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Background: In recent years, germline gain-of-function (GOF) mutations in signal transducer and activator of transcription 3 (STAT3) have been identified as a cause of early-onset multiorgan autoimmune diseases with the widespread use of next-generation sequencing, and targeted therapies such as tocilizumab have been reported to be effective. Objective: We sought to assess whether a novel STAT3 mutation detected by whole-exome sequencing is pathogenic and examine the efficacy of targeted therapy. Methods: A pediatric patient with idiopathic pulmonary hemosiderosis, autoimmune thyroiditis, inflammatory bowel disease unclassified, leukocytosis, thrombocytosis, and severe growth failure was examined. Results: This 7-year-old boy had idiopathic pulmonary hemosiderosis at the age of 6 months. Despite high-dose steroid therapy, pulmonary fibrosis progressed. Furthermore, he presented with severe growth failure, autoimmune thyroiditis, leukocytosis, thrombocytosis, and inflammation bowel disease unclassified. Given the presence of multiple autoimmune diseases, whole-exome sequencing was performed, which detected germline de novo heterozygous STAT3 mutation (NM_139276.2; c.2144C>A, p.(P715Q)). Dual-luciferase reporter assay revealed this novel STAT3 mutation as GOF. After starting tocilizumab therapy at the age of 6, hospital stays decreased, and the progression of pulmonary fibrosis was decelerated without increasing the steroid dose. New autoimmune diseases did not develop, and no apparent adverse effects on growth have been observed. Conclusions: Tocilizumab may be effective for patients with STAT3 GOF mutation, including those requiring long-term management of idiopathic pulmonary hemosiderosis. Diagnosis of patients with early-onset multiorgan autoimmune diseases in which STAT3 GOF is suspected should be confirmed by genetic testing and functional analysis to consider the introduction of targeted therapies.

Abstract Progressive fibrosing interstitial lung diseases (PF-ILDs) have a poor prognosis and may be resistant to corticosteroids and/or immunosuppressants, but antifibrotic therapies such as nintedanib and pirfenidone have been shown to slow the deterioration of lung function. The aim of this study was to identify the characteristic cellular profile of bronchoalveolar lavage fluid at diagnostic bronchoscopy for predicting PF-ILDs, defined as fibrotic diseases on chest high-resolution computed tomography with more than a 5% relative decline in the percent predicted value of forced vital capacity (FVC) over 6 months. The proportions of inflammatory cells, CCR6−CXCR3− T helper type 2 (Th2) cells among conventional CD4+ T cells in bronchoalveolar lavage fluid (BALF) and peripheral blood, were measured by flowcytometry. The proportion of lymphocytes in BALF was significantly higher in non-PF-ILD patients than in PF-ILD patients. The proportion of Th2 cells in BALF, but not in peripheral blood, was significantly higher in PF-ILD patients than in non-PF-ILD patients. Multivariate analysis showed that a greater population of Th2 cells in BALF was the only indicator for PF-ILDs. An increased proportion of Th2 cells in BALF is associated with greater deterioration of lung function in fibrotic interstitial lung diseases.

Abstract Background Endometrial cancer (EC) is a common gynecologic malignancy and patients with advanced and recurrent EC have a poor prognosis. Although chemotherapy is administered for those patients, the efficacy of current chemotherapy is limited. Therefore, it is necessary to develop novel therapeutic agents for EC. In this study, we focused on lipolysis-stimulated lipoprotein receptor (LSR), a membrane protein highly expressed in EC cells, and developed a chimeric chicken–mouse anti-LSR monoclonal antibody (mAb). This study investigated the antitumor effect of an anti-LSR mAb and the function of LSR in EC. Methods We examined the expression of LSR in 228 patients with EC using immunohistochemistry and divided them into two groups: high-LSR (n = 153) and low-LSR groups (n = 75). We developed a novel anti-LSR mAb and assessed its antitumor activity in an EC cell xenograft mouse model. Pathway enrichment analysis was performed using protein expression data of EC samples. LSR-knockdown EC cell lines (HEC1 and HEC116) were generated by transfected with small interfering RNA and used for assays in vitro. Results High expression of LSR was associated with poor overall survival (hazard ratio: 3.53, 95% confidence interval: 1.35–9.24, p = 0.01), advanced stage disease (p = 0.045), deep myometrial invasion (p = 0.045), and distant metastasis (p

ObjectivesSerum levels of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) have been used as useful biomarkers for reflecting the activity of large vessel vasculitides (LVV). However, a novel biomarker that could have a complementary role to these markers is still required. In this retrospective observational study, we investigated whether leucine-rich α-2 glycoprotein (LRG), a known biomarker in several inflammatory diseases, could be a novel biomarker for LVVs.Methods49 eligible patients with Takayasu arteritis (TAK) or giant cell arteritis (GCA) whose serum was preserved in our laboratory were enrolled. The concentrations of LRG were measured with an enzyme-linked immunosorbent assay. The clinical course was reviewed retrospectively from their medical records. The disease activity was determined according to the current consensus definition.ResultsThe serum LRG levels were higher in patients with active disease than those in remission, and decreased after the treatments. While LRG levels were positively correlated with both CRP and erythrocyte sedimentation rate, LRG exhibited inferior performance as an indicator of disease activity compared to CRP and ESR. Of 35 CRP-negative patients, 11 had positive LRG. Among the 11 patients, two had active disease.ConclusionThis preliminary study indicated that LRG could be a novel biomarker for LVV. Further large studies should be required to promise the significance of LRG in LVV.

Background: Epithelial ovarian cancer (EOC) is a lethal malignant tumor, for which new treatment options are urgently required. Lipolysis-stimulated lipoprotein receptor (LSR) is widely expressed in EOC, and it is associated with poor prognosis. In this study, we developed an antibody-drug conjugate (ADC) targeting LSR as a new therapeutic approach to EOC. Methods: We, herein, developed novel anti-LSR monoclonal antibodies (mAbs) and an LSR-ADC by conjugating monomethyl auristatin E as a payload. We subsequently evaluated the in vitro and in vivo (on xenograft models) antitumor effect of the LSR-ADC. Results: An overexpression of LSR was observed not only in the primary EOC tumor but also in its lymph node and omental metastases. The EOC cell lines NOVC7-C and OVCAR3 strongly expressed LSR (as compared to ES2 cells). Both the anti-LSR mAb and the LSR-ADC were able to specifically bind to LSR-positive cells and were rapidly internalized and trafficked to the lysosomes. The LSR-ADC demonstrated a potent antitumor effect against NOVC-7C and OVCAR3, but little activity against ES2 cells. In vitro, the LSR-ADC exhibited a potent antitumor effect against NOVC-7C and OVCAR3. Moreover, in the OVCAR3 xenograft models as well as in the patient-derived xenograft models of LSR-positive EOC, the LSR-ADC significantly inhibited tumor growth. The LSR-ADC also suppressed the omental/bowel metastases in OVCAR3-Luc xenografts and improved the median survival. Conclusion: The developed LSR-ADC demonstrated a significant antitumor activity against LSR-positive EOC cell lines and tumors. Our preclinical data support the use of the LSR-ADC as a novel therapy for patients with LSR-positive ovarian cancer.

An antibody-drug conjugate (ADC) is a promising therapeutic modality because selective and effective delivery of an anti-cancer drug is achieved by drug-conjugated antibody-targeting cancer antigen. Glypican 1 (GPC1) is highly expressed in malignant tumors, including pancreatic ductal adenocarcinoma (PDAC) and esophageal squamous cell carcinoma (ESCC). Herein, we describe the usefulness of GPC1-targeting ADC. Humanized anti-GPC1 antibody (clone T2) was developed and conjugated with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PABC) linkers (humanized GPC1-ADC[MMAE]). Humanized GPC1-ADC(MMAE) inhibited the growth of GPC1-positive PDAC and ESCC cell lines via inducing cycle arrest in the G2/M phase and apoptosis in vitro. The binding activity of humanized GPC1-ADC(MMAE) with GPC1 was comparable with that of the unconjugated anti-GPC1 antibody. The humanized GPC1-ADC(MMAE) was effective in GPC1-positive BxPC-3 subcutaneously xenografted mice but not in GPC1-negative BxPC-3-GPC1-KO xenografted mice. To assess the bystander killing activity of the humanized GPC1-ADC(MMAE), a mixture of GPC1-positive BxPC-3 and GPC1-negative BxPC-3-GPC1-KO-Luc cells were subcutaneously inoculated, and a heterogenous GPC1-expressing tumor model was developed. The humanized GPC1-ADC(MMAE) inhibited the tumor growth and decreased the luciferase signal, measured with an in vivo imaging system (IVIS), which suggests that the suppression of the BxPC-3-GPC1-KO-Luc population. The humanized GPC1-ADC(MMAE) also inhibited the established liver metastases of BxPC-3 cells and significantly improved the overall survival of the mice. It exhibited a potent antitumor effect on the GPC1-positive PDAC and ESCC patient-derived xenograft (PDX) models. Our preclinical data demonstrate that GPC1 is a promising therapeutic target for ADC.

Current xenogeneic mouse models cannot evaluate on-target off-tumor adverse effect, hindering the development of chimeric antigen receptor (CAR) T cell therapies for solid tumors, due to limited human/mouse cross-reactivity of antibodies used in CAR and sever graft-versus-host disease induced by administered human T cells. We have evaluated safety and antitumor efficacy of CAR-T cells targeting glypican-1 (GPC1) overexpressed in various solid tumors. GPC1-specific human and murine CAR-T cells generated from our original anti-human/mouse GPC1 antibody showed strong antitumor effects in xenogeneic and syngeneic mouse models, respectively. Importantly, the murine CAR-T cells enhanced endogenous T cell responses against a non-GPC1 tumor antigen through the mechanism of antigen-spreading and showed synergistic antitumor effects with anti-PD-1 antibody without any adverse effects in syngeneic models. Our study shows the potential of GPC1 as a CAR-T cell target for solid tumors and the importance of syngeneic and xenogeneic models for evaluating their safety and efficacy.

OBJECTIVE:To investigate whether leucine-rich α2-glycoprotein (LRG) can be a biomarker for the disease activity, progression, and prognosis of interstitial pneumonia (IP) in patients with dermatomyositis (DM). METHODS:Correlations between the clinical findings and serum LRG levels were investigated in 46 patients with DM-IP (33 with acute/subacute IP [A/SIP] and 13 patients with chronic IP [CIP], including 10 fatal cases of IP). RESULTS:The median serum LRG level of 18.4 (14.6-25.2) μg/mL in DM-IP patients was higher than that in healthy control subjects. The median levels of serum LRG at baseline and at 2 and 4 weeks after the initiation of treatment in the patients who died were significantly higher than those in the surviving patients (P = 0.026, 0.029, and 0.008, respectively). The median level of serum LRG in the DM-A/SIP patients was significantly higher than that in the DM-CIP patients (P = 0.0004), and that in the anti-MDA5-Ab-positive group was slightly higher than that in the anti-ARS-Ab-positive group. The serum LRG levels correlated significantly with the serum levels of LDH, C-reactive protein, ferritin, AaDO2, %DLco, and total ground-glass opacity score. The survival rate after 24 weeks in patients with an initial LRG level ≥ 17.6 μg/mL (survival rate: 40%) was significantly lower than that in patients with an initial LRG level < 17.6 μg/mL (100%) (P = 0.0009). CONCLUSION:The serum LRG level may be a promising marker of disease activity, progression, and prognosis in patients with DM-IP.

This study aimed to determine the association between umbilical cord leucine-rich alpha-2 glycoprotein (LRG) and fetal infection and investigate the underlying mechanism of LRG elevation in fetuses. We retrospectively reviewed the medical records of patients who delivered at Osaka University Hospital between 2012 and 2017 and selected those with histologically confirmed chorioamnionitis (CAM), which is a common pregnancy complication that may cause neonatal infection. The participants were divided into two groups: CAM with fetal infection (CAM-f[+] group, n = 14) and CAM without fetal infection (CAM-f[-] group, n = 31). Fetal infection was defined by the histological evidence of funisitis. We also selected 50 cases without clinical signs of CAM to serve as the control. LRG concentrations in sera obtained from the umbilical cord were unaffected by gestational age at delivery, neonatal birth weight, nor the presence of noninfectious obstetric complications (all, p > 0.05). Meanwhile, the LRG levels (median, Interquartile range [IQR]) were significantly higher in the CAM-f(+) group (10.37 [5.21-13.7] μg/ml) than in the CAM-f(-) (3.61 [2.71-4.65] μg/ml) or control group (3.39 [2.81-3.93] μg/ml; p < 0.01). The area under the receiver operating characteristic (ROC) curve of LRG for recognizing fetal infection was 0.92 (optimal cutoff, 5.08 μg/ml; sensitivity, 86%; specificity, 88%). In a mouse CAM model established by lipopolysaccharide administration, the fetal LRG protein in sera and LRG mRNA in the liver were significantly higher than those in phosphate-buffered saline (PBS)-administered control mice (p < 0.01). In vitro experiments using a fetal liver-derived cell line (WRL68) showed that the expression of LRG mRNA was significantly increased after interleukin (IL)-6, IL-1β, and tumor necrosis factor- alpha (TNF-α) stimulation (p < 0.01); the induction was considerably stronger following IL-6 and TNF-α stimulation (p < 0.01). In conclusion, LRG is an effective biomarker of fetal infection, and fetal hepatocytes stimulated with inflammatory cytokines may be the primary source of LRG production in utero.

Abstract Background Leucine-rich alpha 2 glycoprotein (LRG) has been identified as a serum protein elevated in patients with active rheumatoid arthritis (RA). Although the function of LRG is ill-defined, LRG binds with transforming growth factor (TGF)-β and enhances Smad2 phosphorylation. Considering that the imbalance between T helper 17 (Th17) cells and regulatory T cells (Treg) plays important roles in the pathogenesis of RA, LRG may affect arthritic pathology by enhancing the TGF-β-Smad2 pathway that is pivotal for both Treg and Th17 differentiation. The purpose of this study was to explore the contribution of LRG to the pathogenesis of arthritis, with a focus on the role of LRG in T cell differentiation. Methods The differentiation of CD4 T cells and the development of collagen-induced arthritis (CIA) were examined in wild-type mice and LRG knockout (KO) mice. To examine the influence of LRG on T cell differentiation, naïve CD4 T cells were isolated from LRG KO mice and cultured under Treg- or Th17-polarization condition in the absence or presence of recombinant LRG. Results In the CIA model, LRG deficiency led to ameliorated arthritis and reduced Th17 differentiation with no influence on Treg differentiation. By addition of recombinant LRG, the expression of IL-6 receptor (IL-6R) was enhanced through TGF-β-Smad2 signaling. In LRG KO mice, the IL-6R expression and IL-6-STAT3 signaling was attenuated in naïve CD4 T cells, compared to wild-type mice. Conclusions Our findings suggest that LRG upregulates IL-6R expression in naïve CD4 T cells by the enhancement of TGF-β-smad2 pathway and promote Th17 differentiation and arthritis development.

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, progressive, motor neuron degenerative disease. Recent evidence indicates that inflammation is associated with many neurodegenerative diseases including ALS. Previously, abnormal levels of inflammatory cytokines including IL-1β, IL-6 and TNF-α were described in ALS patients and/or in mouse ALS models. In addition, one study showed that blocking IL-1β could slow down progression of ALS-like symptoms in mice. In this study, we examined a role for IL-6 in ALS, using an animal model for familial ALS.Mice with mutant SOD1 (G93A) transgene, a model for familial ALS, were used in this study. The expression of the major inflammatory cytokines, IL-6, IL-1β and TNF-α, in spinal cords of these SOD1 transgenic (TG) mice were assessed by real time PCR. Mice were then crossed with IL-6(-/-) mice to generate SOD1TG/IL-6(-/-) mice. SOD1 TG/IL-6(-/-) mice (n = 17) were compared with SOD1 TG/IL-6(+/-) mice (n = 18), SOD1 TG/IL-6(+/+) mice (n = 11), WT mice (n = 15), IL-6(+/-) mice (n = 5) and IL-6(-/-) mice (n = 8), with respect to neurological disease severity score, body weight and the survival. We also histologically compared the motor neuron loss in lumber spinal cords and the atrophy of hamstring muscles between these mouse groups.Levels of IL-6, IL-1β and TNF-α in spinal cords of SOD1 TG mice was increased compared to WT mice. However, SOD1 TG/IL-6(-/-) mice exhibited weight loss, deterioration in motor function and shortened lifespan (167.55 ± 11.52 days), similarly to SOD1 TG /IL-6(+/+) mice (164.31±12.16 days). Motor neuron numbers and IL-1β and TNF-α levels in spinal cords were not significantly different in SOD1 TG /IL-6(-/-) mice and SOD1 TG /IL-6 (+/+) mice.These results provide compelling preclinical evidence indicating that IL-6 does not directly contribute to motor neuron disease caused by SOD1 mutations.

Asthma is a chronic inflammatory disease of airways, but an ideal biomarker that accurately reflects ongoing airway inflammation has not yet been established. The aim of this study was to examine the potential of sputum leucine-rich alpha-2 glycoprotein (LRG) as a new biomarker for airway inflammation in asthma.We obtained induced sputum samples from patients with asthma (N = 64) and healthy volunteers (N = 22) and measured LRG concentration by sandwich enzyme-linked immunosorbent assay (ELISA). Ovalbumin (OVA)-induced asthma model mice were used to investigate the mechanism of LRG production during airway inflammation. The LRG concentrations in the bronchoalveolar lavage fluid (BALF) obtained from mice were determined by ELISA and mouse lung sections were stained with anti-LRG antibody and periodic acid-Schiff (PAS) reagent.Sputum LRG concentrations were significantly higher in patients with asthma than in healthy volunteers (p = 0.00686). Consistent with patients' data, BALF LRG levels in asthma model mice were significantly higher than in control mice (p = 0.00013). Immunohistochemistry of lung sections from asthma model mice revealed that LRG was intensely expressed in a subpopulation of bronchial epithelial cells, which corresponded with PAS-positive mucus producing cells.These findings suggest that sputum LRG is a promising biomarker of local inflammation in asthma.

Constitutively activated signal transducers, activators of transcription (STAT) are reported to cause uncontrolled transmission of growth signals. In this study, we analyzed the roles of an inhibitor of STAT, protein inhibitor of activated STAT (PIAS) 3, in the development of lung cancer. Treatment with an inhibitor of phosphatidylinositol 3-kinase, LY294002, retarded the growth of human lung cancer cells, rendered them more sensitive to chemotherapeutic agents. However, the inhibition of JAK/STAT by AG490 significantly suppressed cell growth but did not increase drug sensitivity at all. Overexpression of PIAS3 not only significantly inhibited cell growth but also rendered cancer cells up to 12.0-fold more sensitive to the above drugs, which was associated with the suppression of Akt phosphorylation. Inhibition of PIAS3 with small interfering RNA, nevertheless, led cancer cells to accelerate cell proliferation, deteriorate chemosensitivity, augment Akt phosphorylation. Although the overexpression of suppressors of cytokine signaling 3 in cancer cells also inhibited cell growth, STAT3 phosphorylation, it neither increased sensitivity to chemotherapeutic drugs nor affected the phosphorylation of Akt. These results indicate that PIAS3 may be an attractive candidate for targeting the JAK/STAT, PI3-K/Akt signaling pathways in cancer treatment.

Karyopherin proteins mediate nucleocytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest KPNA2 expression is induced in tumor cells and is strongly associated with prognosis, although the precise roles and mechanisms of KPNA2 overexpression in proliferative disorders have not been defined. We found that KPNA2 expression is induced in various proliferative disorders of the skin such as psoriasis, Bowen's disease, actinic keratosis, squamous cell carcinoma, Paget's disease, Merkel cell carcinoma, and mycosis fungoides. siRNA-mediated KPNA suppression revealed that KPNA2 is essential for significant suppression of HaCaT proliferation under starvation conditions. Ribosomal RNA transcription and protein synthesis were suppressed by starvation combined with knockdown of KPNA (including KPNA2) expression. KPNA2 localized to the nucleolus and interacted with proteins associated with mRNA processing, ribonucleoprotein complex biogenesis, chromatin modification, and transcription, as demonstrated by tandem affinity purification and mass spectrometry. KPNA2 may be an important promoter of ribosomal RNA and protein synthesis in tumor cells.

OBJECTIVE: Periostin, a novel matricellular protein, is recently reported to play a crucial role in tissue remodeling and is highly expressed under fibrotic conditions. This study was undertaken to assess the role of periostin in scleroderma. METHODS: Using skin from patients and healthy donors, the expression of periostin was assessed by immunohistochemistry and immunoblotting analyses. Furthermore, we investigated periostin(-/-) (PN(-/-)) and wild-type (WT) mice to elucidate the role of periostin in scleroderma. To induce murine cutaneous sclerosis, mice were subcutaneously injected with bleomycin, while untreated control groups were injected with phosphate-buffered saline. Bleomycin-induced fibrotic changes were compared in PN(-/-) and WT mice by histological analysis as well as by measurements of profibrotic cytokine and extracellular matrix protein expression levels in vivo and in vitro. To determine the downstream pathway involved in periostin signaling, receptor neutralizing antibody and signal transduction inhibitors were used in vitro. RESULTS: Elevated expression of periostin was observed in the lesional skin of patients with scleroderma compared with healthy donors. Although WT mice showed marked cutaneous sclerosis with increased expression of periostin and increased numbers of myofibroblasts after bleomycin treatment, PN(-/-) mice showed resistance to these changes. In vitro, dermal fibroblasts from PN(-/-) mice showed reduced transcript expression of alpha smooth actin and procollagen type-I alpha 1 (Col1α1) induced by transforming growth factor beta 1 (TGFβ1). Furthermore, recombinant mouse periostin directly induced Col1α1 expression in vitro, and this effect was inhibited by blocking the αv integrin-mediated PI3K/Akt signaling either with anti-αv functional blocking antibody or with the PI3K/Akt kinase inhibitor LY294002. CONCLUSION: Periostin plays an essential role in the pathogenesis of Bleomycin-induced scleroderma in mice. Periostin may represent a potential therapeutic target for human scleroderma.

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3(-/-) mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3(-/-) mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3(-/-) mice. Lipid metabolism disorders in Sik3(-/-) mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice.

Toll-like receptor (TLR) signaling pathways are strictly coordinated by several mechanisms to regulate adequate innate immune responses. Recent lines of evidence indicate that the suppressor of cytokine signaling (SOCS) family proteins, originally identified as negative-feedback regulators in cytokine signaling, are involved in the regulation of TLR-mediated immune responses. SOCS1, a member of SOCS family, is strongly induced upon TLR stimulation. Cells lacking SOCS1 are hyperresponsive to TLR stimulation. Thus, SOCS1 is an important regulator for both cytokine and TLR-induced responses. As an immune organ, the liver contains various types of immune cells such as T cells, NK cells, NKT cells, and Kupffer cells and is continuously challenged with gut-derived bacterial and dietary antigens. SOCS1 may be implicated in pathophysiology of the liver. The studies using SOCS1-deficient mice revealed that endogenous SOCS1 is critical for the prevention of liver diseases such as hepatitis, cirrhosis, and cancers. Recent studies on humans suggest that SOCS1 is involved in the development of various liver disorders in humans. Thus, SOCS1 and other SOCS proteins are potential targets for the therapy of human liver diseases.