Objective To prepare mouse anti-human lymphocyte activation gene 3 (LAG3) monoclonal antibody (mAb) and perform immunological identification of the antibody. Methods BALB/c mice were immunized with LAG3-mLumin-3T3 cells, which stably express the extracellular and transmembrane regions of human LAG3 in mouse 3T3 cells. The secretion of anti-human LAG3 antibodies in mouse serum was assessed using flow cytometry and immunofluorescence. SP2/0 cells were injected subcutaneously into the mice to elicit solid myelomas, and mouse myeloma cells were subsequently isolated. Spleen cells from the immunized mice were fused with the myeloma cells to establish hybridomas, which were then separated using the limiting dilution method. Flow cytometry was used to detect LAG3 mAbs in the hybridoma culture medium. To map the epitopes recognized by these mAbs, 3T3 cells expressing individual extracellular domains of LAG3(LAG3 domains 1/-2/-3/-4-3T3) were used. Flow cytometry was also applied to analyze LAG3 expression on activated human peripheral blood mononuclear cells (PBMC) before and after co-culture with the LAG3 mAbs. Results Mice immunized with the recombinant eukaryotic cell antigen produced anti-LAG3 antibodies. The generated hybridomas secreted mouse anti-human LAG3 mAbs, with each hybridoma line recognizing different LAG3 antigenic domains. Conclusion Mouse anti-human LAG3 mAbs were successfully generated, with different hybridoma clones secreting antibodies that recognize distinct LAG3 epitopes. These findings lay the groundwork for further studies into the biological properties of LAG3 and the development of diagnostic reagents and therapeutic blocking antibodies for cancer treatment.
Animals ; Humans ; Mice ; Lymphocyte Activation Gene 3 Protein ; Antibodies, Monoclonal/immunology* ; Mice, Inbred BALB C ; Hybridomas/immunology* ; Antigens, CD/genetics* ; Immunization ; Recombinant Proteins/immunology* ; Female ; Eukaryotic Cells/immunology* ; Flow Cytometry ; Epitopes/immunology*

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.
Adult ; Aged ; Aged, 80 and over ; Aging ; genetics ; immunology ; Betacoronavirus ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Lineage ; Chromatin Assembly and Disassembly ; Coronavirus Infections ; immunology ; Cytokine Release Syndrome ; etiology ; immunology ; Cytokines ; biosynthesis ; genetics ; Disease Susceptibility ; Flow Cytometry ; methods ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gene Rearrangement ; Humans ; Immune System ; cytology ; growth & development ; immunology ; Immunocompetence ; genetics ; Inflammation ; genetics ; immunology ; Mass Spectrometry ; methods ; Middle Aged ; Pandemics ; Pneumonia, Viral ; immunology ; Sequence Analysis, RNA ; Single-Cell Analysis ; Transcriptome ; Young Adult

PURPOSE: The purpose of this study was to evaluate the relationships between the resistance of anaplastic lymphoma kinase (ALK)‒positive non-small cell lung cancer (NSCLC) to ALK inhibitors and the programmed cell death-1/programmed cell death–ligand 1 (PD-L1) pathway, we evaluated alterations in PD-L1 following acquisition of resistance to ALK inhibitors in ALK-positive lung cancer. MATERIALS AND METHODS: We established ALK inhibitor-resistant cell lines (H3122CR1, LR1, and CH1) by exposing the parental H3122 ALK-translocated NSCLC cell line to ALK inhibitors. Then, the double-resistant cell lines H3122CR1LR1 and CR1CH1 were developed by exposing the H3122CR1 to other ALK inhibitors. We compared the alterations in PD-L1 expression levels using western blotting, flow cytometry, and quantitative polymerase chain reaction. We also investigated gene expression using RNA sequencing. The expression of PD-L1 in the tumors from 26 ALK-positive metastatic NSCLC patients (11 ALK inhibitor-naïve and 15 ALK inhibitor-resistant patients) was assessed by immunohistochemistry and analyzed. RESULTS: PD-L1 was expressed at higher levels in ALK inhibitor-resistant cell lines than in the ALK inhibitor-naïve parental cell line at the total protein, surface protein, and mRNA levels. Furthermore, PD-L1 expression in the double-resistant cell lines was much higher than that in the single resistant cell lines. RNA sequencing demonstrated that expression of immune-related genes were largely involved in ALK inhibitor resistance. The mean value of the PD-L1 H-score was 6.5 pre-treatment and 35.0 post-treatment, and the fold difference was 5.42 (p=0.163). CONCLUSION: PD-L1 expression increased following acquisition of ALK inhibitor resistance in ALK-positive NSCLC cell lines and tumors.
Antigens, CD274 ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; Cell Line ; Drug Resistance ; Flow Cytometry ; Gene Expression ; Humans ; Immunohistochemistry ; Lung Neoplasms ; Lung ; Lymphoma ; Parents ; Phosphotransferases ; Polymerase Chain Reaction ; RNA, Messenger ; Sequence Analysis, RNA

Isolation-with-migration (IM) models have become popular for explaining population divergence in the presence of migrations. Bayesian methods are commonly used to estimate IM models, but they are limited to small data analysis or simple model inference. Recently three methods, IMa3, MIST, and AIM, resolved these limitations. Here, we describe the major problems addressed by these three software and compare differences among their inference methods, despite their use of the same standard likelihood function.
Bayes Theorem ; Gene Flow ; Likelihood Functions ; Phylogeny ; Statistics as Topic

BACKGROUND: Oxytocin (OXT) has been reported to act as a growth regulator in various tumor cells. However, there is a paucity of data on the influence of OXT on cell proliferation of corticotroph adenomas. This study aimed to examine whether OXT affects cell growth in pituitary tumor cell lines (AtT20 and GH3 cells) with a focus on corticotroph adenoma cells. METHODS: Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were conducted with AtT20 cells to confirm the effects of OXT on hormonal activity; flow cytometry was used to assess changes in the cell cycle after OXT treatment. Moreover, the impact of OXT on proliferating cell nuclear antigen (PCNA), nuclear factor κB, and mitogen-activated protein kinase signaling pathway was analyzed by Western blot. RESULTS: OXT treatment of 50 nM changed the gene expression of OXT receptor and pro-opiomelanocortin within a short time. In addition, OXT significantly reduced adrenocorticotropic hormone secretion within 1 hour. S and G2/M populations of AtT20 cells treated with OXT for 24 hours were significantly decreased compared to the control. Furthermore, OXT treatment decreased the protein levels of PCNA and phosphorylated extracellular-signal-regulated kinase (P-ERK) in AtT20 cells. CONCLUSION: Although the cytotoxic effect of OXT in AtT20 cells was not definite, OXT may blunt cell proliferation of corticotroph adenomas by altering the cell cycle or reducing PCNA and P-ERK levels. Further research is required to investigate the role of OXT as a potential therapeutic target in corticotroph adenomas.
ACTH-Secreting Pituitary Adenoma ; Adrenocorticotropic Hormone ; Blotting, Western ; Cell Cycle ; Cell Line ; Cell Proliferation ; Corticotrophs ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Gene Expression ; Oxytocin ; Phosphotransferases ; Pituitary Neoplasms ; Polymerase Chain Reaction ; Pro-Opiomelanocortin ; Proliferating Cell Nuclear Antigen ; Protein Kinases ; Reverse Transcription

Inflammation can be a causative factor for carcinogenesis or can result from a consequence of cancer progression. Moreover, cancer therapeutic interventions can also induce an inflammatory response. Various inflammatory parameters are used to assess the inflammatory status during cancer treatment. It is important to select the most optimal biomarker among these parameters. Additionally, suitable biomarkers must be examined if there are no known parameters. We briefly reviewed the published literature for the use of inflammatory parameters in the treatment of patients with cancer. Most studies on inflammation evaluated the correlation between host characteristics, effect of interventions, and clinical outcomes. Additionally, the levels of C-reactive protein, albumin, lymphocytes, and platelets were the most commonly used laboratory parameters, either independently or in combination with other laboratory parameters and clinical characteristics. Furthermore, the immune parameters are classically examined using flow cytometry, immunohistochemical staining, and enzyme-linked immunosorbent assay techniques. However, gene expression profiling can aid in assessing the overall peri-interventional immune status. The checklists of guidelines, such as STAndards for Reporting of Diagnostic accuracy and REporting recommendations for tumor MARKer prognostic studies should be considered when designing studies to investigate the inflammatory parameters. Finally, the data should be interpreted after adjusting for clinically important variables, such as age and cancer stage.
Biomarkers ; C-Reactive Protein ; Carcinogenesis ; Checklist ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Gene Expression Profiling ; Humans ; Immune System ; Inflammation ; Lymphocytes

PURPOSE: Chemoresistance is a concern in ovarian cancer patients, in whom survival remains. MicroRNA, a novel class of small RNAs, have frequently been found to be dysregulated in human malignancies and to act as negative regulators of gene expression. This study aimed to explore the function of miR-338-3p in cisplatin resistance in ovarian cancer and potential molecular mechanisms thereof. MATERIALS AND METHODS: The expression levels of miR-338-3p and WNT2B in ovarian cancer tissues and cells were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), transwell, and flow cytometry assays were used to assess biological role of miR-338-3p in vitro. Western blot assay was conducted to measure protein expression of WNT2B, epithelial-mesenchymal transition (EMT)-related proteins, and apoptosis-related proteins. The relationship between miR-338-3p and WNT2B was confirmed by dual-luciferase reporter. Finally, a xenograft tumor model was developed to explore the effects of overexpression of miR-338-3p on tumor growth in ovarian cancer in vivo. RESULTS: MiR-338-3p was downregulated in cisplatin resistant ovarian cancer tissues and cells. Mechanistically, high expression of miR-338-3p enhanced cell sensitivity to cisplatin by inhibiting proliferation, motility, and EMT and by promoting apoptosis via targeting WNT2B expression in vitro. Furthermore, overexpression of miR-338-3p increased cisplatin sensitivity among ovarian cancer in an in vivo xenograft tumor model. CONCLUSION: MiR-338-3p enhances the sensitivity of ovarian cancer cells to cisplatin by downregulating WNT2B.
Apoptosis ; Blotting, Western ; Cisplatin ; Epithelial-Mesenchymal Transition ; Flow Cytometry ; Gene Expression ; Heterografts ; Humans ; In Vitro Techniques ; MicroRNAs ; Ovarian Neoplasms ; Polymerase Chain Reaction ; RNA

BACKGROUND: Glioblastoma multiforme (GBM) is a highly malignant brain tumor with a worst prognosis of less than one year despite advance treatment facilities. Among various signaling pathway genes displaying genetic modifications, aberrant expression of Notch pathway genes is frequent in GBM offering novel therapeutic targets. Herbal extracts having anticancer properties are used in adjuvant therapy that is safe and affordable as compared to chemotherapeutics. Bacopa monnieri has been used for the development of brain cells because of its neuroprotective properties. Its anticancer properties have shown to be promising in cancer treatment. METHODS: The anticancer properties of Bacoside A, an active and abundant component of Bacopa monnieri was assessed on U-87 MG cell line and its effects on expression of Notch pathway genes were studied. Cell cycle arrest and apoptosis were studied using flow cytometry. Expression of Notch pathway genes comprising of Notch receptors (notch1, notch2, notch3 and notch4), ligands (jagged1 and jagged2), a component of gamma-secretase complex (APH1A) and downstream target (HES1) were evaluated by quantitative real-time PCR. RESULTS: Bacoside A exhibited considerable cytotoxicity on U-87 MG cells inducing cell cycle arrest and apoptosis. Cell cycle analysis revealed a significant arrest of 39.21% cells in sub-G0 phase at 80 µg/mL concentration, increasing to 53.21% at a higher concentration of 100 µg/mL. The fraction of early apoptotic cells in control was low (3.48%) that increased substantially to 31.36% and 41.11% after 80 µg/mL and 100 µg/mL of Bacoside A treatment respectively. Additionally, the expression of notch1 gene decreased after exposure to Bacoside A with a fold change of 0.05, whereas HES1 gene expression was increased by 25 fold. CONCLUSION: These data indicate that Bacoside A has a possible anticancer activity that could be inducing cell cycle arrest and apoptosis through Notch pathway in GBM in vitro.
Amyloid Precursor Protein Secretases ; Apoptosis ; Bacopa ; Brain ; Brain Neoplasms ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Line ; Flow Cytometry ; Gene Expression ; Glioblastoma ; Humans ; In Vitro Techniques ; Ligands ; Prognosis ; Real-Time Polymerase Chain Reaction ; Receptors, Notch

Hematopoietic stem cells (HSC) maintain homeostatic hematopoiesis via their multi-lineage differentiation and self-renewal potentials. HSC can be enriched and purified by flow cytometry relying on their cell surface markers and functional characteristics, however, these methods can not meet the need for deep analysis of HSC biological property and function because of the poor purity. Recent studies have successfully purified and tracked HSC using specifically expressed genes, which can enhance the purification efficiency of HSC, thus provide a better tool for the in-vivo study of HSC. This review summarizes the new techniques and discusses their advantages and disadvantages.
Cell Differentiation ; Flow Cytometry ; Gene Expression ; Hematopoiesis ; Hematopoietic Stem Cells

The giant roundworm Ascaris infects pigs and people worldwide and causes serious diseases. The taxonomic relationship between Ascaris suum and Ascaris lumbricoides is still unclear. The purpose of the present study was to investigate the genetic diversity and population genetic structure of 258 Ascaris specimens from humans and pigs from 6 sympatric regions in Ascaris-endemic regions of China using existing simple sequence repeat data. The microsatellite markers showed a high level of allelic richness and genetic diversity in the samples. Each of the populations demonstrated excess homozygosity (Ho < He, Fis > 0). According to a genetic differentiation index (Fst=0.0593), there was a high-level of gene flow in the Ascaris populations. A hierarchical analysis on molecular variance revealed remarkably high levels of variation within the populations. Moreover, a population structure analysis indicated that Ascaris populations fell into 3 main genetic clusters, interpreted as A. suum, A. lumbricoides, and a hybrid of the species. We speculated that humans can be infected with A. lumbricoides, A. suum, and the hybrid, but pigs were mainly infected with A. suum. This study provided new information on the genetic diversity and population structure of Ascaris from human and pigs in China, which can be used for designing Ascaris control strategies. It can also be beneficial to understand the introgression of host affiliation.
Ascaris lumbricoides ; Ascaris suum ; Ascaris ; China ; Gene Flow ; Genetic Structures ; Genetic Variation ; Humans ; Microsatellite Repeats ; Swine