Cell Receptor Signaling Pathway Research Articles

Astragaloside IV ameliorates experimental autoimmune myasthenia gravis by regulating CD4 + T cells and altering gut microbiota

BackgroundMyasthenia gravis (MG) is an antibody-mediated autoimmune disease and its pathogenesis is closely related to CD4 + T cells. In recent years, gut microbiota is considered to play an important role in the pathogenesis of MG. Astragaloside IV (AS-IV) is one of the main active components extracted from Astragalus membranaceus and has immunomodulatory effects. To study the immunomodulatory effect of AS-IV and the changes of gut microbiota on experimental autoimmune myasthenia gravis (EAMG) mice, we explore the possible mechanism of AS-IV in improving MG.MethodsIn this study, network pharmacology was utilized to screen the crucial targets of AS-IV in the treatment of MG. Subsequently, a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to identify potential pathways through which AS-IV acts against MG. Furthermore, experimental investigations were conducted to validate the underlying mechanism of AS-IV in MG treatment. Before modeling, 5 mice were randomly selected as the control group (CFA group), and the other 10 were induced to EAMG model. These mice were randomly divided into EAMG group and EAMG + AS-IV group, n = 5/group. In EAMG + AS-IV group, AS-IV was administered by gavage. CFA and EAMG groups were given the same volume of PBS. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. At the last administration, the feces were collected for 16S RNA microbiota analysis. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected by flow cytometry. The levels of IFN-γ, IL-17 and TGF-β in serum were measured by ELISA. Furthermore, fecal microbial transplantation (FMT) experiments were performed for exploring the influence of changed intestinal flora on EAMG. After EAMG model was induced, the mice were treated with antibiotics daily for 4 weeks to germ-free. Then germ-free EAMG mice were randomly divided into two groups: FMT EAMG group, FMT AS-IV group, n = 3/group. Fecal extractions from EAMG and EAMG + AS-IV groups as gathered above were used to administered daily to the respective groups for 4 weeks. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected at the last administration. The levels of IFN-γ, IL-17 and TGF-β in serum were measured by ELISA.ResultsThe network pharmacology and KEGG pathway analysis revealed that AS-IV regulates T cell pathways, including T cell receptor signaling pathway and Th17 cell differentiation, suggesting its potential in improving MG. Further experimental verification demonstrated that AS-IV administration improved muscle strength and body weight, reduced the level of Th1 and Th17 cells, enhanced the level of Treg cells, and resulted in alterations of the gut microbiota, including changes in beta diversity, the Firmicutes/Bacteroidetes (F/B) ratio, and the abundance of Clostridia in EAMG mice. We further conducted FMT tests and demonstrated that the EAMG Abx-treated mice which were transplanted the feces of mice treated with AS-IV significantly alleviated myasthenia symptoms, reduced Th1 and Th17 cells levels, and increased Treg cell levels.ConclusionThis study speculated that AS-IV ameliorates EAMG by regulating CD4 + T cells and altering the structure and species of gut microbiota of EAMG.

Identification of an Immune-Related Gene Diagnostic Model and Potential Drugs in Sepsis Using Bioinformatics and Pharmacogenomics Approaches.

Sepsis is an organ dysfunction with high mortality. Early identification, diagnosis, and effective treatment of sepsis are beneficial to the survival of patients. This study aimed to find potential diagnosis and immune-related genes, and drug targets, which could provide novel diagnostic and therapeutic markers for sepsis. The GSE69063, GSE154918 and GSE28750 datasets were integrated to evaluate immune infiltration and identify differentially expressed genes (DEGs) and immune-related genes. Weighted gene co-expression network analysis (WGCNA) was applied to find the hub module related to immune score and sepsis. Immune-related key genes were screened out by taking interaction of DEGs, immune-related genes, and genes in hub module. Protein-protein interaction (PPI) analysis was used to further screen immune-related hub genes, followed by construction of a diagnostic model based on immune-related hub genes. Functional analysis and drug prediction of immune-related hub genes were, respectively, performed by David software and DGIdb database, followed by expression validation by reverse transcriptase polymerase chain reaction (RT-PCR). Totally, 93 immune-related key genes were identified between 561 DEGs, 1793 immune-related genes and 12,459 genes in the hub module of WGCNA. Through PPI analysis, a total of 5 diagnose and immune-related hub genes were further obtained, including IL7R, IL10, CD40LG, CD28 and LCN2. Relationship pairs between these 5 genes and immune cell were identified, including LCN2/IL7R/CD28-activated dendritic cell and IL10-immature B cell. Based on pharmacogenomics, 17 candidate drugs might interact with IL 10, including CYCLOSPORINE. Six candidate drugs might interact with CD28 and 11 with CD40LG, CD40LG and CD28 were drug targets of ALDESLEUKIN. Four significantly enriched signaling pathways were identified, such as T cell receptor signaling pathway, NF-kappa B signaling pathway and JAK-STAT signaling pathway. The 5-gene diagnostic model could be used to diagnose and guide clinical immunotherapy for sepsis.

Whole-genome analyses reveal the genomic diversity and selection signatures of Hainan cattle

The equatorial climate of Hainan Island has led to the evolution of Hainan cattle that are well adapted to the local conditions. This study aims to estimate the genetic diversity and selection signatures of Hainan cattle. After analyzing the whole-genome sequencing (WGS) data of 76 cattle from four representative populations of East Asian taurine cattle, India-Pakistan indicine cattle, Chinese indicine cattle, and Hainan locally adapted cattle, a total of 20,075,403 autosomal single nucleotide polymorphisms (SNPs) were detected. The Hainan cattle exhibited the highest level of genetic diversity (Ho=0.31, He=0.3). The increased genetic diversity observed in Hainan cattle population may be attributed to the admixture with Chinese indicine cattle population. The analysis of selective sweep, which was achieved through the utilization of ZFST and log2π, indicated the detection of 92 potential loci in Hainan cattle, which encompassed 141 genes that underwent positive selection and were associated with immune pathways, including bacterial invasion of epithelial cells, the chemokine signaling pathway, and the T cell receptor signaling pathway. Mutations analysis revealed that two missense mutations in SKINT1 (c.458C>T, p.P153L and c.580G>A, p.D194N) and two missense mutations in BCAR1 (c.1765C>G, p.A589P and c.1150C>T, p.G384S) were the homozygous genotypes in Hainan cattle. This finding enhances our understanding of the polygenic basis underlying climatic adaptation in Hainan cattle and holds significant value for the preservation and utilization of these genetic resources.

Discovery and Potential Functional Characterization of Long Noncoding RNAs Associated with Familial Acne Inversa with NCSTN Mutation

Background: Long noncoding RNAs (lncRNAs) are associated with many dermatologic diseases. However, little is known about the regulatory function of lncRNAs in familial acne inversa (AI) patients with nicastrin (NCSTN) mutation. Objectives: The aim of this study was to explore the regulatory function of lncRNAs in familial AI patients with NCSTN mutation. Methods: The expression profiles of lncRNAs and mRNAs in skin tissues from familial AI patients with NCSTN mutation and healthy individuals were analysed in this study via RNA sequencing (RNA-seq). Results: In total, 359 lncRNAs and 1,863 mRNAs were differentially expressed between the two groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the dysregulated mRNAs targeted by lncRNAs were mainly associated with the immune regulation, Staphylococcus aureus infection and B cell receptor signalling pathways. The lncRNA-miRNA-mRNA coexpression network contained 265 network pairs comprising 55 dysregulated lncRNAs, 11 miRNAs, and 74 mRNAs. Conservation analysis of the differentially expressed lncRNAs between familial AI patients with NCSTN mutation and Ncstn keratinocyte-specific knockout (Ncstn^ΔKC) mice identified 6 lncRNAs with sequence conservation; these lncRNAs may participate in apoptosis, proliferation, and skin barrier function. Conclusions: These findings provide a direction for exploring the regulatory mechanisms underlying the progression of familial AI patients with NCSTN mutation.

Identification of cuproptosis-related molecular subtypes and a novel predictive model of COVID-19 based on machine learning.

To explicate the pathogenic mechanisms of cuproptosis, a newly observed copper induced cell death pattern, in Coronavirus disease 2019 (COVID-19). Cuproptosis-related subtypes were distinguished in COVID-19 patients and associations between subtypes and immune microenvironment were probed. Three machine algorithms, including LASSO, random forest, and support vector machine, were employed to identify differentially expressed genes between subtypes, which were subsequently used for constructing cuproptosis-related risk score model in the GSE157103 cohort to predict the occurrence of COVID-19. The predictive values of the cuproptosis-related risk score were verified in the GSE163151 cohort, GSE152418 cohort and GSE171110 cohort. A nomogram was created to facilitate the clinical use of this risk score, and its validity was validated through a calibration plot. Finally, the model genes were validated using lung proteomics data from COVID-19 cases and single-cell data. Patients with COVID-19 had higher significantly cuproptosis level in blood leukocytes compared to patients without COVID-19. Two cuproptosis clusters were identified by unsupervised clustering approach and cuproptosis cluster A characterized by T cell receptor signaling pathway had a better prognosis than cuproptosis cluster B. We constructed a cuproptosis-related risk score, based on PDHA1, PDHB, MTF1 and CDKN2A, and a nomogram was created, which both showed excellent predictive values for COVID-19. And the results of proteomics showed that the expression levels of PDHA1 and PDHB were significantly increased in COVID-19 patient samples. Our study constructed and validated an cuproptosis-associated risk model and the risk score can be used as a powerful biomarker for predicting the existence of SARS-CoV-2 infection.

Identification of key genes and the pathophysiology associated with allergen-specific immunotherapy for allergic rhinitis

BackgroundAllergen-specific immunotherapy (AIT) is a causative treatment in allergic rhinitis (AR), comprising long-term allergen administration and over three years of treatment. This study is carried out for revealing the mechanisms and key genes of AIT in AR.MethodsThe present study utilized online Gene Expression Omnibus (GEO) microarray expression profiling dataset GSE37157 and GSE29521 to analyze the hub genes changes related to AIT in AR. Based on limma package, differential expression analysis for the two groups (samples of allergic patients prior to AIT and samples of allergic patients undergoing AIT) was performed to obtain differentially expressed genes (DEGs). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs were conducted using DAVID database. A Protein-Protein Interaction network (PPI) was built and a significant network module was acquired by using Cytoscape software (Cytoscape, 3.7.2). Utilizing the miRWalk database, we identified potential gene biomarkers, constructed interaction networks of target genes and microRNAs (miRNAs) using Cytoscape software, and explore the cell type-specific expression patterns of these genes in peripheral blood using publicly available single-cell RNA sequencing data (GSE200107). Finally, we are using PCR to detect changes in the hub genes that are screened using the above method in peripheral blood before and after AIT treatment.ResultsGSE37157 and GSE29521 included 28 and 13 samples, respectively. A total of 119 significantly co-upregulated DEGs and 33 co-downregulated DEGs were obtained from two datasets. The GO and KEGG analyses demonstrated that protein transport, positive regulation of apoptotic process, Natural killer cell mediated cytotoxicity, T cell receptor signaling pathway, TNF signaling pathway, B cell receptor signaling pathway and Apoptosis may be potential candidate therapeutic targets for AIT of AR. From the PPI network, 20 hub genes were obtained. Among them, the PPI sub-networks of CASP3, FOXO3, PIK3R1, PIK3R3, ATF4, and POLD3 screened out from our study have been identified as reliable predictors of AIT in AR, especially the PIK3R1.ConclusionOur analysis has identified novel gene signatures, thereby contributing to a more comprehensive understanding of the molecular mechanisms underlying AIT in the treatment of AR.

Discovery of 3-(1H-benzo[d]imidazole-2-yl)-1H-pyrazol-4 -amine derivatives as novel and potent syk inhibitors for the treatment of hematological malignancies

Spleen tyrosine kinase (Syk) is an important oncogene and signal transduction mediator that is mainly expressed in hematopoietic cells. Syk plays a key role in the B cell receptor (BCR) signaling pathway. Abnormal activation of Syk is closely related to the occurrence and development of hematological malignancies. Therefore, Syk is a potential target for the treatment of various hematologic cancers. Starting from compound 6(Syk, IC50 = 15.8 μM), we performed fragment-based rational drug design for structural optimization based on the specific solvent-accessible region, hydrophobic region, and ribose region of Syk. This resulted in the discovery of a series of novel 3-(1H-benzo [d]imidazole-2-yl)-1H-pyrazol-4-amine Syk inhibitors, which led to the identification of 19q, a highly potent Syk inhibitor that exhibited excellent inhibitory activity on Syk enzyme (IC50 = 0.52 nM) and showed potency against several other kinases. In addition, compound 19q effectively reduced phosphorylation of downstream PLCγ2 level in Romos cells. And it also exhibited antiproliferative activity in multiple hematological tumour cells. More gratifyingly, 19q showed impressive efficacy at a low dosage (1 mg/kg/day) in the MV4-11 mouse xenograft model without affecting the body weight of the mice. These findings suggest that 19q is a promising new Syk inhibitor for treating blood cancers.

Expression of tumor-associated macrophages and PD-L1 in patients with hepatocellular carcinoma and construction of a prognostic model.

Hepatocellular carcinoma (HCC) is an inflammation-associated tumor involved in immune tolerance and evasion in the immune microenvironment. Immunotherapy can enhance the immune response of the body, break immune tolerance, and then recognize and kill tumor cells. The polarization homeostasis of M1 and M2 macrophages in tumor microenvironment (TME) is involved in the occurrence and development of tumors and has been considered a hot topic in tumor research. Programmed cell death ligand 1 (PD-L1) plays an important role in the polarity of TAM and affects the prognosis of HCC patients as a target of immunotherapy. To this end, efforts were hereby made to further explore the application value of PD-L1, M1 macrophages (CD86), and M2 macrophages (CD206) in the prognosis assessment of HCC, their correlation with immune cell infiltration in HCC tissues, and their bioenrichment function. The gene expression omnibus (GEO) and the Cancer Genome Atlas (TCGA) database were used to analyze the expression of PD-L1, CD86, and CD206 in different tumor tissues. The correlation between the expression of PD-L1, CD86, and CD206 and the infiltration of immune cells was analyzed using the Tumor Immune Estimation Resource (TIMER). The tissue specimens and clinicopathological data of hepatocellular carcinoma patients having undergone surgical treatment in our hospital were collected. Immunohistochemistry was used to verify the expression of PD-L1, CD86, and CD206, and analyze the relationship with clinicopathological features and prognosis of patients. Besides, nomogram was constructed to predict the overall survival (OS) of patients at 3 and 5years. Finally, the protein-protein interaction network information was analyzed using STRING database, and GO analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis were performed to study the biological functions of PD-L1, CD86, and CD206. Bioinformatics analysis found that PD-L1, CD86, and CD206 were underexpressed in various tumor tissues including liver cancer, while the present immunohistochemical detection found that PD-L1, CD86, and CD206 were overexpressed in liver cancer tissues. Expressions of PD-L1, CD86, and CD206 were positively correlated with the infiltration level of immune cells in liver cancer, while the expression of PD-L1 was positively correlated with the degree of tumor differentiation. Meanwhile, the expression level of CD206 was positively correlated with gender and preoperative hepatitis, and patients with high expression of PD-L1 or low expression of CD86 had poor prognosis. AJCC stage, preoperative hepatitis, and the expression levels of PD-L1 and CD86 in cancer tissues were independent risk factors affecting survival of patients after radical hepatoma surgery. KEGG pathway enrichment analysis showed that PD-L1 was significantly enriched in T cell aggregation and lymphocyte aggregation, and might be involved in the formation of T cell antigen receptor CD3 complex and cell membrane. Besides, CD86 was significantly enriched in positive regulation of cell adhesion, regulation of mononuclear cell proliferation, regulation of leukocyte proliferation, and transduction of T cell receptor signaling pathway, while CD206 was significantly enriched in type 2 immune response, cellular response to LPS, cellular response to LPS, and involvement in cellular response to LPS. In conclusion, these results suggest that PD-L1, CD86, and CD206 may be involved not only in the occurrence and development of HCC, but also in immune regulation, indicating the potential role of PD-L1 and CD86 as potential biomarkers and new therapeutic targets for prognosis assessment of liver cancer.

Gene expression comparison between primary aggressive luminal breast cancer and paired axillary and sentinel lymph node metastasis.

e13060 Background: More aggressive and therapy-resistant hormone receptor (HR)-positive breast cancer (hereafter luminal breast cancer, LBC) remains a great challenge. Few studies examine the genomics of axillary lymph node (ALN) metastasis in LBC. The aim of this study was to characterize and compare the mRNA expression patterns of primary breast cancers and paired ALN metastases. Methods: Aggressive pT1-2N2-3M0 LBC patients aged over 18 years who were treated at Shandong Provincial Hospital Affiliated to Shandong First Medical University and had available matched primary and metastatic tumor samples for gene expression analyses were retrospectively recruited. Bulk mRNA expression levels were determined by RNA sequencing (Illumina NovaSeq 6000 system) and calculated using featureCounts. Differential gene expression analysis was performed using DESeq2. All analyses were carried out with differentially expressed genes (DEGs) with an FDR <0.05. Changes of intrinsic subtypes from primary to metastatic disease were assessed. Results: Ten LBC patients with paired tumor samples were included. Compared with the primary tumor, ALN metastatic tumor had 1509 statistically significant DEGs, including 743 upregulated genes and 766 downregulated genes. Notably, there was an upregulation of genes related to immune response-activating signal transduction, T cell receptor signaling pathway, B cell receptor signaling pathway, and cytokine-cytokine receptor interaction in the ALN metastasis. The downregulation of genes were mainly enriched in the PI3K-Akt signaling pathway, ECM-receptor interaction, Wnt signaling pathway, and TGF-beta signaling pathway. Intrinsic subtype distribution in primary tumors was different from those in ALN metastatic tumors, such as Luminal A (30% vs. 50%), Luminal B (10% vs. 20%), HER2-enriched (20% vs. 10%), Basal-like (10% vs. 10%), and Normal-like (30% vs. 10%). These results suggest that LBC is a highly heterogeneous disease. Conclusions: In aggressive pT1-2N2-3M0 LBC, ALN metastases have a distinct gene expression profile and show upregulation of immune-related genes ( CR2, BLK, PAX5, CD19, FCRL1, TNFRSF13B). Genes associated with signaling pathways are involved in the development of breast cancer ( FST, TNN, LAMC3, SFRP2, FGF18), and are downregulated when compared to autologous primary cancer. These pathways are potential targeted pathways in the treatment of breast cancer. LBC cells metastatic to lymph nodes undergo a change to metastasize and survive in the new microenvironment, which may provide insights into the metastatic process. Besides, a switch towards more aggressive intrinsic subtype from primary to advanced disease is also observed.

Resistance mechanism and microenvironment changes after PARPi treatment: Paired analysis of ovarian cancer samples pre and post treatment.

e17586 Background: PARP inhibitors (PARPi) have been widely used in the maintenance therapy of ovarian cancer (OC) and have shown strong anti-tumor effect in tumors with homologous recombination defects (HRD). However, there is still a lack of systematic clinical data on drug resistance mechanisms and tumor microenvironment changes. Methods: 13 OC patients who were treated with PARPi at West China Second University Hospital were enrolled. PARPi treatment-naive samples (PNS, n = 11) and post-PARPi progression samples (PPS, n = 13) were acquired. Two next-generation sequencing (NGS) panels (Master panel, 563 genes for exons of DNA plus 1831 genes for RNA; HRD panel, 70,000 SNPs for HRD score evaluation, AmoyDx) were used to analyze gene variation, expression, and HRD score changes in tumors. Results: Median duration of treatment for PARPi was 17 months (9-36), and the median lines of chemotherapy before PARPi was 2 (1-3). 38.5% (5/13) patients achieved R0 resection. BRCA reversion mutations were observed in 30.0% (3/10) of the g BRCA+ PPS, including 1 splicing variant and 2 frameshift mutations. High-frequency variants also included MYC amplification (30.8%, 4/13) and ZFHX4 mutations (23.1%, 3/13); Amplification of ch11 q13 were observed in two patients (15.4%). HRD scores and tumor mutation burden (TMB) were significantly increased in PPS. RNA expression profiling showed that the expression of NGFR, CCL18, C7 and other 85 genes in PPS was up regulated. GO/KEGG analysis showed that NF-κb pathway, immune response related cell activation, cytokine - cytokine receptor interaction, B cell receptor signaling pathway, NK cytotoxicity pathways, and etc., were significantly upregulated in PPS, suggesting that PARPi may activated immune-related pathways of the tumor microenvironment (TME). Downregulated pathways in PPS include JAK-STAT signaling pathway, Wnt signaling pathway, and etc. In PPS, the level of memory B cells and neutrophils were significantly up-regulated, while the levels of immune suppression by myeloid cells were significantly down-regulated. Conclusions: Our small-scale study confirmed that BRCA reversion mutation is one of the important resistance mechanisms of PARPi, and the reversion mutation is located near the germline pathogenic mutation to counteract the reading frame change caused by the frameshift mutation. The up-regulation of B-cell receptor, NK cytotoxic cytokine receptor interaction and other related pathways suggested that PARPi may activate immune response, and the changes in the levels of memory B cells, C7 and neutrophils also confirmed the results above. Epithelial mesenchymal transition induced by CCL18 may also lead to PARPi resistance and activate the NF-κb pathway simultaneously, increasing the inflammatory characteristics of TME. These findings can provide ideas for future treatment, and larger samples are needed to verify later.

Comparative transcriptome profiling and functional analysis in the blood of tiger puffer (Takifugu rubripes) in response to acute hypoxia

Dissolved oxygen (DO) is an essential ecological factor encountered by fish and other aquatic animals, and hypoxia can adversely affect the physiology, biochemistry and behavior of aquatic animals. Blood is an essential tissue for oxygen transportation and plays a key role in maintaining homeostasis and adapting the organism to hypoxia. However, studies focusing on the molecular regulatory mechanisms of blood under hypoxia are scant. Therefore, to explore the hypoxia adaptive mechanisms in the blood, we used RNA-Seq technology to investigate the transcriptome changes in the blood of Takifugu rubripes under acute hypoxia. In our study, 20 fish were selected and divided into four groups, including a control group (PPM5.4) and three experimental groups (PPM4, PPM2 and PPM0). In addition to GO and KEGG enrichment analysis, we further used GSEA enrichment analysis to screen the pathways mediated in T. rubripes under hypoxia. Results revealed that hypoxia had a greater effect on blood than other tissues, and T. rubripes could inhibit cell proliferation and down-regulated immune-related pathways (including IL-17 signaling pathway, C-type lectin receptor signaling pathway, B cell receptor signaling pathway and Toll-like receptor signaling pathway). However, the blood of T. rubripes could positively modulate the genes expression involved in energy regulation, angiogenesis and oxidative stress to resist hypoxic damage. Furthermore, T. rubripes could also up-regulate apoptosis and JAK−STAT signaling pathway to conduct self-protection in response to hypoxia. In conclusion, all these results indicated that immune processes, energy metabolism, angiogenesis and oxidative stress were significantly altered in the blood of acute hypoxic T. rubripes, which contributed to maintain cellular energy balance and oxygen supply during hypoxic conditions. The key regulatory genes and pathways identified in this study not only reveal the mechanism of hypoxia tolerance in T. rubripes, but also provide a theoretical basis for breeding hypoxia-tolerant T. rubripes and other fish.

Phase I clinical study of orally-dosed HBW-3220 in patients with relapsed/refractory B-cell lymphoma.

e19532 Background: BTK (Bruton's tyrosine kinase), a central regulator of the B cell receptor signaling pathway, has been targeted for treating a variety of B cell malignancies and autoimmune diseases. The use of the first generation covalent BTK inhibitors (BTKis) have often resulted in the development of resistance, commonly associated with the mutation at the cysteine 481 where the inhibitor binds covalently; therefore, a non-covalent BTKi will offer greater therapeutic benefits. HBW-3220 is a novel reversible BTKi binding to BTK non-covalently. HBW-3220 has potent inhibitory effects on BTK wild-type and BTK mutants (C481S, C481R, T474I, T316A, etc.). HBW-3220 also has a superior preclinical profile, including better anti-tumor efficacy, PK properties and safety, validating its clinical study in cancer patients. Methods: This is a phase I dose-escalation study in patients with r/r B-cell non-Hodgkin's lymphoma. The dose escalation was performed with the accelerated titration and the "3+3" design. Adverse events (AEs) were assigned according to NCI Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Response assessment for chronic lymphocytic leukemia (CLL) was based on the iwCLL 2018 guidelines, for Waldenström macroglobulinemia (WM) was based on the IWWM- 7 consensus, and for all other lymphomas (such as small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), etc.) was based on the Lugano 2014 guidelines. Results: A total of 11 patients were enrolled, and the doses were 15, 30, 60, 90 or 120 mg QD. No dose-limiting toxicity (DLT) occurred in any patient, implicating good tolerability. AEs were similar to other BTKis. Common AEs (≥2 cases, grade 1-3) were neutropenia (n = 2), reduced platelet count (n = 3) and reduced white blood cell count (n = 2). Drug-related grade 3 AEs were neutropenia (n = 1) and pulmonary infection (n = 1). There was no AE leading to discontinuation or dose reduction. 8 patients underwent at least one response assessment, with 1 complete remission (CR) and 5 stable disease (SD). 1 patient (30 mg) with MZL achieved CR at the first response assessment, and her extranodal lesion (2cm*1cm) in the nasopharynx completely disappeared; she remains CR after 20 weeks of medication, and no obvious AEs has been observed! 3 patients in the 60 mg dose group (2 FL and 1 DLBCL) were assessed as SD during the first assessment, and all had tumor shrinkages! 1 MCL patient in the 90 mg dose group has had his lesion reduced by 43%. After oral administration of HBW-3220, the drug exposures show dose-dependent increases, and the time to maximum plasma concentration (Tmax) is 1-4 hours, and elimination half-life (T1/2) is 11-25 hours. Conclusions: The current data show that HBW-3220 is well tolerated and of efficacy in patients with B-cell lymphoma. Its clinical PK characteristics support once-a-day administration. Clinical trial information: ChiCTR20221130 .

Artificial intelligence (AI) –powered H&E whole-slide image (WSI) analysis of tertiary lymphoid structure (TLS) correlates with immune phenotype and related molecular signatures in non–small-cell lung cancer.

e20520 Background: Recent data suggested immune infiltrates in TLS around tumor bed may be a favorable predictive factor for immunotherapy in various cancers. TLS have been suggested to be correlated with inflamed immune phenotype and relevant inflammatory gene signatures of adaptive immunity. To analyze this relationship, here, we developed an AI model to assess TLS objectively in H&E WSI, and assessed its correlation with immune phenotype and immunologic signatures. Methods: H&E images, relevant gene expression profiles and clinical data from The Cancer Genome Atlas (TCGA) lung cancer dataset (LUAD and LUSC, N = 913) were used for the analysis. Lunit SCOPE TLS, an AI-powered H&E WSI analyzer, was developed with 3.59 x 109 μm2 annotated area and 1,439 H&E stained WSI of 18 cancer types to segment TLS within tumor microenvironment (TME). Based on spatial tumor-infiltrating lymphocyte (TIL) density, IPs were classified into inflamed IP (IIP) as high intratumoral TIL (iTIL), immune-excluded IP (IEP) as low iTIL and high sTIL, and immune-desert IP (IDP) as low TIL overall. The infiltration of immune cells, the activity of related pathways and biological processes, and the signature scores of interest gene sets were analyzed by using the CIBERSORT, DESeq2 and GSEA tools. Results: Of 913 samples, 69.7% (636/913) contain TLS. The median value of the proportion of TLS area within TME was 0.12%, which was applied for the cut point of TLS-high vs -low group. The proportion of TLS-high was not significantly different according to EGFR mutation (mutation vs wild type: 45.5% vs 50.2%), KRAS mutation (49.3% vs 50%), but numerically decreased in the merging set of other driver mutations including ALK, ROS1, RET, MET, NTRK1-3 (36% vs 50.3%, p = 0.223). Interestingly, TLS-high proportion was significantly different according to immune phenotype, as TLS were present in 57.6% of IIP (186/323), 49.8% of IEP (241/484), and 28.3% of IDP (30/106, p < 0.001). TLS-high group was positively correlated with memory B cells (fold change [fc] 1.93, p < 0.001), CD8+ T cells (fc 1.20, p < 0.001), and M1 Macrophages (fc 1.17, p < 0.001), and negatively correlated with neutrophils (fc 0.59, p < 0.001) and M2 macrophages (fc 0.91, p = 0.006). This result was additionally supported by GSEA analysis of GO:BP or Hallmark gene sets which showed TLS-high was associated with B cell receptor signaling pathway (normalized enrichment score [NES] 3.16, p = 0.001), immunoglobulin production (NES 3.07, p = 0.002), and interferon-gamma response (NES 2.20, p = 0.002). Epithelial-mesenchymal transition was negatively associated with TLS-high (NES -1.54, p < 0.001). Conclusions: TLS is associated with inflamed immune phenotype and infiltration of memory B cells as well as CD8+ T cells in non-small cell lung cancer.

LncRNA PCBP1-AS1 mediated downregulation of ITGAL as a prognostic biomarker in lung adenocarcinoma.

Integrin alpha L (ITGAL) seemed to play a critical role in carcinogenesis and immune regulation. Nevertheless, the effects of ITGAL on non-small cell lung cancer (NSCLC) remain elusive. The present paper intended to determine the effects of ITGAL in NSCLC via the integration of bioinformatic analyses. In this study, we found that the mRNA and protein levels of ITGAL were downregulated in NSCLC tissues. Significantly, low ITGAL expression was related to poorer prognosis and increased malignancy of NSCLC. In addition, GO analysis and KEGG pathway analysis revealed that the coexpressed genes of ITGAL were predominantly associated with various immune-associated signaling pathways, like the T cell receptor signaling pathway, Th17 cell differentiation, chemokine signaling pathway, and NF-κB signaling pathway. Our result indicated that lncRNA-mediated downregulation of integrin alpha L expression was tightly related to immunocyte infiltration, immune modulators, and chemotactic factors in NSCLC, which potentially serves as a biomarker for clinical prognosis prediction and immunotherapy of NSCLC. This is the first comprehensive analysis of ITGAL in the prognosis, immune microenvironment, and immunotherapy of lung adenocarcinoma. ITGAL are promising biomarkers for predicting clinical outcomes and immunotherapy responses in patients with NSCLC.

Transcriptomic analysis of regenerated skins reveals key genes involved in skin immune response and air-breathing of loach (Paramisgurnus dabryanus)

Fish skin acts as the first immune barrier against pathogens from external environments. Recently, fish skin immunity research has gradually become a hot topic in aquaculture disease control. Interestingly, the skin can do air-breathing in some bimodal respiration fish. However, there is no comprehensive understanding of these two functions. In this study, the skin regeneration of loach (Paramisgurnus dabryanus) was investigated through morphological and histological observations. Then, original skins (OS) and the regenerated skins (RS) when their capillaries were the most abundant during healing, were collected for transcriptomic analysis. 285,899,386 clean reads with a total length of 42.34 Gb were obtained. A total of 1282 differentially expressed genes (DEGs) were detected, including 1030 DEGs up-regulated and 252 DEGs down-regulated in the comparison of RS vs. OS. Fc gamma R-mediated phagocytosis (containing gelsolin (Gsn)), chemokine signaling pathway (containing C–C motif chemokine ligand 28 (Ccl28)), and B cell receptor signaling pathway (containing CD80 molecule (Cd81)) were closely related to skin immune response of the loach. PI3K-Akt signaling pathway [containing fibronectin 1 (Fn1) and lysyl oxidase-like 2b (Loxl2b)], TGF-beta signaling pathway [containing thrombospondin 1 (Thbs1)], ECM-receptor interaction [containing integrin alpha 7 (Itga7), Itgb8, Itgb10 and Itgb5], and dilated cardiomyopathy [containing laminin subunit alpha1 (Lama1)] were closely associated with skin air-breathing of the loach. This study is conducive to explore the molecular mechanisms of skin immune response during its regeneration and air-breathing of bimodal respiration fish. This study will benefit for the aquaculture of P. dabryanus and its related species.

Biologically Interpretable Deep Learning To Predict Response to Immunotherapy In Advanced Melanoma Using Mutations and Copy Number Variations.

Only 30-40% of advanced melanoma patients respond effectively to immunotherapy in clinical practice, so it is necessary to accurately identify the response of patients to immunotherapy pre-clinically. Here, we develop KP-NET, a deep learning model that is sparse on KEGG pathways, and combine it with transfer- learning to accurately predict the response of advanced melanomas to immunotherapy using KEGG pathway-level information enriched from gene mutation and copy number variation data. The KP-NET demonstrates best performance with AUROC of 0.886 on testing set and 0.803 on an unseen evaluation set when predicting responders (CR/PR/SD with PFS ≥6mo) versus non-responders (PD/SD with PFS <6mo) in anti-CTLA-4 treated melanoma patients. The model also achieves an AUROC of 0.917 and 0.833 in predicting CR/PR versus PD, respectively. Meanwhile, the AUROC is 0.913 when predicting responders versus non-responders in anti-PD-1/PD-L1 melanomas. Moreover, the KP-NET reveals some genes and pathways associated with response to anti-CTLA-4 treatment, such as genes PIK3CA, AOX1 and CBLB, and ErbB signaling pathway, T cell receptor signaling pathway, et al. In conclusion, the KP-NET can accurately predict the response of melanomas to immunotherapy and screen related biomarkers pre-clinically, which can contribute to precision medicine of melanoma.

Dasatinib overcomes glucocorticoid resistance in B-cell acute lymphoblastic leukemia

Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.

Profiles of immune infiltration in seasonal allergic rhinitis and related genes and pathways

BackgroundSeasonal allergic rhinitis (SAR) is a chronic inflammatory disease for which the molecular mechanism is unclear. MethodsWhole blood, CD4+ T cells in peripheral blood mononuclear cells (PBMCs), and CD4+ T cells in nasal mucosa from SAR-related datasets (GSE43497, GSE50223, and GSE49782) were downloaded from the Gene Expression Omnibus (GEO) database. Differences in SAR-associated immune cell infiltration in the PBMCs were analyzed using the CIBERSORT algorithm. Differential gene expression analysis was conducted between different groups. Gene set enrichment analysis (GSEA) was performed using the clusterProfiler package to explore functional changes in signaling pathways. ResultsThere was a significant increase in the proportion of CD8+ T cells and a significant decrease in the proportion of neutrophils in the whole blood of SAR patients after allergen challenge compared to SAR patients after diluent challenge. This pattern was also found in SAR patients compared to healthy controls (HCs) by flow cytometry. The NF-κB and Toll-like receptor signaling pathways were enriched in SAR patients following allergen challenge. The expression of CD4+ T cell marker genes and associated cytokines significantly differed between allergen-treated SAR patients, diluent-treated SAR patients and HCs. We also observed heightened CD4+ T cell related genes, cytokines and pathways activation in the nasal mucosa region of SAR patients after allergen challenge. ConclusionOur analysis revealed that T cell receptor signaling pathways, T helper 1 (Th1) /T helper 2 (Th2) cell differentiation may contribute to the development of SAR. The present study is the first bioinformatic analysis to quantify immune cell infiltration and identify underlying SAR mechanisms from combined microarray data and provides insight for further research into the molecular mechanisms of SAR.

Protective effects and mechanisms of Yi Qi Huo Xue Fang in cerebral ischemic stroke based on network pharmacology and experimental verification

Ethnopharmacological relevanceYi Qi Huo Xue Fang (YQHXF) is an effective formula for treating cerebral ischemic stroke (CIS). However, its active ingredients and mechanism of action remain unclear. Aim of the studyThis study aimed to reveal the mechanism of action of YQHXF in the treatment of ischemic stroke based on network pharmacology and experimental validation. Materials and methodsThis study identified the chemical components in YQHXF and the components absorbed by rat serum based on UPLC-Q-TOF/MS technology and used network pharmacology to predict key candidate targets. A protein-protein-interaction (P-P-I) network was constructed using String 11.0 database and Cytoscape, and R software for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Finally, molecular docking combined with animal experiments was used to verify network pharmacology results. ResultsThis study identified and confirmed 36 chemical components of YQHXF and five chemical ingredients that were absorbed into the blood of rats and screened 66 key candidate targets. All targets in the P-P-I network were mainly related to inflammation and vascular processes. KEGG enrichment results revealed that these 66 key candidate targets were primarily involved in the “AGE-RAGE signaling pathway,” “TNF-α signaling pathway, and “T cell receptor signaling pathway.” Molecular docking results revealed that Prostaglandin-endoperoxidase synthase 2(PTGS-2), Nitric oxide synthase, endothelial (NOS3), and peroxisome proliferator-activated receptor gamma (PPARG) were more stably bound to their active ingredients. Animal experiments demonstrated that YQHXF promoted M2 polarization, inhibited M1 polarization in microglia, and promoted angiogenesis, which may be related to the PPARG pathway. ConclusionThis study revealed the key active components and effective targets of YQHXF, identified the mechanism of action of YQHXF, laid the foundation for further research on YQHXF, and provided ideas for developing new drugs for CIS.

Sex, age and menopause differentially impact gut barrier function and proteomic profile

Cardiovascular disease (CVD) and metabolic syndrome (MetS) depends on sex, age and hormonal status and is worsened by comprised gut barrier function. Previous work establishes that loss of estrogen and age comprises intestinal barrier function permeability exacerbating systemic inflammation, increasing gut oxidative stress, and elevating pro-inflammatory cytokines. We hypothesize that loss of estrogen during menopause underlies sex- and age-dependent pathological remodeling in gut barrier function leading to increased risk for CVD and MetS. C57BL-6 male and female mice were fed a high-fat-diet (HFD, 45% fat); menopause was induced via 21 days of 4-vinylcycohexane diepoxide (VCD) intraperitoneal injections (controls were injected with sesame oil) in adult and aged female mice. Diestrus was reached 14+days after the end of VCD injections and menopause was determined by cytology. Upon sacrifice, plasma was collected, and intestines were flash frozen. LPS assays were performed on plasma samples of premenopausal (n=8), menopausal (n=10), aged menopausal (n=6), and male mice (n=11). Male mice, and aged menopausal mice had significantly higher circulating endotoxin levels than premenopausal mice, while menopausal mice did not significantly differ from pre-menopause. These results indicate that age and sex impact barrier leakiness. Further, these results highlight that barrier integrity in menopause is distinctly different from aged menopause mice.Quantitative proteomics of ileum samples in premenopausal (n=5), menopausal (n=5), aged menopausal (n=5), and male mice (n=5) identified 7664 proteins with &gt;1 unique peptide of which 134 proteins were significant (p&lt;0.05) with a max fold change ˂1.27 across all groups. Protein expression of all groups was assessed using principal component analysis. Aged menopausal mice cluster closer to premenopausal mice than menopausal mice. These results indicate that changes in the ileum during menopause are different than those observed in aging. Compared to premenopausal mice, menopausal mice have significantly increased in proteins involved in the Go-biological processes including an enrichment in proteins involved in the innate immune system (6.3 fold enrichment, FE, -log10p=0.00006), phagocytosis recognition (19.82 FE, -log10p=1.14), defense response to bacterium (10.066 FE, -log10p=9.56), and B cell receptor signaling pathways (20.311 FE, -log10p=9.33). In contrast, male mice compared to premenopausal mice show a significant decrease in proteins involved in phagocytosis recognition (22.57 FE, -log10p=4.28), defense response to bacterium (12.898 FE, -log10p=2.76), and B cell receptor signaling pathway (20.56 FE, -log10p=8.12). These results indicate an increase in immune activity in menopausal female mice and a decrease in male mice compared to premenopausal female mice. In summary, these data demonstrate the change within the gut during menopause is dissimilar to those seen in male and aging mice. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.