Serum Of Patients Research Articles

Quantitative evaluation of adalimumab and anti-adalimumab antibodies in sera using a surface plasmon resonance biosensor.

Monitoring of therapeutic antibody adalimumab (ADL) and of anti-adalimumab antibodies (AAA) in autoimmune diseases' patient sera has achieved increased attention since several studies showed a correlation between AAA levels and treatment failure. We evaluated a new surface plasmon resonance (SPR)-based method that, with slight changes in the analysis condition and in the ligand immobilized on the chip surface, allows to monitor both AAA and ADL. This new label-free method does not require sample pretreatments, and it is fully automated, only requiring the preparation of the chip, which can be used for multiple analysis, and the preparation of the sample sets. Sera from ADL-treated patients (n = 47) and controls (n = 13) were included in this study. Quantitative analysis of AAA and ADL were performed separately using a new SPR-biosensor, and a commercially available ELISA kit. Agreement was defined by overall, positive and negative agreement. Wilson Score was used to calculate confidence intervals (CI) on binomial probability and Spearman's rho and Bland-Altman test were used to assess correlations. ; ELISA and SPR-based assay were able to identify circulating AAA in ADL-treated patients, with the percentage of positivity varying among the methods, with an overall agreement of 79%. AAA were detected in 18 (38%) out of the 47 treated patients by the ELISA whereas SPR-based assay detected 10 (21%) out of 47 samples. Real-time label free SPR-based protocol for both AAA and ADL quantification has been set-up. Although quantitative differences were observed when compared with ELISA, the agreement among methodologies was high, particularly for ADL quantification within the therapeutic window of the drug.

The CD244 rs3766379 variant showed no association with susceptibility to rheumatoid arthritis while soluble CD244 was associated with disease activity

Cluster of differentiation 244 (CD244), a member of the signaling lymphocytic activation molecule (SLAM) family, is one of the major cell surface receptors of the SLAM family with activating and inhibitory signaling potentials that is involved in the functional regulation of inflammatory reactions. It has recently been suggested that soluble CD244 levels are dysregulated in patients with rheumatoid arthritis (RA), but the evidence is not conclusive. Furthermore, the CD244 rs3766379 variant showed conflicting results regarding association with disease susceptibility. In this cross-sectional case-control study, serum CD244 concentration was determined in 123 RA patients and 60 controls using an enzyme-linked immunosorbent assay kit. The CD244 rs3766379 variant was also genotyped using real-time polymerase chain reaction principles. Results revealed that Log10-transformed serum concentrations (mean ± standard error) of soluble CD244 were significantly elevated in RA patients compared to controls (0.53 ± 0.04 vs. 0.29 ± 0.02 ng/mL; probability = 0.003), especially in patients with high disease activity (0.73 ± 0.12 vs. 0.29 ± 0.02 ng/mL; probability = 0.003), where CD244 showed reliable discrimination between patients and controls (area under the curve = 0.698; probability = 0.023). The rs3766379 allele and genotype frequencies showed no significant differences between patients and controls. Furthermore, this variant did not affect CD244 concentration. In conclusion, CD244 levels were up-regulated in the serum of patients with RA, particularly those with high disease activity. The CD244 rs3766379 variant was not associated with susceptibility to RA and had no effect on serum CD244 concentration.

Does Eta Protein Differentiate Rheumatoid Arthritis from Psoriatic Arthritis?

The clinical symptoms and laboratory markers of Rheumatoid Arthritis (RA) and Psoriatic Arthritis (PsA) can be very similar, so making a differential diagnosis between these two diseases is often difficult. Serological parameters to be used in differential diagnosis can guide the clinician. This study aimed to investigate the usability of 14-3-3η (eta) protein as a biomarker in the differential diagnosis of PsA and RA, and the relationships between eta protein and disease activity scores and joint erosions in PsA and RA. 54 PsA patients, 53 RA patients, and 56 healthy individuals were included in this study. The ELISA (Enzyme-Linked ImunoSorbent Assay) kit was used as a quantitative sandwich enzyme immunoassay technique to detect human eta protein levels. Receiver- operating Characteristic (ROC) curves analysis was used to determine the sensitivity and specificity of the eta protein. Eta protein levels were found to be significantly higher in the RA group than in the PsA [B: -0.341, OR (95% CI): 0.711 (0.556-0.909), p: 0.007] and control [B: -0.225, OR (95% CI): 0.798 (0.641-0.995), p: 0.045] groups. Eta protein median values were significantly higher in patients with joint erosion than in those without [β= 0.151, OR (95% CI): 1.163 (1.003-1.349), p: 0.046]. Eta protein levels are higher in the serum of RA patients than PsA and are associated with joint erosion. Eta protein may be a potential biomarker in the differential diagnosis of RA and PsA. It may represent a possible therapeutic step in the pathophysiological pathways in the development of joint erosion.

Diagnostic Value of lncRNA FGD5-AS1 Sponge miR-223-3p in Infantile Pneumonia and Its Prognostic Effect on Rehabilitation

Abstract Objective This study aimed to uncover the value of long noncoding RNA FGD5-AS1 (lncRNA FGD5-AS1) in the diagnosis of infantile pneumonia and explore its pathological mechanism in lung fibroblasts. This research may provide a potential biomarker for diagnosing patients and predicting their rehabilitation outcomes. Methods This study included 92 children with infantile pneumonia as the research object, and an equal number of healthy children were introduced as controls. The FGD5-AS1 content was detected using real-time quantitative polymerase chain reaction (RT-qPCR) assay. The diagnostic value of FGD5-AS1 was evaluated by receiver operating characteristic (ROC) and logistic analysis. The molecular mechanism of FGD5-AS1 and miR-223-3p was studied by luciferase activity assays. The impact of abnormal FGD5-AS1 expression on the proliferation and apoptosis of lung fibroblasts was analyzed using the cell counting kit-8 (CCK-8) and flow cytometry. Results FGD5-AS1 expression was decreased in the serum of infantile pneumonia patients, which may be a diagnostic marker for children with pneumonia. Furthermore, FGD5-AS1 has the ability to predict patient outcomes. FGD5-AS1 levels in lung fibroblasts (WI-38) decreased when induced by lipopolysaccharide (LPS). This decline resulted in reduced cell proliferation ability, increased apoptosis rate, and elevated inflammatory factor content. However, upregulated FGD5-AS1 counteracted the effects of LPS on WI-38 cells activity and inflammatory factors. Conclusion FGD5-AS1 may act as a potential marker in infantile pneumonia, and regulate the biological activity and inflammation level of lung fibroblasts by targeting miR-223-3p.

MicroRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway.

The pathogenesis of pulmonary arterial hypertension (PAH) is closely linked to the abnormal proliferation of pulmonary artery smooth muscle cells. Studies have demonstrated that microRNAs play pivotal roles in the progression of pulmonary hypertension. We found that microRNA-637 (miR-637) and microRNA-661 (miR-661) are expressed at low levels in the serum of PAH patients. Moreover, the overexpression of miR-637 or miR-661 inhibited human pulmonary artery smooth muscle cell (HPASMC) proliferation and migration in hypoxic culture. Mechanistically, we overexpressed these two microRNAs in HPASMCs, and the RNA-sequencing (RNA-seq) results demonstrated that TRIM29 mRNA was suppressed, indicating that TRIM29 is a substrate. TRIM29 accumulates in the serum of patients with PAH and promotes cell proliferation and migration by activating AKT/mTOR signalling. In addition, overexpression of miR-637 or miR-661 reversed TRIM29-mediated HPASMC proliferation and migration. This study revealed that miR-637 and miR-661 are able to inhibit the proliferation ability of HPASMCs under hypoxic conditions through targeting TRIM29, suggesting that the microRNA-637/661/TRIM29 axis may act as a target for PAH treatment.

Endodontic treatment modifies circulatory inflammatory mediator levels: A systematic review with meta-analysis.

There is limited and conflicting data on the reduction of circulatory inflammatory mediators in patients with apical periodontitis (AP) following endodontic treatment. To answer the following research question: in adult healthy patients with AP [Population (P)], is there a difference before [Comparator (C)] and after various endodontic treatments (nonsurgical, surgical or retreatment) [Intervention (I)] on systemic levels of inflammatory biomarkers [Outcome (O)] in the follow-up period[Time (T)]? An electronic literature search was conducted in the databases Scopus, PubMed, Clarivate Analytics' Web of Science, Cochrane Database of Systematic Reviews and Grey literature from inception to July 2024 with no language restrictions. Observational studies examining changes in serum levels of inflammatory mediators were included. Two independent reviewers selected studies, extracted data and critically appraised the included studies. Qualitative and quantitative (meta-analysis) data synthesis methods were employed. The Newcastle-Ottawa Scale was used to assess the quality of the included studies. Sixteen studies met the inclusion criteria, of which six were included in the meta-analysis. These studies were published between 1992 and 2024, involving a total of 596 patients (54% females) aged between 16 and 75 years. The meta-analysis of pooled data showed a significant decrease in high-sensitive C-reactive protein (hs-CRP) levels in the serum of patients with AP 6 months after treatment [2.26 ± 1.76 versus 1.28 ± 1.06 mg/L, (Z = 2.03, p = .04)] and a decrease in interleukin-1β (IL-1β) levels 12 months after treatment [13.01 ± 5.95 versus 10.86 ± 3.52 pg/mL, (Z = 3.72, p < .01)]. One study was assessed as poor quality, while all others were considered high quality. Despite the differences in methodologies across the included studies, it has been established that effective endodontic treatment leads to a reduction in systemic inflammatory biomarkers in the body. Following effective endodontic treatment in patients with AP, the systemic levels of hs-CRP and IL-1β exhibit a significant reduction at 6 and 12 months, respectively. Further clinical studies should investigate whether effective endodontic treatment and reduced levels of investigated biomarkers may change the clinical presentation of systemic diseases. PROSPERO database (CRD42024559271).

Exosomal PSM-E inhibits macrophage M2 polarization to suppress prostate cancer metastasis through the RACK1 signaling axis.

It is well-established that understanding the mechanism of prostate cancer (PCa)-associated metastasis is paramount for improving its prognosis. Metastasis is known to involve the communication between tumor-associated macrophages (TAMs) and tumor cells. Exosomes are crucial in mediating this intercellular communication within the tumor microenvironment. Nonetheless, the role of exosomal proteins in PCa metastasis is not yet fully understood. Here, we investigated the mechanisms of prostate cancer-derived exosomal PSM-E on regulating macrophage M2 polarization to suppress tumor invasion and metastasis. PSM-E levels in exosomes were detected by transmission electron microscopy and Western blotting analysis. The diagnostic value of urine-derived exosomal PSM-E in PCa were evaluated by LC-MS/MS, correlation analysis, and ROC curves analysis. The mechanisms underlying the inhibitory effect of exosomal PSM-E on the M2 polarization of macrophages was investigated by co-IP, IHC staining, and PCa tumorigenesis model, etc. RESULTS: We revealed that exosomal PSM-E is upregulated in exosomes derived from the serum and urine of PCa patients. Clinically, an elevated exosomal PSM-E expression in urine is significantly correlated with an advanced pathological tumor stage and a high Gleason score. Our research also revealed that exosomal PSM-E inhibits prostate cancer cell proliferation, invasion, and metastasis by suppressing macrophage polarization in vitro and in vivo. Furthermore, we provided compelling evidence that exosomal PSM-E inhibits M2 polarization of macrophages by recruiting RACK1 and suppressing the FAK and ERK signaling pathways, consequently suppressing PCa invasion and metastasis. Furthermore, we found that the protease-associated domain of PSM-E and the fourth tryptophan-aspartate repeat of RACK1 are crucial for the interaction between PSM-E and RACK1. Notably, exosomes carrying PSM-E from PCa urine could potentially serve as a biomarker for PCa, and targeting exosomal PSM-E may represent a strategy for preventing tumor progression in this patient population.

Complement System and Adhesion Molecule Skirmishes in Fabry Disease: Insights into Pathogenesis and Disease Mechanisms

Fabry disease is a rare X-linked lysosomal storage disorder caused by mutations in the galactosidase alpha (GLA) gene, resulting in the accumulation of globotriaosylceramide (Gb3) and its deacetylated form, globotriaosylsphingosine (Lyso-Gb3) in various tissues and fluids throughout the body. This pathological accumulation triggers a cascade of processes involving immune dysregulation and complement system activation. Elevated levels of complement 3a (C3a), C5a, and their precursor C3 are observed in the plasma, serum, and tissues of patients with Fabry disease, correlating with significant endothelial cell abnormalities and vascular dysfunction. This review elucidates how the complement system, particularly through the activation of C3a and C5a, exacerbates disease pathology. The activation of these pathways leads to the upregulation of adhesion molecules, including vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1), platelet and endothelial cell adhesion molecule 1 (PECAM1), and complement receptor 3 (CR3) on leukocytes and endothelial cells. This upregulation promotes the excessive recruitment of leukocytes, which in turn exacerbates disease pathology. Targeting complement components C3a, C5a, or their respective receptors, C3aR (C3a receptor) and C5aR1 (C5a receptor 1), could potentially reduce inflammation, mitigate tissue damage, and improve clinical outcomes for individuals with Fabry disease.

Targeting PDGF-CC as a promising therapeutic strategy to inhibit cholangiocarcinoma progression.

Cholangiocarcinoma (CCA) is an aggressive malignancy with limited treatment options and poor prognosis. Platelet-Derived Growth Factor CC (PDGF-CC) has been implicated in the progression of various tumors, but its specific role in CCA is not well understood. This study aims to investigate the expression and function of PDGF-CC in CCA and evaluate its potential as a therapeutic target. We conducted gene expression analysis using the GEPIA database to compare PDGF-CC mRNA levels in CCA tissues and normal tissues. Serum samples from CCA patients were analyzed for PDGF-CC protein levels, and immunohistochemistry was used to assess PDGF-CC expression in tissue samples. The impact of PDGF-CC on CCA cell behavior was examined by knocking out PDGF-CC in HuCCT1 and QBC939 cell lines, followed by assessments of cell proliferation, migration, invasion, and colony formation in vitro. Additionally, the effects of PDGF-CC knockout were evaluated in xenograft models. The therapeutic potential of PDGF-CC inhibition was further explored using pharmacological inhibitors and antibodies. PDGF-CC mRNA and protein levels were significantly elevated in CCA tissues and patient sera compared to normal controls. Immunohistochemical analysis confirmed increased PDGF-CC expression in CCA tissues. High PDGF-CC expression correlated with poor overall survival in CCA patients, as shown by Kaplan-Meier analysis. Functional assays revealed that PDGF-CC knockout significantly reduced proliferation, migration, invasion, and colony formation in HuCCT1 and QBC939 cells, the lines with the highest PDGF-CC levels. In vivo, PDGF-CC knockout markedly decreased tumor growth in xenograft models. Pharmacological inhibition of PDGF-CC mirrored the effects of genetic knockout, suggesting it as a viable therapeutic strategy. This study underscores the critical role of PDGF-CC in CCA progression and supports the potential of PDGF-CC inhibitors as a therapeutic approach. Given the association between high PDGF-CC expression and poor prognosis, targeting PDGF-CC may improve outcomes for CCA patients. Further clinical investigations are warranted to develop PDGF-CC-targeted therapies for CCA.

Abstract B047: Monitoring treatment response and toxicity in BRAF V600-mutant metastatic melanoma with circulating cell-free DNA

Abstract Background: The DREAMseq trial (EA6134, NCT02224781) was a national multi-center randomized phase III trial coordinated by ECOG-ACRIN that found that immune checkpoint inhibitor (IO) treatment achieves improved survival outcomes compared to targeted therapy (TT) in patients with BRAF V600-mutant metastatic melanoma. Still, approximately 40% of patients do not respond to IO, and existing biomarkers fail to distinguish this subset of patients who do not benefit from IO therapy. Additionally, as many as 80% of patients may experience immune- related adverse events (irAEs), which range from mild dermatological symptoms to life- threatening myocarditis. Here, we explore the use of the methylation status of cell-free DNA (cfDNA) in serially collected blood samples to measure response and toxicity in the context of IO- and TT-treated metastatic melanoma. Methods: Serial serum samples were collected from patients with BRAF V600-mutant metastatic melanoma treated with ipilimumab/nivolumab (IO) or dabrafenib/trametinib (TT). Circulating cfDNA was isolated from serially collected serum samples, enriched for regions of interest by hybridization capture and sequenced using enzymatic methyl-seq. Cell-type deconvolution was performed to determine the abundance of cell type- specific methylation patterns of cfDNA molecules in patient serum at different time points of treatment. The BRAF V600 mutation abundance in total cfDNA was also assessed. Results: We identified melanocyte lineage-specific DNA methylation regions and demonstrate that the methylation status of these regions remains conserved in malignant melanoma. We characterized the changes in abundance of the melanocyte-lineage cfDNA over the course of treatment and show that these changes distinguish responders from non-responders to either IO or TT. Furthermore, we track the abundance of cell type-specific DNA from normal tissues to identify markers indicative of adverse effects or disease progression. Conclusions: We established melanocyte-lineage methylation markers and evaluated the use of cell-type specific DNA methylation to monitor treatment effects of immune checkpoint or BRAF/MEK inhibitors in metastatic melanoma using serially collected blood samples. Citation Format: Sidharth S Jain, A. Patrick IV McDeed, Megan E McNamara, Amber R Alley, Dori S Rosenstrauch, Harry Sun, Natalie Thompson, John M Kirkwood, Geoffrey T Gibney, Michael B Atkins, Anton Wellstein. Monitoring treatment response and toxicity in BRAF V600-mutant metastatic melanoma with circulating cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B047.

Analysis of allergen positivity rates in relation to gender, age, and cross-reactivity patterns.

This study aims to gain a deeper understanding of the patterns of allergen detection positivity rates among different age and gender groups, and to explore cross-reactivity patterns among allergens. We conducted a retrospective analysis of patients who underwent allergen testing at our hospital. The sample for this study included all patients who underwent allergen testing. We utilized immune blotting to detect specific IgE antibodies to 20 allergens in patient sera. The study results showed a higher proportion of female samples (72.35%) compared to male samples (27.65%). Among all participants, the age distribution was primarily concentrated in the 25-35 age group, accounting for 37.43% of the total sample, followed by the 45 years and older age group, accounting for 27.00%. This indicates that allergic symptoms may occur not only in children and adolescents but also at any time after adulthood. We further observed significant influences of factors such as gender and age on individual sensitivity to specific allergens. For example, compared to males, females were found to be more sensitive to certain allergens such as cat hair, dust mites, and dog epithelium. Similarly, we also found variations in sensitivity to specific allergens among different age groups. Close monitoring of allergen distribution in populations facilitates active engagement of allergic individuals in self-management, while gender and age may be important factors influencing individual sensitivity to specific allergens. These findings provide valuable insights for understanding the pathogenesis of allergic diseases and designing more effective prevention and treatment strategies.

Proteomic Profiling of COVID-19 Patients Sera: Differential Expression with Varying Disease Stage and Potential Biomarkers

Background/Objectives: SARS-CoV-2 is one of the viruses that caused worldwide health issues. This effect is mainly due to the wide range of disease prognoses it can cause. The aim of this study is to determine protein profiles that can be used as potential biomarkers for patients’ stratification, as well as potential targets for drug development. Methods: Eighty peripheral blood samples were collected from heathy as well as SARS-CoV-2 patients admitted at a major tertiary care center in Riyadh, Saudi Arabia. A label-free quantitative mass spectrometry-based proteomic analysis was conducted on the extracted sera. Protein–protein interactions and functional annotations of identified proteins were performed using the STRING. Results: In total, two-hundred-eighty-eight proteins were dysregulated among all four categories. Dysregulated proteins were mainly involved in the network map of SARS-CoV-2, immune responses, complement activation, and lipid transport. Compared to healthy subjects, the most common upregulated protein in all three categories were CRP, LGALS3BP, SAA2, as well as others involved in SARS-CoV-2 pathways such as ZAP70 and IGLL1. Notably, we found fifteen proteins that significantly discriminate between healthy/recovered subjects and moderate/under medication patients, among which are the SERPINA7, HSPD1 and TTC41P proteins. These proteins were also significantly downregulated in under medication versus moderate patients. Conclusions: Our results emphasize the possible association of specific proteins with the SARS-CoV-2 pathogenesis and their potential use as disease biomarkers and drug targets. Our study also gave insights about specific proteins that are likely increased upon infection but are likely restored post recovery.

Abstract 4138633: Infusion of human apolipoprotein A-I (CSL112) promotes several aspects of reverse cholesterol transport

Background: High-density lipoprotein (HDL)-cholesterol is inversely correlated with cardiovascular risk, but increasing its circulating concentration is insufficient to prevent adverse cardiovascular outcomes. Instead, the emerging paradigm is on increasing the function of HDL and its major protein constituent apolipoprotein A-I (apoA-I), to increase reverse cholesterol transport. Objective: To investigate the effect of apoA-I [human] (CSL112) infusion on HDL protein composition, and provide further insights into the mechanism of action of CSL112 administered post-acute myocardial infarction (AMI). Methods: A mass spectrometry (MS)-based proteomic approach was used to evaluate changes in HDL protein composition in patients (n=50) from the AEGIS-I (ApoA-I Event Reducing in Ischemic Syndromes I) study who received either placebo or CSL112 post AMI. HDL was immuno-isolated from patient plasma using anti-apoA-I antibodies. Cholesterol esterification rate (CER) was measured to determine lecithin-cholesterol acyl transferase (LCAT) activity. Cholesterol efflux capacity (CEC) and hepatocyte uptake were assessed using patient serum in ex vivo cell-based assays. Results: CSL112 induced extensive rearrangement of HDL proteins at 4 hours post-infusion. Levels of apolipoproteins A2, B, C, and E as well as the acute phase proteins serum amyloid A1 and A4 were significantly reduced. By contrast, apoA-I, apoM, and LCAT significantly increased. Elevated apoA-I and LCAT levels on HDL were associated with an increase in CEC, plasma HDL-C levels, and CER in CSL112-treated patients. Furthermore, enhanced CEC strongly correlated with cholesterol uptake by hepatic cells (r=0.95 p&lt;0.001). Conclusion: CSL112 altered HDL composition and increased HDL functionality by promoting multiple steps of the reverse cholesterol transport pathway.

Abstract 4119613: Evaluation of Gender, Racial and Ethnic Differences in Time to PCI in the Pre and Post Covid-19 Era

Introduction: Percutaneous Coronary Intervention (PCI) is recommended for reperfusion of patients presenting with ST-segment myocardial infarction (STEMI) within 90 minutes. In this study, we sought to identify differences in PCI timing based on gender, race and ethnicity in the pre- and post-COVID era. Methods: We collected retrospective data on 760 patients admitted with STEMI at our quaternary academic medical center from 2018-2022. We defined our binary outcome as time to PCI less than 90 minutes, and adjusted for transfers from outside hospitals. We utilized univariate logistic regression analysis to analyze the association of demographic, clinical, and cardiac catheterization details on our outcome. We then utilized multivariate logistic regression analysis to determine the association of our covariates of interests with time to PCI. The logistic regression model was adjusted for collinearity which were deemed not significant. Results: Among our study population, COVID did not significantly impact whether or not a patient had a diagnostic cardiac catheterization on univariate analysis (OR 2.68, 95% CI 0.61-18.40, p=0.23). However, the post-COVID era was significantly associated with a delayed time to PCI on multivariate analysis [OR 1.62, 95% CI 1.04-2.55, p=0.035) [Figure 1]. In addition, females were 1.8x more likely to have a delayed PCI than males on multivariate regression [OR 1.80, 95% CI 1.10-2.95, p= 0.019) [Figure 1]. Interestingly, on multivariate analysis, females were more likely to have delayed reperfusion in the pre-COVID era (OR 2.92, 1.29-6.77,p= 0.01) but not the post-COVID era (OR 1.54, 0.78-3.06,p=0.2134). Patients in the post-COVID era had increased risk of having their culprit coronary not revascularized on multivariate analysis (OR 2.85, 1.2-8.03, p= 0.03). Conclusions: At our center, COVID did not significantly impact cardiac catheterization rates. However, COVID was significantly associated with delayed reperfusion timing and not revascularizing culprit vessels. Females were much more likely to have a delayed PCI than males in the pre-COVID era which was not seen following COVID-19.

GNG5 is a novel regulator of Aβ42 production in Alzheimer's disease.

The therapeutic options for Alzheimer's disease (AD) are limited, underscoring the critical need for finding an effective regulator of Aβ42 production. In this study, with 489 human postmortem brains, we revealed that homotrimer G protein subunit gamma 5 (GNG5) expression is upregulated in the hippocampal-entorhinal region of pathological AD compared with normal controls, and is positively correlated with Aβ pathology. In vivo and in vitro experiments confirm that increased GNG5 significantly promotes Aβ pathology and Aβ42 production. Mechanically, GNG5 regulates the cleavage preference of γ-secretase towards Aβ42 by directly interacting with the γ-secretase catalytic subunit presenilin 1 (PS1). Moreover, excessive GNG5 increases the protein levels and the activation of Rab5, leading to the increased number of early endosomes, the major cellular organelle for production of Aβ42. Furthermore, immunoprecipitation and immunofluorescence revealed co-interaction of Aβ42 with GPCR family CXCR2, which is known as the receptor for IL-8, thus facilitating the dissociation of G-proteins βγ from α subunits. Treatment of Aβ42 in neurons combined with structure prediction indicated Aβ42 oligomers as a new ligand of CXCR2, upregulating γ subunit GNG5 protein levels. The co-localizations of GNG5 and PS1, CXCR2 and Aβ42 were verified in eight human brain regions. Besides, GNG5 is significantly reduced in extracellular vesicles (EVs) derived from cerebral cortex or serum of AD patients compared with healthy cognition controls. In brief, GNG5 is a novel regulator of Aβ42 production, suggesting its clinical potential as a diagnosis biomarker and the therapeutic target for AD. The GNG5 content in EVs derived from serum and brain tissue of patients with AD significantly reduced. The GNG5 expression in the hippocampal-entorhinal neurons of donors with pathological AD significantly increased, and can exist in homotrimer subtypes. GNG5 expression positively correlates with Aβ pathology and Aβ42 production. Homotrimer-GNG5 binds to the γ-secretase catalytic subunit PS1 and preferentially generates Aβ42 in early endosome. GNG5 leads to enhanced Rab5 protein and activation levels, increased number of early endosome, promoting Aβ42 production. Further, Aβ42 binds to CXCR2 to upregulate GNG5 levels in a feedback loop.

Complement Molecule C3a Exacerbates Early Brain Injury After Subarachnoid Hemorrhage by Inducing Neuroinflammation Through the C3aR-ERK-P2X7-NLRP3 Inflammasome Signaling Axis.

An important aspect of the pathophysiology of early brain damage (EBI) after subarachnoid hemorrhage (SAH) is inflammasome-mediated neuroinflammation. It has been demonstrated that C3aR activation exacerbates neuronal damage in a number of neurological disorders. This study aims to explore the role of C3a in activating the NLRP3 inflammasome and exacerbating neuroinflammation after SAH. Preprocessing of RNA-seq transcriptome datasets using bioinformatics analysis, and screening of differentially expressed genes between SAH patients and healthy individuals from the GEO database. Internal carotid artery puncture was performed to establish SAH models in rats and mice. SAH grading, neurological scoring, brain water content, behavioral analysis, and assessments using ELISA, Western blot, immunofluorescence, and immunohistochemistry were conducted. An in vitro model of SAH was induced in BV-2 cells treated with heme (200μM). The mechanism of C3a in post-SAH neuroinflammation was studied by interfering with and inhibiting C3aR. Results showed that the expression of C3aR was upregulated in the GEO dataset (serum of SAH patients) and identified as a key differential gene in SAH. Further, elevated levels of C3a were found in the cerebrospinal fluid of clinically collected SAH patients. In the cerebral cortex and/or serum of SAH rats, expression of C3a, IL-1β, IL-6, TNF-α, CD11b, and Ki67 were significantly increased, while IL-10 was significantly decreased. Correlation analysis revealed that C3a showed negative correlation with IL-10 and positive correlation with IL-1β, IL-6, TNF-α, CD11b, and Ki67. After stimulation with heme, protein levels of C3a increased in BV-2 cells. Interfering with C3aR significantly reduced LDH release, IL-1β secretion, Caspase1 activation, levels of NLRP3 expression and ASC oligomerization, and ATP release after heme stimulation in BV-2. Subsequently, the addition of inhibitors of ERK1/2 phosphorylation demonstrated that C3a promotes ATP efflux by activating ERK1/2 phosphorylation, thereby activating P2X7. Further addition of JNJ-55308942 (a P2X7R antagonist) revealed that C3a activated the NLRP3 inflammasome via P2X7. Finally, administering SB290157 (a C3aR inhibitor) in vivo effectively alleviated brain edema, reduced mortality, improved Garcia score, ameliorated motor dysfunction, and suppressed inflammation and NLRP3 inflammasome activation in mice after SAH. Overall, C3a exacerbates EBI-associated NLRP3 inflammasome and neuroinflammation via the C3aR-ERK-P2X7 pathway after SAH. Inhibiting C3aR may serve as a one possible treatment approach to alleviate SAH after EBI.

ASC specks as a single-molecule fluid biomarker of inflammation in neurodegenerative diseases.

Immunotherapeutic strategies for Alzheimer's and Parkinson's disease would be facilitated by better measures of inflammation. Here we established an ultra-sensitive single-molecule pull-down immunoassay combined with direct stochastic optical reconstruction microscopy (dSTORM) to measure the number, size and shape of individual extracellular inflammasome ASC specks. We assayed human post-mortem brain, serum and cerebrospinal fluid of patients with Parkinson's and Alzheimer's as well as healthy elderly. The number of ASC specks increased and showed altered morphology in the blood of early-stage Parkinson's and Alzheimer's patients compared to controls, mimicking those found in the brain and cerebrospinal fluid. In serum samples we also measured the number of Aβ, p-tau and α-syn aggregates and formed a composite biomarker of (ASC + p-tau)/Aβ and (ASC + α-syn)/Aβ ratios that distinguished age-matched healthy controls from patients with early-stage Alzheimer's with AUC of 92% and early-stage Parkinson's with AUC of 97%. Our findings confirm ASC specks as a fluid candidate biomarker of inflammation for neurodegenerative diseases with blood being the main focus for further development as convenient sample for diagnostics and clinical trials.

EXTH-42. GLIOMA-DERIVED CD200 PROMOTES TUMOR GROWTH AND SUPPRESSES NK CELL AND CD8 T CELL ANTI-GLIOMA ACTIVITY

Abstract Immune-checkpoint inhibitors, such as anti-PD1 and anti-CTLA4, have emerged as promising therapeutic options for several malignancies yet show little efficacy against malignant brain cancers. CD200 is a newly recognized immune-checkpoint which is expressed by a marid of cell types, modulating immune homeostasis through multiple receptors. There is currently limited information regarding CD200 effects in brain tumors. Furthermore, while it is well accepted that CD200 binding to its inhibitory receptor induces a pro-tumorigenic environment through its ability to suppress immune responses, there are increasing evidence that CD200 could also have anti-tumor characteristics. Here we evaluated the role of tumor-derived CD200 on anti-glioma immunity. We demonstrate that CD200 is expressed across glioma types, is shed from tumor cells, and increases over time in serum of patients undergoing immunotherapy. Transcriptomic analysis of CD200 knockout (KO) glioma models reveals that glioma-derived CD200 significantly modifies the glioma tumor microenvironment (TME), not only through immune regulation but also through immune-independent pathways, such as tumor metabolism. Furthermore, we show that CD200 KO gliomas have reduced proliferation and are rejected by their hosts. Downstream analysis revealed that rejection of CD200 KO gliomas relied on a functional adoptive immune system, while decreased proliferation was linked to reduced CXCL10 production by the CD200 KO gliomas. Additionally, we demonstrate that glioma-mediated CD200 strongly suppresses anti-glioma NK cell function within the tumor microenvironment (TME), while secreted CD200 inhibits the priming of antigen-specific CD8 T cells in the lymphatic. Notably, NK cells were essential for the initial rejection of CD200 KO gliomas, while CD8 T cells played a critical role in establishing durable anti-tumor responses. Our work provides new mechanistic insights on glioma-derived CD200-mediated immunosuppression and an untapped potential for targeting CD200 by immunotherapies for malignant gliomas.

Exchangeable Copper Excess and Zinc Deficiency in the Serum of Patients with Colorectal Cancer.

Copper (Cu) and Zinc (Zn) are altered in colorectal cancer (CRC) but their association with the clinical classification of the tumor has not been fully explored. To examine the association of Cu and Zn homeostasis in the onset and severity of CRC, we performed an exploratory case-control study comparing the serum levels for Cu, the exchangeable component of Cu in serum (CuExc), Zn, the ratio between them (CuExc:Zn), ceruloplasmin [Cp, concentration (iCp) and its activity (eCp), Cp specific activity (eCp:iCp)], and the Cu:Cp, assessed in 31 consecutive CRC patients before surgical resection to those obtained from 37 healthy controls (CTRL). Additionally, we correlated the analyte levels with the indices of the pathological tumor, node, and metastasis (TNM) staging, namely tumor (T), node (N), and metastasis (M), evaluated at the histopathological examination. We found that Cu, CuExc, CuExc:Zn, iCp, eCp, eCp:iCp, and Cu:Cp ratios increased while Zn decreased in CRC patients. In addition, correlation analyses showed that CuExc and Zn levels confirmed the CRC diagnosis. Specifically, CuExc:Zn further increased the discrimination between the individuals of the two groups, providing an area under the curve (ROC AUC) = 0.94. Elevated CuExc was the strongest factor associated with CRC resulting in 15-fold increased odds. These data were confirmed through a multivariable regression model revealing an effect of Zn and CuExc on the CRC risk, with the CuExc resulting in 11-fold increased odds of having the disease. We also found that most of the Cu biological variables analyzed were associated with T, while the CuExc was associated with M. The current pilot study demonstrates that excess labile Cu pool, Zn deficiency, and even further their combination in the CuExc:Zn provide information about CRC in terms of diagnosis, risk of having CRC, and CRC disease stage.

Connecting the changing trace elements spectrum and survival in sarcoma: a pilot study.

While some metals have been reported as carcinogens or potential carcinogens, only few modern-standard datasets including a large number of elements are available. The present analysis established a first trace elements spectrum by relating the concentration of metals and trace elements in the serum of sarcoma patients with survival data. Patients with sarcoma and controls were retrospectively selected from the International Sarcoma Kindred Study database (ISKS). As part of the ISKS study, blood samples were prospectively collected at the Leon Bérard Cancer Center from February 2012 to July 2019. Stable specimens and copper isotopes (65Cu/63Cu) were analyzed using Triple Quadrupole Inductively Coupled Plasma Mass Spectrometer (ICP-MS) and the Multicollector MC-ICP-MS Nu Plasma HR 500. Wilcoxon rank sum test, log-rank test, and multivariate Cox regression models were used for statistics. In total, 151 patients and 59 healthy controls were included. At the time of blood sample collection, 62% of patients had locally advanced or metastatic disease. Copper (Cu), copper/zinc (Cu/Zn) and potassium/rubidium (K/Rb) ratio were significantly higher in patients compared to controls and were also significantly higher in patients with advanced compared to early-stage sarcoma. Whereas S and Se were significantly correlated in patients, no correlation was observed in controls. Importantly, levels of K, Rb, Se, Fe, P, Si, S, δ65Cu, Cu, S/Se and Cu/Zn ratio were independently associated with overall survival. These results depict the metallomic spectrum in sarcoma and highlight substantial variation associated with survival, enhancing our understanding of sarcoma's biology.