Flow Cytometry Research Articles

The promotive role of reticulocalbin 3 (RCN3) in the pathogenesis of keloid via TGFβ1/Smad2/Smad7 signaling pathway in vitro

Abstract Objectives Keloids, characterized by an excessive accumulation of fibrous tissue, remain a significant dermatological challenge. This study elucidates the role of Reticulocalbin 3 (RCN3) in modulating keloid fibroblasts (KFs) in vitro. Methods The RCN3 expression levels in human KFs and normal dermal fibroblast cells were examined using RT-qPCR and Western blot methods. RCN3 expression was knocked down in keloid fibroblasts after cells were transfected with shRNA targeting RCN3. On the other hand, recombinant RCN3 was added to treat the cells. RT-qPCR, Western blot, flow cytometry, CCK8 assays, and wound healing assays were performed to analyze the fibrotic markers (collagen I, III, MMP2, α-SMA) and TGFβ1/Smad2/6/7 pathway, apoptosis, proliferation and migration. Results RCN3 was upregulated in KFs. Its knockdown reduced fibrotic marker expression (collagen I, III, MMP2, α-SMA), cell proliferation, and migration, while increasing apoptosis. Conversely, recombinant RCN3 treatment enhanced the fibrotic responses. Changes in TGFβ1/Smad2 pathway, especially in Smad2 phosphorylation and Smad7, were evident following RCN3 modulation. Conclusions The study reveals RCN3 as a regulator in keloid pathology, affecting fibrosis, cellular behavior through TGFβ1/Smad2/Smad7 signaling.

The amino acid metabolism pathway of peripheral T lymphocytes and ketamine-induced schizophrenia-like phenotype.

The intricate interplay between peripheral adaptive immune cells and the central nervous system (CNS) has garnered increasing recognition. Given that alterations in cell quantities often translate into modifications in metabolite profiles and that these metabolic changes can potentially traverse the bloodstream and enter the CNS, thereby modulating the progression of mental illnesses, we sought to explore the metabolic profiles of peripheral immune cells in a ketamine-treated mouse model of schizophrenia. We used flow cytometry to scrutinize the alterations in peripheral adaptive immune cells in a ketamine-induced schizophrenia mouse model. Subsequently, we implemented an untargeted metabolomic approach with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to detect the metabolite profiles of peripheral abnormal lymphocytes and identify differential metabolites present in plasma. We then employed targeted metabolomics using UPLC-MS/MS to quantify the common differential metabolites detected in mouse plasma. Flow cytometry analysis detected a notable increase in the count of peripheral CD3+ T cells in a ketamine-induced schizophrenia mouse model. Subsequent untargeted metabolomics analysis revealed that the amino acid metabolism pathway underwent substantial alterations. A detailed quantification of 22 amino acid profiles in the peripheral plasma indicated significant elevation in the levels of glycine, alanine, asparagine, and aspartic acid. Our ongoing research has yet to conclusively identify the precise amino acid metabolism pathway that serves as the pivotal factor in the manifestation of the schizophrenia-like phenotype induced by ketamine. The peripheral amino acid metabolism pathway is involved in the ketamine-induced schizophrenia-like phenotype. The metabolic profile of peripheral immune cells could provide accurate biomarkers for the diagnosis and treatment of psychiatric diseases.

ANTIVIRAL IL-33-STUMULATED GROUP 2 INNATE LYMPHOID CELLS (CD-90 AND CD-117) FROM MOUSE LUNGS

This article represents not only a significant scientific value, but also a great teaching and instruction value for researchers and motivated students to improve the skill spectrum in flow cytometry. This article provides step-by- step procedure description and results based on in vivo studies on murine lung tissue, cell of interest isolation with dual protocols for flow cytometry method as well as IL-33 impact in dynamics. The newly described innate lymphoid cells of group 2 (ILC2) play an important role in type 2 immune reactions, epithelial repair in mucosal tissue and metabolic homeostasis. ILC2 releases large amounts of type 2 cytokines such as interleukin 4 (IL-4), IL-5 and IL-13, which stimulate type 2 immunity, such as protection against worms. However, without strict regulation, the level of ILC2 can cause undesirable type 2 immune pathologies, including allergic inflammation of the respiratory tract, hypersensitivity of the respiratory tract and atopic dermatitis. Viral infections of the respiratory tract, which are typical triggers of type 1 immune reactions, often lead to type 2 pulmonary immune pathologies, such as asthma and its exacerbations. Interestingly, pulmonary virus infections induce the release of IL-33 with subsequent induction of ILC2-mediated type 2 pulmonary immunopathology, which is independent of the adaptive immune system.

Ketogenic diet modulates immune cell transcriptional landscape and ameliorates experimental autoimmune uveitis in mice

BackgroundUveitis manifests as immune-mediated inflammatory disorders within the eye, posing a serious threat to vision. The ketogenic diet (KD) has emerged as a promising dietary intervention, yet its impact on the immune microenvironments and role in uveitis remains unclear.MethodsUtilizing single-cell RNA sequencing (scRNA-seq) data from lymph node and retina of mice, we conduct a comprehensive investigation into the effects of KD on immune microenvironments. Flow cytometry is conducted to verify the potential mechanisms.ResultsThis study demonstrates that KD alters the composition and function of immune profiles. Specifically, KD promotes the differentiation of Treg cells and elevates its proportion in heathy mice. In response to experimental autoimmune uveitis challenges, KD alleviates the inflammatory symptoms, lowers CD4+ T cell pathogenicity, and corrects the Th17/Treg imbalance. Additionally, KD decreases the proportion of Th17 cell and increases Treg cells in the retina. Analysis of combined retinal and CDLN immune cells reveals that retinal immune cells, particularly CD4+ T cells, exhibit heightened inflammatory responses, which KD partially reverses.ConclusionsThe KD induces inhibitory structural and functional alterations in immune cells from lymph nodes to retina, suggesting its potential as a therapy for uveitis.

Lysosomal gene ATP6AP1 promotes doxorubicin resistance via up-regulating autophagic flux in breast cancer

BackgroundBreast cancer remains the most prevalent malignancy in women. Chemotherapy is the primary systemic treatment modality, and the effectiveness of treatment is often hampered by chemoresistance. Autophagy has been implicated in promoting chemoresistance, as elevated autophagic flux supports tumor cell survival under therapeutic stress. Since lysosomes are essential for the completion of autophagy, their role in autophagy-related chemoresistance has been insufficiently studied. This study aims to elucidate the role of the lysosomal gene ATP6AP1 in promoting chemoresistance in breast cancer by upregulating autophagic flux.MethodsDoxorubicin-induced cell death was assessed by cytotoxicity, flow cytometry, lactate dehydrogenase (LDH) release assays in various breast cancer cell lines. Autophagic flux was assessed with western blot and the mRFP-GFP-LC3 fluorescence imaging. Breast cancer cells were infected with shRNA lentivirus targeting ATP6AP1, allowing investigation its tole in doxorubicin-induced cell death. ATP6AP1 expression and its association with prognosis were evaluated using public databases and immunohistochemistry.ResultsDoxorubicin-induced cell death in breast cancer cells is negatively correlated with increased autophagic flux and lysosomal acidification. The lysosomal gene ATP6AP1, which plays a role in autophagic processes, is upregulated in breast cancer tissues. Knocking down ATP6AP1 reduces autophagy-mediated doxorubicin resistance by inhibiting autophagic flux and lysosomal acidification in breast cancer cells. Data analysis from public databases and our cohort indicate that elevated ATP6AP1 expression correlates with poor response to doxorubicin-based neoadjuvant chemotherapy (NAC) and worse prognosis.ConclusionsDoxorubicin-induced cytotoxicity is associated with autophagy flux in breast cancer. The lysosomal gene ATP6AP1 facilitates autolysosome acidification and contributes to doxorubicin resistance in breast cancer.

Homocysteine affects macrophage polarization by altering m6A methylation of scavenger receptors CD209 and CD163L1

ABSTRACT Atherosclerosis is a chronic inflammatory disease characterized by fatty plaque deposits on artery walls. Elevated plasma homocysteine (Hcy) levels are an independent risk factor for atherosclerosis. Research on the mechanism by which Hcy promotes atherosclerosis has gradually turned to epigenetic inheritance, but the correlation between Hcy and m6A (N6-methyladenosine) modification has not been reported. In this study, MeRIP-seq was performed on macrophages and Hcy-treated macrophages. GO and KEGG analyses were used to perform functional analysis of differentially methylated genes. qRT-PCR and western blot were taken to determine the expression of CD209, CD163L1, proinflammatory, and anti-inflammatory factors. Flow cytometry was used to detect the proportion of M2 macrophages. The results showed that after Hcy treatment, the overall m6A methylation of macrophages was down-regulated, and 856 differential methylation peaks were annotated to 781 genes. These included CD209 and CD163L1, whose m6A methylation was inhibited after treatment with Hcy. In addition, mRNA and protein expressions of CD209 and CD163L1 were also inhibited after Hcy treatment. Overexpression of CD209 or CD163L1 prevents the Hcy-induced decrease in the proportion of M2 macrophages. This article identified changes in the modification level of m6A in macrophages by Hcy and revealed the possible mechanism by which Hcy induces macrophage polarization.

Casticin inhibits the hedgehog signaling and leads to apoptosis in AML stem-like KG1a and mature KG1 cells

Abstract Objectives Acute myeloid leukemia (AML) is a severe blood cancer with less than 50 % long-term survival. Despite advancements in treatment options, relapse is still the major obstacle. The main reason of this problem is ineffective targeting of leukemic stem cells (LSCs), which play an important role in tumor development and relapse. In our previous studies, we found that casticin, the major polyphenolic component of Vitex trifolia’s fruit, targets both leukemic cells and LSCs without affecting healthy tissues. Therefore, in this study, we aimed to investigate the effect of casticin-mediated cell death in relation to the LSCs-favored survival pathways at gene and protein expression levels using in vitro LSC-like and parental leukemic cell models. Methods We validated the LSC character of KG1a and KG1 cells (84.55 % CD34+, CD38- and 93.55 % CD34+, CD38+, respectively) by flow cytometry. For the investigation of casticin’s mechanism of action, we employed real time-PCR, western blotting and bioinformatics analyses. Results Our results showed an increase in cleaved PARP/β-actin ratio but no change in LC3BI/II and SQSTM/β-actin ratios. Our gene expression, bioinformatics and immunoblotting analyses represented significant decrease in Shh, Gli and Wnt levels. We also elucidated a possible crosstalk between Hedgehog and other oncogenic cascades via the Gli, Notch, YAP, p38, Mcl-1, and Myc proteins in casticin mediated anti-leukemic effect. Conclusions In conclusion, we found that casticin induces apoptosis in both LSC-like and parental leukemia cells mainly by suppressing Shh signaling, which is crucial for LSC survival and AML relapse.

Genetic Diversity Analysis and Polyploid Induction Identification of Idesia polycarpa

Idesia polycarpa from Sichuan is a valuable germplasm with high economic potential, but it faces variety scarcity. To address this, this study collected 16 varieties (lines), identifying IpHT1 as a promising parent due to its high oil content (38.5%) and red fruits. Polyploid induction via adding 0.50% colchicine to Murashige and Skoog (MS) medium yielded 520 IpHT1 mutagenized seedlings. Subsequently, flow cytometry (FCM) was performed on 401 morphologically variant seedlings which had been initially screened, resulting in the identification of 15 suspected triploids, 35 suspected tetraploids, and 3 chimeras. Furthermore, fluorescence in situ hybridization (FISH) analysis found that the probe (AG3T3)3 had terminal signals at both ends of each chromosome, allowing for the counting of 42 chromosomes in diploids and 84 in tetraploids. The probe 5S rDNA showed 2, 3, and 4 hybridization signals in the interphase nuclei of diploid, triploid, and tetraploid cells, respectively, but the probe (GAA)6 failed to produce any signal on I. polycarpa chromosomes. Ultimately, 18 polyploids were selected, including 7 triploids and 11 tetraploids. Triploids and tetraploids showed significant leaf morphological and physiological differences from diploids. Consequently, this study successfully established a polyploid breeding system for I. polycarpa, thereby enhancing its genetic diversity and breeding potential.

Immunomodulatory Effects of Lactiplantibacillus plantarum Strain RW1 During Salmonella Infection in Murine Intestinal Epithelial Cells and Dextran Sulfate Sodium-Induced Murine Colitis.

Inflammatory diseases resulting from bacterial infections or inflammatory bowel disease pose significant threats to the health of both animals and humans. Although probiotics have emerged as a crucial preventive and adjunctive therapy for these conditions, the precise mechanisms through which probiotics regulate inflammatory diseases remain incompletely understood. In our previous study, animal-derived Lactiplantibacillus plantarum strain RW1 (L. plantarum RW1) with probiotic potential was isolated and characterized. In this study, the signaling pathway of L. plantarum RW1 inhibiting the inflammatory response of mouse intestinal epithelial cells caused by Salmonella infection was studied. Our results revealed that infection of Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC14028 (S. Typhimurium ATCC14028) and Salmonella enterica subsp. enterica serovar Typhimurium strain SL1344 (S. Typhimurium SL1344) significantly increased NF-κB/p65 and TLR4 mRNA levels while decreasing IκB and TLR2 mRNA levels. Whereas L. plantarum RW1 treatment significantly reversed these changes. Western blotting confirmed these findings. Additionally, we explored the protective effects of L. plantarum RW1 in a murine colitis model induced by dextran sulfate sodium (DSS). Treatment with L. plantarum RW1 significantly increased both intestinal length and body weight compared to DSS-treated mice. 16S rRNA sequencing analysis demonstrated that L. plantarum RW1 restored the dysbiosis caused by DSS. Flow cytometry analyses further revealed that L. plantarum RW1 specifically increased regulatory T-cell proportions in Peyer's patches while reducing macrophage and neutrophil proportions, indicating the modulatory effects of L. plantarum RW1 on immune responses in gut-associated lymphatic tissue in the context of colitis. This study sheds light on the intricate interaction between probiotics and hosts, offering valuable insights into their potential application for treating inflammatory diseases in animals and humans.

ADAMDEC1 promotes cervical squamous cell carcinoma by enhancing the JAK/STAT signaling pathway through modulation of TYMP.

Cervical squamous cell carcinoma (CSCC) poses a significant health challenge, especially in developing countries. Identifying novel prognostic biomarkers and potential drug targets is crucial for improving CSCC management. We analyzed ADAMDEC1 expression patterns in CSCC using various bioinformatic datasets. Clinical samples from CSCC patients were evaluated for ADAMDEC1 and TYMP levels through Western blot and qRT-PCR. Multiple in vitro techniques, including flow cytometry, CCK-8, Transwell, Western blot, wound healing assays, and Co-IP, were employed to investigate the role and molecular pathways associated with ADAMDEC1. Our findings reveal that ADAMDEC1 is significantly overexpressed in CSCC tissues compared to adjacent healthy tissues. Functional assays demonstrated that ADAMDEC1 overexpression significantly enhances cell proliferation, migration, and invasion in vitro, while concurrently inhibiting apoptosis. Conversely, the knockdown of ADAMDEC1 reversed these effects, leading to reduced cell proliferation and increased apoptosis. Mechanistically, we identified a direct interaction between ADAMDEC1 and TYMP, which activates the JAK/STAT signaling pathway in CSCC cells. This activation was confirmed through Western blot analysis, indicating increased phosphorylation of JAK1 and STAT3 in response to ADAMDEC1 overexpression. ADAMDEC1 promotes CSCC progression by modulating the JAK/STAT pathway through regulation of TYMP. These findings suggest that ADAMDEC1 could serve as a molecular biomarker for early diagnosis and targeted therapy in CSCC.

Consistent cell‐specific carbon fixation rates by small eukaryotic phytoplankton in contrasting nutrient‐limited conditions

AbstractSmall phytoplankton, consisting of pico and nano size fractions, are diverse in size and taxonomy. Yet, the differences in their productivity and taxonomic diversity are poorly described. Here, we measured the cell‐specific carbon fixation rates of picocyanobacteria Synechococcus, picoeukaryote, and nanoeukaryote populations, while unveiling their taxonomic composition in oligotrophic subtropical and high‐nutrient low‐chlorophyll subantarctic waters. We coupled 24 h in situ radiolabeled 14C incubations to flow cytometry sorting and DNA metabarcoding from the same incubated samples, offering a direct account of the community associated with the carbon fixation rates measured. In both water masses, nanoeukaryotes had the highest cell‐specific carbon fixation rate, followed by picoeukaryotes and Synechococcus (2.24 ± 29.99, 2.18 ± 2.08, and 0.78 ± 0.55 fgC cell−1 h−1, respectively). The cell‐specific carbon fixation rates and growth rates of Synechococcus were threefold higher in subtropical compared to subantarctic waters, while the rates of picoeukaryotes and nanoeukaryotes had no significant difference between the biogeochemically‐contrasting water masses. Sorted picoeukaryote populations were dominated by Mamiellophyceae, Pelagophyceae, Prymnesiophyceae, and Chrysophyceae, while nanoeukaryote populations were dominated by Dinophyceae and Prymnesiophyceae. Despite significant differences in their taxonomic composition, the sorted picoeukaryote populations in subantarctic waters and nanoeukaryote populations in subtropical and subantarctic waters were dominated by taxa reported in the literature as able to engage in phago‐mixotrophic strategies (Prymnesiophyceae, Chrysophyceae, and Dinophyceae), suggesting that such trophic strategy might be applied by discrete small photosynthetic eukaryote populations to alleviate macronutrient and iron stress.

Dynamic three dimensional environment for efficient and large scale generation of smooth muscle cells from hiPSCs

BackgroundChronic ischemic limb disease often leads to amputation, which remains a significant clinical problem. Smooth-muscle cells (SMCs) are crucially involved in the development and progression of many cardiovascular diseases, but studies with primary human SMCs have been limited by a lack of availability. Here, we evaluated the efficiency of two novel protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into SMCs and assessed their potency for the treatment of ischemic limb disease.MethodshiPSCs were differentiated into SMCs via a conventional two-dimensional (2D) protocol that was conducted entirely with cell monolayers, or via two protocols that consisted of an initial five-day three-dimensional (3D) spheroid phase followed by a six-day 2D monolayer phase (3D + 2D differentiation). The 3D phases were conducted in shaker flasks on an orbital shaker (the 3D + 2D shaker protocol) or in a PBS bioreactor (the 3D + 2D bioreactor protocol). Differentiation efficiency was evaluated via the expression of SMC markers (smooth-muscle actin [SMA], smooth muscle protein 22 [SM22], and Calponin-1), and the biological activity of the differentiated hiPSC-SMCs was evaluated via in-vitro assessments of migration (scratch assay), contraction in response to the treatment with a prostaglandin H2 analog (U46619), and tube formation on Geltrex, as well as in-vivo measurements of perfusion (fluorescence angiography) and vessel density in the limbs of mice that were treated with hiPSC-SMCs after experimentally induced hind-limb ischemia (HLI).ResultsBoth 3D + 2D protocols yielded > 5.6 × 107 hiPSC-SMCs/differentiation, which was ~ nine-fold more than that produced via 2D differentiation, and flow cytometry analyses confirmed that > 98% of the 3D + 2D-differentiated hiPSC-SMCs expressed SMA, > 81% expressed SM22, and > 89% expressed Calponin-1. hiPSC-SMCs obtained via the 3D + 2D shaker protocol also displayed typical SMC-like migratory, contraction, and tube-formation activity in-vitro and significantly improved measurements of perfusion, vessel density, and SMA-positive arterial density in the ischemic limb of mouse HLI model.ConclusionsOur dynamic 3D + 2D protocols produced an exceptionally high yield of hiPSC-SMCs. Transplantation of these hiPSC-SMCs results in significantly improved recovery of ischemic limb after ischemic injury in mice.

Nimotuzumab and irinotecan synergistically induce ROS-mediated apoptosis by endoplasmic reticulum stress and mitochondrial-mediated pathway in cervical cancer.

Irinotecan (CPT-11), a chemotherapeutic agent used to treat several types of cancer, induces cytotoxic effects on healthy cells. The epidermal growth factor receptor (EGFR) plays a crucial role in various forms of cancer. Nimotuzumab (NmAb), a monoclonal antibody that targets the EGFR, is utilized in some countries to treat malignancies that have an overexpression of EGFR. Yet, there is a lack of literature on the potential anticancer properties of the CPT-11 and NmAb combination on in vitro human cervical cancer cells. This study investigates the apoptosis mode of the CPT-11 and NmAb combination on cervical HeLa cancer cells. The Annexin V/propidium iodide staining examination demonstrated that the combination of CPT-11 and NmAb resulted in a decrease in the number of viable cells and more potent induction of cell apoptosis than the effects of CPT-11 or NmAb alone in HeLa cells. Furthermore, the combined treatment resulted in elevated levels of reactive oxygen species (ROS) and Ca2+ compared to the treatment with CPT-11 or NmAb alone. Cells that were pretreated with N-acetyl-l-cysteine, a substance that scavenges ROS, and then treated with CPT-11, NmAb, or a combination of CPT-11 and NmAb exhibited higher numbers of viable cells compared to those treated with CPT-11 or NmAb alone. The combination of CPT-11 and NmAb resulted in significantly higher caspase-3, -8, and -9 activity levels than CPT-11 or NmAb alone, as measured by flow cytometer assay. The combination of CPT-11 and NmAb in HeLa cells resulted in elevated endoplasmic reticulum stress-, mitochondria-, and caspase-mediated proteins compared to treatment with CPT-11 or NmAb alone. According to these observations, NmAb enhances the effectiveness of CPT-11 in fighting cancer by stimulating cell death in the HeLa cells. Therefore, NmAb has the potential to improve the efficacy of CPT-11 as a future cervical cancer treatment in humans.

Role of phosphorylated Y1252, Y1336 and Y1472 on NR2B subunits in hypoxia tolerance of neuronal cell in vitro.

The N-methyl-D-aspartate (NMDA) receptors are related to the various functioning of the nervous system. It has been shown that the NR2B subunit plays an important role in neurological hypoxic/ischemic diseases by regulating NMDA receptor function. NR2B tyrosine phosphorylation is also an important regulatory mechanism for NMDA receptor function. However, the mechanism of NR2B tyrosine phosphorylation in hypoxic/ischemic injury is still unclear. Therefore, in the present study, we aimed to further clarify the changes in NR2B tyrosine phosphorylation in hypoxic/ischemic damage in the brain and its relationship with neuronal survival under hypoxic/ischemic conditions. Four types of NR2B tyrosine site mutants (Tyr → Phe at 1252, 1336, and 1472, and all three mutations together, named Y1252F, Y1336F, Y1472F, and Triple) and wild-type plasmids were transfected into HT22 cells. The cells were then exposed to oxygen-glucose deprivation and reoxygenation (OGD/R). NR2B, cell apoptosis-related molecules, and neuronal survival factor CREB-related signaling proteins (CaMKII, ERK, Akt) were measured. Cell viability was assessed using the CCK-8 assay. Cell apoptosis and cell cycle were evaluated using flow cytometry. The death ratio of HT22 cells under OGD conditions was further tested using a live cell analysis platform. The viability of HT22 cells in the Y1252F, Y1336F, Y1472F, Triple mutants, and wild-type groups was elevated. Compared to the wild-type, western blotting and real-time PCR showed that Y1252F, Y1336F, Y1472F, and Triple mutants downregulated the expression of apoptosis factors and upregulated anti-apoptosis factors in the OGD/R model. Flow cytometry and cell cycle analysis demonstrated that Y1252F, Y1336F, Y1472F, and Triple mutants reduced the apoptosis rate. The percentage of cells in the S phase decreased significantly. Live cell analysis illustrated that the Y1252F, Y1336F, Y1472F, and Triple mutants contributed to HT22 cell survival under OGD conditions. Additionally, the Y1252F, Y1336F, Y1472F, and Triple mutants activated the survival signaling pathway. Furthermore, compared to the control group (without plasmid), only the Y1336F, Y1472F, and Triple mutants groups showed significant differences in the above tests. The tyrosine phosphorylation of NR2B at Y1336 and Y1472 plays key roles in hypoxic/ischemic injury. These phosphorylation sites may be potential targets for hypoxic/ischemic neural protection.

Unveiling the interplay between hepatocyte SATB1 and innate immunity in autoimmune hepatitis

BackgroundInvestigating the function of SATB1 in hepatocytes is essential for developing therapeutic strategies for autoimmune hepatitis (AIH). Although SATB1 has been extensively studied in immune cells, its specific activity in hepatocytes within the context of AIH remains unclear. MethodsSATB1 expression in AIH hepatocytes was assessed by qRT-PCR, Western blotting, flow cytometry, and immunohistochemistry. In vivo modulation used RNA interference viruses and overexpression plasmids. SATB1′s proinflammatory effects were analyzed with protein microarray, immunohistochemistry, and flow cytometry. Chemotactic effects on RAW264.7 macrophages were tested in vitro, with mechanisms explored by dual-luciferase assays and CUT&RUN qPCR. Liver injury was evaluated by histopathology and serum biochemistry. ResultsSATB1 was significantly upregulated in hepatocytes of AIH patients and models, showing a stronger increase in hepatocytes than in CD45+ cells, and positively correlated with liver injury severity. In vivo RNAi-mediated SATB1 inhibition reduced liver inflammation, while SATB1 overexpression aggravated AIH progression. Both interference and overexpression experiments confirmed that SATB1 promotes liver injury by facilitating the infiltration of proinflammatory (Ly6Chigh) macrophage. In vitro, supernatant from SATB1-overexpressing hepatocytes enriched chemokine signaling pathways, leading to increased CCL2 expression and release, which attracted macrophages and drove their proinflammatory polarization. Mechanistically, SATB1 promoted CCL2 transcription by binding to its DNA and recruiting p300/CBP. ConclusionsThis study reveals that SATB1 is upregulated in hepatocytes in AIH. Elevated SATB1 levels in liver cells contribute to autoimmune hepatitis by increasing CCL2 expression, promoting the recruitment of inflammatory monocyte-derived macrophage, and reshaping the composition of the liver immune microenvironment.

The Spectrum of B-cell and Plasma Cell Proliferations in Nodal T Follicular Helper Cell Lymphomas.

B-cell and plasma cell proliferations are frequently observed in nodal T follicular helper (nTfh) cell lymphomas and can present a diagnostic challenge. These proliferations can be monotypic or monoclonal and morphologically resemble lymphoma or plasmacytoma, but their clinical behavior is poorly defined. In this study, we reviewed 414 cases of nTfh lymphoma seen over the past decade at our institution. We identified 78 (19%) cases that exhibited B-cell or plasma cell proliferation detected by morphology, flow cytometry, immunohistochemistry, and/or molecular techniques. The B-cell/plasma cell proliferations occurred before (22%), concurrently with (50%), or after (28%) the diagnosis of nTfh lymphoma. We divided them into 3 categories: (1) focal or scattered B-immunoblastic proliferations recognized morphologically without a monotypic/monoclonal B-cell population (17%), (2) monotypic/monoclonal B-cell/plasma cells identified solely by flow cytometry or molecular clonality studies without morphologic confirmation (11%), and (3) unequivocal B-cell/plasma cell expansions recognized by morphologic assessment (72%). We further subdivided group 3 into proliferations associated with and possibly dependent on neoplastic Tfh cells versus those proliferations occurring in the absence of neoplastic Tfh cells and likely bona fide lymphomas. Follow-up biopsy specimens showed persistence of B-cell/plasma cell proliferations in various patient subcategories, with transformation to higher-grade B-cell proliferation or persistence without Tfh cells in some cases. In conclusion, our data support the notion that most B-cell and plasma cell proliferations associated with neoplastic Tfh clones have little impact on the clinical course of patients with nTfh lymphoma and likely do not constitute an independent B-cell lymphoma, especially those of small B cells of plasma cells. However, B-cell expansions exhibiting aggressive morphologic features may represent an independent B-cell lymphoma.

Role of the Lymphocyte Profile in Mediastinal Lymph Nodes in the Differential Diagnosis of Sarcoidosis and Tuberculous Lymphadenitis Patients Undergoing EBUS-TBNA.

The value of lymphocyte profiling (LP) in mediastinal lymph nodes for the differential diagnosis of sarcoidosis has not been extensively studied, and existing literature presents mixed results. This was a prospective study of patients with intrathoracic lymphadenopathy who underwent endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). LP in lymph node puncture fluid (LNPF) was evaluated using flow cytometry. The results of LP in sarcoidosis patients were compared with tuberculous lymphadenitis (TBLA) patients. Receiver operating characteristic (ROC) analysis was performed to determine the optimal cut-offs of the statistically significant parameters for screening for sarcoidosis. Based on the optimal cut-offs and the final diagnosis of sarcoidosis and TBLA, the sensitivity, specificity, and accuracy of every statistically significant parameter and different combinations of the above three parameters were calculated for the diagnosis of sarcoidosis. Forty-five cases of sarcoidosis and 33 cases of TBLA were enrolled in this study. Compared with the LP in TBLA patients, in sarcoidosis patients, the proportion of CD4 T cells and CD4/CD8 ratio increased, and the proportion of CD8 T cells and natural killer (NK) cells decreased. Among all single parameters, the CD4/CD8 ratio had high diagnostic sensitivity (84.4%), specificity (81.8%), and accuracy (83.3%) for sarcoidosis. Among all the combinations of three parameters, the combination of CD4, CD8, and NKT/NK ratio had high diagnostic sensitivity (91.1%), specificity (84.8%), and accuracy (87.2%) for sarcoidosis. Assessment of LP in LNPF may improve the differential diagnostic accuracy of sarcoidosis from TBLA and further strengthen the importance of LP in LNPF in the diagnostic workup of sarcoidosis.

CX43-mediated mitochondrial transfer maintains stemness of KG-1a leukemia stem cells through metabolic remodeling

BackgroundAcute myeloid leukemia (AML) is characterized by abundant immature myeloid cells, relapse and refractory due to leukemia stem cells (LSCs). Bone marrow mesenchymal stem/ stromal cells (BMSCs) supported LSCs survival, meanwhile, chemotherapy improved connexin43 (CX43) expression. CX43, as the most intercellular gap junction, facilitated transmit mitochondria from BMSCs into AML. We hypothesized that increased mitochondria transferred from BMSCs supported metabolic remodeling in LSCs to sustain their stemness.MethodsPrimary BMSCs from AML patients were isolated. CX43-BMSCs, overexpressing CX43, were cocultured with KG-1a cells. Fluorescence and confocal microscopy observed mitochondrial transfer. Flow cytometry, EdU assay, and clonogenicity evaluated cell cycle, proliferation, and clonogenic potential. Xenograft mouse models were used to evaluate the tumorigenicity of KG-1a in vivo. Seahorse, RNA-seq, and LC-MS assessed mitochondrial function, transcriptomes, and metabolites post-coculture.ResultsCX43-BMSCs promoted unidirectional mitochondrial transfer, enhancing KG-1a adhesion and proliferation to maintain LSCs stemness in vitro and vivo. RNA-seq revealed coculture with CX43-BMSCs upregulated genes related to adhesion, proliferation, and migration in KG-1a cells. Elevated CX43 expression strengthened BMSCs-KG-1a interaction, facilitating mitochondrial transfer and nucleoside metabolism, fueling KG-1a cells. This enhanced mitochondrial energy metabolism, promoting metabolic reprogramming and clonogenicity.ConclusionCX43-mediated mitochondrial transfer from BMSCs to KG-1a enhances LSCs adhesion, proliferation, clonogenicity, and metabolic reprogramming. CX43 emerges as a potential therapeutic target for AML by sustaining LSCs stemness through metabolic remodeling.

Circ-TTC17 Promotes Esophagus Squamous Cell Carcinoma Cell Growth, Metastasis, and Inhibits Autophagy-Mediated Radiosensitivity Through miR-145-5p/SIRT1 Axis.

Circular RNA (circRNA) plays a significant role in esophagus squamous cell carcinoma (ESCC) progression. Nevertheless, circ-TTC17 roles in ESCC have not fully understood. The levels of circ-TTC17, miR-145-5p and sirtuin 1 (SIRT1) were determined using qRT-PCR. ESCC cell functions were examined by CCK8 assay, flow cytometry, transwell assay and colony formation assay. The relative protein levels of autophagy marker and SIRT1 were determined by western blot (WB). The interactions among circ-TTC17, miR-145-5p, and SIRT1 were verified by dual-luciferase reporter assay and RIP assay. circ-TTC17 was overexpressed and miR-145-5p was underexpressed in ESCC. circ-TTC17 knockdown restrained ESCC cell proliferation and metastasis, while enhance apoptosis and autophagy-mediated radiosensitivity. Circ-TTC17 could sponge miR-145-5p, and its inhibitor reversed the inhibitory effect of circ-TTC17 knockdown on ESCC cell progression. Additionally, SIRT1 was targeted by miR-145-5p, and SIRT1 overexpression abolished miR-145-5p-mediated the suppressive effect on ESCC cell progression. Also, circ-TTC17 interference reduced ESCC tumor growth via miR-145-5p/SIRT1 axis. circ-TTC17 promoted ESCC cell growth, metastasis and inhibited autophagy-mediated radiosensitivity by miR-145-5p/SIRT1 axis.

Transcriptional reprogramming primes CD8+ T cells toward exhaustion in Myalgic encephalomyelitis/chronic fatigue syndrome

Myalgic encephalomyelitis/chronic fatigue syndrome (ME) is a severe, debilitating disease, with substantial evidence pointing to immune dysregulation as a key contributor to pathophysiology. To characterize the gene regulatory state underlying T cell dysregulation in ME, we performed multiomic analysis across T cell subsets by integrating single-cell RNA-seq, RNA-seq, and ATAC-seq and further analyzed CD8+ T cell subpopulations following symptom provocation. Specific subsets of CD8+ T cells, as well as certain innate T cells, displayed the most pronounced dysregulation in ME. We observed upregulation of key transcription factors associated with T cell exhaustion in CD8+ T cell effector memory subsets, as well as an altered chromatin landscape and metabolic reprogramming consistent with an exhausted immune cell state. To validate these observations, we analyzed expression of exhaustion markers using flow cytometry, detecting a higher frequency of exhaustion-associated factors. Together, these data identify T cell exhaustion as a component of ME, a finding which may provide a basis for future therapies, such as checkpoint blockade, metabolic interventions, or drugs that target chronic viral infections.