Normal Donors Research Articles

Abstract C012: Dissemination of retrotransposable elements from the non-coding genome in tumor-derived extracellular vesicles target stromal and immune cells to induce local and systemic inflammation

Abstract The majority of the human genome (>70%) contains non-coding DNA consisting of retrotransposable elements (RTE), such as LINE-1, SINE/Alu elements, human endogenous retroviruses (HERV), and satellite repeat DNA (Hsat2 and Hsat3). The expression of these elements is suppressed in normal adult tissues but are abnormally expressed in many cancers. Another key mechanism in cancer initiation and progression is the release of tumor-derived extracellular vesicles (EV) that are long-distance communication vehicles propagating signals from tumors inducing metabolic changes, immune dysregulation and promoting metastases across the body. Our work has uncovered a link between RTE, HERV and Hsat expression in cancer and EV in driving tumor progression and immune dysregulation. W hole transcriptome sequencing of EV derived from several different cancer cell lines and patient samples, including Ewing sarcoma, osteosarcoma, prostate, pancreatic, and breast cancer found that RTE, HERV and Hsat2,3 transcripts are highly enriched in EV versus coding region transcripts and in much higher abundance than in the tumor cell themselves. Tumor-derived EV contained dsRNA, dsDNA and RNA:DNA hybrids from these non-coding regions. Plasma EV isolated from both metastatic and non-metastatic patients also showed an enrichment of these RTE, HERV and Hsat2,3 elements and clearly differentiated healthy, normal donor EV that had little or no detectable signal. Evidence of local dissemination of these non-coding elements in EV was also found in vivo in tumor xenografts that showed transfer of Hsat2,3 transcripts into the murine stroma that naturally lack these satellite repeats. Treatment of fibroblasts and myeloid cells with tumor-derived EV triggered innate immune responses via type I IFN and pro-inflammatory cytokines, including IL-1beta, IL-6, IL-8, and TNFalpha owing to the pathogen-like nucleic acid sequences (viral mimicry) of these elements. These EV-induced chronic inflammatory effects were mainly induced through the cGAS-STING nucleic acid sensing pathway. Evidence of DNA damage in stromal fibroblasts and induced senescence was also found as a result of EV treatment. A key element of RTE is the expression of LINE-1 and HERV derived reverse transcriptase (RT) that propagates the action of RTE via RNA:DNA hybrids, dsDNA and retrotransposition in the genome. In a spontaneous estradiol-driven breast cancer model in ACI rats, long-term treatment with an HIV RT inhibitor (lamivudine/3TC) inhibited breast tumor development associated with reduced RTE activation in the mammary gland and in circulating immune cells as well as reduced chronic systemic innate inflammation. Our results suggest that RTE, HERV and Hsat activation, together with their local and systemic dissemination in EV plays an important role in cancer initiation and progression associated with chronic inflammation. We also suggest that abnormal dissemination of these activated non-coding elements in EV may also play a role in "inflammaging" facilitating not only cancer but also other chronic diseases. Citation Format: Laszlo Radvanyi, Valentina Evdokimova, Peter Ruzanov, Zhenbo Zhang, Poul Sorenson, Hendrik Gassmann, Stefan Burdach, Lincoln D. Stein. Dissemination of retrotransposable elements from the non-coding genome in tumor-derived extracellular vesicles target stromal and immune cells to induce local and systemic inflammation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C012.

Interpericyte Tunneling Nanotubes Are Nonuniformly Distributed in the Human Macula.

Pericyte-to-pericyte communication via interpericyte tunneling nanotubes (IP-TNTs) is an important mechanism by which spatial and temporal precision in neurovascular coupling is achieved. This study quantifies the distribution and morphologic characteristics of IP-TNTs in the normal human macula. Ultra high-resolution, three-dimensional microscopic imaging of 11 perfusion-labeled normal human donor eyes was performed. Immunofluorescent markers for collagen IV, glial fibrillary acidic protein, nuclei, α-smooth muscle actin and phalloidin were used to distinguish IP-TNTs from perfused/nonperfused capillaries and glia processes. IP-TNT length, diameter and density in each capillary plexus was quantified and compared. IP-TNTs were present in all capillary plexuses. IP-TNTs bridged capillary segments within and between capillary plexuses but did not connect capillaries to arterioles or venules. Mean length of IP-TNTs was 72.6 ± 39.5µm (range 14.0 to 202µm) and mean diameter was 1.0 ± 0.42µm. IP-TNT length was non-normally distributed with a right-skewed distribution and 43% were 'short' (<55µm). Diameters were greater in the "long" (1.13 ± 0.44µm) than "short" (0.82 ± 0.33µm; P < 0.001) IP-TNTs. Density of IP-TNTs was greater in the superficial vascular plexus (3.80 ± 0.69 per 500µm2) compared to the intermediate (1.85 ± 0.80 per 500µm2; P < 0. 0001) and deep capillary plexus (1.58 ± 0.84 per 500µm2; P < 0.0001). No significant difference in IP-TNT density was found between the four macula quadrants (P = 0.98). The distribution of IP-TNTs in the human macula is non-uniform and is associated with the compartmentalized nature of retinal energy consumption and microvascular perfusion. The nonuniform properties of IP-TNTs may predispose distinct vascular beds to injury in conditions such as diabetes.

Abstract A032: Performance evaluation of a new protein stabilizing blood collection tube with a novel whole blood separation device for clinical plasma proteomics

Abstract Introduction: We have developed a novel blood separation device (BCD) which separates plasma from cellular components by lateral flow and stabilizes the sample for transport to a testing laboratory. Patient blood is drawn into an anticoagulant blood collection tube (BCT) and then applied to the separation device. There is outstanding interest in the field of clinical proteomics regarding the formulation in BCTs and whether they would also additionally the plasma proteome. This is especially critical where hemoglobin may provide interference to the analyte being measured. We evaluated the performance of a newly available plasma protein stabilizing BCT in combination with the BCD and other currently available K2EDTA BCT (no stabilization). Methods: Whole blood from normal healthy donors was drawn into protein stabilizing BCT and a comparator anticoagulant BCT. At baseline, 24-, 48-, 72- and 120-hour time points, a 250 μL aliquot of whole blood from each tube was spotted on to the BCD to allow separation of the cellular components and plasma. All of the test specimens on the spotted devices were allowed to dry overnight to replicate time in transit. After drying, proteins were subjected to MALDI-TOF MS (mass spectrometry) after extraction with water and co- crystallization with sinapinic acid. Spectra were acquired in triplicate and m/z feature intensities for hemoglobin were assessed in R. Results: Visual inspection of the new protein stabilizing and K2EDTA BCTs showed the steady accumulation of hemoglobin over time in the plasma layer in the latter tubes. After spotting on the BCD and MALDI-TOF analysis, plasma from the protein- stabilizing BCT showed no significant increases in either the hemoglobin α or β subunits over the five (5) day period. This was consistent across all donors (heme α p = 0.7933, heme β p = 0.6425) and at all timepoints assessed. In the K2EDTA BCT, both the α and β hemoglobin subunits increased significantly over time and across all donors (heme α p &amp;lt; 0.0001, heme β p = 0.0232). The hemoglobin α subunit accumulated in the plasma component to a greater degree than did the β subunit (heme α slope = 4.863E-5, heme β slope = 1.398E-5). These data suggest that the formulation in the protein stabilizing BCT may attenuate red blood cell hemolysis to a greater degree as compared to conventional K2EDTA blood collection tubes. Additionally, the mean reproducibility for both hemoglobin subunits was lower when the protein stabilizing BCT was used than when the anticoagulant BCT was used across all donors at all time points (protein stabilizing BCT x ̅ CV = 8.5%, anticoagulant BCT x ̅ CV = 8.9%). Conclusions: The use of the newly available protein stabilizing BCT with the BCD reduces the accumulation of hemoglobin in the plasma component while improving reproducibility when compared to the currently used anticoagulant BCT. Interference by heme was significantly attenuated as measured by a sensitive MALDI-ToF assay. Citation Format: Colin McDowell, Nylev Vargas, Gary A Pestano. Performance evaluation of a new protein stabilizing blood collection tube with a novel whole blood separation device for clinical plasma proteomics [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A032.

Cell-Specific Gene Expressions Underlie Selective White Matter Loss Vulnerability in Mild Traumatic Brain Injury.

Traumatic brain injury (TBI), a risk factor for later-life dementia, leads to salient brain atrophy, particularly in the white matter. It is not clear how white matter atrophy progresses or why some brain regions are damaged while others are spared. We hypothesized that spatial variations of cell-specific gene expression contributed to the selective white matter loss vulnerability following mild TBI (mTBI). Gene expression data were sourced from the publicly available Allen Human Brain Atlas, which comprises microarray data spanning nearly the entire brain, derived from six neurologically normal adult donors. A total of 100 patients with acute stage (within 7 days post-injury) mTBI were enrolled. Of these, 60 patients were followed up at 3 months post-injury and 37 were followed up at 6-12 months post-injury. In addition, 59 healthy controls (HCs), matched for age, gender, and education, were included for comparative analysis. White matter volume changes were analyzed at both the acute stage, 3 months, and 6-12 months follow-up in mTBI patients compared with HCs. Patients with mTBI exhibited significant white matter atrophy in the frontal, parietal, and temporal cortices at 3 months post-injury, which even persisted at 6-12 months follow-up. In addition, mTBI patients with cognitive deficits showed more severe brain atrophy compared with those without cognitive deficits. Crucially, the gene expression marking endothelial cells and S1 pyramidal neurons were associated with increased brain atrophy, whereas the gene expression marking microglia and CA1 pyramidal neurons were associated with decreased brain atrophy in mTBI patients at 3 months post-injury. Microglia and endothelial cells can explain 23.6% of regional variations in the white matter atrophy. These findings suggested that modulating cellular activation, especially by promoting microglial activation at 3 months post-injury, might be a promising approach to prevent white matter atrophy, enhance cognitive outcomes, and reduce the risk of later-life dementia.

Developing Advanced Chimeric Cell Therapy for Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal, X-linked disorder leading to muscle degeneration and premature death due to cardiopulmonary complications. Currently, there is no cure for DMD. We previously confirmed the efficacy of human Dystrophin-Expressing Chimeric (DEC) cells created via the fusion of myoblasts from normal and DMD-affected donors. The current study aimed to optimize the development of DEC therapy via the polyethylene glycol (PEG)-mediated fusion protocol of human myoblasts derived from normal, unrelated donors. The optimization of cell fusion assessed different factors influencing fusion efficacy, including myoblast passage number, the efficacy of PKH myoblast staining, the ratio of the single-stained myoblasts in the MIX, and PEG administration time. Additionally, the effect of PEG fusion procedure on cell viability was assessed. A correlation was found between the number of cells used for PKH staining and staining efficacy. Furthermore, the ratio of single-stained myoblasts in the MIX and PEG administration time correlated with fusion efficacy. There was no correlation found between the myoblast passage number and fusion efficacy. This study successfully optimized the myoblast fusion protocol for creation of human DEC cells, introducing DEC as a new Advanced Therapy Medicinal Product (ATMP) for DMD patients.

12419 Single-nuclei Sequencing Of Normal Donor Cadaveric Parathyroid Glands Reveals Distinct Cellular Subpopulations With Key Differences In Gland Anatomical Location And Suppressed Vs Non-suppressed Physiological Status

Abstract Disclosure: T. Glinin: None. E. Khanafshar: None. J.A. Sosa: Advisory Board Member; Self; Novo Nordisk, AstraZeneca, Eli Lilly &amp; Company. Grant Recipient; Self; Exelixis, Inc., Eli Lilly &amp; Company. J. Koh: None. Despite the significant clinical burden of parathyroid disorders, little is known at the molecular level about the cellular composition of normal parathyroid glands (PTGs). We performed a single-nuclei RNAseq study of six cadaveric PTGs (three from the superior anatomical location, three inferior) from four donors (two female). A board-certified pathologist confirmed that the sequenced PTGs were histologically normal. Using the split-pool method, we sequenced 83,693 nuclei, generating 1.7B reads. Data integration was performed with the Harmony algorithm in Seurat 5.0.1. Cluster analysis revealed 14 discrete populations of PTG origin. Gene set enrichment identified clusters corresponding to endothelial cells, neurons, microglia, mast cells, dendritic cells, stromal fibroblasts, macrophages, and smooth muscle. The remaining six clusters were closely related but distinct populations sharing markers including PTH, CaSR, SPOCK3, PRKCE, and PLCB1 (p&amp;lt;2e-300 for group specificity) but with differentially expressed gene (DEG) patterns between clusters suggestive of specialized functions in VEGF signaling (KIAA1549L), GPCR downstream signaling (GRM7, ADGRL3), and subcellular spatial organization (SYNE1) (p&amp;lt;2e-24 for cluster specificity). Superior and inferior PTGs were distinguished by 8 superior-specific DEGs (p&amp;lt;2e-245) including HOXB3, expressed in 71% of superior PTG cells vs 6% of inferior PTG cells. Enhanced expression of HOXB3, a homeobox transcription factor found to inhibit tumorigenesis in multiple tissues, could explain in part why adenomas are three times less likely to arise in superior PTGs. During procurement, one of the presumptive normal donors was found to have a single grossly enlarged, hypercellular PTG, elevated PTH (140 pg/ml), and hypercalcemia (11.2 mg/dL), consistent with primary hyperparathyroidism (PHPT). In PHPT patients with a single tumor, contralateral PTGs are functionally suppressed by the dominant adenoma. We compared the histologically normal suppressed and unsuppressed PTGs. Fifteen genes were more highly expressed in suppressed PTGs, including LRP2, a multi-ligand receptor known to bind PTH (p = 2e-300). Fourteen genes were more highly expressed in unsuppressed PTGs (p&amp;lt;2.14e-296), including PDE11A, NRG2, NRXN3, and PRLR, which are all associated with GABA receptor signaling (q = 2.8e-5). These data independently support our group’s prior work proposing a role for the GABA receptor in PHPT. Differential expression in endothelial cells, neurons, and fibroblasts from suppressed PTGs suggests that non-endocrine cells may also be responsive to changes in PTG activity. Finally, the different proportions of cells with suppressed or unsuppressed gene expression signatures observed between clusters suggests that PTGs incorporate distinct populations of cells reflective of physiological activity or functional suppression. Presentation: 6/2/2024

Hydroxyurea reduces the levels of the fetal globin gene repressors ZBTB7A/LRF and BCL11A in erythroid cells in vitro

Abstract Objectives Hydroxyurea (HU) is the most widely used therapy for adults and children with sickle cell disease (SCD). It is believed to act largely by inducing the transcription of fetal γ-globin genes to generate fetal hemoglobin (HbF), which inhibits the pathological polymerization of sickle hemoglobin (HbS). The mechanisms by which hydroxyurea elevates HbF are unclear. We explored the hypothesis that hydroxyurea induces HbF expression by inhibiting the expression of 2 γ-globin gene repressors, BCL11A and ZBTB7A (also known as LRF), which normally bind the γ-globin gene promoters to inhibit their expression after birth. Methods We treated immortalized murine erythroleukemia cells and normal human donor CD34+ hematopoietic stem and progenitor cell-derived erythroblasts with hydroxyurea and measured the effects on globin, BCL11A and ZBTB7A protein and mRNA expression. Results Treating murine erythroleukemia cells or human CD34+ hematopoietic stem and progenitor cell-derived erythroblasts with hydroxyurea reduced the protein levels of BCL11A and ZBTB7A compared to the vehicle-treated control. BCL11A mRNA levels were reduced in both cell types upon hydroxyurea treatment. However, ZBTB7A mRNA levels were only reduced in human CD34+ hematopoietic stem and progenitor cell-derived erythroblasts. Conclusions Hydroxyurea can act in erythroid cells to reduce the levels and activity of two direct fetal γ-globin transcriptional repressors with accompanying de-repression of the γ-globin genes and induction of HbF, which may explain the mechanism of action leading to amelioration of symptoms in SCD patients treated with this drug.

B-242 Improved Patient Satisfaction With Novel Capillary Blood Collection Devices as an Alternative to Venipuncture for Applications in Clinical Proteomic Assays

Abstract Background Two new clinical diagnostic tests are available that aid in the identification of lung nodules as likely malignant or likely benign. One test is an ELISA that measures the levels of seven autoantibodies to tumor-associated antigens that aids in the identification of patients with likely malignant lung nodules who may benefit from timely intervention. The other is an LC-MS/MS-based (MRM) test that measures the ratio of two proteins (LG3BP and C163A) combined with several clinical and radiological factors to aid in the identification of patients with likely benign lung nodules who may benefit from additional CT surveillance. Since commercialization, we have found that test access may be improved by providing alternatives to traditional venipuncture. Therefore, we conducted evaluation studies with two novel non-fingerstick capillary blood collection devices. These devices puncture the skin by utilizing either microneedles (Device A) or a lancet (Device B) for capillary blood collection. Methods Blood specimens were collected from consented donors using one of the two capillary blood collection devices and by venipuncture into K2EDTA blood collection tubes. Feedback was also solicited from the healthcare professionals and donors via a standard questionnaire. The whole blood specimens were shipped to the testing laboratory where the samples were processed according to the respective clinical assay workflows. Feasibility testing for the ELISA-based assay was conducted with both devices using normal healthy donor specimens (105 donors for Device A and 43 for Device B). Clinical evaluation was executed utilizing only Device B (77 donors with lung cancer and 13 with an indeterminate lung nodule). Feasibility testing for the LC-MS/MS-based assay was conducted with both devices using healthy donor specimens (103 donors for Device A and 41 for Device B), while clinical evaluation utilized only Device B (80 donors with lung cancer and 30 with an indeterminate lung nodule). Results Both capillary blood collection devices revealed good correlations to venous blood draws for the ELISA-based test during feasibility. The two devices also showed good correlations for the LC-MS/MS-based test, with the results from Device A requiring a correction factor. Due to technical and operational challenges throughout feasibility, Device B became the preferred device to be evaluated for the clinical study. Good correlations for both diagnostic tests were observed from the clinical study. During clinical evaluation, healthcare professionals and donors were asked to rate the ease-of-use, pain, and speed of collection when using Device B. Most healthcare professionals (108/126) rated the device as easy to use. Ninety-five percent of the donors reported minimal pain while the remainder reported moderate pain. Ninety-one percent of the donors felt that the time required to collect blood using Device B was acceptable. Sixty percent of the donors preferred the capillary collection device (Device B), 17% preferred venipuncture, while 23% reported no preference for blood collection. Conclusions The novel capillary blood collection devices demonstrated high correlations for two clinical diagnostic tests. Feedback collected from healthcare professionals and donors revealed that the majority preferred the capillary blood collection device as compared to traditional venipuncture for blood collection.

A-168 Comparison of Two Different Lupus Anticoagulant Hexagonal Phase Phospholipid Confirmatory Assays on an Automated Coagulation Analyzer

Abstract Background Antiphospholipid syndrome (aPS) is an autoimmune disorder resulting in venous and/or arterial thrombosis, characterized by presence of lupus anticoagulants (LA) in patient plasma, also referred to as antiphospholipid antibodies. Depending on clinical history and symptoms, or if an unexpected elevated routine aPTT result is observed, LA laboratory testing would be performed, if appropriate. According to published clinical guidelines, LA tests should use aPTT-based and dRVVT-based screening (low phospholipid) and confirmatory (high phospholipid) methods, run in parallel. aPS diagnosis is confirmed from both clinical and laboratory criteria. Laboratory criteria are met if either the aPTT or dRVVT confirmatory test results are positive, and if lab positivity is confirmed following repeated testing 12 weeks following initial results. Due to the multiple tests used, and different manufacturers’ reagents, analysis of comparative performance allows users to identify the optimal tests to use. Objectives of the study were 1) compare intra-run and total precision of two different aPTT-based LA confirmatory tests on a newly available automated coagulation instrument equipped with improved pipetting methodology and preanalytical sample integrity detection and 2) correlate results from the two different aPTT-based LA confirmatory test assays. Methods CRYOcheck Hex LA (Precision BioLogic, Inc., Dartmouth, NS, Canada) and Staclot LA (Diagnostica Stago, Inc., Parsippany, NJ, USA) were run on a STA R Max3 automated coagulation analyzer (Diagnostica Stago, Inc.), using manufacturer recommended protocols. For intra-run precision and total precision analysis, replicates of 5 quality control samples were used (STA Control LA 1+2, Diagnostica Stago, Inc.). For the correlation studies, 20 different LA positive samples were used, supplied by the Specimen Access Team (SAT) from Laboratory Corporation of America Holdings, Burlington, NC, USA. LA positivity was defined as positive by CRYOcheck Hex LA delta result &amp;gt; 11 seconds, which was the cutoff determined at LabCorp’s facility. CRYOcheck Normal Donor Set plasmas were used for normal samples (Precision BioLogic, Inc.). Samples were run in batches containing 5 normal samples and 5 abnormal samples, with quality control run at the beginning and end of each batch. Results Intra-run precision was superior for Staclot LA compared to CRYOcheck Hex LA, with the exception of the high phospholipid “LA Correct” positive tube from CRYOcheck Hex LA. Total precision was either comparable or superior for Staclot LA compared to CRYOcheck Hex LA. In addition, correlation data showed Staclot LA recovered the same result between 3 different aliquots of the same patient mother tube, whereas CRYOcheck Hex LA recovered varying results between runs, when using manufacturer recommended cutoffs. Conclusions Analysis of intra-run and total precision, along with result correlation and recovery between multiple sample aliquots is recommended for when coagulation laboratories evaluate different manufacturers’ reagents. Our data indicate Staclot LA demonstrates superior performance to the CRYOcheck Hex LA on the STA R Max3 automated coagulation analyzer.

Desmoplakin mutations in cardiac fibroblasts cause TGFβ1-mediated pathological fibrogenesis in desmoplakin cardiomyopathy via beclin-1 regulation.

Pathological fibrosis is a major finding in cardiovascular diseases and can result in arrhythmia and heart failure. Desmosome gene mutations can lead to arrhythmogenic cardiomyopathy (ACM). Among ACM, pathogenic desmoplakin ( DSP ) variants cause a distinctive cardiomyopathy with excessive cardiac fibrosis that could precede ventricular dysfunction. DSP variants are also linked to other fibrotic diseases. Whether DSP plays any role in pathological fibrosis remain unknown. Mesenchymal stromal cells (MSCs) are resident fibroblast-like cells that are responsible for fibrogenesis in most organs, including hearts. We first used unbiased genome-wide analyses to generate cardiac fibroblasts-like, induced pluripotent stem cell-derived MSCs from normal donors and ACM patients with DSP mutations. We then studied the fibrogenic responses of cardiac MSCs to transforming growth factor beta-1 (TGF-β1) using Western/Co-IP, autophagy assay, gene knockdowns/over-expressions, genomic analyses, mouse DSP knockdown models, immunostaining, and qPCR. TGFβ1 induced excessive accumulations of vimentin (VIM)/fibrillar collagens, and over-activated fibrotic genes in DSP- mutant MSCs when compared to normal MSCs. In normal MSCs, VIMs bind to wild-type DSP during normal fibrogenesis after TGFβ1. DSP- mutant MSCs exhibited a haplo-insufficient phenotype with increased DSP-unbound VIMs that sequestered beclin-1 (BECN1) from activating autophagy and caveolin-1 (CAV1)-mediated endocytosis. Decreased autophagy caused collagen accumulations and diminished CAV1 endocytosis resulted in abnormal CAV1 plaque formation that over-activated fibrotic genes [ COL1A1, COL3A1, and fibronectin ( FN )] via heightened p38 activities after TGFβ1. Genome-wide analysis and DSP knockdown in mouse fibroblasts confirmed this novel role of DSP mutations in pathological fibrosis. Overexpression of VIM-binding domains of DSP could suppress pathological fibrosis by increasing collagen autophagic degradation and decreasing fibrotic gene expressions. Our data reveal that DSP deficiency in MSCs/fibroblasts leads to exaggerated fibrogenesis in DSP-cardiomyopathy by decreasing BECN1 availability for autophagy and CAV1-endocytosis. Overexpression of VIM binding domains of DSP could be a new strategy to treat pathological fibrosis.

PAI-1 Derived from Alveolar Type 2 Cells Drives Aging-Associated Pulmonary Fibrosis

Pulmonary fibrosis (PF) is a lethal lung disease that predominantly affects older adults; however, whether and how aging triggers fibrosis remains unclear. To pinpoint the predominant initiating factors of PF, we first analyzed single-cell RNA sequencing (scRNA-seq) data from the lung tissues of 45 normal donors and 51 PF patients and found that aging might serve as the primary catalyst for PF development. To further investigate the influence of aging on PF formation, we conducted a comprehensive and thorough study employing a natural aging mouse model. We found that dynamic alterations in the quantity and types of collagen fibers during aging-induced PF progression, especially in collagenous (Col) I, emerged as the predominant driver of PF. We then investigated the regulation of Col I synthesis during aging using primary alveolar type 2 (AT2) cells and A549 cells line through conditioned media and Transwell coculture, and found that secretions—particularly plasminogen activator inhibitor (PAI)-1—from aged AT2 cells promoted fibrosis and enhanced collagen type I alpha 1 (Col1al) production via the transforming growth factor (TGF)-β/small mother against decapentaplegic (Smad)2/3 pathway. Furthermore, scRNA-seq and a histological analysis of human lung tissue demonstrated a significant upregulation of SERPINE1 (the gene encoding PAI-1) and PAI-1 expression in both aging lung tissue and AT2 cells, which was consistent with our findings from animal experiments, providing additional evidence for the pivotal role of PAI-1 during aging and the development of PF. Our research demonstrates that PAI-1, a crucial factor secreted by aging AT2 cells, exerts a pivotal role in promoting the synthesis of Col1a1 in fibroblasts, subsequently leading to Col I deposition, and in driving the progression of PF by mediating the TGF-β/Smad2/3 pathway. Our findings offer critical evidence for the involvement of epithelial dysfunction in age-related PF and provides potential novel therapeutic targets for clinical intervention.

L-Theanine Alleviates Ulcerative Colitis by Regulating Colon Immunity via the Gut Microbiota in an MHC-II-Dependent Manner.

Alterations to the gut microbiota are associated with ulcerative colitis (UC), whereas restoration of normobiosis can effectively alleviate UC. l-Theanine has been shown to reshape the gut microbiota and regulate gut immunity. To investigate the mechanisms by which l-theanine alleviates UC, we used l-theanine and l-theanine fecal microbiota solution to treat UC mice. In this study, we used l-theanine and l-theanine fecal microbiota solution to treat UC mice to explore the mechanism by which l-theanine alleviates UC. By reducing inflammation in the colon, we demonstrated that l-theanine alleviates symptoms of UC. Meanwhile, l-theanine can improve the abundance of microbiota related to short-chain fatty acid, bile acid, and tryptophan production. Single-cell sequencing results indicated that l-theanine-mediated suppression of UC was associated with immune cell changes, especially regarding macrophages and T and B cells, and validated the immune cell responses to the gut microbiota. Further, flow cytometry results showed that the ability of dendritic cells, macrophages, and monocytes to present microbiota antigens to colonic T cells in an MHC-II-dependent manner was reduced after treating normal mouse fecal donors with l-theanine. These results demonstrate that l-theanine modulates colon adaptive and innate immunity by regulating the gut microbiota in an MHC-II-dependent manner, thereby alleviating UC.

Longitudinal Trajectories of Estimated Glomerular Filtration Rate in a European Population of Living Kidney Donors.

A living donor (LD) kidney transplant is the best treatment for kidney failure, but LDs safety is paramount. We sought to evaluate our LDs cohort's longitudinal changes in estimated glomerular filtration rate (eGFR). We retrospectively studied 320 LDs submitted to nephrectomy between 1998 and 2020. The primary outcome was the eGFR change until 15years (y) post-donation. Subgroup analysis considered distinct donor characteristics and kidney function reduction rate (%KFRR) post-donation [-(eGFR6months(M)-eGFRpre-donation)/eGFRpre-donation*100]. Donors had a mean age of 47.3 ± 10.5years, 71% female. Overall, LDs presented an average eGFR change 6M onward of +0.35mL/min/1.73m2/year. The period with the highest increase was 6M-2Y, with a mean eGFR change of +0.85L/min/1.73m2/year. Recovery plateaued at 10years. Normal weight donors presented significantly better recovery of eGFR +0.59mL/min/1.73m2/year, compared to obese donors -0.18L/min/1.73m2/year (p = 0.020). Noteworthy, these results only hold for the first 5years. The subgroup with a lower KFRR (<26.2%) had a significantly higher decrease in eGFR overall of -0.21mL/min/1.73m2/year compared to the groups with higher KFRR (p < 0.001). These differences only hold for 6M-2Y. Moreover, an eGFR<50mL/min/1.73m2 was a rare event, with ≤5% prevalence in the 2-15Y span, correlating with eGFR pre-donation. Our data show that eGFR recovery is significant and may last until 10years post-donation. However, some subgroups presented more ominous kidney function trajectories.

Pilot study of plasma miRNA signature panel for differentiating single vs multiglandular parathyroid disease.

The ability to differentiate sporadic primary hyperparathyroidism (sPHPT) caused by a single parathyroid adenoma (PTA) from multiglandular disease (MGD) pre-operatively, as well as definitely diagnose sPHPT in difficult patients, would enhance surgical decision making. Identify miRNA (miR) signatures for MGD, single- and double-PTA, as well as cell-free miRNA (cfmiR) in plasma samples from patients with single-PTAs to use as biomarkers. 47 patients with sPHPT (single-PTA n=32, double-PTA n=12, MGD n=9). Pre-operative plasma samples from 16 single-PTA and 29 normal healthy donors (NHD). All specimens were processed and analyzed for 2,083 miRs using HTG EdgeSeq miR whole transcriptome assay and normalized using DESeq2 to identify differentially expressed (DE) miRs. MiR classifiers were identified using Random Forest. ROC curves and AUC. MiR signatures distinguished normal parathyroid from MGD and PTA as well as MGD from PTA in tissue samples. Common miRs were found in the single-PTA and double-PTAs. Data integration identified a 27-miR signature in single-PTA tissue samples compared to the rest of the tissue samples. In plasma samples analysis, significant cfmiRs were DE in single-PTA patients compared to NHD. Of those, only 9 miRNAs/cfmiRs were found DE in both tissue and plasma samples from patients diagnosed with a single-PTA (AUC=76%). Twenty-seven miRs were consistently found DE in single-PTA tissue and plasma samples. Data integration showed a 9-cfmiR signature with potential clinical utility to pre-operatively diagnose sPHPT caused by a single-PTA, which could decrease more invasive parathyroid explorations.

Human brain organoids containing microglia that have arisen innately adapt to a β-amyloid challenge better than those in which microglia are integrated by co-culture

BackgroundAlzheimer disease (AD) is a heterogenous and multifactorial disease, and its pathology is partly driven by microglia and their activated phenotype. Brain organoids (BOs) are gaining prominence as a relevant model of the human brain for the study of AD; however, BOs are commonly devoid of microglia. To overcome this limitation, current protocols incorporate microglia through either (1) co-culture (BO co-culture), or (2) molecular manipulation at critical windows of BO development to have microglia arise innately (BO innate cultures). It is currently unclear whether the microglia incorporated into BOs by either of these two protocols differ in function.MethodsAt in vitro day 90, BO innate cultures and BO-co-cultures were challenged with the AD-related β-amyloid peptide (Aβ) for up to 72 h. After Aβ challenge, BOs were collected for immunoblotting. Immunoblots compared immunodensity and protein banding of Aβ and ionized calcium-binding adapter molecule 1 (IBA1, a marker of microglial activation) in BOs. The translational potential of these observations was supported using 56 human cortical samples from neurocognitively normal donors and patients with early-onset AD and late-onset AD. Statistical analyses were conducted using the Kruskal–Wallis test, a two-way ANOVA, or a simple linear regression, and where applicable, followed by Dunn’s or Sidak’s test.ResultsWe show that BO co-cultures promote Aβ oligomerization as early as 24 h and this coincides with a significant increase in IBA1 levels. In contrast, the Aβs do not oligomerize in BO innate cultures and the IBA1 response was modest and only emerged after 48 h. In human cortical samples, we found IBA1 levels correlated with age at onset, age at death, and the putative diagnostic Aβ(1–42)/Aβ(1–40) ratio (particularly in their oligomeric forms) in a sex-dependent manner.ConclusionsOur unique observations suggest that BOs with innate microglia model the response of a healthy brain to Aβ, and by extension the initial stages of Aβ challenge. It would be impossible to model these early stages of pathogenesis in BOs where microglia are already compromised, such as those with microglia incorporated by co-culture.

Biomimetic Proteoglycans Strengthen the Pericellular Matrix of Normal and Osteoarthritic Human Cartilage.

In osteoarthritis (OA), degradation of cartilage pericellular matrix (PCM), the proteoglycan-rich immediate cell microniche, is a leading event of disease initiation. This study demonstrated that biomimetic proteoglycans (BPGs) can diffuse into human cartilage from both normal and osteoarthritic donors and are preferentially localized within the PCM. Applying immunofluorescence (IF)-guided AFM nanomechanical mapping, we show that this localization of BPGs increases the PCM micromodulus of both normal and OA specimens. These results illustrate the capability of BPGs to integrate with degenerative tissues and support the translational potential of BPGs for treating human OA and other diseases associated with proteoglycan degradation.

Combinatorial Genomic Biomarkers Associated with High Response in IgE-Dependent Degranulation in Human Mast Cells.

Mast cells are the major effector cells that mediate IgE-dependent allergic reactions. We sought to use integrated network analysis to identify genomic biomarkers associated with high response in IgE-mediated activation of primary human mast cells. Primary human mast cell cultures derived from 262 normal donors were categorized into High, Average and Low responder groups according to their activation response profiles. Transcriptome analysis was used to identify genes that were differentially expressed in different responder cultures in their baseline conditions, and the data were analyzed by constructing a personalized perturbed profile (PEEP). For upregulated genes, the construction of PEEP for each individual sample of all three responder groups revealed that High responders exhibited a higher percentage of "perturbed" samples whose PEEP values lay outside the normal range of expression. Moreover, the integration of PEEP of four selected upregulated genes into distinct sets of combinatorial profiles demonstrated that the specific pattern of upregulated expression of these four genes, in a tandem combination, was observed exclusively among the High responders. In conclusion, this combinatorial approach was useful in identifying a set of genomic biomarkers that are associated with high degranulation response in human mast cell cultures derived from the blood of a cohort of normal donors.

Chimeric Cell Therapy Transfers Healthy Donor Mitochondria in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by dystrophin gene mutations and mitochondrial dysfunction, leading to progressive muscle weakness and premature death of DMD patients. We developed human Dystrophin Expressing Chimeric (DEC) cells, created by the fusion of myoblasts from normal donors and DMD patients, as a foundation for DT-DEC01 therapy for DMD. Our preclinical studies on mdx mouse models of DMD revealed enhanced dystrophin expression and functional improvements in cardiac, respiratory, and skeletal muscles after systemic intraosseous DEC administration. The current study explored the feasibility of mitochondrial transfer and fusion within the created DEC cells, which is crucial for developing new therapeutic strategies for DMD. Following mitochondrial staining with MitoTracker Deep Red and MitoTracker Green dyes, mitochondrial fusion and transfer was assessed by Flow cytometry (FACS) and confocal microscopy. The PEG-mediated fusion of myoblasts from normal healthy donors (MBN/MBN) and normal and DMD-affected donors (MBN/MBDMD), confirmed the feasibility of myoblast and mitochondrial fusion and transfer. The colocalization of the mitochondrial dyes MitoTracker Deep Red and MitoTracker Green confirmed the mitochondrial chimeric state and the creation of chimeric mitochondria, as well as the transfer of healthy donor mitochondria within the created DEC cells. These findings are unique and significant, introducing the potential of DT-DEC01 therapy to restore mitochondrial function in DMD patients and in other diseases where mitochondrial dysfunction plays a critical role.Graphical

ACE-2 Blockade & TMPRSS2 Inhibition Mitigate SARS-CoV-2 Severity Following Cigarette Smoke Exposure in Airway Epithelial Cells In Vitro.

Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking alters the expression of the SARS-CoV-2 cellular receptor and point of entry, angiotensin converting enzyme-2 (ACE-2), and its cofactors including transmembrane protease serine 2 (TMPRSS2). We investigated the impact of cigarette smoking on the expression of ACE-2, TMPRSS2, and other known cofactors of SARS-CoV-2 infection and the resultant effects on infection severity in vitro. Cigarette smoke extract (CSE) exposure increased ACE-2 and TMPRSS2 mRNA expression compared to air control in ferret airway cells, Calu-3 cells, and primary human bronchial epithelial (HBE) cells derived from normal and COPD donors. CSE-exposed ferret airway cells inoculated with SARS-CoV-2 had a significantly higher intracellular viral load versus vehicle-exposed cells. Likewise, CSE-exposure increased both SARS-CoV-2 intracellular viral load and viral replication in both normal and COPD HBE cells over vehicle control. Apoptosis was increased in CSE-exposed, SARS-CoV-2-infected HBE cells. Knockdown of ACE-2 via an antisense oligonucleotide (ASO) reduced SARS-CoV-2 viral load and infection in CSE-exposed ferret airway cells that was augmented by co-administration of camostat mesylate to block TMPRSS2 activity. Smoking increases SARS-CoV-2 infection via upregulation of ACE2 and TMPRSS2.

Mir-204-5p alleviates mitochondrial dysfunction by targeting IGFBP5 in diabetic cataract.

Cataract contributes to visual impairment worldwide, and diabetes mellitus accelerates the formation and progression of cataract. Here we found that the expression level of miR-204-5p was diminished in the lens epithelium with anterior lens capsule of cataract patients compared to normal donors, and decreased more obviously in those of diabetic cataract (DC) patients. However, the contribution and mechanism of miR-204-5p during DC development remain elusive. The mitochondrial membrane potential (MMP) was reduced in the lens epithelium with anterior lens capsule of DC patients and the H2O2-induced human lens epithelial cell (HLEC) cataract model, suggesting impaired mitochondrial functional capacity. Consistently, miR-204-5p knockdown by the specific inhibitor also attenuated the MMP in HLECs. Using bioinformatics and a luciferase assay, further by immunofluorescence staining and Western blot, we identified IGFBP5, an insulin-like growth factor binding protein, as a direct target of miR-204-5p in HLECs. IGFBP5 expression was upregulated in the lens epithelium with anterior lens capsule of DC patients and in the HLEC cataract model, and IGFBP5 knockdown could reverse the mitochondrial dysfunction in the HLEC cataract model. Our results demonstrate that miR-204-5p maintains mitochondrial functional integrity through repressing IGFBP5, and reveal IGFBP5 may be a new therapeutic target and prognostic factor for DC.