Treatment rationale and protocol design: an investigator-initiated phase II study of combination treatment of nivolumab and TM5614, a PAI-1 inhibitor for previously treated patients with non-small cell lung cancer.

Background: There is no established standard 3rd line treatment for patients with advanced non-small cell lung cancer (NSCLC). Although cytotoxic chemotherapeutic agents that are not used as 1st or 2nd line treatment are administrated as 3rd line treatment, their anti-tumor efficacy is insufficient. Anti-programmed death ligand-1 (PD-L1)/programmed death-1 (PD1) treatment is more effective and less toxic than chemotherapy in anti-PD-L1/PD-1 treatment-naïve patients with NSCLC. Therefore, anti-PD-L1/PD-1 therapy is considered an appropriate 3rd line treatment. However, the anti-tumor efficacy is limited in patients previously treated with anti-PD-L1/PD-1 antibody. Today, new drugs are needed to increase the efficacy of anti-PD-L1/PD-1 antibodies. Methods: This open-label, single-arm, investigator-initiated phase II study is designed to evaluate combination treatment of nivolumab and TM5614, a plasminogen activator inhibitor (PAI-1) inhibitor as 3rd or more line treatment in NSCLC patients who underwent standard treatment. The primary endpoint is the objective response rate and the secondary endpoints are progression-free survival (PFS), overall survival (OS), duration of response (DOR) and safety. Recruitment began in September 2023 and is expected to continue for approximately three years. Discussion: Currently, there is no standard 3rd line treatment for advanced NSCLC, and we hope that the findings of this study will facilitate more effective treatments in this setting. Ethics and dissemination: the study protocol conformed to the ethical principles outlined in the Declaration of Helsinki. All patients will provide written informed consent prior to enrollment. Results will be published in a peer-reviewed publication. Trial Registration: This study is registered to Japan Registry of Clinical Trials with number: jRCT2061230039 (19/July/2023).

D-dimer cut-off value for predicting venous thromboembolism at the initial diagnosis in Japanese patients with advanced lung cancer.

OBJECTIVE: Cancer is a well-known risk factor for venous thromboembolism. The D-dimer level is used to predict venous thromboembolism; however, reports on an appropriate D-dimer cut-off value in Japanese patients with advanced lung cancer are lacking. Therefore, this study aimed to calculate the D-dimer cut-off value for venous thromboembolism at the time of lung cancer diagnosis. METHODS: The Rising-venous thromboembolism/NEJ037 study was a multicenter, prospective observational study. Patients with lung cancer who were contraindicated for radical resection or radiation were enrolled and followed up for 2 years. In the present study (jRCT no. 061180025), a receiver operating characteristic curve for D-dimer levels was created using the dataset of the Rising-venous thromboembolism/NEJ037 study. RESULTS: The Rising-venous thromboembolism/NEJ037 study included a total of 1008 patients, of whom 976, whose D-dimer levels had been measured at the time of cancer diagnosis, were included in the present study. At the time of lung cancer diagnosis, 62 (6.3%) and 914 (93.7%) patients presented with and without venous thromboembolism, respectively. The D-dimer values ranged from 0.1 to 180.1 µg/ml and from 0.1 to 257.2 µg/ml in patients with and without venous thromboembolism, respectively. The receiver operating characteristic curve was discriminative with a cut-off value of 3.3 µg/ml and an area under the curve of 0.794 (sensitivity, 0.742; specificity, 0.782; 95% confidence interval, 0.725-0.863). CONCLUSIONS: This is the first study to calculate the D-dimer cut-off value in Japanese patients with advanced lung cancer. Patients with D-dimer levels ≥3.3 µg/ml at the time of initial diagnosis may have coexisting venous thromboembolism.

Lipocalin-2 as a prognostic marker in patients with acute exacerbation of idiopathic pulmonary fibrosis.

BACKGROUND: Lipocalin-2 (LCN2) is a secretory glycoprotein upregulated by oxidative stress; moreover, patients with idiopathic pulmonary fibrosis (IPF) have shown increased LCN2 levels in bronchoalveolar lavage fluid (BALF). This study aimed to determine whether circulatory LCN2 could be a systemic biomarker in patients with IPF and to investigate the role of LCN2 in a bleomycin-induced lung injury mouse model. METHODS: We measured serum LCN2 levels in 99 patients with stable IPF, 27 patients with acute exacerbation (AE) of IPF, 51 patients with chronic hypersensitivity pneumonitis, and 67 healthy controls. Further, LCN2 expression in lung tissue was evaluated in a bleomycin-induced lung injury mouse model, and the role of LCN2 was investigated using LCN2-knockout (LCN2 -/-) mice. RESULTS: Serum levels of LCN2 were significantly higher in patients with AE-IPF than in the other groups. The multivariate Cox proportional hazards model showed that elevated serum LCN2 level was an independent predictor of poor survival in patients with AE-IPF. In the bleomycin-induced lung injury mouse model, a higher dose of bleomycin resulted in higher LCN2 levels and shorter survival. Bleomycin-treated LCN2 -/- mice exhibited increased BALF cell and protein levels as well as hydroxyproline content. Moreover, compared with wild-type mice, LCN2-/- mice showed higher levels of circulatory 8-isoprostane as well as lower Nrf-2, GCLC, and NQO1 expression levels in lung tissue following bleomycin administration. CONCLUSIONS: Our findings demonstrate that serum LCN2 might be a potential prognostic marker of AE-IPF. Moreover, LCN2 expression levels may reflect the severity of lung injury, and LCN2 may be a protective factor against bleomycin-induced acute lung injury and oxidative stress.

Minimum 10-Year Survivorship of Uncemented Acetabular Reconstruction With Bulk Femoral Head Autografting in Crowe Type IV Developmental Hip Dysplasia.

BACKGROUND: In total hip arthroplasty (THA) for severe dislocations such as Crowe type IV developmental dysplasia of the hip (DDH), sufficient bone volume for stable fixation of the acetabular component can be achieved by placing a reinforcing bone graft prepared from the resected femoral head into the deficient acetabulum. The purpose of the current study was to examine the long-term survivorship of uncemented THA in conjunction with a bulk femoral head autograft in patients who have Crowe type IV DDH. METHODS: A total of 35 patients (42 hips) who have Crowe type IV DDH and underwent THA using uncemented cup fixation with bulk femoral head autografting were followed up for a mean period of 15.0 years (range, 10.0 to 20.0) postoperatively. Anteroposterior pelvic radiographs were used for measurements such as the horizontal coverage of the grafted bone and the center-edge angle. Kaplan-Meier survivorship analyses were performed with revision of the acetabular component as the endpoint. RESULTS: The Kaplan-Meier analysis indicated 15-year survival rates of 90.4%. The mean horizontal coverage of grafted bone was 46.1% (range, 23.7 to 66.0), and there were 16 cases with horizontal coverage of ≥ 50%. There was no difference in the appearance of a thin (< 1 mm) radiolucency line around the cup between cases with < 50% versus ≥ 50% of the horizontal coverage of grafted bone (4 versus 2 hips; P = .446). Trabecular bridging and remodeling were seen in all cases after mean periods of 4.1 and 9.0 months postoperatively, respectively. Trabecular reorientation was seen in 41 of 42 hips (97.6%) at a mean follow-up of 19.9 months. CONCLUSIONS: Acetabular reconstruction with femoral bulk bone grafting for Crowe type IV DDH resulted in high survival rates and was a good method to restore bone stock and obtain long-term fixation.

A case of pleural mesothelioma with immunohistochemical staining positive for Krebs von den Lungen-6.

A 71-year-old male visited a hospital with a chief complaint of exertional dyspnea. A chest CT revealed multiple nodular lesions on the parietal pleura. Thoracoscopic pleural biopsy was performed resulting in a diagnosis of pleural mesothelioma with epithelioid type. When chemotherapy was initially initiated, his serum level of Krebs von den Lungen-6 (KL-6) was high. However, once chemotherapy was started, the serum KL-6 level gradually decreased with tumor shrinkage. Immunohistochemical staining revealed the expression of KL-6 from the tumor cells. This is the first report of KL-6 production directly from tumor cells in epithelial-type pleural mesothelioma.

Scale-preserving shape reconstruction from monocular endoscope image sequences by supervised depth learning.

Reconstructing 3D shapes from images are becoming popular, but such methods usually estimate relative depth maps with ambiguous scales. A method for reconstructing a scale-preserving 3D shape from monocular endoscope image sequences through training an absolute depth prediction network is proposed. First, a dataset of synchronized sequences of RGB images and depth maps is created using an endoscope simulator. Then, a supervised depth prediction network is trained that estimates a depth map from a RGB image minimizing the loss compared to the ground-truth depth map. The predicted depth map sequence is aligned to reconstruct a 3D shape. Finally, the proposed method is applied to a real endoscope image sequence.

Association of immune-related adverse events with durvalumab efficacy after chemoradiotherapy in patients with unresectable Stage III non-small cell lung cancer.

BACKGROUND: Immune-related adverse events (irAEs) have been found to predict PD-L1 inhibitor efficacy in metastatic NSCLC. However, the relation of irAEs to clinical outcome for nonmetastatic NSCLC has remained unknown. METHODS: In this multicenter prospective study of Stage III NSCLC treated with PACIFIC regimen, the relation of irAEs to PFS was evaluated by 8-week landmark analysis to minimise lead-time bias as well as by multivariable analysis adjusted for baseline factors. irAEs were categorised as mild or nonmild according to whether they were treated with systemic steroid. RESULTS: Median PFS was 16.0 months, not reached, and 9.7 months for patients without (85 cases) or with mild (21 cases) or nonmild (21 cases) irAEs, respectively. Multivariable analysis indicated that nonmild irAEs were associated with poor PFS, with HRs of 3.86 (95% CI, 1.31-11.38) compared with no irAEs and 11.58 (95% CI, 2.11-63.63) compared with mild irAEs. This pattern was consistent after irAE grade, the number of durvalumab doses and immune profiles (PD-L1 score, CD8+ tumour-infiltrating lymphocyte density, and tumour mutation burden) were taken into consideration. CONCLUSIONS: The development of mild irAEs might predict a better survival outcome, whereas immunosuppressive steroid-treated irAEs were associated with a worse outcome, regardless of baseline clinical and immune profiles.

High S100A9 level predicts poor survival, and the S100A9 inhibitor paquinimod is a candidate for treating idiopathic pulmonary fibrosis.

BACKGROUND: S100A9 is a damage-associated molecular pattern protein that may play an important role in the inflammatory response and fibrotic processes. Paquinimod is an immunomodulatory compound that prevents S100A9 activity. Its safety and pharmacokinetics have been confirmed in human clinical trials. In this study, we investigated the effects of paquinimod in preventing the development of lung fibrosis in vivo and examined the prognostic values of circulatory and lung S100A9 levels in patients with idiopathic pulmonary fibrosis (IPF). METHODS: The expression and localisation of S100A9 and the preventive effect of S100A9 inhibition on fibrosis development were investigated in a mouse model of bleomycin-induced pulmonary fibrosis. In this retrospective cohort study, the S100A9 levels in the serum and bronchoalveolar lavage fluid (BALF) samples from 76 and 55 patients with IPF, respectively, were examined for associations with patient survival. RESULTS: S100A9 expression was increased in the mouse lungs, especially in the inflammatory cells and fibrotic interstitium, after bleomycin administration. Treatment with paquinimod ameliorated fibrotic pathological changes and significantly reduced hydroxyproline content in the lung tissues of mice with bleomycin-induced pulmonary fibrosis. Additionally, we found that paquinimod reduced the number of lymphocytes and neutrophils in BALF and suppressed endothelial-mesenchymal transition in vivo. Kaplan-Meier curve analysis and univariate and multivariate Cox hazard proportion analyses revealed that high levels of S100A9 in the serum and BALF were significantly associated with poor prognoses in patients with IPF (Kaplan-Meier curve analysis: p=0.037 (serum) and 0.019 (BALF); multivariate Cox hazard proportion analysis: HR=3.88, 95% CI=1.06 to 14.21, p=0.041 (serum); HR=2.73, 95% CI=1.05 to 7.10, p=0.039 (BALF)). CONCLUSIONS: The present results indicate that increased S100A9 expression is associated with IPF progression and that the S100A9 inhibitor paquinimod is a potential treatment for IPF.

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

G protein-biased LPAR1 agonism of prototypic antidepressants: Implication in the identification of novel therapeutic target for depression.

Prototypic antidepressants, such as tricyclic/tetracyclic antidepressants (TCAs), have multiple pharmacological properties and have been considered to be more effective than newer antidepressants, such as selective serotonin reuptake inhibitors, in treating severe depression. However, the clinical contribution of non-monoaminergic effects of TCAs remains elusive. In this study, we discovered that amitriptyline, a typical TCA, directly binds to the lysophosphatidic acid receptor 1 (LPAR1), a G protein-coupled receptor, and activates downstream G protein signaling, while exerting a little effect on ß-arrestin recruitment. This suggests that amitriptyline acts as a G protein-biased agonist of LPAR1. This biased agonism was specific to TCAs and was not observed with other antidepressants. LPAR1 was found to be involved in the behavioral effects of amitriptyline. Notably, long-term infusion of mouse hippocampus with the potent G protein-biased LPAR agonist OMPT, but not the non-biased agonist LPA, induced antidepressant-like behavior, indicating that G protein-biased agonism might be necessary for the antidepressant-like effects. Furthermore, RNA-seq analysis revealed that LPA and OMPT have opposite patterns of gene expression changes in the hippocampus. Pathway analysis indicated that long-term treatment with OMPT activated LPAR1 downstream signaling (Rho and MAPK), whereas LPA suppressed LPAR1 signaling. Our findings provide insights into the mechanisms underlying the non-monoaminergic antidepressant effects of TCAs and identify the G protein-biased agonism of LPAR1 as a promising target for the development of novel antidepressants.

Hypoxia-inducible factor 1α modulates interstitial pneumonia-mediated lung cancer progression.

BACKGROUND: The prognosis of patients with lung cancer accompanied by interstitial pneumonia is poorer than that of patients with lung cancer but without interstitial pneumonia. Moreover, the available therapeutic interventions for lung cancer patients with interstitial pneumonia are limited. Therefore, a new treatment strategy for these patients is required. The aim of the present study was to investigate the pathophysiological relationship between interstitial pneumonia and lung cancer and explore potential therapeutic agents. METHODS: A novel hybrid murine model of lung cancer with interstitial pneumonia was established via bleomycin-induced pulmonary fibrosis followed by orthotopic lung cancer cell transplantation into the lungs. Changes in tumor progression, lung fibrosis, RNA expression, cytokine levels, and tumor microenvironment in the lung cancer with interstitial pneumonia model were investigated, and therapeutic agents were examined. Additionally, clinical data and samples from patients with lung cancer accompanied by interstitial pneumonia were analyzed to explore the potential clinical significance of the findings. RESULTS: In the lung cancer with interstitial pneumonia model, accelerated tumor growth was observed based on an altered tumor microenvironment. RNA sequencing analysis revealed upregulation of the hypoxia-inducible factor 1 signaling pathway. These findings were consistent with those obtained for human samples. Moreover, we explored whether ascorbic acid could be an alternative treatment for lung cancer with interstitial pneumonia to avoid the disadvantages of hypoxia-inducible factor 1 inhibitors. Ascorbic acid successfully downregulated the hypoxia-inducible factor 1 signaling pathway and inhibited tumor progression and lung fibrosis. CONCLUSIONS: The hypoxia-inducible factor 1 pathway is critical in lung cancer with interstitial pneumonia and could be a therapeutic target for mitigating interstitial pneumonia-mediated lung cancer progression.

Liquid biopsy detects genomic drivers in NSCLC without EGFR mutations by single-plex testing: WJOG13620L.

BACKGROUND: Actionable tumor genomic alterations, primarily EGFR mutations, occur in nearly 70% of Japanese advanced nonsquamous non-small cell lung cancer (NSCLC) patients. Standard assessment of tumor tissue includes rapid testing for EGFR mutations, ALK fusions and ROS1 fusions. We conducted a prospective observational study (WJOG13620L) of follow-on next-generation sequencing of circulating tumor DNA (ctDNA) in patients without driver alterations after EGFR testing. METHODS: Patients with untreated advanced (Stage IIIB-IV or relapsed) nonsquamous NSCLC without EGFR mutations according to single-plex testing of tumor tissue, were enrolled into this study. Patients with other known driver mutations or who underwent comprehensive genomic profiling were excluded. Plasma was analyzed by Guardant360, and the primary endpoint was the proportion of patients with pathogenic gene alterations in at least one of nine genes. RESULTS: Among the 72 patients enrolled, ALK and ROS1 fusions were tested in 86.1% and 65.2%, respectively. Alterations in pre-defined genes were detected in 21 patients (29.2%; 95% confidence interval: 19.0-41.1, p < 0.001 [one-sided null hypothesis proportion of 10%]), including RET fusion (n = 1) and mutations in KRAS (n = 11), EGFR (n = 5), ERBB2 (n = 3), and BRAF (n = 1). Median time from sample submission to results was 8 days (range, 5-17 days). CONCLUSION: Rapid follow-on comprehensive testing of ctDNA should be considered prior to first-line treatment for patients with advanced nonsquamous NSCLC when no alterations are detected after single-plex tissue testing.

Significance of micro-EGFR T790M mutations on EGFR-tyrosine kinase inhibitor efficacy in non-small cell lung cancer.

Small amounts of epidermal growth factor receptor (EGFR) T790M mutation (micro-T790M), which is detected using droplet digital PCR (ddPCR) but not conventional PCR, in formalin-fixed and paraffin-embedded (FFPE) samples have been investigated as a predictive factor for the efficacy of EGFR-tyrosine kinase inhibitors (TKIs). However, the predictive value of micro-T790M remains controversial, possibly owing to the failure to examine artificial T790M in FFPE specimens. Therefore, we examined the predictive value of micro-T790M in first-generation (1G), second-generation (2G), and third-generation (3G) EGFR-TKI efficacy using a new method to exclude FFPE-derived artificial mutations in our retrospective cohort. The primary objective was time to treatment failure (TTF) of 1G, 2G, and 3G EGFR-TKIs according to micro-T790M status. In total, 315 patients with EGFR-positive non-small cell lung cancer treated with 1G, 2G, and 3G EGFR-TKIs were included in this study. The proportion of patients positive for micro-T790M in the 1G, 2G, and 3G EGFR-TKI groups was 48.2%, 47.1%, and 47.6%, respectively. In the micro-T790M-positive group, the TTF was significantly longer in the 2G and 3G EGFR-TKI groups than in the 1G TKI group. No differences in the micro-T790M-negative group were observed. Micro-T790M status detected using ddPCR, eliminating false positives, may be a valuable predictor of EGFR-TKI efficacy.

Usefulness of Quantification and Serial Monitoring of Fine Crackles for Early Detection of Treatment-related Lung Injury: A Report of Two Cases.

Early detection and appropriate management of treatment-related interstitial lung disease (ILD) are important in cancer treatment. We established an algorithm for quantifying fine crackles using machine learning and reported that the fine crackle quantitative value (FCQV) calculated by this algorithm was more sensitive than chest radiography for detecting interstitial changes. Using this algorithm, we periodically analyzed respiratory sounds in two patients with lung cancer who developed treatment-related ILDs and found that the FCQV was elevated before the diagnosis of ILD. These cases may indicate the usefulness of the FCQV in the early diagnosis of treatment-related ILDs.

A splice-switching oligonucleotide treatment ameliorates glycogen storage disease type 1a in mice with G6PC c.648G>T.

Glycogen storage disease type 1a (GSD1a) is caused by a congenital deficiency of glucose-6-phosphatase-α (G6Pase-α, encoded by G6PC), which is primarily associated with life-threatening hypoglycemia. Although strict dietary management substantially improves life expectancy, patients still experience intermittent hypoglycemia and develop hepatic complications. Emerging therapies utilizing new modalities such as adeno-associated virus and mRNA with lipid nanoparticles are under development for GSD1a but potentially require complicated glycemic management throughout life. Here, we present an oligonucleotide-based therapy to produce intact G6Pase-α from a pathogenic human variant, G6PC c.648G>T, the most prevalent variant in East Asia causing aberrant splicing of G6PC. DS-4108b, a splice-switching oligonucleotide, was designed to correct this aberrant splicing, especially in liver. We generated a mouse strain with homozygous knockin of this variant that well reflected the pathophysiology of patients with GSD1a. DS-4108b recovered hepatic G6Pase activity through splicing correction and prevented hypoglycemia and various hepatic abnormalities in the mice. Moreover, DS-4108b had long-lasting efficacy of more than 12 weeks in mice that received a single dose and had favorable pharmacokinetics and tolerability in mice and monkeys. These findings together indicate that this oligonucleotide-based therapy could provide a sustainable and curative therapeutic option under easy disease management for GSD1a patients with G6PC c.648G>T.

Exhaled Nitric Oxide and Olfactory Dysfunction in Patients with Asthma: Association with Chronic Rhinosinusitis.

Objectives: Olfactory dysfunction is a clinical sign that is important to detect with coexistent upper airway comorbidities in patients with asthma. This study aimed to investigate the etiology of olfactory dysfunction in patients with asthma and the relationship between fractional exhaled nitric oxide (FeNO) levels. Materials and Methods: This study included 47 asthma patients who were evaluated for olfactory dysfunction at Hiroshima University Hospital between 2012 and 2020. The etiologies of olfactory dysfunction were evaluated, and they were classified according to the FeNO levels of patients with asthma. Results: Olfactory dysfunction was observed in 30 patients with asthma, with chronic rhinosinusitis (77%) being the most prevalent etiology. Eosinophilic chronic rhinosinusitis (ECRS) was the most prevalent etiology of olfactory dysfunction in asthma patients with high FeNO levels (≥25 ppb), while non-eosinophilic chronic rhinosinusitis (NCRS) was the most prevalent etiology in asthma patients with low FeNO levels (<25 ppb). Additionally, the prevalence of ECRS was significantly higher in asthma patients with olfactory dysfunction and high FeNO levels (74%) than in those with either high FeNO levels or olfactory dysfunction and those with low FeNO levels and no olfactory dysfunction (12% and 9%, respectively). Conclusions: We found that ECRS was the predominant cause of olfactory dysfunction in patients with high FeNO levels, while NCRS was more common in those with low FeNO levels. The present study showed that both ECRS and NCRS are common etiologies of olfactory dysfunction in patients with asthma. Additionally, this study supports the link between upper and lower airway inflammation in patients with asthma complicated with olfactory dysfunction.

Proteomic alterations in the brain and blood-brain barrier during brain Aß accumulation in an APP knock-in mouse model of Alzheimer's disease.

BACKGROUND: Blood-brain barrier (BBB) dysfunction is supposed to be an early event in the development of Alzheimer's disease (AD). This study aimed to investigate the relationship between BBB alterations and AD progression in terms of amyloid-ß peptide (Aß) accumulation in the brains of humanized amyloid precursor protein knock-in (APP-KI) mice. METHODS: Brain Aß accumulation was examined using immunohistochemical analysis. Alterations in differentially expressed proteins were determined using sequential window acquisition of all theoretical fragment ion mass spectroscopy (SWATH-MS)-based quantitative proteomics, and Metascape, STRING, Gene Ontology, and KEGG were used for network analyses of altered biological pathways and processes. Statistical significance was determined using the unpaired two-tailed Student's t-test and Welch's t-test for two groups and one-way analysis of variance followed by Tukey's test for more than two groups. Correlations between two groups were determined using Pearson's correlation analysis. RESULTS: Brain Aß accumulation in APP-KI mice was detectable at 2 months, increased significantly at 5 months, and remained elevated at 12 months of age. The levels of differentially expressed proteins in isolated brain capillaries were higher in younger mice, whereas those in the brain were higher in older mice. Network analyses indicated changes in basement membrane-associated and ribosomal proteins in the brain capillaries. There were no significant changes in key proteins involved in drug or Aß transport at the BBB. In contrast, solute carrier transporter levels in astrocytes, microglia, and neurons were altered in the brain of older mice. Moreover, the levels of the lipid transporters Apoe and Apoj were upregulated in both the brain and isolated brain capillaries after Aß accumulation. CONCLUSIONS: Our results suggest that changes in the brain occurred after advanced Aß accumulation, whereas initial Aß accumulation was sufficient to cause alterations in the BBB. These findings may help elucidate the role of BBB alterations in AD progression and predict the distribution of drugs across the BBB in the brain of patients with AD.

Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial-mesenchymal transition in lung endothelial cells.

BACKGROUND: Nestin, an intermediate filament protein, participates in various pathophysiological processes, including wound healing, angiogenesis, endothelial-mesenchymal transition (EndoMT), and fibrosis. However, the pathophysiological roles of lung nestin-expressing cells remain unclear due to conflicting reports. The objective of this study is to elucidate the characteristics and functions of lung nestin-expressing cells. METHODS: We conducted a series of in vitro and in vivo experiments using endothelial cell line MS1 and nestin-GFP mice. This animal model allows for nestin-expressing cell detection without the use of anti-nestin antibodies. RESULTS: Lung nestin-expressing cells occurred in approximately 0.2% of CD45- cells and was co-expressed with epithelial, endothelial, and mesenchymal cell-surface markers. Importantly, virtually all nestin-expressing cells co-expressed CD31. When compared to lung nestin-nonexpressing endothelial cells, nestin-expressing endothelial cells showed robust angiogenesis with frequent co-expression of PDGFRß and VEGFR2. During TGFß-mediated EndoMT, the elevation of Nes mRNA expression preceded that of Col1a1 mRNA, and nestin gene silencing using nestin siRNA resulted in further upregulation of Col1a1 mRNA expression. Furthermore, Notch3 expression was regulated by nestin in vitro and in vivo; nestin siRNA resulted in reduced Notch3 expression accompanied with enhanced EndoMT. Contrary to previous reports, neither Nes mRNA expression nor nestin-expressing cells were increased during pulmonary fibrosis. CONCLUSIONS: Our study showed that (1) lung nestin-expressing cells are an endothelial lineage but are distinct from nestin-nonexpressing endothelial cells; (2) nestin regulates Notch3 and they act collaboratively to regulate angiogenesis, collagen production, and EndoMT; and (3) nestin plays novel roles in lung angiogenesis and fibrosis. Video Abstract.

Denosumab-associated Periprosthetic Atypical Femur Fracture: A Case Report.

Introduction: Denosumab is generally used for 5-10 years to treat postmenopausal osteoporosis. Although atypical fractures caused by bisphosphonate use are well known, reports of denosumab-associated femur fractures are rare. Case Report: Herein, a 75-year-old woman suffered an atypical periprosthetic femoral fracture 31 months after receiving denosumab. The fracture occurred transverse to the stem tip with lateral cortical thickening. The patient underwent revision surgery for conversion to a longer cemented stem. The fracture site healed 10 months after revision surgery. Conclusion: As far as we know, there have been no reports of a case on periprosthetic atypical femur fracture associated with denosumab. Our study shows the potential of periprosthetic atypical femoral fractures in patients using denosumab for a long time.

Wnt/ß­catenin signaling is a novel therapeutic target for tumor suppressor CYLD­silenced glioblastoma cells.

Tumor suppressor cylindromatosis (CYLD) dysfunction by its downregulation is significantly associated with poor prognosis in patients with glioblastoma (GBM), the most aggressive and malignant type of glioma. However, no effective treatment is currently available for patients with CYLD­downregulated GBM. The aim of the present study was to identify the crucial cell signaling pathways and novel therapeutic targets for CYLD downregulation in GBM cells. CYLD knockdown in GBM cells induced GBM malignant characteristics, such as proliferation, metastasis, and GBM stem­like cell (GSC) formation. Comprehensive proteomic analysis and RNA sequencing data from the tissues of patients with GBM revealed that Wnt/ß­catenin signaling was significantly activated by CYLD knockdown in patients with GBM. Furthermore, a Wnt/ß­catenin signaling inhibitor suppressed all CYLD knockdown­induced malignant characteristics of GBM. Taken together, the results of the present study revealed that Wnt/ß­catenin signaling is responsible for CYLD silencing­induced GBM malignancy; therefore, targeting Wnt/ß­catenin may be effective for the treatment of CYLD­negative patients with GBM with poor prognosis.