Flow Cytometry Research Articles

Initial management of patients with acquired aplastic anemia in the United States: results from a large national claims database.

Acquired aplastic anemia (AA) is an immune-mediated disorder leading to bone marrow failure characterized by pancytopenia, with infectious and bleeding complications. The disease course may be complicated by paroxysmal nocturnal hemoglobinuria (PNH), necessitating screening with flow cytometry (FC) at the time of AA diagnosis. Management strategies vary based on disease severity. Severe AA patients are usually heavily transfusion-dependent (HT-AA) and typically treated with antithymocyte globulin, calcineurin inhibitor (CNI) and eltrombopag (EPAG) as triple therapy, while allogeneic hematopoietic stem cell transplant (HSCT) is often reserved for younger patients with matched sibling donors. Moderate AA patients are less transfusion-dependent (LT-AA) and may be observed or treated with CNI or EPAG. We conducted a retrospective cohort study using Blue Cross Blue Shield Axis database, examining adult patients diagnosed with AA between 07/01/2016 and 06/30/2022. We evaluated their management within the first 6months following the diagnosis. Of 793 identified individuals (542 LT-AA, 251 HT-AA), with a median age of 49years, only 42.6% received AA-directed therapy. Triple therapy and HSCT were infrequently used for patients with HT-AA (4.4% and 18.7%, respectively), while the most common treatment was the combination of a CNI and EPAG (LT-AA 37.8%, HT-AA 51.7%). The median time from diagnosis to treatment was 22days, with older patients (age ≥ 40years) experiencing treatment initiation delays (p = 0.03). FC testing was underutilized with only 55.5% of patients undergoing evaluation. These findings highlight the need for better access to diagnostic evaluation and appropriate AA-directed therapy for patients with AA in real-world settings.

Chlorpyrifos Induces Apoptosis in Macrophages by Activating Both Intrinsic and Extrinsic Apoptotic Pathways.

Although chlorpyrifos poses considerable risks to the environment and human health, it is still used in many countries. This pesticide has various toxic effects on humans, including neurotoxicity, reproductive toxicity, genotoxicity, and organ damage caused by oxidative stress and DNA damage. However, its specific toxicity to the immune system remains unclear. In this study, we explored the intrinsic and extrinsic apoptotic pathways through which chlorpyrifos induces apoptosis in macrophages. RAW 264.7 macrophages were treated with chlorpyrifos at concentrations of 0, 2, 4, 10, and 20 ppm for 3 h. Cytotoxicity was assessed using a lactate dehydrogenase assay, whereas apoptosis was evaluated through flow cytometry. The levels of cysteinyl aspartate-specific proteinase (caspase)-3, caspase-8, and caspase-9 were measured. The disruption of mitochondrial function and the expression of the death receptors Fas receptor and tumor necrosis factor-alpha receptor were assessed through JC-1 stain reagent. The release of mitochondrial cytochrome c, expression of Bcl2 family proteins, and level of cleaved caspases were analyzed through Western blotting. Chlorpyrifos induced cytotoxicity and apoptosis in a concentration-dependent manner. It activated caspase-3, caspase-8, and caspase-9, as well as disrupted mitochondrial function and Bcl2 family protein balance. Furthermore, chlorpyrifos induced the release of cytochrome c from the mitochondria and upregulated the expression of Fas receptor and tumor necrosis factor-alpha receptor. These findings suggest that chlorpyrifos induces cytotoxicity through caspase-3-dependent apoptosis via the intrinsic pathway (caspase-8 activation, mitochondrial dysfunction, Bcl2 protein imbalance, and cytochrome c release) and the extrinsic pathway (caspase-9 activation and death receptor expression).

Histotripsy-focused ultrasound treatment abrogates tumor hypoxia responses and stimulates anti-tumor immune responses in melanoma.

Histotripsy focused ultrasound treatment gives rise to systemic anti-tumor immune responses. We investigated whether histotripsy effects on immunosuppressive tumor hypoxia were a potential mechanism for these immunostimulatory effects. Immunocompetent or CD8-deficient C57BL/6 mice with flank B16F10 or YUMM1.7 melanoma tumors underwent sham or subtotal histotripsy. Tumor growth, immune cell infiltration, and intratumoral hypoxia responses were examined using flow cytometry and fluorescence microscopy. Chemokine receptor CXCR3 and hypoxia-inducible factor-1α (HIF-1α) were intercepted with antibodies and inhibitors to assess their roles in immune responses post-histotripsy. Histotripsy-treated tumors exhibited rapid loss of intratumoral hypoxia and suppression of HIF-1α and downstream pro-survival proteins. Histotripsy was followed by intratumoral upregulation of the CXCR3 ligand CXCL10, and CXCR3+/CD8+ T cell infiltration. Tumor growth inhibition by histotripsy was significantly diminished in CD8-deficient mice and mice receiving anti-CXCR3 mAb. Post-histotripsy inhibition of hypoxia and tumor growth eventually receded in parallel with cessation of CD8+ T cell influx, and pharmacological HIF-1α suppression with the MEK inhibitor Trametinib substantially augmented the therapeutic effects of histotripsy. Transient abrogation of intratumoral hypoxia and HIF-1α-associated hypoxia responses is mechanistically linked with intratumoral infiltration of activated CXCR3+/CD8+ T cells via CXCL10:CXCR3 engagement. These findings suggest that the immune effects of histotripsy may be regulated by hypoxia abrogation, and that pharmacological hypoxia abrogation could potentiate the immunotherapeutic effects of histotripsy.

Fibroblast IRF7-mediated chondrocyte apoptosis affects the progression of collapse in steroid-induced osteonecrosis of the femoral head

PurposeThe objective of this study was to identify potential genes implicated in the “peri-collapse” synovium of osteonecrosis of the femoral head through coding gene sequencing and to further clarify their specific mechanisms via in vitro experiments.MethodsSteroid-induced osteonecrosis of the femoral head (SIONFH) (n = 3), femoral neck fracture (FNF) (n = 3), and hip osteoarthritis (HOA) (n = 3) Synovial tissue of the hip joint was collected in total hip arthroplasty. A cellular model of SIONFH constructed from rat synovial fibroblasts by lipopolysaccharide intervention. Lentiviral technology was used to construct a model for fibroblast knockout of the Irf7 gene. HE was used to compare the characteristics of synovial tissue damage, and immunofluorescence and immunohistochemistry were used to compare the expression levels of VIM, IRF7, and IFNα. PCR, WB, and IF were used to examine Irf7 knockdown efficiency, chondrocyte proliferation (Col2a1, Aggrecan, Sox9), cartilage matrix degradation (Mmp13), and apoptosis (Bcl2, Bax, and Caspase3) expression under co-culture conditions. Crystalline violet staining was used to observe the migration rate of fibroblasts, and flow cytometry was used to detect the apoptosis level of chondrocytes under co-culture conditions.ResultsTranscriptome sequencing of synovial tissue and fibroblasts ultimately screened for six differential genes, HOOK1, RNPC3, KCNA3, CD48, IRF7, SAMD9. Compared to FNF and HOA, synovial inflammatory cell recruitment and synovial hyperplasia were more pronounced in SIONFH. IF and IHC confirmed high expression of IRF7 and IFNα in the synovium of SIONFH. PCR and WB results suggested that fibroblasts highly expressed Irf7, Hook1, Rnpc3, Kcna3, Cd48, Samd9, Il-6, and Tnfα after lipopolysaccharide intervention, and the expression levels of Il-6 and Tnfα were significantly reduced after knockdown of Irf7 (P < 0.001). In the co-culture system, fibroblasts intervened with lipopolysaccharide significantly promoted chondrocyte apoptosis, the rate of cartilage matrix degradation, while inhibiting the level of chondrocyte proliferation, and this result was significantly reversed in Irf7 knockout fibroblasts. This was supported by flow cytometry results.ConclusionsIRF7, HOOK1, RNPC3, KCNA3, CD48, and SAMD9 as potential genes affecting the progression of SIONFH collapse. Irf7 mediates the fibroblast inflammatory response and affects the collapse process of SIONFH by influencing chondrocyte apoptosis. Thus, intervention in IRF7 holds promise as one of the key targets for reversing the collapse process of SIONFH.Graphical

Icariin promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by activating PI3K-AKT-UTX/EZH2 signaling in steroid-induced femoral head osteonecrosis

BackgroundDifferentiation of bone marrow mesenchymal stem cells (BMSCs) is pivotal in the pathogenesis of steroid-induced femoral head osteonecrosis. Icariin, an active ingredient in Epimedii herba, has the potential to regulate osteogenic differentiation of BMSCs. Nevertheless, the related mechanism is still unclear. The study aimed to explore whether icariin can affect osteogenic differentiation by activating PI3K/AKT signaling to alter UTX and EZH2 expression and thus regulating osteogenesis-related genes in BMSCs.MethodsBMSCs were collected from Sprague Dawley rats and identified by measuring the positive ratios of cell markers using flow cytometry. Cells were treated with 1 μmol/L dexamethasone (DEX) for 24 h with or without 0.1–10 μM of icariin treatment. Cell counting Kit-8 (CCK-8) assays and flow cytometry analyses were performed to measure cell viability and apoptosis. Western blotting was conducted for measurement of apoptotic markers, factors involved in the PI3K/AKT-UTX/EZH2 pathway, osteogenic markers, and adipogenesis-related factors. Alizarin red S staining and Oil-red O staining were performed to measure the effect of DEX, icariin, UTX overexpression, or EZH2 knockdown on osteogenic and adipogenic differentiation of BMSCs.ResultsIcariin ameliorated DEX-induced rat BMSC injury. Icariin activated the PI3K/AKT signaling, thereby upregulating UTX and phosphorylated EZH2 levels while inhibiting EZH2 and H3K27me3 expression. Additionally, icariin promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. Importantly, overexpressing UTX or silencing EZH2 exerted similar effects on BMSC differentiation as icariin did.ConclusionsIcariin promotes osteogenic differentiation of DEX-treated BMSCs by activating PI3K/AKT signaling to upregulate UTX and inhibit EZH2, finally inducing H3K27me3 depletion.

CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

Diffuse large B-cell lymphoma (DLBCL) is the most prevalent form of lymphoma. The overexpression of CtBP2 in tissues may contribute to tumor occurrence and progression. The expression of EGR1 in DLBCL is elevated, suggesting its potential role as an oncogene that promotes the proliferation of DLBCL cells. Database predictions indicate that CtBP2 can bind to EGR1. The objective of the present study was to investigate whether CtBP2 can influence the proliferation and apoptosis of DLBCL cells by regulating the Wnt signaling pathway through EGR1. Western blot assay showed that CtBP2 expression was upregulated in DLBCL cells. Cell proliferation level was detected by CCK8 assay and EdU staining, and the apoptosis level and cycle distribution were analyzed through flow cytometry. Our data indicated that interference with CtBP2 and EGR1 can inhibit the proliferation and cell cycle progression of DLBCL cells while promoting apoptosis. The predictions from the HDOCK server, along with the results of Co-IP experiments, suggested that EGR1 and CtBP2 can effectively bind to each other, with EGR1 positioned downstream of CtBP2 and regulated by it. Furthermore, interference with CtBP2 could also inhibit the expression of the Wnt/β-catenin signaling pathway. Overexpression of EGR1 counteracted the effects of siRNA-CtBP2, promoting cell proliferation and cycle, inhibiting apoptosis and upregulating the expression of the Wnt/β-catenin signaling pathway. From the above experiments, we found that CtBP2 can regulate the Wnt/β-catenin signaling pathway through EGR1 to influence the proliferation and apoptosis of DLBCL cells. Therefore, EGR1 may be one of the key contributors involved in the regulation of Wnt/β-catenin signaling by CtBP2.

Stromal Cell Subsets Show Model-Dependent Changes in Experimental Colitis and Affect Epithelial Tissue Repair and Immune Cell Activation.

Previous work on inflammatory bowel disease (IBD) revealed changes in the abundance of colonic stromal subsets during intestinal inflammation. However, it is currently unknown whether these stromal cell subset changes are also reflected in different IBD mouse models and how commonly used IBD therapies affect stromal cell subset composition. Stromal subset markers CD55, C-X-C motif chemokine 12 (CXCL12), podoplanin (PDPN), CD90, and CD73 were analyzed by flow cytometry in 3 mouse models for IBD, namely interleukin (IL)-10 knockout (KO), dextran sulfate sodium-induced, and T-cell transfer model for colitis. Next, the effects of IBD therapies on the stromal subset composition were studied. In vitro experiments were performed to study the interaction between stromal cell subsets and epithelial/immune cells. The colitis-induced changes in the abundance of stromal cell subsets differed considerably between the 3 colitis mouse models. Interestingly, treatment with IBD medication affected specific stromal subsets in a therapy and model-specific manner. In vitro experiments showed that specific stromal subsets affected epithelial wound healing and/or T-cell activation. The relative abundance changes of stromal cell subsets during experimental colitis differ between 3 established colitis models. Treatment with IBD therapies influences stromal subset abundance, indicating their importance in IBD pathogenesis, possibly through affecting epithelial migration, and T-cell activation.

Evaluation of D-Dimer, P-Selectin, and miR-17-5p Expression in ICU and Non-ICU COVID-19 Patients: A Cross-sectional Study.

COVID-19, caused by SARS-CoV-2 infection, frequently induces thrombotic complications in affected individuals. P-selectin, a pivotal platelet marker, plays a central role in platelet-leukocyte aggregation, contributing to hemostasis and thrombosis. Additionally, D-dimer serves as an indicator of coagulation system activity, while miR-17-5p exhibits antiviral properties in respiratory infections. This study aimed to evaluate and compare the expression levels of D-dimer, P-selectin, and miR-17-5p in COVID-19 patients hospitalized in intensive care units (ICUs) and those in non-ICU wards. This cross-sectional study included 50 COVID-19 patients, divided into ICU and non-ICU groups. P-selectin expression was assessed using Flow cytometry, D-dimer levels were measured via chemiluminescence, and miR-17-5p expression was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Our analysis revealed no significant difference in P-selectin expression levels between ICU and non-ICU patients (p = 0.1068). However, the expression levels of D-dimer and miR-17-5p were significantly elevated in ICU patients compared to non-ICU patients, with corresponding p-values of 0.032 and 0.0176, respectively. The heightened expression of D-dimer and miR-17-5p in ICU patients suggests their potential utility as predictive biomarkers for assessing the hemostatic status of COVID-19 patients.

Establishment of CD34 + hematopoietic stem cell-derived xenograft model of hyperleukocytic acute myeloid leukemia

BackgroundHyperleukocytic acute myeloid leukemia (HLL) is marked by high early mortality and presents significant therapeutic challenges. Research on HLL is still in its infancy, and comprehensive development of patient-derived xenograft (PDX) models, especially CD34 + hematopoietic stem cell-derived models, remains limited.MethodsWe evaluated the establishment of the HLL model through blood examinations, smear analysis, bone marrow biopsy, flow cytometry, and mutation analysis. Correlation between survival times in mice and patients was assessed using linear regression.ResultsIn the HLL PDX mouse model, leukocyte counts could reach up to 37.35^10⁹/L, and immunophenotyping revealed the presence of hCD45+, hCD15+, and hCD33 + cells in both peripheral blood (PB) and bone marrow (BM) following inoculation with PB-derived cells for the establishment of the HLL PDX model. Similar results were observed with cells derived from the patient’s BM. In the CD34 + hematopoietic stem cell-derived xenograft model, extensive infiltration of CD34 + cells into the BM, liver, and spleen was observed. Additionally, human WT1 and NRAS mutations were identified in the liver, spleen, and BM of the mice. A comparative analysis of multiple experiments revealed that shorter survival times were observed in mice receiving a higher irradiation dose of 2.5 Gy and a greater number of cells derived from PB. Additionally, shorter survival times were observed in model mice injected with cells carrying NRAS, DNMT3A, FLT3, or NPM1 gene mutations. Correlation analysis indicated that the survival times of the mice were significantly associated with the survival status of the patients.ConclusionsWe successfully established a CD34 + hematopoietic stem cell-derived xenograft model of HLL, providing a valuable tool for mechanistic research, drug screening, individualized therapy, and precision medicine.Trial registrationNot application.

Salivary apoptotic microvesicles as biomarkers for prognostic non-healing oral ulcers and oral cancer: a cross-sectional study

Microvesicles (MVs) are membrane vesicles secreted by cells and are present in the saliva of healthy individuals. It has various functions and has been reported to be a biomarker for malignant tumors. The changes in saliva levels of MVs associated with disease(s) is unclear. This study aimed to determine the proportion of salivary apoptotic MVs and their association with oral ulcer(s) in patients with non-healing oral ulcer(s) and reported oral cancer. Saliva (5 mL) was collected from patients with non-healing oral ulcer(s) and reported oral cancer (at the time of saliva collection, the participant have an oral ulcer(s) in the oral cavity and have an oral cancer lesion; n = 73) and healthy volunteers with oral ulcer(s) (n = 62). A standard differential centrifugation protocol was used for the purification of MVs. Dynamic light scattering and transmission electron microscopy were used to characterize MVs. Flow cytometry was used to quantify salivary apoptotic MVs. Immunocytochemistry was performed according to a standard protocol. None of patients with oral cancer has smoking and drinking habit. The majority of saliva samples derived from patients with non-healing oral ulcer(s) and reported oral cancer were more positive for the fluorescent dye carboxyfluorescein succinimidyl ester than those of healthy volunteers with oral ulcer(s). Salivary fluid obtained from patients had membrane-limited vesicles that were round and/or slightly elongated in shape, with diameters of 100–1,000 nm. The number of salivary apoptotic MVs was higher in patients with non-healing oral ulcer(s) than in those derived from healthy volunteers with oral ulcer(s) (p < 0.001). There was an association between salivary apoptotic MVs in patients with non-healing oral ulcer(s) and the degree or severity of oral ulcers (p < 0.001). Levels of salivary apoptotic MVs are elevated in patients with non-healing oral ulcer(s) and confirmed oral cancer. Elevated levels of salivary apoptotic MVs are associated with clinicopathological data of patients with oral cancer. Evidence level: IV. Technical efficacy: stage 3.

Immune modulation in solid tumors: a phase 1b study of RO6870810 (BET inhibitor) and atezolizumab (PD-L1 inhibitor)

PurposeBromodomain and extra-terminal domain (BET) inhibitors (BETi) have demonstrated epigenetic modulation capabilities, specifically in transcriptional repression of oncogenic pathways. Preclinical assays suggest that BETi potentially attenuates the PD1/PD-L1 immune checkpoint axis, supporting its combination with immunomodulatory agents.Patients and methodsA Phase 1b clinical trial was conducted to elucidate the pharmacokinetic and pharmacodynamic profiles of the BET inhibitor RO6870810 as monotherapy and in combination with the PD-L1 antagonist atezolizumab in patients with advanced ovarian carcinomas and triple-negative breast cancer (TNBC). Endpoints included maximum tolerated dosages, adverse event profiling, pharmacokinetic evaluations, and antitumor activity. Pharmacodynamic and immunomodulatory effects were assessed in tumor tissue (by immunohistochemistry and RNA-seq) and in peripheral blood (by flow cytometry and cytokine analysis).ResultsThe study was terminated prematurely due to a pronounced incidence of immune-related adverse effects in patients receiving combination of RO6870810 and atezolizumab. Antitumor activity was limited to 2 patients (5.6%) showing partial response. Although target engagement was confirmed by established BETi pharmacodynamic markers in both blood and tumor samples, BETi failed to markedly decrease tumor PD-L1 expression and had a suppressive effect on antitumor immunity. Immune effector activation in tumor tissue was solely observed with the atezolizumab combination, aligning with this checkpoint inhibitor’s recognized biological effects.ConclusionsThe combination of BET inhibitor RO6870810 with the checkpoint inhibitor atezolizumab presents an unfavorable risk-benefit profile for ovarian cancer and TNBC (triple-negative breast cancer) patients due to the increased risk of augmented or exaggerated immune reactions, without evidence for synergistic antitumor effects.Trial registrationClinicalTrials.gov ID NCT03292172; Registration Date: 2017-09-25.

Oroxylin A reverses SHP-2 oxidative inactivation in GPVI signaling to suppress platelet activation and thrombus formation

BackgroundArterial thrombotic events are the leading causes of death worldwide, and the therapeutic effects of current antiplatelet drugs are not fully satisfactory. Oroxylin A (OroA), a flavone compound extracted from Scutellaria radix, possesses cardioprotective and many other pharmacological effects. While platelets play a crucial role in the development of myocardial infarction, the direct effects of OroA on platelet activation and thrombosis have yet to be investigated.MethodsFeCl₃-induced arteriole thrombosis and whole-blood perfusion were used to assess the inhibitory effect of OroA on thrombus formation. A myocardial ischemia model was employed to evaluate the protective effect of OroA on myocardial injury. Multiple platelet function studies including platelet aggregation, platelet spreading, clot retraction were performed. Network pharmacology, flow cytometry, enzyme-linked immunosorbent assay, co-immunoprecipitation and western blot were utilized to explore the mechanism of OroA on platelet activation.ResultsOroA inhibited thrombus formation with less bleeding risk compared with aspirin. OroA protected against myocardial injury by suppressing microvascular thrombosis and platelet infiltration. OroA suppressed different agonist-induced platelet activation in a concentration-dependent manner, showing greater antiplatelet activity against collagen-induced platelet aggregation compared to ADP or thrombin-induced aggregation. OroA decreased granule release, integrin αIIbβ3 activation, platelet spreading and clot retraction. As a flavone, OroA boosted superoxide dismutase (SOD) and glutathione (GSH) activities and decreased malondialdehyde (MDA), oxidized glutathione (GSSG) and ROS levels in platelets during oxidative stress. OroA binds to SHP-2 and prevents its oxidative inactivation, leading to the tyrosine dephosphorylation of Src, Syk and PLCγ2, as well as the reduction of Ca2+ influx and PKC phosphorylation in GPVI signaling.ConclusionsOroA inhibits platelet activation, thrombus formation and myocardial injury via reversing SHP-2 oxidative inactivation thereby attenuating collagen-induced GPVI signaling. With minor bleeding risk and no obvious pharmacological toxicity, OroA holds promising therapeutic potential as an antithrombotic drug.

Osteosarcoma exosome priming of primary human lung fibroblasts induces an immune modulatory and pro-tumorigenic phenotype.

Osteosarcoma (OS) is the most common malignant bone cancer. 30-40% of all OS patients develop tumor recurrence, almost exclusively in the form of lung metastasis, which is associated with a dismal 20% 5-year survival rate. Cancer-associated fibroblasts are a critical cell type within the lung TME, which promote immune suppression, drug resistance, and tumor cell survival. Prior work shows tumor cells can co-opt fibroblasts to a pro-tumorigenic phenotype via exosome mediated intercellular communication. Currently, the mechanisms by which OS exosomes modulate resident lung fibroblast function has not been evaluated. To investigate this, we isolated exosomes from a panel of 6 OS cell lines. We assessed the uptake and response of human donor-derived primary lung fibroblasts (LF's; n=4) to OS exosome treatment in vitro via flow cytometry, confocal fluorescent microscopy, proliferation assays, phospho-kinase array, multiplex cytokine analysis and RNA-sequencing. We observed that LFs efficiently take up OS exosomes, which is associated with induction of MAPK pathway activation, fibroblast proliferation and significantly enhanced secretion of IL-6, CXCL8 and CCL2 compared to untreated LFs. RNA-seq of exosome treated LFs confirmed these responses and revealed significant enrichment of pathways related to cytokine secretion, proliferation, immune cell chemotaxis, migration, proinflammatory and profibrotic mediators. Finally, in an exosome-educated lung fibroblast-OS co-culture model, exosome educated LFs conferred significantly increased OS cell survival and proliferation as compared to untreated fibroblasts. Together, these data suggest OS-derived exosomes can induce a hallmark, CAF-like inflammatory phenotype in lung fibroblasts providing valuable insights into mechanisms that may promote recurrent OS lung metastasis.

Cannabinoid Receptor-2 Alleviates Sepsis-Induced Neuroinflammation by Modulating Microglia M1/M2 Subset Polarization Through Inhibiting Nogo-B Expression.

Few studies have investigated how Nogo-B affects sepsis-associated encephalopathy (SAE). Cannabinoid receptor 2 (CB2R) plays a critical role in regulating M1/M2 polarization in microglia. This study aimed to explore the association between CB2R and Nogo-B by assessing changes in microglial polarization markers.C57BL/6 mice with SAE induced by cecal ligation and puncture (CLP) surgery were intraperitoneally injected with HU308 for 3 consecutive days at the same time after that, and changes in cognitive function were assessed. After Lipopolysaccharides (LPS) and Interleukin-4 (IL-4) were used to induce BV2 microglial cell models respectively, HU308 and AM630 were applied to assess changes in inflammatory factors, microglial polarization markers, and the expression levels of CB2R and Nogo-B in microglial cells. We established a stable Nogo-B overexpression cell line. ELISA, Western blot, and flow cytometry were utilized to verify whether Nogo-B is a crucial protein in controlling BV2 cell polarization by HU308. There was an increase in Nogo-B protein expression during SAE. HU308 treatment alleviated the cognitive impairment of the CLP mice and markedly decreased the level of Nogo-B in the hippocampus tissues. The efficacy of CB2R activation to promote microglia polarization from M1 to M2 was diminished in BV2 cells overexpressing Nogo-B, although its anti-inflammatory effect was not entirely reversed. Inhibiting the Nogo-B expression, which in turn encourages the conversion of BV2 microglia to M2, attenuates inflammatory responses, and promotes neuronal repair, could be a key mechanism whereby activation of CB2R ameliorates septic encephalopathy.

Characterization, antifungal activity and possible action mechanism of Melissa officinalis essential oil against Candida spp. and Cryptococcus neoformans strains.

This study aimed to characterize Melissa officinalis essential oil (EOMO) from a region in Northeast Brazil and evaluate its antifungal activity against Candida and Cryptococcus neoformans, by analyzing its action mechanism. EOMO was characterized using gas chromatography-mass spectrometry and the main chemical compounds were Geranial (14.10%) and (Z)-Nerolidol (17.75%). The broth microdilution assay was used to determine the minimum inhibitory concentration (MIC) against strains of Candida albicans, Candida parapsilosis, Candida krusei, Candida auris (MIC raging from 256 to 26.7 µg ml-1) and C. neoformans (MIC ranging from 64 to 32 µg ml-1). Flow cytometry and comet assays were employed to investigate EOMO's mechanism of action, which might be related to an increase in the production of reactive oxygen species and damage to fungal DNA. The chemical composition of EOMO from Northeast Brazil showed a higher content of (Z)-Nerolidol and has significant antifungal potential.

Cervical cancer patients with and without HIV infection have unique T cell activation profiles despite similar survival outcomes after chemoradiation.

The global burden of cervical cancer is highest in low- and middle-income countries (LMICs). Women living with HIV infection are particularly impacted by cervical cancer despite availability and adherence to antiretroviral therapy (ART). Immune profile correlates of survival and treatment response have not been widely explored in patients with and without HIV infection. This study recruited women with cervical cancer undergoing definitive chemoradiation (CRT) in Botswana. Clinical characteristics and blood samples were collected. Flow cytometry was performed on samples prior to initiation (Initial), at completion (EOT), and three months after (M3) CRT. Logistic regression analysis identified immune markers that differed by HIV status and correlated with overall survival (OS). The study enrolled 131 consecutive women (HIV+ N=89, HIV- N=42). From Initial to M3, a significant decrease in CD4 frequency (72% to 60.55%, p<0.001) and increase in CD8 frequency (20.9% to 31.5%, p<0.001) was seen in women without HIV whereas no significant changes in CD4 frequency (52.5% to 50.9%) or CD8 frequency (39.9% to 41.4%) were observed in those with HIV. Peripheral T cells underwent similar activation across the cohort regardless of HIV status. Improved OS was associated with reduced frequency of IL-2 expressing CD4 T cell subsets. In women living with HIV, enhanced OS was associated with the presence of proinflammatory CD8 T cells. CRT induces peripheral T cell activation and distinct cytokine profiles that differ by HIV status. Despite similar OS, HIV infection may differentially impact immune response to CRT in women with well-managed disease.

A spiral channel with integrated microelectrodes for label-free particle lateral position and size characterization.

Modified-trident shaped microelectrodes were incorporated into a spiral-shaped microfluidic focusing channel, utilizing impedance flow cytometry to analyze and quantify inertial microfluidic-based separation of homogeneous particles differing in size. Double peak voltage pulses were generated as particles moved across the electrodes, where the ratio of the peak amplitudes indicated the lateral particle positions inside the channel at various flow rates, while the peak amplitude indicated particle size and vertical position. The root mean square error between the optical and electrical position measurements was 11.44µm reflecting the lateral position measurement resolution. The peak amplitudes were used to estimate particle size after being adjusted to account for particle vertical position using a shape parameter, which effectively reduced errors in particle size calculations. The particle size estimate sensitivity was measured to be 2.15μm/mV from the peak amplitudes. The electrodes with the appropriate signal processing were able to detect both the size and location of particles after separation with a spiral channel, showing their utility in potentially controlling the separation conditions for these devices.

Treatment of Acute Liver Injury through Selective Tropism of High Mobility Group Box 1 Gene-Silenced Large Peritoneal Macrophages.

Tissue-resident macrophages (TRMs) are attractive cells to therapeutically deliver oligonucleotide and other gene-expression modifying modalities to treat a wide array of diseases ranging from inflammatory to autoimmune, and even cancer. Here, we focus on TRMs located inside the peritoneal cavity lining the abdomen that selectively express a transcription factor GATA6 called large peritoneal macrophages (GLPMs) and successfully demonstrate functional GLPM-selective delivery of a Cy5-fluorophore-labeled siRNA encapsulated in C12-200 cationic-lipidoid-based nanoparticles (siRNA-Cy5 (C12-200)). Despite being TRMs, GLPMs possess a specific migratory ability to peritoneally located liver tissue upon injury incited by acetaminophen (APAP) overdose in mice. A rapid, liver injury-driven tropism of GLPMs carrying siRNA-Cy5 (C12-200) was seen via systemic circulation, which was elegantly demonstrated by using a noninvasive live-cell tracking technique called diffuse in vivo flow cytometry (DiFC). Finally, RNAi-mediated silencing of a well-known pro-inflammatory damage-associated molecular pattern (DAMP) High Mobility Group Box-1 (HMGB1) gene in GLPMs led to the mitigation of liver injury and inflammation via prevention of GLPM modulation to a pro-inflammatory state, which further translated into significant protection from APAP-driven liver injury and a reduction in liver circulating pro-inflammatory cytokines owing to a muted inflammatory response to acute liver injury. Moreover, silencing HMGB1 by a GalNAc-conjugated hepatocyte-targeting siRNA did not reciprocate the findings, further solidifying our results. Together, our data suggested that GLPMs act as delivery carriers by rapidly bringing lipid nanoparticle-encapsulated RNAi modalities to the injured liver and have emerged as a therapeutically viable strategy to address inflammatory diseases, especially those that are more acute in nature.

CAR-NK cell therapy combined with checkpoint inhibition induces an NKT cell response in glioblastoma.

Glioblastoma is the most aggressive primary brain tumor with limited efficacy of established therapies, and a pronounced immunosuppressive tumor microenvironment. Targeting HER2 with local immunotherapy allows for high tumor specificity in the brain with physiologically very low expression. Monotherapy with CAR-NK cells targeted against HER2 has previously shown efficacy in medium-sized GL261/HER2 tumors. Advanced GL261/HER2 tumors were treated by local CAR-NK cell injection combined with systemic anti-PD-1 checkpoint blockade. Tumor growth and survival were monitored. In-depth characterization of the microenvironment was performed by multiplex immune fluorescence, spectral flow cytometry and RNAseq. Untreated GL261/HER2 tumors were characterized by local immunosuppression and high PD-L1 expression. Combined treatment with NK-92/5.28.z and systemic anti-PD-1 induced robust anti-tumor response and long-term survival. Multiplex immunofluorescence and spectral flow cytometry showed increased CD4+ T cell infiltration in mice treated with CAR-NK cell and anti-PD-1 combination therapy. A cluster of T cells specifically emerging in the combination therapy group expressed markers of NKT cells, which was further verified by immunofluorescence staining. The combination therapy reverted the immunosuppressive tumor microenvironment with increased T and NKT cell infiltration. This resulted in successful treatment of advanced orthotopic tumors refractory to CAR-NK cell monotherapy.

Design, Construction, and Validation of a Yeast-Displayed Chemically Expanded Antibody Library.

In vitro display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library, called the "Clickable CDR-H3 Library", that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photoreactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing O-(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multimodal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.