Normal Donors Research Articles

Prime Editing of the α-Thalassemia Hb Constant Spring Mutation

Hemoglobin Constant Spring (HbCS) is the most common non-deletional α-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. Although the α-globin is expressed from two copies of homologous genes ( HBA2 and HBA1), the constant spring (CS) mutation CD142 (UAA > CAA) occurs only in HBA2 gene that leads to the instability of both mRNA and protein in erythroid cells. The newly developed gene editing technology prime editors (PEs) can generate all types of substitutions, providing therapeutic potential in different types of diseases. However, the efficiency of primer editor in primary hematopoietic stem cells is still a big challenge and lack of study in the HbCS mutation. We first created the endogenous HbCS mutation in human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells, providing as a model for prime editing optimization. The protein of α cs chain (a larger protein with extra 31 amino acids) was detected in the model cells with HBA2 bi-allelic CS mutation and normal HBA1, which exhibited the delay of expansion, higher apoptosis rate and altered erythroid maturation markers after induced differentiation. To correct the CS mutation for converting HbCS to HbA, we performed ex vivo delivery of mRNA encoding the prime editor together with a pegRNA and a nick guide RNA into the Hb CS HUDEP2 model cells and achieved the successful in-situ correction with an efficiency of 39.883±9.720% of CS alleles without enrichment, while no measurable editing in the same loci of HBA1 as evaluated by both Sanger sequencing and NGS. More importantly, the correction resulted in a significant decrease of α cs chain, thereby alleviated erythroid phenotype after induced maturation with reducing the apoptosis rate. After that, we collected the primary Hb H-CS patients derived CD34 + hematopoietic stem/progenitor cells (HSPCs) and confirmed that the defect on erythroid expansion, differentiation in Hb H-CS patients derived CD34 + HSPCs compared to the normal donor, especially demonstrated by lower enucleation rate and higher apoptosis. Moreover, the protein of α cs chain was detected in the erythroblast from Hb H-CS CD34+ HSPCs. We further successfully corrected the CS mutation in Hb H-CS patient derived CD34 + HSPCs with an efficiency of 23.68% by mRNA electroporation. At the same time, the editing efficiency was achieved with minimal generation of insertions and deletions and a low level of Cas9-dependent DNA off-target editing. Importantly, we observed an ~23% decrease of α cs chain protein and significant restoration of the enucleation defect from the differentiated erythroblast after PE correction in CD34 + HSPCs. In summary, these results suggests that the delivery of PE mRNA together with synthesized pegRNA and nick sgRNA can successfully achieve the in-situ correction in Hb HbCS mutation site in the model cells and the primary Hb H-CS patient derived CD34 + HSPCs. Our results provide proof of principle for prime editing for its therapeutic potential in directly and precisely correction of the Hb-CS mutation.

Attenuation of binuclear hepatocytes in the paracancerous liver tissue is associated with short-term recurrence of hepatocellular carcinoma post-radical surgery.

Short-term recurrence of hepatocellular carcinoma (HCC) after radical resection leads to dismal outcomes. To screen high-recurrence risk patients to provide adjuvant treatment is necessary. Herein, based on our previous research, we further focused on the changes in the abundance of binuclear hepatocytes (ABH) in the paracancerous liver tissue to discuss the relationship between the attenuation of binuclear hepatocytes and postoperative short-term recurrence, by combining with the assessment of the value of a reported independent early recurrence risk factor in HCC, protein induced by vitamin K absence or antagonist-II (PIVKA-II). A cohort of 142 paracancerous liver tissues from HCC patients who received radical resection was collected. Binuclear hepatocytes were reduced in the paracancerous liver tissues, compared with the liver tissues from normal donors. ABH was negatively correlated with clinical features such as tumor size, TNM stages, tumor microsatellite formation, venous invasion, and Alpha-fetoprotein (AFP) level, as well as the expression of E2F7 and Anillin, which are two critical regulators concerning the hepatocyte polyploidization. According to the short-term recurrence information, ABH value was laminated, and univariate and multivariate logistic regression was performed to analyze the relationship between paracancerous ABH and short-term tumor relapse. Simultaneously, the predictive effectiveness of the ABH value was compared with the preoperative PIVKA-II value. As observed, the paracancerous ABH value below 1.5% was found to be an independent risk factor for recurrence. In conclusion, the paracancerous ABH is a credible indicator of short-term recurrence of HCC patients after radical resection, and regular assessment of ABH might help to prevent short-term HCC recurrence.

Epigenetic regulation of 15-lipoxygenase-1 expression in human chondrocytes by promoter methylation.

15-Lipoxygenase-1 (15-LOX-1) catalyzes the biosynthesis of many anti-inflammatory and immunomodulatory lipid mediators and was reported to have protective properties in several inflammatory conditions, including osteoarthritis (OA). This study was designed to evaluate the expression of 15-LOX-1 in cartilage from normal donors and patients with OA, and to determine whether it is regulated by DNA methylation. Cartilage samples were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee joint replacement surgery. The expression of 15-LOX-1 was evaluated using real-time polymerase chain reaction (PCR). The role of DNA methylation in 15-LOX-1 expression was assessed using the DNA methyltransferase inhibitor 5-Aza-2'-desoxycytidine (5-Aza-dC). The effect of CpG methylation on 15-LOX-1 promoter activity was evaluated using a CpG-free luciferase vector. The DNA methylation status of the 15-LOX-1 promoter was determined by pyrosequencing. Expression of 15-LOX-1 was upregulated in OA compared to normal cartilage. Treatment with 5-Aza-dC increased 15-LOX-1 mRNA levels in chondrocytes, and in vitro methylation decreased 15-LOX-1 promoter activity. There was no difference in the methylation status of the 15-LOX-1 gene promoter between normal and OA cartilage. The expression level of 15-LOX-1 was elevated in OA cartilage, which may be part of a repair process. The upregulation of 15-LOX-1 in OA cartilage was not associated with the methylation status of its promoter, suggesting that other mechanisms are involved in its upregulation.

Cytokine Profiling of Cyst Fluid and Tumor-Associated Macrophages in Cystic Vestibular Schwannoma.

The vestibular schwannoma (VS) secretome can initiate monocyte recruitment and macrophage polarization to M1 (proinflammatory) and/or M2 (protumorigenic) phenotypes, which in turn secrete additional cytokines that contribute to the tumor microenvironment. Profiling cyst fluid and cerebrospinal fluid (CSF) in cystic VS provides a unique opportunity to understand mechanisms that may contribute to tumor progression and cyst formation. Cystic VSs secrete high levels of cytokines into cyst fluid and express abundant M1 and M2 macrophages. Tumor, CSF, and cyst fluid were prospectively collected from 10 cystic VS patients. Eighty cytokines were measured in fluid samples using cytokine arrays and compared with normal CSF from normal donors. Immunofluorescence was performed for CD80 + M1 and CD163 + M2 macrophage markers. Demographic, audiometric, and radiographic information was obtained through retrospective chart review. Cyst fluid expressed more osteopontin and monocyte chemotactic protein-1 (MCP-1; p < 0.0001), when compared with normal CSF. Cyst fluid also expressed more protein ( p = 0.0020), particularly MCP-1 ( p < 0.0001), than paired CSF from the same subjects. MCP-1 expression in cyst fluid correlated with CD80 + staining in VS tissue ( r = 0.8852; p = 0.0015) but not CD163 + staining. Cyst fluid from cystic VS harbored high levels of osteopontin and MCP-1, which are cytokines important in monocyte recruitment and macrophage polarization. MCP-1 may have a significant role in molding the tumor microenvironment, by polarizing monocytes to CD80 + M1 macrophages in cystic VS. Further investigations into the role of cytokines and macrophages in VS may lead to new avenues for therapeutic intervention.

Simvastatin Preferentially Targets FLT3/ITD Acute Myeloid Leukemia by Inhibiting MEK/ERK and p38-MAPK Signaling Pathways.

The FLT3/ITD mutation exists in many acute myeloid leukemia (AML) patients and is related to the poor prognosis of patients. In this study, we attempted to evaluate the antitumor activity of simvastatin, a member of the statin class of drugs, in vitro and in vivo models of FLT3/ITD AML and to identify the potential mechanisms. Cell Counting Kit-8 (CCK-8) and Annexin V/propidium iodide (PI) staining kits were used to detect cell viability and apoptosis, respectively. Subsequently, Western blot and rescue experiment were applied to explore the potential molecular mechanism. In vivo anti-leukemia activity of simvastatin was evaluated in xenograft mouse models. In vitro experiments revealed that simvastatin inhibited AML progression in a dose- and time-dependent manner, while in vivo experiments showed that simvastatin significantly reduced tumor burden in FLT3/ITD xenograft mouse models. After simvastatin treatment of FLT3/ITD AML cells, intracellular Rap1 was downregulated and the phosphorylation levels of its downstream targets MEK, ERK and p38 were significantly inhibited. The rescue experiment showed that mevalonate, an intermediate product of the metabolic pathway of mevalonate, and its downstream geranylgeranyl pyrophosphate (GGPP) played a key role in this process. Finally, we demonstrate that simvastatin can induce apoptosis of primary AML cells, while having no effect on peripheral blood mononuclear cells from normal donors. Simvastatin can selectively and effectively eradicate FLT3/ITD AML cells in vitro and in vivo, and its mechanism may be related to the disruption of the HMG-CoA reductase pathway and the downregulation of the MEK/ERK and p38-MAPK signaling pathways.

OR23-01 The Nonredundant Role Of Beta-Amyloid In Mediating Tonic PTH Secretion In Physiological And Pathological States

Abstract Disclosure: C. Tu: None. Z. Cheng: None. K.A. Pena: None. N. Szeto: None. J.A. Sosa: None. J. Vilardaga: None. J. Koh: None. W. Chang: None. Understanding the mechanisms driving parathyroid hormone (PTH) hypersecretion in primary hyperparathyroidism (PHPT) is essential for better management of this common endocrinopathy. Prior studies showed that reduced Ca2+-sensing receptor (CaSR) expression and the subsequent increases in heterodimerization of the CaSR with the type B γ-aminobutyric acid receptor 1 (GABAB1R) are causally linked to PTH hypersecretion in PHPT mouse models (Nat Metab 2:243-255). We further showed that expression of the putative GABAB1R ligands, amyloid precursor protein (APP) and its proteolytic product, β-amyloid (Aβ1-42), is significantly upregulated in adenomas of PHPT patients associated with vitamin D insufficiency/deficiency when compared to normal donor controls. The current study aims to delineate the actions of Aβ1-42 in promoting tonic PTH secretion and its interactions with vitamin D receptor (VDR) signaling in parathyroid glands (PTGs) in basal and PHPT states. We show that Aβ1-42 (200 nM) stimulated tonic PTH secretion in cultures of normal human PTGs without shifting the calcium/PTH secretion setpoint (Ca2+-setpoint). In contrast, conditional knockout (KO) of the App gene in the parathyroid cell (PTC) of PTCAppΔflox/Δflox mice significantly reduced serum PTH levels (KO: 64±13 pg/ml vs Control: 109±12 pg/ml; p&amp;lt;0.05, n=8) despite hypocalcemia, indicating hypoparathyroidism. In PTGs cultured from the PTCAppΔflox/Δflox mice, supplementation of Aβ1-42 dose-dependently (EC50=5.6 nM, p&amp;lt;0.001) increased PTH secretion without altering the Ca2+-setpoint. However, the stimulatory effects of Aβ1-42 on tonic PTH secretion were completely abrogated in the PTGs with concurrent deletions of App, Casr and Gabbr1 genes, supporting a direct action of Aβ1-42 on CaSR and/or GABAB1R. The latter notion is further supported by the ability of Aβ1-42 to stimulate cAMP production in cells co-expressing CaSR and GABAB1R. PTC-specific deletion of the Vdr gene in the PTCVdrΔflox/Δflox mice led to elevated serum PTH levels in vivo and increased tonic PTH secretion with unaffected Ca2+-setpoint in PTGs in vitro. Concurrent ablation of the App gene completely normalized serum PTH levels in the PTCVdrΔflox/Δflox mice and prevented PTH hypersecretion in their PTGs in culture. These findings support a critical role of the APP-derived Aβ1-42 in mediating tonic PTH secretion in the normal physiological state and suggest a new mechanism driving PTH hypersecretion in PHPT due to vitamin D deficiency. Presentation: Saturday, June 17, 2023

THU396 Digital Spatial Profiling Of Human Parathyroid Tumors Reveals Distinct Cellular And Molecular Alterations Linked To Vitamin D Deficiency

Abstract Disclosure: C. Tu: None. W. Chang: None. J.A. Sosa: Advisory Board Member; Self; Novo Nordisk, AstraZeneca, Eli Lilly &amp; Company. Grant Recipient; Self; Exelixis, Inc., Eli Lilly &amp; Company. J. Koh: None. Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder characterized by disrupted calcium homeostasis secondary to inappropriately elevated parathyroid hormone (PTH) secretion. Low levels of serum 25-hydroxyvitamin D (25OHD) are significantly more prevalent in PHPT patients than in the general population, but the pathophysiological basis for this association remains unclear. We employed a spatially defined in situ whole-transcriptomics and selective proteomics profiling approach to compare gene expression patterns and cellular composition in parathyroid adenomas from 9 vitamin D-deficient or 34 vitamin D-replete PHPT patients with 12 eucalcemic cadaveric donor parathyroid glands as normal tissue controls. Parathyroid oxyphil cell content is markedly higher in tumors from vitamin D-deficient PHPT patients (Def-Ts) relative to tumors from vitamin D-replete PHPT patients (Rep-Ts) (47.8% vs 17.8%, respectively; p &amp;lt;0.0001) and normal donor parathyroid glands (7.7%). Def-Ts display increased expression of genes encoding electron transport chain (corr. coefficient=0.546), proteasomal catabolism (corr. coefficient=0.587), and oxidative phosphorylation (corr. coefficient=0.982) pathway components. In contrast, Rep-Ts are characterized by upregulation of the ras (corr. coefficient=0.632) and myc (corr. coefficient=0.811) signaling pathways, suggestive of an oncogene-driven etiology. Parathyroid oxyphil cells are comparable to chief cells at the transcriptional level, and vitamin D-status is reflected in the transcriptional profiles of both cell types in a similar manner. Among PHPT patients who did not present as vitamin D deficient, three distinct tumor transcriptional profile clusters showed a dose-dependent relationship to pre-operative 25OHD levels (p &amp;lt;0.0001 by ANOVA). Relative to normal tissue, the abundance of calcium sensing receptor (CaSR) protein is reduced in Def-Ts (mean protein count 253.6 vs 492.3, respectively; p=0.039 for difference), while the type B γ-aminobutyric acid receptor 1 (GABAB1R) is increased (mean protein count 114.5 vs 64.65, respectively; p=0.0099 for difference). These expression changes resulted in a vitamin D dose-dependent reduction in the ratio of CaSR to GABAB1R receptors in Def-T tumor cells (r2 = 0.218, p&amp;lt;0.0001), favoring the formation of calcium sensing-attenuated CaSR:GABAB1R heteromers. The abundance of the amyloid precursor protein (APP) peptide derivative Aβ1-42, a putative GABAB1R ligand, is preferentially increased in Def-Ts compared to normal tissue (mean protein count 5797 vs 4500, respectively; p=0.0318). Collectively, these findings suggest a novel mechanism where chronic vitamin D deficiency can contribute to tonic PTH hypersecretion in PHPT patients by increasing GABAB1R-mediated antagonism of CaSR activity in parathyroid adenomas. Presentation: Thursday, June 15, 2023

Diagnostic miRNA Signatures in Paired Tumor, Plasma, and Urine Specimens From Renal Cell Carcinoma Patients.

The incidence of patients diagnosed with renal cell carcinoma (RCC) is increasing. There are no approved biofluid biomarkers for routine diagnosis of RCC patients. This retrospective study aims to identify cell-free microRNA (cfmiR) signatures in urine samples that can be utilized as biomarkers for early diagnosis of sporadic RCC patients. Tissue, plasma, and urine samples (n = 221) from 56 sporadic RCC patients and respective normal healthy donors were profiled for 2083 microRNAs (miRs) using the next-generation sequencing-based HTG EdgeSeq miR Whole Transcriptome Assay. DESeq2 (FC |1.2|, false discovery rate <0.05) was performed to identify differentially expressed miRs. Data from RCC tissue samples of The Cancer Genome Atlas database were used for miR validation. We found a 10-miR signature that distinguished RCC tissues from remote normal kidney tissue or benign kidney lesion samples. Additionally, we identified subtype-specific miRs (miR-122-5p, miR-210-3p, and miR-21-3p) and miRs specific for all RCC subtypes (miR-106b-3p, miR-629-5p, and miR-885-5p). We observed that miR-155-5p was associated with tumor size. Using The Cancer Genome Atlas data sets, we validated the miRs found in RCC tissue samples. In plasma or urine analysis, we found cfmiRs that were consistently and significantly upregulated in RCC tissue samples. A 15-cfmiR signature was proposed in urine samples of RCC patients, of which miR-1275 was consistently upregulated in tissue, plasma, and urine samples. This integrative study found diagnostic miRs/cfmiRs for RCC patients, which were validated using The Cancer Genome Atlas data sets. Distinctive cfmiR signatures found in urine may have clinical utility for the diagnosis of RCC.

B-044 Analytical validation of an serum glycerol measured by nuclear magnetic resonance spectroscopy NMR

Abstract Background Glycerol kinase deficiency (GKD) is a rare X-linked recessive disorder biochemically characterized by hyperglycerolaemia. Adults with GKD are usually asymptomatic. However, they often present with pseudohypertriglyceridemia because most clinical triglyceride methods measure enzymatically generated glycerol from triglycerides. Currently, assays for measuring plasma glycerol are lacking. We describe a preliminary validation of glycerol measured by nuclear magnetic resonance spectroscopy (NMR). Methods Serum glycerol was measured by NMR (Numares AG, Regensberg, Germany) using a Ascend 600 NMR (Bruker, Billerica, MA). Stability and matrix effects were determined by repeat measurement of 10 freshly collected samples following various storage conditions. Normal donors were recruited for reference interval (n = 143). Analytical specificity was assessed by spiking red cell lysate, bilirubin and intralipid. Precision was assessed by measuring three pools five times for five days. Accuracy was performed by spiking known concentrations of glycerol. Accuracy was assessed using an enzymatic method (Randox Laboratories, Ltd. County Antrim, UK) on a Roche Cobas c501 (Roche Diagnostics, Indianapolis, IN) as reference. Results Measured glycerol increased over time in both serum and plasma, however, prepared samples were stable on the instrument for up to 24 hours. Measured glycerol was not impacted by hemoglobin (1000 mg/dL) or bilirubin (625 mg/dL), however, spiking Intralipid® prevented any measurement. NMR and enzymatic assays strongly correlated, with a constant bias (Table 1). Repeatability and reproducibility were &amp;lt;10% CV and % recovery from glycerol standard were &amp;gt;80% for all concentrations. Conclusion Pre-analytical stability, imprecision and accuracy were acceptable for clinical measurement of glycerol using NMR. While there is a significant systematic bias with enzymatic methods, further studies in normal and disease populations will be necessary to determine clinical impact.

A-149 Performance Characteristics of VerifyNow PRU and ARU Tests During a Clinical Method Verification Exercise

Abstract Background Dual antiplatelet therapy with aspirin and a platelet P2Y12 receptor antagonist is indicated for the prevention of atherothrombotic events in patients with acute coronary syndromes and for those undergoing percutaneous coronary intervention procedure. Two different assays are available in the VerifyNow Test device for monitoring the anti-platelet therapy response. The aspirin assay (ARU Test) utilizes arachidonic acid as an agonist and is sensitive to acyl salicylic acid. The P2Y12 assay (PRU test) is sensitive to thienopyridines (such as clopidogrel, prasugrel, ticagrelor) and uses adenosine diphosphate (ADP) as an agonist. The PRU test is a non-waived test and ARU test is a waived test. A comprehensive method performance verification is required for the PRU test. We evaluated performance characteristics of the VerifyNow PRU Test and ARU Test for clinical use. Methods The standard precision and accuracy study using two levels (low and high) of quality control materials were performed (N = 10) for both the assays. Alternative qualitative method comparison was carried out (N = 49) using the VerifyNow PRU Test in a reference lab and in-house. The in-house inter-instrument comparisons were carried out (N = 42). The blood samples were obtained from patients taking P2Y12 inhibitors and from normal donors not on any drugs. The results of the PRU Test are expressed in P2YI2 reaction units (PRU). The VerifyNow-Aspirin assay results are expressed as aspirin reaction units (ARU). The statistical analysis to evaluate performance characteristics was carried out using the EP Evaluator. Results The VerifyNow device 1561 precision characteristics for low QC (Obs Mean = 1.5 PRU (N = 10), SD = 0.2; 95% CI for Obs Mean 1.0 to 2.0 PRU) and high QC (Obs Mean = 258.3 PRU (N = 10), SD = 8.2; 95% CI for Obs Mean 252.4 to 264.2 PRU) were within acceptable performance limits. The CV for high QC was 3.2%. The ARU test also demonstrated high precision for two levels (Low QC Obs Mean = 350ARU (N = 10), SD = 0.0); High QC Obs Mean = 583.3 (N = 10), SD = 7.2; 95% CI for Obs Mean = 578.1–588.5) with a CV of 1.2%. The PRU Test Method PRU 1561 In House vs Ref Method Verify Now PRU Test (N = 49) demonstrated overall agreement of 93.9% with positive agreement (intended use population on drugs, N = 27) of 92% and negative agreement (normal non-drug group, N = 22) of 95.8% (Cohen’s Kappa = 87.8%; Kappa &amp;gt;75% indicates “"high” agreement). The McNemar Test for Symmetry passed (P &amp;gt; 0.999, Fisher’s Exact test) in this qualitative method comparison. A value of P &amp;lt; 0.05 would suggest the methods are significantly different. The other in-house instrument 1562 also demonstrated comparable or better performance characteristics. Conclusion It is important to confirm the medical decision limit for the intended use population and determine acceptance criteria while considering biologic variability. However, the reference range verifications with drug responders and non-responders are not always feasible and cost-effective. Based on the qualitative method comparison in this study, we have transferred the medical decision limits from the peer group. The in-house multi-instrument comparison should be repeated every six months to monitor the performance of the instruments.

A-004 Analytical Assessment and Expanded Stability Studies of the Abbott Alinity i STAT High Sensitivity Troponin-I Assay

Abstract Background The Mount Sinai Health System has recently integrated the Abbott Alinity i STAT High Sensitivity Troponin-I Assay (HS-Trop) into its “Suspected Acute Coronary Syndrome Algorithm”. Prior to doing so, a comprehensive evaluation of the assay was performed. In particular, the manufacturer’s specifications state that HS-Trop samples (plasma) are stable for 8 h at room temperature (RT), and that plasma and red cells must be separated by centrifugation within 30 min of draw. This centrifugation time window and RT storage requirements are not practical for most large hospital laboratories. Sample delivery, accessioning, and centrifugation are often greater than 30 min, and in most laboratory automation systems, sample storage modules are refrigerated. RT storage would require the removal of the HS-Trop sample from the automation line, and storing the sample elsewhere. This adds additional work to laboratory staff, and makes add-on testing challenging. As such, refrigerated storage and a longer centrifugation time window would be ideal. In this study, we sought to assess the analytical performance of the HS-Trop assay, and validate expanded centrifugation and stability requirements. Methods Inter and Intraday precision was assessed through replicate measurements (n = 10) of low, mid, and high Technopath Quality Controls. Linearity was determined through triplicate analysis of Maine Standards linearity standards. Accuracy was assessed through split sample (n = 40) analysis on two distinct reagent lots. Stability was assessed in centrifuged samples at RT and 4 °C through analysis at 0, 4, and 8 h time points, with an additional 16 h time point assessed at 4 °C. Unspun sample stability was assessed at RT at 0, 2, 4, and 8 h time points. LoQ was assessed through a sensitivity profile, by performing a serial dilution spanning 1:2–1:128, measuring each dilution in triplicate on 10 different days. Reference range verification was performed through the assessment of 20 sex-stratified normal donor samples. Results Intraday precision afforded CVs of 5.1%, 5.6%, and 2.9% for the low, mid, and high controls, respectively, while Interday precision at low, mid, and high controls yielded CVs of 4.3%, 6.3%, and 3.3%. Accuracy assessment resulted in a slope of 0.928, y-intercept of 6.613, and R2 = 0.9943. Linearity was confirmed between 12.3 and 4216.5 ng/mL with a slope of 0.997, y-intercept of 0.261. Spun and Unspun sample stability was confirmed at RT and 4 °C for a maximum of 8 h, affording percent difference values with respect to the initial time point of &amp;lt;20%. Stability studies failed at the 16 h time point. The LoQ was determined to be 4.4 ng/L at 20% CV. The reference range was verified at: Males, &amp;lt;34, and Females, &amp;lt;18 ng/L. Conclusions Our study has shown the Abbott HS-Trop assay performs well analytically, with acceptable precision, accuracy, linearity, and analytical sensitivity for the assessment of HS-Trop. The manufacturer-specified reference range was confirmed. Our study has validated and confirmed an expanded stability and centrifugation window, which will allow for the easy integration of the assay into large hospital networks, and for its use in many laboratory automation systems.

Podocyte Infolding Glomerulopathy: A Special Morphology of Podocyte Injury Caused by Heterogeneous Diseases

Podocyte infolding glomerulopathy (PIG) is a newly recognized rare glomerular injury. The clinical significance and mechanism of this injury pattern remains unclear. We conducted a retrospective study of renal biopsies from January 2018 to December 2020 in Kingmed Diagnostics. The renal biopsy features and clinical data were reviewed. Laser scanning microdissection and mass spectrometry (LMD/MS) was conducted to analyze the potential mechanism. A total of 116 (0.092%) out of 126,086 biopsies were diagnosed as PIG during the period. Of these, 89 (76.7%) cases were found to have PIG coexisting with immune-complex associated glomerulonephritis (IC-PIG) whereas 27 (23.3%) were identified as isolated PIG without immunoglobulin or complement deposition. Systemic lupus erythematosus (SLE), especially with membranous lupus nephritis (LN), was diagnosed in most (70.8%) IC-PIG cases. Of the isolated PIG cases, 51.9% had no known underlying conditions; however, a relatively high positive rate (42.1%) of antinuclear antibody (ANA) was detected. Nearly half (47.5%) of the patients presented with nephrotic syndrome (NS). PIG grade was associated with proteinuria in isolated PIG (P= 0.035). LMD/MS revealed dysregulated cytoskeletal protein α-actinin4 (ACTN4) and tubulin beta-4 chain in PIG compared with normal donor kidney and minimal change disease (MCD). The displacement of ACTN4 into the glomerular basement membrane (GBM) was confirmed by the confocal microscope. PIG is a rare podocyte injury that can exist alone without underlying disease or be concurrent with various diseases, especially SLE. Podocyte cytoskeletal protein ACTN4 and tubulin beta-4 chain were dysregulated, which may be involved in the mechanism of PIG.

The major TMEM106B dementia risk allele affects TMEM106B protein levels, fibril formation, and myelin lipid homeostasis in the ageing human hippocampus

BackgroundThe risk for dementia increases exponentially from the seventh decade of life. Identifying and understanding the biochemical changes that sensitize the ageing brain to neurodegeneration will provide new opportunities for dementia prevention and treatment. This study aimed to determine how ageing and major genetic risk factors for dementia affect the hippocampal proteome and lipidome of neurologically-normal humans over the age of 65. The hippocampus was chosen as it is highly susceptible to atrophy with ageing and in several neurodegenerative diseases.MethodsMass spectrometry-based proteomic and lipidomic analysis of CA1 hippocampus samples from 74 neurologically normal human donors, aged 66–104, was used in combination with multiple regression models and gene set enrichment analysis to identify age-dependent changes in the proteome and lipidome. ANOVA was used to test the effect of major dementia risk alleles in the TMEM106B and APOE genes on the hippocampal proteome and lipidome, adjusting for age, gender, and post-mortem interval. Fibrillar C-terminal TMEM106B fragments were isolated using sarkosyl fractionation and quantified by immunoblotting.ResultsForty proteins were associated with age at false discovery rate-corrected P < 0.05, including proteins that regulate cell adhesion, the cytoskeleton, amino acid and lipid metabolism, and ribosomal subunits. TMEM106B, a regulator of lysosomal and oligodendrocyte function, was regulated with greatest effect size. The increase in TMEM106B levels with ageing was specific to carriers of the rs1990622-A allele in the TMEM106B gene that increases risk for frontotemporal dementia, Alzheimer’s disease, Parkinson’s disease, and hippocampal sclerosis with ageing. Rs1990622-A was also associated with higher TMEM106B fibril content. Hippocampal lipids were not significantly affected by APOE genotype, however levels of myelin-enriched sulfatides and hexosylceramides were significantly lower, and polyunsaturated phospholipids were higher, in rs1990622-A carriers after controlling for APOE genotype.ConclusionsOur study demonstrates that TMEM106B protein abundance is increased with brain ageing in humans, establishes that dementia risk allele rs1990622-A predisposes to TMEM106B fibril formation in the hippocampus, and provides the first evidence that rs1990622-A affects brain lipid homeostasis, particularly myelin lipids. Our data suggests that TMEM106B is one of a growing list of major dementia risk genes that affect glial lipid metabolism.

Levels and in vitro functional effects of circulating anti-hinge antibodies in melanoma patients receiving the immune checkpoint inhibitor pembrolizumab.

The efficacy of PD-1 monoclonals such as pembrolizumab can be modulated by the signals delivered via their Fc region. Tumour/inflammation associated proteases can generate F(ab')2 fragments of therapeutic monoclonals, and subsequent recognition of F(ab')2 epitopes by circulating anti-hinge antibodies (AHA) can then, potentially, link F(ab')2 binding to the target antigen with novel Fc signalling. Although elevated in inflammatory diseases, AHA levels in cancer patients have not been investigated and functional studies utilising the full repertoire of AHA present in sera have been limited. AHA levels in pembrolizumab treated melanoma patients (n = 23) were therefore compared to those of normal donors and adalimumab treated patients. A subset of melanoma patients and the majority of adalimumab patients had elevated levels of AHA reactive with F(ab')2 fragments of IgG4 anti-PD-1 monoclonals (nivolumab, pembrolizumab) and IgG1 therapeutic monoclonals (rituximab, adalimumab). Survival analysis was restricted by the small patient numbers but those melanoma patients with the highest levels (>75% percentile, n = 5) of pembrolizumab-F(ab')2 reactive AHA had significantly better overall survival post pembrolizumab treatment (p = 0.039). In vitro functional studies demonstrated that the presence of AHA+ sera restored the neutrophil activating capacity of pembrolizumab to its F(ab')2 fragment. Neither pembrolizumab nor its F(ab')2 fragments can induce NK cell or complement dependent cytotoxicity (CDC). However, AHA+ sera in combination with pembrolizumab-F(ab')2 provided Fc regions that could activate NK cells. The ability of AHA+ sera to restore CDC activity was more restricted and observed using only one pembrolizumab and one adalimumab patient serum in combination with rituximab- F(ab')2. This study reports the presence of elevated AHA levels in pembrolizumab treated melanoma patients and highlight the potential for AHA to provide additional Fc signaling. The issue of whether tumour associated proteolysis of PD-1 mAbs and subsequent AHA recognition impacts on treatment efficacy requires further study.

Safety and Efficacy of DT-DEC01 Therapy in Duchenne Muscular Dystrophy Patients: A 12 - Month Follow-Up Study After Systemic Intraosseous Administration

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal muscle-wasting disease with no known cure. We previously reported the preliminary safety and efficacy up to six months after the administration of DT-DEC01, a novel Dystrophin Expressing Chimeric (DEC) cell therapy created by fusion of myoblasts of DMD patient and the normal donor. In this 12-month follow-up study, we report on the safety and functional outcomes of three DMD patients after the systemic intraosseous administration of DT-DEC01. The safety of DT-DEC01 was confirmed by the absence of Adverse Events (AE) and Severe Adverse Events (SAE) up to 21 months after intraosseous DT-DEC01 administration. The lack of presence of anti-HLA antibodies and Donors Specific Antibodies (DSA) further confirmed DT-DEC01 therapy safety. Functional assessments in ambulatory patients revealed improvements in 6-Minute Walk Test (6MWT) and timed functions of North Star Ambulatory Assessment (NSAA). Additionally, improvements in PUL2.0 test and grip strength correlated with increased Motor Unit Potentials (MUP) duration recorded by Electromyography (EMG) in both ambulatory and non-ambulatory patients. DT-DEC01 systemic effect was confirmed by improved cardiac and pulmonary parameters and daily activity recordings. This follow-up study confirmed the safety and preliminary efficacy of DT-DEC01 therapy in DMD-affected patients up to 12 months after intraosseous administration. DT-DEC01 introduces a novel concept of personalized myoblast-based cellular therapy that is irrespective of the mutation type, does not require immunosuppression or the use of viral vectors, and carries no risk of off target mutations. This establishes DT-DEC01 as a promising and universally effective treatment option for all DMD patients.Graphical

Glycan node profiling of soluble and membrane glycoproteins in whole cell lysates

Glycan node analysis (GNA) is a molecularly bottom-up glycomics technique based on the relative quantification of glycan linkage-specific monosaccharide units (“glycan nodes”). It was originally applied to blood plasma/serum, where it detected and predicted progression, reoccurrence, and survival in different types of cancer. Here, we have adapted this technology to previously inaccessible membrane glycoproteins from cultured cells. The approach is facilitated by methanol/chloroform precipitation of cell lysates and a “liquid phase permethylation” (LPP) procedure. LPP gave better signal-to-noise, yield and precision for most of the glycan nodes from membrane glycoproteins/glycolipids than the conventional solid phase permethylation approach. This GNA approach in cell lysates revealed that specific glycan features such as antennary fucosylation, N-glycan branching, and α2,6-sialylation were elevated in hepatocellular carcinoma (HepG2) cells relative to leukemia cells (THP-1 and K562) and normal donor PBMCs. Additional nodes commonly associated with glycolipids were elevated in the leukemia cells relative to HepG2 cells and PBMCs. Exposure of HepG2 cells to a fucosyltransferase inhibitor resulted in a significant reduction in the relative abundance of 3,4-substituted GlcNAc, which represents antennary fucosylation—providing further proof-of-concept that downregulation of glycosyltransferase activity is detected by shifts in glycan node expression—now detectable in membrane glycoproteins.

GPER1 Activation Exerts Anti-Tumor Activity in Multiple Myeloma.

G protein-coupled estrogen receptor 1 (GPER1) activation is emerging as a promising therapeutic strategy against several cancer types. While GPER targeting has been widely studied in the context of solid tumors, its effect on hematological malignancies remains to be fully understood. Here, we show that GPER1 mRNA is down-regulated in plasma cells from overt multiple myeloma (MM) and plasma cell leukemia patients as compared to normal donors or pre-malignant conditions (monoclonal gammopathy of undetermined significance and smoldering MM); moreover, lower GPER1 expression associates with worse overall survival of MM patients. Using the clinically applicable GPER1-selective agonist G-1, we demonstrate that the pharmacological activation of GPER1 triggered in vitro anti-MM activity through apoptosis induction, also overcoming the protective effects exerted by bone marrow stromal cells. Noteworthy, G-1 treatment reduced in vivo MM growth in two distinct xenograft models, even bearing bortezomib-resistant MM cells. Mechanistically, G-1 upregulated the miR-29b oncosuppressive network, blunting an established miR-29b-Sp1 feedback loop operative in MM cells. Overall, this study highlights the druggability of GPER1 in MM, providing the first preclinical framework for further development of GPER1 agonists to treat this malignancy.

Identification of Autoantibodies to a Hybrid Insulin Peptide in Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that attacks the insulin-producing b cells of the pancreatic islets. Autoantibodies to b cell proteins typically appear in the circulation years before disease onset, and serve as the most accurate biomarkers of T1D risk. Our laboratory has recently discovered novel b cell proteins comprising hybrid proinsulin:islet amyloid polypeptide peptides (IAPP). T cells from a diabetic mouse model and T1D patients are activated by these hybrid peptides. In this study, we asked whether these hybrid molecules could serve as antigens for autoantibodies in T1D and prediabetic patients. We analyzed sera from T1D patients, prediabetics and healthy age-matched donors. Using a highly sensitive electrochemiluminescence assay, sera were screened for binding to recombinant proinsulin:IAPP probes or truncated derivatives. Our results show that sera from T1D patients contain antibodies that bind larger hybrid proinsulin:IAPP probes, but not proinsulin or insulin, at significantly increased frequencies compared to normal donors. Examination of sera from prediabetic patients confirms titers of antibodies to these hybrid probes in more than 80% of individuals, often before seroconversion. These results suggest that hybrid insulin peptides are common autoantigens in T1D and prediabetic patients, and that antibodies to these peptides may serve as valuable early biomarkers of the disease.

Detection of SARS-CoV-2 Antibodies in Immunoglobulin Products.

For patients with primary antibody deficiency, the first line of therapy is replacement with immunoglobulin (Ig) products. Prior to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, Ig products did not contain antibodies with specificity for this virus, and there have been limited data on the antibodies present in the Ig products in current use. To quantitatively examine SARS-CoV-2 antibodies in current Ig products. We examined 142 unique lots of 11 different Ig products intended for intravenous and/or subcutaneous delivery for IgG-binding activities against recombinant SARS-CoV-2 receptor binding domain, spike, and nucleocapsid proteins by enzyme-linked immunosorbent assays. In addition, to assess functionality, 48 of these unique lots were assessed for their ability to inhibit the variants SARS-CoV-2 Ancestral, Alpha, Beta, Delta, and Omicron spike binding to angiotensin-converting enzyme 2 (ACE2). Significantly increased antibody values were observed for products manufactured after the year 2020 (expiration dates 2023-2024), as compared with Ig products before 2020 (prepandemic). Sixty percent and 85% of the Ig products with expiration dates of 2023 and 2024 were positive for antibody to SARS-CoV-2 proteins, respectively. The area under the curve values were significantly higher in products with later expiration dates. Later dates of expiration were also strongly correlated with inhibition of ACE2-binding activity; however, a decline in inhibition activity was observed with later variants. Overall, more recent Ig products (expiration dates 2023-2025) contained significantly higher binding and inhibition activities against SARS-CoV-2 proteins, compared with earlier, or prepandemic products. Normal donor SARS-CoV-2 antibodies are capable of inhibiting ACE2-binding activities and may provide a therapeutic benefit for patients who do not make a robust vaccine response.

Plasticity of circulating tumor cells in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with low five-year survival rates. Recently described molecular phenotypes of SCLC exhibit differential vulnerabilities heralding potential for stratified treatment. Whilst tumor biopsy in SCLC is challenging, circulating tumor cells in the liquid biopsy are prevalent and can be repeatedly sampled accommodating the dynamic plasticity of SCLC phenotypes. The aim of this study was to characterize the heterogeneity of rare circulating cells with confirmed tumor origin and to explore a liquid biopsy approach for future clinical trials of targeted therapies. This study applied the 3rd generation of a previously validated direct imaging platform to 14 chemo-naive SCLC patients and 10 non-cancerous normal donor (ND) samples. Phenotypic heterogeneity of circulating rare cells in SCLC was observed and a patient-level classification model was established to stratify SCLC patients from non-cancerous donors. Eight rare cell groups, with combinations of epithelial, endothelial, and mesenchymal biomarker expression patterns, were phenotypically characterized. The single-cell genomic analysis confirmed the cancer cell plasticity in every rare cell group harboring clonal genomic alterations. This study shows rare cell heterogeneity and confirms cellular plasticity in SCLC providing a valuable resource for better opportunities to discover novel therapeutic targets in SCLC.