arrest on the inflamed BBB under flow in vitro

IntroductionThe humanized anti-α4 integrin blocking antibody natalizumab (NTZ) is an effective treatment for relapsing–remitting multiple sclerosis (RRMS) that is associated with the risk of progressive multifocal leukoencephalopathy (PML). While extended interval dosing (EID) of NTZ reduces the risk for PML, the minimal dose of NTZ required to maintain its therapeutic efficacy remains unknown.ObjectiveHere we aimed to identify the minimal NTZ concentration required to inhibit the arrest of human effector/memory CD4+ T cell subsets or of PBMCs to the blood–brain barrier (BBB) under physiological flow in vitro.ResultsMaking use of three different human in vitro BBB models and in vitro live-cell imaging we observed that NTZ mediated inhibition of α4-integrins failed to abrogate T cell arrest to the inflamed BBB under physiological flow. Complete inhibition of shear resistant T cell arrest required additional inhibition of β2-integrins, which correlated with a strong upregulation of endothelial intercellular adhesion molecule (ICAM)-1 on the respective BBB models investigated. Indeed, NTZ mediated inhibition of shear resistant T cell arrest to combinations of immobilized recombinant vascular cell adhesion molecule (VCAM)-1 and ICAM-1 was abrogated in the presence of tenfold higher molar concentrations of ICAM-1 over VCAM-1. Also, monovalent NTZ was less potent than bivalent NTZ in inhibiting T cell arrest to VCAM-1 under physiological flow. In accordance with our previous observations ICAM-1 but not VCAM-1 mediated T cell crawling against the direction of flow.ConclusionTaken together, our in vitro observations show that high levels of endothelial ICAM-1 abrogate NTZ mediated inhibition of T cell interaction with the BBB. EID of NTZ in MS patients may thus require consideration of the inflammatory status of the BBB as high levels of ICAM-1 may provide an alternative molecular cue allowing for pathogenic T cell entry into the CNS in the presence of NTZ.

Endothelial mechanical stretch regulates the immunological synapse interface of renal endothelial cells in a sex-dependent manner.

Increased mechanical endothelial cell stretch contributes to the development of numerous cardiovascular and renal pathologies. Recent studies have shone a light on the importance of sex-dependent inflammation in the pathogenesis of renal disease states. The endothelium plays an intimate and critical role in the orchestration of immune cell activation through upregulation of adhesion molecules and secretion of cytokines and chemokines. While endothelial cells are not recognized as professional antigen-presenting cells, in response to cytokine stimulation, endothelial cells can express both major histocompatibility complex (MHC) I and MHC II. MHCs are essential to forming a part of the immunological synapse interface during antigen presentation to adaptive immune cells. Whether MHC I and II are increased under increased mechanical stretch is unknown. Due to hypertension being multifactorial, we hypothesized that increased mechanical endothelial stretch promotes the regulation of MHCs and key costimulatory proteins on mouse renal endothelial cells (MRECs) in a stretch-dependent manner. MRECs derived from both sexes underwent 5%, 10%, or 15% uniaxial cyclical stretch, and immunological synapse interface proteins were determined by immunofluorescence microscopy, immunoblot analysis, and RNA sequencing. We found that increased endothelial mechanical stretch conditions promoted downregulation of MHC I in male MRECs but upregulation in female MRECs. Moreover, MHC II was upregulated by mechanical stretch in both male and female MRECs, whereas CD86 and CD70 were regulated in a sex-dependent manner. By bulk RNA sequencing, we found that increased mechanical endothelial cell stretch promoted differential gene expression of key antigen processing and presentation genes in female MRECs, demonstrating that females have upregulation of key antigen presentation pathways. Taken together, our data demonstrate that mechanical endothelial stretch regulates endothelial activation and immunological synapse interface formation in renal endothelial cells in a sex-dependent manner.NEW & NOTEWORTHY Endothelial cells contribute to the development of renal inflammation and have the unique ability to express antigen presentation proteins. Whether increased endothelial mechanical stretch regulates immunological synapse interface proteins remains unknown. We found that antigen presentation proteins and costimulatory proteins on renal endothelial cells are modulated by mechanical stretch in a sex-dependent manner. Our data provide novel insights into the sex-dependent ability of renal endothelial cells to present antigens in response to endothelial mechanical stimuli.

Endothelial Cell-Selective Adhesion Molecule Enhances Tumor Growth in a HFpEF Mouse Model

Accumulating epidemiological evidence shows that the incidence of multiple types of cancer, such as melanoma and GI cancers, is significantly higher in patients with heart failure with preserved ejection fraction (HFpEF). However, the mechanistic relationships between HFpEF and cancer development are poorly understood. Impaired angiogenesis, upregulation of endothelial adhesion molecules and chronic low-grade inflammation are common in HFpEF. Here we examined the role of endothelial cell-selective adhesion molecule (ESAM) in the progression of malignant melanoma in a HFpEF mouse model. Orthotopic melanoma xenografts (B16F10-Luc2) were used and implanted subcutaneously in mice underwent uninephrectomy and aldosterone infusion (UNX-Aldo with 1% high salt drinking water for 4 weeks) and tumor growth was monitored with bioluminescent imaging. We found that ESAM expression and serum levels were elevated in the UNX-Aldo mice, which was accompanied by enhanced melanoma growth and metastasis rate. ESAM promoted tube formation and increased phospho-Akt, phospho-PKCα and phospho-ERK1/2 in cultured endothelial cells. Flow cytometry analysis showed elevated number of tumor-associated neutrophils (CD11b+Ly6G+Ly6C-) and myeloid-derived suppressor cells (MDSCs, CD11b+F4/80-Gr-1+) in the UNX-Aldo mice. UNX-Aldo mice with genetic deletion of ESAM (ESAM KO) showed a reduced tumor growth and reduced number of tumor-associated neutrophils and MDSCs. Collectively, our results indicate that in HFpEF, a proinflammatory ESAM activation may act to promote tumor angiogenesis, neutrophil and MDSCs tumor infiltration, thereby mitigating a proper immune response and facilitate tumor growth. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vascular traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk

During bloodstream infections, neutrophils home to the liver as part of an intravascular immune response to eradicate blood-borne pathogens, but the mechanisms regulating this crucial response are unknown. Using invivo imaging of neutrophil trafficking in germ-free and gnotobiotic mice, we demonstrate that the intestinal microbiota guides neutrophil homing to the liver in response to infection mediated by the microbial metabolite D-lactate. Commensal-derived D-lactate augments neutrophil adhesion in the liver independent of granulopoiesis in bone marrow or neutrophil maturation and activation in blood. Instead, gut-to-liver D-lactate signaling primes liver endothelial cells to upregulate adhesion molecule expression in response to infection and promote neutrophil adherence. Targeted correction of microbiota D-lactate production in a model of antibiotic-induced dysbiosis restores neutrophil homing to the liver and reduces bacteremia in a model of Staphylococcus aureus infection. These findings reveal long-distance traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk.

Recent Trends on Mitigative Effect of Probiotics on Oxidative-Stress-Induced Gut Dysfunction in Broilers under Necrotic Enteritis Challenge: A Review.

Gut health includes normal intestinal physiology, complete intestinal epithelial barrier, efficient immune response, sustained inflammatory balance, healthy microbiota, high nutrient absorption efficiency, nutrient metabolism, and energy balance. One of the diseases that causes severe economic losses to farmers is necrotic enteritis, which occurs primarily in the gut and is associated with high mortality rate. Necrotic enteritis (NE) primarily damages the intestinal mucosa, thereby inducing intestinal inflammation and high immune response which diverts nutrients and energy needed for growth to response mediated effects. In the era of antibiotic ban, dietary interventions like microbial therapy (probiotics) to reduce inflammation, paracellular permeability, and promote gut homeostasis may be the best way to reduce broiler production losses. The current review highlights the severity effects of NE; intestinal inflammation, gut lesions, alteration of gut microbiota balance, cell apoptosis, reduced growth performance, and death. These negative effects are consequences of; disrupted intestinal barrier function and villi development, altered expression of tight junction proteins and protein structure, increased translocation of endotoxins and excessive stimulation of proinflammatory cytokines. We further explored the mechanisms by which probiotics mitigate NE challenge and restore the gut integrity of birds under disease stress; synthesis of metabolites and bacteriocins, competitive exclusion of pathogens, upregulation of tight junction proteins and adhesion molecules, increased secretion of intestinal secretory immunoglobulins and enzymes, reduction in pro-inflammatory cytokines and immune response and the increased production of anti-inflammatory cytokines and immune boost via the modulation of the TLR/NF-ĸ pathway. Furthermore, increased beneficial microbes in the gut microbiome improve nutrient utilization, host immunity, and energy metabolism. Probiotics along with biosecurity measures could mitigate the adverse effects of NE in broiler production.

Gut-Derived Metabolite, Trimethylamine-N-oxide (TMAO) in Cardio-Metabolic Diseases: Detection, Mechanism, and Potential Therapeutics.

Trimethylamine N-oxide (TMAO) is a biologically active gut microbiome-derived dietary metabolite. Recent studies have shown that high circulating plasma TMAO levels are closely associated with diseases such as atherosclerosis and hypertension, and metabolic disorders such as diabetes and hyperlipidemia, contributing to endothelial dysfunction. There is a growing interest to understand the mechanisms underlying TMAO-induced endothelial dysfunction in cardio-metabolic diseases. Endothelial dysfunction mediated by TMAO is mainly driven by inflammation and oxidative stress, which includes: (1) activation of foam cells; (2) upregulation of cytokines and adhesion molecules; (3) increased production of reactive oxygen species (ROS); (4) platelet hyperreactivity; and (5) reduced vascular tone. In this review, we summarize the potential roles of TMAO in inducing endothelial dysfunction and the mechanisms leading to the pathogenesis and progression of associated disease conditions. We also discuss the potential therapeutic strategies for the treatment of TMAO-induced endothelial dysfunction in cardio-metabolic diseases.

Microphysiological system recapitulating the pathophysiology of adipose tissue in obesity.

A growing body of evidence has indicated that white adipose tissue (AT) remodeling is a major trigger for obesity-associated metabolic complications. However, the scarcity of translational models is an obstacle to the development of medicines that act on adipose restoration. Here, we describe a microphysiological system (MPS) that emulates the unique features of reprogrammed AT as a new in vitro tool for studying AT pathophysiology in obesity. The AT MPS contained mature adipocytes embedded in an extracellular matrix (ECM) hydrogel interfaced with AT microvascular endothelium, which was constantly perfused with fresh media. The unique biochemical signals due to the remodeled ECM in obesity were recapitulated using a decellularized AT ECM (AT dECM) hydrogel, which preserves the features of altered ECM composition in obesity. The mature adipocytes embedded in the AT dECM hydrogel maintained their function and morphology for a week without dedifferentiation. Using the AT MPS, we successfully modeled inflammation-induced AT microvascular dysfunction, the recruitment of immune cells due to the upregulation of cell adhesion molecules, and higher cancer cell adhesion as an indicator of metastasis, which are observed in obese individuals. The AT MPS may therefore represent a promising platform for understanding the dynamic cellular interplay in obesity-induced AT remodeling and validating the efficacy of drugs targeting AT in obesity. STATEMENT OF SIGNIFICANCE: The lack of translational in vitro white adipose tissue (AT) models is one of the main obstacles for understanding the obesity-induced reprogramming and the development of medicines. We report herein the AT microphysiological system (MPS), which recapitulates obesity and normal conditions and yields cell- and AT dECM-derived signals, thereby allowing accurate comparative in vitro analyses. Using the AT MPS, we successfully modeled reprogrammed AT in obesity conditions, including inflammation-induced AT vascular dysfunction, the recruitment of immune cells, and higher cancer cell metastasis, which are observed in obese individuals. Our proposed adipose tissue model providing physiological relevance and complexity may therefore enhance the understanding of obesity-associated disorders and be used to investigate their underlying molecular mechanisms to develop pharmacologic treatment strategies.

Abstract B038: Remodeling the extracellular matrix environment and establishing distinct immune cell profiles enables the formation of brain metastasis in non-small cell lung cancer adenocarcinoma

Abstract Dissemination of tumor cells from the primary site of the lung to distant sites in non-small cell lung cancer (NSCLC) is poorly understood. With distinct mutation signatures in patients, altering pathways is critical to preventing tumor cell dissemination to distant sites. Towards this, we characterized 21 "trios" patient-matched brain metastases (BrM), primary tumors (LT), and normal samples (LN), revealing a pattern of extracellular matrix remodeling which suppressed immune cell infiltration. Our objectives were to (i) determine if clinically sampled primary lung tumors encode gene expression patterns distinct from normal tissue, suggestive of increased tumor invasiveness including brain metastasis, and (ii) determine the biologic underpinning of immune-suppressive information carryover from primary lung tumor to brain metastasis. Transformation of the normal lung cells to primary tumor cells suggested a breakdown in ECM axes primarily governed by upregulation of collagen (COL1A1, COL1A2, COL3A1, COL10A1), fibronectin (FN1), and matrix metalloproteinases (MMP7, MMP9, MMP11, MMP12, MMP13, MMP14) genes. Collectively, the primary tumor cells used MMPs to break down the main components of lung ECM and reorganized collagen and fibronectin in the tumor microenvironment to facilitate migration and invasion. Migration and subsequent cell adhesion in the brain microenvironment revealed upregulation of neural cell adhesion molecule (NCAM1) and phosphoprotein 1 (SPP1). These changes in ECM profiles between LT and BrM signify primary tumor cells' adaptation in the brain microenvironment. Further computational deconvolution revealed reduced HLA class II gene expression (HLA-DMA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA2, HLA-DQB2, HLA-DRA) correlated with lower CIBERSORTx enrichment scores of immune cell populations in BrM. Overall, using the matched sequencing information, we will present the underlying signaling gradients that dictate dissemination of primary lung tumor cells to the brain environment in the background of altered ECM environment using cellular models harboring EGFR, KRAS, and ALK driver mutations. Citation Format: Albert R. Wang, Jared Brown, Andrew Baschnagel, Darya Buehler, Christina Kendziorski, Gopal Iyer. Remodeling the extracellular matrix environment and establishing distinct immune cell profiles enables the formation of brain metastasis in non-small cell lung cancer adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B038.

Perillyl alcohol modulates activation, permeability and integrity of human brain endothelial cells induced by Plasmodium falciparum.

Cerebral malaria (CM) is a severe immunovasculopathy caused for Plasmodium falciparum infection, which is characterised by the sequestration of parasitised red blood cells (pRBCs) in brain microvessels. Previous studies have shown that some terpenes, such as perillyl alcohol (POH), exhibit a marked efficacy in preventing cerebrovascular inflammation, breakdown of the brain-blood barrier (BBB) and brain leucocyte accumulation in experimental CM models. To analyse the effects of POH on the endothelium using human brain endothelial cell (HBEC) monolayers co-cultured with pRBCs. The loss of tight junction proteins (TJPs) and features of endothelial activation, such as ICAM-1 and VCAM-1 expression were evaluated by quantitative immunofluorescence. Microvesicle (MV) release by HBEC upon stimulation by P. falciparum was evaluated by flow cytometry. Finally, the capacity of POH to revert P. falciparum-induced HBEC monolayer permeability was examined by monitoring trans-endothelial electrical resistance (TEER). POH significantly prevented pRBCs-induced endothelial adhesion molecule (ICAM-1, VCAM-1) upregulation and MV release by HBEC, improved their trans-endothelial resistance, and restored their distribution of TJPs such as VE-cadherin, Occludin, and JAM-A. POH is a potent monoterpene that is efficient in preventing P. falciparum-pRBCs-induced changes in HBEC, namely their activation, increased permeability and alterations of integrity, all parameters of relevance to CM pathogenesis.

Amelioration of atherosclerosis in ox-LDL induced HUVEC by sulfated polysaccharides from Gelidium crinale with antihypertensive activity

Red algal polysaccharide is a good potential medical resource. Different red algal polysaccharides have different structural characteristics and rich biological activities. Previous studies have identified some structural information of sulfated polysaccharide (GNP, 25.8 kDa) from red algae, Gelidium crinale and found that GNP has excellent anti-inflammatory, antioxidant and anti-tumor activities. On this basis, this study investigated the effect of GNP on atherosclerosis, which is closely related to antioxidant and anti-inflammatory mechanisms and usually coexists and interacts with hypertension. This study investigated the inhibitory activity of GNP on angiotensin-converting enzyme (ACE) and its mechanism on oxidized low-density lipoprotein (ox-LDL)-induced HUVEC atherosclerosis. The results showed that GNP inhibits the up-regulation of cell adhesion molecules and oxidized low-density lipoprotein receptor-1 (LOX-1). GNP can regulate mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NF-κB) and PI3K/AKT signal pathways, inhibit apoptosis, invasion and migration. Meanwhile, GNP (IC50 = 269.2 μg/mL) antagonizes ACE by competitive binding mode, and it can reduce systolic blood pressure (SBP) of spontaneously hypertensive rats (SHR). It provides a theoretical basis for GNP as a potential substance for the prevention and treatment of atherosclerosis.

MyD88-TLR4-dependent choroid plexus activation precedes perilesional inflammation and secondary brain edema in a mouse model of intracerebral hemorrhage.

The functional neurological outcome of patients with intracerebral hemorrhage (ICH) strongly relates to the degree of secondary brain injury (ICH-SBI) evolving within days after the initial bleeding. Different mechanisms including the incitement of inflammatory pathways, dysfunction of the blood-brain barrier (BBB), activation of resident microglia, and an influx of blood-borne immune cells, have been hypothesized to contribute to ICH-SBI. Yet, the spatiotemporal interplay of specific inflammatory processes within different brain compartments has not been sufficiently characterized, limiting potential therapeutic interventions to prevent and treat ICH-SBI. We used a whole-blood injection model in mice, to systematically characterized the spatial and temporal dynamics of inflammatory processes after ICH using 7-Tesla magnetic resonance imaging (MRI), spatial RNA sequencing (spRNAseq), functional BBB assessment, and immunofluorescence average-intensity-mapping. We identified a pronounced early response of the choroid plexus (CP) peaking at 12-24h that was characterized by inflammatory cytokine expression, epithelial and endothelial expression of leukocyte adhesion molecules, and the accumulation of leukocytes. In contrast, we observed a delayed secondary reaction pattern at the injection site (striatum) peaking at 96h, defined by gene expression corresponding to perilesional leukocyte infiltration and correlating to the delayed signal alteration seen on MRI. Pathway analysis revealed a dependence of the early inflammatory reaction in the CP on toll-like receptor 4 (TLR4) signaling via myeloid differentiation factor 88 (MyD88). TLR4 and MyD88 knockout mice corroborated this observation, lacking the early upregulation of adhesion molecules and leukocyte infiltration within the CP 24h after whole-blood injection. We report a biphasic brain reaction pattern after ICH with a MyD88-TLR4-dependent early inflammatory response of the CP, preceding inflammation, edema and leukocyte infiltration at the lesion site. Pharmacological targeting of the early CP activation might harbor the potential to modulate the development of ICH-SBI.

Visualization of epithelial cell adhesion molecule-expressing renal cell carcinoma xenografts using designed ankyrin repeat protein Ec1 labelled with 99mTc and 125I.

The upregulation of epithelial cell adhesion molecule (EpCAM) expression, found in a substantial fraction of renal cell carcinomas (RCCs), renders it a potential molecular target for the treatment of disseminated RCC. However, the heterogeneous expression of EpCAM necessitates first identifying the patients with sufficiently high expression of EpCAM in tumors. Using the specific radionuclide-based visualization of EpCAM might enable such identification. The designed ankyrin repeat protein, Ec1, is a small (molecular weight, 18 kDa) targeting protein with a subnanomolar affinity to EpCAM. Using a modified Ec1, a tracer was developed for the radionuclide-based visualization of EpCAM in vivo, i.e., an EpCAM-visualizing designed ankyrin repeat protein (EVD). EVD was labelled with either technetium-99m using technetium tricarbonyl or with iodine-125 (as a surrogate for iodine-123) by coupling it to para-[125I]iodobenzoyl ([125I]PIB) groups. Both the 125I-labelled EVD (125I-EVD) and 99mTc-labelled EVD (99mTc-EVD) bound specifically to EpCAM-expressing SK-RC-52 renal carcinoma cells. The binding affinity (KD value) of 99mTc-EVD to SK-RC-52 cells was 400±28 pM. The tracers' uptake in SK-RC-52 ×enografts at 3 h after injection was 5.2±1.4%ID/g for 125I-EVD and 6.0±1.4%ID/g for 99mTc-EVD (no significant difference). These uptake values in SK-RC-52 ×enografts were significantly higher (P<0.001) than those in Ramos lymphoma xenografts (used as EpCAM-negative control). The tumor-to-blood uptake ratio was significantly higher for 99mTc-EVD (25±6) compared with that of 125I-EVD (14±3). However, 125I-EVD was associated with higher tumor-to-liver, tumor-to-salivary gland, tumor-to-spleen and tumor-to-intestinal wall ratios. This makes it the preferable tracer for visualizing EpCAM expression levels in the frequently occurring abdominal metastases of RCC.

A comprehensive assessment of four whole blood stabilizers for flow-cytometric analysis of leukocyte populations.

Though cryopreservation of cell fractions is widely used in flow cytometry studies, whole blood cryopreservation is more challenging due to the presence of erythrocytes and effects of fixatives commonly used for preservation. Here, we evaluated and compared head-to-head the performance of four commercial whole blood cryopreservation kits; (1) Cytodelics, (2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), (3) Proteomic stabilizer (PROT-1), and (4) Transfix. We found that PROT-1, Transfix, and Cytodelics maintained the distribution of major leukocyte subsets-granulocytes, T cells, natural killer cells, and B cells, on a comparable level to unpreserved samples, despite the attenuation of fluorescence intensities in flow cytometric assays. Moreover, these three stabilizers also maintained the activated phenotypes of neutrophils upon stimulation with N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16), and granule proteins (CD66b), as well as the shedding of surface L-selectin (CD62L), was conserved most efficiently in PROT-1 and Cytodelics when compared to samples only treated with erythrocyte lysing. However, none of the stabilizers provided a reliable detection of CCR7 for accurate quantification of T cell maturation stages. We also evaluated the performance of Cytodelics in longitudinal clinical samples obtained from acute COVID-19 patients, where it allowed reliable detection of lymphopenia and granulocyte expansion. These results support the feasibility of whole blood cryopreservation for immunophenotyping by flow cytometry, particularly in longitudinal studies. In conclusion, the performance of different stabilizers is variable and therefore the choice of stabilizers should depend on cell type of interest, as well as antibody clones and experimental design of each study.

RNA-sequencing reveals significant differences in the inflammatory response of iPSC-derived endothelial cells from ACS patients and healthy controls

Abstract Background Coronary artery disease (CAD) and cardiovascular diseases (CVD) in general still represent the leading cause of death worldwide. As a multifactorial disease entity, genetic predisposition and lifestyle strongly impact disease development and progression. Here, induced pluripotent stem cells (iPSC) are an ideal tool to differentiate (epi-)genetic from lifestyle disease risk modulation and identify the underlying molecular mechanisms. Aim This study aims to determine differences in iPSC-derived endothelial cell (iPSC-EC) characteristics and functionality between healthy donors and high-risk CAD patients. Methods IPSC from participants with similar lifestyle but different disease status (healthy &amp;gt;65 y/o, n=5, acute coronary syndrome (ACS) &amp;lt;65y/o, n=6) were differentiated into iPSC-EC. Cells were exposed to inflammatory stimulation with tumour necrosis factor alpha (TNF-a) followed by bulk RNA-seq. Monocyte (THP-1) recruitment was assessed under protective and atherogenic flow conditions. Mitotic age was calculated based on DNA-methylation via Hannum estimate (1) for each donor at the iPSC and iPSC-EC (passage 4) stage. Results ACS-iPSC-EC had significantly higher upregulation of E-selectin upon TNF-a stimulation on mRNA level (ACS: 532.1-fold±48.7 vs healthy: 322.3-fold±55.7 with p=0.02). Similarly, ICAM1 protein upregulation was higher in ACS-iPSC-EC compared to healthy iPSC-EC as determined by flow cytometry (ACS: 1.4-fold±0.01 vs healthy: 1.1-fold±0.0007; p&amp;lt;0.001). ACS-iPSC-EC recruited significantly more THP-1 per view field under protective (high shear stress, laminar) flow conditions (ACS: 78.4±20.7 vs healthy: 33.25±12.3 with p=0.04) compared to healthy iPSC-EC. While both groups recruited significantly more THP-1 under atherogenic than protective conditions (ACS: 302±38.9 with p=0.0055 vs healthy: 340±70.4 with p=0.0005), there was no difference between the donor groups (p=0.43). Analysis of bulk RNA-seq data showed significant differences in TNF-a-induced transcriptome changes. Gene set enrichment analysis identified differences in signalling pathways related to leukocyte and lymphocyte-mediated immunity, adaptive immune response, cytokine activity, and many more as shown in Figure 1 (p.adj ≤1e-10). IPSC of both groups had a negative calculated mitotic age (in years) which significantly increased after endothelial differentiation and four population doublings (ACS-iPSC: −14.1±0.7 vs ACS-EC: −0.1±1.1, p&amp;lt;0.0001 and healthy-iPSC: −13.7±0.8 vs healthy-EC: 2.5±1.5, p=0.0001). Conclusion The higher rate of basal THP-1 recruitment and upregulation of adhesion molecules on mRNA and protein level and the distinct transcriptome changes suggest intrinsic differences between patient and healthy iPSC-EC both in the basal state and in inflammatory response. Furthermore, this demonstrates iPSC as a viable in vitro model also for polygenetic diseases such as CAD. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): German Cardiac Society, Research Scholarship