Serial Serum Samples Research Articles

Biomarker Changes in Response to Tofacitinib Treatment in Patients with Polyarticular Course Juvenile Idiopathic Arthritis.

Examine levels of candidate blood-based biomarkers (CBB) in juvenile idiopathic arthritis (JIA) treated with tofacitinib. JIA patients who participated in clinical trial NCT02592434 received tofacitinib from baseline to week 18. Serial serum samples were assayed for CBB (S100A8/9, S100A12, IL-18, SAA, resistin, VEGF, Angiopoietin-1, Angiopoietin-2, MMP8, MMP2, TIMP1, Leptin, CXCL9, sIL2R, ICAM-1, sTNFr, IL-6, IL-23, MCP1, CCL18, and CCL20). Association of CBB with JIA response to treatment from baseline to week 18 were assessed. This study included 166 patients with polyarticular-course JIA. Paired serum samples from 143 patients were available at both baseline and week 18. There were 35% (50/143) of patients with a JIA-American College of Rheumatology 90 (JIA-ACR90) level improvement while 90/121/137 (63%/85%/96%) achieved JIA-ACR70/50/30 improvement at wk18. Despite small numerical differences by JIA category, there were no baseline CBB values that independently predicted a decrease in JADAS-27 or JIA-ACR90 response by week 18. Decrease in resistin level (baseline to week 18) was significantly associated with wk18 improvement in JADAS-27 and JIA-ACR90 response, after adjusting for age, sex, JIA disease duration and baseline resistin [(r2 0.79, SE, 0.070, p<0.01 and OR(95%CI) = 1.134(1.018,1.264)]. HLA-B27 positivity was significantly associated with not achieving a JIA-ACR90 response at week 18 (p=0.0097). Among the CBB included, only resistin was significantly associated with treatment response, and no CBB was identified that forecasts JIA improvement after initiation of tofacitinib. The association of HLA-B27 positivity with lower response to tofacitinib in JIA is intriguing and merits further study.

The Role of Serum Albumin and Secretory Phospholipase A2 in Sepsis.

Sepsis is caused by a dysregulated host response to an infection that leads to cascading cell death and eventually organ failure. In this study, the role of inflammatory response serum secretory phospholipase A2 (sPLA2) and albumin in sepsis was investigated by determining the activities of the two proteins in serial serum samples collected on different days from patients with sepsis after enrollment in the permissive underfeeding versus standard enteral feeding protocols in an intensive care unit. Serum sPLA2 and albumin showed an inverse relationship with increasing sPLA2 activity and decreasing albumin membrane-binding activity in patients with evolving complications of sepsis. The activities of sPLA2 and albumin returned to normal values more rapidly in the permissive underfeeding group than in the standard enteral feeding group. The inverse sPLA2-albumin activity relationship suggests a complex interplay between these two proteins and a regulatory mechanism underlying cell membrane phospholipid homeostasis in sepsis. The decreased albumin-membrane binding activity in patients' serum was due to its fatty acid-binding sites occupied by pre-bound fatty acids that might alter albumin's structure, binding capacities, and essential functions. The sPLA2-albumin dual serum assays may be useful in determining whether nutritional intervention effectively supports the more rapid recovery of appropriate immune responses in critically ill patients with sepsis.

Krebs von den Lungen-6 as biomarker of the new progressive fibrotic phenotype of interstitial lung disease

BackgroundNovel progressive fibrotic phenotype has recently been proposed characterized by progressive and inexorable worsening of the disease. Krebs von den Lungen-6 (KL-6) has been proposed as fibrotic-ILD biomarker. We aimed to assess the role of KL-6 in fibrotic-ILD and the progressive phenotype in accordance with serial serum KL-6. Methods: 107 patients were enrolled in the study (median age,IQR, 65(54−71)y/o) followed at respiratory diseases and rheumatology units of University of Siena. Thirty-five had diagnoses of IPF, 18 sarcoidosis, 10 PLCH, 5 LAM, 24 fibrotic HP(fHP), 13 RA (4/13 RA-ILD) and 22 SSc (18/22 SSc-ILD). Serial serum samples were collected before therapy (t0) and 24 months later (t1) from IPF, SSc- and RA-ILD patients. Twenty-two healthy controls (HC) were enrolled. Serum samples were assayed for KL-6 concentrations (Fujirebio Europe, Gent, Belgium). Results: Higher KL-6 concentrations were reported in IPF, fHP and SSc-ILD patients than HC (p<0.0001). KL-6 cut-off value of 885 U/mL identified fibrotic-ILD patients. Logistic regression analysis indicated KL-6 (p=0.004) and smoking-habit (p=0.005) affected the ILD diagnosis. The decision tree model showed KL-6>1145 U/mL, DLco≤60.15 %, FVC≤86 % to classify 86 % IPF patients. Inverse correlation between T0-KL-6 and T1-FVC%(r=-0.314, p=0.046) and T1-DLco%(r=-0.327, p=0.038) in the progressive group. Conclusion: KL-6 proved to be a reliable marker for diagnosis and prognosis of fibrotic ILD patients with predictive value in progressive fibrotic patients and a useful marker to identify the new and similar progressive phenotype of IPF and SSc-ILD patients assessing the functional progression in accordance with serial serum KL-6 measurements.

Clinical and immunophenotype correlating with response to immunotherapy in paediatric patients with primary liver carcinoma. A case series

Paediatric hepatocellular carcinomas (HCC) traditionally arise in the context of a normal structural and functional liver and carry a dismal prognosis. While chemotherapy is the frontline standard, there is emerging interest in the study of immunotherapies for paediatric patients with relapsed/refractory disease. There is limited data to support whether immunotherapies will be of utility in this patient population. Six paediatric patients (median age:16 years, range: 12-17at the time of treatment) with advanced hepatocellular neosplams, either conventional hepatocellular or fibrolamellar carcinoma, were treated with immunotherapy. Patients were consented to institutional genomic profiling and biobanking protocols. Baseline samples and serial tissue samples, when available, were evaluated for somatic mutation rate, actionable gene mutations, and pan-immune bulk RNA expression profiling. Results were correlated with clinical course. Three patients responded to checkpoint inhibition: one achieved a complete, durable response and the other two, prolonged stable disease. Three additional patients progressed. Diagnostic tissue from the complete responder demonstrated a higher relative mutational burden and robust immune infiltrate. Pre-treatment samples from the three responders demonstrated decreased expression of genes associated with T-cell dysfunction. A subset of patients with primary paediatric hepatocellular tumours will respond to immunotherapy. Immunotherapies are currently under prospective study for relapsed/refractory liver tumours in paediatric patients. Results from this report support the prospective collection of serial serum and tissue samples which may further identify genomic and immunophenotypic patterns predictive of response. This work was supported by Philanthropic funds (Pan Mass Challenge, Team Angus and Team Perspective).

Evaluating the clinical utility of semi-quantitative luciferase immunosorbent assay using Treponema pallidum antigens in syphilis diagnosis and treatment monitoring

ABSTRACT To assess the clinical applicability of a semi-quantitative luciferase immunosorbent assay (LISA) for detecting antibodies against Treponema pallidum antigens TP0171 (TP15), TP0435 (TP17), and TP0574 (TP47) in diagnosing and monitoring syphilis. LISA for detection of anti-TP15, TP17, and TP47 antibodies were developed and evaluated for syphilis diagnosis using 261 serum samples (161 syphilis, 100 non-syphilis). Ninety serial serum samples from 6 syphilis rabbit models (3 treated, 3 untreated) and 110 paired serum samples from 55 syphilis patients were used to assess treatment effects by utilizing TRUST as a reference. Compared to TPPA, LISA-TP15, LISA-TP17, and LISA-TP47 showed a sensitivity of 91.9%, 96.9%, and 98.8%, specificity of 99%, 99%, and 98%, and AUC of 0.971, 0.992, and 0.995, respectively, in diagnosing syphilis. Strong correlations (rs = 0.89–0.93) with TPPA were observed. In serial serum samples from rabbit models, significant differences in the relative light unit (RLU) were observed between the treatment and control group for LISA-TP17 (days 31–51) and LISA-TP47 (day 41). In paired serum samples from syphilis patients, TRUST titres and the RLU of LISA-TP15, LISA-TP17, and LISA-TP47 decreased post-treatment (P < .001). When TRUST titres decreased by 0, 2, 4, or ≥8-folds, the RLU decreased by 17.53%, 31.34%, 48.62%, and 72.79% for LISA-TP15; 8.84%, 17.00%, 28.37%, and 50.57% for LISA-TP17; 22.25%, 29.79%, 51.75%, and 70.28% for LISA-TP47, respectively. Semi-quantitative LISA performs well for syphilis diagnosis while LISA-TP17 is more effective for monitoring syphilis treatment in rabbit models and clinical patients.

Pharmacokinetics and tolerability of single-dose enteral cannabidiol and cannabidiolic acid rich hemp in horses (Equus caballus).

The pharmacokinetics and tolerability of cannabinoids and their metabolites were determined in eight horses after enteral administration of a commercial CBD/CBDA-rich hemp oil product. Each horse was administered 2 mg/kg or 8 mg/kg CBD/CBDA or no treatment in a randomized cross-over design. Serial serum samples collected over 48 h were analyzed by high performance liquid chromatography with tandem mass spectrometry. Plasma chemistry analysis was performed at 0 h and 24 h. Vital parameters, pedometry, and blinded mentation and gait evaluations were recorded at intervals up to 24 h. Manure production and gastrointestinal transit time were tracked for 48 h after oil administration. The median maximal concentration of CBD and CBDA were 5.2 and 36.95 ng/mL in the 2 mg/kg group, respectively; and 40.35 and 353.56 ng/mL in the 8 mg/kg group. The median half-life of elimination was not calculated for the 2 mg/kg CBD treatment due to lack of time points above the lower quantifiable limit beyond the Cmax while it was 7.75 h in the 8 mg/kg group. CBDA absorption was biphasic. Pharmacokinetic parameters for tetrahydrocannabinol, tetrahydrocannabinolic acid, cannabigerolic acid, and 7-carboxy cannabidiol are also reported. No significant differences in any of the measured tolerability parameters were demonstrated between treatment groups. Single-dose enteral administration of CBD/CBDA-rich hemp extract up to 8 mg/kg does not appear to produce neurologic, behavioral, or gastrointestinal effects in horses.

Arachidonic acid and docosahexaenoic acid levels correlate with the inflammation proteome in extremely preterm infants

Clinical trials supplementing the long-chain polyunsaturated fatty acids (LCPUFAs) docosahexaenoic acid (DHA) and arachidonic acid (AA) to preterm infants have shown positive effects on inflammation-related morbidities, but the molecular mechanisms underlying these effects are not fully elucidated. This study aimed to determine associations between DHA, AA, and inflammation-related proteins during the neonatal period in extremely preterm infants. A retrospective exploratory study of infants (n=183) born below 28 weeks gestation from the Mega Donna Mega trial, a randomized multicenter trial designed to study the effect of DHA and AA on retinopathy of prematurity. Serial serum samples were collected after birth until postnatal day 100 (median 7 samples per infant) and analyzed for phospholipid fatty acids and proteins using targeted proteomics covering 538 proteins. Associations over time between LCPUFAs and proteins were explored using mixed effect modeling with splines, including an interaction term for time, and adjusted for gestational age, sex, and center. On postnatal day one, 55 proteins correlated with DHA levels and 10 proteins with AA levels. Five proteins were related to both fatty acids, all with a positive correlation. Over the first 100 days after birth, we identified 57 proteins to be associated with DHA and/or AA. Of these proteins, 41 (72%) related to inflammation. Thirty-eight proteins were associated with both fatty acids and the overall direction of association did not differ between DHA and AA, indicating that both LCPUFAs similarly contribute to up- and down-regulation of the preterm neonate inflammatory proteome. Primary examples of this were the inflammation-modulating cytokines IL-6 and CCL7, both being negatively related to levels of DHA and AA in the postnatal period. This study supports postnatal non-antagonistic and potentially synergistic effects of DHA and AA on the inflammation proteome in preterm infants, indicating that supplementation with both fatty acids may contribute to limiting the disease burden in this vulnerable population. ClinicalTrials.gov (NCT03201588).

IL-10 Levels Differentially Regulate IFNg Signaling of CART19 and CART22

Introduction: CD19-directed CART (CART19) are highly effective in the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, some pts relapse with CD19 negative disease after CART19, which has led to the development of CD22-directed CART (CART22). While CART22 has produced 70% CR in CD19 negative ALL, this is lower than CART19 and there is less evidence for long-term disease control compared to CART19. Interestingly, toxicity patterns are different with CART22 pts experiencing a later and less severe CRS even at high disease burden. To better understand these differences in efficacy and toxicity we conducted comprehensive serum proteomic profiling on serial samples from pts treated with CART-19 or -22. Methods: Serial serum samples from pts enrolled on two clinical trials of the murine CART19 CTL019 (NCT01626495 &amp; NCT02906371) and CART22 (NCT02650414) were examined. Pts were categorized as having severe (Grade 3 or 4) or minimal CRS (Grade 1 or 2). All statistical analyses were performed using R (v4.2.2) and PRISM (v9). Differential expression analyses were performed between CART19 and CART22 pts. Significance was defined as an absolute fold change &amp;gt; 2, and a false discovery rate (FDR) &amp;lt; 0.05. All in vitro experiments were performed in triplicate. Two-tailed t tests were calculated. Results: 43 pts (CART19 N=26 and CART22 N=17) with 223 unique timepoints were included in this analysis. All CART22 pts had minimal CRS; 13 CART19 pts had minimal and 13 had severe CRS. Both cohorts had high disease burden prior to infusion (CART19 mean = 29.9%, CART22 mean=44.3%, p=0.19). All pts who received CART22 had previously received CD19 directed immunotherapy (CART19 N=16, blinatumomab N=1). At the pre-infusion timepoint, 12 proteins were significantly different between pts in the CART19 and CART22 cohorts. The protein most significantly differentially expressed in patients who subsequently underwent CART22 treatment was the anti-inflammatory cytokine IL-10. IL-10 levels were consistently elevated in patients who received CART22, surpassing levels at all observed timepoints, even when compared with patients treated with CART19 who developed severe CRS (See Fig 1). We therefore hypothesized the IL-10 levels may be impacting the efficacy and CRS associated with CART22. We next tested the impact of IL-10 on IFNγ as a surrogate marker of CRS in a series of in vitro experiments. CART-19 or -22 cells from the same donor were co-cultured with NALM-6 cells in the presence and absence of IL-10. Without IL-10 present, CART22 cells produced higher levels of IFNγ than CART19 cells (8959 vs 6395 pg/mL, p=0.005). Inversely, with IL-10 present, CART19 cells produced more IFNγ than CART22 cells (21006 vs 15157 pg/mL, p=0.05). We evaluated the length of immune synapse (IS) formation, the time between CART cell interaction and blast killing, using live-cell microscopy, and found that CART22 cells had a trend towards a prolonged IS formation relative to CART19 cells (median time to detachment 9.1 min vs. 3.5 min, p=ns). Finally, given the differences in time of onset of CRS following CART19 vs CART22, we looked at levels of IFNγ and its canonical responder CXCL9 between pts with minimal CRS only. As expected, we found a later peak of IFNγ in CART22 pts compared to CART19 pts (Figure 2). Interestingly, despite similar peaks in IFNγ, CXCL9 levels were higher in CART22 pts, implying dysregulation of the IFNγ/CXCL9 axes in CART22 but not CART19 pts. Conclusions: Comparing comprehensive serum proteomic profiling between CART19 and CART22 pts we make several novel observations. First, IL-10 levels are significantly higher in CART22 pts at pre-infusion, suggesting that previous immunotherapy has caused an upregulation of this anti-inflammatory cytokine. Second, we show that IL-10 levels have a differential impact on IFNγ levels in CART19 vs CART22 cells. This may impact both CRS associated with CART cells and efficacy and may contribute to observed differences in IFNγ and CXCL9 levels seen between the two patient populations. These findings are significant for the clinical translation of CART22 and other immunotherapies, as previous immunotherapy may lead to sustained alterations in the cytokine milieu. Furthermore, IL-10 is a targetable cytokine that could be modulated in future studies to improve efficacy of CART22.

Monitoring Minimal Residual Disease in Patients with Multiple Myeloma By Tracking Serum M-Protein Using Mass Spectrometry

Objective： We performed this retrospective real-world study to evaluate the clinical utility of blood-based mass spectrometry for longitudinal M-protein monitoring during the disease course of patients of multiple myeloma（MM）. Methods：We initially analyzed serial serum samples of 56 patients with MM by clonal peptide MS Method named EasyM MS, then we compared the MS results with those determined using serum immunofixation electrophoresis (IFE),multiparameter flow cytometry (MFC), next-generation flow cytometry (NGF), to establish the concordance between different disease-monitoring protocols. A sensitivity for MFC MRD negativity of at least 1×10 -4 anda sensitivity for NGF MRD negativity of at least 1×10 -5.Additionally, we determined the optimal cutoff value of EasyM MS negativity to satisfy both the sensitivity and specificity between MS and NGF, and explored the prognostic impact of MS-MRD status. Results：The median diagnostic age of 56 NDMM patients was 56 years old, including 34 males (60.7%). There were 38 patients with IgG type MP and 18 patients with IgA type MP. 47 patients (83.9%) chose to receive proteasome inhibitor - based induction therapy, and a total of 44 patients (78.6%) received first-line ASCT treatment.Among 56 MM patients，38 patients with IgG type MP can further identify IgG subclasses through mass spectrometry. Additionally, 56 patients with MM have successfully used MS to identify their unique clonal peptides of MP. 56 patients provided a total of 447 samples of serum and all sequential serum samples were successfully measured by using the MS method. The initial comparison of the performance of blood-based MS-MRD with that of the conventional M-protein diagnostic method IFE used . Among the 397 comparable results collected at similar timepoints, 70.8% (281/397) of the samples were both M-MRD- and IFE-positive, 3.3% (13/397) were both MS-MRD- and IFE-negative, 25.7% (102/397) were MS-MRD-positive and IFE-negative.Flow cytometry MRD was performed on 218 bone marrow aspirates in this cohort, including 126 analyzed with MFC and 92 analyzed with NGF. MS-MRD detection was also performed on comparable serum acquired at the same time points. The sensitivity of MS-MRD was 100.0% in comparison with both MFC and NGF, with specificity of 1.9% and 22.2%, respectively. Considering the overly high sensitivity of EasyM at a cutoff of dotp &amp;gt;0.9, we used the sensitivity of NGF as the standard for detection (i.e., a detection sensitivity ≤10 -5) and set the EasyM MS negative cutoff value using ROC analysis, determined to be &amp;lt;1.86% of that at first diagnosis. Using these assumptions, we again performed the aforementioned analyses comparing the sensitivity and specificity between EasyM and IFE, MFC, and NGF. Under this scenario, the sensitivity of EasyM remained high in comparison with IFE (88.3%), MFC (85.1%), and NGF (93.2%), but the specificity was also greatly increased to 90.4%, 55.8%, and 93.2%, respectively. We then divided patients into MS-positive and MS-negative groups according to whether they achieved MS-MRD negativity, and median PFS and OS were calculated for each group. The median PFS of the MS-positive and MS-negative groups was 20.6 (95% CI: 16.6, 24.6) and 61.8 (39.7, 83.9) months, respectively . The median OS was 55.7 (95% CI:31.8, 79.5) months for the MS-positive group and not reached (NR) for the MS-negative group ,Both mPFS and mOS were significantly longer among the MS-negative group compared with the MS-positive group (both p &amp;lt; 0.001). Conclusion：EasyM exhibits high sensitivity and minimally invasive technological advantages, and also has important significance in efficacy evaluation and prognosis evaluation. In the future, EasyM MS detection can be used as a minimally invasive MRD monitoring method based on peripheral blood in clinical practice.

Pharmacokinetic-Targeting and Dose-Adjustment of I.V. Busulfan for Myeloablative Conditioning in Allogeneic Hematopoietic Cell Transplantation

Introduction: Pharmacokinetically (PK)-targeting intravenous busulfan improves outcomes following allogeneic hematopoietic cell transplantation (HCT). However, the optimal busulfan area under the curve (AUC) likely differs based on the specific conditioning regimen, and the target busulfan exposure is unknown in combination with fludarabine and low-dose total body irradiation (TBI). Methods: This study included 1019 adult HCT recipients that received myeloablative conditioning including fludarabine, busulfan, anti-thymocyte globulin, and low-dose (4cGy) TBI. Busulfan was administered as a total dose of ~3.2mg/kg given equally from days -5 to -2 pre-transplant. Total AUC was estimated using measurements of serial serum samples. Multivariate Cox and Fine-Gray regression were used for comparison of AUC subgroups. The primary outcomes of interest were relapse-free survival (RFS) and overall survival (OS). Results: Median AUC was 62.3 mg∙hr/L (range: 39.4-128.0 mg∙hr/L). Total AUC exposure of 49.3-57.5 mg∙hr/L was associated with greater RFS (67% vs. 47%, HR=1.82, P=0.014) and OS (71% vs. 46%, HR=1.99, P=0.008) compared to patients with higher AUCs of 57.5-73.9 mg∙hr/L. Although very low (&amp;lt;49.3 mg∙hr/L) or very high (&amp;gt;73.9 mg∙hr/L) AUCs trended towards worse RFS and OS compared to 49.3-57.5 mg∙hr/L, this analysis was limited by the small number of patients with extreme AUCs and did not reach statistical significance. Except potentially for patients with a high/very high HCT disease risk index, 49.3-57.5 mg∙hr/L appeared to be the optimal AUC regardless of patient sex, age, or primary disease. Conclusion: Within the evaluated AUC range, 49.3-57.5 mg∙h/L appeared to be associated with the most favourable survival. Pharmacokinetic-targeting to this range may improve outcomes.

383. Pharmacokinetics and Safety in Healthy Adults of RSM01, a Novel RSV Monoclonal Antibody, and Population PK Modeling to Support Pediatric Development

Abstract Background RSV is a common cause of infant respiratory infection and hospitalization. RSM01 is a novel anti-RSV mAb targeting the F glycoprotein essential for viral entry with a YTE mutation for half-life extension. Phase 1a PK and safety results are presented, along with an adult population PK model used to predict pediatric exposures via simulation. Methods In this double-blind Phase 1a study, healthy males and non-childbearing females aged 18-49 yrs were randomized 6:1 within dose cohorts to receive a single dose of RSM01 or placebo (PBO) in the following RSM01 dose sequence: 300mg IV, 300mg IM or 1000mg IV, 3000mg IV and cohort at 600mg IM. Serial serum samples were taken to measure RSM01 PK using a validated immunoassay method. A noncompartmental analysis (NCA) was conducted, and a population PK model was developed to characterize adult PK and predict pediatric PK using allometric scaling. Pediatric RSM01 PK was predicted for African infants at different IM doses. Results Overall, 56 participants received RSM01 (n=48) or PBO (n=8): median age 30 yrs (range 19-48), 52% male (n = 29), 66% White (n=37), median BMI 24.6 kg/m2 (range 18.1-29.4). 12/48 (25%) RSM01 recipients and 2/8 (25%) PBO recipients reported an AE, with no hypersensitivity reactions, serious AEs, or deaths reported. The median Tmax was ∼6–7 days after IM injection. Cmax and AUClast increased dose proportionally following IV administration. RSM01 adult PK was characterized using a two-compartment model with first-order elimination. IM absorption was described using a zero-order rate constant. Inter-individual variability was included for clearance (CL), central and peripheral volumes (V2 and V3). The RSM01 model estimated that distribution and a terminal half-life were 1.3 and 79.1 days, respectively, and IM bioavailability was ∼82%. The model predicted that following a 50 mg IM dose, median serum RSM01 concentration on day 150 in African infants 0-12 months old to be ∼ 28 mcg/mL, higher than the target exposure based on preclinical models (6.8 mcg/mL). Serum RSM01 NCA PK summary Note: AUClast and Cmax values are presented as geometric mean (CV). Tmax and Tlast are presented as median (min-max). Arithmetic Mean (±SD) Serum RSM01 Concentration-Time Curve by Treatment Note: Horizontal blue dashed line represents the target exposure based on preclinical models (6.8 mcg/mL). Conclusion RSM01 was shown to be well tolerated in healthy adults. Its long half-life and the predicted infant PK profiles support further pediatric development and indicate the potential for dosing once every RSV season. Disclosures Micha Levi, PhD, Bill &amp; Melinda Gates Medical Research Institute: Salary as a full-time employee Shayne Watson, MSc, Bill &amp; Melinda Gates Medical Research Institute: Salary as a full-time employee Dale Taylor, MD, Sanara Medtech Inc: Stocks/Bonds Luisa M. Stamm, MD, PhD, Bill &amp; Melinda Gates Medical Research Institute: Salary as a full-time employee Michael W. Dunne, MD, Bill &amp; Melinda Gates Medical Research Institute: Salary as a full-time employee

Temporal Profiles of P-Tau, T-Tau, and P-Tau:Tau Ratios in Cerebrospinal Fluid and Blood from Moderate-Severe Traumatic Brain Injury Patients and Relationship to 6-12 Month Global Outcomes.

Traumatic brain injury (TBI) can initiate progressive injury responses, which are linked to increased risk of neurodegenerative diseases known as "tauopathies." Increased post-TBI tau hyperphosphorylation has been reported in brain tissue and biofluids. Acute-to-chronic TBI total (T)-tau and phosphorylated (P)-tau temporal profiles in the cerebrospinal fluid (CSF) and serum and their relationship to global outcome is unknown. Our multi-site longitudinal study examines these concurrent profiles acutely (CSF and serum) and also characterizes the acute- to-chronic serum patterns. Serial serum and CSF samples from individuals with moderate-to-severe TBI were obtained from two cohorts (acute, subacute, and chronic samples from University of Pittsburgh [UPitt] [n = 286 unique subjects] and acute samples from Baylor College of Medicine [BCM] [n = 114 unique subjects]) and assayed for T-tau and P-tau using the Rolling Circle Amplification-Surround Optical Fiber ImmunoAssay platform. Biokinetic analyses described serum T-tau and P-tau temporal patterns. T-tau and P-tau levels are compared with those in healthy controls (n = 89 for both CSF and serum), and univariate/multivariable associations are made with global outcome, including the Disability Rating Scale (DRS) and the Glasgow Outcome Scale-Extended (GOS-E) scores at 3 and 6 months post-TBI (BCM cohort) and at 6 and 12 months post-TBI (UPitt cohort). For both the UPitt and BCM cohorts, temporal increases in median serum and CSF T-tau and P-tau levels occurred over the first 5 days post-injury, while the initial increases of P-tau:T-tau ratio plateaued by day 4 post-injury (UPitt: n = 99, BCM: n = 48). Biokinetic analyses with UPitt data showed novel findings that T-tau (n = 74) and P-tau (n = 87) reached delayed maximum levels at 4.5 and 5.1 days, while exhibiting long serum half-lives (152 and 123 days), respectively. The post-TBI rise in acute (days 2-6) serum P-tau (up to 276-fold) far outpaced that of T-tau (7.3-fold), leading to a P-tau:T-tau increase of up to 267-fold, suggesting a shift toward tau hyperphosphorylation. BCM analyses showed that days 0-6 mean CSF T-tau and P-tau levels and P-tau:T-tau ratios were associated with greater disability (DRS) (n = 48) and worse global outcome (GOS-E) (n = 48) 6 months post-injury. Days 0-6 mean serum T-tau, P-tau, and P-tau:T-tau ratio were not associated with outcome in either cohort (UPitt: n = 145 [DRS], n = 154 [GOS-E], BCM: n = 99 [DRS and GOS-E]). UPitt multivariate models showed that higher chronic (months 1-6) mean P-tau levels and P-tau:T-tau ratio, but not T-tau levels, are associated with greater disability (DRS: n = 119) and worse global outcomes (GOS-E: n = 117) 12 months post-injury. This work shows the potential importance of monitoring post-TBI T-tau and P-tau levels over time. This multi-site longitudinal study features concurrent acute TBI T-tau and P-tau profiles in CSF and serum, and also characterizes acute-to-chronic serum profiles. Longitudinal profiles, along with no temporal concordance between trajectory groups over time, imply a sustained post-TBI shift in tau phosphorylation dynamics that may favor tauopathy development chronically.

Identification of a serum-based microRNA signature that detects recurrent oral squamous cell carcinoma before it is clinically evident

BackgroundSurvival rates for oral squamous cell carcinoma (OSCC) have remained poor for decades, a fact largely attributable to late-stage diagnoses and high recurrence rates. We report analysis of serum miRNA expression in samples from patients with high-risk oral lesions (HRL, including OSCC/carcinoma in situ lesions) and healthy non-cancer controls, with the aim of non-invasively detecting primary or recurrent disease before it is clinically evident.MethodsDiscovery, test, and validation sets were defined from a total of 468 serum samples (305 HRL and 163 control samples). Samples were analysed using multiple qRT-PCR platforms.ResultsA two-miRNA classifier comprised of miR-125b-5p and miR-342-3p was defined following discovery and test analyses. Analysis in an independent validation cohort reported sensitivity and specificity of ~74% for this classifier. Significantly, when this classifier was applied to serial serum samples taken from patients both before treatment and during post-treatment surveillance, it identified recurrence an average of 15 months prior to clinical presentation.ConclusionsThese results indicate this serum miRNA classifier is effective as a simple, non-invasive monitoring tool for earlier detection of recurrent disease when lesions are typically smaller and amenable to a wider array of treatment options to improve survival.

Serum miRNA-based signature indicates radiation exposure and dose in humans: A multicenter diagnostic biomarker study

PurposeMouse and non-human primate models showed that serum miRNAs may be used to predict the biological impact of radiation doses. We hypothesized that these results can be translated to humans treated with total body irradiation (TBI), and that miRNAs may be used as clinically feasible biodosimeters. MethodsTo test this hypothesis, serial serum samples were obtained from 25 patients (pediatric and adults) who underwent allogeneic stem-cell transplantation and profiled for miRNA expression using next-generation sequencing. miRNAs with diagnostic potential were quantified with qPCR and used to build logistic regression models with lasso penalty to reduce overfitting, identifying samples drawn from patients who underwent total body irradiation to a potentially lethal dose. ResultsDifferential expression results were consistent with previous studies in mice and non-human primates. miRNAs with detectable expression in this and two prior animal sets allowed for distinction of the irradiated from non-irradiated samples in mice, macaques and humans, validating the miRNAs as radiation-responsive through evolutionarily conserved transcriptional regulation mechanisms. Finally, we created a model based on the expression of miR-150-5p, miR-30b-5p and miR-320c normalized to two references and adjusted for patient age with an AUC of 0.9 (95%CI:0.83–0.97) for identifying samples drawn after irradiation; a separate model differentiating between high and low radiation dose achieved AUC of 0.85 (95%CI: 0.74–0.96). ConclusionsWe conclude that serum miRNAs reflect radiation exposure and dose for humans undergoing TBI and may be used as functional biodosimeters for precise identification of people exposed to clinically significant radiation doses.

Serum Immune Responses to Group A Streptococcal Antigens following Pharyngeal Acquisitions among Children in Cape Town, South Africa.

There is limited information on the human immune response following infection with group A Streptococcus (Strep A). Animal studies have shown, in addition to the M protein, that shared Strep A antigens elicit protective immunity. This study aimed to investigate the kinetics of antibody responses against a panel of Strep A antigens in a cohort of school-aged children in Cape Town, South Africa. Participants provided serial throat cultures and serum samples at two-monthly follow-up visits. Strep A recovered were emm-typed, and serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA) to assess immune responses to thirty-five Strep A antigens (10-shared and 25-M peptides). Serologic evaluations were performed on serial serum samples from 42 selected participants (from 256 enrolled) based on the number of follow-up visits, the frequency of visits, and throat culture results. Among these, there were 44 Strep A acquisitions, 36 of which were successfully emm-typed. Participants were grouped into three clinical event groups based on culture results and immune responses. A preceding infection was most convincingly represented by a Strep A-positive culture with an immune response to at least one shared antigen and M peptide (11 events) or a Strep A-negative culture with antibody responses to shared antigens and M peptides (9 events). More than a third of participants demonstrated no immune response despite a positive culture. This study provided important information regarding the complexity and variability of human immune responses following pharyngeal acquisition of Strep A, as well as demonstrating the immunogenicity of Strep A antigens currently under consideration as potential vaccine candidates. IMPORTANCE There is currently limited information regarding the human immune response to group A streptococcal throat infection. An understanding of the kinetics and specificity of antibody responses against a panel of Group A Streptococcus (GAS) antigens will serve to refine diagnostic approaches and contribute to vaccine efforts, which together will serve to reduce the burden of rheumatic heart disease, a major source of morbidity and mortality especially in the developing world. This study, utilizing an antibody-specific assay, uncovered three patterns of response profiles following GAS infection, among 256 children presenting with sore throat to local clinics. Overall, the response profiles were complex and variable. Of note, a preceding infection was most convincingly represented by a GAS-positive culture with an immune response to at least one shared antigen and M peptide. Also, more than a third of participants demonstrated no immune response despite a positive culture. All antigens tested were immunogenic, providing guidance for future vaccine development.

Relationship between Body Composition and Serum Immunoglobulin Concentrations after Administration of Intravenous Immune Globulin-Preclinical and Clinical Evidence.

The purpose of this study was to investigate the effect of obesity on immunoglobulin G (IgG) pharmacokinetics in a rat model of obesity, and to collect clinical evidence for an association between the body composition and intravenous immune globulin (IVIG) pharmacokinetic parameters in humans. In a preclinical study, pharmacokinetics of human IgG was evaluated after intravenous (IV) and subcutaneous (SC) delivery to obese and lean rats (n = 6 in each group). Serial serum samples were analyzed using an ELISA. The animal body composition was assessed using computer tomography. Patients with primary immunodeficiency currently managed with IVIG, and at a steady state, were enrolled in the clinical study (n = 8). Serum immune globulin (Ig) concentrations were measured at baseline and immediately after the administration of two consecutive treatments, with an additional measurement at two weeks after the first administration. In addition to the patient demographic and clinical characteristics, body composition was measured using bioelectrical impedance analysis. The pharmacokinetics of human IgG was significantly different between the obese and lean rats after both the IV and SC administration of 0.5 g/kg. Furthermore, a significant difference in endogenous rat IgG was observed between the two strains. In the human study, total serum IgG and subtype (IgG1, IgG2, IgG3, IgG4) half-life negatively correlated with the body mass index and fat mass. The mean change in the total serum IgG concentration was significantly correlated to body mass index and fat mass. The results of the studies corroborated one another. In the animal study, most pharmacokinetic parameters of human IgG following IV and SC administration were significantly affected by obesity and changes in the body composition. In the clinical study, the mean serum IgG change after the IVIG administration strongly correlated to the BMI and body fat mass. Future studies are needed to establish the outcomes achieved with more frequent dosing in obese individuals with primary immunodeficiency.

Abstract WP209: Changes In Serum Superoxide Dismutase 3 (sod3) Following Acute Ischemic Stroke: A Pilot Study

Introduction: Acute ischemic stroke (AIS) elicits peripheral immune activation and reactive inflammatory changes within the lung that contribute to cerebral reperfusion injury. Expression of the antioxidant SOD3 within the lung exerts an intrinsic anti-inflammatory bias on the systemic vasculature. In preclinical models, lung and serum SOD3 levels decline acutely following AIS, while enforced lung SOD3 expression blunts post-stroke inflammation and reperfusion injury. Linking changes in serum SOD3 with stroke severity in a clinical context could have important diagnostic and therapeutic implications. Objectives: 1) Compare serum SOD3 levels among AIS patients vs. controls at initial hospital presentation; 2) Evaluate changes in serum SOD3 in AIS patients during hospitalization; 3) Establish whether serum SOD3 levels correlate with stroke severity. Methods: Inclusion criteria : patients (≥18 years) presenting with AIS (n=7) not treated with either tPA or thrombectomy. Patients with MRI-negative TIA or stroke mimics served as controls (n=8). Exclusion criteria : history of stroke or ICH, seizure at presentation, infection or trauma in the past 30 days, malignancy, autoimmune disease, or major surgery within 90 days. Analyses: Baseline (12-24 hours from LKN) and serial serum samples (1-7 days) were analyzed by automated capillary Western for total SOD3 and compared against the presenting NIH stroke scale score. Results: Analyses revealed 35% (range +9% to -53%) lower initial SOD3 levels in patients with acute ischemic stroke vs. controls, with NIHSS scores ranging from 0 to 10 (median 0.5). Median SOD3 levels in AIS patients were 45,204, 31214, and 41,133 AUC (area under curve) at 12, 24, and 72 hours, respectively. While differences between median peak to nadir SOD3 levels were significant (52117 ± 16052 vs. 27798 ± 7529, p=0.015, two-tailed t-test) in AIS patients, serum SOD3 levels did not correlate with stroke severity. Conclusion: These results suggest minor ischemic stroke is associated with acute reductions in serum SOD3. While further study is required, this finding has potential clinical implications when considering the potential utility of SOD3 as a disease marker and potential therapeutic in the setting of AIS.

Human Proteome Microarray identifies autoantibodies to tumor-associated antigens as serological biomarkers for the diagnosis of hepatocellular carcinoma.

The identification of the high-efficiency and non-invasive biomarkers for hepatocellular carcinoma (HCC) detection is urgently needed. This study aims to screen out potential autoantibodies to tumor-associated antigens (TAAbs) and to assess their diagnostic value for HCC. Fifteen potential TAAbs were screened out from the Human Proteome Microarray by 30 HCC sera and 22 normal control sera, of which eight passed multiple-stage validations by ELISA with a total of 1625 human serum samples from normal controls (NCs) and patients with HCC, liver cirrhosis, chronic hepatitis B, gastric cancer, esophageal cancer, and colorectal cancer. Finally, an immunodiagnostic model including six TAAbs (RAD23A, CAST, RUNX1T1, PAIP1, SARS, PRKCZ) was constructed by logistic regression, and yielded the area under curve (AUC) of 0.835 and 0.788 in training and validation sets, respectively. The serial serum samples from HCC model mice were tested to explore the change in TAAbs during HCC formation, and an increasing level of autoantibodies was observed. In conclusion, the panel of six TAAbs can provide potential value for HCC detection, and the strategy to identify novel serological biomarkers can also provide new clues in understanding immunodiagnostic biomarkers.

Intravenous immunoglobulins, cyclosporine, and best supportive care in epidermal necrolysis: Diverse effects on systemic inflammation.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but potentially life-threatening cutaneous adverse reactions. There is still no consensus on adjuvant treatments, and little is known about their effects on systemic inflammation in SJS/TEN. Our aim was to characterize the systemic and cutaneous immune profiles of SJS/TEN patients and to investigate whether/how intravenous immunoglobulins (IVIG), cyclosporine A (CSA), and best supportive care only (BSCO) affected the systemic immune signature and clinical outcome (6week-mortality, complications, hospitalization stay). We included 16 patients with SJS/TEN, treated with high-dose IVIG (n=8), CSA (n=4) or BSCO (n=4). Serial serum samples were obtained prior-, 5-7 days, and 21 days after treatment onset. Serum levels of inflammation-/immune response-associated proteins were measured by high-throughput proteomics assay (OLINK) and cytotoxic molecules by ELISA. RNA extracted from skin biopsies collected prior treatment was analyzed by Nanostring. Serum inflammatory profiles in SJS/TEN patients were notably characterized by massive upregulation of type 1 immune response and proinflammatory markers. Surprisingly, there was limited overlap between cutaneous and serum immune profiles. Serial serological measurements of immune response markers showed very diverse dynamics between the different treatment groups. IVIG-treated patients showed completely different dynamics and most significant proteomic changes in an early phase (Day 5-7). In all treatment groups, type 1-/inflammatory response markers were dampened at day 21. Clinically, there were no outcome differences. Our study demonstrates that BSCO, CSA, and IVIG have very diverse biological effects on the systemic inflammatory response in SJS/TEN, which may not correlate with clinical outcome differences.

Cerebrospinal fluid concentrations of fluoroquinolones and carbapenems in tuberculosis meningitis.

Background: Tuberculosis meningitis (TBM) is the most lethal form of TB. It is difficult to treat in part due to poor or uncertain drug penetration into the central nervous system (CNS). To help fill this knowledge gap, we evaluated the cerebrospinal fluid (CSF) concentrations of fluoroquinolones and carbapenems in patients being treated for TBM. Methods: Serial serum and CSF samples were collected from hospitalized patients being treated for TBM. CSF was collected from routine lumbar punctures between alternating timepoints of 2 and 6h after drug administration to capture early and late CSF penetration. Rich serum sampling was collected after drug administration on day 28 for non-compartmental analysis. Results: Among 22 patients treated for TBM (8 with confirmed disease), there was high use of fluoroquinolones (levofloxacin, 21; moxifloxacin, 10; ofloxacin, 6) and carbapenems (imipenem, 11; meropenem, 6). Median CSF total concentrations of levofloxacin at 2 and 6h were 1.34mg/L and 3.36mg/L with adjusted CSF/serum ratios of 0.41 and 0.63, respectively. For moxifloxacin, the median CSF total concentrations at 2 and 6h were 0.78mg/L and 1.02mg/L with adjusted CSF/serum ratios of 0.44 and 0.62. Serum and CSF concentrations of moxifloxacin were not affected by rifampin use. Among the 76 CSF samples measured for carbapenem concentrations, 79% were undetectable or below the limit of detection. Conclusion: Fluoroquinolones demonstrated high CSF penetration indicating their potential usefulness for the treatment of TBM. Carbapenems had lower than expected CSF concentrations.