Toxin A-Predominant Pathogenic Clostridioides difficile: A Novel Clinical Phenotype.

BACKGROUND: Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A-negative, toxin B-positive (A-B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. METHODS: An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). RESULTS: There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23-17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (<20 pg/mL; median TcdA:B ratio 17.93). Concentrations of toxin A far exceeded B in in vitro cultures of all 12 recovered isolates (median TcdA:B ratio 26). Of 8 toxin A>>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). CONCLUSIONS: We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.

Differential immunodetection of toxin B from highly virulent Clostridium difficile BI/NAP-1/027.

We developed a simple immunoassay capable of differentially detecting toxin B from highly virulent strains of Clostridium difficile (BI/NAP-1/027) in stool. This assay can simultaneously confirm the presence of in vivo toxin production and provide strain-related information relevant to infection control epidemiology and disease prognosis.

Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool.

The currently available diagnostics for Clostridium difficile infection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenic C. difficile. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinical C. difficile isolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.

Peripheral Blood CD8+ CD28+ T Cells as an Independent Predictor of Treatment Response and Survival After Concurrent Chemoradiotherapy in Pediatric High-Grade Glioma Patients.

Backgroud: The tumor immune microenvironment influences the efficiency of concurrent chemoradiotherapy (CCRT) in high-grade glioma (HGG). This study investigated peripheral blood T lymphocyte subsets as clinical indicators of therapeutic response and prognosis in pediatric high-grade glioma (pHGG). Methods: This retrospective study included 77 patients with postoperative pHGG who were treated concurrently with temozolomide and external beam radiotherapy between January 1, 2012, and December 31, 2018. The median follow-up was 26 (range: 5-106) months. Peripheral venous blood samples were collected before and after CCRT. The proportions of peripheral blood T lymphocytes and their association with treatment outcome and survival were determined. Results: Sixty-four (83.1%) patients achieved complete remission, partial remission, and stable disease, and 13 (16.9%) patients had progressive disease. Higher CD3+ T cell, CD4+ T cell, and CD8+ CD28+ T cell ratios were predictive of better response, while a higher CD8+ CD28- T cell ratio was predictive of poorer response. Binary logistic regression analysis showed that the CD8+ CD28+ T cell ratio was a significant independent predictor of CCRT response (odds ratio [OR] = 53.521, 95% confidence interval [CI] = 4.294-667.119, P = .002). Univariate and multivariate analysis of prognostic factors associated with survival showed that the CD8+ CD28+ T lymphocyte ratio was a significant independent predictor of progression-free survival (hazard ratio [HR] = 1.80, 95% CI = 1.06-3.08, P = .03), but none of the subsets were significantly associated with overall survival. Conclusion: Peripheral blood T lymphocytes have potential as predictors of CCRT response and prognosis in pHGG.

A phase I randomized study to evaluate safety, pharmacokinetics, and pharmacodynamics of SIR2446M, a selective RIPK1 inhibitor, in healthy participants.

Activation of receptor-interacting protein kinase 1 (RIPK1), a broadly expressed serine/threonine protein kinase, by pro-inflammatory cytokines and pathogens can result in apoptosis, necroptosis, or inflammation. RIPK1 inhibition has been shown to reduce inflammation and cell damage in preclinical studies and may have therapeutic potential for degenerative and inflammatory diseases. SIR2446 is a potent and selective novel small molecule RIPK1 kinase inhibitor. This phase I, randomized, double-blind, placebo-controlled study in Australia (ACTRN12621001621808) evaluated the safety (primary objective), pharmacokinetics, and pharmacodynamics of single (3-600 mg) and multiple (5-400 mg for 10 days) ascending oral doses of SIR2446M (SIR2446 magnesium salt form) in healthy adults from Nov 24, 2021, until May 01, 2023. All treatment-emergent adverse events (TEAEs) were mild/moderate. The most reported TEAEs were vascular access site pain, headache, and rash morbilliform. SIR2446M plasma half-lives ranged from 11 to 19 h and there were no major deviations from dose proportionality for maximum concentration and area under the curve across doses. Renal excretion of unchanged SIR2446 was minimal. No marked accumulation was observed (mean accumulation ratio, 1.2-1.6) after multiple daily doses. A high-fat meal mildly reduced the exposure but was not considered clinically significant. SIR2446M had a rapid and sustained inhibitory effect on the activity of RIPK1, with an overall 90% target engagement at repeated doses ranging from 30 to 400 mg in peripheral blood mononuclear cells ex vivo stimulated to undergo necroptosis. The favorable safety, pharmacokinetic, and pharmacodynamic profile of SIR2446M in healthy participants supports its further clinical development in patients with degenerative and inflammatory diseases.

Direct detection of bacterial genomic DNA at sub-femtomolar concentrations using single molecule arrays.

We report a method for the sensitive measurement of genomic DNA based on the direct detection of single molecules of DNA in arrays of femtoliter wells. The method begins by generating short fragments of DNA from large, double-stranded molecules of genomic DNA using either restriction enzymes or sonication. Single-stranded fragments are then generated by melting the duplex, and these fragments are hybridized to complementary biotinylated detection probes and capture probes on paramagnetic beads. The resulting DNA complexes are then labeled with an enzyme (streptavidin-ß-galactosidase), and single enzymes associated with these complexes on beads are detected in single molecule arrays (Simoa). DNA concentration is quantified by determining the average number of enzymes per bead via Poisson statistics (digital) or the average bead intensity (analog). The Simoa DNA assay was used to detect genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, or 2100 DNA molecules per 50 µL sample. We used this assay to detect S. aureus spiked into (a) whole blood, with an average LOD of 1100 bacteria per 25 µL sample (0.074 fM), and (b) water from the Charles River, with an LOD of 1300 bacteria per 50 µL sample (0.042 fM). Bacteria were detected in river water without prior purification of DNA. The Simoa DNA assay, which directly detects target DNA molecules without molecular replication, is an attractive alternative to existing sensitive DNA detection technologies that rely on amplification using polymerases, such as the polymerase chain reaction (PCR).

Soluble PD-L1 as an early marker of progressive disease on nivolumab.

BACKGROUND: Soluble PD-L1 (sPD-L1) has been associated with worse prognosis in numerous solid tumors. We determined sPD-L1 levels before and during nivolumab treatment in two prospective clinical trials of metastatic clear cell renal cell carcinoma (RCC) and melanoma patients, and investigated its relationship to clinical factors, biomarkers, and outcome. METHODS: Using a new Single Molecule Array assay, serum sPD-L1 level were determined in RCC (CheckMate 009, n=91) and melanoma (CheckMate 038-Part 1, n=78) prior to, and at two time points on treatment. Gene expression data was obtained from biopsies taken prior to, and at day 28 on treatment. Results were integrated with clinical variables, tumor PD-L1 status from immuno-histochemistry, and genomic mutation status. RESULTS: In RCC patients, sPD-L1 levels were higher in patients with progressive disease as their best response. For both RCC and melanoma patients, progressive or stable disease was associated with an increase in sPD-L1 on nivolumab therapy, whereas mean sPD-L1 levels did not change or declined in patients with objective responses. By categorizing RCC patients into transcriptomic molecular subtypes, we identified a subgroup where the associations between sPD-L1 and progressive disease were particularly evident. In baseline biopsies, we identified six biological processes that were associated with sPD-L1 level in both RCC and melanoma: higher sPD-L1 is associated with lower tumor expression of the Hallmark gene sets 'hypoxia', 'fatty acid metabolism', 'glycolysis', 'MTORC1 signaling' and 'androgen response', and with higher expression of 'KRAS signaling_Down'. CONCLUSION: Baseline and on-therapy sPD-L1 levels in RCC have the potential to predict progressive disease on PD-1 inhibitor nivolumab. In a hypothesis-generating analysis of tumor gene expression, high baseline sPD-L1 is associated with a tumor metabolic state reflecting potentially targetable processes in both melanoma and RCC. In both trials, we observed associations between change in sPD-L1 on treatment and outcome metrics. sPD-L1 levels may further refine a nivolumab-refractory subtype of RCC within transcriptionally based subtypes of RCC.

Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range.

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ∼1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.

Fifth-generation digital immunoassay for prostate-specific antigen by single molecule array technology.

BACKGROUND: Measurement of prostate-specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a limit of quantification 2 logs lower than current ultrasensitive third-generation PSA assays. METHODS: We developed reagents for a bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients. RESULTS: The assay exhibited a functional sensitivity (20% interassay CV) <0.05 pg/mL, total imprecision <10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA concentrations following surgery were found to be predictive of prostate cancer recurrence risk over 5 years. CONCLUSIONS: The robust 2-log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

Changes in Peripheral Blood Regulatory T Cells and IL-6 and IL-10 Levels Predict Response of Pediatric Medulloblastoma and Germ Cell Tumors With Residual or Disseminated Disease to Craniospinal Irradiation.

PURPOSE: Radiation therapy (RT) modulates immune cells and cytokines, resulting in both clinically beneficial and detrimental effects. The changes in peripheral blood T lymphocyte subsets and cytokines during RT for pediatric brain tumors and the association of these changes with therapeutic outcomes have not been well described. METHODS AND MATERIALS: The study population consisted of children (n = 83, aged 3~18) with primary brain tumors (medulloblastoma, glioma, germ cell tumors (GCT), and central nervous system embryonal tumor-not otherwise specified), with or without residual or disseminated (R/D) diseases who were starting standard postoperative focal or craniospinal irradiation (CSI). Peripheral blood T lymphocyte subsets collected before and 4 weeks after RT were enumerated by flow cytometry. Plasma levels of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-α, interferon-γ, and IL-17A were measured by cytometric bead array. RESULTS: Patients with R/D lesions receiving CSI (n = 32) had a post-RT increase in the frequency of CD3+T and CD8+T cells, a decrease in CD4+T cells, and an increase in regulatory T cells (Tregs) and CD8+CD28- suppressor cells, which was more predominantly seen in these patients than in other groups. In the CSI group with such R/D lesions, consisting of patients with medulloblastoma and germ cell tumors, 19 experienced a complete response (CR) and 13 experienced a partial response (PR) on imaging at 4 weeks after RT. The post/pre-RT ratio of Tregs (P = .0493), IL-6 (P = .0111), and IL-10 (P = .0070) was lower in the CR group than in the PR group. Multivariate analysis revealed that the post/pre-RT ratios of Treg, IL-6, and IL-10 were independent predictors of CR (P < .0001, P = .018, P < .0001, respectively). The areas under the receiver operating curves and confidence intervals were 0.7652 (0.5831-0.8964), 0.7794 (0.5980-0.9067), and 0.7085 (0.5223-0.8552) for IL-6, IL-10, and Treg, respectively. The sensitivities of IL-6, IL-10, and Treg to predict radiotherapeutic responses were 100%, 92.3%, and 61.5%, and specificity was 52.6%, 57.9%, and 84.2%, respectively. CONCLUSIONS: CSI treatment to those with R/D lesions predominantly exerted an effect on antitumor immune response compared with both R/D lesion-free but exposed to focal or CSI RT and with R/D lesions and exposed to focal RT. Such CSI with R/D lesions group experiencing CR is more likely to have a decrease in immunoinhibitory molecules and cells than patients who only achieve PR. Measuring peripheral blood Treg, IL-6, and IL-10 levels could be valuable for predicting radiotherapeutic responses of pediatric brain tumors with R/D lesions to CSI for medulloblastoma and intracranial germ cell tumors.

Plasma Neurofilament Light for Prediction of Disease Progression in Familial Frontotemporal Lobar Degeneration.

OBJECTIVE: We tested the hypothesis that plasma neurofilament light chain (NfL) identifies asymptomatic carriers of familial frontotemporal lobar degeneration (FTLD)-causing mutations at risk of disease progression. METHODS: Baseline plasma NfL concentrations were measured with single-molecule array in original (n = 277) and validation (n = 297) cohorts. C9orf72, GRN, and MAPT mutation carriers and noncarriers from the same families were classified by disease severity (asymptomatic, prodromal, and full phenotype) using the CDR Dementia Staging Instrument plus behavior and language domains from the National Alzheimer's Disease Coordinating Center FTLD module (CDR+NACC-FTLD). Linear mixed-effect models related NfL to clinical variables. RESULTS: In both cohorts, baseline NfL was higher in asymptomatic mutation carriers who showed phenoconversion or disease progression compared to nonprogressors (original: 11.4 ± 7 pg/mL vs 6.7 ± 5 pg/mL, p = 0.002; validation: 14.1 ± 12 pg/mL vs 8.7 ± 6 pg/mL, p = 0.035). Plasma NfL discriminated symptomatic from asymptomatic mutation carriers or those with prodromal disease (original cutoff: 13.6 pg/mL, 87.5% sensitivity, 82.7% specificity; validation cutoff: 19.8 pg/mL, 87.4% sensitivity, 84.3% specificity). Higher baseline NfL correlated with worse longitudinal CDR+NACC-FTLD sum of boxes scores, neuropsychological function, and atrophy, regardless of genotype or disease severity, including asymptomatic mutation carriers. CONCLUSIONS: Plasma NfL identifies asymptomatic carriers of FTLD-causing mutations at short-term risk of disease progression and is a potential tool to select participants for prevention clinical trials. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT02372773 and NCT02365922. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that in carriers of FTLD-causing mutations, elevation of plasma NfL predicts short-term risk of clinical progression.

[Cox proportional hazard model analysis of prognosis in patients with carcinoma of esophagus and gastric cardia after radical resection].

OBJECTIVE: To investigate the factors affecting the long-term survival of patients with carcinoma of esophagus and gastric cardia after curative resection. METHODS: The clinical data of 906 patients with carcinoma of esophagus and gastric cardia treated by radical resection in 1996 - 2004 were analyzed retrospectively. Twelve clinicopathological factors possibly influencing survival were encoded and assessed by Cox regression analysis. RESULTS: The 1-, 3- and 5-year cumulative survival rates were 89.8%, 75.4% and 71.7%, respectively. The univariate analysis showed that age, length of tumor, pathological differentiation, number of metastatic lymph nodes, depth of invasion, involvement of adjacent organs and the TNM stage influenced the prognosis significantly (P < 0.01). However, multivariate analysis showed that pathologic differentiation, number of metastatic lymph nodes, involvement of adjacent organs and TNM stage were independent prognostic factors (P < 0.05). CONCLUSION: The independent prognostic factors of the patients with carcinoma of esophagus and gastric cardia are pathologic differentiation, TNM stage, number of metastatic lymph nodes, and involvement of adjacent organs. The other factors influencing survival are age, length of tumor and depth of invasion. Furthermore, invasion of adjacent organs suggests worse prognosis, and should be followed-up closely.

WITHDRAWN: MicroRNA-18a promotes proliferation and metastasis in oral squamous cell carcinoma via targeting KLF4.

Ahead of Print article withdrawn by publisher.

Glial fibrillary acidic protein elevations relate to neuroimaging abnormalities after mild TBI.

OBJECTIVES: To determine whether a panel of blood-based biomarkers can discriminate between patients with suspected mild traumatic brain injury (mTBI) with and without neuroimaging findings (CT and MRI). METHODS: Study participants presented to the emergency department with suspected mTBI (n = 277) with a CT and MRI scan and healthy controls (n = 49). Plasma concentrations of tau, glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolase L1, and neurofilament light chain (NFL) were measured using the single-molecule array technology. RESULTS: Concentrations of GFAP, tau, and NFL were higher in patients with mTBI, compared with those of controls (p's < 0.01). GFAP yielded an area under the curve (AUC) of 0.93 (95% confidence interval [CI] 0.90-0.96), confirming its discriminatory power for distinguishing mTBI from controls. Levels of GFAP, tau, and NFL were higher in patients with trauma-related intracranial findings on CT compared with those with normal CT, with the only significant predictor being GFAP (AUC 0.77, 95% CI 0.70-0.84). Among patients with mTBI, tau, NFL, and GFAP differentiated subjects with and without MRI abnormalities with an AUC of 0.83, with GFAP being the strongest predictor. Combining tau, NFL, and GFAP showed a good discriminatory power (AUC 0.80, 95% CI 0.69-0.90) for detecting MRI abnormalities, even in patients with mTBI with a normal CT. CONCLUSION: Our study confirms GFAP as a promising marker of brain injury in patients with acute mTBI. A combination of various biomarkers linked to different pathophysiologic mechanisms increases diagnostic subgroup accuracy. This multimarker strategy may guide medical decision making, facilitate the use of MRI scanning, and prove valuable in the stratification of patients with brain injuries in future clinical trials. CLASSIFICATION OF EVIDENCE: Class I evidence that blood concentrations of GFAP, tau, and NFL discriminate patients with mTBI with and without neuroimaging findings.

Phosphopeptide analysis by directly coupling two-dimensional planar electrochromatography/thin-layer chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A novel strategy is presented for the fractionation of complex peptide mixtures using two-dimensional planar electrochromatography/thin-layer chromatography (2D PEC/TLC). Phosphopeptides migrate more slowly in the first dimension, based upon their anionic phosphate residues, and certain predominantly acidic phosphopeptides even migrate in the opposite direction, relative to the bulk of the peptides. Phosphopeptides are further distinguished based upon hydrophilicity in the second dimension. This permits a restricted region of the plate to be directly interrogated for the presence of phosphopeptides by mass spectrometry (MS). Phosphopeptide analysis from the plates by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS and tandem MS enabled peptide sequencing and identification.

Increases of Plasma Levels of Glial Fibrillary Acidic Protein, Tau, and Amyloid ß up to 90 Days after Traumatic Brain Injury.

Glial fibrillary acidic protein (GFAP), microtubule-associated protein tau, and amyloid ß peptide (Aß42) have been proposed as diagnostic and prognostic biomarkers in traumatic brain injury (TBI). Single molecule array (Simoa) is a novel technology that employs highly sensitive immunoassays for accurate measurements of candidate biomarkers found at low concentration in biological fluids. Our objective was to trace the trajectory of tau, GFAP, and Aß42 levels in plasma from the acute through subacute stages after TBI, compared with controls. Samples from 34 TBI subjects enrolled in the Citicoline Brain Injury Treatment Trial (COBRIT) were studied. Injury severity was assessed by Glasgow Coma Scale (GCS) and admission CT. Glasgow Outcome Scale Extended (GOSE) was assessed 6 months after injury. Plasma was collected within 24 h (Day 0), and 30 and 90 days after the TBI. Plasma collected from 69 healthy volunteers was used for comparison. At every time point, increases were noted in plasma GFAP (p < 0.0001 for all comparisons), tau (p < 0.0001, p < 0.0001, and p = 0.0044, at Days 0, 30, and 90, respectively), and Aß42 (p < 0.001, p < 0.0001, and p = 0.0203, respectively) in TBI cases compared with controls. The levels were maximal at Day 0 for GFAP and tau and at Day 30 for Aß42. Area under curve (AUC) analyses for Day 0 GFAP and tau were excellent for discrimination of complicated mild TBI (cmTBI) from controls (0.936 and 0.901, correspondingly). Discriminant component analysis (DCA) for all three biomarkers at Days 0 and 30 differentiated controls from cmTBI (91.1% and 89.7% correctly classified, at each time point). Duration of post-traumatic amnesia (PTA) correlated weakly with tau levels at 30 days (Spearman's r = 0.40; 95% CI 0.0003-0.60, p = 0.044). The Marshall CT Grade on admission correlated weakly with Day 30 tau levels (Spearman's r = 0.41; 95% CI 0.04-0.68, p = 0.027). Day 30 Aß42 correlated with GOSE (standardized ß -0.486, p = 0.042). GFAP, tau and Aß42 were increased up to 90 days after TBI compared with controls. Total tau levels correlated with clinical and radiological variables of TBI severity. Plasma Aß42 correlated with clinical outcome. Combination of all three biomarkers at Days 0 and 30 can be used to differentiate controls from cmTBI populations, and may be useful as biomarkers of TBI in both acute and subacute phases.

Repetitive head impact exposure and later-life plasma total tau in former National Football League players.

INTRODUCTION: Blood protein analysis of total tau (t-tau) may be a practical screening biomarker for chronic traumatic encephalopathy (CTE), a neurodegenerative tauopathy associated with repetitive head impact (RHI) exposure. We examined plasma t-tau in symptomatic former National Football League (NFL) players compared with controls and the relationship between RHI exposure and later-life plasma t-tau. METHODS: Ninety-six former NFL players (age 40-69) and 25 same-age controls underwent blood draw to determine plasma t-tau levels. The cumulative head impact index (CHII) quantified RHI exposure. Subjects completed measures of clinical function. RESULTS: A higher CHII predicted greater plasma t-tau in the former NFL players (P = .0137). No group differences in plasma t-tau emerged, but a concentration ≥3.56 pg/mL was 100% specific to former NFL players. Plasma t-tau did not predict clinical function. DISCUSSION: Greater RHI exposure predicted higher later-life plasma t-tau concentrations, and further study on plasma t-tau as a candidate screening biomarker for CTE is warranted.

A digital enzyme-linked immunosorbent assay for ultrasensitive measurement of amyloid-ß 1-42 peptide in human plasma with utility for studies of Alzheimer's disease therapeutics.

BACKGROUND: Amyloid-ß 1-42 peptide (Aß1-42) is associated with plaque formation in the brain of patients with Alzheimer's disease (AD). Pharmacodynamic studies of AD therapeutics that lower the concentrations of Aß1-42 in peripheral blood require highly sensitive assays for its measurement. A digital enzyme-linked immunosorbent assay (ELISA) using single molecule array (Simoa) technology has been developed that provides improved sensitivity compared with conventional ELISA methods using the same antibody reagents. METHODS: A sensitive digital ELISA for measurement of Aß1-42 using antibodies 3D6 and 21F12 was developed. Assay performance was evaluated by repeated testing of pooled human plasma and buffer diluent quality control samples to determine relative accuracy, intra- and inter-assay precision, limit of detection (LOD), lower limit of quantification (LLOQ), dilutional linearity, and spike recovery. The optimized assay was used to quantify Aß1-42 in clinical samples from patients treated with the ß-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721. RESULTS: The prototype assay measured Aß1-42 with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an Aß1-42 peptide standard from Fujirebio. Assay precision was acceptable with intra- and inter-assay coefficients of variation both being ≤10%. Dilutional linearity was demonstrated in sample diluent and immunodepleted human plasma. Analyte spike recovery ranged from 51% to 93% with a mean of 80%. This assay was able to quantify Aß1-42 in all of the 84 clinical samples tested. A rapid reduction in levels of Aß1-42 was detected within 1 h after drug treatment, and a dose-dependent decrease of Aß1-42 levels was also observed over the time course of sample collection. CONCLUSIONS: This digital ELISA has potential utility in clinical applications for quantification of Aß1-42 in plasma where high sensitivity and precision are required.

Plasma ß-amyloid in Alzheimer's disease and vascular disease.

Implementation of amyloid biomarkers in clinical practice would be accelerated if such biomarkers could be measured in blood. We analyzed plasma levels of Aß42 and Aß40 in a cohort of 719 individuals (the Swedish BioFINDER study), including patients with subjective cognitive decline (SCD), mild cognitive impairment (MCI), Alzheimer's disease (AD) dementia and cognitively healthy elderly, using a ultrasensitive immunoassay (Simoa platform). There were weak positive correlations between plasma and cerebrospinal fluid (CSF) levels for both Aß42 and Aß40, and negative correlations between plasma Aß42 and neocortical amyloid deposition (measured with PET). Plasma levels of Aß42 and Aß40 were reduced in AD dementia compared with all other diagnostic groups. However, during the preclinical or prodromal AD stages (i.e. in amyloid positive controls, SCD and MCI) plasma concentration of Aß42 was just moderately decreased whereas Aß40 levels were unchanged. Higher plasma (but not CSF) levels of Aß were associated with white matter lesions, cerebral microbleeds, hypertension, diabetes and ischemic heart disease. In summary, plasma Aß is overtly decreased during the dementia stage of AD indicating that prominent changes in Aß metabolism occur later in the periphery compared to the brain. Further, increased levels of Aß in plasma are associated with vascular disease.

Plasma tau in Alzheimer disease.

OBJECTIVE: To test whether plasma tau is altered in Alzheimer disease (AD) and whether it is related to changes in cognition, CSF biomarkers of AD pathology (including ß-amyloid [Aß] and tau), brain atrophy, and brain metabolism. METHODS: This was a study of plasma tau in prospectively followed patients with AD (n = 179), patients with mild cognitive impairment (n = 195), and cognitive healthy controls (n = 189) from the Alzheimer's Disease Neuroimaging Initiative (ADNI) and cross-sectionally studied patients with AD (n = 61), mild cognitive impairment (n = 212), and subjective cognitive decline (n = 174) and controls (n = 274) from the Biomarkers for Identifying Neurodegenerative Disorders Early and Reliably (BioFINDER) study at Lund University, Sweden. A total of 1284 participants were studied. Associations were tested between plasma tau and diagnosis, CSF biomarkers, MRI measures, 18fluorodeoxyglucose-PET, and cognition. RESULTS: Higher plasma tau was associated with AD dementia, higher CSF tau, and lower CSF Aß42, but the correlations were weak and differed between ADNI and BioFINDER. Longitudinal analysis in ADNI showed significant associations between plasma tau and worse cognition, more atrophy, and more hypometabolism during follow-up. CONCLUSIONS: Plasma tau partly reflects AD pathology, but the overlap between normal aging and AD is large, especially in patients without dementia. Despite group-level differences, these results do not support plasma tau as an AD biomarker in individual people. Future studies may test longitudinal plasma tau measurements in AD.