Anti-aminoacyl-tRNA synthetase-interacting multifunctional protein-1 antibody improves airway inflammation in mice with house dust mite induced asthma.

Background: Several biologics have been developed and used to treat severe asthma. However, commercialized biologics have limitations in treating T2-low asthma because their main target is the T2 inflammation marker. Therefore, there is an unmet need for treating T2-low severe asthma. Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) is an auxiliary protein in the mammalian multi-aminoacyl-tRNA synthetase complex. AIMP1 also acts as a cytokine and induces the secretion of proinflammatory cytokines. Since anti-AIMP1 has been shown to reduce interleukin (IL)-6, tumor necrosis factor-α, and IL-17A levels in a mouse model, it could be effective in the treatment of T2-low severe asthma. Methods: Wild-type BALB/c mice were sensitized and challenged with intranasal inoculation of a crude HDM extract. Atliximab, a chimeric AIMP1 antibody, was administered once (20 µg, 40 µg, 100 µg) on Day 14. We evaluated airway hyperresponsiveness (AHR), performed cellular analyses of the bronchoalveolar lavage fluid (BALF), measured inflammatory cytokine levels, and examined peribronchial histological features. Results: Atliximab reduced AIMP1 levels in asthmatic mice in a dose-dependent manner. AHR and Inflammatory cells such as neutrophils and eosinophils in the BALF decreased in asthmatic mice treated with atliximab. The levels of IL-6, IL-13, and transforming growth factor-ß (TGF-ß) in the lung tissue decreased in asthmatic mice treated with a high dose of atliximab (100 µg). Atliximab also reduced goblet cell hyperplasia and peribronchial fibrosis. Conclusions: Atliximab improved asthmatic airway inflammation including neutrophilic inflammation in HDM-induced asthma mice. These data suggest that anti-AIMP1 plays an important role in the treatment of severe T2-low asthma.

Intraocular Concentration of Stem Cell Factor/c-KIT and Galectin-1 in Retinal Diseases.

Purpose: To investigate the intraocular concentration profiles of stem cell factor (SCF)/c-KIT, galectin-1 (GAL-1), and vascular endothelial growth factor (VEGF)-A with regard to retinal disease and treatment response. Methods: The study group included 13 patients with dry age-related macular degeneration (AMD), 196 with neovascular AMD (nAMD), 21 with diabetic macular edema (DME), 10 with retinal vein occlusion (RVO), and 34 normal subjects with cataracts. Aqueous humor levels of SCF, c-KIT, GAL-1, and VEGF-A were analyzed by immunoassay according to disease group and treatment response. Results: Increased aqueous levels of SCF, c-KIT, and GAL-1 were observed in eyes with nAMD (2.67 ± 3.66, 296.84 ± 359.56, and 3945.61 ± 5976.2 pg/mL, respectively), DME (1.64 ± 0.89, 238.80 ± 265.54, and 3701.23 ± 4340.54 pg/mL, respectively), and RVO (4.62 ± 8.76, 509.63 ± 647.58, and 9079.60 ± 11909.20 pg/mL, respectively) compared with controls (1.13 ± 0.24, 60.00 ± 0.00, and 613.27 ± 1595.12 pg/mL, respectively). In the eyes of nAMD, the levels of all three cytokines correlated positively with VEGF-A levels. After intravitreal injections of anti-VEGF agents, the levels of GAL-1 and VEGF-A decreased significantly, whereas those of SCF and c-Kit showed no significant change. Eyes of nAMD patients with improved vision after treatment had significantly lower levels of c-KIT, GAL-1, and VEGF-A at baseline. Conclusions: The intraocular levels of cytokines were significantly elevated in eyes with nAMD, DME, and RVO compared to the controls and they showed different response to anti-VEGF treatment. With this result and their known association with angiogenesis, these cytokines may be potential therapeutic targets for future research.

Solidification-induced nonuniformity in U-Zr-RE metallic nuclear fuel rods.

Metallic fuel is being developed for the next generation of sodium-cooled fast reactors due to its safety and fuel cycle economics through spent fuel reprocessing technology. After spent fuel reprocessing, rare earth elements that are immiscible with Uranium and Zirconium are included in the raw material for metallic fuel. Achieving a uniform composition and microstructure of RE inclusions is challenging because phase separation and temperature inhomogeneities during solidification lead to non-uniform composition and microstructure. This study explores the solidification and inclusion formation of U-Zr-RE metallic fuel rods. We show how RE inclusions are formed in the U-Zr matrix and how temperature inhomogeneities during solidification control the inclusion size distribution and edge migration of RE inclusions along the U-Zr-RE rods. Understanding the inhomogeneity of inclusions due to solidification can provide hints for making homogeneous nuclear fuel rods.

Stem cell factor and cKIT modulate endothelial glycolysis in hypoxia.

AIMS: In hypoxia, endothelial cells (ECs) proliferate, migrate, and form new vasculature in a process called angiogenesis. Recent studies have suggested that ECs rely on glycolysis to meet metabolic needs for angiogenesis in ischaemic tissues, and several studies have investigated the molecular mechanisms integrating angiogenesis and endothelial metabolism. Here, we investigated the role of stem cell factor (SCF) and its receptor, cKIT, in regulating endothelial glycolysis during hypoxia-driven angiogenesis. METHODS AND RESULTS: SCF and cKIT signalling increased the glucose uptake, lactate production, and glycolysis in human ECs under hypoxia. Mechanistically, SCF and cKIT signalling enhanced the expression of genes encoding glucose transporter 1 (GLUT1) and glycolytic enzymes via Akt- and ERK1/2-dependent increased translation of hypoxia inducible factor 1A (HIF1A). In hypoxic conditions, reduction of glycolysis and HIF-1α expression using chemical inhibitors significantly reduced the SCF-induced in vitro angiogenesis in human ECs. Compared with normal mice, mice with oxygen-induced retinopathy (OIR), characterized by ischaemia-driven pathological retinal neovascularization, displayed increased levels of SCF, cKIT, HIF-1α, GLUT1, and glycolytic enzymes in the retina. Moreover, cKIT-positive neovessels in the retina of mice with OIR showed elevated expression of GLUT1 and glycolytic enzymes. Further, blocking SCF and cKIT signalling using anti-SCF neutralizing IgG and cKIT mutant mice significantly reduced the expression of HIF-1α, GLUT1, and glycolytic enzymes and decreased the pathological neovascularization in the retina of mice with OIR. CONCLUSION: We demonstrated that SCF and cKIT signalling regulate angiogenesis by controlling endothelial glycolysis in hypoxia and elucidated the SCF/cKIT/HIF-1α axis as a novel metabolic regulation pathway during hypoxia-driven pathological angiogenesis.

CXCL12 Neutralizing Antibody Promotes Hair Growth in Androgenic Alopecia and Alopecia Areata.

We had previously investigated the expression and functional role of C-X-C Motif Chemokine Ligand 12 (CXCL12) during the hair cycle progression. CXCL12 was highly expressed in stromal cells such as dermal fibroblasts (DFs) and inhibition of CXCL12 increased hair growth. Therefore, we further investigated whether a CXCL12 neutralizing antibody (αCXCL12) is effective for androgenic alopecia (AGA) and alopecia areata (AA) and studied the underlying molecular mechanism for treating these diseases. In the AGA model, CXCL12 is highly expressed in DFs. Subcutaneous (s.c.) injection of αCXCL12 significantly induced hair growth in AGA mice, and treatment with αCXCL12 attenuated the androgen-induced hair damage in hair organ culture. Androgens increased the secretion of CXCL12 from DFs through the androgen receptor (AR). Secreted CXCL12 from DFs increased the expression of the AR and C-X-C Motif Chemokine Receptor 4 (CXCR4) in dermal papilla cells (DPCs), which induced hair loss in AGA. Likewise, CXCL12 expression is increased in AA mice, while s.c. injection of αCXCL12 significantly inhibited hair loss in AA mice and reduced the number of CD8+, MHC-I+, and MHC-II+ cells in the skin. In addition, injection of αCXCL12 also prevented the onset of AA and reduced the number of CD8+ cells. Interferon-γ (IFNγ) treatment increased the secretion of CXCL12 from DFs through the signal transducer and activator of transcription 3 (STAT3) pathway, and αCXCL12 treatment protected the hair follicle from IFNγ in hair organ culture. Collectively, these results indicate that CXCL12 is involved in the progression of AGA and AA and antibody therapy for CXCL12 is promising for hair loss treatment.

A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells.

Given that mast cells are pivotal contributors to allergic diseases, various allergy treatments have been developed to inhibit them. Omalizumab, an anti-immunoglobulin E antibody, is a representative therapy that can alleviate allergy symptoms by inhibiting mast cell degranulation. However, omalizumab cannot reduce the proliferation and accumulation of mast cells, which is a fundamental cause of allergic diseases. c-Kit is essential for the proliferation, survival, and differentiation of mast cells. Excessive c-Kit activation triggers various mast cell diseases, such as asthma, chronic spontaneous urticaria, and mastocytosis. Herein, we generated 2G4, an anti-c-Kit antibody, to develop a therapeutic agent for mast cell diseases. The therapeutic efficacy of 2G4 antibody was evaluated in LAD2, a human mast cell line. 2G4 antibody completely inhibited c-Kit signaling by blocking the binding of stem cell factor, known as the c-Kit ligand. Inhibition of c-Kit signaling led to the suppression of proliferation, migration, and degranulation in LAD2 cells. Moreover, 2G4 antibody suppressed the secretion of pro-inflammatory cytokines, including granulocyte-macrophage colony-stimulating factor, vascular endothelial growth factor, C-C motif chemokine ligand 2, brain-derived neurotrophic factor, and complement component C5/C5a, which can exacerbate allergy symptoms. Taken together, these results suggest that 2G4 antibody has potential as a novel therapeutic agent for mast cell diseases.

A Novel Antibody-Drug Conjugate Targeting Nectin-2 Suppresses Ovarian Cancer Progression in Mouse Xenograft Models.

Ovarian cancer is the fifth leading cause of cancer, followed by front line is mostly platinum agents and PARP inhibitors, and very limited option in later lines. Therefore, there is a need for alternative therapeutic options. Nectin-2, which is overexpressed in ovarian cancer, is a known immune checkpoint that deregulates immune cell function. In this study, we generated a novel anti-nectin-2 antibody (chimeric 12G1, c12G1), and further characterized it using epitope mapping, enzyme-linked immunosorbent assay, surface plasmon resonance, fluorescence-activated cell sorting, and internalization assays. The c12G1 antibody specifically bound to the C2 domain of human nectin-2 with high affinity (KD = 2.90 × 10-10 M), but not to mouse nectin-2. We then generated an antibody-drug conjugate comprising the c12G1 antibody conjugated to DM1 and investigated its cytotoxic effects against cancer cells in vitro and in vivo. c12G1-DM1 induced cell cycle arrest at the mitotic phase in nectin-2-positive ovarian cancer cells, but not in nectin-2-negative cancer cells. c12G1-DM1 induced ~100-fold cytotoxicity in ovarian cancer cells, with an IC50 in the range of 0.1 nM~7.4 nM, compared to normal IgG-DM1. In addition, c12G1-DM1 showed ~91% tumor growth inhibition in mouse xenograft models transplanted with OV-90 cells. These results suggest that c12G1-DM1 could be used as a potential therapeutic agent against nectin-2-positive ovarian cancers.

Antibody-immunotoxin Conjugate Using FcBP-mediated Photoconjugation to Treat Cancer.

BACKGROUND/AIM: Cytotoxic payload conjugation to antibodies efficiently suppresses tumors and contributes to the improvement of cancer survival. In our previous study, c-Kit targeting antibody-drug conjugate (2G4-DM1) with DM1, a microtubule inhibitor, efficiently suppressed tumor growth. However, slow-growing c-Kit-positive tumors, such as GIST-48, did not efficiently respond to DM1. In this study, we aimed to treat tumors using 2G4 immunotoxin with Pseudomonas exotoxin A (PE) as a payload. MATERIALS AND METHODS: Modified FcBP-PE24 containing p-benzoyl-L-phenylalanine, unnatural amino acid, was expressed in E. coli and purified. Then, photoconjugation of 2G4 antibody and FcBP-PE24 at 365 nm was carried out and 2G4 immunotoxin was purified using anion exchange chromatography. In vitro cytotoxicity of 2G4 immunotoxins was assessed in HMC-1.2, GIST-48, and MDA-MB-453 cells. Then, in vivo efficacy analysis was performed using C.B-17 SCID mice. RESULTS: 2G4 immunotoxin efficiently induced cytotoxicity in 2G4-DM1-resistant HMC-1.2 and GIST-48 cells by inhibiting protein synthesis but not in c-Kit-negative MDA-MB-453 cells. The results showed ~200-fold or more increase in cytotoxicity against c-Kit-positive cells compared to IC50 of 2G4-DM1. In addition, 2G4 immunotoxin suppressed tumor growth in the in vivo xenograft mouse model. CONCLUSION: 2G4 immunotoxins could be an alternative therapeutic strategy for microtubule inhibitor- resistant cancer cells.

Comparative analysis of mesenchymal stem cells cultivated in serum free media.

Stem cells are attractive candidates for the regeneration of tissue and organ. Mesenchymal stem cells (MSCs) have been extensively investigated for their potential applications in regenerative medicine and cell therapy. For developing effective stem cell therapy, the mass production of consistent quality cells is required. The cell culture medium is the most critical aspect of the mass production of qualified stem cells. Classically, fetal bovine serum (FBS) has been used as a culture supplement for MSCs. Due to the undefined and heterologous composition of animal origin components in FBS, efforts to replace animal-derived components with non-animal-derived substances led to safe serum free media (SFM). Adipose derived mesenchymal stem cells (ADSCs) cultivated in SFM provided a more stable population doubling time (PDT) to later passage and more cells in a shorter time compared to FBS containing media. ADSCs cultivated in SFM had lower cellular senescence, lower immunogenicity, and higher genetic stability than ADSCs cultivated in FBS containing media. Differential expression analysis of mRNAs and proteins showed that the expression of genes related with apoptosis, immune response, and inflammatory response were significantly up-regulated in ADSCs cultivated in FBS containing media. ADSCs cultivated in SFM showed similar therapeutic efficacy in an acute pancreatitis mouse model to ADSCs cultivated in FBS containing media. Consideration of clinical trials, not only pre-clinical trial, suggests that cultivation of MSCs using SFM might offer more safe cell therapeutics as well as repeated administration due to low immunogenicity.

Antibody-Drug Conjugate Targeting c-Kit for the Treatment of Small Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths. Small cell lung cancer (SCLC) accounts for 15-25% of all lung cancers. It exhibits a rapid doubling time and a high degree of invasiveness. Additionally, overexpression of c-Kit occurs in 70% of SCLC patients. In this study, we evaluated an antibody-drug conjugate (ADC) that targets c-Kit, which is a potential therapeutic agent for SCLC. First, we generated and characterized 4C9, a fully human antibody that targets c-Kit and specifically binds to SCLC cells expressing c-Kit with a binding affinity of KD = 5.5 × 10-9 M. Then, we developed an ADC using DM1, a microtubule inhibitor, as a payload. 4C9-DM1 efficiently induced apoptosis in SCLC with an IC50 ranging from 158 pM to 4 nM. An in vivo assay using a xenograft mouse model revealed a tumor growth inhibition (TGI) rate of 45% (3 mg/kg) and 59% (5 mg/kg) for 4C9-DM1 alone. Combination treatment with 4C9-DM1 plus carboplatin/etoposide or lurbinectedin resulted in a TGI rate greater than 90% compared with the vehicle control. Taken together, these results indicate that 4C9-DM1 is a potential therapeutic agent for SCLC treatment.

A novel anti-c-Kit antibody-drug conjugate to treat wild-type and activating-mutant c-Kit-positive tumors.

c-Kit overexpression and activating mutations, which are reported in various cancers, including gastrointestinal stromal tumor (GIST), small-cell lung cancer (SCLC), acute myeloid leukemia, acral melanoma, and systemic mastocytosis (SM), confer resistance to tyrosine kinase inhibitors (TKIs). To overcome TKI resistance, an anti-c-Kit antibody-drug conjugate was developed in this study to treat wild-type and mutant c-Kit-positive cancers. NN2101, a fully human IgG1, was conjugated to DM1, a microtubule inhibitor, through N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (to give NN2101-DM1). The antitumor activity of NN2101-DM1 was evaluated in vitro and in vivo using various cancer cell lines. NN2101-DM1 exhibited potent growth-inhibitory activities against c-Kit-positive cancer cell lines. In a mouse xenograft model, NN2101-DM1 exhibited potent growth-inhibitory activities against imatinib-resistant GIST and SM cells. In addition, NN2101-DM1 exhibited a significantly higher anti-cancer effect than carboplatin/etoposide against SCLC cells where c-Kit does not mediate cancer pathogenesis. Furthermore, the combination of NN2101-DM1 with imatinib in imatinib-sensitive GIST cells induced complete remission compared with treatment with NN2101-DM1 or imatinib alone in mouse xenograft models. These results suggest that NN2101-DM1 is a potential therapeutic agent for wild-type and mutant c-Kit-positive cancers.

A Fully Human Monoclonal Antibody Targeting cKIT Is a Potent Inhibitor of Pathological Choroidal Neovascularization in Mice.

Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.

Real-Life Effectiveness and Safety of Glecaprevir/Pibrentasvir for Korean Patients with Chronic Hepatitis C at a Single Institution.

Background/Aims: Glecaprevir/pibrentasvir (G/P) is a combination of direct-acting antiviral agents that is an approved treatment for chronic infections by all six hepatitis C virus (HCV) genotypes. However, there are limited data on the effect of G/P in Korean patients in actual real-world settings. We evaluated the real-life effectiveness and safety of G/P at a single institution in Korea. Methods: This retrospective, observational, cohort study used sustained virologic response at 12 weeks after treatment completion (SVR12) as the primary effectiveness endpoint. Safety and tolerability were also determined. Results: We examined 267 individuals who received G/P for chronic HCV infections. There were 148 females (55.4%), and the overall median age was 63.0 years (range, 25 to 87 years). Eighty-three patients (31.1%) had HCV genotype-1 and 182 (68.2%) had HCV-2. A total of 212 patients (79.4%) were HCV treatment-naïve, 200 (74.9%) received the 8-week treatment, 13 (4.9%) had received prior treatment for hepatocellular carcinoma, 37 (13.7%) had chronic kidney disease stage 3 or higher, and 10 (3.7%) were receiving dialysis. Intention to treat (ITT) analysis indicated that 256 (95.9%) achieved SVR12. A modified ITT analysis indicated that SVR12 was 97.7% (256/262). Six patients failed therapy because of posttreatment relapse. SVR12 was significantly lower in those who received prior sofosbuvir treatment (p=0.002) and those with detectable HCV RNA at week 4 (p=0.027). Seventy patients (26.2%) experienced one or more adverse events, and most of them were mild. Conclusions: These real-life data indicated that G/P treatment was highly effective and well tolerated, regardless of viral genotype or patient comorbidities.

Efficacy of Ultrasound-Guided Needle Biopsy in the Diagnosis of Kikuchi-Fujimoto Disease.

OBJECTIVES/HYPOTHESIS: Ultrasound-guided fine-needle aspiration cytology (US-FNAC) is a well-established procedure performed to establish the diagnosis of Kikuchi-Fujimoto disease (KFD). Ultrasound-guided core needle biopsy (US-CNB) is an alternative diagnostic tool for KFD. However, the efficacy of US-CNB is not well evaluated. This study aimed to evaluate the efficacy of US-CNB and compare it with that of US-FNAC in the diagnosis of KFD. STUDY DESIGN: Retrospective cohort study. METHODS: We analyzed 170 patients who were diagnosed with KFD between January 2009 and May 2019. US-FNAC, US-CNB, and excisional biopsy were performed in 47, 114, and 9 patients, respectively. Diagnostic accuracies of US-FNAC and US-CNB were analyzed and compared. RESULTS: Of the 170 patients, 45 and 125 were men and women, respectively. The mean age was 26.9 ± 9.1 years. The most common symptom was cervical lymphadenopathy, followed by fever, headache, and myalgia. The diagnosis of KFD was established primarily by US-FNAC in 21 (44.7%) of the 47 patients, by US-CNB in 109 (95.6%) of the 114 patients, and by excisional biopsy in all 9 patients. There was no specific major complication related to US-FNAC and US-CNB. CONCLUSION: US-CNB can be considered safe and effective and used as the primary modality for the pathological diagnosis of KFD. LEVEL OF EVIDENCE: 4. Laryngoscope, 131:E1519-E1523, 2021.

The predictors of sustained virological response with sofosbuvir and ribavirin in patients with chronic hepatitis C genotype 2.

BACKGROUND/AIMS: Real-world, clinical practice data are lacking about sofosbuvir/ ribavirin (SOF/RBV) treatment of Korean patients with hepatitis C virus genotype 2 (HCV GT2) infection. This study investigated the efficacy and safety of SOF/RBV in Korean patients with HCV GT2 infection and clinical factors predicting sustained virological response 12 weeks (SVR12) after the end of SOF/RBV treatment. METHODS: A total of 181 patients with HCV GT2 with/without cirrhosis were treated with SOF/RBV for 16/12 weeks. Rapid virological response (RVR) was defined as non-detectable HCV RNA at 4 weeks. RESULTS: The RVR rate was 80.7% (146/181), the end of treatment response rate was 97.8% (177/181) and the SVR12 rate was 92.8% (168/181). Of eight patients with relapse, four did not achieve RVR. Three patients had a history of hepatocellular carcinoma (HCC). Multivariable analysis showed that RVR (p = 0.015) and no previous history of HCC (p = 0.007) were associated with SVR12. Factors significantly contributing to RVR included cirrhosis, creatinine concentration, and pre-treatment HCV RNA level. SVR12 rate was significantly higher in RVR (+) than RVR (-) patients (95.2% vs. 82.9%, p = 0.011) and also significantly higher in patients without than with a history of HCC (94.1% vs. 72.7%, p = 0.008). During treatment, 80/181 patients (44.2%) experienced mild to moderate adverse events, with 32 (17.7%) requiring RBV dose reductions due to anemia. CONCLUSION: SOF/RBV treatment was effective and tolerable in HCV GT2 patients. RVR and no previous history of HCC were positive predictors of SVR12.

FGF12 (Fibroblast Growth Factor 12) Inhibits Vascular Smooth Muscle Cell Remodeling in Pulmonary Arterial Hypertension.

Loss of BMP (bone morphogenic protein) signaling induces a phenotype switch of pulmonary arterial smooth muscle cells (PASMCs), which is the pathological basis of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Here, we identified FGF12 (fibroblast growth factor 12) as a novel regulator of the BMP-induced phenotype change in PASMCs and elucidated its role in pulmonary vascular remodeling during PAH development. Using murine models of PAH and lung specimens of patients with PAH, we observed that FGF12 expression was significantly reduced in PASMCs. In human PASMCs, FGF12 expression was increased by canonical BMP signaling. FGF12 knockdown blocked the antiproliferative and prodifferentiation effect of BMP on human PASMCs, suggesting that FGF12 is required for the BMP-mediated acquisition of the quiescent and differentiated PASMC phenotype. Mechanistically, FGF12 regulated the BMP-induced phenotype change by inducing MEF2a (myocyte enhancer factor 2a) phosphorylation via p38MAPK signaling, thereby modulating the expression of MEF2a target genes involved in cell proliferation and differentiation. Furthermore, we observed that TG (transgenic) mice with smooth muscle cell-specific FGF12 overexpression were protected from chronic hypoxia-induced PAH development, pulmonary vascular remodeling, and right ventricular hypertrophy. Consistent with the in vitro data using human PASMCs, FGF12 TG mice showed increased MEF2a phosphorylation and a substantial change in MEF2a target gene expression, compared with the WT (wild type) controls. Overall, our findings demonstrate a novel BMP/FGF12/MEF2a pathway regulating the PASMC phenotype switch and suggest FGF12 as a potential target for the development of therapeutics for ameliorating pulmonary vascular remodeling in PAH.

Development and characterization of a fully human antibody targeting SCF/c-kit signaling.

CD117/c-kit, a tyrosine kinase receptor, plays a critical role in hematopoiesis, pigmentation, and fertility. The overexpression and activation of c-kit are thought to promote tumor growth and have been reported in various cancers, including leukemia, glioblastoma and mastocytosis. To disrupt the SCF/c-kit signaling axis in cancer, we generated a c-kit antagonist human antibody (NN2101) that binds to domain 2/3 of c-kit. This completely blocked the SCF-mediated phosphorylation of c-kit and inhibited TF-1 cell proliferation, erythroleukemia. In addition, the examination of binding affinity using surface plasmon resonance (SPR) assay showed that NN2101 can bind to c-kit of monkeys (KD = 2.92 × 10-10 M), rats (KD = 1.68 × 10-6 M), mice (KD = 11.5 × 10-9 M), and humans (KD = 2.83 × 10-12 M). We showed that NN2101 does not cause antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The immunogenicity of NN2101 was similar to that of bevacizumab. Furthermore, the crystal structure of NN2101 Fab was determined and the structure of NN2101 Fab:c-kit complex was modeled. Structural information, as well as mutagenesis results, revealed that NN2101 can bind to the SCF-binding regions of c-kit. Collectively, we generated a c-kit neutralizing human antibody (NN2101) for the treatment of erythroleukemia and characterized its biophysical properties. NN2101 can potentially be used as a therapeutic antibody to treat different cancers.

Atializumab, a humanized anti-aminoacyl-tRNA synthetase-interacting multifunctional protein-1 (AIMP1) antibody significantly improves nephritis in (NZB/NZW) F1 mice.

Aminoacyl-tRNA synthetase (ARS)-interacting multifunctional protein 1 (AIMP1) enhances the expression of proinflammatory cytokines. In our previous study, we have shown that serum AIMP1 in patients with SLE was significantly higher than that of healthy controls. To address whether neutralization of AIMP1 could ameliorate nephritis in lupus-prone mice, we generated atializumab, a humanized antibody against AIMP1 and investigated its therapeutic efficacy. ELISA showed that serum AIMP1 at 23 weeks old was significantly higher than that at 13 weeks old in lupus-prone mice. Therefore, lupus-prone mice were randomly assigned to 5 groups (vehicle, methylprednisolone and 0.5, 2, and 5 mg/kg atializumab). After treatment, disease severity was assessed using a variety of phenotypes, including proteinuria, histological damages, renal deposition of immune-complex. In addition, serum cytokines, anti-dsDNA and IgG subclasses were determined. T cell subsets were analyzed using a fluorescence-activated cell sorter. Atializumab significantly diminished proteinuria, improved glomerular and tubular damages and reduced the renal deposition of immune-complexes. Moreover, atializumab significantly decreased serum interferon (IFN)-γ, interleukin (IL)-17A, and IL-6, whereas it increased serum IL-10. Similarly, atializumab reduced the numbers of TH1, TH2 and TH17 cells in a dose-dependent manner, while atializumab enhanced the number of regulatory T (Treg) cells. Furthermore, atializumab decreased not only splenic plasma cells and serum anti-dsDNA but also pathogenic IgG subclasses for nephritis. It suppressed NF-κB activation by inhibiting IκBα degradation in a dose-dependent manner in vitro. Atializumab alleviated nephritis by inhibiting autoreactive T, B, and plasma cells and decreasing NF-κB-related proinflammatory cytokines in lupus-prone mice. These results suggest that treatment targeting AIMP1 could be a novel and highly immune-modulating therapeutic strategy in lupus nephritis.

SCF (Stem Cell Factor) and cKIT Modulate Pathological Ocular Neovascularization.

OBJECTIVE: Aberrant neovascularization is a leading cause of blindness in several eye diseases, including age-related macular degeneration and proliferative diabetic retinopathy. The identification of key regulators of pathological ocular neovascularization has been a subject of extensive research and great therapeutic interest. Here, we explored the previously unrecognized role of cKIT and its ligand, SCF (stem cell factor), in the pathological ocular neovascularization process. Approach and Results: Compared with normoxia, hypoxia, a crucial driver of neovascularization, caused cKIT to be highly upregulated in endothelial cells, which significantly enhanced the angiogenic response of endothelial cells to SCF. In murine models of pathological ocular neovascularization, such as oxygen-induced retinopathy and laser-induced choroidal neovascularization models, cKIT and SCF expression was significantly increased in ocular tissues, and blockade of cKIT and SCF using cKit mutant mice and anti-SCF neutralizing IgG substantially suppressed pathological ocular neovascularization. Mechanistically, SCF/cKIT signaling induced neovascularization through phosphorylation of glycogen synthase kinase-3ß and enhancement of the nuclear translocation of ß-catenin and the transcription of ß-catenin target genes related to angiogenesis. Inhibition of ß-catenin-mediated transcription using chemical inhibitors blocked SCF-induced in vitro angiogenesis in hypoxia, and injection of a ß-catenin agonist into cKit mutant mice with oxygen-induced retinopathy significantly enhanced pathological neovascularization in the retina. Conclusions; Our data reveal that SCF and cKIT are promising novel therapeutic targets for treating vision-threatening ocular neovascular diseases.

Serum Aminoacyl-tRNA Synthetase-Interacting Multifunctional Protein-1 Can Predict Severe Antineutrophil Cytoplasmic Antibody-Associated Vasculitis: A Pilot Monocentric Study.

We investigated whether serum aminoacyl-tRNA synthetase-interacting multifunctional protein-1 (AIMP1) could predict severe cases of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) based on the Birmingham vasculitis activity score (BVAS). Sixty-one patients with AAV were selected for inclusion from our prospective AAV cohort. AAV-specific indices and clinical manifestations were assessed, and laboratory tests were performed on the day of blood sampling. Patients with severe AAV were defined as those with a BVAS higher than the lower limit of the highest tertile of BVAS (BVAS ≥ 12). We measured serum AIMP1 levels of the stored serum samples. A total of 20 (32.8%) and 41 (67.2%) patients were classified as having severe and nonsevere AAV according to the cut-off of BVAS ≥ 12. Patients with severe AAV showed higher frequencies of general and renal manifestations, along with ANCA positivity, and exhibited a higher mean neutrophil count, erythrocyte sedimentation rate, and C-reactive protein levels, but lower mean haemoglobin and serum albumin levels than those with nonsevere AAV. The mean serum AIMP1 level in patients with severe AAV was significantly higher than that of patients with nonsevere AIMP1 (351.1 vs. 98.4 pg/mL, p = 0.006). Multivariate logistic regression analysis including variables showing significance in univariate analyses revealed that only serum AIMP1 exhibited a significant association with severe AAV (odds ratio 1.004, p = 0.031). When we set the optimal cut-off of serum AIMP1 for severe AAV to 50.28 pg/mL, patients with severe AAV more frequently had AIMP1 levels above the cut-off than those with nonsevere AAV (80.0% vs. 31.7%, relative risk 8.615, p < 0.001). The results from our study suggest that serum AIMP1 can be used to estimate the cross-sectional severe AAV population based on the BVAS.