Identification of inflammatory response genes in ANCA-associated vasculitis by bioinformatics analysis and experimental validation / 安徽医科大学学报

Objective @#To explore the candidate genes and potential molecular mechanisms of anti -neutrophil cyto- plasmic antibodies ( ANCA) -associated vasculitis by bioinformatics and experimental validation , and to provide a scientific theoretical basis for the treatment of potential inflammatory targets for ANCA-associated vasculitis .@*Methods@#The GSE108109 chip data was retrieved from the Gene Expression Omnibus (GEO) database , and the differential genes were processed , analyzed and screened using the R language related program package . Kyoto encyclo- pedia of genes and genomes (KEGG) and gene ontology (GO) enrichment analysis was carried out using DAVID online network cable , and the interaction network of the protein encoded by the selected genes of inflammatory syn- drome was constructed through STRING web site . Further endogenous competitive RNA ( ceRNA) regulatory net- work was predicted and constructed through miRWalk and DIANA-LncBase databases , and key genes were screened from the network to draw ROC curve . The renal biopsy samples of patients with ANCA-associated vasculi- tis confirmed by our hospital were collected as the experimental group , and the renal biopsy samples of IgA ne- phropathy and micro-adaptive nephropathy were collected as the control group . Immunohistochemical staining was performed on the collected renal biopsy samples , and the average optical density was calculated by semi -quantita- tive analysis of immunohistochemical staining to further verify the expression of the key genes screened by the bioin- formatics analysis . Pearson linear correlation analysis was performed between the average optical density results and the clinical inflammatory data of patients . @*Results @#846 differential genes were screened , of which 444 genes were significantly up-regulated and 402 genes were significantly down-regulated . Through KEGG and GO analysis , im- portant differentially expressed genes related to inflammation regulation were obtained . Among them , CSF1R and TNFRSF1B , two differentially expressed genes never reported in ANCA-associated vasculitis , attracted our atten- tion . At the same time , we constructed multiple ceRNA regulatory axes including KCNQ1OT1 -hsa-miR-125 a-5p- TNFRSF1B . There were 15 samples of ANCA-associated vasculitis , 6 samples of IgA nephropathy , and 3 samples of micropathological kidney . Immunohistochemical results of renal biopsy specimens showed that the expression of CSF1R and TNFRSF1B in ANCA-associated vasculitis kidney tissue was higher than that in the control group . Pearson correlation analysis of clinical data of patients in ANCA group showed that the expression of CSF1R was positively correlated with the content of neutrophil count ( r = 0. 587) , and the expression of TNFRSF1B was posi- tively correlated with the content of serum C -reactive protein ( r = 0. 646) . @*Conclusion @#Key genes related to in- flammatory regulation such as CSF1R and TNFRSF1B were investigated by bioinformatics methods , and a rigorous ceRNA regulatory network was constructed . The expression of CSF1R and TNFRSF1B in ANCA vasculitis was high- er than that in the control group through immunohistochemistry . The results provides a scientific theoretical basis for the molecular mechanism of inflammation , and laid a good foundation for new therapeutic targets of ANCA-related vasculitis for inflammation .

Effect Mechanism of Modified Baitouwengtang Treating Colorectal Cancer by Regulating CSF1R/STING/TBK1 Signaling to Polarize Phenotype of Tumor-associated Macrophages （TAMs） / 中国实验方剂学杂志

ObjectiveThis study aims to investigate the effect of modified Baitouwengtang （MBTWD） on tumor growth and the number of tumor-associated macrophages （TAMs） in tumor tissue of MC38 cell tumor-bearing mice with colorectal cancer and explores whether MBTWD mediates the remodeling of TAM phenotype to play an immunologically antitumor effect. MethodFirstly， The C57BL/6 mouse tumor model grafted subcutaneously was established， and then model mice were classified into a model group， positive control group（3 mg·kg-1）， and MBTWD groups with high and low dosages（23.43、46.86 g·kg-1）， with 10 mice in each group. In addition， 10 healthy mice were set as the blank group， and the changes in body weight， tumor volume， and survival status of mice in each group were observed. Tumor tissue， spleen， and peripheral blood were collected to calculate the tumor volume change， tumor inhibition rate， and spleen mass. Hematoxylin-eosin （HE） staining was used to observe the morphological changes of tumor tissue， and an immunofluorescence assay was used to detect the expression levels of CD4， CD8， and CD206 in tumor tissues of tumor-bearing mice. The secretion levels of transforming growth factor （TGF）-β， interleukin （IL）-6， and chemokine （C-C Motif） ligand 2 （CCL2） in peripheral serum were measured by using enzyme-linked immunosorbent assay （ELISA）. Secondly， a co-culture model induced by IL-4 in vitro of MC38 cells and murine monocytic macrophage RAW264.7 cells was established. Cell proliferation and activity assay （CCK-8） was used to detect the inhibitory effect of MBTWD containing serum on cell proliferation. A transwell experiment was used to detect the effect of IL-4-induced M2 macrophages on the invasion of MC38 cells. Flow cytometry was used to detect the expression of CD86 on the membrane of M2 macrophages induced by IL-4 with MBTWD containing serum. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the effect of MBTWD containing serum on the mRNA expression levels of M1 macrophage-related polarization factors CD86， nitric oxide synthase （iNOS）， and IL-12， as well as M2 macrophage-related polarization factors CD206， CD163， and IL-10 after co-cultivation. Finally， the protein expression levels of colony-stimulating factor 1 receptor （CSF1R）， stimulator of interferon genes （STING）， and TANK binding kinase 1 （TBK1） in tumor tissues of tumor-bearing mice were detected by Western blot. ResultIn vivo experimental results show that compared with the model group， the MBTWD can significantly inhibit the tumor growth of tumor-bearing mice. Immunofluorescence experiments show that the MBTWD can increase the number of CD8+ T cell infiltration in tumor tissue of tumor-bearing mice， reduce the number of CD206+ TAMs infiltration， and down-regulate the secretion levels of cytokines IL-6， TGF-β， and CCL2 in peripheral blood of tumor-bearing mice. The results of in vitro experiments show that the MBTWD containing serum has no obvious inhibitory effect on cell proliferation， but the cell supernatant after co-cultivation with RAW264.7 cells can inhibit the proliferation activity of MC38 cells， and the invasion ability of MC38 cells is enhanced by IL-4-induced M2 macrophages. However， this effect can be inhibited in a concentration-dependent manner by the MBTWD containing serum. At the same time， the results of Real-time PCR show that the MBTWD containing serum can up-regulate the mRNA expression levels of M1 macrophage-related polarization factors CD86， iNOS， and IL-12 and down-regulate those of M2 macrophage-related polarization factors CD206， CD163， and IL-10. Flow cytometry results also confirm that the MBTWD containing serum can increase the number of repolarized CD86+ M1 macrophages， indicating that MBTWD can induce M2 macrophages to repolarized M1 macrophages to play an anti-tumor growth role. Finally， Western blot results show that MBTWD can down-regulate the expression of CSF1R protein and up-regulate that of STING and TBK1 proteins in tumor tissue of tumor-bearing mice. ConclusionMBTWD can down-regulate the infiltration number of CD206+ TAMs and increase the infiltration of CD8+ T cells， thereby playing an immunologically antitumor effect on the growth inhibition of colorectal cancer， which may be related to regulating CSF1R signaling and then activating STING/TBK1 signaling pathway to induce phenotypic remodeling of TAMs.

Preclinical and early clinical studies of a novel compound SYHA1813 that efficiently crosses the blood-brain barrier and exhibits potent activity against glioblastoma

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults and is poorly controlled. Previous studies have shown that both macrophages and angiogenesis play significant roles in GBM progression, and co-targeting of CSF1R and VEGFR is likely to be an effective strategy for GBM treatment. Therefore, this study developed a novel and selective inhibitor of CSF1R and VEGFR, SYHA1813, possessing potent antitumor activity against GBM. SYHA1813 inhibited VEGFR and CSF1R kinase activities with high potency and selectivity and thus blocked the cell viability of HUVECs and macrophages and exhibited anti-angiogenetic effects both in vitro and in vivo. SYHA1813 also displayed potent in vivo antitumor activity against GBM in immune-competent and immune-deficient mouse models, including temozolomide (TMZ) insensitive tumors. Notably, SYHA1813 could penetrate the blood-brain barrier (BBB) and prolong the survival time of mice bearing intracranial GBM xenografts. Moreover, SYHA1813 treatment resulted in a synergistic antitumor efficacy in combination with the PD-1 antibody. As a clinical proof of concept, SYHA1813 achieved confirmed responses in patients with recurrent GBM in an ongoing first-in-human phase I trial. The data of this study support the rationale for an ongoing phase I clinical study (ChiCTR2100045380).

An Autopsy Proven Case of CSF1R-mutant Adult-onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) with Premature Ovarian Failure

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a progressive degenerative white matter disorder caused by mutations in the tyrosine kinase domain of the CSF1R gene. ALSP is often misdiagnosed as other diseases due to its rarity and various clinical presentations such as Parkinsonism, pyramidal signs, cognitive impairment and/or psychiatric symptoms. We describe an autopsy case of ALSP with a CSF1R mutation. A 61-year-old woman presented insidious-onset gait difficulty for 12 years since her age of 49, and premature ovarian failure since her age of 35. At initial hospital visit, brain magnetic resonance imaging revealed hydrocephalus. Initially, Parkinson's syndrome was diagnosed, and she was prescribed L-dopa/carbidopa because of spasticity and rigidity of extremities, which had worsened. Subsequently, severe neuropsychiatric symptoms and cognitive impairment developed and radiologically, features of leukoencephalopathy or leukodystrophy were detected. She showed a down-hill course and died, 12 years after initial diagnosis. At autopsy, the brain showed severe symmetric atrophy of bilateral white matter, paper-thin corpus callosum, thin internal capsule, and marked hydrocephalus. Microscopically, diffuse loss of white matter, relatively preserved subcortical U-fibers, and many eosinophilic bulbous neuroaxonal spheroids were noted, but there was no calcification. Pigmented glia with brown cytoplasmic pigmentation were readily found in the white matter, which were positive for Periodic acid-Schiff, p62, and CD163 stains, but almost negative for CD68. Whole-exome and Sanger sequencing revealed a CSF1R mutation (c.2539G>A, p.Glu847Lys) which was reported in prior one ALSP case. This example demonstrates that ALSP could be associated with premature ovarian failure.

Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) with Novel CSF1R Mutation

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a subtype of dominantly inherited leukoencephalopathies caused by novel CSF1R gene mutation predominantly affecting the cerebral white matter. High signal lesions on diffusion weighted image (DWI) are characteristic. Herein, we describe a patent with ALSP with a novel mutation. The patient had persistent DWI lesions, worsening white matter changes associated with rapidly progressive clinical symptoms.