Serum GM-CSF level is a predictor of treatment response to tocilizumab in rheumatoid arthritis patients: a prospective observational cohort study.

BACKGROUND: The aim of this prospective observational cohort study was to unveil the predictors of treatment response to tocilizumab (TCZ) therapy in rheumatoid arthritis (RA) patients, in terms of clinical characteristics and serum proinflammatory cytokines, especially to explore the predictive value of granulocyte macrophage-colony stimulating factor (GM-CSF). METHODS: Active adult RA patients with inadequate response to MTX intending to receive TCZ therapy were recruited prospectively in the study. A total of 174 severe RA patients were included for the identification of the associations between treatment response and the following characteristic features: demographics, medications, disease activity, serum proinflammatory cytokines and so on. RESULTS: Disease duration (OR = 0.996), tender joint count (TJC)/68 (OR = 0.943), neutrophil ratio (W4/baseline) (OR = 0.224), the high level of GM-CSF > 5 ng/ml (OR = 0.414) at baseline were the independent adverse predictors of good response assessed by clinical disease activity index (CDAI) at week 24 (W24) for TCZ therapy in RA patients. Moreover, DAS28-ESR (OR = 2.951, P = 0.002) and the high level of GM-CSF > 10 ng/ml at baseline (OR = 5.419, P = 0.002) were independent predictors of poor response, but not the high level of GM-CSF > 5 ng/ml (OR = 2.713, P = 0.054). The patients in the high GM-CSF group had significantly higher DAS28-ESR and serum levels of cytokines (IL-17A, IL-1ß, IL-6, TNF-α) at baseline, as well as significantly higher rate of non-good response (62.8% vs. 39.4%, P = 0.010) and poor response (27.9% vs. 9.1%, P = 0.004) than the low GM-CSF group at W24. In addition, poor responders had significantly higher levels of GM-CSF with concomitant increase in the serum levels of IL-17A and IL-1ß at baseline than those in moderate and good response groups, while serum levels of IL-6 and TNF-α at baseline were not significantly different in three response groups. CONCLUSION: The high levels of GM-CSF (> 5 ng/ml and > 10 ng/ml) at baseline were the independent predictors of non-good response and poor response to TCZ at W24 respectively. The high level of GM-CSF at baseline is a marker of high disease activity and a predictor of poor response to TCZ in severe RA patients, which may facilitate the development of individualized treatment strategies for refractory RA.

Changes in metabolic parameters and serum YKL-40 levels in Chinese rheumatoid arthritis patients during tocilizumab therapy.

OBJECTIVE: To investigate the metabolic changes during therapy of tocilizumab (TCZ) and methotrexate (MTX) in non-diabetic rheumatoid arthritis (RA) patients and for the first time explore the associations between metabolic parameters and serum YKL-40 (sYKL-40) levels. METHODS: We enrolled active non-diabetic RA patients who were refractory to MTX. Patients received intravenous TCZ (8 mg/kg) once every 4 weeks combined with MTX for 24 weeks. Metabolic parameters and sYKL-40 levels were measured before TCZ infusion at baseline, week 4, week 12, and week 24. Correlations were assessed by the Spearman's rank correlation analysis. RESULTS: A total of 91 non-diabetic RA patients were enrolled in this study. At week 24, we observed a significant elevation in body mass index (BMI), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) levels. In contrast, there was a significant decrease in TC/HDL­C ratio. No apparent changes in insulin resistance were found. Additionally, we detected a significant reduction in sYKL-40 levels during the study. At week 24, changes in sYKL-40 levels showed a significant negative correlation (r = -0.334, p = 0.002) with changes in TC levels. CONCLUSION: The combined therapy of TCZ and MTX resulted in a significant increase in BMI and lipid levels, while an evident decrease in the TC/HDL­C ratio and sYKL-40 levels in RA patients. Additionally, there was a significant correlation between the decrease in sYKL-40 levels and the increase in TC levels during treatment with TCZ and MTX. Key Points • Lipid levels elevated significantly and sYKL-40 levels decreased obviously after therapy of TCZ combined with MTX in Chinese RA patients. • There was a significant correlation between the increase in TC levels and the decrease in sYKL-40 levels during treatment with TCZ and MTX in RA patients.

Microglia-Derived Spp1 Promotes Pathological Retinal Neovascularization via Activating Endothelial Kit/Akt/mTOR Signaling.

Pathological retinal neovascularization (RNV) is the main character of ischemic ocular diseases, which causes severe visual impairments. Though retinal microglia are well acknowledged to play important roles in both physiological and pathological angiogenesis, the molecular mechanisms by which microglia communicates with endothelial cells (EC) remain unknown. In this study, using single-cell RNA sequencing, we revealed that the pro-inflammatory secreted protein Spp1 was the most upregulated gene in microglia in the mouse model of oxygen-induced retinopathy (OIR). Bioinformatic analysis showed that the expression of Spp1 in microglia was respectively regulated via nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor 1α (HIF-1α) pathways, which was further confirmed through in vitro assays using BV2 microglia cell line. To mimic microglia-EC communication, the bEnd.3 endothelial cell line was cultured with conditional medium (CM) from BV2. We found that adding recombinant Spp1 to bEnd.3 as well as treating with hypoxic BV2 CM significantly enhanced EC proliferation and migration, while Spp1 neutralizing blocked those CM-induced effects. Moreover, RNA sequencing of BV2 CM-treated bEnd.3 revealed a significant downregulation of Kit, one of the type III tyrosine kinase receptors that plays a critical role in cell growth and activation. We further revealed that Spp1 increased phosphorylation and expression level of Akt/mTOR signaling cascade, which might account for its pro-angiogenic effects. Finally, we showed that intravitreal injection of Spp1 neutralizing antibody attenuated pathological RNV and improved visual function. Taken together, our work suggests that Spp1 mediates microglia-EC communication in RNV via activating endothelial Kit/Akt/mTOR signaling and is a potential target to treat ischemic ocular diseases.

Prognostic value of lymphadenectomy in node-negative intrahepatic cholangiocarcinoma: A multicenter, retrospectively study.

BACKGROUND: This study aimed to evaluate the prognostic value of lymph node dissection (LND) in node-negative intrahepatic cholangiocarcinoma (ICC) and identify the appropriately total number of lymph nodes examined (TNLE). METHODS: Data from node-negative ICC patients who underwent curative intent resection in ten Chinese hepatobiliary centers from January 2010 to December 2018 were collected. Overall survival (OS), relapse-free survival (RFS) and postoperative complications were analyzed. Propensity score matching (PSM) was performed to reduce the bias due to confounding variables in LND group and non-lymph node dissection (NLND) group. The optimal TNLE was determined by survival analysis performed by the X-tile program using the enumeration method. RESULTS: A total of 637 clinically node-negative ICC patients were included in this study, 74 cases were found lymph node (LN) positive after operation. Among the remaining 563 node-negative ICC patients, LND was associated with longer OS but not RFS before PSM (OS: 35.4 vs 26.0 months, p = 0.047; RFS: 15.0 vs 15.4 months, p = 0.992). After PSM, patients in LND group had better prognosis on both OS and RFS (OS: 38.0 vs 23.0 months, p < 0.001; RFS: 15.0 vs 13.0 months, p = 0.029). There were no statistically differences in postoperative complications. When TNLE was greater than 8, OS (48.5 vs 31.1 months, p = 0.025) and RFS (21.0 vs 13.0 months, p = 0.043) were longer in the group with more dissected LNs. CONCLUSION: Routinely LND for node-negative ICC patients is recommended for it helps accurate tumor staging and associates with better prognosis. The optimal TNLE is more than 8.

Recurrence and prognosis in intrahepatic cholangiocarcinoma patients with different etiology after radical resection: a multi-institutional study.

OBJECTIVE: We aimed to evaluate the prognosis and adjuvant chemotherapy (ACT) in intrahepatic cholangiocarcinoma (ICC) patients with different etiology after radical resection. METHODS: A total of 448 patients with ICC who underwent radical resection between 2010 and 2018 at ten Chinese tertiary hospitals were analyzed in the study. These patients were divided into conventional ICC (Con-ICC, n = 261, 58.2%), hepatitis B virus ICC (HBV-ICC, n = 102, 22.8%) and hepatolithiasis (Stone-ICC, n = 85,19.0%) subtypes according to different etiology. Propensity score matching (PSM) was conducted to mitigate the baseline differences between Con-ICC and HBV-ICC, Con-ICC and Stone-ICC, HBV-ICC and Stone-ICC subtypes. RESULTS: Univariate and multivariate analysis showed that different etiology was a prognostic factor for overall survival and relapse-free survival, and different etiology was an independent risk factor for overall survival in ICC patients, respectively (P < 0.05). In addition, there was a statistical difference for overall survival in early recurrence patients among the three etiological subtypes (P < 0.05). After PSM, the overall survival of patients with Stone-ICC was worse than those of Con-ICC and HBV-ICC subtypes (P < 0.05), while the relapse-free survival of patients with Stone-ICC was equivalent to patients with Con-ICC and HBV-ICC (P > 0.05). In Stone-ICC patients, the median overall survival was 16.0 months and 29.7 months, and the median relapse-free survival was 9.0 months and 20.0 months for non-ACT and ACT patients, respectively (P < 0.05). CONCLUSION: The prognosis of Stone-ICC patients was significantly worse than those of Con-ICC and HBV-ICC patients. Interestingly, postoperative adjuvant chemotherapy can improve the prognosis of Stone-ICC patients effectively.

Comprehensive analysis of genomic mutation signature and tumor mutation burden for prognosis of intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal malignancy of the biliary tract. Analysis of somatic mutational profiling can reveal new prognostic markers and actionable treatment targets. In this study, we explored the utility of genomic mutation signature and tumor mutation burden (TMB) in predicting prognosis in iCCA patients. METHODS: Whole-exome sequencing and corresponding clinical data were collected from the ICGC portal and cBioPortal database to detect the prognostic mutated genes and determine TMB values. To identify the hub prognostic mutant signature, we used Cox regression and Lasso feature selection. Mutation-related signature (MRS) was constructed using multivariate Cox regression. The predictive performances of MRS and TMB were assessed using Kaplan-Meier (KM) analysis and receiver operating characteristic (ROC). We performed a functional enrichment pathway analysis using gene set enrichment analysis (GSEA) for mutated genes. Based on the MRS, TMB, and the TNM stage, a nomogram was constructed to visualize prognosis in iCCA patients. RESULTS: The mutation landscape illustrated distributions of mutation frequencies and types in iCCA, and generated a list of most frequently mutated genes (such as Tp53, KRAS, ARID1A, and IDH1). Thirty-two mutated genes associated with overall survival (OS) were identified in iCCA patients. We obtained a six-gene signature using the Lasso and Cox method. AUCs for the MRS in the prediction of 1-, 3-, and 5-year OS were 0.759, 0.732, and 0.728, respectively. Kaplan-Meier analysis showed a significant difference in prognosis for patients with iCCA having a high and low MRS score (P < 0.001). GSEA was used to show that several signaling pathways, including MAPK, PI3K-AKT, and proteoglycan, were involved in cancer. Conversely, survival analysis indicated that TMB was significantly associated with prognosis. GSEA indicated that samples with high MRS or TMB also showed an upregulated expression of pathways involved in tumor signaling and the immune response. Finally, the predictive nomogram (that included MRS, TMB, and the TNM stage) demonstrated satisfactory performance in predicting survival in patients with iCCA. CONCLUSIONS: Mutation-related signature and TMB were associated with prognosis in patients with iCCA. Our study provides a valuable prognostic predictor for determining outcomes in patients with iCCA.

[Implantation strategy of tissue-engineered liver based on decellularized spleen matrix in rats].

OBJECTIVE: To explore the optimal implantation strategy of tissue-engineered liver (TEL) constructed based on decellularized spleen matrix (DSM) in rats. METHODS: DSM was prepared by freeze-thawing and perfusion with sodium dodecyl sulfate (SDS) of the spleen of healthy SD rats. Primary rat hepatocytes isolated using modified Seglen 2-step perfusion method were implanted into the DSM to construct the TEL. The advantages and disadvantages were evaluated of 4 transplant strategies of the TEL, namely ectopic vascular anastomosis, liver cross-section suture transplantation, intrahepatic insertion and mesenteric transplantation. RESULTS: The planting rate of hepatocytes in the DSM was (74.5∓7.7)%. HE staining and scanning electron microscopy showed satisfactory cell status, and immunofluorescence staining confirmed the normal expression of ALB and G6Pc in the cells. For TEL implantation, ectopic vascular anastomosis was difficult and resulted in a mortality rate of 33.3% perioperatively and massive thrombus formation in the matrix within 6 h. Hepatic cross-section suture failed to rapidly establish sufficient blood supply, and no viable graft was observed 3 days after the operation. With intrahepatic insertion method, the hepatocytes in the DSM could survive as long as 14 days. Mesenteric transplantation resulted in a hepatocyte survival rate of (38.3+7.1)% at 14 days after implantation. CONCLUSION: TEL constructed based on DSM can perform liver-specific functions with a good cytological bioactivity. Mesenteric transplantation of the TEL, which is simple, safe and effective, is currently the optimal transplantation strategy.

SNAI1, an endothelial-mesenchymal transition transcription factor, promotes the early phase of ocular neovascularization.

Ocular neovascularization is a comprehensive process involved in retinal vascular development and several blinding diseases such as age-related macular degeneration and retinopathy of prematurity, with vascular endothelial growth factor (VEGF) regarded as the master regulator. However, the qualified effect of anti-VEGF therapy reveals that the underlying mechanisms are still not clearly identified. To initialize angiogenesis, endothelial cells undergo a phenotype switching to generate highly migratory and invasive cells. This process shares certain similar characters observed in endothelial-mesenchymal transition (EndMT). Here, we found that SNAI1, an EndMT transcription factor, was expressed by endothelial cells in both physiological and pathological ocular neovascularization. SNAI1 overexpression triggered cell morphological change and enhanced cell motility, while loss of SNAI1 attenuated migration, invasion and sprouting. RNA sequence analysis further revealed that SNAI1 knockdown decreased the expression of genes related to cytoskeleton rearrangement and ECM remodeling. Moreover, intravitreal injection of small interfering RNA of SNAI1 suppressed new vessel formation in developing retina as well as mice model of choroidal neovascularization and oxygen-induced retinopathy. Therefore, we propose that the EndMT transcription factor SNAI1 promotes the early phase of ocular neovascularization and may provide a potential therapeutic target.

Macrophages promote vasculogenesis of retinal neovascularization in an oxygen-induced retinopathy model in mice.

To investigate the role of macrophages in oxygen-induced retinal neovascularization (NV) in mice, particularly the involvement of bone marrow-derived cells (BMCs) and the underlying mechanisms, BMCs from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P) 1 mice after irradiation. The mice were exposed to 75 % oxygen from P7 to P12 to initiate oxygen-induced retinopathy (OIR). The macrophages were depleted by injection of clodronate-liposomes (lip) intraperitoneally. The eyes were collected at P12 and P17. Retinal flatmounts and histopathological cross-sections were performed to analyze the severity of retinal NV and BMC recruitment. BMCs immunopositive for CD31 (PECAM-1; endothelial cell marker) and α-SMA (smooth muscle cell marker) antigens were detected using a confocal microscope. Expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) mRNA was detected by RT-PCR. The VEGF, SDF-1, CXCR4 and CD45 protein expression was detected by western blot examination. The retinal avascular area in OIR mice at P12 was unaffected after macrophage depletion carried out twice (38.27 ± 1.92 % reduction) using clodronate-lip. The retinal avascular area and the NV area at P17 were reduced after macrophage depletion four times (79.53 ± 1.02 % reduction); these findings were supported by retinal flatmounts and histopathological cross-sections. Macrophage depletion led to significant inhibition of BMC recruitment into the NV tufts at P17, with decreased expression of retinal VEGF, SDF-1, CXCR4 and CD45. The recruited BMCs differentiated primarily into CD31-positive endothelial cells (ECs) and α-SMA-positive smooth muscle cells (SMCs). This study suggested that macrophages promoted the vasculogenesis of retinal NV, particularly the contribution of BMCs in the mouse OIR model, which might be triggered by VEGF and SDF-1 production.

In-vivo investigation of laser-induced choroidal neovascularization in rat using spectral-domain optical coherence tomography (SD-OCT).

PURPOSE: This study investigated the in-vivo formation process of laser-induced choroidal neovascularization (CNV) in rat using high-resolution spectral-domain optical coherence tomography (SD-OCT), and compared the results to histological methods. METHODS: Brown Norway rats (n = 60, 6-8 weeks of age) received 532-nm diode laser photocoagulation. SD-OCT and fluorescein angiography (FA) were performed in vivo 2, 5, 7, 14, and 21 days post-laser application. Haematoxylin and eosin (H&E) staining and immunohistochemistry for CD31, phosphorylated vascular endothelial factor receptor 2 (pVEGFR2) were conducted at each time point to observe the CNV in vitro. Choroidal flatmount preparations were observed using a confocal laser scanning microscope (CLSM) and a scanning electron microscope (SEM). RESULTS: SD-OCT monitored the longitudinal morphological changes of laser-induced CNV. CNV reached its maximal size on day 7, and began a gradual reduction on day 14. FA revealed similar dynamic changes in leakage. CNV thickness, as assessed by SD-OCT, was consistent with H&E-stained sections at each time point. CLSM and SEM revealed the details of the fibrovascular membrane. CD31 and pVEGFR2 expression supported the results of SD-OCT and histology. CONCLUSIONS: SD-OCT was a convenient and reliable tool for the imaging of the CNV formation process and quantification of the lesion size in vivo.