Flow Cytometric Analysis Research Articles

Resveratrol enhances sensitivity of renal cell carcinoma to tivozanib: An in-vitro study

BackgroundSince tivozanib has many side effects in the treatment of kidney cancer, we decided to use resveratrol as a bioactive molecule with anticancer and antioxidant properties to make tivozanib more effective and also reduce its side effects in kidney cancer cell line. MethodIn this in vitro study, we evaluated the effect of tivozanib, resveratrol and tivozanib- resveratrol combination therapy in ACHN cell line as representatives of human kidney cancer. The assessment includes Hoechst dye staining, scratch-wound assay, 3D spheroid, 2D colony formation assay, flow cytometric analysis of apoptosis and DNA cell cycle, real-time PCR (BAX/BCL2, E-cadherin, Snail, HIF1α, VEGFC and KLK3 genes). ResultTo determine IC50 levels, ACHN cells was exposed to different concentration of tivozanib and resveratrol. Our data indicated that IC50 values for tivozanib (0.5 μM) and resveratrol (30 μM) with MTT in a dose and time-dependent manner. Due to the efficacy of resveratrol in combination with tivozanib, we used 20 μM resveratrol, and 0.25 μM tivozanib instead of 30 μM and 0.25 μM respectively. This data was approved by flow cytometry for ACHN cell line with 38.39, 14.74 and 66.06 percent apoptosis and 8.25, 5.12 and 15.6 percent subG1 for tivozanib, resveratrol and tivozanib-resveratrol combination respectively which was as a consequence of cell cycle arrest at G1/S phase. The treatment also reduced cells’ migration, fragmented nuclei, 3D spheroid and colony formation potentials in analyses. Evaluation of gene expression presented that the effect of the tivozanib and resveratrol combination in ACHN cell lines is completely different during the evaluation of apoptosis genes, BAX, P53 genes and E-Cadherin had significantly increased expression compared to single treatment groups (P < 0.01). Meanwhile, a significant decrease was observed in the expression of VEGFC and HIF1α genes in the combination group compared to the monotherapy groups (P < 0.001). ConclusionConsidering that resveratrol can increase the apoptosis of cancer cells alone and in combination with tivozanib and prevent the proliferation of cancer cells and also reduce the side effects of tivozanib, we suggest that resveratrol as a potential bioactive molecule can be used in treatment of kidney cancer should be used in combination with tivozanib.

Evaluation of the Anti-Inflammatory/Immunomodulatory Effect of Teucrium montanum L. Extract in Collagen-Induced Arthritis in Rats.

The anti-inflammatory/immunomodulatory effects of Teucrium montanum L. (TM), a plant distributed in the Mediterranean region, have been insufficiently examined. The effects of the TM ethanol extract were tested in a rat collagen-induced arthritis (CIA) model of rheumatoid arthritis. LC-MS was used for the phytochemical analysis of the TM extract. Dark Agouti rats were immunized with bovine type II collagen (CII) in incomplete Freund's adjuvant for CIA, and treated with 100 or 200 mg/kg of TM extract daily via oral administration. Clinical and histopathological evaluations and a flow cytometric analysis of the phenotypic and functional characteristics of splenocytes and draining lymph node cells were performed. The cytokines in the paw tissue culture supernatants and anti-CII antibodies in serum were determined by ELISA. The TM extract, with the dominant components verbascoside and luteolin 7-O-rutinoside, reduced the arthritic score and ankle joint inflammation in CIA rats, promoted the antioxidant profile in serum, and lowered pro-inflammatory TNF-α, IL-6 and IL-1β production. It suppressed the activation status of CD11b+ cells by lowering CD86, MHCII and TLR-4 expression, and promoted the Th17/T regulatory cell (Tregs) balance towards Tregs. A lower frequency of B cells was accompanied by a lower level of anti-CII antibodies in treated rats. These findings imply the favorable effect of TM extract on the clinical presentation of CIA, suggesting its anti-inflammatory/immunomodulatory action and potential therapeutic effect.

Flow Cytometric Immunophenotyping: Minimal Differences in Fresh and Cryopreserved Peripheral Blood Mononuclear Cells versus Whole Blood.

Background/Objectives: Flow cytometry is a convenient tool in immunophenotyping for monitoring the status of immunological conditions and diseases. The aim of this study was to investigate the effect of isolation and cryopreservation by flow cytometric analysis on subpopulations of CD4+ T helper (Th), T regulatory (Treg), CD8+ T cytotoxic (Tc), CD56+ NK, CD19+ B and monocytes. Freshly isolated and cryopreserved peripheral blood mononuclear cells (PBMCs) were compared to fresh whole blood. Methods: Peripheral blood was collected from healthy donors and prepared for flow cytometric analysis using the same panels of antibodies throughout the study. Results: Comparisons between fresh (F)- and cryopreserved (C)-PBMCs showed no major differences in percentages of CD4+, Th1, Th2 and CD4+CD25+CD127low Treg cells. No differences in percentage of CD8+ or subpopulations of naive/stem, central or effector memory cells were observed between F- and C-PBMCs. The percentage of CD56+ NK cells, CD19+ B cells or classical and nonclassical monocytes did not differ between F-and C-PBMCs either. On the contrary, whole blood had lower percentages of Th and NK cells but higher percentages of Th1, Th17, Th1Th17, Tregs, Tc and B cells compared to C-PBMCs, while it had a higher proportion of Tc compared to F-PBMCs. Conclusions: Flow cytometric immunophenotyping minimally differs between freshly isolated and cryopreserved PBMCs. This implies the possibility of cryostorage of cohorts for later analysis. Importantly, care must be taken when comparing results from whole blood with isolated and cryopreserved PBMCs. Collectively, these results can contribute to the standardization of flow cytometric protocols in both clinical and research settings.

Integrated bioinformatics analysis and experimental validation on malignant progression and immune cell infiltration of LTBP2 in gliomas

BackgroundGliomas are the highly aggressive brain tumor and also the most devastating human tumors. The latent TGF binding proteins (LTBP) had been found to be involved in malignant biological process and could be used as potent biomarkers in several solid tumors. While the role of LTBP family in human glioma remain to be elucidated.MethodsNormalized gene expression and corresponding clinical data of 2407 gliomas samples in public datasets were downloaded from Gliovis. Kaplan–Meier methods and Cox regression analysis was used for survival analyses.Western blot (WB) and Immunohistochemical (IHC) testing were employed to test LTBPs protein level in 154 gliomas samples. Correlation between LTBP2 expression and immune infiltration was evaluated by immunofluorescence (IF) and IHC in glioma tissues. CCK8 and flow cytometric analysis were used to detect the effect of LTBP2 on glioma cells. Orthotopic glioma- mouse models were utilized to evaluate effects in vivo.ResultsLTBP2 mRNA level was dramatically higher in glioma samples compared with non-tumor brain tissues in XENA-TCGA_GTEx, Gill and Gravendeel datasets (all P < 0.01), and its expression positively correlated with glioma WHO grade, IDH1/2 wildtype and mesenchymal subtypes. These results were confirmed by In-house cohort which was detected by WB and IHC. We found that gliomas patients with high LTBP2 level had shorter OS than those with low LTBP2 level. LTBP2 expression significantly associated with glioma immune score (Spearman r = 0.68, P < 0.01)) and strongly correlated with infiltration degreee of macrophages both in lower grade gliomas (LGG) and GBM. Knocking down LTBP2 obviously reduced proliferation and enhanced sensitivity to temozolomide in U87 and U251 cells. Nude mice with lower expression of LTBP2 had slower tumor growth, and accompanied by less tumor-associated macrophages (TAMs) infiltration detected by IHC staining in vivo. Finally, low LTBP2 expression glioma patients who received chemotherapy survived longer than patients with high LTBP2 expression.ConclusionLTBP2 could be used as a prognostic marker, and high LTBP2 expression related to abundant TAMs infiltration and with a worse response to chemotherapy.

Mesenchymal stem cell-derived exosomal mir-21-5p inhibits YAP1 expression and improves outcomes in myocardial infarction

BackgroundMyocardial infarction (MI) remains a significant global health concern, characterized by cardiomyocyte apoptosis and adverse ventricular remodeling. Nevertheless, the interplay between exosomal miR-21-5p and Yes-associated protein 1 (YAP1) in the context of MI remains unexplored.MethodsRat mesenchymal stem cells (MSCs) and H9c2 cardiomyocytes were cultured, characterized, and instrumental in our experiments. Exosomes were meticulously isolated, and their identity confirmed. The internalization of these exosomes by H9c2 cells was assessed, while RNA and protein expression were quantified using Quantitative Real-Time PCR and Western blot, respectively. MTT assay was implemented for cell viability, and apoptosis was evaluated via flow cytometric analysis. To elucidate gene interactions, we conducted microarray profiling of miRNA expression, dual luciferase reporter assays, and RNA Immunoprecipitation.ResultsMSC-derived exosomes exhibited a remarkable capacity to attenuate hypoxia-induced inflammation and apoptosis in H9c2 cells. Notably, these exosomes significantly upregulated miR-21-5p levels within H9c2 cells, and the abrogation of miR-21-5p function abated their protective effects. Through computational analysis, we unveiled a miR-21-5p binding site in the 3’UTR of YAP1, which directly inhibited YAP1 expression. Importantly, the inhibition of YAP1 effectively reinstated the protective effects of exosomes in cells with impaired exosomal miR-21-5p.ConclusionThis study underscores the pivotal role played by MSC-derived exosomes in safeguarding against MI, primarily by mediating the transfer of miR-21-5p, which targets YAP1 signaling pathways.Clinical trial numberN/A.

Dynamics of Peripheral Lymphocyte Subsets from Birth until Old Age

The immune system is inexperienced before birth and tends to be tolerogenic, rather than immunogenic. After birth, the adaptive immune system develops while facing microbial challenges, but it can become impaired as old age progresses and persistent inflammation can lead to chronic morbidity, disability and frailty. To investigate the potential contributions of lymphocyte subsets to immunity from birth until old age, we enumerated circulating innate and conventional lymphocytes and measured serum cytokine levels in 10 cord blood samples and in peripheral blood from 10 healthy term neonates, 23 healthy school-age children, 25 young adults and 11 older subjects. Flow cytometric analysis revealed that B cell frequencies increase during childhood and gradually decrease into adulthood, whereas natural killer cell frequencies increase throughout life. T cell frequencies remained relatively constant throughout life, as did their expression of CD4 and CD8. However, all four innate T cell populations studied—invariant natural killer T cells, mucosa-associated invariant T cells and the Vδ1 and the Vδ2 subsets of γδ T cells—were extremely rare in cord blood and in peripheral blood of neonates, but they expanded after birth reaching highest levels in adulthood. Analysis of serum cytokine levels revealed that proinflammatory and T helper type 1 (Th1) cytokine levels increase in adulthood, whereas Th2 and Th17 cytokine levels remain relatively constant. These changes in lymphocyte numbers and cytokine levels across the lifetime are likely to affect immunocompetence, leaving newborn and elderly people susceptible to infection, cancer and immune-mediated disease.

AutoGater: a weakly supervised neural network model to gate cells in flow cytometric analyses

Flow cytometry is a useful and efficient method for the rapid characterization of a cell population based on the optical and fluorescence properties of individual cells. Ideally, the cell population would consist of only healthy viable cells as dead cells can confound the analysis. Thus, separating out healthy cells from dying and dead cells, and any potential debris, is an important first step in analysis of flow cytometry data. While gating of debris can be conducted using measured optical properties, identifying dead and dying cells often requires utilizing fluorescent stains (e.g. Sytox, a nucleic acid stain that stains cells with compromised cell membranes) to identify cells that should be excluded from downstream analyses. These stains prolong the experimental preparation process and use a flow cytometer’s fluorescence channels that could otherwise be used to measure additional fluorescent markers within the cells (e.g. reporter proteins). Here we outline a stain-free method for identifying viable cells for downstream processing by gating cells that are dying or dead. AutoGater is a weakly supervised deep learning model that can separate healthy populations from unhealthy and dead populations using only light-scatter channels. In addition, AutoGater harmonizes different measurements of dead cells such as Sytox and CFUs.

The Role of MicroRNA-124-3p in Breast Cancer Stem Cell Inhibition by Benzyl Isothiocyanate.

We have shown previously that benzyl isothiocyanate (BITC) derived from cruciferous vegetables inhibits self-renewal of breast cancer stem-like cells (bCSC). The current study provides insights into the mechanism of bCSC inhibition by BITC. Quantitative real time-polymerase chain reaction and western blot analysis were performed to detect microRNAs(miRNAs) and Forkhead box Q1 (FoxQ1) protein expression, respectively. The bCSC were characterized by aldehyde dehydrogenase 1 activity and flow cytometric analysis of CD49f high/CD133high fraction. BITC treatment resulted in induction of miR-124-3p expression in MDA-MB-231 and MCF-7 cells. miR-124-3p did not affect BITC-mediated inhibition of cell migration or cell proliferation but it significantly regulated bCSC in response to BITC. We also found that miR-124-3p directly targets the 3'untranslated regions (UTR) of FoxQ1 and negatively regulates its expression. The BITC-mediated inhibition of bCSC was partially attenuated by miR-124-3p inhibitor. These findings indicate that miR-124-3p plays an important role in BITC-mediated inhibition of bCSC.

Diagnostic value of IFN-gamma in tuberculous pleural effusion

BackgroundSimple, rapid, and accurate diagnosis of tuberculous pleural effusion (TPE) remains challenging. This study aimed to determine the accuracy of IFN-γ in diagnosing TPE. MethodsWe quantified the expression of interferon-gamma (IFN-γ) in blood (B), adenosine deaminase (ADA), and IFN-γ in pleural effusions (PE) from 25 TPE patients and 31 non-TPE patients using a combination of immunological assays and flow cytometric analysis. The diagnostic performance of these three biomarkers was evaluated using receiver operating characteristic (ROC) curves. ResultsWe found that IFN-γ levels in blood and pleural fluid were higher in the TPE group than in the non-TPE group. The mean concentration of IFN-γ in pleural fluid of the TPE group was 3140.90 (1817.94, 6611.05) pg/mL, while that of the non-TPE group was 4.91 (0.69, 8.6) pg/mL), and the difference was statistically significant (z = 6.39, P < 0.001). The mean blood IFN-γ was 40.19 (16.45, 59.08) pg/mL in the TPE group and 2.76 (1.96, 6.02) pg/mL in the non-TPE group, which was statistically different (z = 5.12, P < 0.001). The area under the ROC curve (AUC) for pleural fluid IFN-γ, blood IFN-γ, and ADA were 0.999 (95 % CI: 0.994–1.00), 0.901 (95 % CI: 0.798–1.00) and 0.996 (95 % CI: 0.987–1.00), respectively. ConclusionThis study confirms that IFN-γ has high diagnostic validity in patients with TPE and can potentially be an excellent biomarker.

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

Therapeutic JAK1 Inhibition Reverses Lupus Nephritis in a Mouse Model and Demonstrates Transcriptional Changes Consistent With Human Disease.

Janus kinase family members are essential for signaling by multiple cytokines, including many implicated in systemic lupus erythematosus (SLE) pathogenesis. To test whether inhibition of JAK1 signaling can be efficacious in SLE, we used a JAK1-selective inhibitor (ABT-317) and evaluated its ability to ameliorate disease in murine SLE. Efficacy of ABT-317 was evaluated using NZB/W-F1 mice treated prophylactically and therapeutically. Primary endpoints were proteinuria, survival, and saliva production. Other endpoints included histological analysis of kidneys and salivary glands, flow cytometric analysis of splenic cell populations, and gene expression analysis by RNA sequencing in the kidneys, salivary glands, and blood. Publicly available human kidney gene transcription data were used to assess the translatability of the mouse findings. ABT-317 was efficacious when dosed prophylactically and prevented disease for up to two months after treatment cessation. When dosed therapeutically, ABT-317 quickly reversed severe proteinuria and restored saliva production, as well as diminished kidney and salivary gland inflammation. ABT-317-induced changes in glomerular morphology coincided with normalization of a human nephrotic gene signature, suggesting translatability to human lupus nephritis (LN). JAK1 inhibition prevented and reversed kidney and salivary gland manifestations of murine lupus with long-lasting effects after treatment cessation. These data, along with the presence of JAK1 and nephrotic gene signatures in human LN glomeruli, suggest that a JAK1-selective inhibitor may be an effective therapeutic in the treatment of human SLE and LN.

Proteomic analysis of plasma-derived extracellular vesicles: pre- and postprandial comparisons

Extracellular vesicles (EVs) are key in intercellular communication, carrying biomolecules like nucleic acids, lipids, and proteins. This study investigated postprandial characteristics and proteomic profiles of blood-derived EVs in healthy individuals. Twelve participants fasted overnight before baseline assessments. After consuming a controlled isocaloric meal, EVs were isolated for proteomic and flow cytometric analysis. Plasma triacylglyceride levels confirmed fasting completion, while protein concentrations in plasma and EVs were monitored for postprandial stability. Proteomic analysis identified upregulated proteins related to transport mechanisms and epithelial/endothelial functions postprandially, indicating potential roles in physiological responses to nutritional intake. Enrichment analyses revealed vesicle-related pathways and immune system processes. Flow cytometry showed increased expression of CD324 on CD9+CD63+CD81+ large extracellular vesicles postprandially, suggesting an epithelial origin. These findings offer valuable insights into postprandial EV dynamics and their potential physiological significance, highlighting the need for stringent fasting guidelines in EV studies to account for postprandial effects on EV composition and function.

Investigating Concomitant RAG-2 and LRBA Mutations in SCID and Autoimmunity.

Inborn errors of immunity (IEI) are a large heterogenous group of diseases characterized by immunodeficiency, immune dysregulation, allergy, auto-inflammation and predisposition for malignancies. Most are inherited in an autosomal recessive trait. We studied a patient with severe combined immunodeficiency (SCID) and immune dysregulation who harbored two distinct bi-allelic IEI-associated genetic mutations. Clinical, immunological and genetic data were collected. Genetic investigation included whole exome sequencing on DNA extracted from skin fibroblasts. Family segregation was performed by Sanger sequencing. Immunological evaluation included absolute and functional evaluation of lymphocytes and chimerism analysis post hematopoietic stem cell transplantation (HSCT). Treg subsets, LRBA and CTLA4 expression levels were measured by flow-cytometric analysis. A nineteen-year-old female patient from a consanguine background underwent unconditioned matched sibling related HSCT during infancy due to clinical presentation of SCID with an Omenn phenotype. At that time her underlying genetic defect was not defined. Years after HSCT, severe auto-immune phenomena were noted, including a systemic lupus erythematosus-like syndrome and ophthalmic manifestations. Genetic evaluation revealed bi-allelic homozygous mutations in RAG-2 (c.685C>T, p.Arg229Trp) and a previously undescribed mutation in LRBA (c.3325G>T, p.Asp1109Tyr). LRBA and CTLA4 expression levels were normal, suggesting that the LRBA variant identified in these kindred is unlikely to be pathogenic. Multiple genetic defects causing complex IEIs may be identified in the same individual in highly consanguineous populations. Functional immunological testing is essential for evaluation of novel genetic variants.

Jieduquyuziyin prescription alleviates lupus development via inhibiting neddylation pathway to promote Bim-induced apoptosis of double negative T cells

Ethnopharmacological relevanceJieduquyuziyin prescription (JP) is an empirical prescription approved for application to treat systemic lupus erythematosus (SLE) in hospital within China. Despite the prominent treatment effect of JP clinically, further investigation is imperative to explore its underlying mechanisms. Aim of the studyWe aim to investigate the impact of JP on DN T cell apoptosis in the treatment of SLE and the specific regulation mechanisms. Materials and methodsFirstly, female MRL/lpr mice were treated with JP and the therapeutic efficacy of JP was evaluated via skin lesions, lymphoid organ enlargement, accumulation of autoantibodies and renal function. Then, flow cytometer analysis was performed to evaluate the proportions and the apoptosis of T cell subpopulations. Based on the above results, double-negative (DN) T cells were subjected to proteomic with subsequent differential screening. The expression of Ube2m and Bim was further validated using real-time PCR and Western blot. Subsequently, DN T cells were incubated with JP-contained serum in vitro, and cell apoptosis was quantified using flow cytometry. Additionally, the expression levels of Ube2m, Bim and other associated proteins were also assessed through western blotting. To further clarify whether Ube2m serves as the key target of JP in regulating DN T cell apoptosis, the mice that Ube2m was specific deleted in T cells with spontaneous lupus (Ube2m−/−lpr) were utilized. JP was administered to WTlpr or Ube2m−/−lpr mice, followed by assessment of the lupus condition and DN T cell apoptosis. ResultsJP administration effectively ameliorated the lupus phenotype. Then flow cytometry assay showed that JP treatment enhanced DN T cell apoptosis to reduce their accumulation and restored the immune homeostasis. Proteomic analysis revealed a significant inhibition of Ube2m for JP treatment, which is essential for maintaining homeostasis of DN T cells. Further experiments confirmed that JP treatment effectively downregulated the expression of Ube2m and subsequently upregulated the level of pro-apoptotic protein Bim with decreased Bim degradation. In vitro experiments also confirmed that JP-contained serum significantly facilitated DN T cell apoptosis and reduced DN T cell accumulation by inhibiting Ube2m expression. Furthermore, Ube2m−/−lpr mice were utilized and the impact of JP treatment on the apoptosis of DN T cells was found to be minimal in the absence of Ube2m. Mechanistic investigation reveals that JP exerts its effects by suppressing the expression of Ube2m, subsequently inhibiting CRL-dependent degradation of Bim, and ultimately promoting Bim-induced apoptosis in DN T cells. Furthermore, the blockade of Ube2m in T cells effectively prevents JP-induced apoptosis in DN T cells, underscoring Ube2m as one crucial therapeutic target of JP in mediating DN T cell apoptosis and managing SLE. ConclusionsOur findings indicate that JP treatment effectively restores the homeostasis of DN T cells in SLE by inhibiting Ube2m expression, thereby reducing Bim ubiquitination degradation. This ultimately enhanced DN T cell apoptosis and alleviated lupus phenotype.

A-152 Enhancing PNH Diagnosis with Multicolor Flow Cytometry Immunophenotyping Protocol in a Medical Center in Taiwan

Abstract Background Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from mutations in the X-linked PIG-A gene, leading to an inability to produce GPI-anchored proteins on RBCs and WBCs. PNH presents in three variants: classic (hemolytic), associated with specified bone marrow disorders, and subclinical. Flow cytometric analysis stands as the gold standard for PNH detection, crucial in diagnosis, monitoring, and clinical management. Recognizing potential variations in PNH testing among labs, it is essential to establish a precise and consistent protocol for accurate diagnosis and patient management. This report aims to demonstrate the effectiveness of a PNH immunophenotyping protocol adhering to ICCS/ESCCA PNH consensus guidelines. Methods A total of 501 samples were analyzed from Jan 1st, 2021 to Dec 31st, 2023. All the acquisitions were accomplished by a 3-laser and 10-PMT Navios EX (Beckman Coulter) flow cytometry. A typical combination of anti-CD235a-FITC (clone KC16) and anti-CD59-PE (clone MEM43) antibodies were adopted to identify the PNH clone size in the RBC tube. To achieve higher sensitivities in PNH granulocyte and PNH monocyte detection, a single tube, 6-color mixture of FLAER, monoclonal antibodies including CD14-APC, CD24-PE, CD33-APC-H7, CD15-PacB, and CD45-KrO, to immunostaining simultaneously was developed. An unstained control was run after the full-stained tube to determine the position of threshold for positivity, and to exam the potential carryover into the test sample. Results RBC and granulocyte assays can detect PNH phenotypes at the 0.009% level or higher with more than 50,000 cells of gated cells acquired, while the monocyte assay can detect at &amp;gt;=0.069% with at least 20,000 cells acquired. Analysis was conducted on five hundred and one patients to investigate potential PNH clones in their blood cells. Among them, 45 patients were identified as having classic PNH, while PNH clones were not detected in 168 cases, constituting 33.5% of total cases. Minor PNH clone (&amp;lt;1.0%) in single cell lineage were observed in 144 cases, making up 28.7% of all cases. Minor PNH clones found in more than one cell lineages were found in 155 cases (30.9% of 501). Additionally, minor PNH clones were identified across RBCs, granulocytes, and monocytes, with percentages of 32.9%, 23.8%, and 43.9%, respectively of all patients. Notably, monocyte clones exceeded granulocyte clones in 23.0% of cases with WBC PNH clones, irrespective of clone sizes. Furthermore, we investigated seven patients exhibiting persistent minor PNH clones in 2 to 3 cell lineages. Among them, five had aplastic anemia or myelodysplastic syndromes, while one underwent evaluation for young stroke and one was investigated for venous thromboembolism. Conclusions Flow cytometry labs aim not only to identify classic PNH patients but also to consistently detect minor or small clones in individuals with bone marrow disorders or subclinical conditions. Our findings are compatible with international reports, underscoring the importance of routinely examining patients with minor clones. Such assays serve as essential tools for ongoing monitoring, facilitating PNH diagnosis and management by physicians.

Therapeutic effects of S-allyl-L-cysteine in a mouse endometriosis model and its immunomodulatory effects via regulation of T cell subsets and cytokine expression.

Endometriosis is a female hormone-dependent gynecological disorder characterized by chronic inflammation. Therefore, the development of novel treatment strategies that can diminish the side effects of the long-term use of hormone-based drugs has been emphasized. S-Allyl-L-cysteine (SAC) is the major constituent of aged garlic extracts. Although the therapeutic effects resulting from the antioxidant properties of SAC have been extensively studied in inflammatory diseases, the therapeutic efficacy of SAC in endometriosis has not been described. In this study, we investigated the therapeutic potential of SAC for endometriosis using a mouse model. An endometriosis mouse model was surgically induced, and oral treatment with 30mg/kg SAC was administered daily for 28 days. The development of endometriotic lesions was assessed by histological analysis, and the expression profiles of adhesion-, apoptosis-, and inflammation-related genes were evaluated by PCR. Flow cytometric analysis of mouse spleen was conducted to assess changes in lymphocyte subpopulations. SAC treatment significantly inhibited endometriotic lesion growth. Transcriptional expression analysis revealed the antiadhesion and apoptosis-promoting effects of SAC. In particular, SAC showed an effective immune modulatory response by altering splenic CD4+ and CD8+ T cell subsets and inflammatory cytokine production in the spleen and endometriotic lesions. This study newly elucidates the inhibitory effects of SAC on the growth of endometriosis in a mouse model and describes its immunomodulatory effects.

SO2 activates Th17 cells through the JAK1,2/STAT3 signaling pathway

ObjectiveTo investigate the effect of SO2 on Th1/Th2/Th17 cells in allergic rhinitis (AR) and the role of JAK1, 2/STAT3 signaling pathways.To Provide potential directions for the treatment of AR. MethodsFifteen AR patients were enrolled as the experimental group, while 15 healthy volunteers served as the normal control group. After collecting venous blood, peripheral blood mononuclear cells (PBMCs) were isolated and cultured, followed by the addition of SO2 derivatives and the JAK inhibitor Ruxolitinib. Flow cytometry was employed to assess alterations in the Th1/Th2 and Th17/Treg cell balance upon stimulation with SO2 and Ruxolitinib. qRT-PCR was utilized to detect the expression of Th1-related cytokines IL-2 and IFN-γ, Th2-related cytokines IL-4 and IL-5, Th17-related cytokines IL-17A and RORγt, as well as genes JAK1, JAK2, and STAT3. Flow cytometric cytokine analysis was conducted for quantitative assessment of the expression levels of inflammation-related cytokines in PBMC culture supernatants after stimulation. In addition, we stimulated the Jurkat T lymphocyte cell line with SO2 derivatives, added Ruxolitinib as an inhibitor, and used Western blot analysis to further determine the effects of SO2 on Th cells and the role of the JAK1,2/STAT3 signaling pathway in this process. ResultsStimulation with SO2 derivatives upregulated the expression levels of Th2 cells and associated cytokines, as well as Th1 cells and associated cytokines. both AR patients and healthy individuals displayed increased percentages of Th17 cells and Th17/Treg ratios in PBMCs. The expression of IL-17A, RORγt, and IL-6 was also elevated. Under SO2 stimulation, the expression of JAK1, JAK2, STAT3, and RORγt in Jurkat cells increased. Moreover, after the application of Ruxolitinib, the JAK/STAT signaling pathway was inhibited. This led to a reduction in Th17 cells and IL-17A levels in both AR patients and healthy individuals, as well as a decrease in RORγt expression in Jurkat cells. Additionally, the expression of IL-5 decreased in healthy individuals. ConclusionSO2 exposure exacerbated Th1/Th2/Th17 inflammation in AR patients and induced Th1 and Th17 inflammation in healthy individuals. The stimulatory effect of SO2 on Th17 cell differentiation could be inhibited by Ruxolitinib. This suggests that the Th17 inflammation induced by SO2 stimulation may be related to the activation of the JAK/STAT signaling pathway, and this has been confirmed in the Jurkat cell line.

Mild therapeutic hypothermic protection activates the PI3K/AKT signaling pathway to inhibit TRPM7 and suppress ferroptosis induced by myocardial ischemia‑reperfusion injury.

The present study aimed to investigate the role of PI3K‑mediated ferroptosis signaling induced by mild therapeutic hypothermia (MTH), which was defined as a temperature of 34˚C, in protecting against myocardial ischemia-reperfusion (I/R) injury (MIRI). To meet this aim, H9C2 cells underwent hypoxia‑reperfusion (H/R) and/or MTH. The MTT assay was used to assess cell viability, cytotoxicity was measured using a lactate dehydrogenase cytotoxicity assay, and Annexin V‑FITC/PI flow cytometric analysis was used to analyze early and late cell apoptosis. In addition, 84 healthy adult male Sprague‑Dawley rats were randomly divided into seven groups (n=12), and underwent I/R and various treatments. Hemodynamics were monitored, and the levels of myocardial injury marker enzymes and oxidative stress markers in myocardial tissue were measured using ELISA. The expression levels of PI3K, AKT, transient receptor potential cation channel subfamily M member 7 (TRPM7), glutathione peroxidase 4 (GPX4) and acyl‑CoA synthetase long chain family member 4 (ACSL4) in animals and cells were measured using western blot analysis. These experiments revealed that MTH could effectively reduce myocardial infarct size, improve hemodynamic performance following MIRI and suppress myocardial apoptosis, thereby contributing to the recovery from H/R injury. Mechanistically, MTH was revealed to be able to activate the PI3K/AKT signaling pathway in cells, upregulating GPX4, and downregulating the expression levels of TRPM7 and ACSL4. Treatment with 2‑aminoethoxydiphenyl borate (an inhibitor of TRPM7) could further strengthen the myocardial protective effects of MTH, whereas treatment with erastin (promoter of ferroptosis) and wortmannin (inhibitor of PI3K) led to the effective elimination of the myocardial protective effects of MTH. Compared with in the I/R group, the PI3K/AKT activation level and the expression levels of GPX4 were both significantly increased, whereas the expression levels of TRPM7 and ACSL4 were significantly decreased in the I/R + MTH group. Taken together, the results of the present study indicated that MTH may activate the PI3K/AKT signaling pathway to inhibit TRPM7 and suppress ferroptosis induced by MIRI.

Loss of synovial tissue macrophage homeostasis precedes rheumatoid arthritis clinical onset.

This study performed an in-depth investigation into the myeloid cellular landscape in the synovium of patients with rheumatoid arthritis (RA), "individuals at risk" of RA, and healthy controls (HC). Flow cytometric analysis demonstrated the presence of a CD40-expressing CD206+CD163+ macrophage population dominating the inflamed RA synovium, associated with disease activity and treatment response. In-depth RNA sequencing and metabolic analysis demonstrated that this macrophage population is transcriptionally distinct, displaying unique inflammatory and tissue-resident gene signatures, has a stable bioenergetic profile, and regulates stromal cell responses. Single-cell RNA sequencing profiling of 67,908 RA and HC synovial tissue cells identified nine transcriptionally distinct macrophage clusters. IL-1B+CCL20+ and SPP1+MT2A+ are the principal macrophage clusters in RA synovium, displaying heightened CD40 gene expression, capable of shaping stromal cell responses, and are importantly enriched before disease onset. Combined, these findings identify the presence of an early pathogenic myeloid signature that shapes the RA joint microenvironment and represents a unique opportunity for early diagnosis and therapeutic intervention.