Activation Markers Research Articles

Ex vivo sensitivity to broadly neutralizing antibodies and anti-CD4 antibody UB-421 of infectious viral isolates from people living with multidrug-resistant HIV

People living with HIV (PLWH) with multidrug-resistant (MDR) viruses have limited therapeutic options and present challenges regarding clinical management. Recent studies have shown that passive transfer of combination broadly neutralizing antibodies (bNAbs) against HIV and anti-domain 1 CD4 antibody UB-421 can sustain virologic suppression in PLWH in the absence of antiretroviral therapy (ART). Yet studies addressing the therapeutic potential of these antibodies and/or detailed characterization of immunologic and virologic parameters in PLWH with MDR HIV are lacking. We examined levels of immune activation and exhaustion markers on CD8+ T cells and the intact HIV proviral DNA burden in 11 PLWH with MDR viruses. For comparison purposes, we included a control group consisting of 27 ART-naïve viremic PLWH. In addition, we determined the sensitivity of infectious viral isolates obtained from the participants against eight bNAbs (3BNC117, 10-1074, VRC01, VRC07, N6, 10E8, PGDM1400, and PGT121) and two anti-CD4 antibodies (ibalizumab and UB-421) using a TZM-bl-based neutralization/suppression assay. The level of intact HIV proviral DNA was comparable between the two groups (P=0.29). The levels of activation and exhaustion markers PD-1 (P=0.0019), TIGIT (P=0.0222), 2B4 (P=0.0015), CD160 (P=0.0015), and CD38+/HLA-DR+ (P=0.0138) were significantly lower in the MDR group. The infectious viral isolates from each study participant with MDR HIV were resistant to at least 2 bNAbs; however, they were sensitive to at least one of the CD4-binding and non-CD4-binding site antibodies. The majority of participants had ibalizumab-sensitive viruses although the isolates from some participants showed reduced sensitivity to ibalizumab. Notably, none of the 93 viral isolates obtained from the participants were resistant to UB-421. Our data suggest that combination therapy with HIV-specific bNAbs and/or UB-421 in the presence of optimized background therapy could potentially provide sustained virologic suppression in PLWH with MDR HIV. However, this therapeutic strategy needs to be evaluated in human clinical trials. Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Exploring the anti-inflammatory and anti-fibrotic activity of NFκB decoy oligodeoxynucleotide-loaded spermine-functionalized acetalated nanoparticles

Chronic inflammation, oxidative stress, and airway remodelling represent the principal pathophysiological features of chronic respiratory disorders. Inflammation stimuli like lipopolysaccharide (LPS) activate macrophages and dendritic cells, with concomitant M1 polarization and release of pro-inflammatory cytokines. Chronic inflammation and oxidative stress lead to airway remodelling causing irreversible functional and structural alterations of the lungs. Airway remodelling is multifactorial, however, the hormone transforming growth factor-β (TGF-β) is one of the main contributors to fibrotic changes. The signalling pathways mediating inflammation and remodelling rely both on the transcription factor nuclear factor-κB (NFκB), underlying the potential of NFκB inhibition as a therapeutic strategy for chronic respiratory disorders. In this study, we encapsulated an NFκB-inhibiting decoy oligodeoxynucleotide (ODN) in spermine-functionalized acetalated dextran (SpAcDex) nanoparticles and tested the in vitro anti-inflammatory and anti-remodelling activity of this formulation. We show that NF-κB ODN nanoparticles counteract inflammation by reversing LPS-induced expression of the activation marker CD40 in myeloid cells and counteracts remodelling features by reversing the TGF-β-induced expression of collagen I and α-smooth muscle actin in human dermal fibroblast. In summary, our study highlights the great potential of inhibiting NFκB via decoy ODN as a therapeutic strategy tackling multiple pathophysiological features underlying chronic respiratory conditions.

Relationship between skeletal mitochondrial function and digital markers of free-living physical activity in older adults.

This study examined the association between in vivo skeletal mitochondrial function and digital free-living physical activity patterns-a measure that summarizes biological, phenotypic, functional, and environmental effects on mobility. Among 459 participants (mean age 68years; 55% women) in the Baltimore Longitudinal Study of Aging, mitochondrial function was quantified as skeletal muscle oxidative capacity via post-exercise phosphocreatine recovery rate (τPCr) in the vastus lateralis muscle of the left thigh, using 31P magnetic resonance spectroscopy. Accelerometry was collected using a 7-day, 24-h wrist-worn protocol and summarized into activity amount, intensity, endurance, and accumulation patterning metrics. Linear regression, two-part linear and logistic (bout analyses), and linear mixed effects models (time-of-day analyses) were used to estimate associations between τPCr and each physical activity metric. Interactions by age, sex, and gait speed were tested. After covariate adjustment, higher τPCr (or poorer mitochondrial function) was associated with lower activity counts/day (β = - 6593.7, SE = 2406.0; p = 0.006) and activity intensity (- 81.5 counts, SE = 12.9; p < 0.001). For activity intensity, the magnitude of association was greater for men and those with slower gait speed (interaction p < 0.02 for both). Conversely, τPCr was not associated with daily active minutes/day (p = 0.15), activity fragmentation (p = 0.13), or endurance at any bout length (p > 0.05 for all). Time-of-day analyses show participants with high τPCr were less active from 6:00 a.m. to 12:00 a.m. than those with low τPCr. Results indicate that poorer skeletal mitochondrial function is primarily associated with lower engagement in high intensity activities. Our findings help define the connection between laboratory-measured mitochondrial function and real-world physical activity behavior.

Human Immunodeficiency Virus-Human Pegivirus Coinfected Individuals Display Functional Mucosal-Associated Invariant T Cells and Follicular T Cells Irrespective of PD-1 Expression.

Human pegivirus (HPgV) appears to alter the prognosis of HIV disease by modulating T cell homeostasis, chemokine/cytokine production, and T cell activation. In this study, we evaluated if HPgV had any 'favorable' impact on the quantity and quality of T cells in HIV-infected individuals. T cell subsets such as CD4lo, CD4hi, and CD8+ T cells, CD4+ MAIT cells, CD8+ MAIT cells, follicular helper T (TFH) cells, and follicular cytotoxic T (TFC) cells were characterized based on the expression of markers associated with immune activation (CD69, ICOS), proliferation (ki67), cytokine production (TNF-α, IFN-γ), and exhaustion (PD-1). HIV+HPgV+ individuals had lower transaminase SGOT (liver) and GGT (biliary) in the plasma than those who were HPgV-. HIV/HPgV coinfection was significantly associated with increased absolute CD4+ T cell counts. HIV+HPgV+ and HIV+HPgV- individuals had highly activated T cell subsets with high expression of CD69 and ICOS on bulk CD4+ and CD8+ T cells, CD4+ MAIT cells, CD8+ MAIT cells, and CXCR5+CD4+ T cells and CXCR5+CD8+ T cells compared with healthy controls. Irrespective of immune activation markers, these cells also displayed higher levels of PD-1 on CD4+ T and CD8+ T cells . Exploring effector functionality based on mitogen stimulation demonstrated increased cytokine production by CD4+ MAIT and CD8+ MAIT cells. Decrease in absolute CD4+ T cell counts correlated positively with intracellular IFN-γ levels by CD4lo T cells, whereas increase of the same correlated negatively with TNF-α in the CD4lo T cells of HIV+HPgV+ individuals. HIV/HPgV coinfected individuals display functional CD4+ and CD8+ MAIT, TFH, and TFC cells irrespective of PD-1 expression.

Chronic inhibition of angiotensin converting enzyme lowers blood pressure in spontaneously hypertensive rats by attenuation of sympathetic tone: The role of enhanced baroreflex sensitivity

Spontaneously hypertensive rats (SHR) are characterized by sympathetic hyperactivity and insufficient parasympathetic activity, and their high blood pressure (BP) can be lowered by long-term inhibition of the renin-angiotensin system. The aim of our study was to determine the influence of chronic inhibition of angiotensin converting enzyme (ACE) by captopril on cardiovascular regulation by the sympathetic and parasympathetic nervous system. Implanted radiotelemetric probes or arterial cannulas were used to measure mean arterial pressure (MAP), heart rate (HR), and arterial baroreflex in adult SHR and Wistar-Kyoto (WKY) rats under basal or stress conditions. MAP and the low-frequency component of systolic blood pressure variability (LF-SBPV, marker of sympathetic activity) were greater in SHR than in WKY rats. Under basal conditions chronic captopril treatment reduced both parameters more effectively in SHR, and the same was true during acute restraint stress. HR was similar in control rats of both strains, but WKY rats showed greater heart rate variability (HRV), indicating higher parasympathetic activity. Captopril administration increased HR in both strains, whereas HRV was decreased only in WKY. Chronic captopril treatment improved the impaired baroreflex-HR control in SHR by increasing the sensitivity but not the capacity of vagal arm of arterial baroreflex. Captopril treatment attenuated BP changes elicited by dimethylphenylpiperazinium (DMPP, agonist of nicotinic acetylcholine receptors), especially in SHR, indicating that sympathetic nerve transmission is facilitated by angiotensin II more in hypertensive than in normotensive animals. Thus, chronic ACE inhibition improves baroreflex sensitivity and lowers BP through both central and peripheral attenuation of sympathetic tone.

A Comprehensive Investigation of Stimulatory Agents on MAIT and Vα7.2+/CD161− T Cell Response and Effects of Immunomodulatory Drugs

Mucosal-associated invariant T (MAIT) cells, a subset of Vα7.2+ T cells, are a crucial link between innate and adaptive immunity, responding to various stimuli through TCR-dependent and independent pathways. We investigated the responses of MAIT cells and Vα7.2+/CD161− T cells to different stimuli and evaluated the effects of Cyclosporin A (CsA) and Vitamin D3 (VitD). Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with various agents (PMA/Ionomycin, 5-OP-RU, 5-OP-RU/IL-12/IL-33) with or without CsA and VitD. Flow cytometric analysis assessed surface markers and intracellular cytokine production. Under steady-state conditions, MAIT cells displayed elevated expression of CCR6 and IL-13. They showed upregulated activation and exhaustion markers after activation, producing IFNγ, TNFα, and TNFα/GzB. CsA significantly inhibited MAIT cell activation and cytokine production. Conversely, Vα7.2+/CD161− T cells exhibited distinct responses, showing negligible responses to 5-OP-RU ligand but increased cytokine production upon PMA stimulation. Our study underscores the distinct nature of MAIT cells compared to Vα7.2+/CD161− T cells, which resemble conventional T cells. CsA emerges as a potent immunosuppressive agent, inhibiting proinflammatory cytokine production in MAIT cells. At the same time, VitD supports MAIT cell activation and IL-13 production, shedding light on potential therapeutic avenues for immune modulation.

Harnessing Metformin’s Immunomodulatory Effects on Immune Cells to Combat Breast Cancer

Metformin, a medication known for its anti-glycemic properties, also demonstrates potent immune system activation. In our study, using a 4T1 breast cancer model in BALB/C WT mice, we examined metformin’s impact on the functional phenotype of multiple immune cells, with a specific emphasis on natural killer T (NKT) cells due to their understudied role in this context. Metformin administration delayed the appearance and growth of carcinoma. Furthermore, metformin increased the percentage of IFN-γ+ NKT cells, and enhanced CD107a expression, as measured by MFI, while decreasing PD-1+, FoxP3+, and IL-10+ NKT cells in spleens of metformin-treated mice. In primary tumors, metformin increased the percentage of NKp46+ NKT cells and increased FasL expression, while lowering the percentages of FoxP3+, PD-1+, and IL-10-producing NKT cells and KLRG1 expression. Activation markers increased, and immunosuppressive markers declined in T cells from both the spleen and tumors. Furthermore, metformin decreased IL-10+ and FoxP3+ Tregs, along with Gr-1+ myeloid-derived suppressor cells (MDSCs) in spleens, and in tumor tissue, it decreased IL-10+ and FoxP3+ Tregs, Gr-1+, NF-κB+, and iNOS+ MDSCs, and iNOS+ dendritic cells (DCs), while increasing the DCs quantity. Additionally, increased expression levels of MIP1a, STAT4, and NFAT in splenocytes were found. These comprehensive findings illustrate metformin’s broad immunomodulatory impact across a variety of immune cells, including stimulating NKT cells and T cells, while inhibiting Tregs and MDSCs. This dynamic modulation may potentiate its use in cancer immunotherapy, highlighting its potential to modulate the tumor microenvironment across a spectrum of immune cell types.

Multistrain probiotics fail to modulate the asthmatic phenotype, respiratory microbiota, and immune responses in cats.

To determine if multistrain probiotics administered to asthmatic cats treated with anti-inflammatory glucocorticoids would attenuate the asthmatic phenotype and beneficially alter respiratory, blood, and oropharyngeal (OP) microbial communities and immune parameters versus placebo. 13 client-owned asthmatic cats. A randomized, blinded, placebo-controlled clinical trial of asthmatic cats receiving anti-inflammatory glucocorticoids with oral multistrain probiotics or placebo assessed owner-perceived improvement and airway eosinophilia at baseline and after 2 weeks of treatment. Bronchoalveolar lavage fluid (BALF), blood, OP, and rectal microbial communities were compared using 16S rRNA amplicon sequencing. Real-time PCR for transcription factors, activation markers and cytokines, and IgA ELISAs were evaluated. Statistical analyses used 2-way repeated-measures ANOVA or permutational ANOVA (significance, P < .05). After treatment, there were no significant differences in owner-perceived clinical signs or mean ± SEM BALF eosinophils between groups. There was a significant decrease in rectal α-diversity but not in α- or β-diversity in BALF, blood, or OP between groups or over time. There were no significant differences in CD25, FoxP3, GATA, Helios, IL-4, IL-5, IL-10, IL-13, IL-17, IFN-γ mRNA, or serum or BALF IgA between groups or over time. In asthmatic cats, oral multistrain probiotics failed to improve owner-perceived signs, reduce airway eosinophilia, modify microbial community composition, or alter assessed immune responses versus placebo or over time. Longer treatment, different probiotic composition or delivery (eg, aerosolized), or larger number of cats would represent the next stages of study.

Melatonin counteracts polyethylene microplastics induced adreno-cortical damage in male albino rats

There are various substances that can disrupt the homeostatic mechanisms of the body, defined as endocrine-disrupting chemicals (EDCs). The persistent nature of microplastics (MPs) is a cause for concern due to their ability to accumulate in food chains and widespread use, making their toxic effects particularly alarming. The potential of MPs for disrupting the endocrine system was observed in multiple tissues. Moreover, the adrenal gland is known to be extremely sensitive to EDCs, while with the effect of MPs on the adrenal gland has not previously been studied. This study aimed to highlight the potential polyethylene microplastics (PE-MPs) induced adreno-toxic effects rather than exploring the implicated mechanisms and concluding if melatonin (Mel) can afford protection against PE-MPs induced adreno-toxicity. To fulfill the goal, six groups of rats were used; control, Mel, PE-MPs (3.75 mg/kg), PE-MPs (15 mg/kg), PE-MPs (3.75 mg/kg) +Mel, and PE-MPs (15 mg/kg) +Mel. PE-MPs induced toxic changes in the adrenal cortex, which was evident by increased adrenal weight, histopathological examination, and ultrastructural changes detected by electron microscope. A reduction in serum cortisol and an increase in serum adrenocorticotropic hormone resulted from the adreno-toxic effects of PE-MPs. Mechanisms may include the reduction of steroidogenesis-related genes, as PE-MPs drastically reduce mRNA levels of StAR, Nr0b1, Cyp11A1, as well as Cyp11B1. Also, oxidative stress that results from PE-MPs is associated with higher rates of lipid peroxidation and decreased superoxide dismutase and glutathione. PE-MPs inflammatory effect was illustrated by elevated expression of IL-1β and NF-ķB, detected by immunohistochemical staining, in addition to increased expression of caspase-3 and mRNA of Bax, markers of proapoptotic activity. The impacts of PE-MPs were relatively dose-related, with the higher dose showing more significant toxicity than the lower one. Mel treatment was associated with a substantial amelioration of PE-MPs-induced toxic changes. Collectively, this study fills the knowledge gap about the MPs-induced adrenal cortex and elucidates various related toxic mechanisms. It also supports Mel’s potential protective activity through antioxidant, anti-inflammatory, anti-apoptotic, and gene transcription regulatory effects.

Differential Gene Expression Involved in Bone Turnover of Mice Expressing Constitutively Active TGFβ Receptor Type I

Transforming growth factor beta (TGF-β) is ubiquitously found in bone and plays a key role in bone turnover. Mice expressing constitutively active TGF-β receptor type I (Mx1;TβRICA mice) are osteopenic. Here, we identified the candidate genes involved in bone turnover in Mx1;TβRICA mice using RNA sequencing analysis. A total of 285 genes, including 87 upregulated and 198 downregulated genes, were differentially expressed. According to the KEGG analysis, some genes were involved in osteoclast differentiation (Fcgr4, Lilrb4a), B cell receptor signaling (Cd72, Lilrb4a), and neutrophil extracellular trap formation (Hdac7, Padi4). Lilrb4 is related to osteoclast inhibition protein, whereas Hdac7 is a Runx2 corepressor that regulates osteoblast differentiation. Silencing Lilrb4 increased the number of osteoclasts and osteoclast marker genes. The knocking down of Hdac7 increased alkaline phosphatase activity, mineralization, and osteoblast marker genes. Therefore, our present study may provide an innovative idea for potential therapeutic targets and pathways in TβRI-associated bone loss.

Investigating protease-mediated peptides of inflammation and tissue remodeling as biomarkers associated with flares in psoriatic arthritis.

Psoriatic arthritis (PsA) is an inflammatory arthritis associated with psoriasis. PsA disease involves flares, which are associated with increased joint inflammation and tissue remodeling. There is a need for identifying biomarkers related to PsA disease activity and flares to improve the management of PsA patients and decrease flares. The tissue turnover imbalance that occurs during the inflammatory and fibro-proliferative processes during flares leads to an increased degradation and/or reorganization of the extracellular matrix (ECM), where increased proteolysis plays a key role. Hence, protease-mediated fragments of inflammatory and tissue-remodeling components could be used as markers reflecting flares in PsA patients. A broad panel of protease-mediated biomarkers reflecting inflammation and tissue remodeling was measured in serum and synovial fluid (SF) obtained from PsA patients experiencing flares (acutely swollen joint[s], PsA-flare). In serum, biomarker levels assessed in PsA-flare patients were compared to controls and in early-diagnosed PsA patients not experiencing flares (referred to as PsA without flare). Furthermore, the biomarker levels assessed in SF from PsA-flare patients were compared to the levels in SF of osteoarthritis (OA) patients. In serum, levels of the PRO-C3 and C3M, reflecting formation and degradation of the interstitial matrix, were found significantly elevated in PsA-flare compared to controls and PsA without flare. The remodeling marker of the basement membrane, PRO-C4, was significantly elevated in PsA-flare compared to PsA without flare. The inflammation and immune cell activity related markers, CRPM, VICM, and CPa9-HNE were significantly elevated in PsA-flare patients compared to controls and PsA without flare. In addition, VICM (AUC = 0.71), CPa9-HNE (AUC = 0.89), CRPM (AUC = 0.76), and PRO-C3 (AUC = 0.86) showed good discriminatory performance for separating PsA-flare from PsA without flare. In SF, the macrophage activity marker, VICM, was significantly elevated whereas the type II collagen formation marker, PRO-C2, was significantly reduced in the PsA-flare compared to OA. The combination of five serum markers reflecting type III and IV collagen degradation (C3M and C4M, respectively), type III and VI collagen formation (PRO-C3 and PRO-C6, respectively), and neutrophil activity (CPa9-HNE) showed an excellent discriminatory performance (AUC = 0.98) for separating PsA-flare from PsA without flares. The serum biomarker panel of C3M, C4M, PRO-C3, PRO-C6, and CPa9-HNE reflecting synovitis, enthesitis, and neutrophil activity may serve as novel tool for quantitatively monitoring flares in PsA patients.

Autoantibodies Targeting Nephrin in Podocytopathies.

Minimal change disease and primary focal segmental glomerulosclerosis in adults, along with idiopathic nephrotic syndrome in children, are immune-mediated podocytopathies that lead to nephrotic syndrome. Autoantibodies targeting nephrin have been found in patients with minimal change disease, but their clinical and pathophysiological roles are unclear. We conducted a multicenter study to analyze antinephrin autoantibodies in adults with glomerular diseases, including minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, antineutrophil cytoplasmic antibody-associated glomerulonephritis, and lupus nephritis, as well as in children with idiopathic nephrotic syndrome and in controls. We also created an experimental mouse model through active immunization with recombinant murine nephrin. The study included 539 patients (357 adults and 182 children) and 117 controls. Among the adults, antinephrin autoantibodies were found in 46 of the 105 patients (44%) with minimal change disease, 7 of 74 (9%) with primary focal segmental glomerulosclerosis, and only in rare cases among the patients with other conditions. Of the 182 children with idiopathic nephrotic syndrome, 94 (52%) had detectable antinephrin autoantibodies. In the subgroup of patients with active minimal change disease or idiopathic nephrotic syndrome who were not receiving immunosuppressive treatment, the prevalence of antinephrin autoantibodies was as high as 69% and 90%, respectively. At study inclusion and during follow-up, antinephrin autoantibody levels were correlated with disease activity. Experimental immunization induced a nephrotic syndrome, a minimal change disease-like phenotype, IgG localization to the podocyte slit diaphragm, nephrin phosphorylation, and severe cytoskeletal changes in mice. In this study, circulating antinephrin autoantibodies were common in patients with minimal change disease or idiopathic nephrotic syndrome and appeared to be markers of disease activity. Their binding at the slit diaphragm induced podocyte dysfunction and nephrotic syndrome, which highlights their pathophysiological significance. (Funded by Deutsche Forschungsgemeinschaft and others.).

A comparative study of blood cell count in four automated hematology analyzers: An evaluation of the impact of preanalytical factors.

Differential white blood cell counts are frequently used in diagnosis, patient stratification, and treatment selection to optimize therapy responses. Referral laboratories are often used but challenged with use of different hematology platforms, variable blood shipping times and storage conditions, and the different sensitivities of specific cell types. To extend the scientific literature and knowledge on the temporal commutability of blood samples between hematology analyzers, we performed a comparative ex-vivo study using four of the most utilized commercial platforms, focusing on the assessment of eosinophils given its importance in asthma management. Whole blood from healthy volunteers with and without atopy (n = 6+6) and participants with eosinophilic asthma (n = 6) were stored under different conditions (at 4, 20, 30, and 37°C, with or without agitation) and analyzed at different time points (3, 6, 24, 48 and 72h post-sampling) in parallel on the Abbott CELL-DYN Sapphire, Beckman Coulter DxH900, Siemens ADVIA 2120i and Sysmex XN-1000V. In the same blood samples, eosinophil-derived neurotoxin (EDN), eosinophil activation and death markers were analyzed. All platforms gave comparable measurements of cell differentials on fresh blood within the same day of sampling. However, by 24 hours, significant temporal and temperature-dependent differences were observed, most markedly for eosinophils. None of the platforms performed perfectly across all temperatures tested during the 72 hours, showing that handling conditions should be optimized depending on the cell type of interest and the hematology analyzer. Neither disease status (healthy vs. asthma) nor agitation of the sample affected the cell quantification result or EDN release. The eosinophil activation markers measured by flow cytometry increased with time, were influenced by temperature, and were higher in those with asthma versus healthy participants. In conclusion, hematology analyzer, time window from sampling until analysis, and temperature conditions must be considered when analyzing blood cell differentials, particularly for eosinophils, via central labs to obtain counts comparable to the values obtained in freshly sampled blood.

Invasive electrochemical impedance spectroscopy with phase delay for experimental atherosclerosis phenotyping.

Distinguishing quiescent from rupture-prone atherosclerotic lesions has significant translational and clinical implications. Electrochemical impedance spectroscopy (EIS) characterizes biological tissues by assessing impedance and phase delay responses to alternating current at multiple frequencies. We evaluated invasive 6-point stretchable EIS sensors over a spectrum of experimental atherosclerosis and compared results with intravascular ultrasound (IVUS), molecular positron emission tomography (PET) imaging, and histology. Male New Zealand White rabbits (n = 16) were placed on a high-fat diet, with or without endothelial denudation via balloon injury of the infrarenal abdominal aorta. Rabbits underwent invivo micro-PET imaging of the abdominal aorta with 68Ga-DOTATATE, 18F-NaF, and 18F-FDG, followed by invasive interrogation via IVUS and EIS. Background signal-corrected values of impedance and phase delay were determined. Abdominal aortic samples were collected for histology. Analyses were performed blindly. EIS impedance was associated with markers of plaque activity including macrophage infiltration (r = .813, p = .008) and macrophage/smooth muscle cell (SMC) ratio (r = .813, p = .026). Moreover, EIS phase delay correlated with anatomic markers of plaque burden, namely intima/media ratio (r = .883, p = .004) and %stenosis (r = .901, p = .002), similar to IVUS. 68Ga-DOTATATE correlated with intimal macrophage infiltration (r = .861, p = .003) and macrophage/SMC ratio (r = .831, p = .021), 18F-NaF with SMC infiltration (r = -.842, p = .018), and 18F-FDG correlated with macrophage/SMC ratio (r = .787, p = .036). EIS with phase delay integrates key atherosclerosis features that otherwise require multiple complementary invasive and non-invasive imaging approaches to capture. These findings indicate the potential of invasive EIS to comprehensively evaluate human coronary artery disease.

Beta-Hydroxybutyrate Mitigates Sensorimotor and Cognitive Impairments in a Photothrombosis-Induced Ischemic Stroke in Mice

The consequences of stroke include cognitive deficits and sensorimotor disturbances, which are largely related to mitochondrial impairments in the brain. In this work, we have shown that the mimetic of the ketogenic diet beta-hydroxybutyrate (βHB) can improve neurological brain function in stroke. At 3 weeks after photothrombotic stroke, mice receiving βHB with drinking water before and after surgery recovered faster in terms of sensorimotor functions assessed by the string test and static rods and cognitive functions assessed by the Morris water maze. At the same time, the βHB-treated mice had lower expression of some markers of astrocyte activation and inflammation (Gfap, Il-1b, Tnf). We hypothesize that long-term administration of βHB promotes the activation of the nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway, which leads to increased expression of antioxidant genes targeting mitochondria and genes involved in signaling pathways necessary for the maintenance of synaptic plasticity. βHB partially maintained mitochondrial DNA (mtDNA) integrity during the first days after photothrombosis. However, in the following three weeks, the number of mtDNA damages increased in all experimental groups, which coincided with a decrease in Ogg1 expression, which plays an important role in mtDNA repair. Thus, we can assume that βHB is not only an important metabolite that provides additional energy to brain tissue during recovery from stroke under conditions of mitochondrial damage but also an important signaling molecule that supports neuronal plasticity and reduces neuroinflammation.

KLF4 Inhibits the Activation of Human Hepatic Stellate Cell In Vitro.

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis. Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs. Kruppel-like factor 4 (KLF4) plays a pivotal role in a wide array of physiological and pathological processes. This study aimed to investigate the effect of KLF4 on the proliferation, apoptosis and phenotype of quiescent HSCs METHODS: We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector, to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection. Cell proliferation was assessed using the CCK-8 assay. Flow cytometry was used to detect the cell cycle distribution and apoptosis rate. Western blotting was used to determine the levels of some quiescence and activation markers of HSCs RESULTS: Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1, which are quiescent HSC markers, while significantly decreased the levels of N-cadherin and a-SMA, known activated HSC markers. In contrast, cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced CONCLUSION: KLF4 inhibits the proliferation and activation of human LX-2 HSCs. It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.

Celastrus orbiculatus Extract Inhibits Immune Inflammatory Thrombotic State of B-Lymphoma.

To investigate the inhibitory effect of Celastrus orbiculatus extracts (COE) on the proliferation of lymphoma cells and the immune regulation ability on inflammation and thrombophilia in vivo. The 38B9 lymphoma cells were treated with COE (160 µ g/mL) and CTX (25 µ mol/L). The apoptosis rate and cell cycle of each group were detected by flow cytometry. The secretion of inflammatory factors, including interleukin (IL)-6, IL-10, and tumor necrosis factor α (TNF-α), in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). In vivo, BALB/c mice were subcutaneously injected with 38B9 lymphoma cells to establish lymphoma model. COE (3 mg·kg-1·d-1) and CTX (40 mg·kg-1·d-1) were administered to the model mice, respectively. The expression of plasma inflammatory factors (IL-6, IL-10 and TNF-α) and thrombus indexes, including D-dimer (D-D), von Willebrand factor (vWF) and tissue factor (TF), were detected by ELISA before tumor bearing (1 d), after tumor formation (14 d) and after intervention (21 d). PicoGreen dsDNA was used to detect the level of serum neutrophil extracellular traps (NETs). Flow cytometry was used to detect the expression of platelet activation marker calcium-dependent lectin-like receptor 2 (CLEC-2). The tumor growth and survival of mice were recorded. The 38B9 lymphoma cells were apoptotic after the intervention of COE and CTX. The ratio of G2-M phase cells decreased in COE intervented cells compared with the control cells (P<0.05), and S phase cells decreased in CTX intervented cells (P<0.05). Also, the secretion level of IL-6 was significantly reduced after COE or CTX intervention (P<0.05), and IL-10 was significantly increased (P<0.05). Furthermore, the tumor mass was reduced, and the median survival time was longer in COE and CTX intervented tumor-bearing mice than in non-intervented mice. The significantly lower levels of TNF-α, IL-6, NETs, TF, DD and CLEC-2, as well as higher IL-10 were observed in COE and CTX treatment mice in comparision with the control mice (P<0.05). COE has a mild and stable anti-tumor effect, which can reduce the secretion of inflammatory factors by lymphoma cells and regulate thrombophilic state caused by tumor inflammatory microenvironment.

MEASUREMENT OF PLATELET ACTIVATION MARKERS CD62P AND CD63 TO DETERMINE THE EFFICIENCY OF DUAL THERAPY WITH ASPIRIN AND CLOPIDOGREL: A PRELIMINARY STUDY

Background: A dual antiplatelet therapy with aspirin and clopidogrel is advised to prevent stent thrombosis. However, the most appropriate assay for evaluating the antiplatelet effects remains undefined. Objective: This study aimed to assess the efficacy of measuring platelet surface activation markers, CD62P and CD63, using flow cytometry to determine the effectiveness of dual antiplatelet therapy. Methods: Thirty patients who received aspirin plus clopidogrel before cerebral artery stent implantation and thirty unrelated healthy controls were enrolled. The expression of CD62P and CD63 was measured using flow cytometry. The diagnostic performances were evaluated and compared with the conventional light-transmitted aggregometry (LTA) using 5.0 and 20.0 µM of adenosine di-phosphate (ADP). Results: The expression ratios of both markers were significantly lower in the patients receiving dual antiplatelet therapy than controls (p &lt; 0.001). The area under the receiver operating characteristic (ROC) curve (AUC) of CD62P using flow cytometry and the LTA using 5.0 and 20.0 µM of ADP were comparable (0.97 vs. 0.95 and 0.97 vs. 0.96, respectively), whereas those of CD63 was lower than the LTA (0.88 vs. 0.96). The sensitivity of CD62P and CD63 were 71.4% and 60.0%, and the specificity of CD62P and CD63 were 100.0% and 96.7%, respectively. Conclusion: Flow cytometry measurements of CD62P could be utilized to identify the efficacy of dual antiplatelet therapy. Additional studies are suggested to support this issue and its effects on clinical outcomes.

#2396 The potential of human-derived macrophages to study autosomal dominant polycystic kidney disease (ADPKD)

Abstract Background and Aims Macrophages are the most prevalent immune cell type in the renal parenchyma in ADPKD. Depletion of macrophages in animal models has shown a dramatic attenuation in cystic burden, suggesting a disease promoting effect of this cell type. So far, studies investigating interaction between epithelial cells and immune cells in the field of ADPKD were performed with mice-derived myeloid cells. We present a new model using human-derived immune cells and tubular epithelial cells for the study of ADPKD. Method We established a technique to isolate CD14+ monocytes from the peripheral blood of young ADPKD patients and healthy controls. Methodology was optimized to create monocyte-derived macrophagesin vitro. Mature macrophages were checked for surface marker expression (CD14, CD11b, CD206, CD163, HLA-DR, D80, and CD86) &amp; cytokine-release after stimulation with LPS, IL-4 and LPS + Nigericin. Co-cultures were performed using human-derived monocytes in combination with double immortalized cystic cells (ciCC) or healthy-kidney derived cell lines (HKT). Differentiation and polarization of macrophages was checked using flowcytometry. Results We observed low expression of PKD1 and PKD2 transcripts in primary, human-derived monocytes of ADPKD patients and controls. The expression of PKD1 and PKD2 increased after differentiation into macrophages. There were no clear differences between ADPKD and control-derived macrophages in the differentiation capacity, cell survival nor surface marker expression before and after differentiation. After stimulation with LPS &amp; LPS + Nigericin ADPKD-derived macrophages showed significantly lower expression of pro-inflammatory cytokines (MIP-1α, IP-10, IL-23 and TNFα) as compared to controls. There was no difference in anti-inflammatory cytokine release. Healthy primary monocytes cultured in direct contact with conditionally immortalized cystic cells (ciCC) had a very good survival and showed differentiation into macrophages with high expression of M2-markers (CD163 and CD206) and the activation marker (HLA-DR). A similar differentiation pattern was seen when monocytes were cultured in the (cell-free) conditioned medium of the ciCC harvested after 72 h, pointing to a soluble factor produced by the cystic cells that induces monocyte differentiation and macrophage polarization. In accordance, unbiased multiplex cytokine analysis (Target 48-panel) of supernatants of ciCC, showed higher levels of macrophage colony-stimulating factor (M-CSF/CSF-1), TNF-α, hepatocyte growth factor (HGF) and MMP-1 as compared to those of HKT. Primary monocytes cultured with the conditioned medium harvested after 72 h from ciCCs (n=4) showed better overall survival than those cultured with medium from and HKT (n=4). In addition, a tendency towards higher expression of the classical M2-markers (CD163, CD206) and the activation marker (HLA-DR) in ciCCs versus HKT was seen. Conclusion There is wealth of data supporting the indispensable role of myeloid cells in disease progression of ADPKD. We established a unique in vitro fully human co-culture model for ADPKD. Cystic cells produce pro-proliferative and pro-angiogenic factors, thus actively shaping their microenvironment and creating the ideal circumstances for infiltrating monocytes/macrophages to proliferate and to destruct the kidney. This mechanism is very similar to what is seen in the cancer microenvironment. Further experiments will help to elucidate the complex interplay between different cell-types in the microenvironment in ADPKD kidneys.

#1467 Pegcetacoplan for post-transplant recurrent C3 glomerulopathy or immune complex membranoproliferative glomerulonephritis in NOBLE: 12-week evolution

Abstract Background and Aims C3 glomerulopathy (C3G) and primary immune complex membranoproliferative glomerulonephritis (IC-MPGN) are rare diseases caused by uncontrolled complement activation, resulting in excess deposition of C3 breakdown products in the kidneys, which leads to kidney damage and eventually kidney failure. Kidney transplantation is an option for end-stage kidney disease, but disease recurrence is common (up to 67% of patients), and up to 50% of transplant recipients lose renal allografts due to recurrence. Pegcetacoplan is a targeted C3 and C3b inhibitor that may prevent excess deposition of C3 and C5 breakdown products and subsequent glomerular inflammation and kidney damage in patients with C3G or IC-MPGN, potentially improving clinical outcomes. NOBLE (NCT04572854) was the first prospective randomized controlled trial of pegcetacoplan vs standard of care (SOC) in post-transplant patients with recurrent C3G or IC-MPGN. Method Adult patients were randomized 3:1 to subcutaneous pegcetacoplan 1080 mg twice weekly plus SOC (n = 10) or SOC only (n = 3) for 12 weeks followed by a 40-week non-controlled period in which all patients received pegcetacoplan. Primary and secondary endpoint results for Week-12 data have been reported. We conducted a patient-level post hoc analysis to describe key histology changes from renal biopsy (C3c staining, C3G histologic index activity score), clinical parameters (urine protein-to-creatinine ratio [uPCR; calculated from triplicate first-morning spot urine], estimated glomerular filtration rate [eGFR]), and biomarkers (serum C3, plasma sC5b-9) through Week 12. The C3G histologic index uses a semiquantitative scale of 0–3 for 7 markers of activity (mesangial hypercellularity, endocapillary proliferation, membranoproliferative morphology, leukocyte infiltration, cellular and/or microcellular crescent formation, fibrinoid necrosis, and interstitial inflammation), for a total activity score of 0–21. eGFR stability was defined as a decrease of no more than 25% versus baseline. Results Among the 10 pegcetacoplan-treated patients, 7 (70%) had 1 previous kidney transplant, 2 (20%) had 2, and 1 (10%) had 3. The mean (range) time to disease recurrence after most recent transplant was 10.8 (0.4–31.8) months and from most recent recurrence to randomization was 12.2 (2.1–37.7) months, with 60% randomized &amp;gt;6 months post-disease recurrence. Among the 3 SOC-only patients, 2 (66.7%) had 1 transplant and 1 (33.3%) had 2. Mean (range) time to recurrence was 32.8 (4.6–63.8) months and to randomization was 33.9 (7.0–65.4) months. At Week 12, 8 (80%) pegcetacoplan-treated patients had a reduction in C3c staining of at least 1 order of magnitude. Of these 8, 7 (70%) had a decrease in activity score, and 6 of these 7 (60%) had a decrease in proteinuria. Four (40%) patients had reduced C3c staining, decreased activity score, and cleared electron microscopy deposits at Week 12. eGFR remained stable in 9 (90%) patients. Six of 9 (67%) patients with available Week-12 data (4 of 5 patients with &amp;gt;1 g/g uPCR at baseline) had a reduction in uPCR with 5 of the 6 achieving ≥50% uPCR reduction (Table). All pegcetacoplan-treated patients had increased serum C3, as expected, and 9 (90%) had decreased sC5b-9. At Week 12, no TEAEs led to study discontinuation, treatment withdrawal, or death. No encapsulated bacterial infections or events of rejection/graft loss related to the study drug were reported. Conclusion As early as Week 12, more than half (6/10 [60%]) of post-transplant patients with recurrent C3G or IC-MPGN treated with pegcetacoplan achieved reductions in C3c staining, activity score of C3G histology index, and proteinuria, suggesting that pegcetacoplan targets the underlying pathophysiology of the C3G/IC-MPGN in kidney transplant recipients. The safety and efficacy of pegcetacoplan will be further evaluated at Week 52 of the NOBLE study and as part of the Phase 3 VALIANT (NCT05067127) trial, which enrolled patients with C3G or primary IC-MPGN who have native kidney or post-transplant disease.