Meso Scale Discovery Electrochemiluminescence Research Articles

Abstract 766: Effects of supplemental calcium and vitamin D on circulating biomarkers of gut barrier function in colorectal adenoma patients: A randomized controlled trial

Abstract Introduction: Recent experimental evidence shows that intestinal barrier disruption initiates colon inflammation, which may promote colorectal carcinogenesis. In experimental models, vitamin D and calcium improved gut barrier function; however, this has not been assessed in humans. Method: To test the effects of supplemental calcium and vitamin D3 on circulating biomarkers of intestinal mucosal damage (intestinal fatty-acid binding protein, IFABP) and exposure to bacterial products due to impaired gut barrier (lipopolysaccharide-binding protein, LBP), we conducted an “adjunct biomarker study” to a larger 11-center, randomized, placebo-controlled, partial 2 × 2 factorial chemoprevention clinical trial, the Vitamin D/Calcium Polyp Prevention Study. Participants were randomized into four different treatment groups: placebo, 1,200 mg/day calcium, 1,000 IU/day vitamin D3, and 1,200 mg/day calcium plus 1,000 IU/day vitamin D3. Circulating concentrations of LBP and IFABP were measured at baseline and 1-year follow-up using the Meso Scale Discovery electrochemiluminescence assays in a subset of 118 participants included in the adjunct biomarker study. Result: Following one year of vitamin D3 treatment, in the vitamin D3 plus calcium and vitamin D3 groups versus placebo and calcium groups, LBP decreased 10% (316 ng/ml; P=0.23) and IFABP 18% (77 ng/ml; P=0.06). A similar decrease of 20% (P=0.10) was observed for IFABP, but not LBP, in the vitamin D3 plus calcium versus the calcium group. There were no appreciable biomarker changes with calcium treatment. In stratified analyses, we found possible effect modifications by baseline serum 25-OH-vitamin D, total calcium intake, body mass index, and interleukin 6 (IL-6) concentration on estimated vitamin D3 treatment effects on LBP and/or IFABP. Conclusion: These preliminary results are consistent with vitamin D’s potential protective effects on intestinal mucosal barrier and support further research of vitamin D against gut barrier disruption and colorectal neoplasms. Citation Format: Sangji Lee, Veronika Fedirko, Roberd M. Bostick, Bradley Pearce, Elizabeth L. Barry, Robin E. Rutherford, March E. Seabrook. Effects of supplemental calcium and vitamin D on circulating biomarkers of gut barrier function in colorectal adenoma patients: A randomized controlled trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 766.

Interleukin-6 trans-signaling regulates monocyte chemoattractant protein-1 production in immune-mediated necrotizing myopathy.

Immune-mediated necrotizing myopathy (IMNM) is pathologically characterized by diffuse myofiber necrosis and regeneration, myophagocytosis, and a sparse inflammatory infiltrate. The monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that regulates monocyte/macrophage infiltration into injured tissues. The interleukin-6 (IL-6) signalling in the induction of MCP-1 expression has not been investigated in IMNM. MCP-1 expression in muscle specimens was assessed using immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR). Levels of multiple serological cytokines were evaluated using the Meso Scale Discovery electrochemiluminescence system. Flow cytometry, RT-qPCR, enzyme-linked immunosorbent assay, western blot, dual-luciferase reporter assays, and chromatin immunoprecipitation-qPCR were performed to explore the effects of IL-6 signalling on MCP-1 production in human myoblasts. MCP-1 was scattered and was positively expressed within myofibers and a few inflammatory cells in the muscles of patients with IMNM. Sarcoplasmic MCP-1 expression significantly correlated with myonecrosis, myoregeneration, and inflammatory infiltration. Serum MCP-1, IL-6, and the soluble form of the IL-6 receptor (sIL-6R) were elevated in patients with IMNM compared with controls. Serological MCP-1 levels were significantly associated with serum IL-6 expression and clinical disease severity in IMNM patients. The IL-6/sIL-6R complex induced MCP-1 expression via the signal transducer and activator of transcription 3 (STAT3) pathway in human myoblasts. Mechanistically, phospho-STAT3 was enriched in the MCP-1 promoter region and promoted the transcription. IL-6 trans-signalling may contribute to the immunopathogenesis of IMNM by augmenting inflammation through regulation of MCP-1 expression in IMNM.

Sex-specific inflammatory markers of risk and presence of depression in adolescents

BackgroundAssociations between inflammatory markers and depression are reported among adults; however, less is known in adolescent depression in particular whether these associations are sex-specific. We aimed to identify inflammatory markers of increased risk and presence of depression in adolescence and their association with severity of depressive symptoms in the entire cohort and separately in boys and girls. MethodsWe measured serum cytokines using a Meso Scale Discovery electrochemiluminescence V-PLEX assay in a cohort of 150 adolescents stratified for risk/presence of depression. Risk group and sex-specific differences in inflammatory markers were assessed with 2-way mixed ANOVA, and sex-moderated associations between inflammatory markers and the severity of depressive symptoms were assessed with moderated multiple hierarchical regression analyses. ResultsWe found a significant interaction between biological sex and the risk group, where boys showed higher interleukin (IL)-2 levels among the depressed group compared with the low-risk group. The severity of depressive symptoms was associated with elevated levels of IL-2 in boys, and of IL-6 in girls. There was a significant moderating effect of sex on the relationship between IL-2 and the severity of depressive symptoms but not for IL-6. LimitationsThe cross-sectional design means that we cannot be certain about the direction of the associations. ConclusionsOur findings suggest sex-specific associations between inflammatory markers and the development of adolescent depression, where IL-2 may increase risk for depression and severity of depressive symptoms in boys, but not in girls. However, IL-6 may increase risk for more severe depressive symptoms in girls.

Evaluation of Plasma Phosphorylated Tau217 for Differentiation Between Alzheimer Disease and Frontotemporal Lobar Degeneration Subtypes Among Patients With Corticobasal Syndrome

Plasma phosphorylated tau217 (p-tau217), a biomarker of Alzheimer disease (AD), is of special interest in corticobasal syndrome (CBS) because autopsy studies have revealed AD is the driving neuropathology in up to 40% of cases. This differentiates CBS from other 4-repeat tauopathy (4RT)-associated syndromes, such as progressive supranuclear palsy Richardson syndrome (PSP-RS) and nonfluent primary progressive aphasia (nfvPPA), where underlying frontotemporal lobar degeneration (FTLD) is typically the primary neuropathology. To validate plasma p-tau217 against positron emission tomography (PET) in 4RT-associated syndromes, especially CBS. This multicohort study with 6, 12, and 24-month follow-up recruited adult participants between January 2011 and September 2020 from 8 tertiary care centers in the 4RT Neuroimaging Initiative (4RTNI). All participants with CBS (n = 113), PSP-RS (n = 121), and nfvPPA (n = 39) were included; other diagnoses were excluded due to rarity (n = 29). Individuals with PET-confirmed AD (n = 54) and PET-negative cognitively normal control individuals (n = 59) were evaluated at University of California San Francisco. Operators were blinded to the cohort. Plasma p-tau217, measured by Meso Scale Discovery electrochemiluminescence, was validated against amyloid-β (Aβ) and flortaucipir (FTP) PET. Imaging analyses used voxel-based morphometry and bayesian linear mixed-effects modeling. Clinical biomarker associations were evaluated using longitudinal mixed-effect modeling. Of 386 participants, 199 (52%) were female, and the mean (SD) age was 68 (8) years. Plasma p-tau217 was elevated in patients with CBS with positive Aβ PET results (mean [SD], 0.57 [0.43] pg/mL) or FTP PET (mean [SD], 0.75 [0.30] pg/mL) to concentrations comparable to control individuals with AD (mean [SD], 0.72 [0.37]), whereas PSP-RS and nfvPPA showed no increase relative to control. Within CBS, p-tau217 had excellent diagnostic performance with area under the receiver operating characteristic curve (AUC) for Aβ PET of 0.87 (95% CI, 0.76-0.98; P < .001) and FTP PET of 0.93 (95% CI, 0.83-1.00; P < .001). At baseline, individuals with CBS-AD (n = 12), defined by a PET-validated plasma p-tau217 cutoff 0.25 pg/mL or greater, had increased temporoparietal atrophy at baseline compared to individuals with CBS-FTLD (n = 39), whereas longitudinally, individuals with CBS-FTLD had faster brainstem atrophy rates. Individuals with CBS-FTLD also progressed more rapidly on a modified version of the PSP Rating Scale than those with CBS-AD (mean [SD], 3.5 [0.5] vs 0.8 [0.8] points/year; P = .005). In this cohort study, plasma p-tau217 had excellent diagnostic performance for identifying Aβ or FTP PET positivity within CBS with likely underlying AD pathology. Plasma P-tau217 may be a useful and inexpensive biomarker to select patients for CBS clinical trials.

Adjuvant DNA vaccine pNMM promotes enhanced specific immunity and anti-tumor effects

ABSTRACT DNA vaccines containing only antigenic components have limited efficacy and may fail to induce effective immune responses. Consequently, adjuvant molecules are often added to enhance immunogenicity. In this study, we generated a tumor vaccine using a plasmid encoding NMM (NY-ESO-1/MAGE-A3/MUC1) target antigens and immune-associated molecules. The products of the vaccine were analyzed in 293 T cells by western blotting, flow cytometry, and meso-scale discovery electrochemiluminescence. To assess the immunogenicity obtained, C57BL/6 mice were immunized using the DNA vaccine. The results revealed that following immunization, this DNA vaccine induced cellular immune responses in C57BL/6 mice, as evaluated by the release of IFN-γ, and we also detected increases in the percentages of nonspecific lymphocytes, as well as those of antigen-specific T cells. Furthermore, immunization with the pNMM vaccine was found to significantly inhibit tumor growth and prolonged the survival of mice with B16-NMM+-tumors. Our data revealed that pNMM DNA vaccines not only confer enhanced immunity against tumors but also provide a potentially novel approach for vaccine design. Moreover, our findings provide a basis for further studies on vaccine pharmacodynamics and pharmacology, and lay a solid foundation for clinical application.

Intrathecal sc-AAV9-CB-GFP: Systemic Distribution Predominates Following Single-Dose Administration in Cynomolgus Macaques.

Biodistribution of self-complementary adeno-associated virus-9 (scAAV9)–chicken β-actin promoter–green fluorescent protein (GFP) was assessed in juvenile cynomolgus macaques infused intrathecally via lumbar puncture or the intracisterna magna (1.0×1013 or 3.0×1013 vg/animal), with necropsy 28 days later. Our results characterized central nervous system biodistribution compared with systemic organs/tissues by droplet digital polymerase chain reaction for DNA and in situ hybridization. Green fluorescent protein expression was characterized by Meso Scale Discovery electrochemiluminescence immunosorbent assay and immunohistochemistry (IHC). Biodistribution was widespread but variable, with vector DNA and GFP expression greatest in the spinal cord, dorsal root ganglia (DRG), and certain systemic tissues (e.g., liver), with low concentrations in many brain regions despite direct cerebrospinal fluid administration. Transduction and expression were observed primarily in perivascular astrocytes in the brain, with a paucity in neurons. Greater GFP expression was observed in hepatocytes, striated myocytes, cardiomyocytes, spinal cord lower motor neurons, and DRG sensory neurons by IHC. These results should be considered when evaluating scAAV9-based intrathecal delivery with the current expression cassette as a modality for neurologic diseases that require widespread brain neuronal expression. This capsid/expression cassette combination may be better suited for diseases that express a secreted protein and/or do not require widespread brain neuronal transduction.

A sensitive and robust bioanalytical assay for pharmacokinetic analysis of ROR1xCD3 bispecific T cell engager (NVG-111) in a first-in-human study.

e19505 Background: Receptor tyrosine kinase-like Orphan Receptor-1 (ROR1) is widely expressed on hematological and solid tumors. NVG-111, a first in class humanized tandem scFv ROR1xCD3 bispecific antibody elicits potent killing of ROR1+ tumor cells in vitro and in vivo. This bispecific T-cell engager (TCE) is being evaluated in a first in human, Phase I trial in patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The predicted therapeutic dose and steady state serum concentrations of NVG-111 were estimated by allometric scaling using relevant doses from a murine PK study. To assess free drug levels in patients following 21 days of continuous infusion of NVG-111, a bespoke, sensitive pharmacokinetics (PK) assay with high levels of specificity and sensitivity was developed. Methods: Anti-idiotype (anti-ID) antibodies directed to anti-ROR1 (αROR1-ID) and anti-CD3 (αCD3-ID) were generated by mouse immunization or by phage display from customized libraries. A proof-of-concept sandwich ELISA assay was developed using αCD3-ID to capture NVG-111 and detection by biotinylated hROR1-streptavidin-HRP. Gyrolab and Quanterix Simoa high sensitivity ELISA platforms were used to detect NVG-111 by αCD3-ID capture and αROR1-ID detection. The mesoscale discovery electrochemiluminescence assay (MSD-ECLA) was developed using a reversed format; NVG-111 capture with αROR1-ID and detection with αCD3-ID. Results: Allometric scaling predicted a theoretically relevant therapeutic dose and steady state serum concentration of 1ng/mL NVG-111 in humans, which was just at the level of sensitivity of a conventional ELISA under non-matrix conditions. Transferring the format to Quanterix Simoa had limited success due to high background levels in all configurations evaluated. The Gyrolab platform increased sensitivity to 75pg/mL, but suboptimal individual human sera matrix selectivity limited assay validity. Assessment of MSD-ECLA provided the best signal/noise, enhanced human disease and healthy sera selectivity, and a dynamic sensitivity range of 250pg/mL to 32ng/mL, which enabled the development of a GCLP qualified PK assay. The MSD-ECLA assay was employed to measure NVG-111 concentrations in CLL or MCL subjects dosed with 0.3-30µg/day NVG-111. MSD-ECLA detected drug in patients receiving NVG-111, with a range of steady-state serum concentrations (Cavg.ss) of 168-610pg/mL. This was in-line with the predicted drug levels from the single species allometric scaling, albeit with observed levels being marginally lower than expected. Conclusions: Development, custom optimization and validation of a highly sensitive MSD-ECLA PK assay has enabled GCLP-compliant measurement of circulating NVG-111 in CLL or MCL patients treated with at least 10µg/day cIV NVG-111.

Plasma P-tau181 and P-tau217 in Patients With Traumatic Encephalopathy Syndrome With and Without Evidence of Alzheimer Disease Pathology.

Traumatic encephalopathy syndrome (TES) has overlapping clinical symptoms with Alzheimer disease (AD). AD pathology commonly co-occurs with chronic traumatic encephalopathy (CTE) pathology. There are currently no validated CTE biomarkers. AD-specific biomarkers such as plasma P-tau181 and P-tau217 may help to identify patients with TES who have AD pathology. We measured plasma P-tau181 and P-tau217 (Meso Scale Discovery electrochemiluminescence) in patients with TES, mild cognitive impairment/dementia with biomarker-confirmed AD ("AD"), and healthy controls ("HC"). Patients underwent amyloid-beta (Aβ)-PET and a subset underwent tau-PET using [18F]Flortaucipir. We compared plasma P-tau levels controlling for age and sex and also performed AUC analyses to evaluate the accuracy of group differentiation. In patients with TES, we evaluated associations between plasma P-tau, years of repetitive head impact exposure, and tau-PET. Four TES patients with autopsy-confirmed CTE were described qualitatively. The sample included 131 participants (TES, N = 18; AD, N = 65; HC, N = 48). Aβ(+) patients with TES (N = 10), but not Aβ(-) TES, had significantly higher plasma P-tau levels than HC (P-tau181: p < 0.001, d = 1.34; P-tau217: p < 0.001, d = 1.59). There was a trend for Aβ(+) TES having higher plasma P-tau than Aβ(-) TES (P-tau181: p = 0.06, d = 1.06; P-tau217: p = 0.09, d = 0.93). AUC analyses showed good classification of Aβ(+) TES from HC for P-tau181 (AUC = 0.87 [0.71-1.00]) and P-tau217 (AUC = 0.93 [0.86-1.00]). Plasma P-tau217 showed fair differentiation of Aβ(+) TES from Aβ(-) TES (AUC = 0.79 [0.54-1.00], p = 0.04), whereas classification accuracy of P-tau181 was slightly lower and not statistically significant (AUC = 0.71 [0.46-0.96], p = 0.13). Patients with AD had higher tau-PET tracer uptake than Aβ(+) TES and were well differentiated using P-tau181 (AUC = 0.81 [0.68-0.94]) and P-tau217 (AUC = 0.86 [0.73-0.98]). Plasma P-tau correlated with the tau-PET signal in Aβ(+) TES but not in Aβ(-) TES, and there was no association between plasma P-tau and years of repetitive head impact exposure. TES patients with severe CTE and no AD at autopsy had low P-tau181 and P-tau217 levels. Measuring P-tau181 and P-tau217 in plasma may be a feasible and scalable fluid biomarker for identifying AD pathology in TES. Low plasma P-tau levels may be used to increase clinical suspicion of CTE over AD as a primary pathology in TES. Currently, there is no support for P-tau181 or P-tau217 as in vivo biomarkers of CTE tau. Larger studies of patients with pathologically confirmed CTE are needed. This study provides Class III evidence that (1) among patients with TES and abnormal Aβ-PET scans, elevated plasma P-tau can differentiate between affected individuals and HCs; (2) low plasma P-tau may help identify patients with TES who do not have Alzheimer; and (3) plasma P-tau181 and P-tau217 are not useful biomarkers of patients with TES who do not have AD.

The impact of baseline peripheral cytokines on survival in metastatic clear cell renal cell carcinoma (RCC) treated with nivolumab: NIVOREN GETUG-AFU 26 Translational study.

379 Background: The NIVOREN GETUG-AFU 26 study reported safety and efficacy of nivolumab in patients with RCC in a “real world setting”. A translational research program was launched to quantify baseline cytokine levels and correlate them with outcomes to nivolumab. Methods: Extreme responder patients (pts) treated with nivolumab as part of the phase II NIVOREN GETUG-AFU 26 were included in this TRAINING cohort. A panel of 14 different plasma cytokines and proteins (VEGF, VCAM-1, IL-6, IL-7, IL-8, IL-10, APRIL, BAFF, 4-1BB, BCA, SDF-1, MDC, IFN-gamma and TNF-alpha) were quantified for each plasma sample using the Elisa-based Meso Scale Discovery electrochemiluminescence assay. The association between baseline cytokine levels and objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) was evaluated. Results: Overall, 80 pts, 40 responders and 40 progressors, were included for cytokine analysis. Baseline characteristics were similar to the overall trial population. The IMDC risk score breakdown was 11.3% good, 56.3% intermediate and 32.5% poor. With a median follow-up of 21.2 months, mOS data was immature at data cut-off. Overall survival rate was 67.3% at 12 months and median PFS was 3.8 months. Increased levels of IL-6 (75th percentile=P75), IL-7 (P75), IL-8 (P50) and VEGF (P50) were significantly negatively associated with survival (IL-6: HR=2.44, p=0.0112; IL-7: HR=2.38, p=0.0123; IL-8: HR=2.80, p=0.0045, and VEGF: HR=2.43, p=0.0133). 4-1BB (P50) was associated with improved OS (HR=0.39, p=0.0375). Higher levels of IL-8 (P50) and VEGF (P50) were associated with worse PFS (IL-8: HR=2.50, p=0.0133, and VEGF: HR=1.96, p=0.0132) and worse ORR (IL-8: p=0.013, and VEGF: p=0.044). Except for IL-8 and VEGF, no other associations with response were observed. Conclusions: Higher baseline plasma levels of IL-6, IL-7, IL-8 and VEGF were significantly associated with worse survival outcomes in mRCC pts treated with nivolumab within TRAINING cohort of the NIVOREN trial. In contrast, 4-1BB was significantly associated with improved OS. IL-8 and VEGF were also associated with worse PFS and ORR. VALIDATION cohort within the entire NIVOREN study is ongoing.

Relative Quantification of NaV1.1 Protein in Mouse Brains Using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) Method.

Densitometric analysis is often used to quantify NaV1.1 protein on immunoblots, although the sensitivity and dilution linearity of the method are usually poor. Here we present a protocol for quantification of NaV1.1 in mouse brain tissues using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) method. MSD-ECL is based on ELISA (enzyme-linked immunosorbent assay) and uses electrochemiluminescence to produce measurable signals. Two different antibodies are used in this assay to capture and detect NaV1.1 respectively in brain tissue lysate. The specificity of the antibodies is confirmed by Scn1a gene knock-out tissue. The calibration curve standards used in this assay were generated with mouse liver lysate spiked with mouse brain lysate, instead of using a recombinant protein. We showed that this method was qualified and used for quantification of NaV1.1 in mouse brain tissues with specificity, accuracy and precision.

Differential effect of interferon-alpha treatment on AEA and 2-AG levels

The endocannabinoid (eCB) system is one of the key players in immunoregulation, and reduced activity of the eCB system has been linked with depressive-like behaviours in animal studies and depression in clinical samples. There is a well-established link between immune activation and depression, such as following the administration of the pro-inflammatory cytokine, interferon-α (IFN-α), used to treat hepatitis C viral (HCV) infection. However, the role of peripheral endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), following immunotherapy with IFN-α and in IFN-α -induced depression, have not been examined yet.In this study, we investigated whether circulating AEA and 2-AG were modified by treatment with IFN-α and whether they were involved in the development of IFN-α-induced depression. We also explored whether circulating eCBs were associated with peripheral cytokines during and after IFN-α treatment. We measured serum concentrations of AEA and 2-AG using High Performance Liquid Chromatography with Tandem Mass Spectrometry, and serum concentrations of cytokines using Meso Scale Discovery electrochemiluminescence V-PLEX assay, in 70 patients with HCV infection and 41 healthy subjects. We assessed HCV patients at baseline, IFN-α-treatment weeks (TW) 4 and 24, end of treatment (END) and at six months follow-up (FU). We assessed depression using M.I.N.I. International Neuropsychiatric Interview.We found a different pattern of change in peripheral AEA and 2-AG during and after IFN-α treatment. Whilst 2-AG increased earlier in immunotherapy (TW4), remained elevated throughout treatment, and reduced at six months follow-up (FU), AEA increased later in treatment (TW24) and remained elevated six months post-treatment. We also found that baseline levels of AEA were lower in HCV patients compared with healthy controls, whereas there were no differences in 2-AG levels. Interestingly, AEA, but not 2-AG, was significantly, negatively correlated with interleukin (IL)-2 and IL-17a at six months follow-up. We did not find any difference in both eCBs between patients with and without IFN-α-induced depression, at any time point.Our findings suggest that AEA and 2-AG are involved in different stages of immunoregulation following IFN-α treatment, where AEA might be involved in chronic inflammation. Lack of association between peripheral eCBs and IFN-α-induced depression suggests that different biological mechanisms may underpin inflammation-induced depression compared with classic “psychiatric” depression, or that any changes in the eCB system in depression may not be captured by peripheral AEA and 2-AG.

Unstimulated Parotid Saliva Sampling in Juvenile Idiopathic Arthritis and Healthy Controls: A Proof-of-Concept Study on Biomarkers.

The aims of this proof-of-concept study were to develop a collecting method for unstimulated parotid saliva in juvenile idiopathic arthritis (JIA) patients and healthy children and to investigate if inflammatory biomarkers could be detected in these samples. Forty-five children with JIA (median age of 12 years and 25th–75th percentile of 10–15 years; 33 girls and 12 boys) and 16 healthy children as controls (median age of 13 years and 25–75th percentile of 10–13 years; 11 girls and 5 boys) were enrolled in this study. Unstimulated parotid saliva was collected with a modified Carlson–Crittenden collector. The salivary flow rate and salivary concentrations of total protein and inflammatory mediators were assessed. The Meso Scale Discovery electrochemiluminescence immunoassay was used for analyzing protein concentrations and the inflammatory biomarkers. Sufficient parotid saliva volumes to be analyzed could be collected with the collection device. JIA patients had a lower sampling saliva volume (p = 0.008) and saliva flow rate (p = 0.039) than controls. The total protein concentrations and inflammatory biomarkers were measured in the last six healthy subjects. The median protein concentration was 1312 µg/mL (25th percentile: 844 µg/mL and 75th percentile: 2062 µg/mL; n = 6) and quantifiable concentrations of 39 inflammatory proteins could be assessed in these samples. In conclusion, this study indicates that the saliva sampling method, as used in the present study, is able to collect sufficient sample volumes in children, and that it is possible to analyze various inflammatory biomarkers in the collected saliva.

A novel sensitive assay for detection of a biomarker of pericyte injury in cerebrospinal fluid.

Blood-brain barrier (BBB) breakdown and loss of brain capillary pericytes contributes to cognitive impairment. Pericytes express platelet-derived growth factor receptor-β (PDGFRβ) that regulates brain angiogenesis and blood vessel stability. Elevated soluble PDGFRβ (sPDGFRβ) levels in cerebrospinal fluid (CSF) indicate pericyte injury and BBB breakdown, which is an early biomarker of human cognitive dysfunction. A combination of reagents and conditions were tested, optimized, and validated on the Meso Scale Discovery electrochemiluminescence platform to develop a new sPDGFRβ immunoassay that was used to measure sPDGFRβ in human CSF from 147 individuals. We developed standard operating procedures for a highly sensitive and reproducible sPDGFRβ immunoassay with a dynamic range from 100 to 26,000 pg/mL, and confirmed elevated CSF sPDGFRβ levels in individuals with cognitive dysfunction. This assay could be applied at different laboratories to study brain pericytes and microvascular damage in relation to cognition in disorders associated with neurovascular and cognitive dysfunction.

Association of LAG3 genetic variation with an increased risk of PD in Chinese female population

BackgroundEmerging evidence suggests that α-synuclein (α-syn) aggregation and intercellular transmission contributes to pathogenesis of Parkinson’s disease (PD) and the toxic fibrillary α-syn binds lymphocyte-activation gene 3 (LAG3) receptor that mediates α-syn transmission. The deletion of LAG3 in animal models was shown to limit α-syn spreading and alleviate the pathological changes of dopaminergic neurons and animal behavioral deficits induced by α-syn aggregation. However, little is known about the genetic association of LAG3 variation with human PD development.ObjectiveHere we investigated LAG3 single nucleotide polymorphisms (SNPs) and examined the levels of soluble LAG3 (sLAG3) of CSF and serum from Chinese PD patients.MethodsWe enrolled 646 PD patients and 536 healthy controls to conduct a case-control study. All the participants were genotyped using Sequenom iPLEX Assay and the partial cerebrospinal fluid (CSF) and serum samples were assessed by Meso Scale Discovery electrochemiluminescence (MSD-ECL) immunoassay to measure sLAG3 concentration.ResultsAs a result, distributions of rs1922452-AA (1.975, 95% confidence interval (CI) 1.311–2.888, p = 0.001) and rs951818-CC (OR = 2.03, 95% CI 1.369–3.010, p = 0.001) genotype frequencies were found higher in the female PD patients than controls, respectively, and a strong linkage disequilibrium (LD) was calculated on the variants. The level of sLAG3 in CSF of PD patients was found to significantly differ from that of controls (51.56 ± 15.05 pg/ml vs 88.49 ± 62.96 pg/ml, p < 0.0001). Meanwhile, the concentration of α-synuclein in CSF of patients was significantly lower than that of controls (939.9 ± 2900 pg/ml vs 2476 ± 4403 pg/ml, p < 0.0001) and the level of sLAG3 was detected to be positive correlation with that of α-synuclein in the control group (r = 0.597, p = 0.0042), but not in PD group (r = 0.111, p = 0.408).ConclusionIn summary, our data suggested that LAG3 SNPs increase the PD risk of Chinese female population and the sLAG3 may be a potential biomarker predicted for PD development.

A host-associated antimicrobial agent exhibits both immunoprotective and immunotherapeutic effects

Abstract Inflammation is a critical component of both the defense against injury and the pathophysiology of disease. Despite advances in anti-inflammatory therapy and critical care, Gram-negative infections remain a significant cause of mortality in multiple species, and are a primary cause of diarrhea, endotoxemia, sepsis and ultimately death in neonatal calves. Given the impact of host response to disease on the health of humans and food animals alike, we examined the effects of supplemental bovine lactoferrin (bLF) and its functional peptide lactoferricin B (LFcin B), on the dysregulated immune response to lipopolysaccharide (LPS). We hypothesized that these antibiotic agents have therapeutic value as low-risk anti-inflammatory agents, a characteristic that is attributable to their regulatory role on intracellular signaling in innate cells. Although present in colostrum, saliva and mucosal secretions, bLF is notably at its highest concentration in the secondary granules of neutrophils. In the host, the 25 AA peptide LFcin B is released following the activity of proteolytic enzymes in the stomach as well as at sites of infection. Viable peripheral monocytes and neutrophils were isolated from calves and stimulated in vitro with LPS, in the presence or absence of bLF/LFcin B. Samples were analyzed using qRT-PCR, Meso Scale Discovery Electrochemiluminescence, immunoprecipitation and western immunoblot detection. The effects of bLF and LFcin B on pro-inflammatory cytokine and inflammatory mediator production will be expressed as the mean ± SD and will be considered significant at P&amp;lt;0.05. Although each exerted differential effects, the p38 MAP kinase pathway appears to be a likely target.

Abstract 5027: Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform

Abstract Identification of soluble biomarkers has become a critical non-invasive approach for disease diagnosis and monitoring in the treatment of solid tumors. Non-Small Cell Lung Cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of all lung cancers worldwide. The EGFR family of genes is known to be highly expressed in NSCLC tumors and play a critical role in the progression of the disease. Although patients initially respond well to EGFR TKI therapy, acquired resistance occurs through several mechanisms, including compensatory cMET pathway activation (Robinson et al. 2013). Previous reports have demonstrated that soluble forms of EGFR, Her2 and cMET are generated through either alternate splicing of mRNA or proteolytic cleavage of the full length receptors (Wilken et al. 2013). Serum concentrations of these receptors can be correlated with prognosis as well as treatment response (Gregorc, 2004, Kasahara, 2010 Heinmoller, 2003). Soluble receptor ligands, including Hepatocyte growth factor (HGF), have also been evaluated in NSCLC and a strong correlation has also been observed between the levels of serum HGF and disease outcome in patients treated with EGFR-TKIs (Kasahara et al. 2010). In this study, we evaluated concentrations of sEGFR, sMET, sHER2,, and HGF in NSCLC versus healthy normal serum samples via meso scale discovery (MSD) platform. Inventoried and custom MSD assays were optimized to measure these proteins in a training set consisting of 19 NSCLC and 16 normal healthy serum samples. Significantly lower concentrations of sEGFR and sHER2 were observed in the NSCLC serum samples compared to normal (p value = 0.0092 for both receptors), whereas, in contrast, the levels of HGF were significantly higher in the NSCLC patient samples (p value &amp;lt;0.0001). No significant difference in sMET concentration was observed between NSCLC and normal serum. Based on this data, it was determined that a ratio of protein concentration of each soluble receptor (EGFR, Her2 and cMET) to HGF yielded a greater differentiation between NSCLC and normal samples (p value &amp;lt;0.0001, = 0.0041,and 0.0327 respectively). Cut-off values to define NSCLC versus normal patients were established for each ratio and validated in a blinded independent cohort, consisting of 50 NSCLC and 12 healthy normal serum samples. PPV and NPV were then calculated to determine sensitivity and specificity for each protein ratio. Results from the validation study show an almost complete separation between healthy and NSCLC patient samples (p value &amp;lt;0.0001, AUC = 1)for all three ratios tested (sEGFR/HGF, sHER2/HGF and sMET/HGF), with a PPV = 100%, 98%, 100% and NPV = 100%, 100%, 85.7%, respectively. Overall, these findings indicate potential clinical applications for these protein pairs as non-invasive biomarkers of NSCLC diagnosis, prognosis and treatment monitoring to support emerging TKI therapies Citation Format: Dana S. Gaffney, Katherine Bell, Gabriela Martinez, Jayaprakash Karkera, Suso Platero. Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5027.

Pittsburgh compound B imaging and cerebrospinal fluid amyloid-β in a multicentre European memory clinic study.

The aim of this study was to assess the agreement between data on cerebral amyloidosis, derived using Pittsburgh compound B positron emission tomography and (i) multi-laboratory INNOTEST enzyme linked immunosorbent assay derived cerebrospinal fluid concentrations of amyloid-β42; (ii) centrally measured cerebrospinal fluid amyloid-β42 using a Meso Scale Discovery enzyme linked immunosorbent assay; and (iii) cerebrospinal fluid amyloid-β42 centrally measured using an antibody-independent mass spectrometry-based reference method. Moreover, we examined the hypothesis that discordance between amyloid biomarker measurements may be due to interindividual differences in total amyloid-β production, by using the ratio of amyloid-β42 to amyloid-β40 Our study population consisted of 243 subjects from seven centres belonging to the Biomarkers for Alzheimer's and Parkinson's Disease Initiative, and included subjects with normal cognition and patients with mild cognitive impairment, Alzheimer's disease dementia, frontotemporal dementia, and vascular dementia. All had Pittsburgh compound B positron emission tomography data, cerebrospinal fluid INNOTEST amyloid-β42 values, and cerebrospinal fluid samples available for reanalysis. Cerebrospinal fluid samples were reanalysed (amyloid-β42 and amyloid-β40) using Meso Scale Discovery electrochemiluminescence enzyme linked immunosorbent assay technology, and a novel, antibody-independent, mass spectrometry reference method. Pittsburgh compound B standardized uptake value ratio results were scaled using the Centiloid method. Concordance between Meso Scale Discovery/mass spectrometry reference measurement procedure findings and Pittsburgh compound B was high in subjects with mild cognitive impairment and Alzheimer's disease, while more variable results were observed for cognitively normal and non-Alzheimer's disease groups. Agreement between Pittsburgh compound B classification and Meso Scale Discovery/mass spectrometry reference measurement procedure findings was further improved when using amyloid-β42/40 Agreement between Pittsburgh compound B visual ratings and Centiloids was near complete. Despite improved agreement between Pittsburgh compound B and centrally analysed cerebrospinal fluid, a minority of subjects showed discordant findings. While future studies are needed, our results suggest that amyloid biomarker results may not be interchangeable in some individuals.

A novel peptide, colivelin, prevents alcohol-induced apoptosis in fetal brain of C57BL/6 mice: signaling pathway investigations

Fetal alcohol exposure is known to induce cell death through apoptosis. We found that colivelin (CLN), a novel peptide with the sequence SALLRSIPAPAGASRLLLLTGEIDLP, prevents this apoptosis. Our initial experiment revealed that CLN enhanced the viability of primary cortical neurons exposed to alcohol. We then used a mouse model of fetal alcohol exposure to identify the intracellular mechanisms underlying these neuroprotective effects. On embryonic day 7 (E7), weight-matched pregnant females were assigned to the following groups: (1) ethanol liquid diet 25% (4.49% v/v) ethanol derived calories; (2) pair-fed control; (3) normal chow; (4) ethanol liquid diet combined with administration (i.p.) of CLN (20 μg/20 g body weight); and (5) pair-fed combined with administration (i.p.) of CLN (20 μg/20 g body weight). On E13, fetal brains were collected and assayed for TdT-mediated dUTP nick end labeling staining, caspase-3 colorimetric assay, enzyme-linked immunosorbent assay, and Meso scale discovery electrochemiluminescence. CLN blocked the alcohol-induced decline in brain weight and prevented alcohol-induced: apoptosis, activation of caspase-3 and increases of cytosolic cytochrome c, and decreases of mitochondrial cytochrome c Analysis of proteins in the upstream signaling pathway revealed that CLN down-regulated the phosphorylation of the c-Jun N-terminal kinase. Moreover, CLN prevented alcohol-induced reduction in phosphorylation of BAD protein. Thus, CLN appears to act directly on upstream signaling proteins to prevent alcohol-induced apoptosis. Further assessment of these proteins and their signaling mechanisms is likely to enhance development of neuroprotective therapies.