Control Subjects Research Articles

The Association Between COVID-19 Infection Severity and Micronutrient Deficiencies in Children

Background: Coronavirus disease 2019 (COVID-19) can cause pulmonary and systemic inflammation in both children and adults. Investigating the potential impact of micronutrient deficiencies on COVID-19 infection is of great importance. Objectives: This case-control study was designed to compare the serum levels of vitamins D and C and zinc between healthy children and children with mild or severe COVID-19 infection, as well as to investigate the impact of micronutrient deficiencies on the clinical symptoms of patients. Methods: This case-control study was conducted at a teaching hospital in Tehran, Iran. Control subjects were recruited from healthy children, and cases were included from children with mild and severe symptoms of COVID-19. Blood samples were obtained from participants to measure the levels of micronutrients and were analyzed using statistical software. Results: A total of 85 subjects were included (33 controls, 25 cases with mild symptoms, and 27 cases with severe symptoms). We found no differences in the means of serum vitamin D and vitamin C levels between the studied groups. However, the mean serum zinc level was significantly lower in severe patients compared to the controls (P < 0.01). Moreover, no significant difference was observed in the frequency of patients with micronutrient deficiencies based on their clinical conditions. Conclusions: Our findings revealed zinc deficiency in children with severe COVID-19 infection, while no significant differences were found in the mean serum levels of vitamins D and C between healthy children and patients. More studies are necessary to provide more robust evidence.

Associations of Clinical Characteristics With Sudden Cardiac Arrest in People With Type 2 Diabetes With and Without Cardiovascular Disease: A Longitudinal Case-Control Study Using Routine Primary Care Data

OBJECTIVE To assess longitudinal associations with sudden cardiac arrest (SCA) of clinical characteristics recorded in primary care in people with type 2 diabetes (T2D), both with and without cardiovascular disease (CVD). RESEARCH DESIGN AND METHODS We performed a case-control study, with SCA case subjects with T2D from the Amsterdam Resuscitation Studies (ARREST) registry of out-of-hospital resuscitation attempts in the Dutch Noord-Holland region (2010–2020) and up to five matched (age, sex, T2D, general practitioner [GP] practice) non-SCA control subjects. We collected relevant clinical measurements, medication use, and medical history from GPs’ electronic health care records. We analyzed the associations of clinical characteristics and medication use with SCA in the total sample and in subgroups with or without CVD using multivariable time-dependent Cox regression (hazard ratios, 95% confidence intervals). RESULTS We included 689 SCA case subjects and 3,230 non-SCA control subjects. In multivariable models, low fasting glucose (<4.5 mmol/mol: 1.91 [1.00–3.64]), antihypertensive (1.80 [1.39–2.33]), glucose lowering (oral only: 1.32 [1.06–1.63]; insulin only: 2.31 [1.71–3.12]; oral and insulin: 1.64 [1.21–2.22]), heart failure (1.91 [1.55–2.35]), and QTc-prolonging prokinetic (1.78 [1.27–2.50]), antibiotic (1.35 [1.05–1.73]), and antipsychotic (2.10 [1.42–3.09]) medication were associated with SCA in the total sample. In subgroup effect modification analyses, QTc-prolonging antibiotic (1.82 [1.26–2.63]) and antipsychotic (3.10 [2.09–4.59]) medication use were associated with SCA only in those without CVD. CONCLUSIONS In people with T2D, low fasting glucose and QTc-prolonging prokinetic, antibiotic, or antipsychotic medication use and a history of heart failure are associated with SCA risk. Subgroup analyses indicate antibiotic and antipsychotic medication use increases SCA risk specifically in those without CVD.

The CML experience to elucidate the role of innate T-cells as effectors in the control of residual cancer cells and as potential targets for cancer therapy

Considering the general view that unconventional immune effectors play a major role in antitumor immunity, we recently postulated that the distinct new innate CD8 T-cell pool (co-expressing the transcription factor Eomesodermin and innate markers such as KIR/NKG2A) may counteract tumor cells, and thereby be potential target for cancer therapy. Here, to test this assumption, we used successfully targeted anti-leukemic therapy discontinuation (TFR) in chronic myeloid leukemia (CML). Numerical and functional status of innate CD8 T-cells, iNKT cells and γδ T-cells, in comparison with NK cells, was compared longitudinally between non-relapsed patients (i.e., with > 12 months TFR) and relapsed patients (i.e., who experienced molecular recurrence during the first 12 months after TKI cessation) in a prospective pilot cohort (n=32), starting from treatment discontinuation (D0). Perforin, a key cytotoxic immune player, was expressed in a significantly higher proportion of both innate CD8 T-cell and NK-cell subsets in non-relapsed patients, compared with relapsed patients at D0. In parallel, we assessed the expression of PD-1, an exhaustion marker used as target in cancer therapy. For all T-cell subsets, surface-expression level of PD-1 decreased in non-relapsed patients compared with relapsed patients at D0. This was particularly the case when considering iNKT cells for which surface-expression level of PD-1 even decreased relative to healthy control subjects. Lastly, we found a negative correlation between the proportion of innate CD8 T-cells expressing PD-1 and those expressing perforin in non-relapsed patients at D0. The fact that this was not the case in conventional CD8 T-cells is compatible with a reprogrammed effector profile preferentially targeting innate CD8 T-cells in non-relapsed patients. All in all, our results highlight NK cells and innate CD8 T-cells harboring cytotoxic content, as well as global downregulation of PD-1-expression on effector T-cells, as potential predictive functional signatures for successful TFR in CML. Considering innate CD8 T-cells, further investigations are needed to determine whether their possible contributory role in cancer surveillance in CML could be extended to other cancers, and also whether their targeting by immune cheek-point inhibitors could enhance their anti-tumoral functions.

Screening electro-oculography protocol as a part of full-field electroretinography.

To simplify the electro-oculography (EOG) method and integrate it into the full-field electroretinogram (ffERG) protocol for screening purposes. 20 control subjects and 5 patients with Best vitelliform macular dystrophy (BVMD) underwent EOG recording according to both the standard protocol and screening EOG protocol that was integrated as part of ffERG testing. Mean values of light peak-to-dark trough ratio (LP:DT ratio) were compared between both protocols using the Student's t-test, sensitivity and specificity for the detection of RPE dysfunction were evaluated with ROC analysis and a survey on the difficulty of each protocol was completed by each subject. With the standard EOG mean LP:DT ratio was 2.67 ± 0.61 in controls and 1.12 ± 0.16 in BVMD patients (p < 0.001). With the screening protocol mean LP:DT ratio was 1.98 ± 0.33 in controls and 1.02 ± 0.14 in BVMD (p < 0.001). A comparison of LP:DT ratios showed significant difference between the standard and the screening protocol (p < 0.01 in controls; p = 0.02 in BVMD), however both protocols showed 100% sensitivity and 100% specificity for detection of BVMD. Patients stated that participation in the screening protocol was easier and less uncomfortable. Screening EOG performed as part of ffERG gives comparable results to standard EOG, examination is patient-friendly, time saving and can be used as a preliminary test for the assessment of RPE function.

Identifying amyotrophic lateral sclerosis using diffusion tensor imaging, and correlation with neurofilament markers.

To determine diagnostic value of diffusion tensor imaging (DTI) in amyotrophic lateral sclerosis (ALS) patients and investigate the association between DTI and neurofilaments (NFs), including serum and cerebrospinal fluid (CSF) levels of neurofilament light chain (NFL) and phosphorylated neurofilament heavy chain (pNFH). Forty-three clinically diagnosed ALS patients and 32 control subjects without neurological disorders underwent routine MRI (magnetic resonance imaging) and DTI scans. DTI parameters (mean diffusivity [MD] and fractional anisotropy [FA]) at axial levels of internal capsules and cerebral peduncles along the corticospinal tract (CST) were measured. The study compared the differences of DTI parameters between ALS patients and controls using the Mann-Whitney U test. Diagnostic efficacy of each DTI metric was evaluated using the receiver operating characteristic (ROC) curve. NFs (NFL and pNFH levels in serum and CSF) were measured by enzyme-linked immunosorbent assay. Correlation analyses were conducted between DTI parameters and NFs. Capsule-MD and Peduncle-MD in ALS patients were higher than those in controls; whereas Capsule-FA and Peduncle-FA in ALS patients were lower than those in controls (all, p < 0.05). The area under curve (AUC) was 0.730 for Capsule-FA, 0.828 for Capsule-MD, 0.890 for Peduncle-FA, and 0.896 for Peduncle-MD. Capsule-FA was negatively correlated with CSF-NFL (r = -0.813, p < 0.001), Serum-NFL (r = -0.493, p = 0.001), CSF-pNFH (r = -0.637, p < 0.001), and Serum-pNFH (r = -0.672, p < 0.001); Peduncle-FA negatively with CSF-NFL (r = -0.562, p < 0.001), CSF-pNFH (r = -0.506, p = 0.001), and Serum-pNFH (r = -0.488, p = 0.001); Peduncle-MD positively with CSF-NFL (r = 0.516, p < 0.001), CSF-pNFH (r = 0.494, p = 0.001). DTI had superior performance in identifying ALS patients and could serve as a reliable predictor. DTI parameters related to neurofilament markers, and Capsule-FA may become a robust surrogate biomarker indicating disease severity and progression rate for ALS patients.

Prevalence of Spinal Meningeal Diverticula in Autosomal Dominant Polycystic Kidney Disease.

Patients with autosomal dominant polycystic kidney disease (ADPKD) develop cysts in the kidneys, liver, spleen, pancreas, prostate, and arachnoid spaces. In addition, spinal meningeal diverticula have been reported. To determine whether spinal meningeal diverticula are associated with ADPKD, we compared their prevalence in subjects with ADPKD with a control cohort without ADPKD. Subjects with ADPKD and age- and sex-matched controls without ADPKD undergoing abdominal MRI from the midthorax to the pelvis from 2003 to 2023 were retrospectively evaluated for spinal meningeal diverticula by 4 blinded observers. The prevalence of spinal meningeal diverticula in ADPKD was compared with that in control subjects, using t tests and correlated with clinical and laboratory data and MR imaging features, including cyst volumes and cyst counts. Identification of spinal meningeal diverticula in ADPKD (n = 285, median age, 47; interquartile range [IQR], 37-56 years; 54% female) and control (n = 285, median age, 47; IQR, 37-57 years; 54% female) subjects had high interobserver agreement (pairwise Cohen κ = 0.74). Spinal meningeal diverticula were observed in 145 of 285 (51%) subjects with ADPKD compared with 66 of 285 (23%) control subjects without ADPKD (P < .001). Spinal meningeal diverticula in ADPKD were more prevalent in women (98 of 153 [64%]) than men (47 of 132 [36%], P < .001). The mean number of spinal meningeal diverticula per affected subject with ADPKD was 3.6 ± 2.9 compared with 2.4 ± 1.9 in controls with cysts (P < .001). The median volume (IQR, 25%-75%) of spinal meningeal diverticula was 400 (IQR, 210-740) mm3 in those with ADPKD compared with 250 (IQR, 180-440) mm3 in controls (P < .001). The mean spinal meningeal diverticulum diameter was greater in the sacrum (7.3 [SD, 4.1] mm) compared with thoracic (5.4 [SD, 1.8] mm) and lumbar spine (5.8 [SD, 2.0] mm), (P < .001), suggesting that hydrostatic pressure contributed to enlargement. ADPKD has a high prevalence of spinal meningeal diverticula, particularly in women.

Mechanisms underlying the roles of leukocytes in the progression of cystic fibrosis

Recent advances in cystic fibrosis (CF) treatments have led to improved survival, with life expectancy for Australians living with CF at 57yo. As life expectancy improves, long-term cardiovascular disease risk factors (as for the general population) will become an issue in these patients. We hypothesized that increased leukocyte expression of vasoconstriction and pro-fibrotic mediators may contribute to CF severity in adults with CF. We recruited 13 adult and 24 pediatric healthy controls, and 53 adults and 9 children living with CF. Leukocyte expression/release of endothelin-1 (ET1) and members of the TGF-β/Smad signaling were measured by multifluorescence quantitative confocal microscopy, Western blotting, ELISA, and real-time quantitative polymerase chain reaction. The association between plasma ET1 levels and lung function was assessed. Leukocytes from adults living with CF expressed higher ET1 levels (p = 0.0033), and TGF-β (p = 0.0031); the phosphorylation ratio increased for Smad2/3 (p = 0.0136) but decreased for Smad1/5/8 (p = 0.0007), vs. control subjects. Plasma ET1 levels were significantly increased in adults with CF with FEV1<50% (p = 0.002) vs. controls, and adults with CF with normal lung function. The release of ET1 in adult plasma inversely correlated with CF severity (-0.609, p = 0.046). Our data indicates that upregulated ET1 and TGF-β/Smad signaling in leukocytes may contribute to CF severity, highlighting the need for further investigations into their impact on the clinical outcomes of people living with CF.

Individuals with SATB2-associated syndrome have impaired vitamin and energy metabolism pathways.

Special AT-rich sequence-binding protein 2 (SATB2) is a master regulator of gene expression. Mutations of the SATB2 gene results in the SATB2-associated syndrome (SAS), a genetic disorder characterized by neurodevelopmental disabilities and autism-related phenotype. The importance of plasma as an indicator of SAS phenotypes is unknown. We aim to investigate if pathogenic variants in SATB2 are associated with alteration to relevant pathways in the plasma of SAS patients and identify key differentially regulated proteins which may serve as biomarkers to improve diagnostic and future pharmacological approaches. We used well-validated proteomic technologies to determine the proteomic profile of plasma from SAS patients compared to healthy control subjects. Bioinformatical analysis was performed to identify significant proteins and functionally enriched pathways. We identified differentially expressed proteins in the plasma of SAS patients that are significantly involved in metabolism-related pathways. Energy metabolism, glucose metabolism and vitamin metabolism pathways are significantly enriched in SAS patients as compared to healthy controls. Our study linked SATB2 mutations to the impairment of plasma proteins involved in different metabolic pathways in SAS patients.

The abundance of regulatory T cell subsets is associated with the clinical outcomes of sarcoidosis.

Some patients with sarcoidosis achieve spontaneous remission, whereas others repeatedly experience relapse. We examined differences in the clinical course of active sarcoidosis according to peripheral blood immunophenotypes before treatment. This retrospective study compared peripheral blood immunophenotypes between patients with active sarcoidosis (n=28) and healthy control subjects (n=10). Patients with sarcoidosis were divided into the spontaneous remission group without treatment (n=9), the non-relapsed group after treatment (n=13), and the relapsed group after treatment (n=6) and were compared for peripheral blood immunophenotypes and background characteristics at baseline. Patients with sarcoidosis showed increases in activated T helper (Th) 1 cells, activated Th17 cells, and regulatory T (Treg) cell subsets. The proportion of effector Treg cells was highest in the spontaneous remission group, and the proportion of non-suppressive Treg cells was highest in the relapsed group. No differences were observed in the proportions of other CD4+ T cell subsets. The cut-off values for predicting spontaneous remission and relapse were calculated for the effector Treg/non-suppressive Treg ratio. As a result, A ratio ≥1.469 predicted spontaneous remission (75%), while ≤0.722 predicted relapse (66.7%). Effector and non-suppressive Treg cell proportions before treatment may predict spontaneous remission and relapse in active sarcoidosis.

Depletion by Hemodialysis of the Antioxidant Ergothioneine

Background: Hemodialysis may excessively remove valuable solutes. Untargeted metabolomics data from a prior study suggested that ergothioneine was depleted in the plasma of hemodialysis subjects. Ergothioneine is a dietary-derived solute with antioxidant properties. The presence of a highly specific ergothioneine uptake transporter suggests that it is valuable. Ergothioneine levels are high in tissues susceptible to oxidative stress, particularly erythrocytes. We compared erythrocyte and plasma ergothioneine levels in subjects receiving hemodialysis to those in subjects with advanced chronic kidney disease (CKD) and subjects without known kidney disease (controls). We further examined the extent to which indiscriminate removal by hemodialysis could contribute to ergothioneine depletion. Methods: Liquid chromatography tandem mass spectrometry with stable isotope dilution was used to measure the erythrocyte and plasma levels of ergothioneine in 12 control, 12 CKD, and 11 hemodialysis subjects. We also measured the urinary excretion of ergothioneine in control and CKD subjects, and the dialytic removal of ergothioneine in hemodialysis subjects. Results: Erythrocyte ergothioneine levels were markedly reduced in CKD and hemodialysis subjects. Erythrocyte levels in CKD subjects were on average 24% of the levels in control subjects and were even lower in hemodialysis subjects, averaging 8% of control subjects. Plasma ergothioneine levels were also reduced in CKD and hemodialysis subjects but to a lesser extent than the erythrocyte levels. Kidney tubular reabsorption of ergothioneine was avid. In contrast, hemodialysis cleared ergothioneine at a rate of 146±36 ml/min so that removal of ergothioneine by hemodialysis greatly exceeded the amount excreted in the urine in both CKD and control subjects. Conclusions: Ergothioneine, a potentially valuable antioxidant, is severely depleted in people maintained on hemodialysis. Future studies are required to assess the consequences of ergothioneine depletion.

Abstract A075: Circulating T-cell receptor repertoire analysis improves cancer early detection

Abstract Introduction: Cancer screening by liquid biopsy promises to detect cancer early when it can be cured. However, current ctDNA-based approaches detect only a minority of early-stage cancers. For liquid biopsy to realize its full potential for cancer early detection, sensitivity for earliest stage disease must be improved. Methods: We set out to improve sensitivity of liquid biopsy by exploiting tumor recognition by T cells through sequencing of the circulating T-cell receptor repertoire. Using gDNA extracted from blood buffy coats, we studied a cohort of 471 lung cancer patients (85% stage I) and 600 subjects without cancer enriched for older individuals with a history of smoking. We performed TCR beta chain sequencing to yield a median of 101,899 TCR clonotypes per sample (median 81,602 cancer / 112,966 controls) and built a TCR sequence similarity graph to cluster clonotypes into TCR repertoire functional units (RFUs). The TCR frequencies of RFUs were tested for association with cancer status and significantly associated RFUs were combined using an SVM model into a cancer score. Cancer RFU levels were also correlated to imputed subject HLA types for 32 HLA Class I and 38 Class II alleles. The model was evaluated by 10-fold cross-validation and compared to a ctDNA panel of 237 mutation hotspots in 154 lung cancer driver genes and to 17 lung cancer related protein biomarkers in 86 subjects. Results: We identified 372 cancer-associated TCR RFUs at FDR≤0.1, including 198 enriched in cancer samples (fold-change range 1.03 to 3.13) and 174 enriched in controls (fold- change 0.96 to 0.30). Levels of 265/372 (71%) RFUs were correlated to presence of an HLA allele at FDR ≤ 0.1. Of the total 2,770 significant HLA-RFU associations, 479 (17%) involved an HLA Class I allele, while 2,291 (83%) involved an HLA Class II allele, highlighting a potential role for CD4 helper or regulatory T cell antigen recognition in the cancer RFUs found. The RFU cancer score achieved a cross-validated test ROC AUC of 0.71 for cancer status prediction (0.71 Stage I / 0.70 Stage II-IV) with 49% of Stage I cancers detected at a specificity of 80%. Importantly, the cancer score was not dependent on sample source site, technical or biological variation in TCR repertoire depth, or lung cancer histology subtype. The TCR RFU score also distinguished lung cancer patients from control subjects with other conditions such as benign lung nodules and COPD. We observed an up to ∼20%-point gain in sensitivity for stage I cancer when TCR RFUs were added to ctDNA and proteins in a potential multi-analyte cancer screening test. By contrast, TCR analysis did not provide additional sensitivity for stage II-IV disease. Conclusion: We demonstrate detection of a significant fraction of early lung cancer cases from blood by analyzing the circulating TCR repertoire and show that this signal is complementary to established analytes such as proteins and ctDNA. A similar study for the detection of early colorectal cancer and advanced adenomas is under way. Citation Format: Yilong Li, Michelle Nahas, Dennis Stephens, Kate Froburg, Emma Hintz, Devin Champagne, Amaneet Lochab, Markus Brown, Jasper Braun, María Antonia Fortuño, María-del-Mar Ocón, Andrea Pasquier, Inés María Luque, Christopher W. Seder, Jeffrey A. Borgia, Luis Miguel Seijo, Luis M Montuenga, Roman Yelensky. Circulating T-cell receptor repertoire analysis improves cancer early detection [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A075.

Miodynamic and Radiographic Evaluation in Recreative Athletes with Patellofemoral Pain

Background and Objectives: Patellofemoral pain (PFP) is frequent in the young and active population. The effect of muscle strength in the lower extremities after aerobic activity in patients with this condition has yet to be detailed. Our objective was to determine if patients with PFP show alterations in lower extremity muscle strength measurements after performing a session of ten minutes of aerobic activity on a treadmill compared to people without patellofemoral pain. Materials and Methods: We conducted a prospective experimental study with a stratified, non-randomized, and non-blinded population sample with group matching, including an experimental group with PFP and a control group with no pain. Subjects completed self-reported functional questionnaires (IKDC, Kujala, KOOS, SF-12), underwent radiographic studies, and were evaluated by measuring the strength of hip and knee muscles and the Single-Leg Triple-Hop (SLTH) test before and after ten minutes of exercise on a treadmill. Results: Seventeen subjects diagnosed with PFP and seventeen control subjects were evaluated. Both groups were homogeneous and had no significant differences in the demographic variables. A wider sulcus angle at 30° (136.8 ± 3.8° vs. 132.5 ± 5.6°, p = 0.0140), a decrease strength post-exercise in the hip abductor (37.9 ± 7.1 N⋅m vs. 45.6 ± 7.7 N⋅m, p &lt; 0.05) and knee extensor (36.0 ± 9.1 N⋅m vs. 47.7 ± 14.0 N⋅m, p &lt; 0.05), and a shorter distance in the SLTH test (337.9 ± 74.9 cm vs. 438.6 ± 65.8 cm, p &lt; 0.01) was recorded in subjects with patellofemoral pain. Conclusions: Subjects with PFP had an overall lower strength of hip and knee muscles, showing significant differences in the hip abductors and knee extensors between people with PFP and healthy matched controls after aerobic exercise.

Screening for mitochondrial tRNA variants in 200 patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a common autoimmune disease with unknown etiology. Recently, a growing number of evidence suggested that mitochondrial dysfunctions played active roles in the pathogenesis of SLE, but its detailed mechanism remains largely undetermined. The aim of this study was to analysis the frequencies of mitochondrial tRNA (mt-tRNA) variants in Chinese individuals with SLE. We carried out a mutational screening of mt-tRNA variants in a cohort of 200 patients with SLE and 200 control subjects by PCR-Sanger sequencing. The potential pathogenicity of mt-tRNA variants were evaluated by phylogenetic conservation and haplogroup analyses. In addition, trans-mitochondrial cybrid cell lines were established, mitochondrial functions including ATP, reactive oxygen species (ROS), mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) and mt-RNA transcription were analyzed in cybrids with and without these putative pathogenic mt-tRNA variants. We identified five possible pathogenic variants: tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G that only found in SLE patients but were absent in controls. Interestingly, these variants were located at extremely conserved nucleotides of the corresponding tRNAs and may alter tRNAs' structure and function. Furthermore, cells carrying these tRNA variants had much lower levels of ATP, mtDNA copy number, MMP and SOD than controls, by contrast, the levels of ROS increased significantly (p<0.05 for all). Furthermore, a significantly reductions in mt-ND1, ND2, ND3, ND5 and A6 mRNA expression were observed in cells with these mt-tRNA variants, while compared with controls. Thus, failures in tRNAs metabolism caused by these variants would impair mitochondrial translation, and subsequently lead to mitochondrial dysfunction that was involved in the progression and pathogenesis of SLE. Our study suggested that mt-tRNA variants were important causes for SLE, screening for mt-tRNA pathogenic variants was recommended for early detection and prevention for this disorder.

Differentiating Multiple Sclerosis and Glaucoma With Sectoral Pattern Analysis of Peripapillary Nerve Fiber Layer.

To distinguish between multiple sclerosis (MS) and glaucoma by nerve fiber layer (NFL) thinning patterns. MS patients were diagnosed by the 2017 McDonald Criteria; glaucoma patients had disc rim thinning or an NFL defect, with or without perimetric defect. The peripapillary NFL thickness was divided into eight sectors, and percentage reduction (% reduction) was calculated relative to normative reference values. The MS and glaucoma eyes were grouped based on the severity of NFL thinning in the worst sector: significant reduction (<1 percentile of normal reference), borderline reduction (1%∼5%), and no reduction (>5%). We devised four diagnostic indexes, and the area under the curve of receiver operating characteristics (AROC) and accuracy were used to evaluate the indexes. We enrolled 58 control subjects (58 eyes), 56 MS subjects (112 eyes), and 92 glaucoma subjects (92 eyes) at two centers. The most pronounced percent reduction in MS eyes occurred in the temporal-upper and temporal-lower sectors. In glaucoma eyes, this occurred in the inferior-temporal, inferior-nasal, and superior-temporal sectors. The temporal pattern index had the best AROC (0.96, 0.91-1.00) and accuracy (92.6%) in the significant reduction group. It had good AROC (0.88, 0.78-0.99) and accuracy (76.7%) in the borderline reduction group. Normalizing NFL reduction as a percentage of normal reference accentuated patterns characteristic of MS and glaucoma. Quantitative pattern indexes were effective in differentiating the two diseases. The utility of optical coherence tomography in the differential diagnosis of optic neuropathies is enhanced by analyzing the retinal nerve fiber layer percentage reduction pattern.

Abstract 4131736: Myocardial Work in Patients with Systemic Arterial Hypertension: Correlation with Left Ventricular Mass and Aortic Strain Lidia Capotosto 1,2 , Enrico Mangieri 2 , Gaetano Tanzilli 2 , Giuseppe Placanica 2 , Attilio Placanica 2 , Fabio Miraldi 2 , Antonio Vitarelli 1,21 Department of Echocardiography, Cardiodiagnostica CS, Rome, Italy, 2 Department of Cardiology and Cardiac Surgery, Sapienza

Background: Myocardial work (MW) is a new echocardiography-based diagnostic tool, which allows to quantify left ventricular performance based on pressure-strain loops and has been validated against invasively derived pressure-volume measurements. The purpose of the present study was to analyze whether systemic arterial hypertension is associated with abnormal left ventricular (LV) function assessed by myocardial work and how these changes are related to left ventricular hypertrophy (LVH) and aortic strain. Methods: We examined 86 hypertensive (mean age, 66±14 years, 51% male) and 86 sex- and age-matched healthy controls (mean age, 64±15 years, 53% male). Hypertensive patients were divided into two groups: patients without LVH (group-A) and patients with LVH (group-B, LVMI&gt;115g/m2 men, LVMI&gt;95g/m2 women). All of them had preserved LV ejection fraction. Global LV longitudinal strain (GLS) was calculated by speckle tracking echocardiography. The following indices of MW were assessed: global work index (GWI), global constructive work (GCW), global wasted work (GWW), and global work efficiency (GWE). Corrected circumferential aortic strain (Ao-S) was calculated in the ascending aorta by two-dimensional speckle tracking echocardiography as global aortic strain /pulse pressure. Data analysis was performed offline (GE EchoPAC). Results: GWI and GCW were increased in group-A compared to controls (p&lt;0.05) and decreased in group-B (p=0.04). GWW was increased in group-A(p=0.02) and group-B(p&lt;0.001). GWE was decreased in group-A(p&lt;0.01) and group-B(p&lt;0.005). There was a positive correlation between GWE and Ao-S(r=0.51,p=0.03). GWE was independently associated with GLS(β=0.26,p=0.034), LV mass(β=0.31,p=0.02) and Ao-S(β=0.37,p=0.01) in the whole hypertensive population. At the receiver operating characteristic (ROC) analysis, optimal cutoff values of GWI, GLS, Ao-S, GWE and (GWE+Ao-S) discriminating LV hypertrophy were 0.7329, 0.7613, 0.8011, 0.8442 and 0.8986, respectively. Conclusions: Decreased GWE in systemic hypertension was related to aortic strain and occurred even in the absence of LV hypertrophy. Additional assessment of aortic strain improved the ability of myocardial work variables to differentiate patients with LVH, patients without LVH, and control subjects.

Abstract 4121527: Empagliflozin Improves Mitochondrial Biogenesis in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by endothelial proliferation and progressive pulmonary vascular (PV) remodeling. Empagliflozin (EMPA), a sodium glucose cotransporter 2 (SGLT2) inhibitor, benefits heart failure patients, potentially mediated by improved mitochondrial function. This study aimed to explore the impact of EMPA in PAH. Methods and Results: Immunohistochemistry of pulmonary arteries revealed the expression of SGLT2 in the intima of PAH patients. Microvascular endothelial cells (MVECs) were isolated from lungs of control subjects (CTRL) and PAH patients and found intense expression of SGLT2 in PAH MVECs. Western blot showed significantly higher expression of SGLT2, while PGC-1α, a transcriptional coactivator known as a master regulator of mitochondrial biogenesis, was lower in PAH MVECs compared to CTRL (Image 1). PAH MVECs were treated with EMPA (1 μM) and observed a significant increase of PGC-1α and BMPR2-pSmad1/5/9. Similarly, knockdown of SGLT2 using siRNA resulted in enhanced expression of PGC-1α. Moreover, Seahorse analysis revealed increased oxygen consumption rate and reduced extracellular acidification rate by EMPA, suggesting that EMPA improved mitochondrial respiration. In addition, EMPA attenuated reactive oxygen species and proliferation of PAH MVECs, as revealed by MitoTracker analysis and MTT assay respectively. For the in vivo study, Sprague-Dawley rats (120-180 gram) were injected with SU5416 (Week-0) and exposed to hypoxia for 3 weeks followed by normoxia. After randomization at Week-4, rats were treated with EMPA (300 mg/kg in chow, n=12) or placebo (PLAC, n=12). Catheterization and histological analysis were performed at Week-8. EMPA decreased PV resistance index (0.21±0.02 vs. 0.37±0.05 mmHg/mL/min/mm 2 , p=0.01), ameliorated intima thickening (21.0±7.5 vs. 40.0±7.8%, p&lt;0.01) and vessel occlusion (Image 2). We further conducted a single-center, open-label, single-arm interventional proof-of-concept study to assess the tolerability of 12 weeks of treatment with 10mg of EMPA as add-on therapy in eight patients with a prior PAH diagnosis. There were no treatment related adverse events during the study time course. Conclusions: EMPA directly improved mitochondrial biogenesis and attenuated proliferation of MVECs from PAH patients and reversed PV remodeling in experimental PAH. EMPA treatment was feasible in patients with PAH. EMPA may serve as a new therapeutic option for PAH.

Abstract 4137186: No Room for Chronotropic Incompetence in Living Fontan Patients: Enhanced Heart Rate Increase in Response to Exercise to Compensate Low Stroke Volume Reserve

Background: Fontan patients reveal decreased peak exercise performance in combination with low peak heart rate (HR), which is frequently interpreted as chronological incompetence. This is counterproductive as HR increase has to be enhanced to compensate limited stroke volume increase in Fontan physiology. Methods: Cardiopulmonary exercise testing (CPET) data in asymptomatic Fontan patients and age-matched control subjects were retrospectively studied. Peak and submaximal CPET parameters were obtained by cycle ergometer. Chronotropic capacity was assessed by peak HR/peak [WR/kg] and submaximal slope of HR/[work rate (WR)/kg] (ΔHR/Δ[WR/kg]) for males and females, separately. Results: Table 1 presents CPET data of Fontan patients (51) and controls (65). Fontan patients showed significantly lower peak HR, peak WR, peak oxygen consumption (VO2)/kg, %predicted maximum VO2, and peak oxygen pulse (pOP)/kg than controls in both sexes whereas peak HR/peak [WR/kg] was significantly higher in Fontan patients, indicating higher HR at the maximum effort in Fontan patients than in controls. Fontan patients showed higher ΔHR/Δ[WR/kg], a marker for HR-dependency, and lower Δ[VO2/kg]/ΔHR, a surrogate of stroke volume, suggesting higher HR was compensating lower stroke volume during submaximal phase of exercise in Fontan patients. Figure 1 demonstrates a linear relationship between ΔHR/ΔWR and peak HR/peak WR in both sexes. Male Fontan patients revealed a comparable trend with controls, whereas female Fontan patients showed higher HR increase at the peak exercise effort than controls with the same ΔHR/ΔWR, suggesting more limited stroke volume reserve than male Fontan patients. Conclusions: Fontan patients showed higher HR-response to exercise than controls in both sexes due to limitation in stroke volume increase. Our data indicate that lower peak HR in Fontan patients results from early exhaustion due to limited stroke volume reserve rather than primary chronotropic incompetence.

Abstract 4118444: Right Ventricular Contractile Reserve and Right Ventricular-Pulmonary Arterial Coupling Are Impaired in Long-Term Survivors of Childhood Cancers

Background: Childhood cancer survivors are at risk of right ventricular (RV) dysfunction in relation to cardiac toxicity due to chemotherapy and radiation therapy. This study tested the hypothesis that RV contractile reserve and RV-pulmonary arterial (PA) coupling are altered in long-term survivors of childhood cancers. Methods: Thirty survivors (60% men) aged 24.3 ± 5.2 years at 15.3 ± 6.3 years after completion of chemotherapy and thirty healthy control subjects (47% men) were studied. Resting and submaximal supine bicycle stress echocardiography was performed for assessment of RV fractional area change (FAC), tricuspid annular plane systolic excursion (TAPSE), left ventricular (LV) and RV longitudinal strain, mitral and tricuspid annular velocities and myocardial acceleration during isovolumic contraction (IVA). The slope of the RV force-frequency relationship was derived from the change in IVA with the change in heart rate during exercise (△IVA/△heart rate), while RV-PA coupling was determined by the ratio between TAPSE and pulmonary arterial acceleration time indexed to RV ejection time (PAATi). Results: At rest, tricuspid annular systolic velocity and RV systolic strain were significantly lower in survivors ( P &lt; 0.05 for both), while RV FAC, TAPSE and IVA were similar between the two groups ( P &gt; 0.05 for all). During submaximal exercise testing, all RV systolic functional indices were significantly lower in survivors than controls ( P &lt; 0.05 for all). The slope of the RV force-frequency relationship was significantly flatter in survivors compared to controls (0.038 ± 0.002 vs 0.059 ± 0.003 m/sec 2 beats/min, P &lt; 0.001). For RV-PA coupling, TAPSE/PAATi was similar between survivors and controls at baseline but became significantly lower in survivors during submaximal exercise ( P = 0.002). Conclusions: The RV contractile reserve and RV-PA coupling are impaired during exercise in long-term survivors of childhood cancers. Future studies aimed at determining the clinical and prognostic significance of these impairments are warranted.

Scleroderma Renal Crisis and Musculoskeletal Corticosteroid Injections.

Inflammatory arthritis frequently affects patients with systemic sclerosis (SSc) but musculoskeletal corticosteroid (MSKC) injections are often avoided due to concerns of scleroderma renal crisis (SRC). This study investigated the incidence of SRC following MSKC injections. In a 136-SSc cohort, 46 subjects underwent a total of 330 MSKC injections each receiving a significant dosage of triamcinolone acetonide (mean, 95.2 ± 44.2 mg per injection session). Blood pressure (BP), serum creatinine and glucose, urine protein, and complications were retrospectively reviewed before and after injection. MSKC and control subjects were similar in age (MSKC: 58.9 ± 12.1 vs. 55.5 ± 14.9 years), female (MSKC: 97.8% [45/46] vs. 89.9% [81/90]), antinuclear antibody (MSKC: 71.7% [33/46] vs. 81.1% [73/90]), anti-centromere antibody (MSKC: 47.8% [22/46] vs. 37.8% [34/90]), anti-topoisomerase antibody (MSKC: 26.1% [12/46] vs. 26.7% [24/90]), and anti-RNA polymerase III antibody (MSKC: 17.4.1% [8/46] vs. 24.4% [22/90]) (all p > 0.05). Pre- and post-MSKC demonstrated nonsignificant changes in systolic BP (pre: 127 ± 22 vs. post: 127 ± 21 mm Hg, p = 1.0), diastolic BP (pre: 71 ± 13 vs. post: 71 ± 11 mm Hg, p = 1.0), creatinine (pre: 0.78 ± 0.56 vs. post: 0.76 ± 0.20 mg/dL, p = 0.64), glucose (pre: 100 ± 21 vs. post: 99 ± 24 mg/dL, p = 0.67), and urine protein-creatinine ratio (pre: 0.14 ± 0.12 vs. post: 0.12 ± 0.11 mg/mg, p = 0.41). One case of SRC with mortality occurred in the controls and none in the MSKC group. No infections, hematologic abnormalities, or tendon rupture were noted. MSKC injections in established SSc are generally safe with low incidences of SRC and complications. However, it is still prudent to monitor high-risk individuals and recent-onset SSc post-MSKC injection.

BIOM-76. EXPLORING CSF MICRORNA SIGNATURES AS DIAGNOSTIC BIOMARKERS IN IDH-WILDTYPE DIFFUSE GLIOMAS

Abstract The most prevalent malignant tumors of the central nervous system are adult-type diffuse gliomas. Isocitrate dehydrogenase (IDH) mutations are identified in a subset of diffuse gliomas, leading to two distinct subtypes of gliomas: IDH-wildtype and IDH-mutant. In comparison to IDH-mutant gliomas, IDH-wildtype gliomas tend to be associated with a more rapid progression and lower overall survival rates. The analysis of differentially expressed microRNAs (DE-miRNAs) in cerebrospinal fluid (CSF) of IDH-wildtype diffuse gliomas could provide an effective and non-invasive approach to assist in the early diagnosis, classification, and management of the disease. Extracellular vesicle (EV)-derived miRNAs obtained from the CSF of 11 patients with IDH-wildtype diffuse gliomas and 8 controls were analyzed by microRNA sequencing (miRNA-seq). DE-miRNAs were defined using p-value≤0.05 and absolute log2 fold change (log2FC) ≥1. Candidate DE-miRNAs were validated by qRT-PCR in 24 samples (15 IDH-wildtype vs. 9 control) using the 2-ΔΔCT method, followed by receiver operating characteristic (ROC) curve analysis. Hsa-miR-24-3p was used as the internal control. miRNA-seq analysis identified hsa-miR-484, hsa-miR-223-3p, hsa-miR-1246, hsa-miR-323a-3p, hsa-miR-106b-3p, hsa-miR-21-5p, hsa-miR-15a-5p, has-miR-15b-5p, and hsa-miR-92a-3p among the top 20 upregulated miRNAs in the CSF of patients with IDH-wildtype diffuse gliomas. Further analysis by qPCR showed that patients with IDH-wildtype diffuse gliomas had significantly higher levels of has-miR-21-5p (2.28 ± 2.33) compared to control subjects (-2.22e-007 ± 1.08, p=0.012). The ROC curve analysis revealed an area under the curve (AUC) of 0.84 (95% CI, 0.68-1.0) for has-miR-21-5p CSF levels in distinguishing IDH-wildtype diffuse gliomas from control subjects. EV-derived miRNAs in CSF, including has-miR-21-5p, have the potential to distinguish IDH-wildtype diffuse gliomas from control individuals in a minimally-invasive manner and serve as diagnostic biomarkers.