Carboxyfluorescein Diacetate Succinimidyl Ester Research Articles

The Effect and Mechanism of Astragalus Polysaccharides on T Cells and Macrophages in Inhibiting Prostate Cancer

BackgroundThe impact and underlying mechanisms of astragalus polysaccharide (APS) on prostate cancer, particularly its role in immunomodulation, remain inadequately elucidated. MethodsThis study employed the XTT assay for assessing proliferation in prostate cancer cells and macrophages. T cell proliferation was determined using the Carboxyfluorescein diacetate succinimidyl ester labeling assay. APS's effect on T cells and macrophages was scrutinized via flow cytometry, Western blot analysis, ELISA, quantitative PCR and cytokine membrane arrays. The effect of APS on interaction between PD-L1 and PD-1 was investigated by the PD-L1/PD-1 homogeneous assay. Additionally, the impact of conditioned medium from T cells and macrophages on PC-3 cell migration was explored through migration assays. ResultsIt was observed that APS at concentrations of 1 and 5 mg/mL enhanced the proliferation of CD8+ T cells. At a concentration of 5 mg/mL, APS activated both CD4+ and CD8+ T cells, attenuated PD-L1 expression in prostate cancer cells stimulated with interferon gamma (IFN-γ) or oxaliplatin, and moderately decreased the population of PD-1+ CD4+ and PD-1+ CD8+ T cells. Furthermore, APS at this concentration impeded the interaction between PD-L1 and PD-1, inhibited the promotion of prostate cancer migration mediated by RAW 264.7 cells, THP-1 cells, CD4+ T cells, and CD8+ T cells, and initiated apoptosis in prostate cancer cells treated with conditioned medium from APS (5 mg/mL)-treated CD8+ T cells, RAW 264.7 cells, or THP-1 cells. ConclusionThe findings indicate a potential role of 5 mg/mL APS in modulating the PD-1/PD-L1 pathway and influencing the immune response, encompassing T cells and macrophages. Consequently, further in vivo research is recommended to assess the efficacy of APS.

A Flexible Mouse Model of Autoimmune Thyroiditis Induced by Immunization with an Adenovirus Containing Full-Length Thyroglobulin cDNA.

The main challenge in the "post-GWAS" era is to determine the functional meaning of genetic variants and their contribution to disease pathogenesis. Development of suitable mouse models is critical because disease susceptibility is triggered by complex interactions between genetic, epigenetic, and environmental factors that cannot be modeled by in vitro models. Thyroglobulin (TG) is a key gene for autoimmune thyroid disease (AITD) and several single nucleotide polymorphisms (SNPs) in the TG coding region have been associated with AITD. The classical model of experimental autoimmune thyroiditis (EAT), based on immunization of genetically susceptible mouse strains with purified TG protein in adjuvant, does not allow testing the impact of TG sequence variants on the development of autoimmune thyroiditis. Here we describe a protocol for the induction of EAT by immunization of mice susceptible to thyroiditis with an adenovirus vector carrying full-length human TG cDNA (Ad-TG EAT). We also provide support protocols for evaluation of autoimmune thyroiditis including serological assessment of TG antibodies, in vitro splenocyte proliferation assay and cytokines secretion, thyroid histology, and evaluation of thyroid lymphocytic infiltration by immunostaining. This protocol for EAT induction allows manipulation of the TG cDNA to introduce variants associated with AITD, enabling the testing of the functional effects of susceptible variants and their haplotypes on the immunogenicity of TG. Furthermore, the Ad-TG EAT mouse model is a valuable model for studying the interactions of the TG variants with non-genetic factors influencing AITD development (e.g., cytokines, iodine exposure) or with variants of other susceptible genes (e.g., HLA-DRβ1). © 2024 Wiley Periodicals LLC. Basic Protocol: Development of a mouse model of autoimmune thyroiditis induced by immunization with adenovirus containing full-length thyroglobulin cDNA Support Protocol 1: Splenocytes isolation Support Protocol 2: T cell stimulation and carboxyfluorescein diacetate succinimidyl ester (CFSE) based cell proliferation assay Support Protocol 3: Cytokine assays: measuring levels of interferon gamma (IFNγ) and interleukins IL-2, IL-4, and IL-10 in splenocyte supernatants Support Protocol 4: Evaluating thyroid histology and infiltration with immune cells: hematoxylin-eosin staining of mice thyroid glands Support Protocol 5: Immunohistochemistry of thyroid tissues: Immunofluorescence protocol of paraffin-embedded thyroid sections Support Protocol 6: Anti-thyroglobulin antibody measurement in mice sera by enzyme-linked immunosorbent assay (ELISA).

The dynamics of bacterial proliferation, viability, and extracellular polymeric substances in oral biofilm development

ObjectivesThis study investigated the relationship between bacterial growth, viability, and extracellular polymeric substances (EPS) formation in biofilms, particularly regarding resistance development. It also examined the impact of chemical factors on the EPS matrix and bacterial proliferation in oral biofilms. MethodsThree multi-species oral biofilms were incubated in anaerobic conditions. Three strains of Enterococcus faecalis were incubated in aerobic conditions. The incubation periods ranged from 0 h to 7 days for short-term biofilms, and from 3 to 90 days for long-term biofilms. Fluorescent labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) and flow cytometry were used to track EPS and bacterial growth. Confocal laser scanning microscopy (CLSM) assessed bacterial viability and EPS structure. Biofilms aged 7, 14, and 21 days were treated with 2 % chlorhexidine (CHX) and 1 % sodium hypochlorite (NaOCl) to evaluate their effects on EPS and bacterial proliferation. ResultsShort-term biofilms showed rapid bacterial proliferation and a gradual increase in EPS, maintaining stable viability. In the first two weeks, a significant rise in CFSE indicated growing maturity. From 14 to 90 days, EPS and CFSE levels stabilized. Following treatment, CHX significantly reduced bacterial proliferation, while NaOCl decreased EPS volume. ConclusionsBiofilm development involves a balance between bacterial proliferation and EPS production. The complexity of this process poses challenges in treating biofilm-associated infections, requiring strategies tailored to the biofilm's developmental stage. Clinical significanceFor effective root canal treatment, it is imperative to focus on reducing bacterial proliferation during the early stages of oral infections. In contrast, strategies aimed at minimizing EPS production could be more beneficial for long-term management of these conditions.

Probing cell proliferation: Considerations for dye selection.

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.

Anti-Inflammatory Effects of Alphitolic Acid Isolated from Agrimonia coreana Nakai Extracts Are Mediated via the Inhibition of ICRAC Activity in T Cells.

Agrimonia pilosa Ledeb., an important medicinal herb in traditional East Asian medicine, is primarily used to treat abdominal pain, dysentery, and hemostasis. There are ten other reported species of Agrimonia plants, including Agrimonia coreana Nakai-a naturally growing species in South Korea-and Agrimonia eupatoria Linn. Although recent studies have isolated numerous active constituents and investigated their effects, the medicinal utility of this herb is not yet fully explored. Through patch-clamp recording, a previous study reported that Agrimonia plant extracts inhibit the function of Ca2+ release-activated Ca2+ channels (CRACs). Herein, we aimed to identify and isolate the main compounds in A. coreana responsible for CRAC inhibition while assessing the anti-inflammatory effects mediated by this inhibition. We demonstrated for the first time that alphitolic acid isolated from A. coreana has a dose-dependent inhibitory effect on CRAC activity and, thus, an inhibitory effect on intracellular calcium increase. Furthermore, analysis of human CD4+ T cell proliferation via the carboxyfluorescein diacetate succinimidyl ester method revealed that alphitolic acid inhibited T cell proliferation in a concentration-dependent manner. Our findings provide a theoretical basis for the potential therapeutic use of alphitolic acid in the treatment of inflammatory diseases.

Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EV-labelling methods.

Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

The CDK-Inhibitor p27 Kip1 Regulates Fetal Hemoglobin Expression

γ-globin upregulation has been shown to be beneficial for individuals with Sickle Cell Disease, leading to improvement in morbidity and mortality rates. Intriguingly, small molecules that modulate the cell cycle have been observed to impact γ-globin expression in erythroid cells, though a causal relationship has not been definitively established. Understanding the connection between cell cycle dynamics/regulation and γ-globin production may shed light into a novel molecular mechanism by which γ-globin expression is regulated, with potential therapeutic implications for Sickle Cell Disease. To evaluate if cell cycle speed correlates with γ-globin expression, we labeled HUDEP-2 cells (which express beta-globin) with carboxyfluorescein diacetate succinimidyl ester (CFSE) at different days of differentiation. With each cell division, CFSE dye intensity is expected to be reduced by half; therefore, faster cycling cells retain less CFSE than slower cycling cells. Forty-eight hours post-CFSE labeling, HUDEP-2 cells were analyzed for γ-globin expression by intracellular flow cytometry at days 2, 4, 6, and 8 of differentiation. Starting at Day 6 of differentiation, the slowest cycling (Top 5% CFSE) HUDEP-2 cells exhibited >2-fold increase in γ-globin expressing cells (F-cells) compared to the fastest cycling (bottom 5% CFSE) cells. Notably, these results were confirmed in erythroid cells differentiated from primary human CD34+ hematopoietic stem and progenitor cells (HSPCs), therefore ruling out the possibility that the above findings were an artifact of the immortalized HUDEP-2 cell line. The correlation between slow cell cycling speed and increased γ-globin expression prompted us to genetically perturb the cell cycle machinery and define the impact of these genetic perturbations on γ-globin expression. Using CRISPR activation (CRISPRa), we increased the expression of 8 Cyclin Dependent Kinase Inhibitors (CDKN2A, CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C and CDKN3) in HUDEP-2 cells. To do so, we first generated a HUDEP-2-MPH cell line that stably expresses the transcriptional activator complex MPH (MS2-P65-HSF1). We then transduced this cell line with a virus that expresses one of 3 sgRNAs targeting each of the 8 CDKIs, a VP64 activation domain fused to catalytically dead Cas9 (dCas9-VP64), and a blasticidin resistance cassette. We found that activation of CDKN1b (P27 Kip1) in particular, resulted in increased F-cell percentage. Indeed, compared to cells transduced with control sgRNAs, HUDEP-2-MPH cells transduced with a CDKN1B-targeting sgRNA (resulting in a 2-fold higher CDKN1B mRNA level; p= 0.006) exhibited an increased proportion of F-cells, from a baseline of ~6% up to ~20% (p= 0.024). Increased CDKN1B expression resulted in a ~14-fold increase in γ-globin mRNA levels (p= 0.026), associated with a mild reduction in beta-globin mRNA level and a reduction in BCL11A mRNA level to ~65% of normal. These results suggest that the increased γ-globin expression resulting from CDKN1B overexpression may result, at least in part, from reduced BCL11A expression (which we are currently validating). To rule out an off-target effect of the CDKN1B targeting sgRNA, we overexpressed CDKN1B in HUDEP-2 cells, using a cDNA expression construct, and found comparable results as observed with CDKN1B CRISPRa. We next overexpressed CDKN1B cDNA in erythroid cells differentiated from CD34+ HSPCs. In early preliminary results, increased CDKN1B expression in the latter cells resulted in increased γ-globin expression, both at the mRNA and protein level, validating the HUDEP-2 data. Additional studies are ongoing to define the role of CDKN1B in the regulation of γ-globin expression, and to define the impact of CDKN1B overexpression on erythroid differentiation. In summary, our preliminary studies have uncovered a link between cell cycle regulation and γ-globin production. These findings may lay the foundation for the development of new therapeutic strategies for Sickle Cell Disease.

Synergistic Anti-Tumor Effects in RAS Mut Multiple Myeloma By Targeting MAP4K2 and RAS Pathways

Mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) is an important key regulator of the stress-activated MAPK core signaling pathways. Our previous work demonstrated that MAP4K2 is highly expressed in multiple myeloma (MM) cells, and MM cells carrying a RAS mutation are more vulnerable to MAP4K2 inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins ( Li et al. Blood 2021). Targeting the aberrant RAS/MAPK signaling, MEK inhibitors have been shown to induce cytotoxicity in MM (De la Puente, P. et al. Blood Cancer J. 2016). Thus, we used a recently developed MEK inhibitor (MEKi) Trametinib to investigate the therapeutic potential of combined MEK and MAP4K2 signaling inhibition in RAS-driven MM. Tet-on sh-MAP4K2 lentivirus was introduced into RAS Mut MM cells to establish the inducible MAP4K2 knockdown cells upon doxycycline treatment. To examine the combined effects, Tet-on sh-MAP4K2 RAS Mut MM cells were treated with doxycycline to silence MAP4K2 expression, followed by treatment of MEKi Trametinib at different dosages. Cell proliferation, apoptosis, and cell cycle were analyzed five days later. Data showed that MAP4K2 silencing combined with MEK inhibition significantly decreased cell proliferation to 10% compared with MEKi alone 32% ( p<0.01) by MTS assays, and enhanced cell apoptosis (MEKi alone vs. with MAP4K2 silencing: 26% vs 91%, p<0.01). Cell cycle assay demonstrated that MEKi induced G1 arrest in RAS Mut MM cells (MEKi alone:76% vs vehicle control: 54%, p<0.01), which was also significantly higher in combination with MAP4K2 silencing (92%). Western blot assay confirmed that MAP4K2 silencing combined with MEKi significantly enhanced the downregulation of IKZF1 and c-MYC compared to the MEKi treatment alone. These data suggest that the MEKi and MAP4K2 inhibition combination has synergetic anti-MM effects. To further confirm the synergetic effects, we tested the combined effects of MEKi with the MAP4K2 kinase inhibitor TL4-12. H929 (N-Ras Mut) cells were treated by vehicle control, TL4-12 (2 uM) alone, MEKi (200 nM) alone, or TL4-12 + MEKi for 4 days, followed by an apoptosis assay. The combination of TL4-12 and MEKi strongly enhanced the cytotoxicity effects compared to the single-agent treatments. Consistently, the carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation assay confirmed that dual treatment significantly delayed H929 cell proliferation compared to the single-agent treatments (percentage of proliferating cells: TL4-12 alone: 86.2%; MEKi alone: 89.03%; dual treatment: 18.9%. p<0.01). Western blotting assay further confirmed that dual treatment of MEKi and MAP4K2i enhanced the decrease in MEK1/2 and ERK1/2 phosphorylation and downregulation of transcriptional factors IKZF1 and c-MYC. Taken together, our findings demonstrate that the combination of MAP4K2 inhibition with MEKi results in synergetic anti-MM effects in RAS mutation myeloma, therefore could be a potent novel therapeutic regimen for patients with relapsed/refractory multiple myeloma.

Exosomes derived from human umbilical cord mesenchymal stem cells loaded with RVG-Lamp2b and Netrin-1 promotes Schwann cell invasion and migration

BackgroundNetrin-1 has a neuroprotective effect by regulating angiogenesis, autophagy, apoptosis, and neuroinflammation. This study investigated the effects of netrin-1 delivery to mouse Schwann cells and vascular endothelial cells using exosomes modified with rabies virus glycoprotein (RVG) peptides. Materials and methodsRVG-Lamp2b and/or Netrin-1 were overexpressed in human umbilical cord mesenchymal stem cells to obtain exosomes modified with RVG-Lamp2b and/or loaded with Netrin-1. Then, exosomes were labeled with carboxyfluorescein diacetate succinimidyl ester and co-cultured with mouse Schwann cells and endothelial cells. Netrin-1 expression in Schwann cells and endothelial cells was measured using quantitative polymerase chain reaction and immunoblotting. Moreover, methyl thiazolyl tetrazolium assays and Transwell assays were used to detect proliferation, migration, and invasion of Schwann cells and endothelial cells. ResultsExosomes with RVG-Lamp2b entered Schwann cells more readily compared with the exosomes without RVG-Lamp2b. Meanwhile, this was not the case in endothelial cells. Netrin-1-loaded exosomes significantly promoted Netrin-1 expression, cell proliferation, migration, invasion, and epithelial-mesenchymal transition in Schwann cells and endothelial cells. These effects were further enhanced by Netrin-1-loaded exosomes modified with RVG-Lamp2b in Schwann cells, but not in endothelial cells. ConclusionHucMSC-derived exosomes loaded with RVG-Lamp2b and Netrin-1 promote proliferation, migration, and invasion of Schwann cells.

Hypoxia promotes immune escape of pancreatic cancer cells by lncRNA NNT-AS1/METTL3-HuR-mediated ITGB1 m6A modification

Pancreatic cancer (PC) cell immune escape is a crucial element in PC malignant development. Some previous studies have reported that LncRNA NNT-AS1 played a carcinogenic role in various tumors. However, the effect of lncRNA NNT-AS1 in PC cell immune escape remains unclear. To evaluate PC cell immune escape, PC cells were co-cultured with CD8+ T cells under a hypoxic condition. PC cell proliferation and migration were evaluated using the colony formation assay and transwell assay. CD8+ T cell proliferation and aoptosis were measured using the carboxy fluorescein diacetate succinimidyl ester (CFSE) assay and flow cytometry. The secretion of antitumor cytokines was assessed using enzyme-linked immunosorbent assay (ELISA). The molecular interactions were analyzed using chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), or dual-luciferase reporter gene assays. A tumor xenograft model was established to evaluate the effects of lncRNA NNT-AS1 on PC in vivo. It was found that lncRNA NNT-AS1 was highly expressed in PC, and its silencing inhibited hypoxia-induced PC cell growth and immune escape in vivo and in vitro. Mechanically, HIF-1α transcriptionally activated NNT-AS1 expression and NNT-AS1 increased ITGB1 stability and expression in a METTL3-HuR dependent manner. ITGB1 overexpression reversed the inhibitory effects of NNT-AS1 knockdown on hypoxia-induced PC cell immune escape. In conclusion, Hypoxia promoted PC cell immune escape through lncRNA NNT-AS1/METTL3-HuR-mediated m6A modification to increase ITGB1 expression, which provided a theoretical foundation and a potential therapeutic target for PC.

Xanthatin suppresses pancreatic cancer cell growth via the ROS/RBL1 signaling pathway: In vitro and in vivo insights

BackgroundAs a malignant digestive system tumor, pancreatic cancer has a high mortality rate. Xanthatin is a sesquiterpene lactone monomer compound purified from the traditional Chinese herb Xanthium strumarium L. It has been reported that Xanthatin exhibits inhibitory effects on various cancer cells in retinoblastoma, glioma, hepatoma, colon cancer, lung cancer, as well as breast cancer. However, in pancreatic cancer cells, only one report exists on the suppression of Prostaglandin E2 synthesis and the induction of caspase 3/7 activation in Xanthatin-treated MIA PaCa-2 cells, while systematic in vitro and in vivo investigations and related mechanisms have yet to be explored. PurposeThis research aims to explore the in vitro and in vivo effects of Xanthatin on pancreatic cancer and its molecular mechanisms. MethodsThe anticancer effects and mechanisms of Xanthatin on pancreatic cancer cells were assessed through employing cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, carboxyfluorescein diacetate succinimidyl ester (CFDA SE) cell proliferation assay, colony formation assay, wound healing assay, transwell assay, Annexin V-FITC/propidium iodide (PI) dual staining, Hoechst nuclear staining, Western blot analysis, phosphoproteomics, and reactive oxygen species (ROS) measurement. The in vivo anticancer effects of Xanthatin on pancreatic cancer cells were studied using a nude mouse model. ResultsThe present study showed that Xanthatin can prevent the proliferation and metastasis of pancreatic cancer cells and trigger the exposure of phosphatidylserine (PS), chromatin condensation, and caspase activation, thereby inducing apoptosis. Phosphoproteomic analysis indicated that Xanthatin inhibits the phosphorylation of the proliferation-associated protein RBL1, and oxidative stress can lead to RBL1 dephosphorylation. Further investigation revealed that Xanthatin significantly upregulates ROS levels in pancreatic cancer cells, and the antioxidant N-acetylcysteine (NAC) can reverse Xanthatin-induced cell proliferation inhibition and apoptosis. In addition, Xanthatin can suppress pancreatic cancer cell growth in a xenograft nude mouse model with low toxicity to the mice. ConclusionXanthatin may inhibit the proliferation of pancreatic cancer cells and trigger apoptosis through the ROS/RBL1 signaling pathway.

Glucose metabolism and function of CD4+ Tregs are regulated by the TLR8/mTOR signal in an environment of SKOV3 cell growth.

To investigate the role of mammalian target of rapamycin (mTOR) signal in Toll-like receptor (TLR) 8-mediated regulation of glucose metabolism and its effect on reversing immunosuppression in CD4+ regulatory T-cells (Tregs) in ovarian cancer (OC). Fluorescence-activated cell sorting was used to detect the expression levels of mTOR+ and 4E-BP1+ cells in CD4+ Tregs. The prognosis and immune infiltration analysis of mTOR mRNA in OC were performed using the TIMER and Kaplan-Meier plotter database. Furthermore, real-time polymerase chain reaction (RT-PCR) and western blot (WB) were used to detect expression levels of glucose metabolism-related genes and proteins in CD4+ Tregs. Glucose uptake and glycolysis levels were detected by colorimetry, while the effects of CD4+ Tregs on the proliferation of CD4+ T-effector cells (Teffs) were evaluated by carboxyfluorescein diacetate succinimidyl ester (CFSE). mTOR expression in CD4+ Tregs was significantly higher in patients with OC compared with controls and in CD4+ Tregs than in CD4+ Teffs in OC. Additionally, the expression level of mTOR mRNA was related to prognosis and immune infiltration levels in patients with OC. Blocking the mTOR signal resulted in downregulation of glucose metabolism in CD4+ Tregs. Simultaneous inhibition of the mTOR signal while activation of the TLR8 signal had a coordinated inhibitory effect on glucose metabolism and the immunosuppressive function of CD4+ Tregs. Furthermore, the mTOR signal played an essential role in TLR8-mediated reversal of immunosuppressive function in CD4+ Tregs. These findings imply that activation of the TLR8 signal inhibits glucose metabolism in CD4+ Tregs by downregulating mTOR signaling, thereby reversing the immunosuppressive function of these cells in an OC cell growth environment.

HIV Infection Elicits Differential Transcriptomic Remodeling in CD4+ T Cells with Variable Proliferative Responses to the T Cell Receptor Stimulus.

Identification of a cellular biomarker of latent HIV infection will facilitate the latent reservoir detection, quantification, and targeting for elimination. Unfortunately, the latency biomarkers reported in the literature define only a fraction of the entire reservoir. The latent HIV reservoir may be established in dividing cells that subsequently return to quiescence and in resting cells. The strength of the T cell receptor (TCR) signaling at the time of infection affects characteristics of the established reservoir, such as the ability to reactivate with latency reversing agents. To better understand the cellular environments before latency establishment, we characterized transcriptomic remodeling induced by the initial HIV infection in cells with differential proliferative responses to the TCR stimulus. Cell proliferation was monitored using the viable dye carboxyfluorescein diacetate succinimidyl ester. Cells that divided many times, a few times, or remained non-dividing were subjected to single-cell RNA sequencing. A subset of identified transcriptional changes induced by HIV infection was independent of the number of cell divisions; however, responses unique to different cell subsets were also detected. Some of these early gene expression changes were consistent with reported markers of latently infected cells. We pose that the latency biomarkers may depend on the cellular proliferative state at the time of infection.

The effect of microvesicles derived from K562 cells on proliferation and apoptosis of human bone marrow mesenchymal stem cells.

Microvesicles (MVs) are small membrane-bound particles that act as a vehicle to transfer their contents, such as proteins, RNAs, and miRNAs, to the target cells, making them undergo several changes. Depending on the origin and the target cell, MVs may cause cell survival or apoptosis. This study investigated the effects of MVs released from the leukemic K562 cell line on the human bone marrow mesenchymal stem cells (hBM-MSCs) to evaluate changes in the survival or apoptosis of the cells in an in vitro system. In this experimental study, we added the isolated MVs from the K562 cell line to hBM-MSCs, and after three and then seven days, subsequently cell count, cell viability, transmission electron microscopy, tracing MVs by carboxyfluorescein diacetate, succinimidyl ester (CFSE) solution, flow cytometry analysis for Annexin-V/PI staining and qPCR for the evaluation of BCL-2, KI67, and BAX expression were carried out. On the 10th day of the culture, hBM-MSCs were examined by Oil red O and Alizarin Red staining to evaluate their differentiation into adipocytes and osteoblasts. There was a significant decrease in cell viability and KI67 and BCL-2 expression; however, BAX was significantly upregulated in the hBM-MSCs compared to control groups. Annexin-V/PI staining results also showed the apoptotic effects of K562-MVs on hBM-MSCs. Moreover, the differentiation of hBM-MSCs into adipocytes and osteoblasts was not observed. MVs from the leukemic cell line could affect the viability of normal hBM-MSCs and induce cell apoptosis.

Extensively Hydrolyzed Hypoallergenic Infant Formula with Retained T Cell Reactivity.

Immunoglobulin E (IgE)-mediated cow's milk allergy (CMA) can be life-threatening and affects up to 3% of children. Hypoallergenic infant formulas based on hydrolyzed cow's milk protein are increasingly considered for therapy and prevention of cow's milk allergy. The aim of this study was to investigate the allergenic activity and ability to induce T cell and cytokine responses of an infant formula based on extensively hydrolyzed cow's milk protein (whey) (eHF, extensively hydrolyzed formula) supplemented with Galactooligosaccharides (GOS) and Limosilactobacillus fermentum CECT5716 (LF) to determine its suitability for treatment and prevention of CMA. eHF and standard protein formula based on intact cow's milk proteins (iPF) with or without Galactooligosaccharide (GOS) and Limosilactobacillus fermentum CECT5716 (LF) were investigated with allergen-specific antibodies and tested for IgE reactivity and allergenic activity in basophil degranulation assays with sera from cow's milk (CM)-allergic infants/children. Their ability to stimulate T cell proliferation and cytokine secretion in cultured peripheral blood mononuclear cells (PBMC) from CM-allergic infants and children was studied with a FACS-based carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay and xMAP Luminex fluorescent bead-based technology, respectively. An eHF supplemented with GOS and LF exhibiting almost no IgE reactivity and allergenic activity was identified. This eHF induced significantly lower inflammatory cytokine secretion as compared to an intact protein-based infant formula but retained T cell reactivity. Due to strongly reduced allergenic activity and induction of inflammatory cytokine secretion but retained T cell reactivity, the identified eHF may be used for treatment and prevention of CMA by induction of specific T cell tolerance.

FASCIA Method in the Assessment of Lymphocyte Mitogen Responses in the Laboratory Diagnostics of Primary Immunodeficiencies

Lymphocyte responses to mitogens constitute a key part of the diagnostics of combined immunodeficiency (CID). Currently, mostly radioactive thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution methods are used. Flow-cytometric assay for specific cell-mediated immune-response in activated whole blood (FASCIA) has been put forth as an easy-to-perform option for the measurement of lymphocyte responses with the advantage of recognizing different lymphocyte subtypes and avoiding the use of radioactive reagents. Our aim was to analyze retrospectively the usefulness of FASCIA in the diagnostics of CID. We included all lymphocyte stimulation tests done with FASCIA in HUSLAB (Helsinki, Finland) between February 2015 and September 2018 in our analysis. The cohort was divided into two groups according to the patients’ final diagnoses: CID (n = 30) or non-CID (n = 159). We evaluated the stimulation responses with a combined FASCIA score (the average of all mitogen responses). The FASCIA score was significantly lower among the CID group compared to the other patients (p = 0.002), and in the ROC analysis, the AUC was 0.75 (p < 0.001) for the FASCIA score. When the three mitogens were analyzed separately, phytohemagglutinin (PHA) was best in separating patients with CID from non-CID (in the ROC analysis AUC 0.71, p = 0.001). Immunosuppressive medication affected the FASCIA result significantly and needs to be considered when evaluating the results. In conclusion, FASCIA can reliably detect the CID patients in the absence of immunosuppressive medication. It emerges as a method with many benefits compared to tests requiring radioactive reagents or the complicated CFSE staining.

In Vitro Drug Studies of Inhibiting MDSC to Improve Efficacy of ICI (immune check point inhibitor) Therapy in Ph(-) myeloproliferative Neoplasm

Although there has been much progress in the use of immune check-point inhibitor (ICI) therapy to treat cancers, very few studies have been published regarding the ICI therapy in Ph(-) myeloproliferative neoplasm (MPN). One clinical trial by Mascarenhas and his associates (ASH annual meeting 2020) in treating nine myelofibrosis patients with pembrolizumab was a anegative trial with no patients responded. Recent studies suggest a leading role for MDSCs in the immunosuppressive tumor micro-environments (TME) which could be the main cause of therapeutic resistance to ICI therapy. Therefore, we perform in vitro experiments to test some of potential drug to inhibit MDSC which may be added to the ICI therapy for future clinical ICI therapy in MPN. Material & Methods: 1) in vitro culture for MDSC formation. Peripheral blood mononuclear cells (PBMC) were isolated from patients with ph(-) MPN by Ficoll Hypaque (GE Healthcare) density gradient centrifugation. Monocytes were isolated using CD14 MicroBeads (Miltenyi Biotec) and cultured in RPMI1640 media supplemented with 10% heat- inactivated FBS and 10 ng/mL IL-6 and GM-CSF (R&D Systems Inc) for 5 days. 2) Drug treatment. Where indicated, monocytes were treated with 0.01% DMSO or 1 umol/L each of Ibrutinib (in 0.01% DMSO), Ruxolitinib (in 0.01% DMSO). Lenalidomide (in 0.01% DMSO) for 1 hour every day during the MDSC cultures. 3) Flow cytometry: Cultured MDSC cells were subject for flow cytometric assessment on BD Accuri C6 flow cytometer. Monocytic MDCS was determined as HLA-DRdim/- and CD14+; guanulocytic MDCS as HLA-DRdim/-, CD14-, and CD33+, (BD Biosciences Inc). The flow data were processed using FlowJo vX. 4) T cell proliferation assay. T cells were isolated from a T4 or T8 isolation kits then T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies). T cells were nonspecifically activated with anti-CD3/CD28 beads (Life Technologies) and cocultured at 1:1 ratios of T cells to the MDSC obtained from in vitro culture, in the presence of 10 ng/mL IL2 (Invitrogen). After 3 days, T-cell proliferation levels (CFSE) in gated CD4+ and CD8+ cells was assessed respectively by flow cytometry. The flow data were processed using FlowJo vX. Results: After 5 days cultures, Fig 1a ,b, c, shows Ruxolitinib, Ibrutinib, Lenalidomide significantly inhibits M-MDSC formation presented as (mean + SE ( % of DMSO )) of 79.72 + 4.66 ( n=27) , 73.19 + 4.71 (n=31), and 72.48 + 5.89 (n=20)respectively; Inhibits G-MDSC as of 77.82 + 5.66 (n=27) ,66.34 + 5.79 (n=31), (73.59 + 7.03) (n=20) respectively. Fig 2 showed CFSE levels in proliferated CD4+ (Fig. 2 A-D) and CD8+ (Fig.2 E-H) cells in a representative MF patient. Conclusion: Ibrutinib, Ruxolitinib, Lenalidomide significantly inhibits MDSC formation in vitro in patients with MPN and improves T cell function. A further clinical trial to evaluate the combination of ICI therapy with MDSC inhibitor such as Ibrutinib , Ruxolitinib or Lenalidomide may be worthwhile in treating patients with Myelofibrosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification and characterization of slow‑cycling cells in Ewing sarcoma.

Ewing sarcoma (ES) is an aggressive primary malignant bone tumor that predominantly affects children and young adults. Multimodal treatment approaches have markedly improved the survival of patients with localized ES. However, local recurrence and distant metastasis following curative therapies remain a main concern for patients with ES. Recent studies have suggested that slow-cycling cells (SCCs) are associated with tumor progression, local recurrence and distant metastasis in various types of cancers. According to the results of these studies, it was hypothesized that SCCs may play a critical role in tumor progression, chemoresistance and local/distal recurrence in patients with ES. The present study applied a label-retaining system using carboxyfluorescein diacetate succinimidyl ester (CFSE) to identify and isolate SCCs in ES cell lines. In addition, the properties of SCCs, including sphere formation ability, cell cycle distribution and chemoresistance, in comparison with non-SCCs were investigated. RNA sequencing also revealed several upregulated genes in SCCs as compared with non-SCCs; the identified genes not only inhibited cell cycle progression, but also promoted the malignant properties of SCCs. On the whole, the present study successfully identified SCCs in ES cells through a label-retaining system using CFSE. Moreover, to the best of our knowledge, the present study is the first to describe the characteristic properties of SCCs in ES. The findings of this study, if confirmed, may prove to be useful in elucidating the underlying molecular mechanisms and identifying effective therapeutic targets for ES.

Evaluation of T Cell Proliferation Using CFSE Dilution Assay: A Comparison between Stimulation with PHA and Anti-CD3/Anti-CD28 Coated Beads.

A decrease in T cell count or reduced T cell function can be indicative of T cell immunodeficiency. In the present study, T-cell function was assessed using Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution test after stimulation with commonly used Phytohaemagglutinin (PHA) or anti-CD3/anti-CD28 coated beads in pediatric patients with recurrent infections. Seven infants with recurrent infections and seven sex/age-matched healthy infants were included in this study. A blood cell count, immunophenotyping, and serum immunoglobulin level were performed. The proliferation of T cells was also assessed with CFSE dilution after stimulation with PHA or anti-CD3/anti-CD28 coated beads. This study showed increased IgA, IgG, and IgM levels in patients compared to the controls. In contrast to the controls, the immunophenotyping results showed a significant decline in the number of CD4+ T cells in patients. Although there was no difference in CD3+ T cell proliferation between patients and controls, the CD4+ and CD8+ T cell proliferation rates were significantly decreased in patients when stimulated with PHA. As a mitogen with the potential for maximum proliferation of T cells, PHA is better able to distinguish between patients with recurrent infections and controls than anti-CD3/anti-CD28, which mimics only the TCR pathway for stimulation of T cells.

Predicting response of micrometastases with MIRDcell V3: proof of principle with 225Ac-DOTA encapsulating liposomes that produce different activity distributions in tumor spheroids.

The spatial distribution of radiopharmaceuticals within multicellular clusters is known to have a significant effect on their biological response. Most therapeutic radiopharmaceuticals distribute nonuniformly in tissues which makes predicting responses of micrometastases challenging. The work presented here analyzes published temporally dependent nonuniform activity distributions within tumor spheroids treated with actinium-225-DOTA encapsulating liposomes (225Ac-liposomes) and uses these data in MIRDcellV3.11 to calculate absorbed dose distributions and predict biological response. The predicted responses are compared with experimental responses. Four types of liposomes were prepared having membranes with different combinations of release (R) and adhesion (A) properties. The combinations were R-A-, R-A+, R+A-, and R+A+. These afford different penetrating properties into tissue. The liposomes were loaded with either carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) or 225Ac. MDA-MB-231 spheroids were treated with the CFDA-SE-liposomes, harvested at different times, and the time-integrated CFDA-SE concentration at each radial position within the spheroid was determined. This was translated into mean 225Ac decays/cell versus radial position, uploaded to MIRDcell, and the surviving fraction of cells in spherical multicellular clusters was simulated. The MIRDcell-predicted surviving fractions were compared with experimental fractional-outgrowths of the spheroids following treatment with 225Ac-liposomes. The biological responses of the multicellular clusters treated with 225Ac-liposomes with physicochemical properties R+A+, R-A+, and R-A- were predicted by MIRDcell with statistically significant accuracy. The prediction for R+A- was not predicted accurately. In most instances, MIRDcell predicts responses of spheroids treated with 225Ac-liposomes that result in different tissue-penetrating profiles of the delivered radionuclides.