Tyrosine Kinase Binding Protein Research Articles

Primary microgliopathy presenting as degenerative dementias: A case series of novel gene mutations from India

Introduction Microglia exert a crucial role in homeostasis of white matter integrity and several studies highlight the role of microglial dysfunctions in neurodegeneration. Primary microgliopathy are disorders where the pathogenic abnormality of the microglia causes white matter disorder and leads to a neuropsychiatric diseases. Triggering Receptor Expressed on Myeloid Cells (TREM2), TYRO protein tyrosine kinase binding protein (TYROBP), Colony-stimulating factor 1 receptor (CSF1R) are genes implicated in primary microgliopathy. Clinical manifestations of primary microgliopathy are myriad ranging from neuropsychiatric syndrome, motor disability, gait dysfunction, ataxia, pure dementia, frontotemporal dementia, Alzheimer dementia and so on. It becomes imperative to establish diagnosis of microgliopathy masquerading as degenerative dementia, especially with promising therapies on horizon for the same. We aim to describe a case series of subjects with dementia harboring novel genes of primary microgliopathy, along with their clinical, neuropsychological, and cognitive profile and radiological patterns. Methods: The prospective study was conducted in a university referral hospital in South India, as a part of an ongoing clinic-genetic research on Dementia subjects and was approved by the institutional ethics committee. All patients underwent detailed assessment including socio demographic profile, clinical and cognitive assessment, pedigree analysis and comprehensive neurological examination. Subjects consenting for blood sampling underwent genetic testing by Whole exome sequencing (WES). Results: 100 patients of dementia underwent genetic analysis using whole exome sequencing and three pathogenic variants, one each of TREM2, TYROBP and CSF1R and two variants of uncertain significance in CSF1R were identified as cause of primary microgliopathy .TREM 2 and TYROBP presented as frontotemporal syndrome whereas CSF1R presented as frontotemporal syndrome and Alzheimer dementia. Conclusion :WES has widened the spectrum of underlying neuropathology of degenerative dementias and diagnosing primary microglial dysfunction with emerging therapeutic options is of paramount importance. Cases of primary microgliopathy due to Novel mutations in TREM2, TYROBP and CSF1R with phenotype of degenerative dementia are being first time reported from Indian cohort. Our study enriches the spectrum of genetic variants implicated in degenerative dementia, and provides the basis for exploring complex molecular mechanisms like microglial dysfunction, as underlying cause for neurodegeneration.

Higher TYROBP and lower SOX6 as predictive biomarkers for poor prognosis of clear cell renal cell carcinoma: A pilot study

Clear cell renal carcinoma (ccRCC) is the most common subtype of renal cancer, accounting for approximately 75% of all histological types of renal cancer, and is the leading cause of death from renal cancer. However, the molecular mechanism of tyrosine kinase binding protein (TYROBP) and sex-determining region Y Box-6 (SOX6) in the ccRCC was not precise. Bioinformatics analysis was performed to explore the hub role of TYROBP and SOX6 on the ccRCC. A total of 6 patients with clear cell renal cell carcinoma (ccRCC) were recruited. HE staining was performed to observe the pathology result of ccRCC. Immunohistochemistry and Immunofluorescence assay was made to detect the protein expression of TYROBP. Total RNA was extracted using TRIzol to examine the mRNA expression of TYROBP via the Real time quantitative polymerase chain reaction. The strong correlation between the expression of TYROBP and the survival time of ccRCC patients was performed by the BP neural network and support vector machine. Compared with the control group, the expression of SOX6 was downregulated in the samples with ccRCC. However, the expression of TYROBP was higher in the samples with ccRCC than in the control group. Compared with the patients with high SOX6 expression, the patients with low SOX6 expression have a poor survival prognosis (HR=0.39, P < .05). However, the patients with high TYROBP expression have a shorter survival time than the patients with low TYROBP expression (HR=1.66, P < .05). The genes related with TYROBP and SOX6 are mainly enriched in the regulation of cell activation, leukocyte activation, negative regulation of cell activation, myeloid leukocyte activation, positive regulation of response to external stimulus, immune response-regulating signaling pathway. The interaction between TYROBP, SOX6, and kidney neoplasms was drawn, and the inference score of TYROBP and SOX6 on the kidney neoplasms was high. In conclusion, TYROBP is highly expressed in renal clear cell carcinoma, and when this molecule is highly expressed, the survival prognosis of renal carcinoma is poor. TYROBP and SOX6 may be potential targets for diagnosing and treating renal clear cell carcinoma.

TYRO protein tyrosine kinase-binding protein predicts favorable overall survival in osteosarcoma and correlates with antitumor immunity.

To explore the prognostic significance and underlying mechanism of TYRO protein tyrosine kinase-binding protein (TYROBP) in osteosarcoma. Firstly, the expression of TYROBP was analyzed using the t test. The Kaplan–Meier plotter analysis and a receiver operating characteristic curve were performed to evaluate the influence of TYROBP on overall survival (OS). Further, Cox regression analysis was conducted to predict the independent prognostic factors for OS of osteosarcoma patients, and a nomogram was constructed. Then, the relationship between TYROBP and clinicopathological characteristics was determined using statistical methods. Enrichment analyses were conducted to evaluate the biological functions of TYROBP. Finally, the ESTIMATE algorithm was used to assess the association of TYROBP with immune cell infiltration. TYROBP was significantly increased in osteosarcoma (all P < .001). However, the high expression of TYROBP was related to better OS in osteosarcoma patients. Cox regression analysis showed that TYROBP was an independent prognostic factor for predicting OS (P = .005), especially in patients of the male sex, age <18 years, metastasis, and tumor site leg/foot (all P < .05). Besides, TYROBP mRNA expression was significantly associated with the tumor site (P < .01) but had no remarkable relationship with age, gender, and metastasis status (all P > .05). Functional annotation and gene set enrichment analysis (GSEA) revealed that TYROBP was mainly involved in immune-related pathways. Importantly, TYROBP positively correlated with immune scores (P < .001, R = .87). TYROBP served as an independent prognostic biomarker for OS in osteosarcoma. High TYROBP expression might prolong the survival of osteosarcoma patients mainly through promoting antitumor immunity.

MRNA and lncRNA expression profiles of liver tissues in children with biliary atresia

Progressive liver fibrosis is the most common phenotype in biliary atresia (BA). A number of pathways contribute to the fibrosis process so comprehensive understanding the mechanisms of liver fibrosis in BA will pave the way to improve patient's outcome after operation. In this study, the differentially expressed profiles of mRNAs and long non-coding RNAs from BA and choledochal cyst (CC) liver tissues were investigated and analyzed, which may provide potential clues to clarify hepatofibrosis mechanism in BA. A total of two BA and two CC liver tissue specimens were collected, the expression level of mRNAs and lncRNAs was detected by RNA sequencing. Differentially expressed mRNAs (DEmRNAs) were functionally annotated and protein-protein interaction networks (PPI) was established to predict the biological roles and interactive relationships. Differentially expressed lncRNAs (DElncRNAs) nearby targeted DEmRNA network and DElncRNA-DEmRNA co-expression network were constructed to further explore the roles of DElncRNAs in BA pathogenesis. The expression profiles of significant DEmRNAs were validated in Gene Expression Omnibus database. A total of 2,086 DEmRNAs and 184 DElncRNAs between BA and CC liver tissues were obtained. DEmRNAs were enriched in 521 Gene Ontology terms and 71 Kyoto Encyclopedia of Genes and Genomes terms which were mainly biological processes and metabolic pathways related to immune response and inflammatory response. A total of five hub proteins (TYRO protein tyrosine kinase binding protein, C-X-C motif chemokine ligand 8, pleckstrin, Toll-like receptor 8 and C-C motif chemokine receptor 5) were found in the PPI networks. A total of 31 DElncRNA-nearby-targeted DEmRNA pairs and 2,337 DElncRNA-DEmRNA co-expression pairs were obtained. The expression of DEmRNAs obtained from RNA sequencing were verified in GSE46960 dataset, generally. The present study identified key genes and lncRNAs participated in BA associated liver fibrosis, which may present a new avenue for understanding the patho-mechanism for hepatic fibrosis in BA.

Antibody Characterization Report for TYRO protein tyrosine kinase-binding protein (TYROBP)

Antibody Characterization Report for TYRO protein tyrosine kinase-binding protein (TYROBP)

Long-term running exercise improves cognitive function and promotes microglial glucose metabolism and morphological plasticity in the hippocampus of APP/PS1 mice

BackgroundThe role of physical exercise in the prevention of Alzheimer’s disease (AD) has been widely studied. Microglia play an important role in AD. Triggering receptor expressed in myeloid cells 2 (TREM2) is expressed on microglia and is known to mediate microglial metabolic activity and brain glucose metabolism. However, the relationship between brain glucose metabolism and microglial metabolic activity during running exercise in APP/PS1 mice remains unclear.MethodsTen-month-old male APP/PS1 mice and wild-type mice were randomly divided into sedentary groups or running groups (AD_Sed, WT_Sed, AD_Run and WT_Run, n = 20/group). Running mice had free access to a running wheel for 3 months. Behavioral tests, [18]F-FDG-PET and hippocampal RNA-Seq were performed. The expression levels of microglial glucose transporter (GLUT5), TREM2, soluble TREM2 (sTREM2), TYRO protein tyrosine kinase binding protein (TYROBP), secreted phosphoprotein 1 (SPP1), and phosphorylated spleen tyrosine kinase (p-SYK) were estimated by western blot or ELISA. Immunohistochemistry, stereological methods and immunofluorescence were used to investigate the morphology, proliferation and activity of microglia.ResultsLong-term voluntary running significantly improved cognitive function in APP/PS1 mice. Although there were few differentially expressed genes (DEGs), gene set enrichment analysis (GSEA) showed enriched glycometabolic pathways in APP/PS1 running mice. Running exercise increased FDG uptake in the hippocampus of APP/PS1 mice, as well as the protein expression of GLUT5, TREM2, SPP1 and p-SYK. The level of sTREM2 decreased in the plasma of APP/PS1 running mice. The number of microglia, the length and endpoints of microglial processes, and the ratio of GLUT5+/IBA1+ microglia were increased in the dentate gyrus (DG) of APP/PS1 running mice. Running exercise did not alter the number of 5-bromo-2′-deoxyuridine (BrdU)+/IBA1+ microglia but reduced the immunoactivity of CD68 in the hippocampus of APP/PS1 mice.ConclusionsRunning exercise inhibited TREM2 shedding and maintained TREM2 protein levels, which were accompanied by the promotion of brain glucose metabolism, microglial glucose metabolism and morphological plasticity in the hippocampus of AD mice. Microglia might be a structural target responsible for the benefits of running exercise in AD. Promoting microglial glucose metabolism and morphological plasticity modulated by TREM2 might be a novel strategy for AD treatment.

To Investigate the Molecular Mechanism of Wugedan Pill in Improving Insulin Resistance Based on Network Pharmacology

Objective To explore the network pharmacological mechanism of Wugedan Pill in improving insulin resistance (IR). Methods The main active ingredients and corresponding target genes of Wugedan Pill were retrieved from the Traditional Chinese Medicine System Pharmacology Technology Platform (TCMSP), and the related targets of insulin resistance were obtained through the Human Gene Database (GeneCards) and the Human Online Mendelian Inheritance Database (OMIM). Gene, the active ingredient target of the drug is mapped with the insulin resistance target, and the intersection target is obtained, which is the predicted target of Wugedan Pill acting on insulin resistance. The "pharmaceutical active ingredient-target" network model was constructed by Cytoscape 3.7.0 software, and the key active ingredients were selected. The STRING database was used to construct an intersecting target-protein interaction network (PPI), and key proteins were selected. Intersection targets were subjected to Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using the Metascape platform. Results There are 94 active ingredients and 163 related targets of Wugedan Pill acting on insulin resistance. The key active ingredients are quercetin, β-sitosterol and kaempferol, and the key proteins are estrogen receptor 1 (ESR1), aldose reductase (AR), nuclear receptor coactivator protein 1 (NCOA1), Fos protein et al.; GO enrichment analysis showed that Wugedan Pill could affect the activities of oxidoreductase, tyrosine kinase-binding protein, steroid-binding protein and other proteins in improving insulin resistance; KEGG pathway enrichment analysis showed that the effect was the most Notably lipid and atherosclerotic pathways, also significantly affected diabetes complications AGE-RAGE signaling pathway, PI3K-Akt signaling pathway, neuroactive ligand-receptor interactions, alcoholic liver disease, prostate cancer, cancer miRNAs expression, pancreatic cancer, thyroid hormone signaling and other pathways. Conclusion Wugedan Pill may play a role in improving insulin resistance, regulating lipid metabolism, reducing blood glucose and preventing complications by regulating the above targets and pathways with the effective ingredients quercetin, β-sitosterol and kaempferol.

Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

CD33/Sialic acid-binding Ig-like lectin 3 (SIGLEC3) is an innate immune receptor expressed on myeloid cells and mediates inhibitory signaling via tyrosine phosphatases. Variants of CD33 are associated with Alzheimer’s disease (AD) suggesting that modulation of CD33 signaling might be beneficial in AD. Hence, there is an urgent need for reliable cellular CD33 reporter systems. Therefore, we generated a CD33 reporter cell line expressing a fusion protein consisting of the extracellular domain of either human full-length CD33 (CD33M) or the AD-protective variant CD33ΔE2 (D2-CD33/CD33m) linked to TYRO protein tyrosine kinase binding protein (TYROBP/DAP12) to investigate possible ligands and antibodies for modulation of CD33 signaling. Application of the CD33-specific antibodies P67.6 and 1c7/1 to the CD33M-DAP12 reporter cells resulted in increased phosphorylation of the kinase SYK, which is downstream of DAP12. CD33M-DAP12 but not CD33ΔE2-DAP12 expressing reporter cells showed increased intracellular calcium levels upon treatment with CD33 antibody P67.6 and partially for 1c7/1. Furthermore, stimulation of human induced pluripotent stem cell-derived microglia with the CD33 antibodies P67.6 or 1c7/1 directly counteracted the triggering receptor expressed on myeloid cells 2 (TREM2)-induced phosphorylation of SYK and decreased the phagocytic uptake of bacterial particles. Thus, the developed reporter system confirmed CD33 pathway activation by CD33 antibody clones P67.6 and 1c7/1. In addition, data showed that phosphorylation of SYK by TREM2 activation and phagocytosis of bacterial particles can be directly antagonized by CD33 signaling.

Elevated TYROBP expression predicts poor prognosis and high tumor immune infiltration in patients with low-grade glioma

BackgroundTyrosine protein tyrosine kinase binding protein (TYROBP) binds non-covalently to activated receptors on the surface of various immune cells, and mediates signal transduction and cellular activation. It is dysregulated in various malignancies, although little is known regarding its role in low-grade glioma. The aim of this study is to explore the clinicopathological significance, prognostic value and immune signature of TYROBP expression in low-grade glioma (LGG).MethodsThe differentially expressed genes (DEGs) between glioma samples and normal tissues were identified from two GEO microarray datasets using the limma package. The DEGs overlapping across both datasets were functionally annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. STRING database was used to establish the protein-protein interaction (PPI) of the DEGs. The PPI network was visualized by Cytoscape and cytoHubba, and the core module and hub genes were identified. The expression profile of TYROBP and patient survival were validated in the Oncomine, GEPIA2 and CGGA databases. The correlation between TYROBP expression and the clinicopathologic characteristics were evaluated. Gene Set Enrichment Analysis (GSEA) and single-sample GSEA (ssGSEA) were performed by R based on the LGG data from TCGA. The TIMER2.0 database was used to determine the correlation between TYROBP expression and tumor immune infiltrating cells in the LGG patients. Univariate and multivariate Cox regression analyses were performed to determine the prognostic impact of clinicopathological factors via TCGA database.ResultsSixty-two overlapping DEGs were identified in the 2 datasets, and were mainly enriched in the response to wounding, focal adhesion, GTPase activity and Parkinson disease pathways. TYROBP was identified through the PPI network and cytoHubba. TYROBP expression levels were significantly higher in the LGG tissues compared to the normal tissues, and was associated with worse prognosis and poor clinicopathological parameters. In addition, GSEA showed that TYROBP was positively correlated to neutrophil chemotaxis, macrophage activation, chemokine signaling pathway, JAK-STAT signaling pathway, and negatively associated with gamma aminobutyric acid signaling pathway, neurotransmitter transport, neuroactive ligand receptor intersection etc. TIMER2.0 and ssGSEA showed that TYROBP expression was significantly associated with the infiltration of neutrophils, macrophages, myeloid dendritic cells and monocytes. The infiltration of the M2 phenotype macrophages, cancer-associated fibroblasts and myeloid dendritic cells correlated to worse prognosis in LGG patients. Finally, multivariate analysis showed that elevated TYROBP expression is an independent risk factor for LGG.ConclusionTYROBP is dysregulated in LGG and correlates with immune infiltration. It is a potential therapeutic target and prognostic marker for LGG.

Triggering Receptor Expressed on Myeloid Cell 2 R47H Exacerbates Immune Response in Alzheimer's Disease Brain.

The R47H variant in the microglial triggering receptor expressed on myeloid cell 2 (TREM2) receptor is a strong risk factor for Alzheimer’s disease (AD). To characterize processes affected by R47H, we performed an integrative network analysis of genes expressed in brains of AD patients with R47H, sporadic AD without the variant, and patients with polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), systemic disease with early-onset dementia caused by loss-of-function mutations in TREM2 or its adaptor TYRO protein tyrosine kinase-binding protein (TYROBP). Although sporadic AD had few perturbed microglial and immune genes, TREM2 R47H AD demonstrated upregulation of interferon type I response and pro-inflammatory cytokines accompanied by induction of NKG2D stress ligands. In contrast, PLOSL had distinct sets of highly perturbed immune and microglial genes that included inflammatory mediators, immune signaling, cell adhesion, and phagocytosis. TREM2 knockout (KO) in THP1, a human myeloid cell line that constitutively expresses the TREM2- TYROBP receptor, inhibited response to the viral RNA mimetic poly(I:C) and phagocytosis of amyloid-beta oligomers; overexpression of ectopic TREM2 restored these functions. Compared with wild-type protein, R47H TREM2 had a higher stimulatory effect on the interferon type I response signature. Our findings point to a role of the TREM2 receptor in the control of the interferon type I response in myeloid cells and provide insight regarding the contribution of R47H TREM2 to AD pathology.

Progress in experimental research on SPRED protein family.

The Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology-1 (EVH-1) domain (SPRED) family of proteins was discovered in 2001. These Sprouty-related tyrosine kinase-binding proteins negatively regulate a variety of growth factor-induced Ras/ERK signaling pathways. In recent years, SPRED proteins have been found to regulate vital activities such as cell development, movement, and proliferation, and to participate in pathophysiological processes such as tumor metastasis, hematopoietic regulation, and allergic reactions. The findings of these studies have important implications regarding the involvement of SPRED proteins in disease. Early studies of SPRED proteins focused mainly on various tumors, cardiovascular diseases, and organ development. However, in recent years, great progress has been made in elucidating the role of SPRED proteins in neuropsychiatric, inflammatory, endocrine, and ophthalmic diseases. This article provides a review of the experimental studies performed in recent years on the SPRED proteins and their role in the pathogenesis of certain diseases.

Expressions of triggering receptor expressed on myeloid cell 2 in different brain regions in tyrosine kinase binding protein gene knockout mice

Objective To compare the expression of myeloid cell trigger receptor expressed on myoid cell 2 (TREM2) in different brain regions of tyrosine kinase binding protein(TYROBP) knockout mice and wild-type mice at different months of age, and to explore the relationship between TREM2, TYROBP and early onset Alzheimer's disease(EOAD). Methods Healthy TYROBP gene knockout mice were divided into three groups according to the results of gene sequencing: the homozygous (TYROBP-/-) group, the heterozygous (TYROBP-/+ ) group, and the wild type (WT) group.Western blot and RT-qPCR were used to detect the expression of TREM2 in prefrontal cortex and hippocampus of 2, 4 and 6 month old mice in the three groups and with 10 in each group at each time point. Results (1) In the prefrontal cortex: Western blot and RT-qPCR results showed that compared with WT mice (2-month-old: (0.993±0.048), (1.654±0.033); 4-month-old: (0.503±0.019), (2.169±0.023); 6-month-old: (0.600±0.036), (1.468±0.057)), the levels of TREM2 protein and mRNA in 2-month-old TYROBP-/+ group ((0.746±0.062), (1.137±0.067)) and TYROBP-/- group ((0.661±0.028), (0.644±0.012)) were decreased.While in 4-month-old and 6-month-old TYROBP-/+ group (4-month-old: (1.140±0.006), (5.483±0.088); 6-month-old: (0.827±0.043), (3.020±0.082)) and TYROBP-/- group (4-month-old: (1.071±0.010), (3.012±0.150); 6-month-old: (0.627±0.026), (1.633±0.027)) were increased, especially in 4-month-old mice and the differences were statistically significant (F=12.946, 134.445; 725.318, 289.202; 12.172, 202.791; all P<0.05). (2) In the hippocampus: Western blot results showed that compared with WT mice (2-month-old: (1.268±0.036); 4-month-old: (0.813±0.010); 6-month-old: (0.312±0.021)), the level of TREM2 protein in 2-month-old TYROBP-/+ group ((0.804±0.034)) and TYROBP-/- group ((0.534±0.020)) were decreased.While in 4-month-old and 6-month-old TYROBP-/+ group ((0.932±0.011); (0.769±0.031)) and TYROBP-/- group ((0.910±0.014); (0.609±0.018)) were increased, especially in 4-month-old mice and the differences were statistically significant (F=142.807; 27.884; 94.067; all P<0.05). Conclusion The expression level of TREM2 decreases in 2-month-old TYROBP gene knockout mice while increases in 4-month-old and 6-month-old TYROBP gene knockout mice.It is presumed that TREM2/TYROBP signal pathway participates in the pathological process of EOAD and plays different roles in different pathological stages of EOAD. Key words: Tyrosine kinase binding protein; Triggering receptor expressed on myeloid cell 2; Alzheimer's disease; Mice

IGFBP6 Is Downregulated in Unstable Carotid Atherosclerotic Plaques According to an Integrated Bioinformatics Analysis and Experimental Verification.

Aims: To investigate the differentially expressed genes (DEGs) and molecular interaction in unstable atherosclerotic carotid plaques.Methods: Gene expression datasets GSE41571, GSE118481, and E-MTAB-2055 were analyzed. Co-regulated DEGs in at least two datasets were analyzed with the enrichment of Gene Ontology Biological Process (GO-BP), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) networks, interrelationships between miRNAs/transcriptional factors, and their target genes and drug-gene interactions. The expression of notable DEGs in human carotid artery plaques and plasma was further identified.Results: The GO-BP enrichment analysis revealed that genes associated with inflammatory response, and extracellular matrix organization were altered. The KEGG enrichment analysis revealed that upregulated DEGs were enriched in the tuberculous, lysosomal, and chemokine signaling pathways, whereas downregulated genes were enriched in the focal adhesion and PI3K/Akt signaling pathway. Collagen type I alpha 2 chain (COL1A2), adenylate cyclase 3 (ADCY3), C-X-C motif chemokine receptor 4 (CXCR4), and TYRO protein tyrosine kinase binding protein (TYROBP) might play crucial roles in the PPI networks. In drug–gene interactions, colony-stimulating factor-1 receptor had the most drug interactions. Insulin-like growth factor binding protein 6 (IGFBP6) was markedly downregulated in unstable human carotid plaques and plasma. Under a receiver operating characteristic curve analysis, plasma IGFBP6 had a significant discriminatory power (AUC, 0.894; 95% CI, 0.810–0.977), with a cutoff value of 142.08 ng/mL.Conclusions: The genes COL1A2, ADCY3, CXCR4, and TYROBP are promising targets for the prevention of unstable carotid plaque formation. IGFBP6 may be an important biomarker for predicting vulnerable plaques.

Primary Alcohol-Activated Human and Mouse Hepatic Stellate Cells Share Similarities in Gene-Expression Profiles.

Alcoholic liver disease (ALD) is a leading cause of cirrhosis in the United States, which is characterized by extensive deposition of extracellular matrix proteins and formation of a fibrous scar. Hepatic stellate cells (HSCs) are the major source of collagen type 1 producing myofibroblasts in ALD fibrosis. However, the mechanism of alcohol‐induced activation of human and mouse HSCs is not fully understood. We compared the gene‐expression profiles of primary cultured human HSCs (hHSCs) isolated from patients with ALD (n = 3) or without underlying liver disease (n = 4) using RNA‐sequencing analysis. Furthermore, the gene‐expression profile of ALD hHSCs was compared with that of alcohol‐activated mHSCs (isolated from intragastric alcohol‐fed mice) or CCl4‐activated mouse HSCs (mHSCs). Comparative transcriptome analysis revealed that ALD hHSCs, in addition to alcohol‐activated and CCl4‐activated mHSCs, share the expression of common HSC activation (Col1a1 [collagen type I alpha 1 chain], Acta1 [actin alpha 1, skeletal muscle], PAI1 [plasminogen activator inhibitor‐1], TIMP1 [tissue inhibitor of metalloproteinase 1], and LOXL2 [lysyl oxidase homolog 2]), indicating that a common mechanism underlies the activation of human and mouse HSCs. Furthermore, alcohol‐activated mHSCs most closely recapitulate the gene‐expression profile of ALD hHSCs. We identified the genes that are similarly and uniquely up‐regulated in primary cultured alcohol‐activated hHSCs and freshly isolated mHSCs, which include CSF1R (macrophage colony‐stimulating factor 1 receptor), PLEK (pleckstrin), LAPTM5 (lysosmal‐associated transmembrane protein 5), CD74 (class I transactivator, the invariant chain), CD53, MMP9 (matrix metallopeptidase 9), CD14, CTSS (cathepsin S), TYROBP (TYRO protein tyrosine kinase‐binding protein), and ITGB2 (integrin beta‐2), and other genes (compared with CCl4‐activated mHSCs). Conclusion: We identified genes in alcohol‐activated mHSCs from intragastric alcohol‐fed mice that are largely consistent with the gene‐expression profile of primary cultured hHSCs from patients with ALD. These genes are unique to alcohol‐induced HSC activation in two species, and therefore may become targets or readout for antifibrotic therapy in experimental models of ALD.

Gene Expression Microarray Data Meta-Analysis Identifies Candidate Genes and Molecular Mechanism Associated with Clear Cell Renal Cell Carcinoma.

Objective We aimed to explore potential molecular mechanisms of clear cell renal cell carcinoma (ccRCC) and provide candidate target genes for ccRCC gene therapy. Materials and Methods This is a bioinformatics-based study. Microarray datasets of GSE6344, GSE781 and GSE53000 were downloaded from Gene Expression Omnibus database. Using meta-analysis, differentially expressed genes (DEGs) were identified between ccRCC and normal samples, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) function analyses. Then, protein-protein interaction (PPI) networks and modules were investigated. Furthermore, miRNAs-target gene regulatory network was constructed. Results Total of 511 up-regulated and 444 down-regulated DEGs were determined in the present gene expression microarray data meta-analysis. These DEGs were enriched in functions like immune system process and pathways like Toll-like receptor signaling pathway. PPI network and eight modules were further constructed. A total of 10 outstanding DEGs including TYRO protein tyrosine kinase binding protein (TYROBP), interferon regulatory factor 7 (IRF7) and PPARG co-activator 1 alpha (PPARGC1A) were detected in PPI network. Furthermore, the miRNAs-target gene regulation analyses showed that miR-412 and miR-199b respectively targeted IRF7 and PPARGC1A to regulate the immune response in ccRCC. ConclusionTYROBP, IRF7 and PPARGC1A might play important roles in ccRCC via taking part in the immune system process.

Abstract C104: Therapeutic targeting of TREM2+ tumor-associated macrophages as a means of overcoming checkpoint inhibitor resistance

Abstract Tumor-associated macrophages (TAMs) are a major sources of innate checkpoint inhibitor (CPI) resistance, as they subvert anti-tumor immunity through immunosuppression and supporting tumor growth. In patients, high levels of TAMs predict poor prognosis across multiple solid tumor indications. Therefore, therapeutic targeting of TAMs by impacting their survival and/or modulating their suppressive function is a promising strategy to augment response rates in solid tumor indications, as well as overcome resistance to CPI therapies. We and others have identified the transmembrane protein triggering receptor expressed on myeloid cells-2 (TREM2) as a highly enriched TAM target. TREM2 is comprised of one IgV domain but lacks any known intracellular signaling motifs. However, TREM2 associates with TYRO protein tyrosine kinase-binding protein (TYROBP), which contains an intracellular immunoreceptor tyrosine-baased activation motif (ITAM), allowing for TREM2/TYROBP-mediated signaling to occur. Screening of human tumor tissue microarrays for TREM2 protein demonstrated that TREM2+ TAMs were present in multiple human solid tumors and correlated with disease severity in some indications. Furthermore, in a variety of tumor types, TREM2 mRNA expression negatively correlated with patient survival. To target TAMs, we developed an anti-TREM2 monoclonal antibody (mAb), termed PY314m, that demonstrated compelling anti-tumor activity either as single agent in CPI-sensitive syngeneic tumor models or in combination with anti-PD-1 in CPI-resistant mouse syngeneic tumor models. Mechanistically, PY314m reduced a subset of TAMs, namely the MHCIILow ArginaseHigh M2-like TAMs, expanded MHCIIHigh M1-like TAMs, and induced both innate and adaptive pro-inflammatory cytokine production. By depleting M2-like TAMs and inducing expansion of M1-like TAMs, PY314m treatment removes and modulates multiple immunosuppressive mechanisms. The combination treatment of PY314m and anti-PD-1 mAb produced long-term immunological memory as evidenced by the resistance to tumor rechallenge in mice cured of their tumors. Collectively these findings suggest that anti-TREM2 mAb therapy could be used to overcome CPI resistance in humans. To this end, we developed a humanized version of PY314m termed PY314, which was well-tolerated and showed good pharmacokinetic properties in pilot, repeat dose non-GLP studies in non-human primates. Our ongoing efforts are aimed at better defining CPI-resistant patient populations that will maximally benefit from PY314 therapy. Citation Format: Mikhail Binnewies, Marwan Abushawish, Tian Lee, Tiep Le, Joshua L. Pollack, Erick Lu, Amanda Chen, Ranna Mehta, Nadine Jahchan, Vicky Huang, Xiaoyan Du, Subhadra Dash, Manith Norng, Aritra Pal, Kevin P. Baker, Venkataraman Sriram. Therapeutic targeting of TREM2+ tumor-associated macrophages as a means of overcoming checkpoint inhibitor resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C104. doi:10.1158/1535-7163.TARG-19-C104

Identification of key genes and pathways in chronic rhinosinusitis with nasal polyps using bioinformatics analysis.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a prevalent inflammatory disease of yet unknown etiology. The purpose of this study was to uncover key genes and pathways related to the pathogenesis of CRSwNP via bioinformatics approaches. The gene expression profile of GSE36830 extracted from Gene Expression Omnibus database was used to screen differentially expressed genes (DEGs) between nasal polyp samples and control samples. Furthermore, functional and pathway enrichment analysis was performed using the clusterProfiler package in R language. In addition, protein-protein interaction (PPI) network was constructed by STRING database and functional modules were detected using Molecular Complex Detection algorithm. A total of 538 DEGs (326 up-regulated and 212 down-regulated) were identified. The most significantly enriched pathways for up-regulated and down-regulated genes were hematopoietic cell lineage and salivary secretion, respectively. Moreover, twenty hub genes with high connectivity degrees were selected from the PPI network, such as TYRO protein tyrosine kinase binding protein (TYROBP), G protein subunit gamma 2 (GNG2), CCR7, and CCR3. Besides, six important modules were obtained, which were highly associated with chemokine signaling pathway, Th1 and Th2 cell differentiation, complement and coagulation cascades, cell cycle, systemic lupus erythematosus, and Staphylococcus aureus infection. The results of this study may provide new insights into potential molecular mechanisms of CRSwNP. Nevertheless, further experiments are needed to confirm these findings.

Comparison of the Transcriptomic Signature of Pediatric Vs. Adult CML and Normal Bone Marrow Stem Cells

IntroductionPediatric chronic myeloid leukemia (CML) accounts for 10 to 15% of children with myeloid leukemia and 2 to 9% of all pediatric leukemias. Prior to the discovery of tyrosine kinase inhibitors (TKI) such as imatinib, stem cell transplantation was the only curative treatment for both adults and children with CML. However, due to the small numbers of patients, standardized treatment approaches for pediatric CML have not been established. There are several unique characteristics of CML diagnosed in children and adolescents, and young adults (AYA; 16-29 years), compared to adults. Children and AYA with CML present with a higher white blood count and have larger spleens, higher peripheral blast counts, and lower hemoglobin levels, suggesting that the biology of pediatric CML is different than adult CML. In addition, potential side effects of TKIs unique to pediatric CML patients include impaired bone growth, fertility and immune function, however none have been extensively studied. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that are absent in adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells.MethodsCD34+ cells were isolated from pediatric CML (n=7), adult CML (n=8), pediatric normal (n=2) and adult normal (n=3) bone marrow samples. Total RNA was isolated from cells, and then cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. We aligned reads using the HISAT2 alignment software, and mapped to genes with HT-Seq. We removed genes that had zero reads across all the samples, resulting in a set of 4,696 genes that were detected in one or more samples. In case of technical replicates, we used mean of replicates. We performed three differential expression comparisons with edgeR: (1) Pediatric CML vs Adult CML, (2) Adult CML vs Adult Normal, and (3) Pediatric CML vs Pediatric Normal. We used a False Discovery Rate (FDR) of £ 20% and absolute log2 fold-change ³ 1 for selecting differentially expressed genes in each comparison. We used Fisher's exact test to identify significant KEGG pathways for the differentially expressed genes in each comparison.ResultsPediatric CML vs Adult CMLWe found 24 differentially expressed genes (15 over- and 9 under-expressed). Though no pathway was found to be significant at the false discovery rate (FDR) £ 20%, we identified a number of sub-pathways that are relevant. For example, the Chemokine Signaling pathway shows at the top of the list (ordered by raw p-value) because of two genes, XCR1 and HCK, associated with VEGF and MAPK pathways involved in cell proliferation, angiogenesis, DNA repair, and cancer pathogenesis.Adult CML vs Adult NormalWe found 60 genes (30 over- and 30 under-expressed) differentially expressed when comparing adult CML patients to normal adults. Ten genes overlapped with 24 genes we identified when comparing pediatric and adult CML patients. We found 11 pathways as significant at FDR £ 10%. Multiple pathways, including Cell adhesion, allograft rejection, Graft versus Host Disease, and Type I diabetes pathways, showed downregulation of MHC, with subsequent downstream reduction in expression of apoptosis-related genes. The IL-17 pathway makes sense, as MAPK, well-known to be associated with various cancers, is down-regulated. Lastly, in the NK pathway the gene DAP12 is up-regulated. This gene is known as a tyrosine kinase binding protein, and although tyrosine kinase inhibitors are the standard treatment for CML, the role of DAP12 in relation to leukemia has not yet been described.Pediatric CML vs Pediatric NormalWe found 509 genes (350 over- and 159 under-expressed) differentially expressed in pediatric CML patients compared to normal. Interestingly, transcriptional regulators are differentially enriched in the hematopoietic stem cell differentiation function group including GATA1, GATA2, KLF1 and KLF2. RFC is down-regulated. RFC is a mismatch repair gene known to be involved in colorectal cancer. Many of the significant pathways are involved in glucose and fatty acid metabolism.Our pilot study identified novel molecular features of pediatric CML bone marrow stem cells, providing new insights into the novel biomarkers and pathogenesis of pediatric CML. DisclosuresGotlib:Blueprint Medicines: Consultancy, Honoraria, Research Funding; Promedior: Research Funding; Deciphera: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Bioinformatics analysis to identify key genes and pathways influencing synovial inflammation in osteoarthritis.

Osteoarthritis (OA) is a chronic arthropathy that occurs in the middle-aged and elderly population. The present study aimed to identify gene signature differences between synovial cells from OA synovial membrane with and without inflammation, and to explain the potential mechanisms involved. The differentially expressed genes (DEGs) between 12 synovial membrane with inflammation and 12 synovial membrane without inflammation from the dataset GSE46750 were identified using the Gene Expression Omnibus 2R. The DEGs were subjected to enrichment analysis, protein-protein interaction (PPI) analysis and module analysis. The analysis results were compared with text-mining results. A total of 174 DEGs were identified. Gene Ontology enrichment results demonstrated that functional molecules encoded by the DEGs primarily had extracellular location, molecular functions predominantly involving ‘chemokine activity’ and ‘cytokine activity’, and were associated with biological processes, including ‘inflammatory response’ and ‘immune response’. The Kyoto Encyclopedia of Genes and Genomes results demonstrated that DEGS may function through pathways associated with ‘rheumatoid arthritis’, ‘chemokine signaling pathway’, ‘complement and coagulation cascades’, ‘TNF signaling pathway’, ‘intestinal immune networks for IgA production’, ‘cytokine-cytokine receptor interaction’, ‘allograft rejection’, ‘Toll-like receptor signaling pathway’ and ‘antigen processing and presentation’. The top 10 hub genes [interleukin (IL)6, IL8, matrix metallopeptidase (MMP)9, colony stimulating factor 1 receptor, FOS proto-oncogene, AP1 transcription factor subunit, insulin-like growth factor 1, TYRO protein tyrosine kinase binding protein, MMP3, cluster of differentiation (CD)14 and CD163] and four gene modules were identified from the PPI network using Cytoscape. In addition, text-mining was used to identify the commonly used drugs and their targets for the treatment of OA. It was initially verified whether the results of the present study were useful for the study of OA treatment targets and pathways. The present study provided insight for the molecular mechanisms of OA synovitis. The hub genes and associated pathways derived from analysis may be targets for OA treatment. IL8 and MMP9, which were validated by text-mining, may be used as molecular targets for the OA treatment, while other hub genes require further validation.

Alzheimer's disease pathology in Nasu-Hakola disease brains.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of either triggering receptor expressed on myeloid cells 2 (TREM2) or TYRO protein tyrosine kinase binding protein (TYROBP), alternatively named DNAX-activation protein 12 (DAP12), both of which are expressed on microglia in the brain and form the receptor-adaptor complex that chiefly recognizes anionic lipids. TREM2 transmits the signals involved in microglial survival, proliferation, chemotaxis, and phagocytosis. A recent study indicated that a loss of TREM2 function causes greater amounts of amyloid-β (Aβ) deposition in the hippocampus of a mouse model of Alzheimer's disease (AD) owing to a dysfunctional response of microglia to amyloid plaques, suggesting that TREM2 facilitates Aβ clearance by microglia. TREM2/DAP12-mediated microglial response limits diffusion and toxicity of amyloid plaques by forming a protective barrier. However, the levels of Aβ deposition in postmortem brains of NHD, where the biological function of the TREM2/DAP12 signaling pathway is completely lost, remain to be investigated. By immunohistochemistry, we studied the expression of Aβ and phosphorylated tau (p-tau) in the frontal cortex and the hippocampus of five NHD cases. Although we identified several small Aβ-immunoreactive spheroids, amyloid plaques were almost undetectable in NHD brains. We found a small number of p-tau-immunoreactive neurofibrillary tangle (NFT)-bearing neurons in NHD brains. Because AD pathology is less evident in NHD than the full-brown AD, it does not play an active role in the development of NHD.