Maturation Markers Research Articles

Nutrient transporter pattern in CD56dim NK cells: CD16 (FcγRIIIA)-dependent modulation and association with memory NK cell functional profile

BackgroundHuman memory NK cells represent a heterogeneous CD56dim population that expands and persists in human cytomegalovirus (HCMV)-seropositive healthy individuals. They are characterized by the preferential, not fully overlapping, expression of NKG2C (activating receptor for HLA-E) and CD57 maturation marker, and by the lack of FcεRIγ adaptor chain. Hyperresponsiveness to Fcγ receptor IIIA (CD16) engagement represents the distinctive functional signature of memory NK cells. Although CD16 engagement was shown to acutely enhance glycolytic and oxidative pathways, its capability to induce a persisting metabolic reprogramming of human NK cells is poorly understood yet.ResultsHere, we describe the peculiar nutrient transporter expression pattern of FcεRIγ- memory NK cells, characterized by higher levels of CD98 neutral amino acid antiporter and CD71 transferrin receptor, and lower expression of GLUT1 glucose transporter, with respect to FcεRIγ+ conventional NK cells. Although CD16 engagement acutely enhances glycolytic and oxidative pathways, its capability to induce a persisting metabolic reprogramming of human NK cells is poorly understood yet. Our results firstly show that sustained CD16 engagement by contact with IgG-opsonized target cells induces the mTORC1-dependent upregulation of CD98 and CD71 nutrient receptors on CD56dim NK cells, in a transporter-specific fashion, that is finely tuned by cell-dependent (grade of functional maturation, and memory or conventional lineage) and stimulus-dependent (time length and cooperation with cytokines) factors. We also demonstrate that CD98 antiporter function is required for CD16-dependent IFN-γ production, and that enhanced CD98-mediated neutral amino acid uptake associates with heightened memory NK cell functional response.ConclusionCollectively, our work documents that CD16 engagement leads to a metabolic rewiring of human NK cells and suggests that a distinct nutrient transporter expression pattern may contribute to memory NK cell peculiar functional features.

Ray‐Resorption Syndrome in European Seabass, Dicentrarchus labrax (Linnaeus, 1758)

ABSTRACTFin abnormalities are common in reared fish. They mainly consist of partial to complete lack of rays and severe abnormalities of fin‐supporting skeletal elements, which develop during the larval stage, up to the completion of fin skeleton ontogeny. This study reports a new abnormal condition, ray‐resorption syndrome (RSS), which developed after the completion of fin ontogeny in initially normal European seabass larvae, leading to extensive loss of fin rays. At 49 days post‐fertilisation (dpf) (15 mm standard length, SL) all fish presented normal fins. However, nineteen days later (21 mm SL), significant fin damage, characterised by ray loss or fractures, was observed in all studied populations. The dorsal fin was most affected (55%–84%), followed by the pelvic (27%–53%) and anal fins (7%–17%). Microscopically, multiple non‐mineralised areas resembling resorption lacunae were evident along all fin lepidotrichia. By 98 dpf (40 mm SL), the fin‐ray loss had advanced, reaching its higher frequency in the dorsal (74%–83%) and caudal fins (71%–94%). Gene expression analysis revealed a significant upregulation of bglap (osteoblast maturation marker), acp5a (osteoclast maturation marker) and mmp13a (extracellular‐matrix remodelling marker) in RSS specimens. The results are discussed in respect of the possible causative factors of RSS.

DDDR-26. IDENTIFICATION OF MODULATORS OF IDH INHIBITOR SENSITIVITY INIDH-MUTATED DIFFUSE GLIOMAS

Abstract Driver mutations in IDH1 and IDH2 characterize a substantial proportion of lower-grade diffuse gliomas in adults. Mutated IDH molecules cause the accumulation of D-2-hydroxyglutarate (D-2-HG), which perturbs many cellular functions, including metabolism, epigenetic regulation, and the ability to differentiate. This is notably associated with DNA and histone hyper-methylation. Recently, the newly developed IDH inhibitor, vorasidenib, was shown to delay tumor progression and the need for additional treatment in the groundbreaking INDIGO clinical trial. Nevertheless, the responses to vorasidenib were variable between patients, and so far have been established only for less aggressive disease. In addition, it is unclear to what extent vorasidenib can cause tumor regression. This highlights the need to identify molecules and pathways that control IDH inhibitor sensitivity. Towards this goal, we generated a new mouse model that combines Idh1R132H and Trp53 loss-of-function, targeted to oligodendrocyte progenitors. All the mutant mice develop diffuse gliomas that recapitulate the cardinal features of the corresponding human disease. We established multiple cell lines from these tumors, which retained expression of mutant IDH. The cells robustly produced D-2-HG, which was concentration-dependently suppressed by vorasidenib. Accordingly, vorasidenib could lower DNA methylation, and induce the expression of mature glial cell markers. To identify determinants of vorasidenib sensitivity, we performed genome-wide loss-of-function CRISPR/Cas9 functional genomics screens. In those experiments, targeting genes associated with astrocyte differentiation or Notch signaling enhanced cell fitness in the presence of vorasidenib, consistent with the anticipated effects of the drug on inducing cell differentiation. Conversely, vorasidenib-treated cells were more sensitive to the loss of regulators of cell growth and metabolism, and of some epigenetic regulators. These studies point to strategies that may enhance the efficacy of IDH inhibitors for the treatment of IDH mutated gliomas.

IMMU-61. THE LAST OF US: A FUNGI BASED DENDRITIC CELL VACCINE

Abstract BACKGROUND Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults. The current standard treatment for newly diagnosed GBM involves maximal surgical resection followed by radiotherapy and temozolomide. Despite these interventions, the median overall survival for GBM patients remains approximately 15 months, highlighting the critical need for innovative therapeutic approaches. OBJECTIVE Our laboratory has previously developed a subcutaneous autologous tumor vaccine incorporating irradiated (IR) tumor cells with phagocytosis-stimulating ligands (Mannan-BAM), toll-like receptor (TLR) agonists, and an immunostimulant anti-CD40 antibody (collectively termed MBT). This combination has shown efficacy in mouse models of colon carcinoma, breast cancer, melanoma, and, more recently, gliomas. Our study aimed to create a dendritic cell (DC) vaccine using this strategy. METHODS To evaluate whether MBT could induce the maturation of DCs in vitro, we harvested BMDCs from mice and co-cultured them in the presence of MBT-tagged irradiated tumor cells. We then analyzed the maturation markers using flow cytometry and assessed cytokine production via ELISA. To test the efficacy of this approach in vivo, we utilized two different murine glioma models (SB28 and GL261). RESULTS Using flow cytometry, we observed a significant upregulation of important co-stimulatory molecules. Additionally, ELISA analysis demonstrated that MBT-treated DCs significantly increased the secretion of Type 1 cytokines while limiting the secretion of the regulatory cytokine IL-10. MBT-primed DCs combined with immune checkpoint inhibitor PD-1 increased mouse survival in a glioma model compared to WT mice. However, mice eventually succumbed to cerebral edema. Histology demonstrated significant treatment effects. CONCLUSION Our results demonstrate that MBT effectively matures DCs ex-vivo. Further research is required to immunophenotype the response to vaccination in glioma models and explore ways to overcome resistance.

Transcriptomic heterogeneity of non-beta islet cells is associated with type 2 diabetes development in mouse models.

The aim of this work was to understand the role of non-beta cells in pancreatic islets at early stages of type 2 diabetes pathogenesis. Specific clustering was employed to single-cell transcriptome data from islet cells of obese mouse strains differing in their diabetes susceptibility (diabetes-resistant B6.V.Lepob/ob [OB] and diabetes-susceptible New Zealand Obese [NZO] mice) on a diabetogenic diet. Refined clustering analysis revealed several heterogeneous subpopulations for alpha cells, delta cells and macrophages, of which 133 mapped to human diabetes genes identified by genome-wide association studies. Importantly, a similar non-beta cell heterogeneity was found in a dataset of human islets from donors at different stages of type 2 diabetes. The predominant alpha cell cluster in NZO mice displayed signs of cellular stress and lower mitochondrial capacity (97 differentially expressed genes [DEGs]), whereas delta cells from these mice exhibited higher expression levels of maturation marker genes (Hhex and Sst) but lower somatostatin secretion than OB mice (184 DEGs). Furthermore, a cluster of macrophages was almost twice as abundant in islets of OB mice, and displayed extensive cell-cell communication with beta cells of OB mice. Treatment of beta cells with IL-15, predicted to be released by macrophages, activated signal transducer and activator of transcription (STAT3), which may mediate anti-apoptotic effects. Similar to mice, humans without diabetes possess a greater number of macrophages than those with prediabetes (39 mmol/mol [5.7%] < HbA1c < 46 mmol/mol [6.4%]) and diabetes. Our study indicates that the transcriptional heterogeneity of non-beta cells has an impact on intra-islet crosstalk and participates in beta cell (dys)function. scRNA-seq data from the previous study are available in gene expression omnibus under gene accession number GSE159211 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159211 ).

Dendritic cell maturation is induced by p53-armed oncolytic adenovirus via tumor-derived exosomes enhancing systemic antitumor immunity.

Dendritic cells (DCs) are crucial in cancer immunity, because they activate cytotoxic T cells by presenting tumor antigens. Recently, oncolytic virus therapy has been recognized as a systemic immune stimulator. We previously developed a telomerase-specific oncolytic adenovirus (OBP-301) and a p53-armed OBP-301 (OBP-702), demonstrating that these viruses strongly activate systemic antitumor immunity. However, their effects on DCs remained unclear. In the present study, the aim was to elucidate the mechanisms of DC activation by OBP-702, focusing particularly on tumor-derived exosomes. Exosomes (Exo53, Exo301, or Exo702) were isolated from conditioned media of human or murine pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and PAN02) after treatment with Ad-p53, OBP-301, or OBP-702. Exo702 derived from Panc-1 and MiaPaCa-2 cells significantly upregulated CD86, CD80, CD83 (markers of DC maturation), and IFN-γ in DCs in vitro. Similarly, Exo702 derived from PAN02 cells upregulated CD86 and IFN-γ in bone marrow-derived DCs in a bilateral PAN02 subcutaneous tumor model. This DC maturation was inhibited by GW4869, an inhibitor of exosome release, and anti-CD63, an antibody targeting the exosome marker. Intratumoral injection of OBP-702 into PAN02 subcutaneous tumors significantly increased the presence of mature DCs and CD8-positive T cells in draining lymph nodes, leading to long-lasting antitumor effects through the durable activation of systemic antitumor immunity. In conclusion, tumor-derived exosomes play a significant role in DC maturation following OBP-702 treatment and are critical for the systemic activation of antitumor immunity, leading to the abscopal effect.

Preterm infant attachment is independent of breast milk intake during neonatal care

Background Following preterm birth, breast milk feeding is recommended because it confers broad health benefits to preterm infants. Breast milk has been suggested to promote secure attachment in infancy, but this is uncertain. Elucidating the relationship between breast milk intake and infant attachment is crucial for supporting parents of preterm infants to make feeding choices and providing accurate counselling when breast milk is not available. We aimed to investigate if breast milk exposure during neonatal care associates with attachment outcomes derived from infants’ responses to the Still-Face Paradigm (SFP) at nine months of age. Methods We studied 77 preterm (&lt; 33 weeks’ gestation) and 71 term infants. Preterm infant feeding data were collected from birth until hospital discharge. Infant responses to the SFP were videocoded at nine months corrected age. Infants’ distress, fretfulness, attentiveness to caregivers and attachment classification were compared between preterm infants who received exclusive breast milk for ≥ 75% of inpatient days, those who received exclusive breast milk for &lt; 75% of inpatient days, and term infants. Milk intake thresholds were chosen based on our previous work demonstrating positive associations between breast milk intake and MRI markers of brain maturation. Results There were no significant differences in infant distress, fretfulness, attentiveness to caregivers (p-values &gt; 0.19), or in the odds of secure versus insecure attachment classification (relative risk ratio = 0.57–0.89, p-values = 1.00) between preterm infants with high or low breast milk exposure, or term comparators. Conclusions Attachment outcomes at nine months were similar between term and preterm infants, and breast milk exposure did not associate with attachment in the preterm group. Although breast milk feeding has clear benefits, these findings may alleviate some of the pressures experienced by parents who are unable to provide breast milk for infants cared for in the neonatal unit.

Development and intra-renal delivery of renal progenitor organoids for effective integration in vivo.

Renal progenitor organoids have been proposed as a source of tissue for kidney regeneration; however, their clinical translatability has not been demonstrated due to an inability to mass-produce comprehensive renal progenitor organoids and the lack of an effective intra-renal delivery platform that facilitates rapid integration into functionally meaningful sites. This study addresses these shortcomings. Human-induced pluripotent stem cells were differentiated into renal progenitor cells using an established protocol and aggregated using a novel assembly method to produce high yields of organoids. Organoids were encapsulated in collagen-based scaffolds for in vitro study and in vivo implantation into mouse renal cortex. In vitro, the organoids demonstrated sustained cell viability and renal structure maturation over time. In vivo delivered organoids showed rapid integration into host renal parenchyma while showing tubular and glomerular-like structure development and maturity markers. This proof-of-concept study presents many promising results, providing a system of renal organoid formation and delivery that may support the development of clinically translatable therapies and the advancement of in vitro renal organoid studies.

Engineering hiPSC-derived tissue models for advanced amyloidosis research and therapy development

Abstract Introduction Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease currently lacking an optimal therapy, stemming from the absence of representative models. Our work aims to stablish for the first time an all-human 3D model of ATTR-CM where the main cellular and extracellular players of the pathology are present. Methodology A human induced pluripotent stem cell (hiPSC) line was derived from a patient carrying a p.Ser43Asn TTR mutation and differentiated into hepatocytes (hiPSC-HEPs), cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs). Cells were embedded in a hydrogel (tailored for each cell type) and combined with a designed 3D printed scaffold using Melt Electro Writing (MEW) technology. Tissues were maintained in culture for up to 4 weeks and characterized by staining, albumin production, electrophysiological, plus genomic analysis. Exposure to TTR fibrils (TTR-FIB) was evaluated. Results Human hepatic and cardiac functional tissues can be generated using MEW-designs. These constructs exhibited albumin production and an active spontaneous contraction, respectively. Moreover, tissues displayed well distributed cells throughout the 3D-structure, positive for specific hepatic and cardiac mature markers. Interestingly, electrophysiological analysis showed that cardiac tissues displayed shortened calcium transients after TTR-FIB exposure compared to unexposed tissues. However, no differences were found in terms of beat rate, metabolic activity and gene expression for the concentrations used. Conclusion We have developed an advanced model of ATTR-CM capable of recapitulating the multisystem complexity of the disease, which will contribute to advancing our understanding of the mechanisms of the disease, and help discovering and formulating new treatments.

Safety, Growth and Development After Dapagliflozin or Saxagliptin in Children With Type 2 Diabetes (T2NOW Follow-Up).

The T2NOW trial of dapagliflozin or saxagliptin versus placebo in pediatric patients with type 2 diabetes (T2D) demonstrated promising efficacy data for dapagliflozin and did not raise any safety concerns over 52 weeks. Assess long-term effects of prior dapagliflozin/saxagliptin administration on safety, growth and development. Multicenter, randomized, double-blind phase 3 trial (T2NOW). 210 children with T2D aged 10-17 years, followed for up to one year after treatment. Previous treatment with once-daily dapagliflozin (5, 10 mg), saxagliptin (2.5, 5 mg) or placebo as add-on to diet, exercise, metformin and/or insulin for 52 weeks, plus a 52-week non-treatment follow-up period. Change in height, weight, body mass index (BMI), Tanner staging, growth and maturation markers, bone biomarkers and adverse events (AEs) from baseline to Week 104. As expected in a pediatric population, mean height and weight slightly increased from baseline to Week 104. BMI remained generally stable; Changes were similar across treatment groups. Sexual maturation progressed normally to Week 104, with similar shifts between Tanner stages and changes in growth and maturation markers and bone biomarkers across groups. The proportion of patients reporting ≥1 AE during the non-treatment follow-up period was similar across groups previously treated with dapagliflozin (18.5%) or saxagliptin (15.9%) compared to placebo (21.1%). No deaths occurred. Prior treatment with dapagliflozin or saxagliptin for 52 weeks did not raise any safety concerns relating to height, weight, BMI, Tanner staging, growth and maturation markers, bone biomarkers or AEs for up to 52 weeks following treatment discontinuation, in pediatric patients with T2D.

Forearm Fractures in Older Children and Adolescents: ORIF is Safer Than IMN With Equivalent Outcomes.

Greater understanding of the impact of skeletal maturity on outcomes is needed to guide operative treatment of diaphyseal forearm fractures in children and adolescents. The purpose of this study was to compare the complications and outcomes of pediatric diaphyseal forearm fractures treated with intramedullary nailing (IMN) or open reduction internal fixation (ORIF) and to identify a radiographic marker of skeletal maturity that will aid in selecting between treatment options. A retrospective review of patients aged 10 to 16 years treated operatively for diaphyseal forearm fractures was performed. Markers of skeletal maturity including the olecranon apophysis score, the presence of the thumb adductor sesamoid, and radial epiphyseal capping. Complications were graded with the modified Clavien-Dindo system. Outcomes were scored based on final postoperative range of motion combined with complication grade. A total of 260 patients were included: 163 treated with IMN, 97 treated with ORIF, mean age 12.7 years, 72% male. Among closed forearm fractures treated with IMN, open reduction was required in 45% (53/118). Patients treated with IMN had a higher complication rate than ORIF (27.0% vs. 9.3%, P<0.05), including when stratified by age. Complication rates were not impacted by greater skeletal maturity as indicated by the presence of thumb sesamoid or radial epiphyseal capping. There was no significant difference in outcomes between the ORIF and IMN groups. More skeletally immature patients, as identified by a lack of either the thumb adductor sesamoid or radial epiphyseal capping, had significantly better outcomes with ORIF than patients with greater maturity. Across all age groups and levels of skeletal maturity, ORIF had a significantly lower rates of complications compared with IMN with equivalent outcomes. More skeletally immature patients had significantly better outcomes with ORIF treatment when compared with older patients. The thumb adductor sesamoid, radial epiphyseal capping, and the olecranon apophysis score did not provide useful information to select between ORIF over IMN in this population. Level III-retrospective comparative study.

Mice with Lymphatic Dysfunction Develop Pathogenic Lung Tertiary Lymphoid Organs that Model an Autoimmune Emphysema Phenotype of COPD.

We have previously shown that mice with loss of C-type lectin-like type II (CLEC2), which have lymphatic dysfunction due to the role of CLEC2 in platelets for maintaining separation between the venous and lymphatic system, develop lung tertiary lymphoid organ (TLO) formation and lung injury that resembles an emphysema phenotype of chronic obstructive pulmonary disease (COPD). We now sought to investigate whether and how TLOs in these mice may play a pathogenic role in lung injury that is relevant to human disease. We found that inhibiting TLO formation using an anti-CD20 antibody in CLEC2-deficient mice partially blocked the development of emphysema. TLOs in CLEC2-deficient mice were rich in plasma cells and were a source of a broad array of autoantibodies. Chronic cigarette smoke exposure increased the size and number of lung TLOs in CLEC2-deficient mice, and was associated with increased markers of antigen presentation and maturation, leading to increased autoantibody deposition. Using lung tissue from COPD patients, we found an increase in lymphatic markers in patients with an emphysema phenotype and autoreactive TLOs compared to COPD patients without emphysema that lack prominent TLOs. Taken together, these results demonstrate that emphysema in mice with lymphatic dysfunction can be partially rescued by blocking TLO formation, and that these TLOs are source of autoantibodies that are exacerbated by cigarette smoke. Our work suggests that lymphatic dysfunction in mice may recapitulate some aspects an autoimmune emphysema phenotype that is seen in a subset of patients with COPD.

Abstract A052: RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction

Abstract RNF43_p.G659fsV*41 mutation (RNF43659mut) is found in approximately 8% of colorectal cancers (CRCs) and is enriched in microsatellite-instability high (MSI-high) tumors. Previously, we reported that RNF43659mut promotes tumor growth independent of WNT signaling and activates PI3K/AKT signaling in CRC, conferring vulnerability to PI3K/MTOR inhibition. In addition, RNF43659mut was associated with decreased interferon signaling suggesting a link with anti-tumor immunity. Thus, in the current work we investigated the role of RNF43659mut in CRC immunomodulation. The immune-cell landscape was evaluated in RNF43659mut compared with RNF43-wild type (wt) MSI-high CRCs in two cohorts; single-cell (sc)-RNA-sequencing data from treatment-naïve primary tumors from 34 patients (cohort-1) and multiplex immunofluorescence / whole exome sequencing data from an independent cohort of 91 patients (cohort-2). Healthy donor blood-derived natural killer (NK) cell responses and cytotoxicity were investigated in co-cultures with RNF43659mut isogenic tumor lines and with patient-derived organoids harboring RNF43659mut or overexpressing RNF43wt. A third patient cohort with MSI-high CRCs treated with immune checkpoint blockade was analyzed to understand the impact of RNF43659mut on immunotherapy efficacy. Interrogation of sc-RNASeq data in cohort-1 revealed a significant enrichment of NK cells in the RNF43659mut patient samples compared to the RNF43wt ones. This finding was also confirmed by multiplex immunofluorescence in cohort-2. Single-cell transcriptomic analyses in cohort-1 showed increased inhibitory markers on NK cells despite their enrichment in RNF43659mut tumors. RNAseq analysis of sorted NK cells co-cultured with RNF43659mut isogenic tumor lines further supported their dysfunctional state. Flow cytometric analyses of NK cell maturation, activation, and inhibitory markers in NK cells co-cultured with RNF43659mut isogenic tumor lines and organoids highlighted decreased levels of NCAM1 (CD56)dim and NCAM1 (CD56)bright subsets, FCGR3A (CD16), and KLRK1 (NKG2D), along with increased levels of KLRC1 (NKG2A), KIR3DL1 (NKB1) and TIGIT inhibitory markers. There was reduced NK cell mediated tumor cell killing in RNF43659mut isogenic tumor lines and organoids. We postulated that aberrant signaling of the PI3K/AKT pathway with RNF43659mut could lead to NK cell dysfunction. Indeed, siRNA-mediated blocking of PI3K/AKT pathway restored the NK-cell dysfunctional phenotype and tumor cell killing in co-cultures with RNF43659mut isogenic tumor lines and organoids. Conversely, overexpression of WT RNF43 in RNF43659mut organoids downregulated PI3K/AKT signaling and increased NK cell cytotoxicity. Lastly, MSI-high CRC tumors harboring RNF43659mut in patients treated with immunotherapy showed less benefit from immune checkpoint blockade. PI3K/AKT activation due to RNF43659mut in MSI-high CRC leads to NK cell dysfunction and reduced NK cell cytotoxicity. This could be responsible for immunotherapy failure, warranting further investigation. Citation Format: Pushpamali De Silva, Samantha Fitzgerald, Jules Cazaubiel, Matan Hofree, Tomotaka Ugai, Juha P Väyrynen, Dane Ford-Roshon, Lishan Fang, Ana Garrido-Castro, Nir Hacohen, Kimmie Ng, Shuji Ogino, Jonathan A Nowak, Marios Giannakis. RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A052.

A stepwise approach to deriving functional β-cells from human embryonic or induced pluripotent stem cells

Abstract Our understanding of β-cell differentiation from pluripotent stem cells (PSCs) is rapidly evolving. Although progress has been made, challenges remain, particularly in achieving glucose-stimulated insulin secretion (GSIS). Human embryonic stem cells (hESCs) are valuable due to their pluripotent ability. A fixed protocol targeting master regulatory genes initiates stem cells into pancreatic lineage commitment. Due to the observations that a single stem cell can differentiate into multiple cell types depending on various factors and conditions, non-linear differentiation pathways exist. Co-expression of key factors remains essential for successful β-cell differentiation. The mature β-cell marker MAFA plays a critical role in maintaining the differentiation state and preventing dedifferentiation. Recapitulating pancreatic islet clustering enhances physiological responses, offering potential avenues for diabetes treatment. On the other hand, several enhanced differentiation protocols from induced pluripotent stem cells (iPSCs) have improved the functional insulin producing β-cells generated. These findings, with their potential to revolutionize diabetes treatment, highlight the complexity of β-cell differentiation and guide further advancements in regenerative medicine.

Slight and hidden hearing loss in young rats is associated with impaired recognition memory and reduced myelination in the corpus callosum.

Slight and hidden hearing loss in children have been linked to cognitive and social difficulties, and yet the neurobiological mechanisms behind these issues remain poorly understood. Most animal models focus on severe hearing loss, leaving the effects of hidden or slight hearing loss largely unexplored. To uncover the neural mechanisms connecting slight/hidden hearing loss to cognitive and social challenges, we induced hearing loss in young (4-week-old) Wistar rats through noise exposure. We then examined cognitive function (object recognition test) and social behavior (juvenile play behavior and social interaction). Changes in brain anatomy were assessed using cortical thickness and hippocampal size measurements, while (immuno)histochemical staining investigated neuronal circuitry maturation (myelin basic protein, parvalbumin, and perineuronal nets) and neurogenesis (doublecortin). Noise-exposed rats displayed slight high-frequency hearing loss (around 20 dB) and hidden hearing loss at other tested frequencies. This slight/hidden hearing loss was associated with impaired object recognition but did not alter social behavior. Slight/hidden hearing loss was associated with reduced myelin basic protein expression in the corpus callosum but no other alterations in cortical thickness, hippocampal size, or other markers of maturation and neurogenesis were found. These findings show that even slight/hidden hearing loss can lead to subtle brain alterations tied to cognitive deficits. This study emphasizes the need for further research to fully understand the brain changes associated with slight/hidden hearing loss and to pinpoint the mechanisms connecting these changes to behavioral deficits. This information is crucial to develop interventions to prevent the cognitive and social consequences of hearing loss. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Transcriptional coactivator MED15 is required for beta cell maturation

Mediator, a co-regulator complex required for RNA Polymerase II activity, interacts with tissue-specific transcription factors to regulate development and maintain homeostasis. We observe reduced Mediator subunit MED15 expression in endocrine hormone-producing pancreatic islets isolated from people living with type 2 diabetes and sought to understand how MED15 and Mediator control gene expression programs important for the function of insulin-producing β-cells. Here we show that Med15 is expressed during mouse β-cell development and maturation. Knockout of Med15 in mouse β-cells causes defects in β-cell maturation without affecting β-cell mass or insulin expression. ChIP-seq and co-immunoprecipitation analyses found that Med15 binds β-cell transcription factors Nkx6-1 and NeuroD1 to regulate key β-cell maturation genes. In support of a conserved role during human development, human embryonic stem cell-derived β-like cells, genetically engineered to express high levels of MED15, express increased levels of maturation markers. We provide evidence of a conserved role for Mediator in β-cell maturation and demonstrate an additional layer of control that tunes β-cell transcription factor function.

7004 Systems genetics analysis to identify candidate genes for fat distribution in BXD mice

Abstract Disclosure: N. Kim: None. D. Ryu: None. C. Oh: None. Obesity is defined as the accumulation of excess fat in the body. Although obesity is the main cause of metabolic disorders, not all obese people have metabolic disorders. Among many factors identified, the ratio of visceral (vWAT) to subcutaneous white adipose tissue (sWAT), representing body fat distribution, has been elucidated to be closely associated with metabolic health. Body fat distribution has been proposed as a possible explanation for this discrepancy. To investigate the regulation of body fat distribution, we performed a genetic analysis using the phenotype data and male subcutaneous fat microarray data from the BXD mouse genetic reference population. Our analysis identified H2-Ke6 (Hsd17b8), encoding hydroxysteroid 17-beta dehydrogenase, as a candidate target for adipose tissue plasticity. To further explore its role, we employed multiple approaches to reveal the effect of Hsd17b8 inhibition. First, lipid accumulation was attenuated in 3T3-L1 preadipocytes where Hsd17b8 was inhibited by CRISPER/Cas9 as well as lasalocid. Furthermore, we confirmed that the expression of adipogenesis-related transcription factor as well as mature adipocyte marker was decreased in terminally differentiated 3T3-L1. Specifically, CCAAT/enchancer binding protein α (Cebpa), peroxisome proliferator-activated receptor γ (Pparg), adiponectin (Adipoq), and fatty acid binding protein 4 (Fabp4) were transcriptionally downregulated. Consistently, in C. elegans, we used RNA interference (RNAi) to inhibit the expression of the Hsd17b8 ortholog, dhs-25, and observed a reduction in lipid accumulation. All these data suggest that the reduction in lipid accumulation in differentiated 3T3-L1 cells by the inhibition of Hsd17b8 is attributed to the suppression of early differentiation during the adipogenesis process. On top of that, to determine whether Hsd17b8 indeed influences body fat distribution in vitro, we utilized IngWAT Mouse Immortalized Preadipocyte Cell Line derived from subcutaneous white adipose tissue (sWAT) of a wild-type mouse. In contrast to the previous findings, we observed an increase in lipid accumulation of IngWAT when Hsd17b8 was inhibited. Surprisingly, the elevation of lipid accumulation in sWAT, as seen in gynoid obesity, has been revealed to be indicative of robust metabolic health compared to android obesity. Collectively, these data suggest that Hsd17b8 may represent a novel therapeutic target for the treatment of obesity. Our study provides valuable insights into the mechanisms underlying metabolic health in obesity and highlights the potential of Hsd17b8 as a therapeutic target for modulating adipose tissue plasticity. Presentation: 6/3/2024

The human gut Bacteroides eggerthii expresses a new galactofuranose-containing lipooligosaccharide with weak immunostimulatory properties

Lipopolysaccharides (LPS) decorating the cell surface of Gram-negative bacteria exhibit nuanced functionalities linked to their precise structural composition. However, despite their critical role in health and disease, information on the structure and function of LPS from members of the human gut microbiota is still limited. Here, we deciphered the complete structure of the LPS isolated from the human gut bacterium Bacteroides eggerthii 1_2_48FAA. We showed that B. eggerthii 1_2_48FAA produces an R-type LPS (or lipooligosaccharide, LOS) composed of a heterogeneous mixture of tetra- and penta-acylated lipid A species with different degree of phosphorylation, and a compact galactofuranose-containing core oligosaccharide. Using in vitro human cell lines, we showed that B. eggerthii 1_2_48FAA LOS acts as a weak activator of TLR4-mediated signaling. Moreover, we observed that expression of maturation markers CD40, CD80 and CD86 on monocytes-derived dendritic cells upon B. eggerthii 1_2_48FAA LOS exposure was significantly lower compared to pro-inflammatory Escherichia coli LPS. Taken together, these data provide new structural and biological insights into LPS from gut bacteria, underscoring the importance of structural features in modulating host immunity.

Immunometabolic Mechanisms of LANCL2 in CD4+ T Cells and Phagocytes Provide Protection from Systemic Lupus Erythematosus.

Lanthionine synthetase C-like 2 (LANCL2) is an immunoregulatory therapeutic target for autoimmune diseases. NIM-1324 is an investigational new drug aimed at addressing the unmet clinical needs of patients with systemic lupus erythematosus (SLE) by targeting the LANCL2 immunometabolic pathway. In R848 and bm12 adoptive transfer models of systemic inflammation that share pathologies with SLE, Lancl2-/- mice experienced greater mortality, increased spleen weight, and reduced CD25hi FOXP3+ CD4+ regulatory T cells compared with the wild type. Conversely, treatment with NIM-1324 in the wild type increased CD25hi FOXP3+ regulatory T cells while reducing inflammatory IL-17+ and IL-21+ CD4+ T cell subsets in the spleen. In traditional mouse models of SLE (NZB/W F1 and MRL/lpr), oral treatment with NIM-1324 protected against weight loss and proteinuria, decreased anti-dsDNA titers, and provided similar changes to the CD4+ T cell compartment in the spleen. The pharmacological activation of LANCL2 by NIM-1324 rescued hypocomplementemia, reduced kidney histopathological scores, and decreased blood IFN response genes and inflammatory cytokines. The loss of LANCL2 in phagocytes impairs phagosome processing, leading to increased uptake of material and inflammatory cytokine production, yet decreased markers of endosomal maturation, phagosome turnover, and lysozyme activity. Treatment with NIM-1324 increases metabolic and lysozyme activity in the phagosome, providing support for increased markers of early phagosome function. This efficacy translated to human PBMCs from patients with SLE, because ex vivo treatment with NIM-1324 resulted in reduced levels of IFN-α, IL-6, and IL-8. Consequently, the activation of LANCL2 effectively modulates CD4+ T cell differentiation and phagocyte activation, supporting immune tolerance in SLE.

Pseudo-3D Topological Alignments Regulate Mechanotransduction and Maturation of Smooth Muscle Cells.

Smooth muscle cells (SMCs) sense and respond to mechanical stimuli in their extracellular microenvironments (ECMs), playing a crucial role in muscle tissue engineering. Increasing evidence from topological cues-mediated mechanotransduction of SMCs in ECMs has suggested some potential underlying mechanisms of how SMC functions and maturation are regulated by their mechanosensing leading to transduction. However, how the expression of yes-associated protein 1 (YAP) influences the phenotypic shift from synthetic to contractile is still controversial. Here, pseudo-3D topological alignments mimicking native muscle tissues are generated using laser-cutter engraving to explore the influence of topological cues on SMC mechanotransduction and maturation. The analysis of topological cue-mediated mechanotransduction and maturation marker expression revealed YAP is involved in mechanotransduction for SMCs cultured on cross-patterned substrates in the presence of cell-cell interactions. Moreover, these SMCs with YAP-linked mechanosensing showed higher expression of calponin, indicating a shift toward contractile phenotypes in vitro and in vivo. Furthermore, it showed skeletal muscle cells has different mechanosensing and maturation mechanisms compared to SMCs, revealing muscle type-dependent different sensing of topological cues, and converting into maturation-associated signaling cascades. This study provides insights into the regulation of SMC mechanotransduction and maturation by topological cues, suggesting the involvement of YAP-linked signaling pathways in this process.