Downregulation of GPX8 in hepatocellular carcinoma: impact on tumor stemness and migration.

PURPOSE: GPX8, which is found in the endoplasmic reticulum lumen, is a member of the Glutathione Peroxidases (GPXs) family. Its role in hepatocellular carcinoma (HCC) is unknown. METHODS: Immunohistochemical staining was used to detect the protein levels of GPX8 in HCC tissue microarrays. A short hairpin RNA lentivirus was used to knock down GPX8, and the main signaling pathways were investigated using transcriptome sequencing and a phosphorylated kinase array. The sphere formation assays, cloning-formation assays and cell migration assays were used to evaluate the stemness and migration ability of HCC cells. Identifying the GPX8-interacting proteins was accomplished through immunoprecipitation and protein mass spectrometry. RESULTS: The GPX8 protein levels were downregulated in HCC patients. Low expression of GPX8 protein was related to early recurrence and poor prognosis in HCC patients. GPX8 knockdown could enhance the stemness and migration ability of HCC cells. Consistently, Based on transcriptome analysis, multiple signaling pathways that include the PI3K-AKT and signaling pathways that regulate the pluripotency of stem cells, were activated after GPX8 knockdown. The downregulation of GPX8 could increase the expression of the tumor stemness markers KLF4, OCT4, and CD133. The in vivo downregulation of GPX8 could also promote the subcutaneous tumor-forming and migration ability of HCC cells. MK-2206, which is a small-molecule inhibitor of AKT, could reverse the tumor-promoting effects both in vivo and in vitro. We discovered that GPX8 and the 71-kDa heat shock cognate protein (Hsc70) have a direct interaction. The phosphorylation of AKT encouraged the translocation of Hsc70 into the nucleus and the expression of the PI3K p110 subunit, thereby increasing the downregulation of GPX8. CONCLUSION: The findings from this study demonstrate the anticancer activity of GPX8 in HCC by inactivating the Hsc70/AKT pathway. The results suggest a possible therapeutic target for HCC.

Development and Comparison of Treatment Decision Tools for Glucocorticoid-Induced Osteoporosis.

Long-term Glucocorticoid (GC) use results in compromised bone strength and fractures, and several treatment recommendations have been developed to prevent fractures, but none have been validated in a real-world setting. This study aims to create a treatment decision tool and compares this tool to the treatment suggestions from the American College of Rheumatology (ACR), International Osteoporosis Foundation and European Calcified Tissue Society (IOF-ECTS), and GC-adjusted Fracture Risk Assessment Tool (GC-FRAX), above the intervention threshold. We utilized registry data gathered at Chang Gung Memorial Hospital at Kaohsiung, Taiwan, between September 2014 and April 2021. This research is a single-center, observational, and case-controlled study. We recruited participants using prednisone for at least 2.5 mg/day or the equivalent dose for over 3 months, excluding those younger than 40, those with malignancies, or those currently undergoing anti-osteoporosis therapy. The primary endpoint was new fragility fractures within 3 years, including morphometric vertebral fractures detected at baseline and with a follow-up thoracic-lumbar spine X-ray. Participants were randomly allocated into derivation and validation sets. We developed the Steroid-Associated Fracture Evaluation (SAFE) tool in the derivation cohort by assessing the weights of exploratory variables via logistic regression. Prediction performance was compared in the validation set by the receiver operating characteristic (ROC) curve, the area under the curve (AUC), and sensitivity and specificity. A total of 424 treatment-naïve subjects were enrolled, and 83 (19.6%) experienced new fractures within 3 years. The final formula of the SAFE tool includes osteoporosis (1 point), an accumulated GC dose ≥ 750 mg within 6 months (or equivalent prednisolone of ≥4.5 mg/day for 6 months) (1 point), a BMI ≥ 23.5 (1 point), previous fractures (1 point), and elderliness of ≥70 years (2 points). In the validation set, a treatment decision based on the SAFE ≥ 2 points demonstrated an AUC of 0.65, with a sensitivity/specificity/accuracy of 75.9/54.0/58.9, with an ACR of 0.56 (100.0/11.0/31.0), IOF-ECTS 0.61 (75.9/46.0/52.7), and GC-FRAX 0.62 (82.8/42.0/51.2). Among current GIOP recommendations, the SAFE score serves as an appropriate treatment decision tool with increased accuracy and specificity.

Autophagy suppression facilitates macrophage M2 polarization via increased instability of NF-κB pathway in hepatocellular carcinoma.

The tumor microenvironment is a highly heterogeneous circumstance composed of multiple components, while tumor-associated macrophages (TAMs) are major innate immune cells with highly plastic and are always educated by tumor cells to structure an advantageous pro-tumor immune microenvironment. Despite emerging evidence focalizing the role of autophagy in other immune cells, the regulatory mechanism of autophagy in macrophage polarization remains poorly understood. Herein, we demonstrated that hepatocellular carcinoma (HCC) cells educated macrophages toward M2-like phenotype polarization under the condition of coculture. Moreover, we observed that inhibition of macrophage autophagy promoted M2-like macrophage polarization, while the tendency was impeded when autophagy was motivated. Mechanistically, macrophage autophagy inhibition inactivates the NF-κB pathway by increasing the instability of TAB3 via ubiquitination degradation, which leads to the M2-like phenotype polarization of macrophages. Both immunohistochemistry staining using human HCC tissues and experiment in vivo verified autophagy inhibition is correlated with M2 macrophage polarization. Altogether, we illustrated that macrophage autophagy was involved in the process of HCC cells domesticating M2 macrophage polarization via the NF-κB pathway. These results provide a new target to interfere with the polarization of macrophages to M2-like phenotype during HCC progression.

Disease-modifying anti-rheumatic drugs associated with different diabetes risks in patients with rheumatoid arthritis.

OBJECTIVES: Patients with rheumatoid arthritis are prone to developing diabetes, which may lead to various sequelae and even cardiovascular diseases, the most common cause of death in such patients. Previous research has shown that some rheumatoid arthritis treatments may help prevent the development of diabetes. This study aimed to investigate whether patients using disease-modifying anti-rheumatic drugs (DMARDs) may have different levels of risk for diabetes and to analyse other risk factors for diabetes. METHODS: This cohort study used data from the Chang Gung Research Database. 5530 adults with rheumatoid arthritis but without diabetes were eligible for the analysis. The endpoint of this study was new-onset diabetes, defined as an HbA1c value ≥7% during follow-up. The entire follow-up period was divided into monthly subunits. These 1-month units were then divided into methotrexate (MTX) monotherapy, any biological DMARDs (bDMARDs), MTX combination, other conventional DMARDs (cDMARDs) and non-DMARDs. RESULTS: A total of 546 participants (9.87%) developed diabetes between 2001 and 2018. The risk of diabetes was significantly lower in the bDMARD periods (HR 0.51; 95% CI 0.32 to 0.83), MTX combination periods (HR 0.50; 95% CI 0.32 to 0.78) and other cDMARD periods (HR 0.56; 95% CI 0.37 to 0.84) than in the MTX monotherapy periods. Individual drug analysis showed that hydroxychloroquine (HR 0.52; 95% CI 0.42 to 0.65) reduced the risk of diabetes. Tumour necrosis factor-α inhibitors (HR 0.69; 95% CI 0.46 to 1.03) tended to be protective. CONCLUSION: Patients with rheumatoid arthritis may have different levels of risk of diabetes depending on the treatment options.

Transcriptomic analysis reveals the potential biological mechanism of AIS and lung adenocarcinoma.

Introduction: Acute ischemic stroke (AIS) and lung adenocarcinoma (LUAD) are associated with some of the highest morbidity and mortality rates worldwide. Despite reports on their strong correlation, the causal relationship is not fully understood. The study aimed to identify and annotate the biological functions of hub genes with clinical diagnostic efficacy in AIS and LUAD. Methods: Transcriptome and single-cell datasets were obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). We identified the differentially expressed genes (DEGs) upregulated in AIS and LUAD and found 372 genes intersecting both datasets. Hub genes were identified using protein-protein interaction (PPI) networks, and the diagnostic and prognostic utility of these hub genes was then investigated using receiver operating characteristic (ROC) curves, survival analysis, and univariable Cox proportional hazard regression. Single-cell analysis was used to detect whether the hub genes were expressed in tumor epithelial cells. The immune microenvironment of AIS and LUAD was assessed using the CIBERSORT algorithm. The protein expression of these hub genes was tracked using the Human Protein Atlas (HPA). We calculated the number of positive cells using the digital pathology software QuPath. Finally, we performed molecular docking after using the Enrichr database to predict possible medicines. Results: We identified the molecular mechanisms underlying hub genes in AIS and LUAD and found that CCNA2, CCNB1, CDKN2A, and CDK1 were highly expressed in AIS and LUAD tissue samples compared to controls. The hub genes were mainly involved in the following pathways: the cell cycle, cellular senescence, and the HIF-1 signaling pathway. Using immunohistochemical slices from the HPA database, we confirmed that these hub genes have a high diagnostic capability for AIS and LUAD. Further, their high expression is associated with poor prognosis. Finally, curcumin was tested as a potential medication using molecular docking modeling. Discussion: Our findings suggest that the hub genes we found in this study contribute to the development and progression of AIS and LUAD by altering the cellular senescence pathway. Thus, they may be promising markers for diagnosis and prognosis.

Factors associated with subclinical inflammation of wrist joints in rheumatoid arthritis patients with low or no disease activity- a RA ultrasound registry study.

BACKGROUND: To evaluate the factors to predict subclinical inflammation of wrist joints in patients with RA who are in clinical remission or low disease activity. METHODS: Gray scale and power Doppler ultrasound were performed on the dorsal radio-lunate of both wrists. The presence of synovitis, comorbidities, and use of disease modifying anti-rheumatic drugs were recorded. A Multivariable forward logistical regression model was used to identify factors associated with subclinical inflammation. RESULTS: There were 1248 patients (1010 females, 238 males; mean age: 60.0 ± 10.5 years ). 57.4% of patients in complete remission and low disease activity had sonographic inflammation. Multivariable forward logistic regression analysis indicated that male sex, smoking are positively associated with inflammation and that age, alcohol consumption, and use of methotrexate, glucocorticoid, or a biological therapy are negatively associated with inflammation. Use of biological agents decreased the risk of inflammation by 40.9%. CONCLUSIONS: There was evidence of subclinical inflammation in most patients who were in low or no disease activity, those with biological therapy had lower risk of subclinical inflammation.

Development of a deep pathomics score for predicting hepatocellular carcinoma recurrence after liver transplantation.

BACKGROUND AND PURPOSE: Tumor recurrence after liver transplantation (LT) impedes the curative chance for hepatocellular carcinoma (HCC) patients. This study aimed to develop a deep pathomics score (DPS) for predicting tumor recurrence after liver transplantation using deep learning. PATIENTS AND METHODS: Two datasets of 380 HCC patients who underwent LT were enrolled. Residual convolutional neural networks were used to identify six histological structures of HCC. The individual risk score of each structure and DPS were derived by a modified DeepSurv network. Cox regression analysis and Concordance index were used to evaluate the prognostic significance. The cellular exploration of prognostic immune biomarkers was performed by quantitative and spatial proximity analysis according to three panels of 7-color immunofluorescence. RESULTS: The overall classification accuracy of HCC tissue was 97%. At the structural level, immune cells were the most significant tissue category for predicting post-LT recurrence (HR 1.907, 95% CI 1.490-2.440). The C-indices of DPS achieved 0.827 and 0.794 in the training and validation cohorts, respectively. Multivariate analysis for recurrence-free survival (RFS) showed that DPS (HR 4.795, 95% CI 3.017-7.619) was an independent risk factor. Patients in the high-risk subgroup had a shorter RFS, larger tumor diameter and a lower proportion of clear tumor borders. At the cellular level, a higher infiltration of intratumoral NK cells was negatively correlated with recurrence risk. CONCLUSIONS: This study established an effective DPS. Immune cells were the most significant histological structure related to HCC recurrence. DPS performed well in post-LT recurrence prediction and the identification of clinicopathological features.

Inhibition of autophagy in macrophage promotes IL-1ß-mediated hepatocellular carcinoma progression via inflammasome accumulation and self-recruitment.

Hepatocellular carcinoma (HCC) has a complex and changeable tumor microenvironment. Despite emerging evidence focusing on autophagy process within immune cells, the function and regulatory mechanism of macrophage autophagy in tumor progression remains unclear. Our results of multiplex-immunohistochemistry and RNA-sequencing identified the reduced levels of autophagy in tumor macrophages in the HCC microenvironment, associated with a poor prognosis and increased microvascular metastasis in HCC patients. Specifically, HCC suppressed the macrophage autophagy initiation through the up-regulation of mTOR and ULK1 phosphorylation at Ser757. Knockdown of autophagy-related proteins to further inhibit autophagy significantly boosted the metastatic potential of HCC. Mechanistically, the accumulation of NLRP3 inflammasome mediated by autophagy inhibition promoted the cleavage, maturation, and release of IL-1ß, which facilitated the HCC progression, eventually accelerating HCC metastasis via the epithelial-mesenchymal transition. Autophagy inhibition provoked macrophage self-recruitment through the CCL20-CCR6 signaling was also a crucial account of HCC progression. Recruited macrophages mediated the cascade amplification of IL-1ß and CCL20 to form a novel pro-metastatic positive feedback loop through promoting HCC metastasis and increased macrophage recruitment, respectively. Notably, targeting IL-1ß/IL-1 receptor signaling impaired lung metastasis induced by macrophage autophagy inhibition in a mice HCC lung metastasis model. In summary, this study highlighted that inhibition of tumor macrophage autophagy facilitated HCC progression by increasing IL-1ß secretion via NLRP3 inflammasome accumulation and by macrophage self-recruitment through the CCL20 signaling pathway. Interruption of this metastasis-promoting loop by IL-1ß blockade may provide a promising therapeutic strategy for HCC patients.

Danazol in Refractory Autoimmune Hemolytic Anemia or Immune Thrombocytopenia: A Case Series Report and Literature Review.

Danazol is a treatment option for autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP). Three patients with AIHA and eight patients with ITP between 2008 and 2022 were enrolled in the Rheumatology Outpatient Clinic of Chang Gung Memorial Hospital, Kaohsiung. Those patients were refractory or intolerant to conventional therapy and were treated with danazol. All the patients received an initial dose of danazol (200-400 mg). The observation period was 6 months. Three patients (100%) with AIHA and six (75%) with ITP achieved treatment response after 6 months of danazol therapy. The dose of glucocorticoid for responders could be reduced to ≤5 mg/day of prednisolone, and the immunosuppressants, except hydroxychloroquine and azathioprine for systemic lupus erythematosus, could be discontinued. Adverse events were acne in two (18.2%) patients and transient dose-related liver function impairment in one (9.1%) patient in the current series. Danazol therapy appears to be a favorable alternative for refractory AIHA and ITP by altering the erythrocyte membrane to resist osmotic lysis and protecting platelets against complement-mediated lysis. In this report, we also performed a literature review and searched the PubMed/Cochrane Library for articles published from 1984 to January 2022 on danazol therapy for patients with AIHA and ITP.

Effectiveness and Safety of Avatrombopag in Liver Cancer Patients with Severe Thrombocytopenia: Real-World Data and Challenges.

Background: Avatrombopag has been approved in patients who have severe thrombocytopenia (<50 × 109/L) and chronic liver disease (CLD) while receiving invasive procedures. The real-world application and effectiveness of avatrombopag in the subgroup patients with liver cancer remain unknown. Methods: Liver cancer patients (including primary liver cancer and colorectal cancer liver metastasis) who had severe thrombocytopenia and received avatrombopag were retrospectively enrolled. Avatrombopag dose, peak and absolute platelet count increase, combination treatment with other thrombopoietic agents, responder (peak count ≥50 × 109/L with absolute increase ≥20 × 109/L) rate, and anticancer treatment effect were analyzed. Thrombosis and bleeding events were assessed. Results: In total, 93 patients were enrolled, with 72 and 21 in the CLD and non-CLD groups, respectively. Patients with CLD had hepatitis B or C, larger spleen volume, and a higher cirrhosis degree. Baseline platelet counts were similar between two groups (median, 37.0 × 109/L vs. 39.0 × 109/L; P=0.594), while patients without CLD had higher peak platelet (median, 134.0 × 109/L vs. 74.0 × 109/L; P=0.015) and absolute increase (median, 101.0 × 109/L vs. 41.0 × 109/L; P=0.020) after avatrombopag treatment. The responder rate was higher in patients without CLD (100% vs. 76.4%; P=0.010). Combined avatrombopag with other thrombopoietic agents significantly increased platelet count; repeated use of avatrombopag produced similar effects with that of initial treatment. Concerning anticancer treatment effect, patients who responded to avatrombopag had a higher disease control rate. No thrombosis or hemorrhagic events were observed, even in patients with portal vein tumor thrombosis. Conclusion: Avatrombopag was safe and effective and ensured successful implementation of anticancer treatment in liver cancer patients with severe thrombocytopenia, accompanied with or without CLD.

Hip BMD is associated with visceral fat change: a registry study of osteoporosis and sarcopenia.

Background: Osteoporosis increases the risk of fractures. Visceral fat is associated with cardiovascular disease (CVD). There is inadequate knowledge on the relationship between osteoporosis and visceral fat. The study aimed to evaluate the relationship between bone mineral density (BMD) and visceral fat mass in the elderly. Methods: This was a prospective cohort study. Subjects were enrolled from the Rheumatology Clinic. All subjects underwent baseline bone mineral density and body composition measurements using dual-energy X-ray absorptiometry. Results: A total of 321 patients including 288 females and 33 males were enrolled in this study. We followed up DEXA for 1 year for fat and muscle mass change and found that 162 (50.5%) had a decrease in fat mass, 129 (40.2%) had decreased visceral fat, and 138 (43%) had decreased muscle mass. Furthermore, we found that the baseline hip T score was correlated with visceral fat decrease. Using visceral fat decrease as the outcome, we found that hip T score could predict visceral fat loss: the higher the T score, the more visceral fat loss was found [p < 0.001, OR: 1.6, CI: (1.3-2.1)]. Conclusion: A high hip T score was associated with a future decrease in visceral fat, which may decrease the risk of atherosclerosis and CV risk. Therefore, evaluation of visceral fat may be useful for assessing CVD risk in patients with osteoporosis. Effective management of the risk of atherosclerosis and CVD is important in improving the life expectancy of these patients.

Establishing the Competency Development and Talent Cultivation Strategies for Physician-Patient Shared Decision-Making Competency Based on the IAA-NRM Approach.

Shared decision making (SDM) is a collaborative process involving patients and their healthcare workers negotiating to reach a shared decision about medical care. However, various physician stakeholders (attending physicians, medical residents, and doctors in post-graduate years) may have different viewpoints on SDM processes. The purpose of this study is to explore the core competence of physicians in performing SDM tasks and to investigate the significant competency development aspects/criteria by applying the literature research and expert interviews. We adopt the IAA (importance awareness analysis) technique for different stakeholders to evaluate the status of competency development aspects/criteria and to determine the NRM (network relation map) based on the DEMATEL (decision-making trial and evaluation laboratory) technique. The study combines the IAA and NRM methods and suggests using the IAA-NRM approach to evaluate the adoption strategies and common suitable paths for different levels of physicians. Our findings reveal that SDM perception and practice is the primary influencer of SDM competence development for all stakeholders. The current model can help hospital administrators and directors of medical education understand the diverse stakeholders' perspectives on the core competence of SDM tasks and determine common development plans. It provides strategic directions for SDM competency development and talent cultivation programs.

Determining the Development Strategy and Suited Adoption Paths for the Core Competence of Shared Decision-Making Tasks through the SAA-NRM Approach.

Shared decision making (SDM) is an interactive process that involves patients and their healthcare professionals reaching joint decisions about medical care through negotiation. As the initiators of medical decision-making in daily routine, physicians should be aware of and concerned about the SDM process. Thus, professional competency development for SDM has become increasingly critical for physicians' training. Therefore, this study investigates the professional competency and the important competency development aspects/criteria of SDM tasks through expert interviews and literature research. The study adopts the SAA (satisfaction-attention analysis) method to assess the status of competency development aspects/criteria and determine the NRM (network relation map) based on the DEMATEL (decision-making trial and evaluation laboratory) technique. The results demonstrate that the CE (concept and evaluation) aspect is the dominant aspect, and the CR (communication and relationship) aspect is the aspect being dominated. The CE aspect influences the aspects of SP (skill and practice), JM (joint information and decision making) and CR, and the SP aspect affects the aspects of JM and CR. Then, the JM aspect affects the CR aspect. The study also suggests suitable adoption paths of competency development for SDM tasks using the NRM approach. It provides recommendations and strategic directions for SDM competency development and sustainable training programs.

The Impact of Seropositivity on Systemic Bone Loss in Rheumatoid Arthritis-A 3-Year Interim Analysis of a Longitudinal Observational Cohort Study.

Objective: To explore the impact of seropositivity on systemic bone loss in rheumatoid arthritis (RA). Methods: We conducted an interim analysis of the RA registry. Patients were examined with dual-energy X-ray absorptiometry at baseline and again 3 years later. Participants were grouped into seropositive (SPRA) and seronegative (SNRA) based on the presence or absence of rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (ACPA). After matching (1:2) for age and sex, SNRA and SPRA patients were divided into groups A and B. Each matched group (A or B) was further subdivided according to the number of antibodies present (0, group I; 1, group II; 2, group III). Multiple ordinary least squares regression was used with the dependent variables to develop a model to predict bone mineral density (BMD) change. Results: A total of 477 participants who completed a 3-year observation period were included. After matching, 312 participants were enrolled (group A, 104; group B, 208). Three years later, group B had significant BMD reduction in the femoral neck (FN) (p < 0.001), total hip (TH) (p = 0.001), and first through fourth lumbar vertebrae (L1-4) (p = 0.006), while group A had bone loss only at FN (p = 0.002). Groups I, II, and III included 104, 52, and 156 participants, respectively. Compared to baseline, BMD decreased significantly at FN (p = 0.002) in group I, FN (p < 0.001) in group II, and FN (p < 0.001), TH (p = 0.002), and L1-4 (p = 0.016) in group III. In terms of regression-adjusted percent change in BMD, more significantly negative changes were found at all measured sites in group B (p < 0.001, all) and at TH and L1-4 within groups I-III (p for trend < 0.001 and < 0.001, respectively). Regardless of antibodies, anti-osteoporotic therapy can preserve bone density in RA patients. Conclusion: After 3 years, SPRA patients lost more bone density than SNRA patients. More attention should be paid to SPRA patients, especially those with double-positive antibodies, including a vigorous evaluation of BMD and fracture risk. Anti-osteoporotic therapy can prevent BMD loss irrespective of autoantibodies.

Establishment of a preliminary FRAX®-based intervention threshold for rheumatoid arthritis-associated fragility fracture: a 3-year longitudinal, observational, cohort study.

BACKGROUND: To establish a FRAX®-based prediction model for rheumatoid arthritis (RA)-associated fragility fracture. METHODS: This study is a longitudinal, real-world, registry cohort study. Patients with RA were registered to start in September 2014. The baseline demographics, bone mineral density (BMD), and risk factors of osteoporosis or fragility fracture were recorded. Subsequent fragility fractures during the 3-year observation period were also recorded. We developed a fixed intervention threshold (FITD) to identify fractures by choosing an optimal cut-off point on the receiver operating characteristic (ROC) curve and FRAX®. Several models for intervention thresholds (IT), including fixed intervention threshold (Taiwan) (FITT), age-specific individual intervention threshold (IIT), and hybrid intervention threshold (HIT), were compared to evaluate which IT model will have better discriminative power. RESULTS: As of December 2020, a total of 493 RA participants have completed the 3-year observation study. The mean age of the participants was 59.3 ± 8.7, and 116 (23.5%) new fragility fractures were observed during the study period. In terms of pairwise comparisons of area under the curve (n, 95% confidence interval) in the ROC curve, the FITD (0.669, 0.610-0.727, p < 0.001) with a value of 22% in major osteoporotic fracture and FITT (0.640, 0.582-0.699, p < 0.001) is significantly better than reference, but not for IIT (0.543, 0.485-0.601, p = 0.165) and HIT (0.543, 0.485-0.601, p = 0.165). CONCLUSION: An optimal FIT is established for intervention decisions in RA-associated fragility fractures. This model can offer an easy and simple guide to aid RA caregivers to provide interventions to prevent fragility fractures in patients with RA.

KRAS acting through ERK signaling stabilizes PD-L1 via inhibiting autophagy pathway in intrahepatic cholangiocarcinoma.

BACKGROUND: While the correlation between PD-L1 expression and KRAS mutation has been previously reported in other solid tumors such as non-small cell lung cancer (NSCLC), whether PD-L1 can be modulated by ERK signaling downstream of KRAS in intrahepatic cholangiocarcinoma (iCCA) and the underlying molecular regulatory mechanism remain unclear. METHODS: The expression of ERK, p-ERK, PD-L1 and autophagy markers following KRAS knockdown or Ras/Raf/MEK/ERK signaling inhibitors treatment was examined in two human iCCA cell lines (HuCCT1 and RBE) using western blotting and immunofluorescence. Both pharmacological autophagy inhibitors and short-interfering RNA against ATG7 were applied to inhibit autophagy. The apoptosis rates of iCCA cell lines were detected by flow cytometry and CCK-8 after co-culturing with CD3/CD28-activated human CD8+ T lymphocytes. Immunohistochemistry was applied to detect the correlation of ERK, p-ERK and PD-L1 in 92 iCCA tissues. RESULTS: The present study demonstrated that the PD-L1 expression level was distinctly reduced in KRAS-mutated iCCA cell lines when ERK signaling was inhibited and ERK phosphorylation levels were lowered. The positive association between p-ERK and PD-L1 was also verified in 92 iCCA tissue samples. Moreover, ERK inhibition induced autophagy activation. Both inhibiting autophagy via autophagy inhibitors and genetically silencing the ATG7 expression partially reversed the reduced PD-L1 expression caused by ERK inhibition. In addition, ERK-mediated down-regulation of PD-L1 via autophagy pathways induced the apoptosis of iCCA cells when co-cultured with CD3/CD28-activated human CD8+ T lymphocytes in vitro. CONCLUSIONS: Our results suggest that ERK signaling inhibition contributes to the reduction of PD-L1 expression through the autophagy pathway in iCCA. As a supplement to anti-PD-1/PD-L1 immunotherapy, ERK-targeted therapy may serve as a potentially novel treatment strategy for human KRAS-mutated iCCA.

Targeted activation of androgen receptor signaling in the periosteum improves bone fracture repair.

Low testosterone level is an independent predictor of osteoporotic fracture in elderly men as well as increased fracture risk in men undergoing androgen deprivation. Androgens and androgen receptor (AR) actions are essential for bone development and homeostasis but their linkage to fracture repair remains unclear. Here we found that AR is highly expressed in the periosteum cells and is co-localized with a mesenchymal progenitor cell marker, paired-related homeobox protein 1 (Prrx1), during bone fracture repair. Mice lacking the AR gene in the periosteum expressing Prrx1-cre (AR-/Y;Prrx1::Cre) but not in the chondrocytes (AR-/Y;Col-2::Cre) exhibits reduced callus size and new bone volume. Gene expression data analysis revealed that the expression of several collagens, integrins and cell adhesion molecules were downregulated in periosteum-derived progenitor cells (PDCs) from AR-/Y;Prrx1::Cre mice. Mechanistically, androgens-AR signaling activates the AR/ARA55/FAK complex and induces the collagen-integrin α2ß1 gene expression that is required for promoting the AR-mediated PDCs migration. Using mouse cortical-defect and femoral graft transplantation models, we proved that elimination of AR in periosteum of host mice impairs fracture healing, regardless of AR existence of transplanted donor graft. While testosterone implanted scaffolds failed to complete callus bridging across the fracture gap in AR-/Y;Prrx1::Cre mice, cell-based transplantation using DPCs re-expressing AR could lead to rescue bone repair. In conclusion, targeting androgen/AR axis in the periosteum may provide a novel therapy approach to improve fracture healing.

Simple Self-Assessment Tool to Predict Osteoporosis in Taiwanese Men.

Background: Although the self-assessment tools for predicting osteoporosis are convenient for clinicians, they are not commonly used among men. We developed the Male Osteoporosis Self-Assessment Tool for Taiwan (MOSTAi) to identify the patients at risk of osteoporosis. Methods: All the participants completed a questionnaire on the clinical risk factors for the fracture risk assessment tool. The risk index was calculated by the multivariate regression model through the item reduction method. The receiver operating characteristic (ROC) curve was used to analyze its sensitivity and specificity, and MOSTAi was developed and validated. Results: A total of 2,290 men participated in the bone mineral density (BMD) survey. We chose a model that considered two variables (age and weight). The area under the curve (AUC) of the model was 0.700. The formula for the MOSTAi index is as follows: 0.3 × (weight in kilograms) - 0.1 × (years). We chose 11 as the appropriate cut-off value for the MOSTAi index to identify the subjects at the risk of osteoporosis. Conclusions: The MOSTAi is a simple, intuitive, and country-specific tool that can predict the risk of osteoporosis in Taiwanese men. Due to different demographic characteristics, each region of the world can develop its own model to identify patients with osteoporosis more effectively.

Risk factor analysis of fragility fractures in rheumatoid arthritis: A 3-year longitudinal, real-world, observational, cohort study.

OBJECTIVES: To explore the risk factors for fragility fractures in rheumatoid arthritis (RA) patients using a 3-year longitudinal, observational cohort study. METHODS: This RA registry study included consecutive RA patients in the outpatient clinic of Chang Gung Memorial Hospital since September 1, 2014. The demographics, clinical characteristics, lifestyle, evidence of previous fracture, risk factors according to the Fracture Risk Assessment Tool (FRAX®), and the FRAX score of each participant were recorded. The participants were categorized into the new incident fracture (group A) and no incident fracture (group B) groups based on evidence or absence of new incident fractures and propensity score matching (age and gender, 1:2). RESULTS: Overall, 477 participants completed the 3-year observation period. After matching, 103 and 206 participants were allocated to groups A and B, respectively. The non-adjusted model revealed, presented as hazard ratio (HR) (95% confidence interval [CI]), that the presence of co-morbidity (1.80 [1.17-2.78], p = 0.008), Health Assessment Questionnaire Disability Index (1.35 [1.07-1.69], p = 0.010), lower baseline hip bone mineral density (0.11 [0.02-0.48], p = 0.004), longer disease duration (1.02 [1.00-1.04], p = 0.026), higher FRAX score of major fracture (1.03 [1.02-1.04], p<0.001) or hip fracture (1.03 [1.02-1.04], p<0.001), and previous fracture history (2.65 [1.79-3.94], p<0.001) were associated with new incident fracture. After adjustment, it was disclosed that a previous fracture is an independent risk factor for fragility fractures in RA patients (2.17 [1.20-3.90], p = 0.010). CONCLUSIONS: In addition to aging and disease-related factors, previous fracture history is the most important risk factor for fragility fractures in RA patients.