Development of a novel bispecific antibody GR1801 for rabies.

Rabies is a zoonotic viral disease characterized by an almost 100% fatality rate once symptoms appear. However, it can be prevented through timely postexposure prophylaxis (PEP). Currently, there is a growing trend to replace polyclonal rabies immune globulin (RIG) with monoclonal antibodies (mAbs) in rabies PEP. In this study, we developed a human bispecific antibody, GR1801, by combining two mAbs, A2 and B353, which target distinct epitopes. GR1801 is an asymmetric immunoglobulin G1 molecule, with one arm (A2 targeting epitope III) in fragment antigen-binding (Fab) form and the other arm (B353 targeting epitope I) in single-chain variable fragment (scFv) form, constructed using Knobs-into-Holes technology. GR1801 demonstrated the ability to neutralize 90 naturally occurring rabies virus (RABV) glycoprotein antigenic variants, 21 pseudotyped, and 18 live street RABVs, exhibiting broad-spectrum neutralizing activity. In vivo, GR1801 provided protection equivalent to that of human RIG in golden hamsters challenged with lethal RABV. In conclusion, these findings demonstrate the neutralization potency and breadth of GR1801, which can be a promising candidate drug for rabies PEP, and a comprehensive testing against a broad spectrum of Chinese prevalent RABVs will be investigated in great detail in the future for the in vitro and in vivo studies.

ILC2 regulates hyperoxia-induced lung injury via an enhanced Th17 cell response in the BPD mouse model.

BACKGROUD: Recent research has focused on the role of immune cells and immune responses in the pathogenesis of bronchopulmonary dysplasia (BPD), but the exact mechanisms have not yet been elucidated. Previously, the key roles of type 2 innate lymphoid cells (ILC2) in the lung immune network of BPD were explored. Here, we investigated the role Th17 cell response in hyperoxia-induced lung injury of BPD, as well as the relationship between ILC2 and Th17 cell response. METHODS: A hyperoxia-induced BPD mouse model was constructed and the pathologic changes of lung tissues were evaluated by Hematoxylin-Eosin staining. Flow cytometry analysis was conducted to determine the levels of Th17 cell, ILC2 and IL-6+ILC2. The expression levels of IL-6, IL-17 A, IL-17 F, and IL-22 in the blood serum and lung tissues of BPD mice were measured by ELISA. To further confirm the relationship between ILC2 and Th17 cell differentiation, ILC2 depletion was performed in BPD mice. Furthermore, we used immunomagnetic beads to enrich ILC2 and then flow-sorted mouse lung CD45+Lin-CD90.2+Sca-1+ILC2. The sorted ILC2s were injected into BPD mice via tail vein. Following ILC2 adoptive transfusion, the changes of Th17 cell response and lung injury were detected in BPD mice. RESULTS: The expression levels of Th17 cells and Th17 cell-related cytokines, including IL-17 A, IL-17 F, and IL-22, were significantly increased in BPD mice. Concurrently, there was a significant increase in the amount of ILC2 and IL-6+ILC2 during hyperoxia-induced lung injury, which was consistent with the trend for Th17 cell response. Compared to the control BPD group, ILC2 depletion was found to partially abolish the Th17 cell response and had protective effects against lung injury after hyperoxia. Furthermore, the adoptive transfer of ILC2 enhanced the Th17 cell response and aggravated lung injury in BPD mice. CONCLUSIONS: This study found that ILC2 regulates hyperoxia-induced lung injury by targeting the Th17 cell response in BPD, which shows a novel strategy for BPD immunotherapy.

IL-33-ST2 pathway regulates AECII transdifferentiation by targeting alveolar macrophage in a bronchopulmonary dysplasia mouse model.

Evidence points to the indispensable function of alveolar macrophages (AMs) in normal lung development and tissue homeostasis. However, the importance of AMs in bronchopulmonary dysplasia (BPD) has not been elucidated. Here, we identified a significant role of abnormal AM proliferation and polarization in alveolar dysplasia during BPD, which is closely related to the activation of the IL-33-ST2 pathway. Compared with the control BPD group, AMs depletion partially abolished the epithelialmesenchymal transition process of AECII and alleviated pulmonary differentiation arrest. In addition, IL-33 or ST2 knockdown has protective effects against lung injury after hyperoxia, which is associated with reduced AM polarization and proliferation. The protective effect disappeared following reconstitution of AMs in injured IL-33 knockdown mice, and the differentiation of lung epithelium was blocked again. In conclusion, the IL-33-ST2 pathway regulates AECII transdifferentiation by targeting AMs proliferation and polarization in BPD, which shows a novel strategy for manipulating the IL-33-ST2-AMs axis for the diagnosis and intervention of BPD.

[The 161th lysine of C/EBPα in alveolar type II epithelial cells is modified by small ubiquitin-related modification].

Objective To clarify the interaction between CCAAT enhancer-binding protein α (C/EBPα) and small ubiquitin-related modification (SUMO) in human alveolar type II epithelial cells (AECII), and further identify its modification sites. Methods The expression of C/EBPα and SUMO1 in human AECII was detected by immunofluorescence double labeling. Co-IP was used to detect the interaction of C/EBPα and SUMO1 in AEC II. The SUMO site of C/EBPα was predicted to be the 161st lysine (K161) by the SUMOsp software. The wild-type GFP-C/EBPα plasmids and mutant GFP-K161R plasmids were constructed and transfected into AECII. The SUMO site of C/EBPα was identified by Co-IP. Results Immunofluorescence double staining found that SUMO1 and C/EBPα were co-located in the nucleus. C/EBPα-SUMO band could be marked by Co-IP, which suggested that C/EBPα could interact with SUMO1.When AECII was transfected by wild-type GFP-C/EBPα plasmids. C/EBPα-SUMO1 band could be detected by immunoprecipitation (IP), but could not be detected when transfected by mutant GFP-C/EBPα plasmids. These suggested that the SUMO site of C/EBPα was the 161st lysine. Conclusion C/EBPα can be modified by SUMO1 and the site of its modification is the 161st lysine in human AECII.

[Dynamic expression and role of SUMO-modified C/EBPα in preterm rats with bronchopulmonary dysplasisa induced by hyperoxia exposure].

OBJECTIVE: To study the expression of SUMO-modified CCAAT enhancer binding protein α (C/EBPα) in preterm rat model of bronchopulmonary dysplasisa (BPD) induced by hyperoxia exposure and its role. METHODS: Eighteen preterm rats were randomly divided into an air group and a hyperoxia group (n=9 each). The model of BPD was prepared in preterm rats exposed to hyperoxia. The rats from the two groups were sacrificed on postnatal days 4, 7 and 14 respectively (3 rats at each time) and lung tissues were harvested. Periodic acid-Schiff (PAS) staining was used to observe the differentiation of rat lung tissues. Ki67 expression was detected by immunohistochemistry. Western blot was used to measure the protein expression of small ubiquitin-related modifier-1(SUMO1) and C/EBPα. A co-immunoprecipitation assay was performed to measure the protein expression of SUMO-modified C/EBPα. RESULTS: Compared with the air group, the hyperoxia group showed a decreased glycogen content in the lung tissue on postnatal day 4, and an increased content on postnatal days 7 and 14. Over the time of hyperoxia exposure, the hyperoxia group showed an increased expression of Ki67 in the lung tissue compared with the air group at all time points. Compared with the air group, the protein expression of C/EBPα increased on postnatal day 4 and decreased on postnatal days 7 and 14 in the hyperoxia group (P<0.05). The hyperoxia group had significantly upregulated expression of SUMO1 and SUMO-modified C/EBPα compared with the air group at all time points (P<0.05). In the hyperoxia group, the protein expression of SUMO-modified C/EBPα was positively correlated with the glycogen content (r=0.529, P<0.05) and the expression of Ki67 (r=0.671, P<0.05). CONCLUSIONS: Hyperoxia may induce over-proliferation and differentiation disorders of alveolar epithelial cells in preterm rat model of BPD, possibly through an increased expression of SUMO-modified C/EBP&alpha.

Expression profile analysis of long non-coding RNA associated with vincristine resistance in colon cancer cells by next-generation sequencing.

Vincristine (VCR) is widely used in tumor treatment. However, long-term use of this drug can make tumor cells resistant to it. Furthermore, the mechanisms underlying resistance development are unclear. The aim of this study was to investigate the long non-coding RNAs (lncRNAs) associated with colon cancer drug resistance using next-generation sequencing. A cDNA library of HCT-8 VCR-resistant colon cancer cell was established through PCR amplification. Using HiSeq 2500 sequencing and bioinformatic methods, we identified lncRNAs showing different expression levels in drug-resistant and non-resistant cells, and constructed expression profiles of the lncRNA differences. The pretreatment of data was quality controlled using FastQC software. Transcription of lncRNA was calculated using Fragments Per Kilobase of transcript per Million fragments mapped (FPKM). To reveal the potential functions of these lncRNAs, we applied GO analysis to study the differentially expressed lncRNAs. Total transcript number was higher in resistant cells than in non-resistant colon cancer cells, and high-quality transcripts constituted the major portion of the total. In addition, 121 transcripts showed significantly different expression in VCR-resistant and non-resistant cells. Of these, we observed 23 up-regulated and 20 down-regulated lncRNAs (fold change >10.0). This is the first report of the expression profile of lncRNA of VCR-resistant colon cancer cells. Abnormal lncRNA expression was associated with VCR resistance in colon cancer cells and these expression differences may play a key role in VCR resistance of these cells.