scholarly journals Pro-Inflammatory Activation of a New Immortalized Human Microglia Cell Line

2019 ◽  
Vol 9 (5) ◽  
pp. 111
Author(s):  
Marta Chiavari ◽  
Gabriella Maria Pia Ciotti ◽  
Pierluigi Navarra ◽  
Lucia Lisi
Keyword(s):  
Cell Line ◽  
Mtor Pathway ◽  
Microglia Cell ◽  
Human Microglia ◽  
Cytokines And Chemokines ◽  
Inflammatory Activation ◽  
Cell Sources ◽  
Retinal Degenerative Diseases

The characterization of human microglia has been hampered by poor availability of human cell sources. However, microglia is involved in the physiopathology of multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, HIV dementia, retinal degenerative diseases, cancer, and many other conditions. Therefore, there is an important need to have experimental paradigms of human microglia characterized and usable to study the role of microglia in the different pathologies in which it is involved. In the present work, we carried out an extensive characterization of Immortalized Human Microglia—SV40 cell line (IMhu), marketed by Applied Biological Material. The functional response of IMhu to a large variety of stimuli was studied. In particular, we investigated morphology, mortality, and changes in the production of different cytokines and chemokines, both under basal conditions and after stimulation. Moreover, western blotting analysis was conducted on phospho-mTOR (Ser 2448) and downstream parameters, p-P70S6K and 4EBP1, in order to understand if IMhu can be used for evaluations of mTOR pathway. In conclusion, IMhu cells proved to be a useful experimental model to investigate the physiopathology of inflammatory disease that involved microglia cells, including pathological conditions that involved the mTOR pathway.

scholarly journals Modeling Marginal Zone Lymphomagenesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Victor Yazbeck ◽  
Ian McConnell ◽  
Emily Harris ◽  
Joseph Lownik ◽  
Ariel Sindel ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
B Cell ◽  
Marginal Zone ◽  
Pi3k Pathway ◽  
Marginal Zone Lymphoma ◽  
Mtor Pathway ◽  
Genetically Engineered Mouse Model ◽  
Hodgkin's Lymphomas

Introduction: Indolent B-Cell Non-Hodgkin's Lymphomas (NHL) represent a heterogeneous group of lymphoproliferative malignancies, that remain largely incurable. Marginal zone lymphomas (MZL) are the second most common subtype of indolent NHL, and lack a unique cytogenetic identifying abnormality. The B-cell receptor signaling pathway is activated in B-cell malignancy and mediates its activity mainly through the Phosphoinositide 3-kinase (PI3K) pathway. Furthermore, novel PI3K inhibitors, such as copanlisib and parsaclisib, have shown impressive clinical activity in several indolent lymphomas including MZL. This further supports the important role of the PI3K pathway in the pathogenesis of this tumor. Therefore, we hypothesized that the PI3K-mTOR pathway is sufficient for driving the pathogenesis of MZL. Methods: In order to test our hypothesis, we generated a genetically engineered mouse model carrying heterozygous global knockout alleles of both the tumor suppressor genes Phosphatase and Tensin Homolog (PTEN) and Liver Kinase B1 (LKB1). This led to over-activation of the PI3K-mTOR pathway in all mouse tissues. We closely monitored these mice for tumor formation via weekly physical examinations for several months. Upon tumor detection, the mouse was sacrificed, and tumors were sectioned for histological characterization. In order to generate a more specific model of B cells, and more accurately mimic the underlying human disease, we used the Cre-LoxP system to create the CD19-Cre-PTENfl/fl-LKB1fl/fl. Results: Thirty mice of global KO PTEN+/- LKB1 +/- died or were sacrificed due to disease progression, defined as either lymph node enlargement and/or splenomegaly. All mice showed either abnormal lymphadenopathy or splenomegaly. By Kaplan-Meier analysis, we saw a steady decrease in both tumor-free and overall survival after 3 months of age. Utilizing the product limit method, the median survival time was 6 months (95% CI: 6, 8). A total of 51 lymph nodes were sent for immunohistochemistry and pathological characterization. Of the 51 nodes, 61.5% (N=32) showed indolent Non-Hodgkin's Lymphoma, 25% (N=13) were atypical, and 11.5% (N=6) were reactive. All lymph nodes with indolent NHL were of MZL subtype. Compared to wild type (n=3), the new CD19-Cre-PTENfl/fl -LKB1fl/fl (n=3) showed an overall increase in spleen mass (120 vs 196 mg, p=0.0564), % B1 cells (4% vs 59%, p= 0.0075), % MZ cells (5% vs 30%, p=0.0547), % plasma cells (1% vs 12%, p=0.0729), and decrease in % FO cells (80% vs 12%, p=0.0003) by flowcytometry. Further characterization of the new model is currently underway. Conclusion: Marginal zone lymphoma remains an incurable lymphoma that lacks reliable preclinical models. Our data provides, for the first time, a proof of concept on the role of the PI3K-mTOR pathway in the pathogenesis of marginal zone lymphoma and paves the way for future studies understanding the biology of this disease, and developing rational therapies for this incurable malignancy. Disclosures Yazbeck: Celgene: Consultancy; AstraZeneca: Consultancy; Gilead: Research Funding; Seattle Genetics: Consultancy; Verastem: Speakers Bureau.

scholarly journals A chloroquine-resistant Swiss 3T3 cell line with a defect in late endocytic acidification

1988 ◽  
Vol 106 (2) ◽  
pp. 269-277 ◽  
Author(s):  
CC Cain ◽  
RF Murphy
Keyword(s):  
Acridine Orange ◽  
Cell Line ◽  
Second Phase ◽  
Lysosomal Fraction ◽  
New Class ◽  
Swiss 3T3 Cell

To investigate the role of acidification in cell proliferation, several cell lines resistant to chloroquine were isolated with the expectation that some would express altered endocytic acidification. The preliminary characterization of one of these lines, CHL60-64, is described. In contrast to endocytic mutants described previously, the initial phase of endocytic acidification, as measured by transferrin acidification, is normal in this cell line. However, a difference in subsequent endocytic acidification was observed in CHL60-64. In the parental cells, internalized dextran was fully acidified to approximately pH 5.5 within 1 h. In CHL60-64, the pH in the endocytic compartment was only 6.1 after 1 h and remained as high as 5.8 for at least 4 h. After an 8-h incubation, the pH decreased to 5.5, indicating that the second phase of acidification is only slowed in CHL60-64, and not blocked. Consistent with this retarded acidification, ATP-dependent acidification in vitro (as measured by acridine orange accumulation) was reduced in both the lysosomal fraction and the endosomal fraction isolated from CHL60-64. A decrease in the in vivo rate of acridine orange accumulation after perturbation with amine was also observed. In addition to amine resistance and defective acidification, CHL60-64 was found to be resistant to vacuolation in the presence of chloroquine and ammonium chloride, and was resistant to ouabain. Further studies on this new class of endocytosis mutant, in combination with existing mutants, should help to clarify the mechanisms responsible for the regulation of endocytic acidification.

scholarly journals Cromolyn inhibits the secretion of inflammatory cytokines by human microglia (HMC3)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yi-Jun Wang ◽  
Alina Monteagudo ◽  
Matthew A. Downey ◽  
Philip G. Ashton-Rickardt ◽  
David R. Elmaleh
Keyword(s):  
Wide Spectrum ◽  
Neuroinflammatory Response ◽  
Microglia Cell ◽  
Human Microglia ◽  
Slowing Down ◽  
Cytokines And Chemokines ◽  
Tnf Α ◽  
Ifn Γ ◽  
Early Alzheimer’S Disease ◽  
Future Treatment Option

AbstractCromolyn is a known mast cell stabilizer and is approved for treatment of asthma and for other allergic indications. Cromolyn, in a new redesigned dry powder formulation, is being tested in a pivotal clinical trial in combination with low dose ibuprofen to treat early Alzheimer’s Disease (AD) subjects. To better understand the mechanistic effect cromolyn has in slowing down or halting the neuroinflammatory response associated with AD progression, we tested the effect of cromolyn to dampen the inflammatory response in the human HMC3 microglia cell line. The direct effect of cromolyn on HMC3 microglia is on cytokines and chemokines production following their activation by the inflammatory cytokine TNF-α. Cromolyn and a new fluorinated analog dramatically reduced the secretion of a wide spectrum of inflammatory mediators, which included cytokines such as IL-1β, IL-6, IL-8 and IFN-γ, and chemokines such as CXCL10, CCL2, CCL3 and CCL4. These results bolster our understanding of how our cromolyn platform modulates toxic microglia behavior as a dynamic future treatment option for neurodegenerative disorders.

scholarly journals Cromolyn Inhibits the Secretion of Inflammatory Cytokines by Human Microglia (HMC3)

2020 ◽  
Author(s):  
Yi-Jun Wang ◽  
Alina Monteagudo ◽  
Matthew Downey ◽  
Philip Ashton-Rickardt ◽  
David Elmaleh
Keyword(s):  
Wide Spectrum ◽  
Neuroinflammatory Response ◽  
Microglia Cell ◽  
Human Microglia ◽  
Slowing Down ◽  
Cytokines And Chemokines ◽  
Tnf Α ◽  
Ifn Γ ◽  
Early Alzheimer’S Disease ◽  
Future Treatment Option

Abstract Cromolyn is a known mast cell stabilizer and is approved for treatment of asthma and for other allergic indications. Cromolyn, in a new redesigned dry powder formulation, is being tested in a pivotal clinical trial in combination with low dose ibuprofen to treat early Alzheimer’s Disease (AD) subjects. To better understand the mechanistic effect cromolyn has in slowing down or halting the neuroinflammatory response associated with AD progression, we tested the effect of cromolyn to dampen the inflammatory response in the human HMC3 microglia cell line. The direct effect of cromolyn on HMC3 microglia is on cytokines and chemokines production following their activation by the inflammatory cytokine TNF-α. Cromolyn and a new fluorinated analog dramatically reduced the secretion of a wide spectrum of inflammatory mediators, which included cytokines such as IL-1β, IL-6, IL-8 and IFN-γ, and chemokines such as CXCL10, CCL2, CCL3 and CCL4. These results bolster our understanding of how our cromolyn platform modulates toxic microglia behavior as a dynamic future treatment option for neurodegenerative disorders.

Characterization of a novel regulator of AKT/mTOR signaling in gastric cancer.

2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 68-68
Author(s):  
Shikha Satendra Singh
Keyword(s):  
Gastric Cancer ◽  
Cell Invasion ◽  
Poor Prognosis ◽  
Mtor Pathway ◽  
Physical Interaction ◽  
Dual Inhibitor ◽  
Dead Box ◽  
Inhibitor Treatment

68 Background: Gastric cancer (GC) is a leading cause of cancer-related death with poor prognosis and “one-size fits all approach”. Dysregulation of PI3K/Akt/mTOR pathway is a common event in GC with PIK3CAmutations reported to correlate with poor prognosis. With prognosis far from satisfactory, we aimed to identify novel surrogate biomarkers. We hypothesize the role of DP103, a DEAD-box RNA helicase, as a novel surrogate biomarker for GC by regulating the PI3K/Akt/mTOR pathway. Additionally, we shed light on high DP103 GC cells responding to Akt/mTOR dual inhibitor treatment, indicating its potential role as a surrogate biomarker for GC treatment. Methods: Tissue microarray data from GC cohorts were used in correlation analysis between DP103 and PIK3CA expression. Effect of DP103 knockdown was assessed on functions downstream of PI3K/Akt/mTOR pathway including apoptosis, autophagy and cell invasion. Next, DP103 depletion studies were done and PI3K/Akt/mTOR pathway proteins were assessed. Further, immunoprecipitation studies assessed for DP103 interacting partners from PI3K/Akt/mTOR pathway in GC. Results: Herein, we provide clinical evidence of DP103 upregulation in GC patients’ tissues compared to normal gastric tissues. Additionally, we showed a positive correlation between DP103 and PIK3CA expression in GC patients’. DP103 knockdown resulted in decrease in cell invasion and an increase in apoptosis and autophagy. Further, decrease in phosphorylation of PI3K/Akt/mTOR pathway proteins was observed with DP103 depletion. Mechanistically, we discovered a novel physical interaction between DP103 and PI3K in GC. Conclusions: Here we summarized, current understanding of DP103 regulating PI3K/Akt/mTOR pathway in GC and discuss potential use as a new surrogate biomarker for better clinical application.

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