Human Tissue Samples Research Articles

Clinical functional proteomics of intercellular signalling in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has an atypical, highly stromal tumour microenvironment (TME) that profoundly contributes to its poor prognosis1. Here, to better understand the intercellular signalling between cancer and stromal cells directly in PDAC tumours, we developed a multidimensional proteomic strategy called TMEPro. We appliedTMEPro to profile the glycosylated secreted and plasma membrane proteome of 100 human pancreatic tissue samples to a great depth, define cell type origins and identify potential paracrine cross-talk, especially that mediated through tyrosine phosphorylation. Temporal dynamics during pancreatic tumour progression were investigated in a genetically engineered PDAC mouse model. Functionally, we revealed reciprocal signalling between stromal cells and cancer cells mediated by the stromal PDGFR-PTPN11-FOS signalling axis. Furthermore, we examined the generic shedding mechanism of plasma membrane proteins in PDAC tumours and revealed that matrix-metalloprotease-mediated shedding of the AXL receptor tyrosine kinase ectodomain provides an additional dimension of intercellular signalling regulation in the PDAC TME. Importantly, the level of shed AXL has a potential correlation with lymph node metastasis, and inhibition of AXL shedding and its kinase activity showed a substantial synergistic effect in inhibiting cancer cell growth. In summary, we provide TMEPro, a generically applicable clinical functional proteomic strategy, and a comprehensive resource for better understanding the PDAC TME and facilitating the discovery of new diagnostic and therapeutic targets.

Integrating gene expression and imaging data across Visium capture areas with visiumStitched.

Visium is a widely-used spatially-resolved transcriptomics assay available from 10x Genomics. Standard Visium capture areas (6.5mm by 6.5mm) limit the survey of larger tissue structures, but combining overlapping images and associated gene expression data allow for more complex study designs. Current software can handle nested or partial image overlaps, but is designed for merging up to two capture areas, and cannot account for some technical scenarios related to capture area alignment. We generated Visium data from a postmortem human tissue sample such that two capture areas were partially overlapping and a third one was adjacent. We developed the R/Bioconductor package visiumStitched, which facilitates stitching the images together with Fiji (ImageJ), and constructing SpatialExperiment R objects with the stitched images and gene expression data. visiumStitched constructs an artificial hexagonal array grid which allows seamless downstream analyses such as spatially-aware clustering without discarding data from overlapping spots. Data stitched with visiumStitched can then be interactively visualized with spatialLIBD. visiumStitched provides a simple, but flexible framework to handle various multi-capture area study design scenarios. Specifically, it resolves a data processing step without disrupting analysis workflows and without discarding data from overlapping spots. visiumStitched relies on affine transformations by Fiji, which have limitations and are less accurate when aligning against an atlas or other situations. visiumStitched provides an easy-to-use solution which expands possibilities for designing multi-capture area study designs.

Abstract 4137500: The Histone Methyltransferase MLL1 Regulates Notch Signaling and T Cell Phenotype During Pathologic Abdominal Aortic Aneurysm Development

Objective: Abdominal aortic aneurysms (AAA) are characterized by pathological vascular remodeling and an imbalance between anti-inflammatory regulatory T H cells (Tregs) and pro-inflammatory T H 17 cells within the aortic wall. The mechanisms regulating CD4 + T H cell differentiation during AAA development remain unknown. Recently, the histone methyltransferase, mixed lineage leukemia 1 (MLL1), has been shown to influence T H cell phenotype by directly altering key transcription factors for T H 17 (RORγ) and Treg (FOXp3) differentiation in varying disease states. As such the objective of this study was to evaluate the role of MLL1 in promoting T H 17 activity within AAA development and to determine key T H 17 cell signaling pathways that are altered Methods: Single-cell sequencing was conducted on human AAAs and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and fed a saturated fat diet followed by either AngII infusion to induce AAAs (1 µg/kg/min) or saline. Mice with T-cell specific deletion of Notch1 signaling (Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) were subjected to the AngII-induced AAA model and AAA diameters quantified. CD4+ T-cells were isolated by MACs and gene expression analyzed by qPCR as well as T-cell phenotype by flow cytometry. ChIP was used to evaluate histone 3 lysine trimethylation (H3K4me3). Results: Single-cell RNA sequencing of human aortic tissue revealed that the epigenetic enzyme MLL1 and Notch1 signaling were profoundly upregulated in human AAA CD4+ cells compared to non-aneurysmal control samples. Congruently, CD4 + T H cells isolated from the murine AngII-induced AAA model displayed a predominance of T H 17 phenotype and elevated expression of MLL1. Histone methylation was evaluated by ChIP in AngII-induced AAA CD4+ cells and demonstrated increased levels of the MLL1-mediated activation histone methylation mark, H3K4me3, on the Notch 1 promoter. Mice deficient in CD4 + T H cell Notch signaling ( Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) subjected to the AngII-induced murine model displayed significant reduction AAA development (n=10/group, p<0.05) with decreased CD4+ T H 17 cell activity and reciprocal increase in Treg expression. Conclusions: MLL1, a histone methyltransferase, regulates Notch1 signaling and T-cell differentiation in AAA development, causing pathologic inflammation. This pathway may serve as a potential therapeutic target to prevent aortic dilation.

Ischemia/Reperfusion Upregulates Genes Related to PANoptosis in Human Lung Transplants.

Activation of multiple programmed cell death (PCD) pathways has been reported in cellular and animal studies of ischemia/reperfusion injury in lung transplantation. However, the status of these pathways in human lung transplants remains unknown. This study investigates the involvement of PCD pathways and their relationship with inflammation and signaling pathways in human lung transplants. Transcriptomic analysis was conducted on 54 paired human lung tissue samples at the end of cold preservation time and 2 h after reperfusion, collected between 2008 and 2011. Gene Set Enrichment Analysis (GSEA) and single-sample GSEA were used to examine the activation of genes in 6 PCD pathways. The relationships between PCD pathways and inflammation, as well as signaling pathways, were assessed via single-gene GSEA. GSEA results indicated that apoptosis and necroptosis were significantly upregulated after reperfusion in human lung transplants, whereas the gene sets related to pyroptosis, ferroptosis, autophagy, and cuproptosis were not significantly upregulated. Notably, single-sample GSEA demonstrated an intricate interplay among pyroptosis, apoptosis, and necroptosis, collectively referred to as PANoptosis, which is further supported by enrichment of genes related to PANoptosome, inflammatory response, and nuclear factor-κB and interferon signaling pathways, via single-gene GSEA assays. This study demonstrated the genes of PANoptosis are upregulated in human lung grafts during reperfusion. The discovery of PANoptosis as an underlying mechanism of cell death in human lung grafts implies that effective therapeutics to prevent or reduce PANoptosis may alleviate ischemia/reperfusion injury and improve clinical lung transplant outcomes.

Blockade of the Platelet-Driven CXCL7-CXCR1/2 Inflammatory Axis Prevents Murine Cerebral Aneurysm Formation and Rupture.

Platelet aggregation is intimately associated with vascular inflammation and is commonly seen on routine histology studies of cerebral aneurysms. Platelets, when activated, have been shown to augment neutrophil response and the pro-inflammatory cascade. Platelet-neutrophil complexes have been found to aggravate atherosclerosis through a positive feedback loop. We hypothesized that targeting platelet aggregation and downstream inflammation could be used to prevent aneurysm formation and progression. First, we induced cerebral aneurysm formation in a previously described intracranial aneurysm model via carotid artery ligation, hypertension, and stereotactic elastase injection in C57BL/6 mice and analyzed vessels for lesion and thrombus formation. Raybiotech cytokine arrays were used to analyze 96 cytokines in induced murine aneurysms and 120 cytokines in human tissue samples. Cerebral aneurysm formation and inflammatory pathway were then studied in animals treated with IgG2 antibody (control), anti-GpIb antibody (platelet depletion), 1:10 DMSO:PBS (control), clopidogrel, anti-CXCR1/2 small molecule inhibitor, or anti-CXCL7 antibody. Bleeding assays and flow cytometry were used to evaluate platelet function in treated groups. CD31 + platelet aggregates are a common feature in human and mouse cerebral aneurysm specimens. Platelet ablation in mice prevents cerebral aneurysm formation (20% vs 100% in control antibody-treated mice, n = 5 each, p = 0.0476). Mice treated with 1mg/kg clopidogrel develop significantly less aneurysms than controls (18% vs 73%, n = 11 and 11, respectively, p = 0.03). Semi-quantitative analysis of 96 different cytokines using Raybiotech arrays shows increased protein expression of CXCL7 in murine cerebral aneurysms when compared to controls. Treatment with clopidogrel results in reciprocal decrease in detected CXCL7. Targeting CXCL7-CXCR1/2 axis with 10mg/kg reparixin (CXCR1/2 antagonist) significantly decreases cerebral aneurysm formation (11% vs 73%, n = 9 and 11, p = 0.0098) while treatment with 10mg/kg SB225002 tends to decrease aneurysm formation (36% vs 73%, n = 11 vs n = 7, p = 0.11). Lastly, specific antibody blockade against CXCL7 using anti-CXCL7 antibody at 100 ug/mL significantly decreases cerebral aneurysm formation (29% vs 75%, n = 7 vs n = 8, p = 0.046). Platelet inflammation has an important role in cerebral aneurysm formation. Small molecule inhibitors targeting platelet CXCL7-CXCR1/2 inflammatory axis could be used to prevent cerebral aneurysm formation or progression.

A Necessary Role for Cyclin D2 Induction During Colon Cancer Progression Mediated by L1.

The cell adhesion molecule L1CAM (L1), mainly known for its function in brain cells, is a Wnt/β-catenin signaling target gene in colorectal cancer (CRC) cells, where it promotes invasion and liver metastasis. We interrogated which genes are expressed at increased levels in human CRC tissue and induced in CRC cell lines overexpressing L1. We found increased cyclin D2 levels in CRC tissue and LS 174T and HCT 116 human CRC cells overexpressing L1. Increased cyclin D2 in CRC cells was associated with higher proliferation rates, faster motility, tumorigenesis, and liver metastasis. The suppression of cyclin D2 expression by shRNA to cyclin D2 blocked the increase in these cellular properties of L1-expressing cells. The overexpression of cyclin D2 in the absence of L1 also conferred tumorigenic properties similar to L1 expression. The pathways involved in the elevation of cyclin D2 by L1 include NF-κB, Akt, and β-catenin signaling but not the Erk pathway. We found that in a significant percentage of human CRC tissue samples, cyclin D2 is expressed at high levels in the nuclei of cancer cells. At the same time, the adjacent normal mucosa was negative for cyclin D2 staining. The results suggest that the increased cyclin D2 expression by L1 is required to induce proliferative, motile tumor development in CRC tissue and can serve as a diagnostic marker and a target for CRC therapy.

Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells.

Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) play a critical role in resistance to immunotherapy. In this study, we identified epidermal growth factor-like 6 (Egfl6) as a regulator of myeloid cell functions. Our analyses indicated that Egfl6, via binding with β3 integrins and activation of p38 and SYK signaling, acts as a chemotactic factor for myeloid cell migration and promotes their differentiation toward an immunosuppressive state. In syngeneic mouse models of ovarian cancer (OvCa), tumor expression of Egfl6 increased the intratumoral accumulation of polymorphonuclear (PMN) MDSCs and TAMs and their expression of immunosuppressive factors, including CXCL2, IL-10, and PD-L1. Consistent with this, in an immune 'hot' tumor model, Egfl6 expression eliminated response to anti-PD-L1 therapy, while Egfl6 neutralizing antibody decreased the accumulation of tumor-infiltrating CD206+ TAMs and PMN-MDSCs and restored the efficacy of anti-PD-L1 therapy. Supporting a role in human tumors, in human OvCa tissue samples, areas of high EGFL6 expression colocalized with myeloid cell infiltration. scRNA-Seq analyses revealed a correlation between EGFL6 and immune cell expression of immunosuppressive factors. Our data provide mechanistic insights into the oncoimmunologic functions of EGFL6 in mediating tumor immune suppression and identified EGFL6 as a potential therapeutic target to enhance immunotherapy in patients with OvCa.

Spatial proteomics of single cells and organelles on tissue slides using filter-aided expansion proteomics.

Hydrogel-based tissue expansion combined with mass spectrometry (MS) offers an emerging spatial proteomics approach. Here, we present a filter-aided expansion proteomics (FAXP) strategy for spatial proteomics analysis of archived formalin-fixed paraffin-embedded (FFPE) specimens. Compared to our previous ProteomEx method, FAXP employed a customized tip device to enhance both the stability and throughput of sample preparation, thus guaranteeing the reproducibility and robustness of the workflow. FAXP achieved a 14.5-fold increase in volumetric resolution. It generated over 8 times higher peptide yield and a 255% rise in protein identifications while reducing sample preparation time by 50%. We also demonstrated the applicability of FAXP using human colorectal FFPE tissue samples. Furthermore, for the first time, we achieved bona fide single-subcellular proteomics under image guidance by integrating FAXP with laser capture microdissection.

Tumor-Associated Tractography Derived from High-Angular-Resolution Q-Space MRI May Predict Patterns of Cellular Invasion in Glioblastoma.

The invasion of glioblastoma cells beyond the visible tumor margin depicted by conventional neuroimaging is believed to mediate recurrence and predict poor survival. Radiomic biomarkers that are associated with the direction and extent of tumor infiltration are, however, non-existent. Patients from a single center with newly diagnosed glioblastoma (n = 7) underwent preoperative Q-space magnetic resonance imaging (QSI; 3T, 64 gradient directions, b = 1000 s/mm2) between 2018 and 2019. Tumors were manually segmented, and patterns of inter-voxel coherence spatially intersecting each segmentation were generated to represent tumor-associated tractography. One patient additionally underwent regional biopsy of diffusion tract- versus non-tract-associated tissue during tumor resection for RNA sequencing. Imaging data from this cohort were compared with a historical cohort of n = 66 glioblastoma patients who underwent similar QSI scans. Associations of tractography-derived metrics with survival were assessed using t-tests, linear regression, and Kaplan-Meier statistics. Patient-derived glioblastoma xenograft (PDX) mice generated with the sub-hippocampal injection of human-derived glioblastoma stem cells (GSCs) were scanned under high-field conditions (QSI, 7T, 512 gradient directions), and tumor-associated tractography was compared with the 3D microscopic reconstruction of immunostained GSCs. In the principal enrollment cohort of patients with glioblastoma, all cases displayed tractography patterns with tumor-intersecting tract bundles extending into brain parenchyma, a phenotype which was reproduced in PDX mice as well as in a larger comparison cohort of glioblastoma patients (n = 66), when applying similar methods. Reconstructed spatial patterns of GSCs in PDX mice closely mirrored tumor-associated tractography. On a Kaplan-Meier survival analysis of n = 66 patients, the calculated intra-tumoral mean diffusivity predicted the overall survival (p = 0.037), as did tractography-associated features including mean tract length (p = 0.039) and mean projecting tract length (p = 0.022). The RNA sequencing of human tissue samples (n = 13 tumor samples from a single patient) revealed the overexpression of transcripts which regulate cell motility in tract-associated samples. QSI discriminates tumor-specific patterns of inter-voxel coherence believed to represent white matter pathways which may be susceptible to glioblastoma invasion. These findings may lay the groundwork for future work on therapeutic targeting, patient stratification, and prognosis in glioblastoma.

The myocardium of human dilated nonischemic cardiomyopathy harbors unique tissue-resident lymphocyte subsets

Abstract Purpose The etiology of dilated nonischemic cardiomyopathy (NICM) is often unclear, but thought to be multifactorial, involving an interplay between genetic factors and direct myocardial damage caused by infection, autoimmunity, or toxins. Tissue-resident lymphocytes are non-circulating immune cells which are retained within various tissues and play integral roles in maintaining homeostasis, responding to infection, regulating inflammation, and mediating tissue repair. Despite their likely involvement in various cardiac pathologies, the presence of tissue-resident lymphocytes in the human heart has not yet been well-characterized, neither in health nor in disease. Methods Human left ventricular tissue and paired peripheral blood samples were obtained from six adult organ donors with normal heart function who died of noncardiac causes and from the explants of six adult patients with end-stage nonischemic cardiomyopathy. Heart tissue was processed using mechanical and enzymatic digestion. Mononuclear cells were isolated from both heart and paired blood by density centrifugation. Isolated mononuclear cells were surface stained with an antibody cocktail and interrogated using multidimensional flow cytometry. Results The adult human heart contains a diverse immune cell population (Fig 1A). Both healthy and NICM cardiac lymphocytes express distinct phenotypic profiles which resemble those of resident lymphocytes found across various tissues, distinct from those isolated from paired blood. Frequencies of effector memory T (Tem) cells (CD45RA- CCR7-) were higher in heart than in blood among both CD4+ and CD8+ T-cells. In heart compared to blood, significantly more Tem expressed CD69, a canonical marker of tissue-resident memory T (Trm) cells (Fig 1B). Cardiac Trm, especially CD8+ Trm, expressed classical Trm signature markers including integrins CD103 (epithelial binding) and CD49a (collagen binding). NICM Trm expressed more CD49a than healthy heart Trm (Fig 1C). NK-cells also demonstrated higher CD69 expression (trNK) in heart than in blood, with associated expression of CD103, CD49a, and CXCR6 (Fig 1D). Compared to healthy hearts, NICM trNK were predominantly CD56bright/CD16- (Fig 1E and 1F) and expressed less CD57 (Fig 1D), overall indicating a less mature state with lower cytotoxic potential. Confocal imaging of cardiac tissue revealed CD69-expressing tissue-resident lymphocytes within the cardiac interstitium. Conclusions The adult human heart contains diverse populations of lymphocytes which express canonical markers of tissue residency, similar to lymphocytes resident in other tissues. Compared to healthy hearts, NICM hearts have more CD49a+ Trm, perhaps related to greater fibrosis, and also harbor a distinct subset of immature tissue-resident NK cells which may shed light on pathophysiology. Future work is focused on better exploring the functional phenotypes of these resident cells and better localizing them within the myocardium.FA - Figure 1

Polyethylene Terephthalate Microplastic Exposure Induced Reproductive Toxicity Through Oxidative Stress and p38 Signaling Pathway Activation in Male Mice

Polyethylene terephthalate (PET) is a type of polymer plastic that is often used to make plastic bags, bottles, and clothes. However, the waste of such plastic products is decomposed into microplastics (MPs), which are plastic fragments smaller than 5 mm, by various external forces such as wind, UV radiation, mechanical wear, and biodegradation. PET MPs have been widely detected in the environment and human tissue samples; however, the toxicity and mechanism of PET MPs in mammals are still unclear. In this study, we investigated the male reproductive toxicity of PET MPs and their underlying mechanism. A total of 80 male mice were orally exposed to 0.01, 0.1, and 1 mg/d of PET MPs (with a diameter of 1 μm) for 42 days. The results showed that 1 μm PET MPs induced different degrees of pathological damage to testicular tissues, decreased sperm quality, and increased the apoptosis of spermatogenic cells via oxidative stress and p38 signaling pathway activation. To further illustrate and verify the mechanistic pathway, oxidative stress was antagonized using N-acetylcysteine (NAC), and the activation of the p38 signaling pathway was blocked using SB203580. The results revealed that the male reproductive injury effects after exposure to PET MPs were significantly ameliorated. Specifically, the testicular tissue lesions were relieved, the sperm quality improved, and the apoptosis of spermatogenic cells decreased. These results demonstrated that PET MP exposure induced male reproductive toxicity through oxidative stress and the p38 signaling pathway. This study provides new insights into the reproductive toxicity of MPs in males, as well as valuable references for public health protection strategies.

Sensing and Imaging Agents for Cyclooxygenase Enzyme.

In this concept, we present a comprehensive study on the development and application of COX-2-specific fluorescent probes for cancer imaging and diagnosis. Targetting cancer cells and measuring cancer-related activities in specific organelles quickly and accurately are crucial factors for early diagnosis and research on cancer pathology and treatment. This concept explores a variety of probes which include BDIMC1-2, TPI-IMC, YZP1, IQ-1, ANQ-IMC-6, Niblue-C6-IMC, AZB-IMC2, CMP, CF3-Fluorocoxib, IBPC1, CoxFluor, etc. and each one demonstrates unique mechanisms and high selectivity towards COX-2 enzymes. These probes were designed to enhance fluorescence upon binding to COX-2 which enable precise visualization of tumor and inflamed tissues. The research emphasizes the importance of COX-2 as a biomarker in cancer diagnostics, particularly in identifying cancer stem cells and inflamed tissues. This concept highlights the potentiality of these probes in non-invasive imaging techniques which offering significant advancements in cancer diagnosis and monitoring. The in vivo and in vitro experiments, including applications in mouse models and human tissue samples, confirm the efficacy of these probes in providing detailed imaging for clinical and research applications.

Current Applications of Raman Spectroscopy in Intraoperative Neurosurgery.

Neurosurgery demands exceptional precision due to the brain's complex and delicate structures, necessitating precise targeting of pathological targets. Achieving optimal outcomes depends on the surgeon's ability to accurately differentiate between healthy and pathological tissues during operations. Raman spectroscopy (RS) has emerged as a promising innovation, offering real-time, in vivo non-invasive biochemical tissue characterization. This literature review evaluates the current research on RS applications in intraoperative neurosurgery, emphasizing its potential to enhance surgical precision and patient outcomes. Following PRISMA guidelines, a comprehensive systematic review was conducted using PubMed to extract relevant peer-reviewed articles. The inclusion criteria focused on original research discussing real-time RS applications with human tissue samples in or near the operating room, excluding retrospective studies, reviews, non-human research, and other non-relevant publications. Our findings demonstrate that RS significantly improves tumor margin delineation, with handheld devices achieving high sensitivity and specificity. Stimulated Raman Histology (SRH) provides rapid, high-resolution tissue images comparable to traditional histopathology but with reduced time to diagnosis. Additionally, RS shows promise in identifying tumor types and grades, aiding precise surgical decision-making. RS techniques have been particularly beneficial in enhancing the accuracy of glioma surgeries, where distinguishing between tumor and healthy tissue is critical. By providing real-time molecular data, RS aids neurosurgeons in maximizing the extent of resection (EOR) while minimizing damage to normal brain tissue, potentially improving patient outcomes and reducing recurrence rates. This review underscores the transformative potential of RS in neurosurgery, advocating for continued innovation and research to fully realize its benefits. Despite its substantial potential, further research is needed to validate RS's clinical utility and cost-effectiveness.

DHA plays a protective role in cerebral ischemia–reperfusion injury by affecting macrophage/microglia type polarization

A close correlation exists between the macrophage/microglia(MΦ/MG) polarization states and the development of cerebral ischemia and reperfusion (I/R). Therefore it is of great significance to research on how to modulate the MΦ/MG states for improved patient outcomes. In particular, regulatory mechanisms involved in this process remain to be identified. Hereby, we aim to shed light on how docosahexaenoic acid (DHA) actively modulates the switch between M1 and M2 macrophage states by restraining the NACHT-LRR-PYD-containing protein three inflammasome (NALP3). We found that NALP3-positive cells were detected in clinical human cerebral infarction tissue samples and the mouse tMCAO model. In mice after DHA treatment, the number of NALP3-positive cells was significantly reduced, significantly decreasing infarct volume and improving the postoperative physical status of mice. NALP3-positive cells were found to be MΦ/MG after co-staining with CD11b. By extracting peritoneal macrophages, it was verified that DHA inhibited the activation of NALP3 and regulated the transformation of M1 and M2 cells, thereby reducing I/R injury.

Influence of Donor-Specific Characteristics on Cytokine Responses in H3N2 Influenza A Virus Infection: New Insights from an Ex Vivo Model.

Influenza A virus (IAV) is known for causing seasonal epidemics ranging from flu to more severe outcomes like pneumonia, cytokine storms, and acute respiratory distress syndrome. The innate immune response and inflammasome activation play pivotal roles in sensing, preventing, and clearing the infection, as well as in the potential exacerbation of disease progression. This study examines the complex relationships between donor-specific characteristics and cytokine responses during H3N2 IAV infection using an ex vivo model. At 24 h post infection in 31 human lung explant tissue samples, key cytokines such as interleukin (IL)-6, IL-10, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) were upregulated. Interestingly, a history of lung cancer did not impact the acute immune response. However, cigarette smoking and programmed death-ligand 1 (PD-L1) expression on macrophages significantly increased IL-2 levels. Conversely, age inversely affected IL-4 levels, and diabetes mellitus negatively influenced IL-6 levels. Additionally, both diabetes mellitus and programmed cell death protein 1 (PD-1) expression on CD3+/CD4+ T cells negatively impacted TNF-α levels, while body mass index was inversely associated with IFN-γ production. Toll-like receptor 2 (TLR2) expression emerged as crucial in mediating acute innate and adaptive immune responses. These findings highlight the intricate interplay between individual physiological traits and immune responses during influenza infection, underscoring the importance of tailored and personalized approaches in IAV treatment and prevention.

SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation.

Background/Objectives: Damage to renal tubular cells (RTCs) represents a critical pathological manifestation in calcium oxalate (CaOx) stone disease, but the underlying mechanism remains elusive. Energy metabolism reprogramming is a vital influencer of RTC survival, and SMYD2 is a histone methylation transferase that has been extensively implicated in various metabolic disorders. Hence, this research aimed to identify whether SMYD2 induces the reprogramming of energy metabolism in RTCs exposed to CaOx nephrolithiasis. Methods: Kidney samples were obtained from patients who underwent laparoscopic nephrectomy for non-functioning kidneys caused by nephrolithiasis. The glyoxylate-induced CaOx stone mice model was established and treated with AZ505. The SMYD2-knockout HK-2 cell line was constructed. Histological changes were evaluated by HE, VK, Tunel, Masson stainings. The molecular mechanism was explored through co-immunoprecipitation and western blotting. Results: The results found that SMYD2 upregulation led to energy reprogramming to glycolysis in human kidney tissue samples and in mice with CaOx nephrolithiasis. We also identified the substantial involvement of glycolysis in the induction of apoptosis, inflammation, and epithelial-mesenchymal transition (EMT) in HK-2 cells caused by calcium oxalate monohydrate (COM). In vivo and in vitro results demonstrated that SMYD2 inhibition reduces glycolysis, kidney injury, and fibrosis. Mechanistically, SMYD2 was found to promote metabolic reprogramming of RTCs toward glycolysis by activating the AKT/mTOR pathway via methylated PTEN, which mediates CaOx-induced renal injury and fibrosis. Conclusions: Our findings reveal an epigenetic regulatory role of SMYD2 in metabolic reprogramming in CaOx nephrolithiasis and associated kidney injury, suggesting that targeting SMYD2 and glycolysis may represent a potential therapeutic strategy for CaOx-induced kidney injury and fibrosis.

Myelination potential and injury susceptibility of grey versus white matter human oligodendrocytes.

Increasing evidence indicates heterogeneity in functional and molecular properties of oligodendrocyte lineage cells both during development and under pathologic conditions. In multiple sclerosis, remyelination of grey matter lesions exceeds that in white matter. Here we used cells derived from grey matter versus white matter regions of surgically resected human brain tissue samples, to compare the capacities of human A2B5-positive progenitor cells and mature oligodendrocytes to ensheath synthetic nanofibers, and relate differences to the molecular profiles of these cells. For both cell types, the percentage of ensheathing cells was greater for grey matter versus white matter cells. For both grey matter and white matter samples, the percentage of cells ensheathing nanofibers was greater for A2B5-positive cells versus mature oligodendrocytes. Grey matter A2B5-positive cells were more susceptible than white matter A2B5-positive cells to injury induced by metabolic insults. Bulk RNA sequencing indicated that separation by cell type (A2B5-positive vs mature oligodendrocytes) is more significant than by region but segregation for each cell type by region is apparent. Molecular features of grey matter versus white matter derived A2B5-positive and mature oligodendrocytes were lower expression of mature oligodendrocyte genes and increased expression of early oligodendrocyte lineage genes. Genes and pathways with increased expression in grey matter derived cells with relevance for myelination included those related to responses to external environment, cell-cell communication, cell migration, and cell adhesion. Immune and cell death related genes were up-regulated in grey matter derived cells. We observed a significant number of up-regulated genes shared between the stress/injury and myelination processes, providing a basis for these features. In contrast to oligodendrocyte lineage cells, no functional or molecular heterogeneity was detected in microglia maintained in vitro, likely reflecting the plasticity of these cells ex vivo. The combined functional and molecular data indicate that grey matter human oligodendrocytes have increased intrinsic capacity to myelinate but also increased injury susceptibility, in part reflecting their being at a stage earlier in the oligodendrocyte lineage.

6885 Glucose Utilization Differs Between RAS-like and BRAF-like Thyroid Cancer

Abstract Disclosure: S. Kumari: None. E. Chuki: None. J. Klubo-Gwiezdzinska: None. Background: RAS-like thyroid cancer (TC) tends to be more differentiated and less [1][8]Fluorodeoxyglucose Positron Emission Tomography-avid as compared with BRAF-like TC. Therefore, we hypothesized that glucose metabolism may differ between RAS-like and BRAF-like tumors and performed a study aimed at metabolomic profiling of TC. Methods: Based on Institutional Review Board approved protocol, we obtained fresh frozen human TC samples with corresponding normal tissue and performed targeted metabolomics including oxidative phosphorylation (OXPHOS), glycolysis and tricarboxylic acid (TCA) markers using mass spectrometry (MS). The Trusight 500 gene panel was used to determine molecular signature of tumors. Similarly, we performed targeted metabolomics of 4 BRAF-like and 2 RAS-like human TC cell lines. The data were analyzed using Ingenuity Pathway Analysis (Qiagen). We also analyzed mRNA expression of OXPHOS and glycolytic enzymes in 496 human TC tissue samples of The Cancer Genome Atlas (TCGA). The division into BRAF-like and RAS-like TC was based on well-established BRAF-RAS score (BRS) ranging from -1 to +1 for BRAF-like and RAS-like TC, respectively. Results: The study cohort consisted of 20 patients, 13 with BRAF-like and 7 with RAS-like tumors, aged 43±12 years old, 90% (18/20) females, with an average tumor size of 3±1.9 cm, multifocal growth in 55% (11/20) patients, central neck lymph node (LN) metastases in 55% (11/20), lateral neck LN metastases in 15% (3/20) patients and no distant metastases. The tumor size tended to be larger in RAS-like cohort (BRAF-like 2.27±1.28 vs RAS-like 4.34±2.52, p=0.08), and LN metastases more prevalent in BRAF-like cohort (BRAF-like 45% vs RAS-like 10%, p=0.08). Metabolomics data revealed that compared with normal thyroid, human TC tissue samples were characterized by an increased glucose metabolism (p<0.001), TCA (p<0.001) and gluconeogenesis (p<0.001). RAS-like tumors were characterized by a significantly higher TCA (p<0.001) and lower gluconeogenesis (p<0.001) as compared with BRAF-like tumors. Consistently, we found that TCA (p<0.001) and glucose metabolism (p<0.001) was higher in RAS-like vs BRAF-like cell lines. Transcriptomic profiling revealed that RAS-like TC was characterized by an increased expression of majority of tested OXPHOS enzymes compared to BRAF-like tumors, as evidenced by positive correlation with BRS, ranging from r = 0.28 to 0.54, p<0.001. Consistently, BRAF-like TC was associated with an upregulated mRNA expression of anaerobic glycolysis markers, with lactate dehydrogenase A, characterized by the highest negative correlation (r = -0.6, p<0.001) with BRS. Conclusion:RAS-like TC is more likely to utilize glucose through Krebs cycle, while BRAF-like tumors are more prone towards anaerobic glycolysis. Therapeutic strategies based on diverse metabolic profiles may provide a personalized approach to TC therapy. Presentation: 6/3/2024

8159 Single Nuclei RNA-Sequencing Reveals Novel Cellular Diversity in Human Neck Adipose Tissue

Abstract Disclosure: H. Camara: None. S.D. Kodani: Employee; Self; Hoxton Farm. S. Yang: None. V. Efthymiou: None. K. An: None. A. Gupta: None. F. Shamsi: None. A. Streets: None. M.E. Patti: Advisory Board Member; Self; Fractyl Laboratories. Consulting Fee; Self; AstraZeneca, Hamni, MBX Biosciences. Grant Recipient; Self; Dexcom. Y. Tseng: Consulting Fee; Self; LyGenesis. Other; Self; BioHaven. Adipose tissue plays a crucial role in maintaining metabolic health. It fulfills distinct functions through two types of fat: white adipose tissue (WAT), the primary site of triglyceride storage, and brown adipose tissue (BAT) along with related beige fat, specializing in thermogenic energy expenditure. Despite the scarcity of BAT in humans, these thermogenic adipocytes efficiently utilize glucose and triglycerides and act as an endocrine organ by secreting batokines. Hence, increased BAT activity is associated with improved metabolic health in humans. In adults, thermogenic adipose tissue is localized in deep neck planes, exhibiting a gradient from white to beige/brown from superficial to deep layers. Mounting evidence suggests the diverse cell types within the adipose tissue and their dynamic interactions play an important role in its versatile functions. To understand the cellular compositions of human adipose tissue and directly compare the differences between human BAT vs. WAT, we collected 30 paired samples of superficial, intermediate and deep human neck adipose tissue from 15 participants who underwent anterior cervical discectomy fusion or thyroid surgery and subjected the frozen tissues to single-nucleus RNA sequencing (snRNA-seq). The integration of the samples yielded 41,337 good-quality nuclei. As expected, white adipocytes (WAd) were the dominant cell type, comprising 38% of the total cells captured. Further analysis of the WAd cluster revealed increased expression of brown adipocyte-associated genes, such as EBF2 and COBL, and enrichment of oxidative phosphorylation pathway score in cells from deep neck samples, indicating increased thermogenic potential. Bona fide brown adipocytes (BAds), marked by the expression of PPARGC1A, were predominantly located in the deep neck samples (>80%) but comprised less than 2% of the dataset. Despite their low numbers, cell-cell communication analysis by CellChat predicted that BAds account for most of the communication events within this niche, highlighting their endocrine importance. snRNA-seq also captured non-adipocyte cells, including adipocyte progenitors, smooth muscle, immune, Schwann and neuron-associated cells. Among these, a novel Neuron-Associated Cell-1, marked by the expression of CNTNAP2, emerged as the second most abundant in the dataset (∼10% of total). Another neuron-associated cell population marked by expression of DNAH11, though less abundant (∼1%), was enriched in deep neck samples, potentially regulating the high levels of thermogenic potential in this region. Taken together, the identified subpopulations and their gene expression profiles offer new insights into the tissue's role in metabolic regulation. This knowledge could be pivotal for developing targeted therapies for obesity and related disorders. Presentation: 6/1/2024

Analysis of tetrahydroisoquinolines formed after simultaneous consumption of ethanol and amphetamine or its derivatives by LC-MS/MS in human blood, brain, and liver tissue.

The effects of the simultaneous consumption of amphetamine or amphetamine derivatives and alcohol have not yet been adequately clarified, particularly concerning potential condensation products resulting from the endogenous reaction between these substances and their metabolites (e.g., acetaldehyde, a metabolite of ethanol). In this study, we developed an LC-MS/MS method employing liquid-liquid extraction for the qualitative detection of some relevant condensation products belonging to the class of tetrahydroisoquinolines and their derivatives in human blood, brain, and liver samples. This includes the analysis of the substrates amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, as well as the condensation products 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, 1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline, and N-methyl-1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline. Therefore, the reference standards of the mentioned tetrahydroisoquinolines were synthesized in advance and the method was validated with regard to the question of the qualitative detection of these compounds. The validation parameters included selectivity, specificity, limit of detection, lower limit of quantification, recovery, matrix effects, and stability for blood, brain, and liver samples. Following the analysis of human blood and post-mortem tissue samples, evidence of the condensation product 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline originating from the interaction between amphetamine and acetaldehyde was identified in two liver samples. On the contrary, no evidence of this or other tetrahydroisoquinolines was found in the remaining tissue and serum samples.