Anti-TIM1 suppresses airway inflammation and hyperresponsiveness via the STAT6 /STAT1 pathways in mice with allergic asthma.

T cell immunoglobulin and mucin domain-1 (TIM-1) is a transmembrane glycoprotein and has been reported as an molecular mechanism of allergic diseases. This study aimed to explore the effects of anti-TIM-1 monoclonal antibodies (anti-TIM1) on the development of allergic asthma. Female C57BL/6 mice were induced and challenged with ovalbumin (OVA) and received subsequent intranasal administration of anti-TIM1. The airway resistance of all mice was evaluated using a Buxco PFT system. Flow cytometry was used to detect the expression of TIM-1 in peripheral blood mononuclear cells. The level of cytokine production in the bronchial alveolar lavage fluid and serum was determined using ELISA. Mucous cells were observed using Alcian blue and periodic acid-Schiff staining. In addition, B-cell lymphoma gene 2(BCL2), T-box transcription factor (T-bet), GATA binding protein-3(GATA3), signal transducer and activator of transcription (STAT) 1, STAT6 were analyzed by western blot analysis. Their corresponding mRNA expression levels were determined by quantitative PCR. The mRNA expression level of Mucin 5AC in the lung tissues was also detected using quantitative RT-PCR. The results showed that the intranasal administration of anti-TIM1 ameliorated airway inflammation and hyperresponsiveness in an acute model of asthma. Following administration of anti-TIM1, both the mRNA and protein levels of T-bet were upregulated, while those of BCL2 and GATA3 were downregulated. Moreover, the phosphorylation levels of STAT1 and STAT6 were increased. Taken together, these findings demonstrated that intranasal administration of anti-TIM1 ameliorated allergic lung inflammation and remodeling in mouse models of asthma by repairing both the STAT1 and STAT6 pathways.

Amyloid deposition in a mouse model humanized at the transthyretin and retinol-binding protein 4 loci.

Familial amyloidotic polyneuropathy is an autosomal dominant disorder caused by a point mutation in the transthyretin (TTR) gene. The process of TTR amyloidogenesis begins with rate-limiting dissociation of the TTR tetramer. Thus, the TTR stabilizers, such as Tafamidis and Diflunisal, are now in clinical trials. Mouse models will be useful to testing the efficacy of these drugs. Although several mouse models have been generated, they all express mouse Rbp4. Thus, human TTR associates with mouse RBP4, resulting in different kinetic and thermodynamic stability profiles of TTR tetramers. To overcome this problem, we previously produced humanized mouse strains at both the TTR and Rbp4 loci (Ttr hTTRVal30 , Ttr hTTRMet30 , and Rbp4 hRBP4 ). By mating these mice, we produced double-humanized mouse strains, Ttr hTTRVal30/hTTRVal30 :Rbp4 hRBP4/hRBP4 and Ttr hTTRVal30/Met30 :Rbp4 hRBP4/hRBP4 . We used conventional transgenic mouse strains on a wild-type (Ttr +/+ :Tg[6.0hTTRMet30]) or knockout Ttr background (Ttr-/-:Tg[6.0hTTRMet30]) as reference strains. The double-humanized mouse showed 1/25 of serum hTTR and 1/40 of serum hRBP4 levels. However, amyloid deposition was more pronounced in Ttr hTTRVal30/Met30 :Rbp4 hRBP4/hRBP4 than in conventional transgenic mouse strains. In addition, a similar amount of amyloid deposition was also observed in Ttr hTTRVal30/ hTTRVal30 :Rbp4 hRBP4/ hRBP4 mice that carried the wild-type human TTR gene. Furthermore, amyloid deposition was first observed in the sciatic nerve without any additional genetic change. In all strains, anti-TTR antibody-positive deposits were found in earlier age and at higher percentage than amyloid fibril deposition. In double-humanized mice, gel filtration analysis of serum revealed that most hTTR was free of hRBP4, suggesting importance of free TTR for amyloid deposition.

Abhd2, a Candidate Gene Regulating Airway Remodeling in COPD via TGF-ß.

Purpose: The typical characteristic of COPD is airway remodeling, affected by environmental and genetic factors. However, genetic studies on COPD have been limited. Currently, the Abhd2 gene is found to play a critical role in maintaining alveolar architecture and stability. The research aims to investigate the predictive value of Abhd2 for airway remodeling in COPD and its effect on TGF-ß regulation. Methods: In humans, Abhd2 protein was obtained from peripheral blood monocytes. Peripheral blood TGF-ß, pulmonary surfactant proteins (SPs), metalloproteinases, inflammatory indicators (WBC, NEU, NLR, EOS, CRP, PCT, D-Dimer), chest CT (airway diameter and airway wall thickness), pulmonary function, and blood gas analysis were used to assess airway remodeling. In animals, Abhd2 deficient mice (Abhd2Gt/Gt) using gene trapping and C57BL6 mice were injected intraperitoneally with CSE to construct COPD models. HE staining, Masson staining and immunohistochemistry were used to observe the pathological changes of airway in mice, and RT-PCR, WB, ELISA and immunofluorescence were used to detect the expression of secreted proteins and EMT markers. Results: COPD patients with worse pulmonary function and higher airway remodeling-related inflammatory factors had lower Abhd2 protein expression. Moreover, indicators followed the same trend for COPD patients grouped by prognosis (Group A vs Group B). Serum TGF-ß was negatively correlated with Abhd2 protein expression, FEV1/FVC, FEV1, and FEV1% PRED. In mice, Abhd2 depletion promoted deposition of TGF-ß, leading to more pronounced emphysema, airway thickening, increased alveolar macrophage infiltration, decreased AECII number and SPs, and EMT phenomenon. Conclusion: Downregulation of Abhd2 can promote airway remodeling in COPD by modulating repair after injury and EMT via TGF-ß. This study suggests that Abhd2 may serve as a biomarker for assessing airway remodeling and guiding prognosis in COPD.

Complex Evaluation of Surfactant Protein A and D as Biomarkers for the Severity of COPD.

Purpose: Pulmonary surfactant proteins A (SP-A) and D (SP-D) are lectins, involved in host defense and regulation of pulmonary inflammatory response. However, studies on the assessment of COPD progress are limited. Patients and Methods: Pulmonary surfactant proteins were obtained from the COPD mouse model induced by cigarette and lipopolysaccharide, and the specimens of peripheral blood and bronchoalveolar lavage (BALF) in COPD populations. H&E staining and RT-PCR were performed to demonstrate the successfully established of the mouse model. The expression of SP-A and SP-D in mice was detected by Western Blot and immunohistochemistry, while the proteins in human samples were measured by ELISA. Pulmonary function test, inflammatory factors (CRP, WBC, NLR, PCT, EOS, PLT), dyspnea index score (mMRC and CAT), length of hospital stay, incidence of complications and ventilator use were collected to assess airway remodeling and progression of COPD. Results: COPD model mice with emphysema and airway wall thickening were more prone to have decreased SP-A, SP-D and increased TNF-α, TGF-ß, and NF-kb in lung tissue. In humans, SP-A and SP-D decreased in BALF, but increased in serum. The serum SP-A and SP-D were negatively correlated with FVC, FEV1, FEV1/FVC, and positively correlated with CRP, WBC, NLR, mMRC and CAT scores (P < 0.05, respectively). The lower the SP-A and SP-D in BALF, the worse the lung function and the increased probability of complications and ventilator use. Moreover, the same trend emerged in COPD patients grouped according to GOLD severity grade (Gold 1-2 group vs Gold 3-4 group). The worse the patient's condition, the more pronounced the change. Conclusion: This study suggests that SP-A and SP-D may be related to the progression and prognostic evaluation of COPD in terms of airway remodeling, inflammatory response and clinical symptoms, and emphasizes the necessity of future studies of surfactant protein markers in COPD.

The lower the eosinophils, the stronger the inflammatory response? The relationship of different levels of eosinophils with the degree of inflammation in acute exacerbation chronic obstructive pulmonary disease (AECOPD).

BACKGROUND: Blood eosinophil levels are a known marker for the effects of therapy in patients with chronic obstructive pulmonary disease (COPD). This study aimed to clarify the cutoff values for blood eosinophils (EOS) to predict exacerbation risk and prognosis of acute exacerbation COPD (AECOPD) and investigate their correlation using inflammatory indicators and clinical characteristics. METHODS: In this observational study of 174 patients with AECOPD, we assessed the relationship between EOS and COPD. According to the percentage of blood EOS, patients were grouped into two groups (Group 1: EOS <2%, n=98; Group 2: EOS ≥2%, n=76), and Group 2 was further divided into Group A (2%≤ EOS <4%) and Group B (EOS ≥4%) based on a cutoff value of 4%. Patients received standardized treatment after collection of peripheral blood specimen. Associations of EOS with laboratory indicators before any treatment in hospital and with clinical data were compared. RESULTS: Patients in Group 1 showed significantly severe inflammation, worse pulmonary function, longer length of stay (LOS) (P<0.001), higher mMRC score (P<0.05), higher CAT score (P<0.05), higher rates of mortality (P<0.05), and greater noninvasive mechanical ventilation usage (P<0.05) compared with Group 2. Intriguingly, the CRP, total mMRC and CAT scores of patients in Group A were significantly lower than those in Group B (P<0.001; P<0.01; P<0.05, respectively). Pearson correlation analysis showed that a low percentage blood eosinophil level was negatively associated with higher WBC count (r=-0.155, P<0.05), NLR (r=-0.227, P<0.01) and CRP (r=-0.308, P<0.01). CONCLUSIONS: Different cutoff values for percentage blood EOS might be useful biomarkers for predicting the severity of exacerbation and prognosis of inpatients with AECOPD.

Identification of Prognostic miRNAs Associated With Immune Cell Tumor Infiltration Predictive of Clinical Outcomes in Patients With Non-Small Cell Lung Cancer.

BACKGROUND: A detailed means of prognostic stratification in patients with non-small cell lung cancer (NSCLC) is urgently needed to support individualized treatment plans. Recently, microRNAs (miRNAs) have been used as biomarkers due to their previously reported prognostic roles in cancer. This study aimed to construct an immune-related miRNA signature that effectively predicts NSCLC patient prognosis. METHODS: The miRNAs and mRNA expression and mutation data of NSCLC was obtained from The Cancer Genome Atlas (TCGA). Immune-associated miRNAs were identified using immune scores calculated by the ESTIMATE algorithm. LASSO-penalized multivariate survival models were using for development of a tumor immune-related miRNA signature (TIM-Sig), which was evaluated in several public cohorts from the Gene Expression Omnibus (GEO) and the CellMiner database. The miRTarBase was used for constructing the miRNA-target interactions. RESULTS: The TIM-Sig, including 10 immune-related miRNAs, was constructed and successfully predicted overall survival (OS) in the validation cohorts. TIM-Sig score negatively correlated with CD8+ T cell infiltration, IFN-γ expression, CYT activity, and tumor mutation burden. The correlation between TIM-Sig score and genomic mutation and cancer chemotherapeutics was also evaluated. A miRNA-target network of 10 miRNAs in TIM-Sig was constructed. Further analysis revealed that these target genes showed prognostic value in both lung squamous cell carcinoma and adenocarcinoma. CONCLUSIONS: We concluded that the immune-related miRNAs demonstrated a potential value in clinical prognosis.

CSP-1103 (CHF5074) stabilizes human transthyretin in healthy human subjects.

Hereditary amyloid polyneuropathy is a type of protein misfolding disease. Transthyretin (TTR) is a homotetrameric serum protein and TTR tetramer dissociation is the limiting step in amyloid fibril formation. Thus, prevention of TTR dissociation is a promising therapeutic approach and some TTR stabilizers have been approved for the treatment of TTR amyloidosis. CSP-1103 (CHF5074) is a non-steroidal anti-inflammatory derivative that lacks cyclooxygenase inhibitory activity. In vitro, CSP-1103 stabilizes the TTR tetramer by binding to the thyroxine (T4) binding site. We have previously shown that serum TTR levels were increased by oral CSP-1103 administration through stabilization of TTR tetramers in humanized mice at both the Ttr locus and the Rbp4 locus. To determine whether CSP-1103 stabilizes TTR tetramers in humans, multiple CSP-1103 oral doses were administered for two weeks to 48 healthy human volunteers in a double-blind, placebo-controlled, parallel-group study. CSP-1103 treatment stabilized TTR tetramers in a dose-dependent manner under normal or denaturing stress conditions, thereby increasing serum TTR levels. Preincubation of serum with CSP-1103 or diflunisal in vitro increased the TTR tetramer stability. Computer simulation analysis revealed that the binding affinities of CSP-1103 with TTR at pH 7.0 were similar to those of tafamidis, thus confirming that CSP-1103 has potent TTR-stabilizing activity.

Associations of ABHD2 genetic variations with risks for chronic obstructive pulmonary disease in a Chinese Han population.

The human α/ß hydrolase domain-containing protein 2 gene (ABHD2) plays a critical role in pulmonary emphysema, a major subset of the clinical entity known as chronic obstructive pulmonary disease (COPD). Here, we evaluated genetic variation in the ABHD2 gene in a Chinese Han population of 286 COPD patients and 326 control subjects. The rs12442260 CT/CC genotype was associated with COPD (P < 0.001) under a dominant model. In the former-smoker group, the rs12442260 TT genotype was associated with a decreased risk of developing COPD after adjusting for age, gender and pack-years (P = 0.012). Rs12442260 was also associated with pre-FEV1 (the predicted bronchodilator forced expiratory volume in the first second) in controls (P = 0.027), but with FEV1/ forced vital capacity (FVC) ratios only in COPD patients (P = 0.012) under a dominant model. Results from the current study suggest that ABHD2 gene polymorphisms contribute to COPD susceptibility in the Chinese Han population.

CHF5074 (CSP-1103) stabilizes human transthyretin in mice humanized at the transthyretin and retinol-binding protein loci.

Familial amyloidotic polyneuropathy is one type of protein misfolding disease. Transthyretin (TTR) tetramer dissociation is the limiting step for amyloid fibril formation. CHF5074 (CSP-1103) stabilizes TTR tetramer in vitro by binding to the T4 binding site. Here, we used three strains of double humanized mice (mTtr(hTTRVal30/hTTRVal30), mTtr(hTTRVal30/hTTRMet30), and mTtr(hTTRMet30/hTTRMet30)) to assess whether CHF5074 stabilizes TTR tetramers in vivo. Treatment of mice with CHF5074 increased serum TTR levels by stabilizing TTR tetramers. Although the binding affinities of CHF5074 and diflunisal with TTRMet30 were similar, CHF5074 bound TTRVal30 more strongly than did diflunisal, suggesting the potent TTR-stabilizing activity of CHF5074.