CD68-positive Cells Research Articles

Sgr-2921, a Potent CDC7 Inhibitor, Demonstrates Significant Anti-Leukemic Responses in Patient-Derived AML Models Representing Difficult-to-Treat Disease

Introduction: Elevated replication stress (RS) is a common feature of many highly proliferative cancers including AML. CDC7 has been shown to activate key components of the RS response, including ATR, and the BRCA1 and cohesin complexes, thereby mitigating accumulation of DNA damage and genomic instability. Inhibition of CDC7 in cancer cells results in an impaired response to RS, leading to accumulation of DNA damage and cell death. SGR-2921 is a potent inhibitor of CDC7 and a clinical trial to evaluate the safety, tolerability and preliminary antitumor activity of SGR-2921 in patients with AML and MDS is expected to begin in 2023 (NCT 05961839). We have previously shown that SGR-2921 has potent anti-proliferative activity in 12 out of 16 patient-derived AML samples ex vivo. Importantly, 6 out of 6 AML patient samples with mutations in TP53 responded to SGR-2921. Here we show that SGR-2921 results in potent, dose-dependent, anti-leukemic activity in multiple disseminated in vivo AML patient-derived xenograft (PDX) models representing difficult-to-treat disease. Methods: NOG-EXL mice were inoculated with primary human AML blast cells after sublethal irradiation. Engraftment of human AML blast cells were measured in surrogate mice by flow cytometry using human CD45 vs. mouse CD45 markers. When human CD45 engraftment reached >20% of bone marrow cells in surrogate mice, the study mice were randomized based on weight and dosed with vehicle, SGR-2921 at 3 dose levels or a standard of care agent. SGR-2921 was administered via oral gavage (PO) twice daily in cycles of 5 days of dosing followed by 9 days of no compound dosing over 2 cycles. In a separate study, phosphorylated MCM2 (a direct substrate of CDC7) was measured in the same cohort of mice engrafted with human AML cells following 3 doses of SGR-2921 to determine target engagement. Results: In vivo, SGR-2921 showed strong anti-leukemic activity in the AML PDX models at tolerated doses. Specifically, engrafted AML cells derived from a patient with TP53 mutated AML, a relapsed and refractory patient and a patient with KTM2a rearrangements showed significant and dose-dependent reduction in human CD45 positive AML blast cells. The reduction of blast cells was observed in both peripheral blood and bone marrow. The reduction in human CD45 positive blast cells were associated with an increase in mouse CD45 positive cells. In addition, SGR-2921 demonstrated dose-dependent inhibition of phosphorylated MCM2 in the spinal cord and spleen of the AML PDX models as surrogates of the bone marrow and extravascular compartments, respectively. Conclusions: Our data show remarkable dose-dependent in vivo activity of SGR-2921 in AML PDX models, including in those representing difficult-to-treat disease. Direct inhibition of CDC7 by SGR-2921 in AML blasts was demonstrated by a dose-dependent reduction of phosphorylated MCM2. Together, these data demonstrate that SGR-2921-mediated CDC7 inhibition is an attractive novel treatment opportunity in AML, with potential utility in patients with high risk mutations and relapsed and refractory AML.

Improvement of Inflammation and Abnormal Vascularization by TSP1 Treatment Combined with ADSCs Transplantation in Mice with Induced Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disease with a certain degree of chronic inflammation and abnormal ovarian angiogenesis in reproductive women. Mesenchymal stem cells (MSCs) have potent immunomodulatory properties to regulate ovarian function, while thrombospondin 1 (TSP1) improves the abnormal formation of ovarian vessels. The present study investigated the efficacy of the combined use of adipose-derived mesenchymal stem cells (ADSCs) and TSP1 in PCOS mice. The PCOS model is established using dehydroepiandrosterone (DHEA) by subcutaneous injection. Ovarian apoptosis is assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Real-time quantitative PCR (RT-PCR) and western blotting are used to detect the expression of inflammatory factors and the levels of angiogenesis-related factors in ovarian tissues. Inflammatory cells count and ovarian angiogenesis are evaluated by immunofluorescence staining. This research shows that TSP1 and ADSCs treatment can significantly reduce the inflammatory state of PCOS mice, relieve the degree of ovarian cell apoptosis, optimize the ovarian histological manifestations, and restore the levels of related hormones. The proportion of CD31-positive cells in PCOS mice returned to near-normal levels. The synergistic use of ADSCs and TSP1 therapy can exert a more impressive effect by inhibiting the ovarian inflammatory response and regulating the balance of angiogenesis than the single application in PCOS mice.

IL-1β-mediated adaptive reprogramming of endogenous human cardiac fibroblasts to cells with immune features during fibrotic remodeling

The source and roles of fibroblasts and T-cells during maladaptive remodeling and myocardial fibrosis in the setting of pulmonary arterial hypertension (PAH) have been long debated. We demonstrate, using single-cell mass cytometry, a subpopulation of endogenous human cardiac fibroblasts expressing increased levels of CD4, a helper T-cell marker, in addition to myofibroblast markers distributed in human fibrotic RV tissue, interstitial and perivascular lesions in SUGEN/Hypoxia (SuHx) rats, and fibroblasts labeled with pdgfrα CreERt2/+ in R26R-tdTomato mice. Recombinant IL-1β increases IL-1R, CCR2 receptor expression, modifies the secretome, and differentiates cardiac fibroblasts to form CD68-positive cell clusters. IL-1β also activates stemness markers, such as NANOG and SOX2, and genes involved in dedifferentiation, lymphoid cell function and metabolic reprogramming. IL-1β induction of lineage traced primary mouse cardiac fibroblasts causes these cells to lose their fibroblast identity and acquire an immune phenotype. Our results identify IL-1β induced immune-competency in human cardiac fibroblasts and suggest that fibroblast secretome modulation may constitute a therapeutic approach to PAH and other diseases typified by inflammation and fibrotic remodeling.

Increased expression of the P2Y12 receptor is involved in the failure of autogenous arteriovenous fistula caused by stenosis

Objective This study investigated the role of the P2Y12 receptor in autogenous arteriovenous fistula (AVF) failure resulting from stenosis. Methods Stenotic venous tissues and blood samples were obtained from patients with end-stage renal disease (ESRD) together with AVF stenosis, while venous tissues and blood samples were collected from patients with ESRD undergoing initial AVF surgery as controls. Immunohistochemistry and/or immunofluorescence techniques were utilized to assess the expression of P2Y12, transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein 1 (MCP-1), and CD68 in the venous tissues. The expression levels of P2Y12, TGFβ1, and MCP-1 were quantified using quantitative reverse transcription–polymerase chain reaction and western blot analyses. Double and triple immunofluorescence staining was performed to precisely localize the cellular localization of P2Y12 expression. Results Expression levels of P2Y12, TGFβ1, MCP-1, and CD68 were significantly higher in stenotic AVF venous tissues than in the control group tissues. Double and triple immunofluorescence staining of stenotic AVF venous tissues indicated that P2Y12 was predominantly expressed in α-SMA-positive vascular smooth muscle cells (VSMCs) and, to a lesser extent, in CD68-positive macrophages, with limited expression in CD31-positive endothelial cells. Moreover, a subset of macrophage-like VSMCs expressing P2Y12 were observed in both stenotic AVF venous tissues and control venous tissues. Additionally, a higher number of P2Y12 +/TGF-β1+ double-positive cells were identified in stenotic AVF venous tissues than in the control group tissues. Conclusion Increased expression of P2Y12 in stenotic AVF venous tissues of patients with ESRD suggests its potential involvement in the pathogenesis of venous stenosis within AVFs.

Effects of SU5416 on angiogenesis and the ERK-VEGF/MMP-9 pathway in rat endometriosis.

SU5416 is a small molecule vascular endothelial growth factor (VEGF) receptor signal transduction inhibitor, which can block the VEGF re-ceptor autophosphorylation and inhibit receptor tyrosine kinase signal trans-duction, thereby reducing VEGF activity. However, there are few reports about the correlation of SU5416 to the occurrence and angiogenesis in endometrio-sis. In this study, we observed the effects of VEGF receptor inhibitor SU5416 on angiogenesis in endometriosis in rats. Thirty femalespecific-pathogen-free Sprague-Dawley rats were randomly divided into sham operation group (SOG), model group (MG), and SU5416 group (n=10 for each group). In the SOG, only the uterus was cut and sutured, and endometriosis models were established in the MG and SU5416 group by autologous transplantation. The SU5416 group was injected with 15 mg/kg SU5416 intraperitoneally, and the SOG and MG were intraperitoneally injected with an equal volume of normal saline for 6 weeks. The volume of ectopic lesions was lower in the SU5416 group at 42 d postoperatively thanin the MG (p<0.05). The proportion of CD31-positive cells in the endometrial tissue of the SU5416 group was lower than that of the MG (p<0.05); angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), laminin-5γ2 (LN-5γ2) and phosphorylation of ERK (P-ERK), VEGF, matrix metalloproteinase (MMP)-2, and MMP-9 protein expressions were lower in the SU5416 groupthan in the MG (p<0.05).VEGFreceptor inhibitor SU5416 can inhibit endometrio-sis angiogenesis and reduce inflammatory response in rats, and its mechanism of action may be related to the down-regulation of the ERK-VEGF/MMP-9 path-way expression.

Abstract 13603: Inhibition of Exercise-Regulated Long Noncoding RNA SALTe1 Reverses Vascular Aging and Age-Related Cardiac Dysfunction

Rationale: Age is a major risk factor for cardiovascular disease. Recent work suggests vascular aging is an important determinant of longevity and end-organ function, although its role in age-related cardiac dysfunction and how exercise mitigates these phenotypes remain unclear. Objective: To identify exercise-regulated, cardiac long noncoding RNAs (lncRNAs) that modulate vascular aging. Methods and Results: Twenty-month-old mice underwent voluntary wheel running for eight weeks. RNAseq identified a novel set of lncRNAs altered in exercised aged hearts, termed S enescence A ssociated L ncRNA T ranscripts in E xercise (SALTes). SALTe1 was conserved and primarily expressed in endothelial cells (ECs). SALTe1 increased in aged hearts but decreased with exercise. Myocardial perfusion and CD31 positive cells were reduced in 20-month-old vs 10-week-old mice while EC senescence (CD31 and p16 or γH2AX double-positive cells) and cardiac diastolic dysfunction increased (p<0.05 for all). In 20-month-old mice, SALTe1 inhibition using antisense GapmeR or EC-specific knockdown in CAS9-mice reversed multiple hallmarks of vascular aging. Recovery from hindlimb ischemia improved (post-surgery day 28 perfusion ratio, Control: 0.58±0.03 vs GapmeR: 0.73±0.03, p=0.04, n=12/group) and aortic stiffness was reduced to levels seen in young animals. Myocardial perfusion (time-to-peak, Control: 2.585±0.08 vs GapmeR: 2.048±0.11 seconds, p=0.03, n=12/group) improved. Cardiac EC number increased, and EC senescence decreased (p<0.05 for all). Inhibition of SALTe1 reduced E/E’ (-20.16±1.60 vs -15.4±1.10, p=0.04, n=12/group) and increased global longitudinal strain (-15.88±0.84 vs 20.66±1.02, p=0.02, n=12/group). Mechanistic studies in vitro and in vivo revealed that SALTe1 works through regulation of Poly (ADP-Ribose) Polymerase Family Member 9 (PARP9) and RNA-binding protein with multiple splicing (RBPMS). Conclusions: SALTe1 regulates EC senescence through PARP9 and RBPMS. SALTe1 inhibition reverses multiple hallmarks of vascular aging and improves age-related cardiac dysfunction. These findings suggest a primary role for EC senescence in age-related cardiac dysfunction and identify SALTe1 as a potential new therapeutic target.

Abstract 18443: Early Inflammation in βII-Spectrin Deficient Hearts Promote Cardiac Remodeling and Heart Failure

Background: Dysregulation of cardiac cytoskeletal proteins such as βII-spectrin has been recognized to promote accelerated heart failure (HF) and arrhythmias in human and mouse models. However, the mechanism by which βII-spectrin dysregulation underlies HF is poorly understood. Hypothesis: We hypothesized that cardiomyocyte loss of βII-spectrin leads to recruitment of inflammatory macrophages that augment cardiac remodeling by promoting arrhythmogenesis and increased susceptibility to HF. Methods: Cardiomyocyte-specific βII-spectrin knockout (βII-cKO) and control mice (βII-flox) were generated and harvested for RNA-sequencing (RNA-seq), qRT-PCR (to validate candidate inflammatory genes), immunohistochemistry, immunoblotting, and flow cytometry. siRNA was also used to knockdown βII-spectrin in cultured cells to validate that direct loss of βII-spectrin contributes to immune cell recruitment. Transaortic constriction (TAC) was used to induce pressure overload. Results: RNA-seq demonstrated an early significant increase of inflammatory macrophages and T cell markers following βII-spectrin loss. Consistent with RNA-seq and qRT-PCR results, immunohistochemistry demonstrated an increase in CD45 positive cells in juvenile βII-cKO mouse hearts compared to control. siRNA targeted depletion of βII-spectrin in cultured cardiomyocyte led to increased expression and secretion of TGFβ-1 protein, which can promote infiltrating of immune cells. Notably, accelerated HF at two weeks was observed in βII-cKO mice compared to controls after TAC. Conclusions: These data suggest that βII-spectrin loss may mediate inflammation via the TGFβ-1 pathway, which in part contributes to βII-spectrin-mediated accelerated HF. Furthermore, these are the first data implicating βII-spectrin in the TGFβ1-immune signaling pathway in cardiomyocytes.

Wound-healing Processes After Pulpotomy in the Pulp Tissue of Type 1 Diabetes Mellitus Model Rats

IntroductionPatients with type 1 diabetes mellitus (DM1) tend to have delayed wound healing, even in the pulp tissue. We hypothesized that hyperglycemia affects odontoblast-like cell (OLC) differentiation and is involved in macrophage polarization. Accordingly, we evaluated dental pulp stem cell differentiation and macrophage phenotypes after pulpotomy. MethodsAfter modifying DM1 rat models by streptozotocin, 8-week-old rats’ upper left first molars were pulpotomized with mineral trioxide aggregate. Meanwhile, the control group was administered saline. Immunohistochemical localization of nestin, osteopontin, α-smooth muscles (α-SMAs), and CD68 (pan-macrophage marker) was conducted 7 days after pulpotomy. The OLC differentiation stage was determined using double immunofluorescence of nestin and α-SMA. Double immunofluorescence of CD68 and iNOS was counted as M1 macrophages and CD68 and CD206 as M2 macrophages. Proliferating cell nuclear antigen and Thy-1 (CD90) were evaluated by immunofluorescence. ResultsIn DM1 rats, the reparative dentin bridge was not complete; however, the osteopontin-positive area did not differ significantly from that in controls. Proliferating cell nuclear antigen, indicative of cell proliferation, increased in positive cells in DM1 rats compared with controls. Double-positive cells for α-SMA and nestin indicated many immature OLCs in DM1. CD90 was positive only in controls. CD68-positive cells, especially M1 macrophages, were increased in DM1 rats, allowing the inflammatory stage to continue 7 days after pulpotomy. ConclusionsThe condition of DM1 model rats can interfere at various stages of the wound healing process, altering OLC differentiation and macrophage polarization. These findings highlight the importance of normal blood glucose concentrations during pulp wound healing.

Artificial Intelligence (AI)-Predicted Medical Diagnosis in Suspected Mature B-Cell Neoplasms Based on Flow Cytometric Raw Data

Introduction: AI is becoming an integral part of diagnostic workup solutions worldwide. Using our vast database of flow cytometry data of hematologic neoplasms, we develop AI-powered workflows to support high-throughput routine diagnostics. As one of our most frequent requests is proof / exclusion of B-cell lymphoma, we initially focus on mature B-cell neoplasms (B-NHL), aiming at reliably classifying lymphoma cases and distinguish such from normal controls. Methods: An AI model was developed to decipher flow cytometry data in order to classify B-NHL. To that end, uniformly processed samples analyzed by human experts during standardized routine diagnostics were employed (Navios / Cytoflex cytometers, Kaluza software, Beckman Coulter). The model used 3,830 cases in order to distinguish the lymphoma categories diagnosed in routine setting. The model was built with XGBoost using raw cytometric data and expert-informed features. Recall (R, corresponds to sensitivity), precision (P, proportion of truly positive cases) and prediction probability (PP, confidence of the model in its prediction) were recorded. The predicted diagnosis was compared with the human expert diagnosis and this information was used for further model improvement. The process comprised various steps adapted to the flow cytometric data structure. These include essential technicalities such as data transformation and scaling, cluster formation, careful definition of light chain restriction and feature engineering. Results: Focusing on B-NHL (all categories) versus no lymphoma, the model initially resulted in an average R of 88% and P of 87%. The model distinguished categories 1) no lymphoma (R 95%, P 95%), 2) CLL (R 62%, P 71%), 3) HZL (R 96%, P 96%), 4) FL (R 88%, P 100%), 5) LPL (R 78%, P 70%), 6) MCL (R 97%, P 76%), 7) MZL (R 71%, P 71%) and 8) SBLPN (R 100%, P 100%). We found that 364 out of 3830 (9.5%) cases in our cohort could not be processed by the AI model due to technical limitations. To make these accessible for preprocessing and analysis, downstream improve R and P values, and make the same strategy usable for further Flow-AI models, several technical foundations were revisited. Flow cytometry data uses vastly different data ranges considering e.g. scatter vs. fluorescence properties. Since the model needs to be able to identify main populations of interest (such as lymphocytes, granulocytes, monocytes) in a dynamic fashion, data display was modified laying foundation for all further analysis. Our data show that transformation of the raw values followed by scaling to 0 to 1 enables superior clustering results. Normalization is achieved by the RobustScaler from the scikit-learn library. Implementing proper data transformation and scaling, all 3830 cases could now be successfully processed. The next step was to revisit cluster formation options as they are prerequisite for lymphoma identification in the downstream classifier. We found best results with HDBSCAN after optimizing the hyperparameters for the minimum cluster size and noise eradication to adapt automatically based on the number of CD45 positive cells or other intermediate gating results. We could reduce the rate of failure of correct classification from 20% to 17% by HDBSCAN clustering. Essential further steps included the dynamic definition of the light chain separation enabling the model to adapt to each case´s specificities in terms of expression strength of kappa and lambda. This was followed by updating the features in order to make all new information useful. These processes allowed for higher P values for CLL (71% to 80%) and LPL (70% to 74%) as well as higher R values for follicular lymphoma FL (88% to 89%) MZL (71% to 76%). Conclusion: AI is applicable to analyze flow cytometric data allowing prediction of B-NHL. By fine-tuning data transformation, scaling and clustering we massively improved the analysis pipeline gaining 12.5% of cases that had not been accessible for analysis, and in context with proper light chain definition and feature adaptation we were able to improve R and P values of several lymphoma categories. The improvements are a result of explainable feature engineering which enables us to track the weight and actual value of a feature down to the events in the dot plots and expands on our explainable AI (XAI) strategy. Our work sets the groundwork to fully embark on AI-based B-cell lymphoma identification.

LSD1 Inhibition Synergizes with Venetoclax in Acute Myeloid Leukemia By Targeting Cellular Metabolism

Introduction: Acute myeloid leukemia (AML) is a hematological neoplasm with poor clinical outcomes. The introduction of the BCL2 inhibitor venetoclax in combination with hypomethylating agent has improved response rates in older patients with AML by targeting the metabolism of leukemic stem cells (LSCs). Resistance, however, is unfortunately still common. Thus, there remains a need to enhance the clinical efficacy of venetoclax by combining it with novel agents. In diverse AML models, lysine-specific demethylase 1 (LSD1) inhibition promotes cellular differentiation and reduces LSCs. We therefore asked if LSD1 inhibition could enhance the efficacy of venetoclax in the treatment of AML. Methods: We have studied the effect of single-agent and co-treatment with venetoclax and LSD1 inhibitor bomedemstat on cell proliferation in human AML cell lines (N=3), Hoxa9/ Meis1(H9M)- and MN1-transformed murine myeloid progenitor lines as well as primary AML patient samples (N=5). We have analyzed the effect of co-treatment with these two compounds on functional cellular bioenergetics in MOLM-13 cells using Seahorse extracellular flux analyzer and followed up by performing NMR spectrometry to study changes in metabolite abundance. Further, we have studied the efficacy of this drug combination in vivo in patient-derived xenograft (PDX) AML models (N=2). Results: Human AML cell lines showed a differential cytocidal activity at 96 hours (h) to venetoclax with MOLM-13 (IC 50 0.04 µM) being most sensitive and OCI-AML3 most resistant (IC 50 9.81 µM). A similar range (between 0.15 µM and 7.3 µM) was also observed in the primary AML samples. For bomedemstat, we observed a time-dependent response where the IC 50 decreased over time, in particular, in the most sensitive model systems. In murine H9M cells, the IC 50 decreased from 30.43 µM at 48 h to 0.034 µM at 96 h and further down to 0.0068 µM at 168 h. Combining bomedemstat and venetoclax, the IC 50 for venetoclax decreased; and synergism was observed in MOLM-13 cells, the most sensitive AML model, but no synergism was observed in venetoclax-resistant cells. Synergism was also observed in MN1 cells as well as 3 of the 5 primary AML samples studied (Table 1). To investigate the mechanism of the observed synergism, we explored the effect of these treatments on metabolic pathways using the Seahorse extracellular flux analyzer. LSD1 inhibition had no effect on oxidative phosphorylation (measured as oxygen consumption rate (OCR)). However, glycolysis, measured as proton efflux rate (PER), was significantly reduced from 102.88 ± 11.03 pmol/min to 54.17 ± 6.18 pmol/min (P <0.01) after 96 h of treatment with 1μM bomedemstat. With the combined treatments, we observed a reduction in both OCR and PER. This synergistic effect was further investigated by studying metabolite abundance using NMR spectrometry where co-treatment showed a reduction in metabolite abundance across a broad spectrum of metabolic pathways (Fig. 1). To determine if the observed combined effect of bomedemstat and venetoclax could enhance activity against AML cells in vivo, we employed a PDX model with two primary AML samples. In the first model system (monosomy 7), co-treatment resulted in a significant reduction in human CD45-positive cells as compared to vehicle (P <0.001) and single-agent treatments with venetoclax (P= 0.011) and bomedemstat (P <0.001). Similarly, in the second PDX model ( NPM1-mutated, FLT3-ITD positive), the co-treatment exhibited a significant reduction in leukemic burden in bone marrow as compared to vehicle (P=0.003) and single-agent treatments with venetoclax (P= 0.024) and bomedemstat (P < 0.001) (Fig. 1). Conclusion: The combination of bomedemstat and venetoclax had synergistic cytocidal effects on AML cell line and primary AML cells in vitro. It significantly reduced the leukemic burden in PDX AML models and synergistically downregulated cellular energy metabolism. These findings suggest that combining venetoclax with LSD1 inhibition holds promise as a combination treatment in AML and warrants further clinical investigation.

IL-6 Mediates a Switch in Free Fatty Acid Metabolism from the Liver to the Bone Marrow to Support HSC Expansion during Infection

The hematopoietic stem cell (HSC) pool is tightly controlled in both normal and stressed conditions. Under normal conditions, HSCs reside quiescently in their hypoxic niche with minimal mitochondrial activity, instead favouring glycolysis to meet their energy needs. During acute infection HSCs switch to oxidative phosphorylation over glycolysis due to increased energy requirements. This metabolic switch enables HSCs to expand and differentiate into downstream progenitors to increase the immune cell pool. We recently showed that fatty acid (FA) uptake and metabolism play a central role in providing HSCs with sufficient energy for their expansion during acute infection. However, how infection drives changes in available FAs for HSC expansion is not known. This study investigated the role of liver in regulating lipid availability for HSC expansion. We have previously shown that circulating free fatty acids (FFAs) within the serum of mice increased in response to infection using both lipopolysaccharide (LPS) (6 hours) and S. Typhimurium (72 hours) models. This correlated with an expansion of bone marrow HSCs 16 hours after LPS and 72 hours after S. Typhimurium confirmed using flow cytometry. To investigate real-time fatty-acid uptake by hematopoietic cells in response to infection, an in vivo transplant model was used. CD45.1 lineage negative, CD117-positive cells tagged with firefly luciferase (LK+FF) were transplanted into CD45.2 mice. After transplantation was confirmed, mice were injected with LPS for 16 hours. Mice were then subsequently injected with a luciferin molecule conjugated to a long-chain FFA (FFA-luc), which is visible via bioluminescent imaging if the FFA-luc is taken up into the transplanted LK+FF engrafted cells. Live animal imaging using this method confirmed that long-chain FFA is taken up by hematopoietic cells in response to LPS in vivo. As the liver is the master regulator of FA in the serum we isolated livers from control and LPS (6 hour) treated mice. RNAseq revealed down-regulation of genes involved in FA uptake and metabolism as well genes involved in ketogenesis and up-regulation in genes involved in glucose uptake. Further kinetic studies showed that FA uptake and metabolism genes were downregulated within 2 hours of LPS treatment. To understand if this was a direct LPS effect on hepatocytes which express the receptor for LPS (TLR4) or mediated through unknown factors, we isolated primary hepatocytes and treated them with LPS or media conditioned with serum from control or LPS treated mice. Only the media conditioned with serum from LPS treated mice downregulated genes associated with FA uptake and metabolism. To identify the factors driving this metabolic switch in hepatocytes, a cytokine array of serum from control or LPS treated mice (90 minutes) was performed. This proteome cytokine array revealed a significant upregulation of several cytokines and chemokines, including IL-6, TNFα and CXCL1 and CXCL2. Primary mouse hepatocytes were then treated with factors identified in the array for 2 and 4 hours. RT-qPCR confirmed that IL-6 downregulated FA uptake and metabolism genes in primary hepatocytes. To understand if this was a LPS specific response or induce by infection mice were treated with S. Typhimurium for various time points and livers extracted. RT-qPCR confirmed that S. Typhimurium treated mice down-regulated genes associated with FA uptake and metabolism. To study the role of IL-6 in regulating liver FA metabolism in response to LPS, mice were treated with IL-6 neutralising antibody (MP5-20F3). Pre-treatment of mice with MP5-20F3 inhibited LPS induced downregulation of liver FA uptake and metabolic genes, which lead to reversing LPS-induced increase in serum FA. In conclusion we have shown a role for IL-6 in switching fatty acid metabolism from the liver to the bone marrow to allow HSC expansion during infection.

Altered expression of 15-hydroxyprostaglandin dehydrogenase in chronic rhinosinusitis with nasal polyps

Objective:To investigate the expression level and regulatory mechanism of 15-hydroxyprostaglandin dehydrogenase（HPGD） in chronic rhinosinusitis with nasal polyps（CRSwNP）. Methods:The expression pattern and level of HPGD in CRSwNP and control was observed using immunofluorescence, and western blot was used for analysis of HPGD expression in nasal polyp tissues. The effect of recombinant human high mobility group box-1（HMGB1） on HPGD expression in primary human nasal epithelial cells was observed, and the potential blocking effect of RAGE neutralizing antibody on HMGB1-induced HPGD expression was investigated. Results:The expression of HPGD was elevated in CRSwNP patients compared to the control, while the protein mainly localized at CD68-positive cells and epithelial cells. Recombinant human HMGB1 stimulated an increase in HPGD expression in primary human nasal mucosal epithelial cells at a time-dependent manner. Additionally, increased phosphorylation levels of MEK and elevated RAGE expression were also observed at 12 hours, but decreased at 24 hours after the incubation of HMGB1. The increase in the expression of HPGD induced by HMGB1 in primary human nasal epithelial cells was partly inhibited with RAGE neutralizing antibody. Conclusion:Elevated HPGD expression is observed in CRSwNP, predominantly in macrophages and epithelial cells. HMGB1 regulates HPGD expression through the RAGE-MEK signaling pathway, potentially providing a new target for future regulation of PGE2levels in CRSwNP.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

How fixation affects the results of lymph node immunophenotyping by flow cytometry

<b>Aim: </b>Flow cytometric diagnosis of lymphoma and leukemia is of high clinical and research importance. However, performing flow cytometry analysis on the day of biopsy might be of challenge due to several reasons, including late sample delivery, problems of preparing the reliable panel for immunophenotyping based on other diagnostic studies, etc. This problem could be partially solved if cell suspension could be fixed and stained on another day or after several days after standard FFPE (formalin-fixed and paraffin-embedded) procedure.<br /> <b>Material and methods: </b>Addressing this issue, we compared staining of live lymphocytes in suspension obtained from lymph node biopsies and same specimens fixed using 2-4%-paraformaldehyde, 1-3%-glyoxal, and 0.1-1% glutaraldehyde with subsequent immunostaining on the next day or later.<br /> <b>Results: </b>Staining after fixation could be partially representative only after paraformaldehyde fixation for 20 min and subsequent storage of cell suspension in phosphate-buffer saline within not more than 3 days. Probes stained after fixation always shows lower stain index compared to staining of live cells.<br /> <b>Conclusion:</b> Staining after fixation cannot be used for determining of the percentage of CD45-positive cells and for testing B-cell lymphomas since antigens against light chains of IgG cannot be properly detected in fixed specimens.

Examining the Aryl Hydrocarbon Receptor Network in the Placental Tissues of Pregnancies Complicated by Pre-Eclampsia: An Explorative Case-Control Analysis.

Severe maternal and newborn morbidity and mortality associated with pre-eclampsia, which are caused partly by premature delivery, affect a factual proportion of pregnancies. Despite its prevalence, the underlying causes of pre-eclampsia remain elusive, with emerging evidence implicating the aryl hydrocarbon receptor (AhR) in its pathogenesis. This study sought to elucidate the involvement of the AhR and its associated pathway in pre-eclampsia by comparing placental components of the AhR pathway in pregnant individuals with and without pre-eclampsia. This case-control investigation was conducted at the University Hospital of Udine from May 2021 to February 2023. The AhR was assessed using immunohistochemistry and immunofluorescence, and its mRNA was evaluated using a Real-Time Quantitative Reverse Transcription PCR. Levels of mRNA expression were also estimated for other components of the AhR pathway (CYP1B1, IDO1, ARNT, TIPARP, S100A4, and AHRR). Our findings show decreased levels of expression of AhR, IDO1, ARNT, TiPARP, and S100A4 in the placental tissues of individuals with pre-eclampsia compared to controls (p < 0.05). The AhR exhibited a distinct localization within the syncytiotrophoblast (nuclei and cytoplasm) and CD45-positive cells (nuclei and cytoplasm). Furthermore, a significant positive correlation between the AhR and S100A4 (rho = 0.81) was observed in normal placentas, while CYP1B1 displayed a significant negative correlation with the AhR (rho = -0.72), within addition to its negative correlation with TiPARP (rho = -0.83). This study illuminates pre-eclampsia's molecular aberrations, suggesting new diagnostic, therapeutic, and mechanistic approaches. This study emphasizes the need for more research to validate and broaden these findings to improve the management of this complex pregnancy condition.

The activated complement pathway in the fibrous process of benign prostatic hyperplasia.

To elucidate the changes in activated complement pathway in the fibrous process of benign prostatic hyperplasia (BPH), we analyzed the correlation between complement component expression and histological types of fibrosis using human BPH tissue. Fifty-six histological BPH patients who underwent prostate needle biopsy at our institution (mean age 68.6 ± 6.5 years), divided into two histological groups, fibromuscular and fibrous, were compared. Inflammatory cell infiltration in BPH tissue was evaluated by immunohistochemical staining using CD45, with complement expression analysis performed using C3, factor B, and C5b-9 antibody, and the occupancy ratio of the stained region was calculated. Further, correlation between the histological types of fibrous components in BPH tissue and lower urinary tract symptoms questionnaires was analyzed. Twenty-seven (48.2%) and 29 (51.8%) cases were classified in the fibromuscular and fibrous groups, respectively. The proportion of CD45-positive cells in BPH tissue was significantly higher in the fibromuscular group. In complement component analysis, factor B did not significantly differ between groups, while C3 (fibromuscular group; 10.7 ± 8.2%, fibrous group; 16.4 ± 12.7%) and C5b-9 (fibromuscular group; 15.9 ± 6.2%, fibrous group; 17.6 ± 9.2%) were significantly higher in the fibrous group (p = 0.04, p = 0.04, respectively). International Prostate Symptom Score Q5 subscore, indicating slow stream, was significantly higher in the fibrous group (p = 0.04). In fibrous BPH with abundant fibrosis, the late complement pathway in addition to alternative pathway was activated compared to fibromuscular BPH. These results suggested that the alternative and late complement pathways were involved in the histological fibrous process of BPH.

Interactions of Neuronally Induced Stem Cells from Apical Papilla Spheres, Stems Cells from Apical Papilla, and Human Umbilical Vascular Endothelial Cells on Vasculogenesis and Neurogenesis

IntroductionStem cell–based dental pulp regeneration has been extensively studied, mainly focusing on exploiting dental stem cells’ osteogenic and angiogenic potentials. Dental stem cells’ neurogenic role is often overlooked. Stem cells from apical papilla (SCAPs), originating from the neural crest and capable of sphere formation, display potent neurogenic capacity. This study aimed to investigate the interactions of neuronally induced stem cells from apical papilla (iSCAP) spheres, SCAPs, and human umbilical vascular endothelial cells (HUVECs) on vasculogenesis and neurogenesis. MethodsSCAPs were isolated and characterized using flow cytometry and multilineage differentiation assays. SCAP monolayer culture and spheres were neuronally induced by a small molecule neural induction medium, and the neural gene expression and neurite formation at days 0, 3, and 7 were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and using phase-contrast light and fluorescence microscopy. Direct coculture or pulp-on-chip was used to investigate iSCAP sphere interaction with SCAPs and HUVECs. RT-qPCR, fluorescence microscopy, and immunostaining with β-tubulin III, alpha-smooth muscle actin, and CD31 were used to study neural gene expression, neurite formation, and neurovascular cell interactions. ResultsNeural induction medium with small molecules rapidly induced SCAP differentiation toward neural-like cells. Gene expression of Nestin, β-tubulin III, microtubule-associated protein 2, neuron-specific enolase, and NeuN was higher in iSCAP spheres than in iSCAPs. iSCAP spheres formed more and longer neurites compared with iSCAPs. iSCAP sphere, HUVEC, and SCAP direct coculture significantly enhanced vessel formation along with up-regulated VEGF (P < .001) and multiple neural markers, such as Nestin (P < .01), microtubule-associated protein 2 (P < .001), S100 (P < .001), and NG2 (P < .001). iSCAP spheres, SCAPs, and HUVECs cultured in a pulp-on-chip system promoted endothelial and neural cell migration toward each other and alpha-smooth muscle actin–positive and CD31-positive cells assembling for the vascular constitution. ConclusionsiSCAP-formed spheres interact with SCAPs and HUVECs, promoting vasculogenesis and neurogenesis.

Severe preeclampsia complicated by HELLP syndrome alters the structure of Hofbauer and syncytiotrophoblast cells: ultrastructural and immunohistochemical study.

Hemolysis, elevated liver enzymes, low platelet count (HELLP) syndrome is generally considered to be a variant or complication of preeclampsia. It is a life-threatening obstetric complication. To evaluate the immunohistochemistry and ultrastructural of syncytiotrophoblastand Hoffbauer cells in placental villi of patients with HELLP syndrome. Two groups of patients with a total of 50 full-term human placentas (n = 25 in each group) were assigned as the control (normotensive) and HELLP syndrome. Placental tissue samples were fixed in 10% neutral formalin and paraffin-embedding protocol was performed. We prepared 5 μm sections for histological and immunohistochemical staining. Sections were immunostained with Hoffbauer cell marker CD68. For transmission electron microscopy (TEM), placental tissue samples were fixed in 2.5% buffered glutaraldehyde and then, in 1% osmium tetroxide for routine ultrastructural examinations. When the HELLP group fetal placental sections were examined, intracytoplasmic edema in syncytiotrophoblast, degenerative vacuoles, and degenerative findings on cell surface membranes were observed. Moreover, villous edema was remarkable. The number of CD68-positive Hoffbauer cells per villus control group sections was 0.23 ± 0.02 and the number of CD68-positive cells per villus in HELLP group placenta sections was 0.83 ± 0.12. The increase in the number of Hoffbauer cells per villus in the HELLP group was significant (P < 0.001). Compared with the control group, there was a significant increase in the number of Hoffbauer cells and syncytiotrophoblasts in the HELLP group, and degenerative changes were also observed in the ultrastructure of these cells. Pathology of the HELLP syndrome is in relation to CD68-positive placental macrophages.

Tissue formation and host remodeling of an elastomeric biodegradable scaffold in an ovine pulmonary leaflet replacement model.

In pursuit of a suitable scaffold material for cardiac valve tissue engineering applications, an acellular, electrospun, biodegradable polyester carbonate urethane urea (PECUU) scaffold was evaluated as a pulmonary valve leaflet replacement in vivo. In sheep (n = 8), a single pulmonary valve leaflet was replaced with a PECUU leaflet and followed for 1, 6, and 12 weeks. Implanted leaflet function was assessed in vivo by echocardiography. Explanted samples were studied for gross pathology, microscopic changes in the extracellular matrix, host cellular re-population, and immune responses, and for biomechanical properties. PECUU leaflets showed normal leaflet motion at implant, but decreased leaflet motion and dimensions at 6 weeks. The leaflets accumulated α-SMA and CD45 positive cells, with surfaces covered with endothelial cells (CD31+). New collagen formation occurred (Picrosirius Red). Accumulated tissue thickness correlated with the decrease in leaflet motion. The PECUU scaffolds had histologic evidence of scaffold degradation and an accumulation of pro-inflammatory/M1 and anti-inflammatory/M2 macrophages over time in vivo. The extent of inflammatory cell accumulation correlated with tissue formation and polymer degradation but was also associated with leaflet thickening and decreased leaflet motion. Future studies should explore pre-implant seeding of polymer scaffolds, more advanced polymer fabrication methods able to more closely approximate native tissue structure and function, and other techniques to control and balance the degradation of biomaterials and new tissue formation by modulation of the host immune response.

A-075 Development and Validation of Image-based Cell Counter Microscanner C3 for CD34-positive Hematopoietic Stem Cell Enumeration

Abstract Background Accurate measurement of CD34-positive hematopoietic stem cells (HSCs) is critical for successful peripheral blood stem cell transplantation (PBSCT). Flow cytometry is by far the gold standard for measuring CD34-positive stem cells. However, its complicated procedure requires time, cost and highly-trained operators, thus the necessity of simpler and cheaper alternative method is emerging. We have developed a new image-based cell counter, Microscanner C3 (MSC3, Biozentech, South Korea) as an alternative method for enumeration of CD34-positive HSCs and evaluated its performance. Methods 100 µl of apheresis product was stained with CD45-BB515 and CD34-PE antibodies (Becton Dickinson, USA) at room temperature in the dark. Red blood cell lysis with 500 µl of OptiLyze C solution (Beckman Coulter, USA) was followed by centrifugation. After the removal of supernatants, the sample was resuspended with 500 µL PBS, which then infused to filter paper imbedded BZ-1 microchip (Biozentech, South Korea). Thirty-fields of the loaded microchip was auto-scanned by MSC3 and its dedicated software analyzed the images and yielded CD34-positive cell fraction out of CD45-positive cells as percentage. Results of thirty-six samples were compared to that of FacsLyric (Beckton Dickinson, USA) flow cytometry and accuracy and imprecision (pentaplicate measurements for three samples), repeatability (two experimenters performed triplicate measurements for three samples each), linearity (triplicate measurements for five levels), limit of detection (hexaplicate measurements for five levels) and specificity (using whole blood specimen from three donors with monoclonal gammopathy, thrombocytosis or monocytosis) was evaluated. Results CV% values of inaccuracy, imprecision and repeatability did not exceed 15%. The method was found to be linear across the measuring range. LOD was evaluated to be 7 cells/µL and no background interference was detected. The difference between the measured fraction of MSC3 and FacsLyric was within allowable limit, with an R-value greater than 0.98. Conclusion We developed a reliable method for quantifying CD34-positive HSCs based on image analysis. The limitation involved in MSC3 applications in CD34-positive HSCs is that, like dual-platform flow cytometry, the results should be combined with the results of conventional hematology analyzer in order to define absolute HSC numbers. However, with its simpler and faster procedures, MSC3 exhibited a great potential to be used as an alternative to conventional flow cytometry in CD34-positive HSCs enumeration and other applications as well.