Plasma Of Healthy Controls Research Articles

Blood-based microRNAs as the potential biomarkers for Alzheimer's disease: evidence from a systematic review.

Alzheimer's disease (AD) is a neurodegenerative disorder that progresses over time and is identified by the development of neurofibrillary tangles and amyloid deposits in the brain. Mounting evidence has revealed that microRNAs (miRNAs) are significantly involved in AD progression, and may be used as promising biomarkers for diagnosis and prognosis. Nevertheless, the existing body of data regarding dysregulated circulating miRNAs in AD and their therapeutic applications are characterized by a lack of consistency. A comprehensive search was performed across various databases (PubMed, EMBASE, Web of Science, Scopus, Google Scholar, Cochrane, and ProQuest), starting from its inception and ending in January 2023. The criteria for inclusion consisted of original research studies written in English, which utilized Real-Time PCR to analyze miRNA expression in the blood, serum, or plasma of AD patients and healthy controls. The extracted data included the miRNA(s) investigated, dysregulation status, study type, human sample(s), and major findings. The search produced 608 records, which after careful examination, resulted in 48 suitable articles for data extraction. The research revealed a wide range of sample types used, with whole blood (39.59%) and serum (27.09%, including serum-exosome at 4.17%) emerging as the most prominent. The compiled dataset featured 4001 AD patients and 3886 healthy controls, revealing intricate regulatory patterns among 83 up-regulated (35.78%), 66 down-regulated (28.44%), and 83 not significantly altered (35.78%) miRNAs. Our results demonstrated that specific circulating miRNAs are consistently dysregulated in AD and could serve as non-intrusive biomarkers for the identification, prognosis, and prediction of cognitive decline. Further large-scale prospective studies are required to validate their clinical applications.

Application of EXODUS system combined with allosteric DNA nanoswitches in the detection of miR-107 among plasma exosomes of Parkinson's disease patients

This study aimed to achieve rapid detection of Parkinson's disease (PD) plasma exosome miR-107. A case-control design was used to collect ten Parkinson's disease and ten healthy control plasma samples from the Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from December 2023 to January 2024. Exosome detection via the ultrafast-isolation system (EXODUS) was used to isolate plasma exosomes. The nanoparticle tracking analysis technology and electron microscopy were used to identify exosome particle size and morphology. The Qiagen miRNeasy Micro Kit was used to extract RNA. The microRNA-activated conditional looping of engineered switches (miRacles) was used to detect miR-107, and the relative expression was analyzed by agarose gel electrophoresis. Thermo Fisher RevertAid RT Reverse Transcription Kit was used to perform reverse transcription of RNA, and real-time PCR was used to detect miR-107. The independent samples t-test was used for comparison between groups. EXODUS system completed the isolation of exosomes from 500 μl plasma within 1.5 hours. The exosome concentration (mean±SD) was (4.82±2.02)×1010 particles/ml in the control group and (5.08±2.34)×1010 particles/ml in the PD group. There was no significant difference in exosome concentration between PD patients and healthy controls (t=-0.168, P=0.872). The morphology of exosomes was confirmed by electron microscopy. The miRacles nanoswitch could detect fM-level miR-107 and also effectively distinguish miR-107 from its family members, including miR-15a and miR-16. Agarose gel electrophoresis showed that the mean±SD of relative grey value content was 1.00±0.26 in the control group and 1.86±0.21 in the PD group. The miR-107 in the PD group was significantly higher than that in the control (t=-8.143, P<0.001), which was consistent with the result of real-time PCR. EXODUS combined with miRacles could achieve rapid, non-enzymatic and cheap detection of plasma exosomal miR-107 in PD patients.

Abstract Tu020: Contrasting effect of Ox-PAPC and PC-KLH in atherosclerotic plaques or peripheral blood T cell, macropahges and dendritic cell activation

Background: Atherosclerosis is a chronic inflammation characterized by accumulation of immune cells, oxidized low density lipoprotein (OxLDL) and dead cells in the lesions, both of the latter have the hapten phosphorylcholine (PC) as an important antigen. PC is also exposed on microorganisms and other compounds and needs a carrier to be antigenic. Antibodies against PC (anti-PC) ameliorates deleterious effects of both OxLDL and dead cells. Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (Ox-PAPC) is a major compound in the lipid moiety of OxLDL exposing PC. Methods: We studied effect of Ox-PAPC and PC with keyhole limpet cyanid (KLH) on T cells, dendritic cells (DC) and macrophages (MQ). We examined the influence of Ox-PAPC and PC conjugated to KLH (PC-KLH) on T cells, DC, and MQ from peripheral blood or atherosclerotic plaques. We investigated phospholipid and glutathione (GSH) levels in the blood plasma of healthy controls (n=30) and patients (n=30). Results: Ox-PAPC, in contrast to PAPC and PC-KLH, independently of DC, induced proinflammatory T cell activation, which was inhibited by mito tempo, inhibitor of reactive oxygen species (ROS). Ox-PAPC triggered inflammatory activities while reducing metabolic activities in T cells. PC-KLH, on the other hand, reduced CD86 and CD11C, proinflammatory cytokines, and induced anti-inflammatory IL-10 in DC. Ox-PAPC induced ROS, GSH, and proinflammatory MQ activation, inhibited by Mito tempo or GSH inhibitor. Additionally, Ox-PAPC, in the presence of PCSK9, elevated CD86 expression and lipid peroxidation. Patients exhibited higher GSH levels comapre to control groups, associated with phospholipid levels. Conclusions: The study reveals the differential effects of Ox-PAPC on various immune cell types, showcasing T cell activation independent of DC and heightened macrophage activation in the presence of PCSK9. Ox-PAPC-induced redox imbalance regulates T cell activation, apoptosis, and MQ activation, potentially rendering plaques more vulnerable and contributing to lesion formation and thus plaque rupture. In contrast, PC-KLH had anti-inflammatory effects, inducing anti-inflammatory cytokines from T cells, DC and MQ. PC-effects thus vary from proinflammatory to anti-inflammatory depending on presentation. Inhibition of OxPAPC effects by agents such as anti-PC could thus be anti-atherogenic, while on the other hand PC presented on a protein as KLH could be anti-atherogenic and anti-inflammatory itself.

Evaluation of the Plasma Expression Levels of miR-21 and miR-145 as Potential Non-Invasive Biomarkers for Early Detection of Colorectal Cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in the world. Early detection would be greatly enhanced if accurate and cost-effective diagnostic biomarkers for CRC were accessible. The development of blood tests would evidently lower the screening cost of CRC detection. The aim of the present study was to examine the prospective of plasma miRNAs as non-invasive biomarkers for CRC screening. The expressions of miR-21 and miR-145 in the plasma of colorectal adenocarcinomas and normal healthy controls were quantified by using TaqMan miRNA assays. MiRNA expression levels were also correlated with commonly used clinicopathological features of CRC. Out of 30 CRC patients, 19 were male and 11 were female. The Mean age of patients was 51.3 ±14.6 years. A statistically significant increase in expression of miR-21 was observed in CRC patients' as compared to healthy controls (p<0.001). A significant association between miR-21 expression and age group (p=0.002) was noticed. Also, a statistically significant difference (p=0.015) between miR-21 expression and tumor location in the proximal and distal sites of the colon was observed in CRC patients. Further, a statistically significant downregulation of miR-145 expression was observed in the plasma of CRC patients as compared to healthy controls (p<0.05). This is the first study to report a significant association between miR-21 expression, age group, and tumor location in CRC patients. The present study thus emphasises that the appraisal of miR-21 and miR-145 plasma levels may serve as a promising non-invasive screening tool for the early detection of colorectal cancer.

Quantitative Detection and Integrality Analysis of Plasma Circulating Cell-Free DNA in Multiple Myeloma

To investigate the role of plasma circulating cell-free DNA (cf-DNA) in the screening and diagnosis of patients with newly diagnosed multiple myeloma (MM) and explore the changes of cf-DNA in terms of content and integrality in the assessment of disease in patients treated with chemotherapy. Peripheral blood specimens were collected from 35 newly diagnosed MM patients and 18 healthy volunteers, and 13 of the 35 patients who had finished 3 courses of standard induction chemotherapy were selected as follow-up group. The ALU247 and ALU115 fragments were used as the target genes, and the cf-DNA content in the plasma of patients and healthy controls was measured by quantitative polymerase chain reaction (qPCR). The integrality of cf-DNA was calculated by the ratio of ALU247 to ALU115. Both the concentration of ALU247 and ALU115 fragments and the integrality of cf-DNA in patients were significantly higher than those in healthy controls (all P < 0.05). Patients who had underwent 3 courses of induction chemotherapy had significantly decreased concentration of ALU247 and ALU115 fragments and integrality of cf-DNA after treatment (all P < 0.05), and strong positive correlations were manifested between cf-DNA integrality and serum M protein content, as well as proportion of abnormal plasma cells in bone marrow before and after treatment (r =0.703, 0.705). Cf-DNA has certain positive significance for the screening and diagnosis of MM. Furthermore, cf-DNA may be a synergism or alternative to serum M-protein content and proportion of abnormal plasma cells in bone marrow in assessing the condition, curative effect and prognosis of patients.

Analysis of potential microRNA biomarkers for multiple sclerosis

Multiple sclerosis (MS) is a chronic demyelinating autoimmune neurodegenerative disorder for which no specific blood biomarker is available. MicroRNAs (miRNAs) have been investigated for their diagnostic potential in MS. However, MS-associated miRNAs are rarely replicated in different MS populations, thus impeding their use in clinical testing. Here, we evaluated the fold expression of seven reported MS miRNAs associated with MS incidence and clinical characteristics in 76 MS patients and 75 healthy control plasma samples. We found miR-23a-3p to be upregulated in relapsing-remitting MS (RRMS), while miR-326 was downregulated. MiR-150-5p and -320a-3p were significantly downregulated in secondary progressive MS (SPMS) patients compared to RRMS. High disability was associated with low miR-320a-3p, whereas low BDNF levels were associated with upregulation of miR-150-5p and downregulation of miR-326 expression in the total cohort. MiR-23a-3p and miR-326 showed significant diagnostic sensitivity, specificity, and accuracy for RRMS diagnosis. In addition, miR-150-5p and miR-320a-3p had comparable significant diagnostic test performance metrics distinguishing SPMS from RRMS. Therefore, there is potential for including miR-23a-3p and miR-326 in an RRMS diagnostic miRNA panel. Moreover, we have shown that miR-150-5p and miR-320a-3p could be novel RRMS conversion to SPMS biomarkers. The use of these miRNAs in MS diagnosis and prognosis warrants further investigation.

A Novel RP-UHPLC-MS/MS Approach for the Determination of Tryptophan Metabolites Derivatized with 2-Bromo-4'-Nitroacetophenone.

Many biologically active metabolites of the essential amino acid L-tryptophan (Trp) are associated with different neurodegenerative diseases and neurological disorders. Precise and reliable methods for their determination are needed. Variability in their physicochemical properties makes the analytical process challenging. In this case, chemical modification of analyte derivatization could come into play. Here, we introduce a novel fast reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with tandem mass spectrometry (MS/MS) method for the determination of Trp and its ten metabolites in human plasma samples after derivatization with 2-bromo-4'-nitroacetophenone (BNAP). The derivatization procedure was optimized in terms of incubation time, temperature, concentration, and volume of the derivatization reagent. Method development comprises a choice of a suitable stationary phase, mobile phase composition, and gradient elution optimization. The developed method was validated according to the ICH guidelines. Results of all validation parameters were within the acceptance criteria of the guideline, i.e., intra- and inter-day precision (expressed as relative standard deviation; RSD) were in the range of 0.5-8.2% and 2.3-7.4%, accuracy was in the range of 93.3-109.7% and 94.7-110.1%, limits of detection (LODs) were in the range of 0.15-9.43 ng/mL, coefficients of determination (R2) were higher than 0.9906, and carryovers were, in all cases, less than 8.8%. The practicability of the method was evaluated using the blue applicability grade index (BAGI) with a score of 65. Finally, the developed method was used for the analysis of Alzheimer's disease and healthy control plasma to prove its applicability. Statistical analysis revealed significant changes in picolinic acid (PA), anthranilic acid (AA), 5 hydroxyindole-3-acetic acid (5-OH IAA), and quinolinic acid (QA) concentration levels. This could serve as the basis for future studies that will be conducted with a large cohort of patients.

P140 IgG Autoantibodies with Increased Affinity for Epithelial Cells in Severe Systemic Sclerosis

Abstract Background/Aims In health IgG immunoglobulins form a network through interaction with cell surface proteins and controlled by regulatory cells, whereas in autoimmune diseases IgG responses become skewed towards higher affinity and potentially damaging responses against self-antigens. Moreover, recent studies have demonstrated activation of the epithelial layer of skin in systemic sclerosis (SSc), implicating keratinocytes as a target of the autoimmune process. We investigated the presence of plasma IgG autoantibodies directed against keratinocytes and sought correlation with clinical parameters in well-characterized patients. Methods Diffuse SSc (dcSSc), limited SSc (lcSSc) and healthy control (HC) plasma samples (each n = 30), were assayed in an anti-epithelial cell antibody assay (AEpCA), using fixed keratinocytes to evaluate binding of IgG . Clinical characteristics of patients with positive versus negative AEpCA were compared. The capacity for SSc IgG to activate epithelial cells was assessed using keratinocytes in vitro. Results Preliminary serial dilution assay of SSc and HC plasma (n = 10) showed enhanced affinity of SSc IgG for keratinocytes (ANOVA p &amp;lt; 0.001 for overall effect SSc vs HC IgG p &amp;lt; 0.0048 for dose effect)(Figure 1A). Dilution of 1/80 gave optimal differential binding in the AEpCA assay (corrected p &amp;lt; 0.027 for SSc vs HC at 1/80). Further HC, dcSSc, and lcSSc plasma samples (each n = 30) were screened, demonstrating increased AEpCA titre in SSc (Figure 1B) (Kruskall-Wallace test p &amp;lt; 0.014 for lcSSc vs HC, p &amp;lt; 0.041 for dcSSc vs HC). SSc patients with positive AEpCA had higher skin scores (mRSS) (AEPCA neg 5(0-24); AEpCA pos 9(0-40) p &amp;lt; 0.0032), higher frequency of ATA antibody (AEpCA neg ATA pos 6/30; AEpCA pos ATA pos 13/30, chi squared p = 0.052) more interstitial lung disease (AEPCA neg 7/30, AEpCA pos 13/30, p NS)(Figure 1C) and trend towards higher use of immunosuppression. SSc but not HC IgG activated keratinocytes in vitro, triggering IL-1α release. Conclusion In this study, we have shown evidence of IgG autoantibodies with increased affinity for keratinocytes in the plasma of SSc patients. Positive testing for the AEpCA was associated with more severe skin disease and lung involvement. The perturbed IgG network in such patients could be activating through common epithelial cells antigens in the skin and lung. Disclosure C. Moezinia: None. V. Wong: None. J. Watson: None. L. Nagib: None. S. Lopez Garces: None. H. Lopez: None. G. Martin: None. S. Zhang: None. B. Ahmed Abdi: None. C. Denton: None. D. Abraham: None. R.J. Stratton: None.

P141 The Roles and Therapeutic Implications of Macrophages in Inducing Calcinosis Via Activin-A Pathway and CD206 Expression in Patients with Systemic Sclerosis

Abstract Background/Aims Calcinosis may result from localised trans-differentiation of tissue resident stem cells in the subcutaneous layer of affected skin in systemic sclerosis (SSc), as a severe disabling manifestation linked to ischaemia and local trauma. Polarised macrophages synergistically promote osteogenesis: M1 macrophages recruit mesenchymal stem cells leading to osteogenic differentiation, while M2 are responsible for osteoblast formation. Previous studies have implicated two pathways-Activin-A signalling and CD206 expression by macrophages - in heterotopic osteogenesis. We investigated the pathogenesis of calcinosis through macrophages, using a novel tissue culture model created by stimulation of adipose derived mesenchymal stem cells (ASCs) with SSc macrophages and explored the relevance of the Activin-A pathway. Methods Activin-A was measured by ELISA in diffuse cutaneous systemic sclerosis (dcSSc) patients' (n = 72), and healthy control (HC) plasma samples (n = 42). Macrophages were derived from peripheral blood monocytes of dcSSc patients (n = 4) and cultured under basal and BzATP-stimulated conditions. dcSSc macrophages were maintained in co-culture with ASCs in osteogenic media ±anti-activin-A neutralising antibody (anti-AA). Osteogenesis was assessed using Alizarin Red. Results Activin-A was increased in dcSSc patients’ plasma when compared to HC, most notably in anti-centromere antibody (ACA) subgroup (plasma activin-A 521±199pg/ml vs 255 ± 170pg/ml [p = 0.000065]) (Figure 1A). Moreover, there was synthesis of Activin A by SSc macrophages, enhanced by stimulation with BzATP (0.1µM) (basal Activin A 3.3 (0-14.5), BzATP stimulated 21.7 (7.7-418) pg/ml p NS) (Figure 1B). In co-cultures, the addition of SSc macrophages to ASCs led to significantly enhanced synthesis of Activin A, which was reduced by the addition of anti-AA (ASC monoculture 987+/-87, SSc macrophages+AScs 1599+/- 122, p &amp;lt; 0.0065, SSc macrophages+ASCs+anti-AA 1034+/-108 pg/ml, p &amp;lt; 0.013) (Figure 1C). Moreover, adding SSc macrophages to ASC cultures significantly enhanced calcinosis, as measured by Alizarin red stain, but anti-AA did not fully supress the Alizarin staining (ASC: 0.5, 0.4-0.7; ASC+M: 13.0, 5.8-17.2 p = 0.0022; ASC+M+anti-AA p NS median, IQR, red pixels per field) (Figure1D). Conclusion Elevated plasma Activin-A, most notable in ACA subgroup, is consistent with systemic upregulation of the Activin-A pathway in SSc. The interaction of activated macrophages with ASCs represents a potential model for calcinosis. Targeting Activin-A might benefit in this severe complication. Disclosure K. Liang: None. S. Lopez Garces: None. T. Searle: None. H. Lopez: Corporate appointments; CEO of Murigenics. G. Martin: Corporate appointments; Senior Scientific Officer Riptide. C. Denton: None. D. Abraham: None. R.J. Stratton: None.

Blood-Based Proteomics for Adult-Onset Focal Dystonias.

The adult-onset focal dystonias are characterized by over-active muscles leading to abnormal movements. For most cases, the etiology and pathogenesis remain unknown. In the current study, unbiased proteomics methods were used to identify potential changes in blood plasma proteins. A large-scale unbiased proteomics screen was used to compare proteins (N = 6,345) in blood plasma of normal healthy controls (N = 49) with adult-onset focal dystonia (N = 143) consisting of specific subpopulations of cervical dystonia (N = 45), laryngeal dystonia (N = 49), and blepharospasm (N = 49). Pathway analyses were conducted to identify relevant biological pathways. Finally, protein changes were used to build a prediction model for dystonia. After correction for multiple comparisons, 15 proteins were associated with adult-onset focal dystonia. Subgroup analyses revealed some proteins were shared across the dystonia subgroups while others were unique to 1 subgroup. The top biological pathways involved changes in the immune system, metal ion transport, and reactive oxygen species. A 4-protein model showed high accuracy in discriminating control individuals from dystonia cases [average area under the curve (AUC) = 0.89]. These studies provide novel insights into the etiopathogenesis of dystonia, as well as novel potential biomarkers. ANN NEUROL 2024;96:110-120.

Circulating microbiome DNA as biomarkers for early diagnosis and recurrence of lung cancer

Lung cancer mortality is exacerbated by late-stage diagnosis. Emerging evidence indicates the potential clinical significance of distinct microbial signatures as diagnostic and prognostic biomarkers across various cancers. However, circulating microbiome DNA (cmDNA) profiles are underexplored in lung cancer (LC). Here, whole-genome sequencing is performed on plasma of LC patients and healthy controls (HCs). Differentially enriched microbial species are identified between LC and HC. A diagnostic model is developed, which has a high sensitivity of 87.7% and achieves an AUC of 93.2% in the independent validation dataset. Crucially, this model demonstrates the capability to detect early-stage LC, achieving a sensitivity of 86.5% for stage I and 87.1% for tumors <1cm. In addition, we construct a cmDNA model for recurrence, which precisely predicts LC recurrence after surgery. Overall, this study highlights the significant alterations of cmDNA profiles in LC, indicating its potential as biomarkers for early diagnosis and recurrence.

Increased interleukin-9 and Th9 cells in patients with refractory Graves' disease and interleukin-9 polymorphisms are associated with autoimmune thyroid diseases.

Autoimmune thyroid diseases (AITDs) are prevalent disorders, primarily encompassing Graves' disease (GD) and Hashimoto's thyroiditis (HT). Despite their common occurrence, the etiology of AITDs remains elusive. Th9 cells, a new subset of CD4+T cells with immunomodulatory properties, have been linked to the development of various autoimmune diseases. However, research on the role of Th9 cells in AITDs is limited. We investigated the expression of Th9 cells,their functional cytokine IL-9, and transcription factor IRF4 in peripheral blood mononuclear cells (PBMCs) and plasma of AITD patients and healthy controls. Additionally, we explored the genetic association between four loci polymorphisms (rs31564, rs2069879, rs1859430, and rs2069868) of the IL-9 gene and AITDs. We reported, for the first time, that refractory GD patients exhibited elevated mRNA levels of IL-9 and IRF4 in PBMCs, increased IL-9 protein levels in plasma, and a higher proportion of Th9 cells in peripheral blood when compared to normal controls. Furthermore, human recombinant IL-9 protein was found to enhance IFN-g secretion in PBMCs from both GD patients and normal controls. At the genetic association level, after adjusting for age and sex, the rs2069879 polymorphism exhibited a significant association with AITDs under an additive model (P<0.001, OR= 0.05, 95% CI=0.03-0.08). Our results reveal that Th9 cells may exert a pivotal role in the pathogenesis and progression of refractory GD and HT, and IL-9 holds promise as a novel therapeutic target for the management of AITDs.

Integrated plasma metabolomic and cytokine analysis reveals a distinct immunometabolic signature in atopic dermatitis.

Disease models for atopic dermatitis (AD) have primarily focused on understanding underlying environmental, immunologic, and genetic etiologies. However, the role of metabolic mechanisms in AD remains understudied. To investigate the circulating blood metabolomic and cytokine profile of AD as compared to healthy control patients. This study collected plasma from 20 atopic dermatitis with moderate-to-severe itch (score of ≥5 on the itch Numeric Rating Scale and IGA score ≥3) and 24 healthy control patients. Mass-spectrometry based metabolite data were compared between AD and healthy controls. Unsupervised and supervised machine learning algorithms and univariate analysis analyzed metabolic concentrations. Metabolite enrichment and pathway analyses were performed on metabolites with significant fold change between AD and healthy control patients. To investigate the correlation between metabolites levels and cytokines, Spearman's rank correlation coefficients were calculated between metabolites and cytokines. Patients were recruited from the Johns Hopkins Itch Center and dermatology outpatient clinics in the Johns Hopkins Outpatient Center. The study included 20 atopic dermatitis patients and 24 healthy control patients. Fold changes of metabolites in AD vs healthy control plasma. In patients with AD, amino acids isoleucine, tyrosine, threonine, tryptophan, valine, methionine, and phenylalanine, the amino acid derivatives creatinine, indole-3-acrylic acid, acetyl-L-carnitine, L-carnitine, 2-hydroxycinnamic acid, N-acetylaspartic acid, and the fatty amide oleamide had greater than 2-fold decrease (all P-values<0.0001) compared to healthy controls. Enriched metabolites were involved in branched-chain amino acid (valine, leucine, and isoleucine) degradation, catecholamine biosynthesis, thyroid hormone synthesis, threonine metabolism, and branched and long-chain fatty acid metabolism. Dysregulated metabolites in AD were positively correlated cytokines TARC and MCP-4 and negatively correlated with IL-1a and CCL20. Our study characterized novel dysregulated circulating plasma metabolites and metabolic pathways that may be involved in the pathogenesis of AD. These metabolic pathways serve as potential future biomarkers and therapeutic targets in the treatment of AD.

Sickle cell disease iPSC-derived sensory neurons exhibit increased excitability and sensitization to patient plasma

AbstractIndividuals living with sickle cell disease (SCD) experience severe recurrent acute and chronic pain. Challenges to gaining mechanistic insight into pathogenic SCD pain processes include differential gene expression and function of sensory neurons between humans and mice with SCD, and extremely limited availability of neuronal tissues from patients with SCD. Here, we used induced pluripotent stem cells (iPSCs), derived from patients with SCD, differentiated into sensory neurons (SCD iSNs) to begin to overcome these challenges. We characterize key gene expression and function of SCD iSNs to establish a model to investigate intrinsic and extrinsic factors that may contribute to SCD pain. Despite similarities in receptor gene expression, SCD iSNs show pronounced excitability using patch clamp electrophysiology. Furthermore, we find that plasma taken from patients with SCD during acute pain associated with a vaso-occlusive event increases the calcium responses to the nociceptive stimulus capsaicin in SCD iSNs compared with those treated with paired plasma from patients with SCD at steady state baseline or healthy control plasma samples. We identified high levels of the polyamine spermine in baseline and acute pain states of plasma from patients with SCD, which sensitizes SCD iSNs to subthreshold concentrations of capsaicin. Together, these data identify potential intrinsic mechanisms within SCD iSNs that may extend beyond a blood-based pathology.

3D Holo-tomographic Mapping of COVID-19 Microclots in Blood to Assess Disease Severity.

The coronavirus disease 2019 (COVID-19) has impacted health globally. Cumulative evidence points to long-term effects of COVID-19 such as cardiovascular and cognitive disorders, diagnosed in patients even after the recovery period. In particular, micrometer-sized blood clots and hyperactivated platelets have been identified as potential indicators of long COVID. Here, we resolve microclot structures in the plasma of patients with different subphenotypes of COVID-19 in a label-free manner, using 3D digital holo-tomographic microscopy (DHTM). Based on 3D refractive index (RI) tomograms, the size, dry mass, and prevalence of microclot composites were quantified and then parametrically differentiated from fibrin-rich microclots and platelet aggregates in the plasma of COVID-19 patients. Importantly, fewer microclots and platelet aggregates were detected in the plasma of healthy controls compared to COVID-19 patients. Our imaging and analysis workflow is built around a commercially available DHT microscope capable of operation in clinical settings with a 2 h time period from sample preparation and data acquisition to results.

Validation of an Ultra-Sensitive Method for Quantitation of Phospho-Tau 217 (pTau217) in Human Plasma, Serum, and CSF using the ALZpath pTau217 Assay on the Quanterix HD-X Platform.

Both blood (plasma and serum) and CSF tau phosphorylated at Threonine 217 (pTau217) have been shown to be able to discriminate early to mild Alzheimer's disease (AD) from non-AD neurodegenerative disorders and healthy controls with high sensitivity and specificity. We report the full validation of the ALZpath ultra-sensitive pTau217 research-use only (RUO) assays for human EDTA plasma, serum, and CSF using the Quanterix Simoa HD-X platform, in support of clinical trials and early diagnosis of Alzheimer's disease. The ALZpath pTau217 Simoa Advantage v2 kit from ALZpath/Quanterix was used for method development and validation of pTau217 in human plasma, serum, and CSF. All three method validations have followed the 2018 FDA BMV and 2022 ICH M10 guidelines. Validation of pTau217 quantitation in human plasma, serum, and CSF demonstrated that the validated pTau217 assay has an analytical LLOQ of 0.00977 pg/mL. It is one of the most sensitive assays with a minimal required sample volume of 33.3 µl for plasma and serum, and 5 µl for CSF, and is relatively cost-effective, compared to the currently published data. The minimum required dilution (MRD) for plasma, serum, and CSF were determined. The analyte passed short-term stability tests. It was also tolerant of moderate hemolytic, and lipemic interferences in plasma and serum. pTau217 was detected in 100% of tested healthy control plasma, 90.0% of healthy control serum, 92.3% of healthy control CSF samples, and 100% of AD plasma and CSF samples. The validated assay was successfully applied in the analysis of AD samples, with data showing dramatic differences in the pTau217 levels between healthy control subjects and AD patients in both plasma and CSF. Validation of the ALZpath pTau217 Simoa assay in human plasma, serum, and CSF demonstrates ultra sensitivity, high accuracy, and assay robustness. Together with other established and sensitive assays for pTau181, GFAP, and NfL, these assays have immediate utility for application in clinical trials and can play a role in the early diagnosis of neurodegenerative diseases.

Diagnostic value of plasma circular RNA based on droplet digital polymerase chain reaction in lung adenocarcinoma.

Plasma circular (circ)RNAs detected by droplet digital polymerase chain reaction (ddPCR) may be ideal markers for liquid biopsy. However, ddPCR detection of circRNAs in plasma for diagnosis of lung adenocarcinoma has been rarely reported. An RNA sequencing analysis was performed in plasma from patients with early lung adenocarcinoma and healthy individuals. Droplet digital PCR was used to verify the differentially expressed genes. The copy numbers of circle RNALZIC (circLZIC)and circle RNACEP350 (circCEP350) in the plasma of lung adenocarcinoma patients were significantly higher than in plasma of healthy people, and the copy numbers in postoperative plasma of the same patients were significantly lower than those in preoperative plasma. CircLZIC and circCEP350 alone and in combination had diagnostic value in lung adenocarcinoma and early lung adenocarcinoma. CircLZIC and circCEP350 had more binding sites with multiple microRNAs. Their target genes were enriched in several signaling pathways. The copy numbers of circLZIC and circCEP350 were higher in plasma of lung adenocarcinoma patients than in plasma of healthy controls, significantly correlated with tumor size and TNM stage, and closely related to the occurrence and development of tumors. These circRNAs may serve as molecular markers for the diagnosis of lung adenocarcinoma.

Neuron‐derived extracellular vesicles blood‐based diagnosis of synuclein TDP43 autophagy and synaptic dysfunction pathologies

AbstractBackgroundStratifying dementia into different subgroups based on specific pathologies can facilitate the development of more effective treatments for subgroups of individuals with dementia. Effective stratification relies on reliable biomarkers. Neuron‐derived extracellular vesicles (NDEs) are lipid structures released by neurons into the extracellular space, and some get into the bloodstream. NDEs carry proteins and RNA that reflect their cell of origin and thus provide a promising biomarker platform.MethodNeuroDex developed two methods. ExoSORT is an optimized NDE isolation platform by immunoaffinity, where NDEs are captured with antibodies against the neuronal‐specific markers GAP43 and NLGN3. LuminEV is an intact extracellular vesicle (EV) immunoassay based on the Luminex platform. Antibodies against a potential biomarker, general EV marker, and IgG (negative control) are multiplexed to generate a robust assay.ResultThe vesicular nature and size of NDEs were confirmed by electron microscopy and nanoparticle tracking. EV markers were ascertained by western blot and proteomic analyses, and capture specificity was demonstrated by significantly elevated neuron‐specific proteins and mRNA levels compared to procedural control (IgG). Capture efficiency (∼80%) and precision (CV &lt; 20%) demonstrated recovery of spiked NDEs. Measurements of αSYN following ExoSORT with an MSD assay and on EVs with the LuminEV generated a separation with 83% sensitivity and 86% specificity (45 Parkinson and 30 healthy controls plasma samples). LC3, an autophagy marker, was also measured following ExoSORT and was significantly (P&lt;0.03) elevated in PD and Alzheimer’s disease (AD) (P&lt;0.02, N = 30) plasma samples. TDP43 was also significantly (P&lt;0.004) higher in AD plasma samples following ExoSORT. The synaptic marker GLUR2 was measured by the LuminEV assays and was significantly (P&lt;0.001) reduced in AD and PD plasma samples. Its level was also significantly (P&lt;0.03) correlated with the patient’s cognitive score.ConclusionImmunoaffinity isolation of EVs from plasma samples according to their cell of origin is a promising biomarker platform. We demonstrate the NDEs isolation selectivity, specificity, and precision. We also show the platform’s potential to identify pathologies like αSYN, TDP43, autophagy, and synaptic dysfunction; this reflects the potential for patient stratification.

BIOM-62. DETECTION OF EXCLUSIVE MUTATIONS AND GENE-GENE FUSIONS IN GLIOBLASTOMA THROUGH ANALYSIS OF CIRCULATING CELL-FREE DNA (CFDNA) AS AN EMERGING DIAGNOSTIC TOOL

Abstract Tumor heterogeneity is a significant obstacle for developing tailored therapies for glioblastoma (GBM). We collected blood and tumors from 20 GBM patients at 4-time points and 20 blood samples from healthy controls. cfDNA was extracted from the plasma of GBM patients and healthy controls. Tumor DNA was extracted from fresh tumor samples. Extracted DNA was sequenced using a whole-genome sequencing procedure. We found that plasma cfDNA concentrations in GBM patients were significantly elevated (22.6 ± 5 ng·mL-1), as compared to healthy controls (1.4 ± 0.4 ng·mL-1). We identified unique mutations in cfDNA of GBM patients, including some of the most frequently mutated genes in GBM (TP53, 18.75%; EGFR, 37.5%; NF1, 12.5%; LRP1B, 25%; IRS4, 25%) and built a unique clinical panel for further patients monitoring. Using our gene fusion database, ChiTaRS 5.0, we identified gene fusions in both tumor DNA and cfDNA, such as KDR-PDGFRA and NCDN-PDGFRA, which correspond to previously reported alterations of PDGFRA in GBM (44% of all samples). Interestingly, the PDGFRA protein fusions can be targeted by tyrosine kinase inhibitors. We identified ROS1 fusions (CEP85L-ROS1 and GOPC-ROS1) in 8% of samples, and those might be targeted by crizotinib analogs. Moreover, we analyzed TCGA/ICGC data of 180 GBM samples and observed FGFR3-TACC3, EGFR-SEPT14 fusions in silico. Next, we measured cfDNA levels in vitro in response to Temozolomide treatment and discovered a positive correlation between unique 2000bp cfDNA fragments (R = 0.8961, P-value = 0.015632) and treatment responses in vitro. We extracted exosomes from the in-vitro experiments and validated the concentrations of both the encapsulated cfDNA as well as the free cfDNA. We found that half of cfDNA was exosome encapsulated and it varied according to the tumor stage. CONCLUSION: cfDNA may be used for the detection of exclusive patient mutations and gene fusions and is an emerging diagnostic tool in GBM.

Single-cell mass cytometric analysis of peripheral immunity and multiplex plasma marker profiling of non-small cell lung cancer patients receiving PD-1 targeting immune checkpoint inhibitors in comparison with platinum-based chemotherapy.

The effect of platinum-based chemotherapy (Chem.) and second- or multiple- line immune checkpoint PD-1 blocking therapy by Nivolumab or Pembrolizumab (ICI) was assayed in the peripheral blood of non-small cell lung cancer (NSCLC) patients. Flow cytometry was used to detect NSCLC-related antigen binding IgG antibodies. The Luminex MagPix multiplex bead-based cytokine/chemokine detecting system was used to quantitatively measure 17 soluble markers in the plasma samples. Single-cell mass cytometry was applied for the immunophenotyping of peripheral leukocytes. The incubation of patient derived plasma with human NSCLC tumor cell lines, such as A549, H1975, and H1650, detected NSCLC-specific antibodies reaching a maximum of up to 32% reactive IgG-positive NSCLC cells. The following markers were detected in significantly higher concentration in the plasma of Chem. group versus healthy non-smoker and smoker controls: BTLA, CD27, CD28, CD40, CD80, CD86, GITRL, ICOS, LAG-3, PD-1, PD-L1, and TLR-2. The following markers were detected in significantly higher concentration in the plasma of ICI group versus healthy non-smoker and smoker controls: CD27, CD28, CD40, GITRL, LAG-3, PD-1, PD-L1, and TLR-2. We showed the induction of CD69 and IL-2R on CD4+ CD25+ T-cells upon chemotherapy; the exhaustion of one CD8+ T-cell population was detected by the loss of CD127 and a decrease in CD27. CD19+CD20+, CD79B+, or activated B-cell subtypes showed CD69 increase and downregulation of BTLA, CD27, and IL-2R in NSCLC patients following chemotherapy or ICI. Peripheral immunophenotype caused by chemotherapy or PD-1 blocking was shown in the context of advanced NSCLC.