Targeted heart repair by Tß4-loaded cardiac-resident macrophage-derived extracellular vesicles modified with monocyte membranes.

Recent studies have demonstrated the critical role of cardiac-resident macrophages (cMacs) in the maintenance of physiological homeostasis. However, recruitment of circulating monocyte-derived macrophages decreases cMac levels post-myocardial infarction (MI). Transplanting cMacs is not an ideal option due to their low survival rates and the risk of immunological rejection. However, extracellular vesicle therapy has the potential to provide a feasible and safe alternative for cardiac repair. In this study, cell membrane-modified extracellular vesicles (MmEVs) were developed for heart repair by modifying cMac-derived extracellular vesicles (mEVs) with monocyte membranes, resulting in immune evasion and sequential targeted localization to damaged regions through expression of CD47 on MmEVs and strong affinity between monocyte membrane proteins and CCL2. Additionally, to fully exploit the potential clinical application of MmEVs and achieve a better curative effect, thymosin ß4 (Tß4) was loaded into the nanoparticles, resulting in Tß4-MmEVs. In vitro experiments indicated that both the MmEVs and Tß4-MmEVs promoted cardiomyocyte proliferation and endothelial cell migration. Animal experiments suggested that MI mice treated with MmEVs and Tß4-MmEVs exhibited reduced myocardial fibrosis and increased vascular density compared to the control group. Thus, we posit that these targeted nanoparticles hold significant potential for MI adjuvant therapy and may open new avenues for cardiac repair and regeneration. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) derived from bioactive parent cell sources involved in pathological and repair processes for cardiovascular disease have emerged as a compelling strategy for regenerative therapy. In this study, we constructed monocyte membrane-modified extracellular vesicles loaded with a drug (Tß4-MmEVs) for heart repair that exhibit extraordinary abilities of immune evasion and sequential localization to damaged regions owing to the presence of CD47 and the strong affinity between monocytes and damaged cardiomyocytes and endothelial cells. The bioactivities of Tß4-MmEVs on enhancing cardiomyocyte and endothelial cell proliferation were validated both in vitro and in vivo. Effective development and implementation of therapeutically membrane-modified nanoparticles from homologous origins can provide a reference for adjuvant therapy in clinical MI management.

Hsa_circ_0063329 inhibits prostate cancer growth and metastasis by modulating the miR-605-5p/tgif2 axis.

Circular RNAs play crucial regulatory roles in the progression of various cancers. Nevertheless, the underlying mechanisms of circRNAs in prostate cancer (PCa) proliferation and metastasis remain largely uncertain. Here, we performed circRNA microarray analyses to identify differentially expressed circRNAs in a normal prostate epithelial cell line and PCa cell lines. We found that hsa_circ_0063329 was significantly downregulated in PCa. A series of in vitro and in vivo functional assays showed that overexpression of hsa_circ_0063329 inhibits PCa cell progression, while silencing of hsa_circ_0063329 achieved the opposite effects. Mechanistically, bioinformatics analysis, RNA pulldown, RNA-seq and dual-luciferase reporter assays demonstrated that hsa_circ_0063329 exerts its effect by sponging miR-605-5p to derepress TG-interacting factor 2 (TGIF2) and inactivate the TGF-ß pathway. In conclusion, hsa_circ_0063329 inhibits the proliferation and metastasis of PCa via modulation of the miR-605-5p/TGIF2 axis, and targeting hsa_circ_0063329 may provide a promising treatment strategy for aggressive PCa.

Advances in Renal Cell Carcinoma Drug Resistance Models.

Renal cell carcinoma (RCC) is the most common form of kidney cancer. Systemic therapy is the preferred method to eliminate residual cancer cells after surgery and prolong the survival of patients with inoperable RCC. A variety of molecular targeted and immunological therapies have been developed to improve the survival rate and prognosis of RCC patients based on their chemotherapy-resistant properties. However, owing to tumor heterogeneity and drug resistance, targeted and immunological therapies lack complete and durable anti-tumor responses; therefore, understanding the mechanisms of systemic therapy resistance and improving clinical curative effects in the treatment of RCC remain challenging. In vitro models with traditional RCC cell lines or primary cell culture, as well as in vivo models with cell or patient-derived xenografts, are used to explore the drug resistance mechanisms of RCC and screen new targeted therapeutic drugs. Here, we review the established methods and applications of in vivo and in vitro RCC drug resistance models, with the aim of improving our understanding of its resistance mechanisms, increasing the efficacy of combination medications, and providing a theoretical foundation for the development and application of new drugs, drug screening, and treatment guidelines for RCC patients.

Heat Shock Protein A6 Is Especially Involved in Enterovirus 71 Infection.

Hand foot and mouth disease (HFMD) caused by Enterovirus 71 (EV71) infection is still a major infectious disease threatening children's life and health in the absence of effective antiviral drugs due to its high prevalence and neurovirulence. A study of EV71-specific host response might shed some light on the reason behind its unique epidemiologic features and help to find means to conquer EV71 infection. We reported that host heat shock protein A6 (HSPA6) was induced by EV71 infection and involved infection in both Rhabdomyosarcoma (RD) cells and neurogliocytes. Most importantly, we found that EV71 did not induce the expression of other heat shock proteins HSPA1, HSPA8, and HSPB1 under the same conditions, and other HFMD-associated viruses including CVA16, CVA6, CVA10, and CVB1-3 did not induce the upregulation of HSPA6. In addition, EV71 infection enhanced the cytoplasmic aggregation of HSPA6 and its colocalization with viral capsid protein VP1. These findings suggest that HSPA6 is a potential EV71-specific host factor worthy of further study.

Drug Repositioning for Hand, Foot, and Mouth Disease.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease in children caused by a group of enteroviruses. HFMD currently presents a major threat to infants and young children because of a lack of antiviral drugs in clinical practice. Drug repositioning is an attractive drug discovery strategy aimed at identifying and developing new drugs for diseases. Notably, repositioning of well-characterized therapeutics, including either approved or investigational drugs, is becoming a potential strategy to identify new treatments for virus infections. Various types of drugs, including antibacterial, cardiovascular, and anticancer agents, have been studied in relation to their therapeutic potential to treat HFMD. In this review, we summarize the major outbreaks of HFMD and the progress in drug repositioning to treat this disease. We also discuss the structural features and mode of action of these repositioned drugs and highlight the opportunities and challenges of drug repositioning for HFMD.

Immune Checkpoints in Viral Infections.

As evidence has mounted that virus-infected cells, such as cancer cells, negatively regulate the function of T-cells via immune checkpoints, it has become increasingly clear that viral infections similarly exploit immune checkpoints as an immune system escape mechanism. Although immune checkpoint therapy has been successfully used in cancer treatment, numerous studies have suggested that such therapy may also be highly relevant for treating viral infection, especially chronic viral infections. However, it has not yet been applied in this manner. Here, we reviewed recent findings regarding immune checkpoints in viral infections, including COVID-19, and discussed the role of immune checkpoints in different viral infections, as well as the potential for applying immune checkpoint blockades as antiviral therapy.

Constitutively expressed MHC class Ib molecules regulate macrophage M2b polarization and sepsis severity in irradiated mice.

Macrophages can change their physiology in response to microenvironmental signals. This differentiation into classically activated M1 or alternatively activated M2 macrophages is known as polarization. In this study, we isolated bone marrow-derived macrophages from ß2m-deficient (deficient in both MHC class Ia and Ib) and Kb Db -deficient (deficient only in MHC class Ia) mice and found that ß2m-deficient macrophages showed a significantly lower M2b polarization efficiency. In addition, the absence of constitutive MHC class Ib expression decreased the stability of the Notch-1 intracellular domain. Finally, we found that ß2m-deficient mice exposed to irradiation showed reduced bacterial translocation and sepsis severity. Overall, our study demonstrates that MHC class Ib molecules are essential for M2b macrophage polarization and suggests that MHC class Ib molecules play an important role during infection-induced innate immunity.

PIK3CD induces cell growth and invasion by activating AKT/GSK-3ß/ß-catenin signaling in colorectal cancer.

The catalytic subunit p110δ of phosphoinositide 3-kinase (PI3K) encoded by PIK3CD has been implicated in some human solid tumors. However, its roles in colorectal cancer (CRC) remain largely unknown. Here we found that PIK3CD was overexpressed in colon cancer tissues and CRC cell lines and was an independent predictor for overall survival (OS) of patients with colon cancer. The ectopic overexpression of PIK3CD significantly promoted CRC cell growth, migration and invasion in vitro and tumor growth in vivo. In contrast, inhibition of PIK3CD by specific small-interfering RNA or idelalisib dramatically suppressed CRC cell growth, migration and invasion in vitro and tumor growth in vivo. Moreover, PIK3CD overexpression increased AKT activity, nuclear translocation of ß-catenin and T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activity and decreased glycogen synthase kinase 3ß (GSK-3ß) activity, whereas PIK3CD inhibition exhibited the opposite effects. Furthermore, PIK3CD-mediated cell growth, migration and invasion were reversed by blockade of AKT signaling or depletion of ß-catenin. In addition, PIK3CD expression in colon cancer tissues positively correlated with ß-catenin abnormal expression, which was an independent predictor for OS of colon cancer patients. Taken together, our findings demonstrate that PIK3CD is an independent prognostic factor in CRC and that PIK3CD induces CRC cell growth, migration and invasion by activating AKT/GSK-3ß/ß-catenin signaling, suggesting that PIK3CD might be a novel prognostic biomarker and a potential therapeutic target for CRC.

Serum miR-146a and miR-150 as Potential New Biomarkers for Hip Fracture-Induced Acute Lung Injury.

BACKGROUND: Acute lung injury (ALI) and subsequent pulmonary infection are the most severe and usually fatal complications for elderly hip fracture patients. It is necessary to find some biomarkers for early diagnosis and prognosis of it. OBJECTIVE: This study is aimed at examining the differential expression of miR-146a, miR-150, and cytokines (IL-6 and IL-10) between younger and elderly rats suffering from hip fracture and investigating the possible meaning of them in early diagnosis and prognosis of ALI after hip fracture. METHODS AND SUBJECTS: Elderly rats and younger rats were randomly divided into sham group and fracture group, respectively. Two fracture groups received hip fracture operations. The damage degree of ALI was evaluated by histological observation and pathological score. Cytokines were measured by ELISA; miR-146a and miR-150 were analysed by qRT-PCR. RESULTS: After treatment, compared with the corresponding sham groups, the pulmonary histological score, the serum miR-146a concentrations, and the cytokine (IL-6 and IL-10) levels in serum and BALF were significantly higher (the miR-150 were lower) in the fracture groups (with the exception of IL-6 of the younger fracture group at 72 h, all P < 0.05). Meanwhile, compared with the younger fracture group, the aforementioned variables were significantly higher (the miR-150 levels were lower) in the elderly fracture group (with the exception of serum IL-10 and pulmonary histological score at 8 h, all P < 0.05). The results of linear regression analysis showed that serum miR-146a and miR-150 were significantly associated with pulmonary histological score. CONCLUSION: Hip fracture can result in significant systemic inflammation and ALI in the rats. Compared to the younger rats, the elderly rats suffered a more remarkable ALI after hip fracture. It may be related to the abnormal expression of miR-146a and miR-150. Serum miR-146a and miR-150 are potential biomarkers for diagnosis and prognosis of ALI after hip fracture.

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10-8 M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL-1, respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL-1), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Production and Characterization of Monoclonal Antibody Broadly Recognizing Cry1 Toxins by Use of Designed Polypeptide as Hapten.

In this study, by use of synthesized polypeptides as haptens, a monoclonal antibody with broad recognition against seven major Cry1 toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, and Cry1F) has been produced and characterized. First, by comparing the three-dimensional structures of seven Cry1 toxins, analyzing the conserved sequences, and considering the antigenicity and hydrophilicity, three polypeptides (T1, T2, and T3) have been chosen and coupled to keyhole limpet hemocyanin as immunogens for the generic monoclonal antibody (Mab) generation. Thereafter, a double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) was developed for simultaneous determination of seven Cry1 toxins. The results revealed that the haptens T1, T2, and T3 had different effects in the production of antibodies. Among them, the obtained Mab (strain 2D3) generated by T2 can recognize seven Cry1 toxins simultaneously. Equilibrium dissociation constant (KD) values for seven Cry1 toxins with Mab 2D3 were 1.198 × 10(-8) M for Cry1Aa, 2.197 × 10(-8) M for Cry1Ab, 1.367 × 10(-8) M for Cry1Ac, 2.092 × 10(-8) M for Cry1B, 5.177 × 10(-8) M for Cry1C, 4.016 × 10(-8) M for Cry1E, and 3.497 × 10(-8) M for Cry1F. For 2D3-based DAS-ELISA, the limits of detection (LOD) and limits of quantification (LOQ) can reach 15 and 30 ng·mL(-1) for each Cry1 toxin, respectively. Our study is the first report of a broadly specific immunoassay for multidetermination of seven major Cry1 toxins, and it will provide a new idea and technical routes for development of multidetermination immunoassays.

Fracture initiates systemic inflammatory response syndrome through recruiting polymorphonuclear leucocytes.

Fracture, a common type injury in trauma patients, often results in the development of the systemic inflammatory response syndrome (SIRS). Though the mechanism of the fracture-initiated SIRS still remains not well characterized, it is well documented that the polymorphonuclear leucocytes (PMN) play an important role in the inflammatory process. We hypothesize that fractures recruit PMN to the local tissue, which is followed by an increase in the number of peripheral PMN and initiation of SIRS. In the current study, we established a closed femoral fracture rat model. We evaluated the levels of MPO, IL-1ß and CINC-1 in fractured tissue homogenate, and we measured the levels of IL-6 and IL-10, the biomarkers for systemic inflammatory response, in the rat sera. In clinical part of the study, we collected blood from patients with isolated closed femoral fractures and evaluated PMN-related chemoattractants (IL-8, IL-1ß and G-CSF) and the number of peripheral PMN. We further evaluated the level of mitochondrial DNA in the local haematoma of fracture and the circulating plasma of the patients with fracture. In the animal model of closed femoral fracture, we found a significant recruitment of PMN to the local tissue after fracture, which correlates with the elevated MPO level. We also showed that the concentration of IL-1ß and CINC-1 in local tissue is significantly increased and might be responsible for the PMN recruitment. Recruitment of PMN to the local tissue was accompanied with a significant increase in the systemic levels of IL-6 and IL-10 in serum. In the patients with closed femoral fracture, we observed an increase in the number of peripheral PMN and PMN-related chemoattractants, including IL-8, IL-1ß and G-CSF. The level of mitochondrial DNA in the local haematoma of fracture and the circulating plasma of patients were significantly higher compared to the healthy volunteers. Our data suggest that fracture released mitochondrial DNA into the local haematoma of fracture, which recruited the PMN into the local tissue via chemokines (IL-1ß and CINC-1), then increased the numbers of peripheral PMN and SIRS related cytokines in serum (IL-6 and IL-10). This might be the mechanism of the fracture-initiated SIRS.

The Immediate Intramedullary Nailing Surgery Increased the Mitochondrial DNA Release That Aggravated Systemic Inflammatory Response and Lung Injury Induced by Elderly Hip Fracture.

Conventional concept suggests that immediate surgery is the optimal choice for elderly hip fracture patients; however, few studies focus on the adverse effect of immediate surgery. This study aims to examine the adverse effect of immediate surgery, as well as to explore the meaning of mtDNA release after trauma. In the experiment, elderly rats, respectively, received hip fracture operations or hip fracture plus intramedullary nail surgery. After fracture operations, the serum mtDNA levels as well as the related indicators of systemic inflammatory response and lung injury significantly increased in the rats. After immediate surgery, the above variables were further increased. The serum mtDNA levels were significantly related with the serum cytokine (TNF-α and IL-10) levels and pulmonary histological score. In order to identify the meaning of mtDNA release following hip fracture, the elderly rats received injections with mtDNA. After treatment, the related indicators of systemic inflammatory response and lung injury significantly increased in the rats. These results demonstrated that the immediate surgery increased the mtDNA release that could aggravate systemic inflammatory response and lung injury induced by elderly hip fracture; serum mtDNA might serve as a potential biomarker of systemic inflammatory response and lung injury following elderly hip fracture.

Significance of Serum mtDNA Concentration in Lung Injury Induced by Hip Fracture.

Acute lung injury is the most serious and fatal complication of the elderly patients with hip fracture, but the mechanisms are unknown. Recent studies demonstrated the mitochondrial DNA (mtDNA) release was associated with lung injury after trauma. This study aimed to examine the differential release of mtDNA between younger and elderly rats suffering from hip fracture and to investigate the possible mechanism of mtDNA in the lung injury induced by hip fracture. In the first part of the study, we investigated the effects of hip fracture on the rats. The elderly and younger rats, respectively, received hip fracture operations. The degree of lung injury was evaluated, toll-like receptor 9 (TLR9) and nuclear factor kappa B (NF-κB) were determined using Western blot, and mtDNA were analyzed by fluorescent quantitative polymerase chain reaction. In the second part of the study, we investigated the effects of mtDNA on the rats. The elderly and younger rats directly received intravenous injections with mtDNA. After 24 h, the specimens were collected and detected as the first part. Hip fracture resulted in significant mtDNA release, TLR9 and NF-κB p65 expression, and lung injury in the rats. Meanwhile, the mtDNA injection could indirectly induce lung injury. Compared to the younger ones, the elderly rats suffered more serious lung injury after hip fracture and mtDNA injection. These results suggest that the lung injury induced by hip fracture may be involved with the mtDNA release and its TLR9/NF-κB pathway.

Reduced expression of microRNA-134 correlates with malignancy and poor prognosis in human glioma.

MicroRNA-134 (miR-134) has been demonstrated to be dysregulated in glioma tissues. However, its clinical significance in this tumor type has not been fully elucidated. This study was designed to explore the association of miR-134 expression with clinicopathological features and prognosis in human glioma. Using real-time quantitative polymerase chain reaction, miR-134 expression was detected in 162 glioma specimens with various World Health Organization (WHO) grades and compared to the expression in 36 normal brain tissue samples. Glioma tissues exhibited significantly reduced expression of miR-134 (mean 2.15 ± standard deviation 0.82 versus 4.37 ± 1.16, p<0.001) compared with normal brain tissues. In addition, miR-134 expression was notably associated with WHO grade (p<0.001) and Karnofsky Performance Scale score (KPS; p=0.02) in glioma tissues. Low miR-134 expression occurred more frequently in glioma tissues with high WHO grades and low KPS scores. In univariate analysis, both progression-free survival (PFS) and overall survival (OS) of glioma patients with low miR-134 expression were significantly shorter than those with high miR-134 expression (both p<0.001). Additionally, glioma patients with high WHO grades, low KPS scores and subtotal resection attained significantly poorer PFS (p<0.001, 0.02 and 0.01, respectively) and OS (p<0.001, 0.01 and 0.01, respectively). In multivariate analysis, miR-134 expression, WHO grade, KPS score and extent of resection were identified as the independent prognostic factors for both PFS and OS. Collectively, our data prove that the reduced expression of miR-134 may predict aggressive progression and poor prognosis in human gliomas. miR-134 may represent both a prognostic marker and a novel therapeutic target for this malignancy.