Fully human anti-B7-H3 recombinant antibodies inhibited tumor growth by increasing T cell infiltration.

Mortality due to malignant tumors is one of the major factors affecting the life expectancy of the global population. Therapeutic antibodies are a cutting-edge treatment method for restricting tumor growth. B7-H3 is highly expressed in tumor tissues, but rarely in normal tissues. B7-H3 is closely associated with poor prognosis in patients with tumors. B7-H3 is an important target for antitumor therapy. In this study, the fully human anti-B7H3 single-chain antibodies (scFvs) were isolated and screened from the fully human phage immune library with B7H3 as the target. The antibodies screened from a fully human phage library had low immunogenicity and high affinity, which was more beneficial for clinical application. Leveraging B7-H3 scFvs as a foundation, we constructed two distinct recombinant antibody formats, scFv-Fc and IgG1, characterized by elevated affinity and a prolonged half-life. The results demonstrated that the recombinant antibodies had high specificity and affinity for the B7-H3 antigen and inhibited tumor cell growth by enhancing the ADCC. After treatment with anti-B7H3 recombinant antibody, the number of infiltrating T cells in the tumor increased and the secretion of IFN- γ by infiltrating T cells increased in vivo. Additionally, the use of pleural fluid samples obtained from tumor-afflicted patients revealed the ability of anti-B7-H3 recombinant antibodies to reverse CD8+ T cell exhaustion. In summary, we screened the fully human anti-B7H3 recombinant antibodies with specificity and high affinity that increase immune cell infiltration and IFN-γ secretion, thereby inhibiting tumor cell growth to a certain extent. This finding provides a theoretical basis for the development of therapeutic tumor antibodies and could help promote further development of antibody-based drugs.

An Aggrephagy-Related LncRNA Signature for the Prognosis of Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a common, highly malignant, and aggressive gastrointestinal tumor. The conventional treatment of PAAD shows poor results, and patients have poor prognosis. The synthesis and degradation of proteins are essential for the occurrence and development of tumors. Aggrephagy is a type of autophagy that selectively degrades aggregated proteins. It decreases the formation of aggregates by degrading proteins, thus reducing the harm to cells. By breaking down proteins, it decreases the formation of aggregates; thus, minimizing damage to cells. For evaluating the response to immunotherapy and prognosis in PAAD patients, in this study, we developed a reliable signature based on aggrephagy-related genes (ARGs). We obtained 298 AGGLncRNAs. Based on the results of one-way Cox and LASSO analyses, the lncRNA signature was constructed. In the risk model, the prognosis of patients in the low-risk group was noticeably better than that of the patients in the high-risk group. Additionally, the ROC curves and nomograms validated the capacity of the risk model to predict the prognosis of PAAD. The patients in the low-risk and high-risk groups showed considerable variations in functional enrichment and immunological analysis. Regarding drug sensitivity, the low-risk and high-risk groups had different half-maximal inhibitory concentrations (IC50).

Novel targets for immunotherapy associated with exhausted CD8 + T cells in cancer.

In response to prolonged stimulation by tumour antigens, T cells gradually become exhausted. There is growing evidence that exhausted T cells not only lose their potent effector functions but also express multiple inhibitory receptors. Checkpoint blockade (CPB) therapy can improve cancer by reactivating exhausted effector cell function, leading to durable clinical responses, but further improvements are needed given the limited number of patients who benefit from treatment, even with autoimmune complications. Here, we suggest, based on recent advances that tumour antigens are the primary culprits of exhaustion, followed by some immune cells and cytokines that also play an accomplice role in the exhaustion process, and we also propose that chronic stress-induced hypoxia and hormones also play an important role in promoting T-cell exhaustion. Understanding the classification of exhausted CD8+ T-cell subpopulations and their functions is important for the effectiveness of immune checkpoint blockade therapies. We mapped the differentiation of T-cell exhausted subpopulations by changes in transcription factors, indicating that T-cell exhaustion is a dynamic developmental process. Finally, we summarized the novel immune checkpoints associated with depletion in recent years and combined them with bioinformatics to construct a web of exhaustion-related immune checkpoints with the aim of finding novel therapeutic targets associated with T-cell exhaustion in malignant tumours, aiming to revive the killing ability of exhausted T cells and restore anti-tumour immunity through combined targeted immunotherapy.

Systematic Pan-Cancer Analysis Identifies CDK1 as an Immunological and Prognostic Biomarker.

Cyclin-dependent kinase 1 (CDK1) plays an important role in cancer development, progression, and the overall process of tumorigenesis. However, no pan-cancer analysis has been reported for CDK1, and the predictive role of CDK1 in immune checkpoint inhibitors (ICIs) therapy response remains unexplored. Thus, in this study, we first investigated the potential oncogenic role of CDK1 in 33 tumors by multidimensional bioinformatics analysis based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Bioinformatic analysis and immunohistochemical experiments confirmed that CDK1 is significantly upregulated in most common cancers and is strongly associated with prognosis. Further analysis indicated that CDK1 may influence tumor immunity mainly by mediating the degree of tumor infiltration of immune-associated cells, and the effect of CDK1 on immunity is diverse across tumor types in tumor microenvironment. CDK1 was also positively correlated with tumor mutational burden (TMB) and microsatellite instability (MSI) in certain cancer types, linking its expression to the assessment of possible treatment response. The results of the pan-cancer analysis study showed that the CDK1 gene was positively associated with the expression of three classes of RNA methylation regulatory proteins, and affects RNA function through multiple mechanisms of action and plays an important role in the posttranscriptional regulation of the tumor microenvironment. These findings shed light on the role of the CDK1 gene in cancer progression and provide information to further study the CDK1 gene as a potential target for pan-cancer.

EphA2 recognizes Dermatophagoidespteronyssinus to mediate airway inflammation in asthma.

Most of the asthma with low Th2 is severe steroid-resistant asthma, the exact pathogenesis of which has not yet been fully elucidated. We found that IL-6 and IL-8 were highly expressed in the sputum supernatant of severe asthma and ephrin type-A receptor 2 (EphA2) was highly expressed on bronchial epithelial cells. So, is there a connection between these two phenomena? To clarify this issue, we stimulated bronchial epithelial cells 16HBE with Dermatophagoides pteronyssinus and its compontents LPS, respectively, and detected the activation of EphA2, activation of downstream pathways and secretion of inflammatory cytokines. A mouse asthma model was established, and the therapeutic effects of inhibiting or blocking EphA2 on mouse asthma were investigated. The results showed that D. pteronyssinus and its component LPS phosphorylated EphA2 on 16HBE, activated downstream signaling pathways STAT3 and p38 MAPK, and promoted the secretion of IL-6 and IL-8. After knockout of EphA2 on 16HBE, the activation of inflammatory pathways was attenuated and the secretion of IL-6 and IL-8 was significantly reduced. Inhibition or blockade of EphA2 on mouse airways resulted in a significant reduction in airway hyperresponsiveness and airway inflammation, and a significant decrease in the expression levels of IL-6, IL-17F, IL-1α, IL-1ß and TNF in bronchoalveolar lavage fluid and lung tissue. Our study uncovers a novel role for EphA2 expressed on airway epithelial cells in the pathogenesis of asthma; EphA2 recognizes D. pteronyssinus or its component LPS and promotes the secretion of IL-6 and IL-8 by airway epithelial cell, thereby mediating airway inflammation. Thus, it is possible to provide a new molecular therapy for severe asthma.

A novel fully human recombinant antibody neutralizing α-hemolysin of Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to almost all ß-lactam antibiotics. Hence, new ways to control MRSA infection, such as antibacterial antibodies, need to be explored. α-hemolysin is the most important virulence factor widely expressed in S. aureus. This study aimed to develop a new fully human antibody against α-hemolysin of S. aureus and research its neutralizing effect. The single-chain antibody fragments (scFvs) against S. aureus were screened from a fully human scFv library using phage display technology. The selected scFvs had good binding affinities to α-hemolysin and S. aureus. The IgG-like scFv-Fc inserted into the pcDNA3.1 or pMH3 vector was expressed in HEK293F suspension cells to extend the half-life and restore Fc function. The size of purified scFv-Fc was about 55 kDa. The functions of expressed scFv-Fcs against α-hemolysin were validated. The cytotoxicity assays showed that scFv555-Fc had better protective effects on A549 cells than other scFv-Fcs. The results of anti-rabbit erythrocyte lysis and A549 cell apoptosis assay confirmed that scFv555-Fc had a significant neutralizing effect on α-hemolysin. The scFv555-Fc was used to construct the docking model of antigen-antibody complexes using Discovery Studio software. It predicted that the key binding sites of α-hemolysin were TYR28, LYS37, PHE39, ARG56, and LYS58, which might be the key toxic sites of α-hemolysin. A novel fully human scFv-Fc antibody neutralizing the α-hemolysin toxin of S. aureus was successfully developed. The findings might provide a new theoretical basis and treatment method for preventing MRSA infection.

Changes in the phenotype and function of mucosal-associated invariant T cells in neutrophilic asthma.

Asthma is a chronic heterogeneous inflammatory disease. Most neutrophilic asthma (NA) cases are severe asthma involving many inflammatory cells and mediators, although the specific pathogenesis is not clear. Mucosal-associated invariant T (MAIT) cells as innate-like T lymphocytes play an important role in the immune response in asthma by producing cytokines. In this study, we evaluated the phenotype and function of circulating MAIT cells in patients with NA and inflammatory-related cytokines in plasma and induced sputum supernatants using flow cytometry. The results showed that the frequency of circulating MAIT cells in asthma patients, particularly NA patients, decreased significantly, and CD8+ MAIT and MAIT Temra cells also decreased significantly. Increased expression of CD69 and PD-1 on MAIT cells indicated excessive activation and depletion, leading to the decrease in MAIT cells. Levels of IL-17A and TNF-α secreted by MAIT cells of NA patients increased, whereas IFN-γ levels decreased, indicating that MAIT cells in NA are biased to the Th17 subtype. MAIT cells were also negatively correlated with clinical parameters, indicating that these cells are related to asthma severity. Pro-inflammatory cytokines in plasma and sputum supernatant increased to varying degrees, whereas IL-10 declined, corresponding with asthma severity. We speculate that increased IL-17A and TNF-α synergistically stimulated respiratory epithelial cells to secrete IL-6 and IL-8, thereby recruiting neutrophils to inflammatory sites and aggravating asthma symptoms. Therefore, MAIT cells could serve as a potential therapeutic target in NA immunity, thus providing a new strategy for the treatment of asthma.

The onset, development and pathogenesis of severe neutrophilic asthma.

Bronchial asthma is divided into Th2 high, Th2 low and mixed types. The Th2 high type is dominated by eosinophils while the Th2 low type is divided into neutrophilic and paucigranulocytic types. Eosinophilic asthma has gained increased attention recently, and its pathogenesis and treatment are well understood. However, severe neutrophilic asthma requires more in-depth research because its pathogenesis is not well understood, and no effective treatment exists. This review looks at the advances made in asthma research, the pathogenesis of neutrophilic asthma, the mechanisms of progression to severe asthma, risk factors for asthma exacerbations, and biomarkers and treatment of neutrophilic asthma. The pathogenesis of neutrophilic asthma is further discussed from four aspects: Th17-type inflammatory response, inflammasomes, exosomes and microRNAs. This review provides direction for the mechanistic study, diagnosis and treatment of neutrophilic asthma. The treatment of neutrophilic asthma remains a significant challenge for clinical therapists and is an important area of future clinical research.

The Emerging Roles of T Helper Cell Subsets and Cytokines in Severe Neutrophilic Asthma.

Neutrophilic asthma (NA) is a severe type of steroid resistant asthma, and so far the immune mechanisms underlying NA are not clear. In this article, we performed a comprehensive assessment of Th-cell subsets and cytokines in severe NA patients. A total of 13 healthy individuals and 31 severe asthma patients were enrolled in this study. Refractory asthma patients were defined as those with eosinophilic asthma (EA, accounted for 32% of asthmatic patients) or NA (68%) according to sputum neutrophil/eosinophil counts or blood eosinophils. Th-cell subsets in peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry, and cytokines were detected by cytometric bead array (CBA). The results showed significant differences were observed in Th-cell phenotypes, where the number of Th1 cells were reduced and the numbers of Th2 cells were increased in NA and EA groups, respectively, when compared with healthy controls. Th17 cells were not strongly associated with severe neutrophilic asthma. The frequencies of mucosal-associated invariant T (MAIT) cells were strikingly reduced in severe asthma patients, especially in the NA group. This NA group also showed increased levels of IL-17A, IL-17F, TNF-α, and IL-6 in serum and increased levels of IL-17A, IL-17F, IFN-γ, TNF-α, IL-1ß, IL-5, IL-6, and IL-8 in sputum. In addition, sputum IL-6 was positively correlated with TNF-α, IFN-γ, IL-17A, and IL-8. Our results uncovered a controversial role for Th17 cells, which were reduced in severe asthma patients. Severe neutrophilic asthma was associated with a striking deficiency of MAIT cells and high pro-inflammatory cytokine levels.

[Preparation and functional identification of human single chain recombinant antibody targeting α-hemolysin of Staphylococcus aureus].

Objective To prepare a new fully human antibody against α-hemolysin of Staphylococcus aureus (S. aureus) and to investiagete its neutralizing effect. Methods The IgG-like scFv-Fc inserted into the pcDNA3.1 vector by homologous recombination was expressed in HEK293F suspension cells and purified. ELISA was used to detect the purified scFv538-Fc's binding activity and specificity to S. aureus. The cell proliferation & toxicity assay and rabbit erythrocyte hemolysis assay were used to identify the scFv538-Fc against α-hemolysin of S. aureus. Results A new fully human recombinant antibody scFv-Fc against S. aureus. α-hemolysin was successfully prepared. The mass of the purified scFv-Fc was about 55 kDa. The purified antibody had binding activity to scFv538-Fc, and the antibody bound to Staphylococcus aureus specifically. The results of A549 cytotoxicity assays showed that scFv538-Fc had protective effects on A549 cells. The result of anti-rabbit erythrocyte hemolysis assay confirmed that scFv538-Fc had a significant neutralizing effect on toxins. Conclusion A novel fully human scFv-Fc antibody neutralizing the α-hemolysin toxin of S. aureus is successfully prepared.

Fully human recombinant antibodies against EphA2 from a multi-tumor patient immune library suitable for tumor-targeted therapy.

Enhanced EphA2 expression is observed in a variety of epithelial-derived malignancies and is an important target for anti-tumor therapy. Currently, Therapeutic monoclonal antibodies against immune checkpoints have shown good efficacy for tumor treatment. In this study, we constructed an immune single-chain fragment variable (scFv) library using peripheral blood mononuclear cells (PBMCs) from 200 patients with a variety of malignant tumors. High affinity scFvs against EphA2 can be easily screened from the immune library using phage display technology. Anti-EphA2 scFvs can be modified into any form of recombinant antibody, including scFv-Fc and full-length IgG1 antibodies, and the recombinant antibody affinity was improved following modification. Among the modified anti-EphA2 antibodies the affinity of 77-IgG1 was significantly increased, reaching a pmol affinity level (10-12). We further demonstrated the binding activity of recombinant antibodies to the EphA2 protein, tumor cells, and tumor tissues using macromolecular interaction techniques, flow cytometry and immunohistochemistry. Most importantly, both the constructed scFvs-Fc, as well as the IgG1 antibodies against EphA2 were able to inhibit the growth of tumor cells to some extent. These results suggest that the immune libraries from patients with malignant tumors are more likely to screen for antibodies with high affinity and therapeutic effect. The constructed fully human scFv immune library has broad application prospects for specific antibody screening. The screened scFv-Fc and IgG1 antibodies against EphA2 can be used for the further study of tumor immunotherapy.

Integrative Analysis of the Expression of SIGLEC Family Members in Lung Adenocarcinoma via Data Mining.

Background: Sialic acid-binding immunoglobulin-type lectin (SIGLEC) family members are involved in regulating immune-cell activation, proliferation, and apoptosis, and they play an important role in tumor development. However, their expression and correlation with immune molecules in lung adenocarcinoma (LUAD) remain unclear. Methods: We utilized Gene Expression Profiling Interactive Analysis, Kaplan-Meier analysis, the limma package in R/Bioconductor, the University of California Santa Cruz Cancer Genome Browser, cBioPortal, STRING, Cytoscape, DAVID, and the Tumor Immune Estimation Resource for gene and protein profiling and analyses. Results: The results showed that SIGLEC10 and SIGLEC15 levels were upregulated in LUAD, whereas SIGLEC1, CD22 (SIGLEC2), CD33, myelin-associated glycoprotein (SIGLEC4), SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC11, and SIGLEC14 levels were significantly downregulated, with their low expression associated with poor overall survival. Moreover, we observed high SIGLEC-mutation rates (22%) in LUAD patients, with SIGLEC functions determined as primarily involved in regulating the immune response, signal transduction, inflammatory response, and cell adhesion. Furthermore, we found that SIGLEC expression was significantly correlated with immune-cell infiltration, especially macrophages, neutrophils, and dendritic cells, and highly associated with immune molecules such as CD80, CD86, CD28, B-cell-activating factor, programmed cell death 1 ligand 2, and colony stimulating factor 1 receptor. Conclusion: These results provide insight into the potential molecular mechanism associated with SIGLEC-related development of LUAD, as well as clues for screening biomarkers and therapeutic targets.

Title of article: Mucosal-associated invariant T cells in lung diseases.

The lungs are directly connected to the external environment, which makes them more vulnerable to infection and injury. They are protected by the respiratory epithelium and immune cells to maintain a dynamic balance. Both innate and adaptive immune cells are involved in the pathogenesis of lung diseases. Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells, which have attracted increasing attention in recent years. Although MAIT cells account for a small part of the total immune cells in the lungs, evidence suggests that these cells are activated by T cell receptors and/or cytokine receptors and mediate immune response. They play an important role in immunosurveillance and immunity against microbial infection, and recent studies have shown that subsets of MAIT cells play a role in promoting pulmonary inflammation. Emerging data indicate that MAIT cells are involved in the immune response against SARS-CoV-2 and possible immunopathogenesis in COVID-19. Here, we introduce MAIT cell biology to clarify their role in the immune response. Then we review MAIT cells in human and murine lung diseases, including asthma, chronic obstructive pulmonary disease, pneumonia, pulmonary tuberculosis and lung cancer, and discuss their possible protective and pathological effects. MAIT cells represent an attractive marker and potential therapeutic target for disease progression, thus providing new strategies for the treatment of lung diseases.

[House dust mite extract activates EphA2-STAT3/p38 MAPK signaling pathway in airway epithelial cells to mediate airway inflammation].

Objective To investigate the effect of ephrin type-A receptor 2 (EphA2) on the expression of inflammatory cytokines in airway epithelial cells induced by house dust mite extract (HDM) and the underlying mechanism. Methods The cell model of EphA2 knockdown was established by transfection of EphA2 siRNA into airway cell line 16HBE cells. After the 16HBE cells were stimulated with HDM, the mRNA levels of EphA2, interleukin 6 (IL-6) and IL-8 were determined by real-time quantitative PCR (qPCR), and the protein levels of IL-6, IL-8, IL-17A, IL-17F and tumor necrosis factor-α (TNF-α) were measured by cytometric bead array (CBA). Western blotting was used to analyze the protein expression of EphA2, phosphorylated EphA2 (p-EphA2), signal transducer and activator of transcription (STAT3), phosphorylated STAT3 (p-STAT3), p38 mitogen-activated protein kinases (p38 MAPK), phosphorylated p38 MAPK (p-p38 MAPK), nuclear factor κ-B p65 (NF-κB p65) and phosphorylated NF-κB p65 (p-NF-κB p65). Then, in the 16HEB cells stimulated by STAT3 inhibitor Stattic or p38 MAPK inhibitor SB203580 in combination with HDM, the mRNA and protein expression levels of IL-6 and IL-8 were detected by qPCR and CBA. Results Knockdown of EphA2 significantly inhibited the expression of IL-6 and IL-8 in HDM-induced 16HBE, and reduced the total protein and phosphorylated levels of STAT3 and p38 MAPK, but had no significant effect on the total protein and phosphorylated levels of NF-κB p65. After stattic inhibited the expression and activation of STAT3, the mRNA and protein levels of IL-6 and IL-8 significantly decreased in HDM-induced 16HBE cells. Interestingly, while SB203580 inhibited the activation of p38 MAPK signaling pathway, it only inhibited the mRNA levels of IL-6 and IL-8 in HDM-induced 16HBE cells, but had no effect on their protein levels. Conclusion HDM can induce the expression of IL-6 and IL-8 in 16HBE cells to participate in airway inflammation by activating the EphA2-STAT3/p38 MAPK pathway.

[Establishment of a mouse neutrophil-dominated house dust mite allergic asthma model].

Objective To establish a mouse neutrophil-dominated asthma model using house dust mite (HDM). Methods With a random number table, a total of 16 specific pathogen-free (SPF) BALB/c mice were divided into control group and model group. The model group was sensitized and stimulated by HDM nasal drip, and the control group mice were given the same amount of saline. In 24 hours after the last stimulation, the enhanced pause (Penh) was measured by the BUXCO noninvasive lung function detector to determine the airway resistance; the bronchoalveolar lavage fluid (BALF) was collected to count cells and classify inflammatory cells. The infiltration degree of inflammatory cells in the lung tissue was observed by HE staining. The expression of IL-6, IL-17F and IL-1ß mRNA was detected by real-time quantitative PCR, and the levels of IL-4, IL-6, IL-17F and tumor necrosis factor α (TNF-α) in BALF were detected by cytometric bead array (CBA). Results Compared with the control group, the value of Penh in the model group was significantly higher; the tracheal and paravascular inflammation significantly aggravated; the levels of IL-6, IL-17F, IL-1ß mRNA, the cell count and neutrophil count in BALF, and the levels of neutrophil cytokines IL-6, IL-17F and TNF-α in the BALF increased significantly. Conclusion The mouse neutrophil-dominated house dust mite allergic asthma model has been successfully established.

Analogous and Diverse Functions of APSES-Type Transcription Factors in the Morphogenesis of the Entomopathogenic Fungus Metarhizium rileyi.

APSES-type transcription factors (TFs) have analogous and diverse functions in the regulation of fungal morphogenesis processes. However, little is known about these functions in microsclerotium formation. In this study, we characterized two orthologous APSES genes (MrStuA and MrXbp) in the entomopathogenic fungus Metarhizium rileyi Deletion of either MrStuA or MrXbp impaired dimorphic transition, conidiation, fungal virulence, and microsclerotium formation. Compared with the wild-type strain, ΔMrStuA and ΔMrXbp mutants were hypersensitive to thermal and oxidative stress. Furthermore, transcriptome sequencing analysis revealed that MrStuA and MrXbp independently regulate their own distinctive subsets of signaling pathways during dimorphic transition and microsclerotium formation, but they also show an overlapping regulation of genes during these two distinct morphogenesis processes. These results provide a global insight into vital roles of MrStuA and MrXbp in M. rileyi and aid in dissection of the interacting regulatory mechanisms of dimorphism transition and microsclerotium development.IMPORTANCE Transcription factors (TFs) are core components of the signaling pathway and play an important role in transcriptional regulation of gene expression during fungal morphogenesis processes. A prevailing theory suggests an interplay between different TFs regulating microsclerotial differentiation; however, the persisting issue remains that these interplay mechanisms are not clear. Here, we analyzed two members of the APSES-type TFs in Metarhizium rileyi using a gene deletion strategy and transcriptome analysis. Mutants were significantly impaired in microsclerotium formation and dimorphic transition. Transcriptome analysis provided evidence for interacting regulatory mechanisms by the two TFs in microsclerotium formation and dimorphic transition. Furthermore, we investigated their overlapping roles in mediating the expression of genes required for different fungal morphogenesis processes. Characterization of TFs in this study will aid in dissecting the interplay between regulatory mechanisms in fungal morphogenesis processes.

Affinity improvement of the fully human anti­TSLP recombinant antibody.

Thymic stromal lymphopoietin (TSLP) is a potentially important target for the treatment of asthma and malignancies. However, a fully human antibody reactive with TSLP is currently unavailable for clinical use. In a previous study, a human anti­TSLP­single­chain antibody variable fragment (anti­TSLP­scFv) 84 was selected by phage display from a constructed human scFv library. In the present study, a computer simulation method was developed using Discovery Studio 4.5 software, to increase the affinity of anti­TSLP­scFv­84. Specific primers were designed and mutated DNA sequences of anti­TSLP­scFvs were obtained by overlap extension PCR. The mutant scFvs were expressed in pLZ16 and affinity­enhanced anti­TSLP­scFv­M4 was screened using ELISA. However, in general the scFvs have low stability and short half­lives in vivo. Therefore, scFv­84 and scFv­M4 were inserted into eukaryotic expression vectors (pcDNA3.1­sp­Fc and PMH3EN­sp­Fc) and then transfected into 293F cells to express anti­TSLP­scFv­Fc. ELISA and western blotting results indicated the size of the anti­TSLP­scFv­Fc to be ~50 kDa. Binding of anti­TSLP­scFv­Fc­M4 to TSLP was enhanced compared with the pre­mutated scFv­Fc­84. The affinity of the mutated recombinant antibody was determined using the BIAcore technique and found to be ~10­fold greater than the pre­mutated antibody.

blaNDM-5 carried by a hypervirulent Klebsiella pneumoniae with sequence type 29.

Background: A carbapenem-resistant hypermucoviscous Klebsiella pneumoniae isolate was recovered from human sputum. Methods: Whole genome sequencing of this isolate was carried out to reveal its clonal background, antimicrobial resistance determinants and virulence factors. Virulence assays were performed using wax moth larvae. The transfer of blaNDM-5 between bacterial strains was tested using conjugation. 59 genome assemblies of ST29 K. pneumoniae and 230 IncX3 plasmids regardless of the carriage of resistance gene were employed for phylogenetic analysis, respectively. Results: The strain carried a virulence plasmid pVir-SCNJ1 bearing the virulence gene rmpA and exhibited a high virulence in wax moth. This hypervirulent strain belongs to sequence type 29 and carries blaNDM-5, which is located on a conjugative plasmid, designated pNDM5-SCNJ1, belonging to type IncX3. pNDM5-SCNJ1 was fully sequenced and shows high similarity with pNDM_MGR194, except some deletion inside the ISAba125 region. Phylogenetic analysis of IncX3 plasmids revealed that although blaNDM-5 can be evolved from blaNDM-1 via point mutations within some IncX3 plasmids, most of blaNDM-5-carrying IncX3 plasmids probably have acquired blaNDM-5 in multiple events. Conclusions: In this study, we characterized a blaNDM-5-positive hypervirulent K. pneumoniae of sequence type 29 in China. Our results highlight the need for active surveillance on this lineage of carbapenem-resistant K. pneumoniae.

Development and identification of a fully human single-chain variable fragment 29 against TSLP.

Thymic stromal lymphopoietin is a key initiator for inducing Th2-type inflammation and a potential therapeutic target for allergic disease. In the present study, the naive human antibody library was enriched using human thymic stromal lymphopoietin (hTSLP) as an antigen by phage display. Single clones were randomly picked from the enriched antibody library after three rounds of selection, and these were expressed for enzyme-linked immunosorbent assay (ELISA). The positive single-chain fragment variables (scFvs) determined by ELISA were further identified by Western blot, Biacore, and flow cytometry. After three rounds of phage display, 35% of the scFv clones were positive by ELISA and could bind well with hTSLP. Further identification revealed that scFv29 had satisfactory characteristics. The scFv29 was specific to hTSLP, and had no cross-reaction with hIL-33, hIL-4, and hIL-13. The scFv29 could bind to hTSLP in competition with the TSLP receptor and could also bind to mouse TSLP. Cellular experiments revealed that mTSLP could stimulate myeloid dendritic cell (DC) to mature, and scFv29 blocking could reduce the maturation rate of DC. These findings suggest that scFv29 could be used as a neutralizing antibody to block the signaling of TSLP, and this work provides the foundation for further study of the therapeutic roles of TSLP in allergic inflammation diseases.

[The efficiently expression of soluble CD20 in HEK293T cells].

Objective To construct the eukaryotic expression vector of CD20 and overexpress soluble CD20 proteins in HEK293T cells. Methods The total RNA from human peripheral blood mononuclear cells (PBMCs) was used to amplify the coding sequence of CD20 by reverse transcription PCR, and then the CD20 gene was cloned into the expression vector pCMV3-C-His. After PCR and sequencing validation, the recombinant CD20 protein was transiently expressed in HEK293T cells and detected by ELISA and Western blot analysis. Moreover, the expression conditions were optimized to improve the expression level of CD20. Results The coding sequence of CD20 was successfully cloned into eukaryotic expression vector pCMV3-C-His. After 72 hours of transfection, the expression of CD20 was detected both in intracellular and supernatant by Western blot analysis. The passage number of HEK293T cells was related to the expression level of CD20 in supernatant. Conclusion The coding sequence of CD20 was successfully cloned into the eukaryotic expression vector, and CD20 was over expressed in HEK293T cells.