Peripheral Plasma Research Articles

Cancer Genomic Profiling and Minimal Residual Disease Monitoring By Cell-Free DNA Sequencing in Pediatric Leukemia

Introduction: Cell-free DNA (cfDNA) sequencing has been reported as a promising non-invasive tool for detecting cancer and monitoring treatment response. We sought to evaluate the potential of a cfDNA-based strategy for detecting diverse classes of somatic mutations relevant to pediatric leukemia and cancers. Methods: Plasma samples collected at diagnosis from 135 acute lymphoblastic leukemia (ALL) and 34 acute myeloid leukemia (AML) patients were evaluated, including 114 B-ALL and 21 T-ALL. We also studied diagnostic plasma samples from 38 solid tumor and 19 brain tumor patients, and plasma collected from 12 patients with B-ALL at days 15, 22 and end of induction chemotherapy (EOI). Plasma cfDNA extraction and library preparation were performed with the NucleoSnap cfDNA kit and ThruPLEX Tag-Seq HV kit, respectively. Next-generation sequencing was performed using a capture panel covering 355 pediatric cancer genes (including noncoding regions covering hotspots of structural variation) and 8,103 heterozygous population polymorphisms. We developed analytic approaches for de novo detection of all types of somatic mutations, including focal copy number alterations, rearrangements, and large chromosomal abnormalities. Whole-genome sequencing (WGS) was also performed for 10 cfDNA samples from leukemia. Data were compared with corresponding whole-genome, exome and transcriptome tumor cell sequencing data at diagnosis. Results: At diagnosis, plasma cfDNA yield was higher for leukemias than for solid and brain tumors (median, 142 ng/ml, 7.16 ng/ml, and 7.07 ng/ml, respectively. The average coverages of leukemia cfDNA sequencing were 790X by capture panel post error suppression and 69X by WGS. We found that 100% cases of leukemia at diagnosis had detectable circulating tumor DNA (ctDNA) in plasma cfDNA, and the median ctDNA purity was 77%, comparable to primary tumor purity from diagnostic sequencing. Overall, our panel covered 86% somatic variants present in the tumor samples. Of those covered events, capture-based cfDNA sequencing detected 98% SNVs/indels, 95% focal copy gains/losses, 100% internal tandem duplications, 95% chromosomal translocations, and 99% aneuploidy. In addition, capture-based sequencing identified cfDNA-specific variants such as NRAS/KRAS SNVs and a IKZF1 deletion that were subclonal in plasma cfDNA. Of those uncovered events, 85% were structural variants with their breakpoints in intronic/noncoding regions, and cfDNA WGS identified all somatic events present in the 10 sequenced tumor samples. To detect minimal residual disease (MRD) in B-ALL patients during induction, we increased cfDNA sequencing depth for low VAF events, and the detection limit was 0.4% for SNV/indel and structural variations. Compared to the MRD results from flow cytometry, somatic mutations were detected in 7/8 MRD-positive and 1/4 MRD-negative patients on day 15, 3/4 MRD-positive patients on day 22, and 0/2 MRD-positive patients at EOI. We also sequenced cfDNA from peripheral blood and bone marrow plasma for 2 B-ALL patients on day 0, day 15, and EOI, with comparable ctDNA purity and dynamics observed between the two compartments. In a patient with concurrent ALL and neuroblastoma (NBL) who experienced NBL relapse four months after the primary diagnosis, cfDNA genomic profiling identified ctDNA derived from ALL (52% of total plasma cfDNA), primary NBL (20%), and relapsed NBL (10%), suggesting that genetic variations of multiple tumors could be detected in a single cfDNA sample. Conclusion: Our study has demonstrated the utility of cfDNA sequencing to identify both sequence and structural somatic mutations in childhood acute leukemia. This approach will be useful to monitor disease, as well as providing a non-invasive diagnostic approach.

JAK inhibitors improve ATP production and mitochondrial function in rheumatoid arthritis: a pilot study

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease associated by inflammation of the synovial tissue and autoantibody production. Oxidative stress and free radicals are known to be indirectly implicated in joint damage and cartilage destruction in RA. Several studies describe the presence of mitochondrial dysfunction in RA, but few of them follow the dynamics in energy parameters after therapy. The aim of our investigation is to evaluate the direct effect of JAK inhibitors on cellular metabolism (and under induced oxidative stress) in RA patients. Ten newly diagnosed RA patients were included in the study. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated before and 6 months after therapy with JAK inhibitors. A real-time metabolic analysis was performed to assess mitochondrial function and cell metabolism in PBMCs. Sonographic examination, DAS28 and conventional clinical laboratory parameters were determined also prior and post therapy. A significant decrease in proton leak after therapy with JAK inhibitors was found. The increased production of ATP indicates improvement of cellular bioenergetics status. These findings could be related to the catalytic action of JAK inhibitors on oxidative phosphorylation which corresponds to the amelioration of clinical and ultra-sonographic parameters after treatment. Our study is the first to establish the dynamics of mitochondrial parameters in PBMCs from RA patients before and after in vivo therapy with JAK inhibitors.

Extracellular vesicle small RNA cargo discriminates non-cancer donors from pediatric B-lymphoblastic leukemia patients.

Pediatric B-acute lymphoblastic leukemia (B-ALL) is a disease of abnormally growing B lymphoblasts. Here we hypothesized that extracellular vesicles (EVs), which are nanosized particles released by all cells (including cancer cells), could be used to monitor B-ALL severity and progression by sampling plasma instead of bone marrow. EVs are especially attractive as they are present throughout the circulation regardless of the location of the originating cell. First, we used nanoparticle tracking analysis to compare EVs between non-cancer donor (NCD) and B-ALL blood plasma; we found that B-ALL plasma contains more EVs than NCD plasma. We then isolated EVs from NCD and pediatric B-ALL peripheral blood plasma using a synthetic peptide-based isolation technique (Vn96), which is clinically amenable and isolates a broad spectrum of EVs. RNA-seq analysis of small RNAs contained within the isolated EVs revealed a signature of differentially packaged and exclusively packaged RNAs that distinguish NCD from B-ALL. The plasma EVs contain a heterogenous mixture of miRNAs and fragments of long non-coding RNA (lncRNA) and messenger RNA (mRNA). Transcripts packaged in B-ALL EVs include those involved in negative cell cycle regulation, potentially suggesting that B-ALL cells may use EVs to discard gene sequences that control growth. In contrast, NCD EVs carry sequences representative of multiple organs, including brain, muscle, and epithelial cells. This signature could potentially be used to monitor B-ALL disease burden in pediatric B-ALL patients via blood draws instead of invasive bone marrow aspirates.

Untargeted metabolomic and lipidomic analyses reveal lipid dysregulation in the plasma of acute leukemia patients.

Acute leukemias (AL) are aggressive neoplasms with high mortality rates. Metabolomics and oxidative status have emerged as important tools to identify new biomarkers with clinical utility. To identify the metabolic differences between healthy individuals (HI) and patients with AL, a multiplatform untargeted metabolomic and lipidomic approach was conducted using liquid and gas chromatography coupled with quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS or GC-QTOF-MS). Additionally, the total antioxidant capacity (TAC) was measured. A total of 20 peripheral blood plasma samples were obtained from patients with AL and 18 samples from HI. Our analysis revealed 135 differentially altered metabolites in the patients belonging to 12 chemical classes; likewise, the metabolic pathways of glycerolipids and sphingolipids were the most affected in the patients. A decrease in the TAC of the patients with respect to the HI was evident. This study conducted with a cohort of Colombian patients is consistent with observations from other research studies that suggest dysregulation of lipid compounds. Furthermore, metabolic differences between patients and HI appear to be independent of lifestyle, race, or geographic location, providing valuable information for future advancements in understanding the disease and developing more global therapies.

Associations of maternal exposure to multiple plasma metals with the risk of fetal congenital heart defects: a prospective nested case-control study

Abstract Background Congenital heart defects (CHDs) are the most common congenital anomalies with complex etiologies. There is knowledge gap with respect to the association between single and combined exposures to multiple heavy metals during pregnancy and the risk of CHDs in the fetus. Thus, we aimed to examine the association between maternal plasma heavy metal concentrations in mid-pregnancy and the risk of CHDs in the fetus. Methods A prospective nested case-control study was conducted in a cohort of 11,578 newborns. Exposure odds ratios (ORs) were compared between 164 CHD cases and 164 non-malformed controls delivered at the same hospital, individually matched by maternal age (±5 years) and parity. Concentrations of 21 metals were determined in maternal peripheral blood plasma via an ultra-performance liquid chromatography inductively coupled to mass spectrometry (ICP-MS). Single- and multiple-metal logistic regressions, the adaptive least absolute shrinkage and selection operator (LASSO) penalized regression analysis and restricted cubic spline (RCS) analysis were applied to explore the associations and dose-response relationships of plasma metals with CHD. We also applied Bayesian Kernel Machine Regression (BKMR) model to evaluate the cumulative effect of the exposure metals. Results Median (interquartile range, IQR) Vanadium (V) [0.3 (0.3-0.4) vs. 0.3 (0.3-0.4) μg/L, P = 0.027] and cadmium (Cd) [0.1 (0.0-0.1) vs. 0.1 (0.0-0.1) μg/L, P = 0.036] levels were different between the CHDs and control groups. Compared with the first quartile, the Directed acyclic graph(DAG) model-adjusted ORs (95% CI) were 3.12 (1.72-5.64) for kalium (K), 2.21 (1.23-3.94) for manganese (Mn), 0.55 (0.33-0.94) for cerium (Ce), 1.96 (1.06-3.64) for lead (Pb) in the second quartile, and ORs (95%CIs) were 1.88 (1.08-3.26) for V, 0.53 (0.30-0.93) for Cr, 1.92 (1.10-3.35) for copper (Cu), 2.09 (1.18-3.70) for Cd, 2.14 (1.24-3.70) for stannum (Sn) and 3.05 (1.69-5.49) for Pb in the third quartile. In the multi-metal model, compared to the lowest quartile, the ORs for Sn and Pb in the second quartile were 1.32 (95% CI: 0.69-2.55) and 1.94 (95% CI: 0.98-3.84), respectively, and the ORs for both metals in the third quartiles were 2.15 (95% CI: 1.16-3.98) and 3.32 (OR=0.66, 95% CI: 1.68-6.55), suggesting that Sn and Pb had relatively stable positive results with a linear dose progression. BKMR models with 2 cluster for 9 metals showed that the overall effect of metal mixture was not statistically significant at or below 35th percentile and at or above the 70th percentile, compared to their median levels. Pb was the major contributor to the combined effect. Conclusions Elevated level of plasma plumbum was associated with increased risk of CHDs. Further investigations in larger perspective cohorts are needed to confirm our findings.fig 1fig 2

Abstract 15922: Integrative Analysis of Extracellular Vesicles in Heart Transplant Rejection: From Biomarker Discovery to Novel Immune Mechanisms

Introduction: At the forefront of post-heart transplant care is early detection of transplant rejection. However, the current gold standard for detecting acute cellular transplant rejection is recurrent invasive endomyocardial biopsies (EMB). We aimed at developing a non-invasive biomarker for acute cellular heart transplant rejection (ACR) using a plasma extracellular vesicle (EV) platform. Methods: Peripheral and coronary sinus plasma samples were prospectively collected from 183 (31.7% female) patients from 2020 to 2023 during routine surveillance EMB. A discovery cohort comprised five patients who displayed ISHLT 0-1R biopsy followed by an ISHLT 2R grade rejection event. Plasma EVs were isolated from the baseline (0-1R) and rejection (2R) time points. Subsequently, integrative analysis of plasma EV proteomic, miRNomic, and metabolomics was performed pairwise with the samples. Results: The study collected 943 sample time points from 183 patients. Of these sample collections, 43 (4.5%) cases were 2R rejections. Our proteomic analysis of the plasma EVs identified 28 proteins found explicitly during a grade 2R rejection and 87 differentially expressed proteins during a 2R rejection event. An Ingenuity Pathway Analysis (IPA) analysis of the upregulated proteins highlighted EVs mediated mTOR signaling and antigen presentation as central to rejection events. Although all patients in the discovery cohort were negative for antibody-mediated rejection (AMR), the proteomic findings also revealed increased B-cell receptor signaling, implying a mechanistic intersection of ACR and AMR. Conclusions: A distinct set of proteins were found in plasma EVs during ACR events, providing potentially specific and sensitive biomarkers to reduce the need for EMB that could be exploited for ACR diagnosis. The proteins identified formed an immune signaling network that suggests a novel role for EVs in ACR pathogenesis.

A Novel Comprehensive Tumor-Informed Plasma cfDNA Assay to Monitor Minimal Residual Disease for Hematological and Solid Malignancies

Background: Cell-free DNA (cfDNA) in peripheral blood plasma is a promising tool for monitoring minimal residual disease (MRD) in hematologic and solid malignancies. Unlike conventional biopsy methods, cfDNA extraction is non-invasive, and the short turnaround time enables the analysis of patient samples across multiple time points over an extended period to monitor disease progression. Currently available tests are difficult to scale because they detect only a limited number of mutations, require patient- and mutation-specific customization, and demand constant review by experts. Here, we describe a novel and comprehensive cfDNA testing platform encompassing all major clinically reliable genetic hotspots that we validated using plasma samples from a cancer patient cohort. We also analyzed plasma samples collected from a follicular lymphoma (FL) patient at multiple time points to illustrate disease relapse before clinical manifestation and to show the applicability of our assay in MRD testing. Methods: We constructed a panel of 216 clinically actionable genes, including exonic and intronic regions, capable of tracking point somatic mutations (SNVs/indels), structural variants, and microsatellite instability (MSI). To identify mutational events, we used deep sequencing (median exon coverage of at least 2000×) and a tumor-informed variant calling algorithm developed in-house. Next, we determined the limit of detection (LOD), sensitivity, and specificity of our assay using plasma samples from 74 patients with various solid cancer diagnoses. We tested our platform on standard reference dilutions (Seraseq® ctDNA Complete™ Mutation Mix 0.1%, 0.25%, 0.5%, 1%, 2.5%; Twist cfDNA Pan-cancer Reference Standard 0.1%, 0.25%, 0.5%, 1%, 2%) to validate our results. Our assay was validated by sequencing samples from the internal cohort (n = 74) in replicates and by repeat sequencing of the reference materials in various input amounts in triplicate. Finally, we analyzed plasma samples from an FL patient at five timepoints to showcase our ability to detect mutations in disease recurrence before clinical manifestation. Results: We established the capability of our assay using plasma samples from a cancer patient cohort (n = 74) and two sets of dilutions of reference materials. With an LOD > 0.05% variant allele frequency (VAF) and sequencing depth of more than 2000x our assay achieved a sensitivity of 93-97% and a specificity of 98-99%. It also detected a clonal hematopoiesis of indeterminate potential (CHIP) mutation in one plasma sample, implying that it was more sensitive than variant calling from solid tumors and may allow us to distinguish true somatic mutations from CHIP mutations. VAF comparison between two plasma replicates showed high correlation (Pearson's r > 0.99, p < 0.0001), indicating that our assay is robust and suitable for comparing samples from the same patient across multiple time points. Using different amounts of the reference cfDNA as the starting material for library preparation (15 ng, 25 ng, 50 ng), we assessed the variability of VAF to determine the error range associated with each amount used. By identifying the appropriate amount of starting material needed in order to maintain the LOD at 0.05% VAF across all samples analyzed, we were able to account for variations in cfDNA levels in plasma samples collected at different time points from the same patient. As such, it ensured the reliability of our test across all analyzed samples. We used our assay to analyze plasma samples from a patient diagnosed with stage III FL in 2014 who underwent six cycles of therapy with bendamustine and rituximab. The five plasma samples analyzed were collected in 2021, 2022, and 2023 ( Figure 1). Our assay detected clinically relevant mutations in samples from Mar 2021, which was 14 months earlier than mutation calling from blood cells. Conclusions: At a LOD of 0.05% VAF and with all performance metrics greater than 95%, our assay meets the requirements for MRD testing. Analysis of plasma samples from an FL patient also shows that our assay can detect clinically relevant mutations in plasma samples much earlier than mutation calling from blood cells. We will further validate our assay with more clinical samples and expand our scope to include structural variants and MSI, to further lower the LOD.

Multi-Omics Exploration of Adaptive Mechanism to BTK Inhibition By Ibrutinib in CLL Identified TMBIM6/BI-1 As a Poor Prognosis Variable and Potential Therapeutic Target

Introduction: Ibrutinib is currently part of the therapeutic armamentarium employed in patients with CLL. Despite being an active drug, its usage is not devoid of adverse effects and resistances leading to discontinuation. In addition, current evidence suggests that ibrutinib improves the T cell disfunction associated with CLL progression. The objective of our study was to longitudinally assess the T cell immune profile of ibrutinib-treated CLL patients, and to identify potential adaptive mechanisms to BTK inhibition in B cells in the first 12 months of continuous treatment. Methods: Blood samples from 28 patients from GELLC7 trial (NCT03280160, ibrutinib followed by ofatumumab consolidation) were collected before treatment (BT), and 1, 3, 6 and 12 months on treatment (at least two timepoints). Peripheral blood mononuclear cells and plasma were isolated and stored to perform multi-omics analyses including flow cytometry, RNA sequencing, cytokine determination, migration assays and targeted DNA sequencing. Results: T cell immunophenotyping revealed a decrease of CD4+ and CD8+ T cells expressing PD1 and CD244 and a reduction of regulatory T cells (Tregs) and T follicular helper (Tfh) cells after ibrutinib treatment. In addition, bulk-RNAseq of purified T cells revealed 170 differentially expressed genes (DEG) after 6 months of ibrutinib treatment. These genes participate in pathways related to cell stimulation and regulation of cell activation. Gene set enrichment analysis (GSEA) showed a downregulation of oxidative metabolism, proliferation, and activation pathways, indicating a reduction of the CLL inflammatory burden. Furthermore, there was a significant reduction of several inflammatory/migration chemokines in plasma at 6 months (CCL2/3/4, CXCL10, CXCL12, CXCL13, CCL19). In CLL cells, a decreased expression of adhesion (CD44/CD62L), immunosuppression- (CD200/BTLA), and migration-related markers (CXCR5/CCR7) at 6 months of ibrutinib treatment was observed. However, CXCR4 expression increased upon ibrutinib treatment, and the proliferative CXCR4dimCD5br population was reduced. We investigated CXCR4-mediated migration as a potential mechanism of CLL cell adaptation to ibrutinib in 14 patients at BT and 6 months of BTK inhibition. Reduced CXCR4-migratory capacity was observed, but some cells retained migration under BTK inhibition. Targeted-DNAseq and bulk-RNAseq were performed at BT and 6 months of ibrutinib to characterize CLL cells with retained migratory capacity. There was an alteration of the subclonal composition of CLL cells that retained migratory capacity under BTK inhibition in 7/12 patients with a recurrent enrichment of subclones with mutations in ATM and NFKBIE gene. BTK p.Cys481Ser mutation was acquired in one patient after 6 months of treatment with ibrutinib, while no PLCG2 mutations were detected. GSEA showed an upregulation of mTORC1 and Myc pathways in cells with retained migration. Among the DEG, TMBIM6 was the most significantly upregulated gene. TMBIM6 gene is associated with apoptosis, mTOR-Akt axis and poor prognosis in solid tumors. We evaluated the prognostic impact of TMBIM6 expression using public data from the CLL-map portal (Knisbacher et al. 2022 Nat Genet). TMBIM6 high expression was associated with poor overall survival in both univariate and multivariate analyses including age, gender, IGHV mutation status (Figure 1) and recurrent genetic alterations. When TMBIM6 was targeted with BIA, a TMBIM6 antagonist (Kim et al. 2020 Nat Commun), its effect on CXCR4-mediated migration was limited but it induced CLL cell apoptosis in suspension and co-cultured CLL cells (Figure 2). Moreover, BIA addition to ibrutinib treatment induced synergistic apoptosis. Collectively, these results indicate that TMBIM6 may be a potential novel target for CLL and that combination with ibrutinib could be a valid approach for time-limited therapy. Conclusions: Ibrutinib in T cells reduced the expression of exhaustion markers, Tregs and Tfh cells. In CLL cells, we observed a downregulation of markers related to adhesion, immunosuppression and migration, but an overexpression of TMBIM6 in CLL cells that retained migrative capacity towards CXCL12 under ibrutinib. Finally, we identified TMBIM6 expression as an independent poor prognostic factor and a potential novel target for CLL.

Ruxolitinib Promotes Clinical Responses in Large Granular Lymphocytic Leukemia Via Suppression of JAK/STAT-Dependent Inflammatory Cascades

Background: Large granular lymphocytic leukemias (LGL) are T or NK-cell lymphoproliferative disorders that are characterized by myelosuppression and frequent mutations in STAT3. Commonly used agents include low doses of methotrexate or cyclophosphamide. Among 5 patients (pts) with LGL treated on our phase II study of ruxolitinib in T-cell lymphoma (Moskowitz, et al. Blood 2021), 2 achieved partial response (PR) and 2 achieved stable disease, suggesting oncogenic dependence on JAK/STAT pathway activity in LGL. Methods: To further explore the efficacy and mechanism of JAK1/2 blockade in LGL, we expanded our phase II study to include 23 pts with relapsed/refractory LGL. This sample size was chosen to allow for narrowing of the 95% confidence interval to 54-87% (Wilson score method) if similar efficacy was observed. Pts received ruxolitinib 20mg PO BID continuously. Responses were assessed after cycles 2, 5, and every 3 cycles thereafter using the response assessment from the ECOG-5998 study for LGL. Furthermore, stable disease (SD) was defined as improvement in cytopenia(s) by 1 grade (without meeting criteria for PR) for a minimum of 6 months. Overall response rate (ORR) included complete response (CR) plus PR; clinical benefit rate (CBR) included ORR plus SD. In parallel, we performed single-cell RNA sequencing (scRNA-seq) on paired C1D1 and C1D8 peripheral blood mononuclear cells (PBMCs) from 7 patients (5 PR, 1 SD, and 1 non-responder) and plasma proteomic profiling on 5 patients (3 PR, 1 SD, and 1 POD). Results: Among 23 pts, median age was 58 (31-86), 13 (57%) were male, 19 (83%) were white, 1 (4%) was black. Median number of prior therapies was 2 (range 0-5). 12/22 (54%) had STAT3 mutations (of whom 1 also had a JAK1 mutation); 1 had a JAK2 mutation, 1 pt was missing sequencing. Baseline cytopenias included anemia (n=14, 61%), thrombocytopenia (n=12, 52%), and/or neutropenia (n=16, 70%). Among 20 pts evaluable for response (3 too early), there were 5 (25%) CRs, 6 (30%) PRs, and 4 (20%) SD. ORR and CBR were 55% and 75% respectively. Median event free survival (EFS) was not reached and was 68% (95% CI: 50-93) at 14 months ( Fig. 1A). STAT3 mutation status predicted for improved EFS (14-month EFS 100% vs 40%, p=0.007). After a median duration of treatment of 13.1 months, there were no unexpected toxicities. Severe adverse events included febrile neutropenia (n=3, 13%) and herpes zoster (n=1, 4%). To understand the mechanism by which ruxolitinib improved hematologic parameters in LGL, we performed unbiased proteomic analysis of peripheral blood plasma samples using the OLink 96 Inflammation and Immune Response Platforms. Ruxolitinib led to a decrease in several soluble mediators known to be driven by interferon-driven JAK/STAT activation, including CXCL9, CXCL10, and PD-L1, as well as TNF ( Fig. 1B). Several of these factors have been shown to suppress hematopoietic stem cell function, suggesting a JAK/STAT-dependent paracrine mechanism by which LGL leads to myelosuppression. ScRNA-seq of PBMCs identified LGL cells based on expression of cytotoxic genes, including NKG7, CST7, CCL5, GZMB and PRF1. Analysis of cell type proportions within the peripheral blood showed that patients achieving a PR to Ruxolitinib showed a decrease in the proportion of circulating myeloid cells. Gene set enrichment analysis (GSEA) of LGL cells revealed substantial downregulation of both JAK/STAT signaling and TNF signaling in LGL cells in PR but not SD patients, whereas persistently elevated TNF signaling was seen not only in LGL cells but also circulating myeloid and naïve T-cells in SD patients, suggesting that LGL cells might promote bone marrow suppression via paracrine effects on the bystander host immune system. Network analysis of LGL cells and non-malignant peripheral blood immune cells is ongoing. Conclusions: Ruxolitinib induced durable responses in pts with LGL and therefore may represent an option for pts requiring treatment. STAT3 mutation status was predictive of EFS and responses were associated with JAK/STAT pathway down-regulation, demonstrating on-target effects. Integrated immune profiling suggests that ruxolitinib may interrupt JAK/STAT-dependent paracrine inflammatory signals that promote bone marrow suppression. An ongoing phase II study of ruxolitinib in LGL will aid in clarifying optimal dosing and confirming both efficacy and mechanism of action (NCT05592015).

Evaluation of Metabolic Profile in Monoclonal Gammopathy of Undetermined Significance and Asymptomatic Multiple Myeloma

Background: Multiple myeloma (MM) is an incurable, heterogeneous neoplasm of clonal plasma cells preceded by the precursor conditions monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic multiple myeloma (AMM). Features of metabolic syndrome including obesity and diabetes mellitus (DM) have been linked to MM development and mortality. A study of serum metabolomics in patients with clinical MM revealed alterations in the citrate cycle, pyrimidine, purine, and amino acid metabolism (Chanukuppa V, et al, 2019). However, the role of dysregulated metabolism in progression of monoclonal gammopathy across the disease spectrum requires further investigation. We performed a pilot study to compare metabolic profiles in patients with MGUS and AMM. Methods: Clinical data and peripheral blood plasma samples were collected on 20 patients with non-immunoglobulin M MGUS and 20 patients with AMM. Global metabolomic studies by liquid chromatography (LC) tandem mass spectrometry (MS) were performed on plasma using a SCIEX TripleTOF 6600+ mass spectrometer. Each sample was run on Hypercarb and Reverse phase LC columns in both positive and negative MS modes. Standard targeted and untargeted metabolomic platforms were used with assessment of changing metabolites between the two groups in a one-to-one comparison. Correlation analysis and differential expression analysis were completed and top changing metabolites determined based on ranking of log fold change and p value cutoff (at least <0.05). Pathway enrichment data was generated with advanced data analysis. Results: Median age was 70 and 67 years in the MGUS and AMM groups, respectively. 65% of patients in the AMM group were male versus (vs) 40% in the MGUS group. Black individuals represented 15% of MGUS and 30% of AMM patients. Mean serum monoclonal (M) protein was higher in AMM patients (0.87 g/dL vs 0.28 g/dL, p=0.009); mean bone marrow clonal plasma cells in AMM was 17% (range 10-40%). Three AMM patients had high risk cytogenetics inclusive of chromosome 1 abnormalities. We observed that more individuals in the AMM group had a diagnosis of DM (35% vs 15%), were on hyperlipidemia (HLD) medications (80% vs 45%) and were on ≥3 hypertension (HTN) medications (35% vs 20%). Increasing weight and hemoglobin a1c values were seen with rise in serum M protein in the combined groups. For global metabolomics, 2,513 metabolites were annotated from targeted and untargeted peak curation and results underwent data normalization and standardization with exclusion of outliers from the data set. Using targeted data, Partial Least Squares-Discriminant Analysis showed separation of the MGUS and AMM groups (Figure 1). There were 8 significantly increased metabolites (dihydrouracil, quinate, melatonin, azelate, phosphonoacetate, malate, L-alanine, and lysine) and 9 significantly decreased metabolites (diethanolamine, xanthosine, nervonate, quinolinate, L-dopa, ethylmalonate, butanoate, carnosine, and thyroxine) in AMM when compared to MGUS. Pathway enrichment analysis revealed several pathways increased in AMM (Figure 2), including D-glutamine and D-glutamate metabolism and the citrate cycle. Pathways decreased in AMM included purine, pyrimidine, and glutathione metabolism. Conclusions: In our study, patients with AMM were more likely to demonstrate features of metabolic syndrome, including higher rate of DM and need for HTN and HLD medications. Additionally, a trend of higher weight and hemoglobin a1c values occurred as serum M protein increased. Global metabolomics identified unique metabolites that differed in AMM relative to MGUS patients. The unsaturated fatty acid nervonate, decreased in AMM in our study, was previously shown to have potential preventive effects on obesity-related metabolic disorders (Oda E, et al, 2005). Our findings in these precursor cohorts are concordant with prior serum metabolomics in MM showing alterations in amino acid metabolism including glutamine (Chanukuppa V, et al, 2019), with glutaminase inhibitors currently under study in relapsed/refractory MM (Bolzoni M, et al, 2016). Additional investigation of metabolic profile in larger groups across the spectrum of monoclonal gammopathy will help further elucidate the role of this dysregulated metabolism in myelomagenesis and could lead to preventative interventions targeting the associated metabolic syndrome earlier in disease course.

Cell-Free DNA from Blood or Bone Marrow as Alternatives to Bone Marrow Cells for Molecular Diagnostics and Monitoring of Multiple Myeloma

Introduction: Molecular profiling of bone marrow (BM) tumor cells in multiple myeloma (MM) can be limited by low tumor cell content for analysis by multiple assays. We therefore evaluated the concordance of molecular profiles of cancer cells enriched from BM versus cell-free DNA (cfDNA) derived from peripheral blood (PB) and BM plasma collected at diagnosis. Use of cfDNA has the potential to enable the prioritization of scarce cancer cells for single-cell RNA-sequencing or other applications that require intact cells. Methods: The MM Molecular Monitoring (M4) Study is a prospective cohort study of newly diagnosed MM patients (pts) receiving standard-of-care treatment at 12 sites across Canada. From 27 pts in this study, we analyzed diagnostic BM-derived CD138+ enriched plasma cell (PC) DNA (n=27, 15-20 mL BM per pt), BM plasma cfDNA (n=26 same tube as BM sampling), PB cfDNA (n=27, 15-20 mL), and PB buffy coat DNA (n=27, 4-6mL) using a combination of a targeted next generation sequencing (NGS) panel (10,000X coverage) and shallow (0.1-1X) whole-genome sequencing (sWGS). PB and BM samples collected in STRECK and EDTA tubes, respectively incurred overnight shipping to a central lab for initial processing. The targeted panel includes exons from 37 genes of potential clinical relevance, all immunoglobulin (Ig) loci, and 5 MM-specific translocation hotspots. CD138+ PCs were profiled by clinical FISH for recurrent MM translocations and copy number variations (CNVs). Results: All PB and BM cfDNA samples yielded sufficient DNA quantity for analyses (>20ng), yet only 19/26 BM cfDNA samples passed quality control and were sequenced. Various strategies such as shearing, dilution, and the use of heparinase were not effective in salvaging the failed BM cfDNA. However, the high success rate in a separate local study (24/25 BM cfDNA samples) suggests shipping in non-cell stabilizing tubes may have contributed to failures. The average DNA yield for PB and BM cfDNA samples was 1408ng (sd = 2752ng) and 2462ng (sd = 4301ng), respectively. We estimated tumor fraction (TF) by examining the proportion of genome altered by CNVs using ichorCNA in samples with sWGS. 14/24 (58%) of PB cfDNA samples and 10/18 (56%) of BM cfDNA samples had a high TF (>5%). TF in PB and BM cfDNA were significantly correlated (rho = 0.85, p < 0.001, n = 18 pairs), but were consistently higher in PB cfDNA (p = 0.03, n = 18 pairs). We first compared mutations between BM-derived PCs and cfDNA. We found 12 distinct somatic mutations (variant allele fraction >1%) that were previously reported to have a pathogenic effect in 11/27 (41%) PC samples. This consisted of 10 single nucleotide variants (SNVs) and 2 deletions across NRAS, BRAF, KRAS, CYLD and PRDM1. We found 9/12 mutations in matched PB cfDNA samples [sensitivity (SN)=75%] and 4/8 mutations in matched BM cfDNA (SN = 50%). We next searched for FISH-validated chromosome rearrangements among enriched BM PCs. From these, we correctly detected 5/8 (63%) fusions using the targeted panel that were called out by clinical FISH. These were further detected in matched PB cfDNA (4/5) and BM cfDNA (2/4) samples. Recurrent MM CNVs (amplification at chromosome 1q or deletions at chromosomes 1p, 13p or 17p) were identified in 18/27 (67%) PC samples by sWGS. These CNVs were successfully detected in 8/9 matched PB cfDNA samples with high TF (by sWGS) and in 2/7 samples with low TF. In BM cfDNA, CNVs were successfully detected in 7/8 high TF samples and 1/6 low TF samples. Lastly, we evaluated BCR Ig rearrangements. We detected a clonal Ig rearrangement in 26/27 (96%) BM-derived PC samples. These clones were detected in 25/26 (96%) matched PC to PB cfDNA pairs and in 16/18 (89%) PC to BM cfDNA pairs. The proportion of fragments at Ig loci which corresponded to productive Ig rearrangements was significantly correlated with TF in PB cfDNA (rho = 0.62, p = 0.002, n = 23) but not in BM cfDNA (rho = 0.37, p = 0.14, n = 17). Conclusions: PB cfDNA yielded 78% of the aberrations detected in BM-derived PCs, whereas BM cfDNA yielded 62%. The lower rate of detection in BM cfDNA may reflect DNA contamination from dying white blood cells as a result of shipping BM samples in non-cell stabilizing preservative tubes. BM and PB cfDNA contain highly concordant CNV calls to PCs when cfDNA TF estimates were >5%. Tumor Ig rearrangements were more readily detected in cfDNA compared to other sources of variation and may provide a less-invasive alternative for disease monitoring.

Evaluation of GBT021601 As a Therapeutic Agent to Restore Bone Marrow Health and Effective Erythropoiesis in a Sickle Mouse Model

Introduction: Hypoxia-mediated damage resulting from vaso-occlusion in sickle cell disease (SCD) leads to abnormal angiogenesis, such as occurs with moyamoya disease and proliferative retinopathy. Abnormal angiogenesis in mouse models of SCD has been observed to impact the bone marrow niche, resulting in a structurally abnormal vascular network. A dysfunctional bone marrow environment results in ineffective erythropoiesis and can damage hematopoietic stem cells (HSCs). Healthy HSCs are essential to the maintenance of hemoglobin (Hb) levels regardless of the rapid turnover of red cells and because of their potential as material for gene therapy. Recently, red cell transfusions were shown to reverse pathogenic angiogenesis in a sickle cell mouse model. However, chronic red cell transfusions are difficult to sustain in individuals with SCD due to complications including iron overload, risk of alloimmunization, and the time and cost involved. GBT021601 is a potent sickle Hb polymerization inhibitor that improves the rheology of red blood cells (RBCs) and has been shown to increase total Hb by as much as 3 g/dL. Here, we explore the effectiveness of GBT021601 for restoring bone marrow health and decreasing proangiogenic factors while maintaining the stemness of marrow cells to preserve erythrocyte maturation. Understanding the impact of GBT021601 on aberrant angiogenesis and the function of HSCs will indicate if it has a role in improving the bone marrow niche and HSC fitness in an era of expanded cell-based therapies for SCD. Methods: Nine-week-old, humanized Townes HbSS SCD mice were split into 3 cohorts (n=8 per group: 4 male, 4 female) and for 12 weeks were fed either standard chow (control) or chow containing 0.1% or 0.2% of GBT021601. After 12 weeks, the mice were phlebotomized and euthanized. Spleens were collected and weighed. Whole bone marrow was collected via centrifugation from both tibias and one femur; the other femur was saved for subsequent 3D confocal microscopy. Bone marrow was analyzed using flow cytometry to measure erythroid cell maturation (Annexin V, TER119, and CD44) and HSC markers (Lin, Sca-1, c-kit). Immunoblot analysis was performed on peripheral plasma to measure hypoxia-induced signaling and markers of angiogenesis (VCAM-1, VEGF-A, ANG-1, and -2). Results: Hb increased by 3 g/dL on average with administration of GBT021601, and spleen weights were significantly reduced ( P=0.0023 and P=0.0015 for control vs 0.1% and 0.2%, respectively). Flow cytometry of the extracted bone marrow revealed a significant increase in mature RBCs and a decrease in erythroid progenitors at all stages ( P<0.001 for RBCs and basophils; P=0.04, P=0.002, and P=0.001 for pro-, poly-, and ortho-chromatic erythroblasts, respectively) (Figure 1). HSCs, defined as Lin-Sca-1 +c-kit +, were not significantly different between groups (control vs 0.1%, control vs 0.2%, and for 0.1% vs 0.2%). Immunoblot analysis revealed a reduction in VCAM-1 and ANG-1 markers with administration of GBT021601 relative to control. However, no difference between ANG-2 and VEGF-A markers was observed between treated and control mice (Figure 2). Conclusions: Treatment with GBT021601 was effective at reducing extramedullary hematopoiesis as determined by the reduction in spleen size compared with control mice. VCAM-1 and ANG-1 are increased in HbSS compared with HbAA mice; treatment with GBT021601 reduced factors that contribute to pathogenic angiogenesis. Treatment with GBT021601 did not change ANG-2 and VEGF-A expression; notably, VEGF-A levels are similar between HbSS and HbAA mice. Our findings support GBT021601 as a sustainable treatment to reduce ineffective erythropoiesis and abnormal angiogenesis-which can otherwise damage the bone marrow niche-thereby suggesting a potential benefit for individuals with SCD participating in future cell-based therapies with curative intent and improved outcomes. More work is planned to examine the ability of GBT021601 to normalize the bone marrow environment and reduce the risk of deleterious effects on allogenic or autologous edited HSCs.

Disturbance of Adaptive Immunity System Was Accompanied by a Decrease in Plasma Short-Chain Fatty Acid for Patients Hospitalized During SARS-CoV-2 Infection After COVID-19 Vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to disorders of immune function and a decrease in the diversity of intestinal flora. We aimed to explore the changes of circulating immune cell subsets and the plasma level of intestinal short-chain fatty acids (SCFAs) in patients with Coronavirus disease 2019 (COVID-19), further understanding the pathogenesis of COVID-19. The study included 83 newly diagnosed COVID-19 patients and 39 non-COVID-19 controls. All have completed a full course of vaccination against SARS-CoV-2. The levels of peripheral lymphocyte subsets and plasma cytokines were detected by flow cytometry. Targeted metabolomics was used to explore the level of SCFAs in plasma. Compared with the non-COVID-19 group, COVID-19 patients showed a decrease in CD19+B cells, CD4+T cells, CD8+T cells, NK cells, CD4+CD8+T cells and CD4-CD8-T cells (all p<0.001) and concomitantly an increase in sIL-2R, IL-6 and IL-10 (all p<0.005). These alterations were more pronounced in those critical patients. In addition, COVID-19 patients had lower levels of propanoic acid (PA), butyric acid (BA), isobutyric acid (IBA) and isohexanoic acid (ICA) (all p<0.01). Among them, the level of ICA is positively correlated with the absolute number of immune cells. Our study suggests the immune cell subsets in COVID-19 patients who had completed vaccination were still severely disturbed and concomitantly lower SCFAs, especially in severe patients with poor prognosis. Lower levels of plasma SCFAs may contribute to lymphopenia in COVID-19. The potential relationship between plasma SCFAs and immune cell reduction provides a new direction for the treatment of COVID-19.

Changes in the level of metallothioneins, zinc and copper in blood plasma of patients with chronic lymphocytic leukemia

The relationship between the content of metallothioneins, copper and zinc ions in peripheral blood plasma in chronic lymphocytic leukemia (CLL) depending on the disease progression was studied. It was shown that in patients with CLL in blood plasma with a progressive disease, partial and complete remission, the values of the zinc-to-copper ratios significantly decreased in comparison to similar indicators of practically healthy donors. It was established that in patients with CLL, the level of metallothioneins in blood plasma decreases with the disease progression compared to other studied groups, which indicates a reduced function of cysteine-containing metallothionein proteins. Based on the obtained data, it can be concluded that the zinc-to-copper concentration ratio and the level of metallothionenins in peripheral blood plasma are potential diagnostic and prognostic markers in chronic leukemia.

MiR-135b-5p targets ADAM12 to suppress invasion and accelerate trophoblast apoptosis in preeclampsia

IntroductionPreeclampsia was a serious complication often leaded to adverse pregnancy outcomes. Abnormal placental miR-135b-5p expression in preeclampsia was observed in our preliminary investigation. However, the role of miR-135b-5p in preeclampsia was unclear. MethodsWe determined the miR-135b-5p expression pattern at the fetomaternal interface and levels in placental tissue and exosomes. MiR-135b-5p expression in the trophoblast cell line HTR8/SVneo was manipulated by transient agomir or antagomir transfection or establishment of HTR8/SVneo cell line stably overexpressing miR-135b or miR-135b-5p-sponger. Then the function of miR-135b-5p on the motility of HTR8/SVneo cells, and its effects on cell viability was determined. Finally, we confirmed the relationship between miR-135b-5p and ADAM12. ResultsMiR-135b-5p exclusively expressed in the villous cytotrophoblast, and extravillous trophoblast. Significant miR-135b-5p upregulation was observed in the placenta and peripheral plasma exosomes in preeclampsia, and could be a highly sensitive molecular marker for preeclampsia. Elevated miR-135b-5p expression significantly promoted apoptosis and inhibited HTR8/SVneo cell invasion and migration. Binding of miR-135b-5p to the ADAM12 mRNA 3ʹ-untranslated region was predicted by bioinformatics analysis and confirmed using a dual-luciferase reporter assay. High miR-135-5p levels inhibit the invasion and migration of trophoblastic cells, possibly by directly binding to the 3ʹ-UTR of DADM12 and suppressing its translation efficiency, thereby nullifying the promotion of trophoblast invasion and migration via ADAM12. DiscussionAbnormal upregulation of miR-135b-5p may be involved in preeclampsia through triggering trophoblast apoptosis and impeding trophoblast invasion and migration by targeting ADAM12.

THU213 Posterior Reversible Encephalopathy Syndrome As A Rare Presentation Of ACTH-dependent Cushing Syndrome In A Pediatric Patient

Abstract Disclosure: E.E. Bell-Sambataro: None. C. Tatsi: None. P. Chittiboina: None. S.A. Bowden: None. Background: Cushing disease (CD), caused by ACTH-secreting pituitary adenoma, is the most common cause of Cushing Syndrome (CS) in children &amp;gt; 5 years of age. Hypertension is reported in up to 50% of pediatric cases with CS. We report a challenging pediatric case with a rare presentation of hypertensive emergency associated with CD. Clinical Case: A 6-year-old female with 3 month-history of rapid weight gain (8 kg), round face, and hypertrichosis presented to emergency department for abdominal pain from nephrolithiasis and was noted to have severe hypertension. While in ED, she developed status epilepticus and altered mental status. Brain MRI findings were consistent with posterior reversible encephalopathy syndrome (PRES). Her hypertensive encephalopathy gradually improved with aggressive anti-hypertensive and anti-epileptic therapy. Further studies confirmed hypercortisolism with markedly elevated urine free cortisol of 1288 ug/day (&amp;lt;18 ug/day) and lack of diurnal variation of serum cortisol (8 AM cortisol of 47.9 ug/dL, midnight cortisol of 20.3 ug/dL). ACTH level of 23 pg/mL suggested ACTH-dependent CS. Low dose (1 mg) dexamethasone suppression test (DST) failed to suppress 8 am cortisol (31.4 ug/dL). High dose DST (120 mcg/kg) suppressed serum cortisol to 67%- borderline threshold for CD, likely due to increased dexamethasone clearance by anticonvulsant. Pituitary MRI did not reveal a visible lesion. Desmopressin stimulation test showed stimulation of ACTH by 700% and cortisol by 160% suggesting pituitary source. Inferior petrosal sinus sampling showed a mildly increased gradient in central to peripheral plasma ACTH of 1.7 at baseline (&amp;gt;2) and post-stimulation of 2.5 (&amp;gt;3). ACTH gradient did not reach cutoffs for pituitary source, likely due to insufficient catheterization of petrosal sinus from anatomically small blood vessels. Given her severity, she underwent surgical pituitary exploration. Postoperative morning cortisol was &amp;lt;5 mcg/dL, with decreased midnight salivary cortisol level obtained 2 months after surgery. She developed post-operative central hypothyroidism but did not have diabetes insipidus. She had a 4 kg weight loss with increased height percentile 5 months after surgery and remains under close monitoring. Conclusion: PRES is a clinical-radiographic disorder characterized by seizures, encephalopathy with neuroimaging findings of reversible cortical and subcortical vasogenic edema. Hypertension induced by marked hypercortisolism precipitated by pain is likely the etiologic factor. Prompt recognition and treatment of PRES are crucial to prevent further life-threatening complications and permanent neurological damage. Early diagnosis and treatment of CD are critical to prevent associated morbidities. Pituitary exploration by expert neurosurgery team should be considered in patients with MRI-negative CD. Presentation: Thursday, June 15, 2023

THU269 Ethnic Differences In Hepatic Insulin Clearance: A Cross-Sectional Study Comparing South Asians And Non-Hispanic Whites

Abstract Disclosure: A.L. Ramanathan: None. H. Fan: None. R. Muniyappa: None. Background: Hepatic insulin clearance homeostaticallyregulates peripheral plasma insulin concentration. Reduced or impaired insulinclearance promotes insulin resistance and hyperinsulinemia, which increase therisk of type 2 diabetes mellitus (T2DM). South Asians (SA) have an elevatedrisk and prevalence of T2DM compared to other ethnic groups, but insulinclearance by ethnicity has not yet been investigated. Our cross-sectional studyexamines insulin clearance in SA compared to non-Hispanic whites (NHW) todetermine ethnic differences in hepatic extraction in both the fasting andpostprandial states. Methods: Thirty healthy SA (age: 39.1 ± 9.4; female: n=12;BMI: 27.7 ± 4.8 kg/m2) and thirty healthy NHW (age: 39.2 ± 11.4;female: n=12; BMI: 26.7 ± 5.3 kg/m2) underwent mixed meal tolerancetests (MMTT) to determine plasma glucose, insulin, and C-peptide levels. Wederived parameters of hepatic clearance using a C-peptide kinetics model, aninsulin secretion model, and an insulin kinetics three-compartmental model. Model parameters included distribution volume of insulin (Vp), control ofplasma glucose on hepatic extraction (aG), basal hepatic extraction levels(HEb), and total hepatic extraction levels (HEtot). Differences by ethnicity werecalculated using an unpaired t-test for parametric variables and a Mann-Whitneytest for nonparametric data. Results: Fasting glucose (93.7 ± 9.77 vs. 89.0 ± 6.96 mg/dL,p=0.06) and fasting insulin (13.30 ± 8.04 vs. 6.58 ± 6.61 mu/mL, p&amp;lt;0.001)were higher in SA. Insulin area under the curve during MMTT was also elevatedin SA (12608 ± 6031 vs. 5892 ± 3533 min*mU/mL, p&amp;lt;0.001). Calculated mean Vp(0.0365 ± 0.0133 vs. 0.0564 ± 0.0130 kg-1, p&amp;lt;0.0001), mean aG(0.2001 ± 0.0409 vs. 0.2285 ± 0.0556 mmol-1, p=0.03), and basalhepatic extraction (54.9 ± 22.6 vs. 65.6 ± 18.8%, p=0.04) were lower in SA.Total hepatic extraction during MMTT was not different between SA and NHW(p=0.33). Conclusions: SA have reduced basal hepatic clearancecompared to NHW, although total extraction remained similar between the twogroups during MMTT. Further investigations are needed to derive accurateinsulin clearance parameters for ethnic groups with elevated T2DM risk. Presentation: Thursday, June 15, 2023

Expression and Significance of IL-17A and IL-22 in Children with Infectious Mononucleosis Complicated with Liver Damage

Aim: To investigate the expression and clinical significance of IL-17A and IL-22 in the plasma of children with infectious mononucleosis complicated with liver damage caused by Epstein–Barr virus (EBV). Method: Peripheral plasma was collected from 64 children with infectious mononucleosis (IM). We compared IL-17A, IL-22, the clinical data and laboratory examination indicators between liver damage and non-liver damage groups. Results: We found that the levels of IL-17A and IL-22 were higher in the liver damage group and positively correlated with liver function indicators. CD4+/CD8+ was lower in the liver damage group. Conclusion: The study provides valuable insights into the clinical significance of IL-17A and IL-22 in children with IM complicated with liver damage caused by EBV.

Metabolite profiling of peripheral blood plasma in pigs in early postnatal life fed whole bovine, caprine or ovine milk.

Ruminants' milk is commonly used for supplying nutrients to infants when breast milk is unavailable or limited. Previous studies have highlighted the differences between ruminants' milk composition, digestion, absorption, and fermentation. However, whether consuming different ruminants' milk impact the appearance of the circulatory blood metabolites in the early postnatal life is not well understood. The analysis conducted here aimed to determine the effect of feeding exclusively whole milk from bovine, caprine or ovine species to pigs, approximately 7 days-old for 15 days, on circulatory blood plasma metabolites. Relative intensities of plasma metabolites were detected using a liquid chromatography-mass spectrometry based metabolomic approach. Seven polar and 83 non-polar (lipids) metabolites in plasma were significantly different (false discovery rate < 0.05) between milk treatments. These included polar metabolites involved in amino acid metabolism and lipids belonging to phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, and triglycerides. Compared to the caprine or bovine milk group, the relative intensities of polar metabolites and unsaturated triglycerides were higher in the peripheral circulation of the ovine milk group. In contrast, relative intensities of saturated triglycerides and phosphatidylcholine were higher in the bovine milk group compared to the ovine or caprine milk group. In addition, correlations were identified between amino acid and lipid intake and their appearance in peripheral blood circulation. The results highlighted that consuming different ruminants' milk influences the plasma appearance of metabolites, especially lipids, that may contribute to early postnatal life development in pigs.

EVALUATION OF COMPONENTS OF THE EXTRACELLULAR PURINERGIC SIGNALING SYSTEM IN HUMAN SEPSIS.

Objective: Extracellular purines such as adenosine triphosphate (ATP), uridine triphosphate (UTP), and uridine diphosphate (UDP) and the ATP degradation product adenosine are biologically active signaling molecules, which accumulate at sites of metabolic stress in sepsis. They have potent immunomodulatory effects by binding to and activating P1 or adenosine and P2 receptors on the surface of leukocytes. Here we assessed the levels of extracellular purines, their receptors, metabolic enzymes, and cellular transporters in leukocytes of septic patients. Methods: Peripheral blood mononuclear cells (PBMCs), neutrophils, and plasma were isolated from blood obtained from septic patients and healthy control subjects. Ribonucleic acid was isolated from cells, and mRNA levels for purinergic receptors, enzymes, and transporters were measured. Adenosine triphosphate, UTP, UDP, and adenosine levels were evaluated in plasma. Results: Adenosine triphosphate levels were lower in septic patients than in healthy individuals, and levels of the other purines were comparable between the two groups. Levels of P1 and P2 receptors did not differ between the two patient groups. mRNA levels of ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1 or CD39 increased, whereas those of NTPDase2, 3, and 8 decreased in PBMCs of septic patients when compared with healthy controls. CD73 mRNA was lower in PBMCs of septic than in healthy individuals. Equilibrative nucleoside transporter (ENT) 1 mRNA concentrations were higher and ENT2, 3, and 4 mRNA concentrations were lower in PBMCs of septic subjects when compared with healthy subjects. Concentrative nucleoside transporter (CNT) 1 mRNA levels were higher in PBMCs of septic versus healthy subjects, whereas the mRNA levels of CNT2, 3, and 4 did not differ. We failed to detect differences in mRNA levels of purinergic receptors, enzymes, and transporters in neutrophils of septic versus healthy subjects. Conclusion: Because CD39 degrades ATP to adenosine monophosphate (AMP), the lower ATP levels in septic individuals may be the result of increased CD39 expression. This increased degradation of ATP did not lead to increased adenosine levels, which may be explained by the decreased expression of CD73, which converts AMP to adenosine. Altogether, our results demonstrate differential regulation of components of the purinergic system in PBMCs during human sepsis.