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BACKGROUND: Skewed T helper (Th)2 response plays a crucial role in the pathogenesis of allergic diseases. The therapeutic efficacy for allergic diseases is unsatisfactory currently. This study aims to regulate the skewed Th2 response with CARsomes. METHODS: The CARsome consisted of an epitope of Dermatophagoides farina-1 (Derf1), a segment of the anti-DEC205 antibody, the scFv, and an open reading frame of perforin. This fusion protein binds to DEC205 molecule on the surface of exosomes derived from dendritic cells (DC). The effects of CARsome on inducing antigen (Ag)-specific Th2 cell apoptosis were assessed both in vivo and in vitro. RESULTS: Exposure to CARsomes in the culture induced Ag-specific Th2 cell apoptosis. Injection of CARsomes through the vein puncture also induced Ag-specific Th2 cell apoptosis in the lungs of sensitized mice. CARsomes could induce Ag-specific regulatory T cells. Administration of CARsomes efficiently inhibited experimental allergic airway inflammation. CONCLUSIONS: The CARsomes can inhibit allergic airway inflammation by inducing Ag-specific Th2 cell apoptosis and induce Ag-specific regulatory T cells. The data suggest that CARsomes have the translational potential to be used to treat allergic airway inflammation.
Subject(s)
Asthma , Th2 Cells , Animals , Antigens , Apoptosis , Dendritic Cells , Inflammation , Mice , Mice, Inbred BALB C , OvalbuminABSTRACT
Background: Ventilator-Associated Pneumonia (VAP) severely impacts stroke patients' prognosis after endovascular treatment. Hence, this study created a nomogram to predict the occurrence of VAP after endovascular treatment. Methods: The individuals with acute ischemic stroke and large vessel occlusion (AIS-LVO) who received mechanical ventilation and endovascular therapy between July 2020 and August 2023 were included in this retrospective study. The predictive model and nomogram were generated by performing feature selection optimization using the LASSO regression model and multifactor logistic regression analysis and assessed the evaluation, verification and clinical application. Results: A total of 184 individuals (average age 61.85 ± 13.25 years, 73.37% male) were enrolled, and the rate of VAP occurrence was found to be 57.07%. Factors such as the Glasgow Coma Scale (GCS) score, duration of stay in the Intensive Care Unit (ICU), dysphagia, Fazekas scale 2 and admission diastolic blood pressure were found to be associated with the occurrence of VAP in the nomogram that demonstrating a strong discriminatory power with AUC of 0.862 (95% CI, 0.810-0.914), and a favorable clinical net benefit. Conclusion: This nomogram, comprising GCS score, ICU duration, dysphagia, Fazekas scale 2 and admission diastolic blood pressure, can aid clinicians in predicting the identification of high-risk patients for VAP following endovascular treatment in large vessel occlusion stroke.
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BACKGROUND: Autoimmune hepatitis (AIH) is a chronic liver disease caused by inflammation of the liver. The etiology of AIH remains elusive, and there are no reliable serum biomarkers. METHODS: In order to identify candidate biomarkers, 2-DE analysis of serum proteins was performed using a mouse model of AIH induced by treatment with concanavalin A (ConA). To enrich samples for low abundance molecules a commercial albumin removal reagent was used. In an independent analysis, candidate biomarkers were identified in AIH patient's serum by a targeted iTRAQ (isobaric tags for relative and absolute quantification) identification. Candidates were validated in independent cohorts of ConA treated mice and AIH patients by ELISA (enzyme-linked immuno sorbent assay). RESULTS: Nine proteins were differentially expressed in AIH mice treated with con-A. Two of these, the third component of complement (C3) and alpha-2-macroglobulin (A2M) were also up-regulated in AIH patient's sera by a targeted iTRAQ identification. In separate validation studies, serum C3 and A2M levels were increased in mice with ConA treatment after 20-40 h and in 34 AIH patients in a subgroup analysis, females with AIH aged 20-50 years old displayed the largest increases in serum A2M level. Biological network analysis implements the complement cascade and protease inhibitors in the pathogenesis of AIH. CONCLUSION: The serum proteins C3 and A2M are increased both in a mouse model and in patients with AIH by both 2-DE and iTRAQ methods. This integrated serum proteomics investigation should be applicable for translational researchers to study other medical conditions.
Subject(s)
Blood Proteins/metabolism , Hepatitis, Autoimmune/blood , Proteomics , Amino Acid Sequence , Animals , Complement C3/metabolism , Computational Biology , Disease Models, Animal , Electrophoresis, Gel, Two-Dimensional , Enzyme-Linked Immunosorbent Assay , Female , Humans , Isotope Labeling , Male , Mass Spectrometry , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptides/chemistry , Proteome/metabolism , Reproducibility of Results , Signal Transduction , Software , alpha-Macroglobulins/metabolismABSTRACT
To determine the factors influencing the resulting molecular weight of polysialic acid (PSA), batch fermentations by using Escherichia coli were conducted. It was found that temperature and pH were significant factors affecting the PSA production and its resulting molecular weight. When pH was set at 6.4, temperature of 37 °C was suitable for cell growth and PSA production while 33 °C facilitated production of higher molecular weight of PSA. pH 6.4 was favorable for PSA production while pH 7.4 was good for higher molecular weight of PSA at 37 °C. Intramolecular self-cleavage of PSA might lead to relatively low molecular weight under mild acidic condition. Our data suggest that the PSA molecular weight is significantly affected by the pH condition rather than the temperature. It is concluded that the resulting PSA molecular weight not only depends on fermentation conditions but also relates to cell growth rate and PSA production rate. Higher PSA molecular weight was made when its production rate was faster than degradation rate. A novel two-stage pH control fermentation process for production of high molecular weight PSA was developed. At the first stage, pH was set at 6.4 to encourage cell growth and PSA production, whereas pH was set at 7.4 at the second stage to promote the formation of higher molecular weight PSA. PSA yield up to 5.65 g/L and its resulting molecular weight of 260 kDa was attained, the highest level ever reported.
Subject(s)
Biotechnology/methods , Culture Media/chemistry , Escherichia coli/metabolism , Sialic Acids/chemistry , Sialic Acids/metabolism , Escherichia coli/drug effects , Escherichia coli/growth & development , Escherichia coli/radiation effects , Fermentation , Hydrogen-Ion Concentration , Molecular Weight , TemperatureABSTRACT
Background: Acute kidney injury (AKI) is a common and serious medical condition with high morbidity and mortality. Recent research has highlighted ferroptosis, a novel form of programmed cell death, as a potential therapeutic target in mitigating renal tubular injury in AKI. Ferrostatin-1, a specific ferroptosis inhibitor, has been demonstrated to prevent renal injury through ferroptosis inhibition. Methods: Utilizing a murine AKI model, we investigated the effects of Ferrostatin-1 by administering it post-injury. Through high-throughput sequencing and pathological analysis, we focused on the critical role of ferroptosis-related pathways in the treatment. Results: Ferrostatin-1 post-conditioning effectively mitigated oxidative damage and reduced iron content associated with AKI. Additionally, critical ferroptosis-related proteins, such as GPX4, SLC7A11, NRF2, and FTH1, exhibited increased expression levels. In vitro, Ferrostatin-1 treatment of HK-2 cells significantly diminished lipid peroxidation and iron accumulation. Furthermore, Ferrostatin-1 was found to downregulate the PI3K signalling pathway. Conclusion: Ferrostatin-1 acted as a potential ferroptosis inhibitor with the capacity to enhance antioxidant defences. This study suggests that Ferrostatin-1 could serve as a promising novel strategy for improving the treatment of AKI and promoting recovery from the condition.
Subject(s)
Acute Kidney Injury , Ferritins , Animals , Mice , Iron , Cyclohexylamines/pharmacology , Acute Kidney Injury/drug therapyABSTRACT
PURPOSE: Tong Sheng tablets (TSTs) have long been used for treating cerebral ischemic reperfusion injury (CIRI) in clinic, but the underlying mechanism remains unknown. Therefore, in this study, TSTs were evaluated systematically using chemical analysis, network pharmacology and classical pharmacology. METHODS: The first part was TSTs quality control including TSTs fingerprint establishment and chemicals identification. In the second part, network pharmacology analysis and bioinformatics were combined to construct a compound-target-disease network, which can screen out key targets or pathways, revealing complex molecule mechanism of TSTs. The last part was experiment verification. Classical pharmacology of TSTs was investigated in vivo to verify the results of network pharmacology. RESULTS: (1) Fingerprints of TSTs were established, and 11 characteristic peaks were identified using HPLC. (2) Network pharmacology and bioinformatics suggested that the protection of TSTs in treating CIRI might be related to regulation of oxidative stress, inflammation and apoptosis, and some key molecules such as Nrf2, IL-1ß, TNF, Bcl-2 and Cyt-C involved in the pathways. (3) TSTs significantly improved neurologic behavior scores, decreased the areas of ischemic necrosis and neuronal necrosis, and increased Nissl body counts. Besides, TSTs significantly decreased pro-inflammatory cytokine (IL-1ß, TNF-α) and pro-oxidative product levels (LPO, MDA) and increased anti-oxidative product levels (NO, SOD). TSTs downregulated the protein expressions of Nrf2 and HO-1. Meanwhile, TSTs reduced apoptotic cell counts, downregulated the protein expressions of Cyt-C and Bax, and upregulated the protein expression of Bcl-2. In terms of autophagy, TSTs enhanced LC-3B protein expression. CONCLUSION: The present results illustrated that TSTs effectively alleviated CIRI, and the underlying mechanism might be associated with multiple molecular pathways. Herein, we established a primary pattern for studying Chinese herbal compounds and provided basic guidance for future investigation.
ABSTRACT
Daptomycin (DAP) is a novel lipopeptide antibiotic which exhibits excellent antibacterial activity against most clinically relevant Gram-positive bacteria, but the DAP-targeting protein molecules against host bacterial infection are far from clear. In order to discover bacterial protein response to DAP treatment, an iTRAQ-based quantitative proteomic analysis was applied to identify differential bacterial proteome profiling of Staphylococcus aureus (S. aureus) ATCC 25923 to 0.125µg/ml DAP exposure. Totally 51 bacterial proteins were significantly changed with DAP treatment, among which 34 proteins were obviously up-regulated and 17 proteins were down-regulated. Meanwhile, 139 bacterial cell membrane (CM) proteins were identified, and 7 CM proteins were significantly altered to decrease CM potential to disrupt bacterial cell membrane. Especially the up-regulation of NDK and down-regulation of NT5 in several S. aureus strains are validated to be a universal variation tendency response to DAP treatment. Under DAP exposure, bacterial membrane potential is decreased and cell membrane is disrupted, and bacterial chromosome is aggregated, which contributes to bacterial DNA rapid release and induces bacteria death within 2-5h. In general, multiple bacterial protein expressions are changed in response to DAP antibiotic exposure, which disrupts host bacterial physiology by multiple cellular levels. To our knowledge, this is the first time to exactly identify infectious bacterial proteins in response to DAP antibiotic action. Our findings help better understand DAP antibacterial mechanism and develop novel DAP derivatives against the upcoming antibiotic-resistant bacterial infection. BIOLOGICAL SIGNIFICANCE: DAP is a novel lipopeptide antibiotic that it exhibits excellent in vitro activity against most clinically relevant Gram-positive bacteria, and the investigations on its pharmaceutical action mode of DAP have dramatically increased in the past decade due to its unique antimicrobial mechanism. However, the target molecules of DAP acting on the infectious bacteria, are far from clear. The state-of-the-art quantitative proteomic technologies provide new avenues to uncover underlying mechanism of antibiotics. Our research main aims to identify bacterial proteome profiling of host strain S. aureus response to DAP treatment through an iTRAQ-based quantitative proteomic analysis, which contributes to understand DAP efficient antibacterial activity and the microbial-antibiotic interactions.
Subject(s)
Anti-Bacterial Agents/pharmacology , Daptomycin/pharmacology , Proteome/drug effects , Proteomics/methods , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Microbial Sensitivity Tests , Proteome/analysis , Proteome/metabolism , Staphylococcus aureus/geneticsABSTRACT
Epstein-Barr virus (EBV)-related non-Hodgkin's lymphoma (NHL) represents a major problem in hematological clinical studies due to its drug tolerance and refractoriness. EBV infection is a key factor driving the process of tumor growth. Immune therapy is an important biotherapeutic method of treating cancer, which is attracting increasing attention. We hypothesized that combining conventional chemotherapy with immune therapy in the treatment of EBV-related NHL may achieve better outcomes. First, we successfully cloned large numbers of EBV-specific T cells by immune stimulation ex vivo. Subsequently, the combined therapy was applied in a murine model of human EBV-related NHL. As expected, combined therapy inhibited tumor growth more effectively compared with monotherapy. In addition, we continuously tested the tumor-associated immune microenvironment and observed that the numbers of tumor-infiltrating cytotoxic T lymphocytes (CTLs) and macrophages were elevated following combined therapy. These effects suggest that EBV-specific CTLs may indirectly promote an innate immune reaction in lymphoma by activating tumor-infiltrating macrophage proliferation. Our findings may provide a guide for the prospective treatment of EBV-related NHL.
ABSTRACT
Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity tags.