The effect of hydatid cyst protoscolex somatic antigens on full-thickness skin wound healing in mouse.

BACKGROUND: Wound healing has evolved in recent years, resulting in diverse therapeutic options. OBJECTIVE: This study evaluated the effects of the somatic antigen of the hydatid cyst protoscolex on wound healing in mice with full-thickness skin wounds. METHODS: Fifty-four adult mice, weighing 25 ± 5 g and approximately 60 days old, were divided into three groups (A, B, and C), each further divided into three subgroups. Subgroups A1, A2, and A3 were assigned negative controls. B1, B2, and B3 received hydatid cyst somatic antigen tests at 10 µg/SC, whereas C1, C2, and C3 received somatic antigen tests at 20 µg/SC. Under general anesthesia, a wound biopsy puncture of 9.8 mm in diameter was performed on the mice's back and spine. In the experimental group, antigen and alum adjuvant were administered subcutaneously around the wound, while the control group received Phosphate-Buffered Saline (PBS). Using digital images, a geometric assessment was conducted on days 0, 1, 3, 6, 9, 12, 15, 18, and 21 post-wounding. The obtained images were analyzed by Image J software and after analyzing the data by SPSS software. RESULTS: A significant difference in terms of epithelization was observed in the antigen treatment group with a dose of 20 µg on days 3 and 6 (P < 0.05). Furthermore, the 20 µg antigen group was significantly higher than the 10 µg antigen group in terms of this factor on day 3 (P < 0.05). Skin samples were taken from all wounds on days 3, 10 and 21 for microscopic evaluation. Regarding epithelization, on day 10, a significant difference was observed in the treatment group with a concentration of 10 µg with the control group and the treatment group with a concentration of 20 µg (P < 0.05). CONCLUSION: Based on the results of the present study, it can be concluded that somatic antigens of protoscolex hydatid cyst are dose-dependent and antigens with a dose of 20 µg by subcutaneous injection accelerate wound healing and epithelialization.

Personalized Cancer Vaccines Directed against Tumor Mutations: Building Evidence from Mice to Humans.

Personalized vaccines directed to tumor mutations have recently gained significant momentum. On the basis of the concept of stimulating T-cell responses against neoantigens encoded by a tumor's host of personal mutations, these vaccines utilize genome or exome sequencing, mutation calling, and epitope prediction followed by manufacturing of a customized vaccine for each patient. In their 2012 Cancer Research publication, Castle and colleagues provided evidence that vaccinating with long peptide vaccines encompassing neoantigens can generate robust immune responses and induce antitumor activity in a mouse B16F10 melanoma. This approach, harnessing the exquisite specificity of mutations to the tumor and thus providing an effective target for cancer vaccines, was subsequently shown to be safe and immunogenic in a series of small first in man trials in patients with melanoma. The field has accelerated and expanded substantially over the last 5 years, propelled by increasing evidence for vaccine-mediated clinical efficacy, leading to ongoing registrational trials using personalized RNA neoantigen vaccines in patients with melanoma and several other malignancies. See related article by Castle and colleagues, Cancer Res 2012;72:1081-91.

Circadian disturbances by altering the light-dark cycle negatively affects hematopoietic function of bone marrow in mice.

Circadian rhythms in metabolically active tissues are crucial for maintaining physical health. Circadian disturbance (CD) can cause various health issues, such as metabolic abnormalities and immune and cognitive dysfunctions. However, studies on the role of CD in immune cell development and differentiation, as well as the rhythmic expression of the core clock genes and their altered expression under CD, remain unclear. Therefore, we exposed C57bl/6j mice to repeated reversed light-dark cycles for 90 days to research the effects of CD on bone marrow (BM) hematopoietic function. We also researched the effects of CD on endogenous circadian rhythms, temporally dependent expression in peripheral blood and myeloid leukocytes, environmental homeostasis within BM, and circadian oscillations of hematopoietic-extrinsic cues. Our results confirmed that when the light and dark cycles around mice were frequently reversed, the circadian rhythmic expression of the two main circadian rhythm markers, the hypothalamic clock gene, and serum melatonin, was disturbed, indicating that the body was in a state of endogenous CD. Furthermore, CD altered the temporally dependent expression of peripheral blood and BM leukocytes and destroyed environmental homeostasis within the BM as well as circadian oscillations of hematopoietic-extrinsic cues, which may negatively affect BM hematopoiesis in mice. Collectively, these results demonstrate that circadian rhythms are vital for maintaining health and suggest that the association between CD and hematopoietic dysfunction warrants further investigation.

IL-17 induces NSCLC cell migration and invasion by elevating MMP19 gene transcription and expression through the interaction of p300-dependent STAT3-K631 acetylation and its Y705-phosphorylation.

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer (NSCLC). Although researchers have disclosed that interleukin 17 (IL-17) can increase matrix metalloproteinases (MMPs) induction causing NSCLC cell metastasis, the underlying mechanism remains unclear. In the study, we found that IL-17 receptor A (IL-17RA), p300, p-STAT3, Ack-STAT3, and MMP19 were up-regulated both in NSCLC tissues and NSCLC cells stimulated with IL-17. p300, STAT3 and MMP19 overexpression or knockdown could raise or reduce IL-17-induced p-STAT3, Ack-STAT3 and MMP19 level as well as the cell migration and invasion. Mechanism investigation revealed that STAT3 and p300 bound to the same region (-544 to -389 nt) of MMP19 promoter, and p300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity, p-STAT3 or MMP19 expression and the cell mobility exposed to IL-17. Meanwhile, p300-mediated STAT3-K631 acetylation and its Y705-phosphorylation could interact, synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion. Besides, the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300, STAT3 or MMP19 gene plus IL-17 treatment, the nodule number, and MMP19, Ack-STAT3, or p-STAT3 production in the lung metastatic nodules were all alleviated. Collectively, these outcomes uncover that IL-17-triggered NSCLC metastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and its Y705-phosphorylation, which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy.

miR-125b reverses cisplatin resistance by regulating autophagy via targeting RORA/BNIP3L axis in lung adenocarcinoma.

The platinum-based chemotherapy is one of the most frequently used treatment protocols for lung adenocarcinoma (LUAD), and chemoresistance, however, usually results in treatment failure and limits its application in the clinic. It has been shown that microRNAs (miRNAs) play a significant role in tumor chemoresistance. In this study, miR-125b was identified as a specific cisplatin (DDP)-resistant gene in LUAD, as indicated by the bioinformatics analysis and the real-time quantitative PCR assay. The decreased serum level of miR-125b in LUAD patients was correlated with the poor treatment response rate and short survival time. MiR-125b decreased the A549/DDP proliferation, and the multiple drug resistance- and autophagy-related protein expression levels, which were all reversed by the inhibition of miR-125b. In addition, xenografts of human tumors in nude mice were suppressed by miR-125b, demonstrating that through autophagy regulation, miR-125b could reverse the DDP resistance in LUAD cells, both in vitro and in vivo. Further mechanistic studies indicated that miR-125b directly repressed the expression levels of RORA and its downstream BNIP3L, which in turn inhibited autophagy and reversed chemoresistance. Based on these findings, miR-125b in combination with DDP might be an effective treatment option to overcome DDP resistance in LUAD.

Age-related Morphofunctional Changes in Sickle Cell Mice Bone Marrow Mesenchymal Stromal Cells.

BACKGROUND AND OBJECTIVES: Bone marrow mesenchymal stromal cells (BM-MSCs) are key elements of the hematopoietic niche and participate in the regulatory mechanisms of hematopoietic stem cells (HSCs). Hematological diseases can affect MSCs and their functions. However, the dysregulations caused by sickle cell disease (SCD) are not fully elucidated. This work explored changes in BM-MSCs and their relationship with age using sickle cell mice (Townes-SS). MATERIALS AND METHODS: BM-MSCs were isolated from Townes-SS, and control groups 30- and 60-day-old Townes-AA and C57BL/6 J. RESULTS: The BM-MSCs showed no morphological differences in culture and demonstrated a murine MSC-like immunophenotypic profile (Sca-1+, CD29+, CD44+, CD90.2+, CD31-, CD45-, and CD117-). Subsequently, all BM-MSCs were able to differentiate into adipocytes and osteocytes in vitro. Finally, 30-day-old BM-MSCs of Townes-SS showed higher expression of genes related to the maintenance of HSCs (Cxcl12, Vegfa, and Angpt1) and lower expression of pro-inflammatory genes (Tnfa and Il-6). However, 60-day-old BM-MSCs of Townes-SS started to show expression of genes related to reduced HSC maintenance and increased expression of pro-inflammatory genes. CONCLUSION: These results indicates age as a modifying factor of gene expression of BM-MSCs in the context of SCD.

Integrin αvß3 and LHRH Receptor Double Directed Nano-Analogue Effective Against Ovarian Cancer in Mice Model.

Introduction: The high mortality rate of malignant ovarian cancer is attributed to the absence of effective early diagnosis methods. The LHRH receptor is specifically overexpressed in most ovarian cancers, and the integrin αvß3 receptor is also overexpressed on the surface of ovarian cancer cells. In this study, we designed LHRH analogues (LHRHa)/RGD co-modified paclitaxel liposomes (LHRHa-RGD-LP-PTX) to target LHRH receptor-positive ovarian cancers more effectively and enhance the anti-ovarian cancer effects. Methods: LHRHa-RGD-LP-PTX liposomes were prepared using the thin film hydration method. The morphology, physicochemical properties, cellular uptake, and cell viability were assessed. Additionally, the cellular uptake mechanism of the modified liposomes was investigated using various endocytic inhibitors. The inhibitory effect of the formulations on tumor spheroids was observed under a microscope. The co-localization with lysosomes was visualized using confocal laser scanning microscopy (CLSM), and the in vivo tumor-targeting ability of the formulations was assessed using the IVIS fluorescent imaging system. Finally, the in vivo anti-tumor efficacy of the formulations was evaluated in the armpits of BALB/c nude mice. Results: The results indicated that LHRHa-RGD-LP-PTX significantly enhanced cellular uptake in A2780 cells, increased cytotoxicity, and hand a more potent inhibitory effect on tumor spheroids of A2780 cells. It also showed enhanced co-localization with endosomes or lysosome in A2780 cells, improved tumor-targeting capability, and demonstrated an enhanced anti-tumor effect in LHRHR-positive ovarian cancers. Conclusion: The designed LHRHa-RGD-LP-PTX liposomes significantly enhanced the tumor-targeting ability and therapeutic efficacy for LHRH receptor-positive ovarian cancers.

Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection.

Purpose: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the lingering threat to public health has fueled the search for effective therapeutics to treat SARS-CoV-2. This study aimed to develop lipid nanoparticle (LNP) inhibitors of SARS-CoV-2 entry to reduce viral infection in the nose and upper airway. Methods: Two types of LNP formulations were prepared following a microfluidic mixing method. The LNP-Trap consisted of DOPC, DSPC, cholesterol, and DSPE-PEG-COOH modified with various spike protein binding ligands, including ACE2 peptide, recombinant human ACE2 (rhACE2) or monoclonal antibody to spike protein (mAb). The LNP-Trim consisted of ionizing cationic DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG lipids encapsulating siACE2 or siTMPRSS2. Both formulations were assayed for biocompatibility and cell uptake in airway epithelial cells (Calu-3). Functional assessment of activity was performed using SARS-CoV-2 spike protein binding assays (LNP-Trap), host receptor knockdown (LNP-Trim), and SARS-CoV-2 pseudovirus neutralization assay (LNP-Trap and LNP-Trim). Localization and tissue distribution of fluorescently labeled LNP formulations were assessed in mice following intranasal administration. Results: Both LNP formulations were biocompatible based on cell impedance and MTT cytotoxicity studies in Calu-3 cells at concentrations as high as 1 mg/mL. LNP-Trap formulations were able to bind spike protein and inhibit pseudovirus infection by 90% in Calu-3 cells. LNP-Trim formulations reduced ACE2 and TMPRSS2 at the mRNA (70% reduction) and protein level (50% reduction). The suppression of host targets in Calu-3 cells treated with LNP-Trim resulted in over 90% inhibition of pseudovirus infection. In vivo studies demonstrated substantial retention of LNP-Trap and LNP-Trim in the nasal cavity following nasal administration with minimal systemic exposure. Conclusion: Both LNP-Trap and LNP-Trim formulations were able to safely and effectively inhibit SARS-CoV-2 pseudoviral infection in airway epithelial cells. These studies provide proof-of-principle for a localized treatment approach for SARS-CoV-2 in the upper airway.

Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation.

Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1ß, IL-6, IL-12p40, IFN-ß) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.

Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility.

Background: Genetic knockout-based studies conducted in mice provide a powerful means of assessing the significance of a gene for fertility. Forkhead-associated phosphopeptide binding domain 1 (FHAD1) contains a conserved FHA domain, that is present in many proteins with phospho-threonine reader activity. How FHAD1 functions in male fertility, however, remains uncertain. Methods: Fhad1-/- mice were generated by CRISPR/Cas9-mediated knockout, after which qPCR was used to evaluate changes in gene expression, with subsequent analyses of spermatogenesis and fertility. The testis phenotypes were also examined using immunofluorescence and histological staining, while sperm concentrations and motility were quantified via computer-aided sperm analysis. Cellular apoptosis was assessed using a TUNEL staining assay. Results: The Fhad1-/-mice did not exhibit any abnormal changes in fertility or testicular morphology compared to wild-type littermates. Histological analyses confirmed that the testicular morphology of both Fhad1-/-and Fhad1+/+ mice was normal, with both exhibiting intact seminiferous tubules. Relative to Fhad1+/+ mice, however, Fhad1-/-did exhibit reductions in the total and progressive motility of epididymal sperm. Analyses of meiotic division in Fhad1-/-mice also revealed higher levels of apoptotic death during the first wave of spermatogenesis. Discussion: The findings suggest that FHAD1 is involved in both meiosis and the modulation of sperm motility.

Laurencia johnstonii extract reverses early lesions in the K14E7HPV16 murine cervical carcinogenesis model.

Persistent infection with high-risk human papillomavirus (HR-HPV) is a well-established risk factor to the development of cervical intraepithelial neoplasia (CIN), a condition that can progress to cervical cancer (CC) a major health problem worldwide. Recently, there has been growing interest in exploring alternative therapies utilizing natural products, among which is the algae species Laurencia johnstonii Setchell & Gardner, 1924 (L. johnstonii), proposed for the management of precancerous lesions. The aim of this work was to determine the effect of an organic extract from L. johnstonii (ELj) in early cervical lesions (CIN 1). These CIN 1 lesions were generated in a murine model expressing the HR-HPV16 E7 oncoprotein (K14E7HPV transgenic mice) with a single exogenous hormonal stimulus using 17ß-estradiol. The histopathological studies, the determination of cell proliferation and of the apoptotic levels in cervical tissue, showed that, seven doses of ELj (30 mg/kg weight per day diluted in a DMSO-saline solution [1:7]) lead to recovery the architecture of cervical epithelium. Accordingly, in the transgenic mice it was observed a statistically significant decrease of the PCNA expression levels, a marker of cell proliferation, and a statistically significant increase in the apoptosis levels using Caspase 3 as a marker. In addition, we determined the expression levels of the tumor suppressor miR-218 and the oncomiRNA miR-21. Interestingly, our results may suggest that ELj treatment tended to restore the normal expression of both miRNAs as compared with controls being more evident in the non-transgenic induced mice. Differences of p < 0.05 were considered statistically significant through the whole study. Based on these results, we propose that the use of ELj could be an alternative for the treatment of cervical early lesions.

Lung-delivered IL-10 therapy elicits beneficial effects via immune modulation in organic dust exposure-induced lung inflammation.

Efficacious therapeutic options capable of resolving inflammatory lung disease associated with environmental and occupational exposures are lacking. This study sought to determine the preclinical therapeutic potential of lung-delivered recombinant interleukin (IL)-10 therapy following acute organic dust exposure in mice. Here, C57BL/6J mice were intratracheally instilled with swine confinement organic dust extract (ODE) (12.5%, 25%, 50% concentrations) with IL-10 (1 µg) treatment or vehicle control intratracheally-administered three times: 5 hr post-exposure and then daily for 2 days. The results showed that IL-10 treatment reduced ODE (25%)-induced weight loss by 66% and 46% at Day 1 and Day 2 post-exposure, respectively. IL-10 treatment reduced ODE (25%, 50%)-induced lung levels of TNFα (-76%, -83% [reduction], respectively), neutrophil chemoattractant CXCL1 (-51%, -60%), and lavage fluid IL-6 (-84%, -89%). IL-10 treatment reduced ODE (25%, 50%)-induced lung neutrophils (-49%, -70%) and recruited CD11cintCD11b+ monocyte-macrophages (-49%, -70%). IL-10 therapy reduced ODE-associated expression of antigen presentation (MHC Class II, CD80, CD86) and inflammatory (Ly6C) markers and increased anti-inflammatory CD206 expression on CD11cintCD11b+ cells. ODE (12.5%, 25%)-induced lung pathology was also reduced with IL-10 therapy. In conclusion, the studies here showed that short-term, lung-delivered IL-10 treatment induced a beneficial response in reducing inflammatory consequences (that were also associated with striking reduction in recruited monocyte-macrophages) following acute complex organic dust exposure.

STUB1 exacerbates calcium oxalate-induced kidney injury by modulating reactive oxygen species-mediated cellular autophagy via regulating CFTR ubiquitination.

The formation of calcium oxalate (CaOx) crystals in the kidneys leads to renal epithelial damage and the progression of crystalline nephropathy. This study investigated the role of STIP1 homology and U-box protein 1 (STUB1), an E3 ubiquitin ligase, and cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel, in CaOx-related renal damage and autophagy regulation. HK-2 cells were treated with various doses of CaOx monohydrate (COM) to simulate kidney injury in vitro. Cell viability, reactive oxygen species (ROS) production, and apoptosis were assessed. The regulation of CFTR ubiquitination by STUB1 was confirmed by immunoprecipitation. An in vivo model was established by injecting mice with glyoxylate. COM treatment dose-dependently decreased cell viability, increased TNF-α and ROS production, and induced apoptotic cell death in HK-2 cells. COM-treated cells also showed decreased CFTR protein expression. CFTR overexpression improved cell viability and reduced ROS production in COM-stimulated HK-2 cells. Bioinformatics analysis predicted CFTR's ubiquitination binding site for STUB1. Further analysis confirmed the role of STUB1 as a ubiquitin ligase in CFTR degradation. Knockdown of STUB1 upregulated CFTR expression, while STUB1 overexpression had the opposite effect. Knockdown of CFTR reversed the impact of STUB1 deficiency on autophagy. The in vivo experiments showed that CFTR overexpression attenuated kidney tissue damage and CaOx deposition in mice. STUB1-mediated CFTR ubiquitination plays a crucial role in mitigating calcium oxalate-related renal damage by regulating autophagy. Targeting the STUB1/CFTR axis may hold therapeutic potential for treating kidney injury associated with calcium oxalate deposition.

Surface modification of a commercial bone plate (Ti6Al4V) implant for improved antibacterial and cytocompatibility via thermal dewetting of a silver thin film.

The high demand for bone grafts has motivated the development of implants with excellent osteogenic activity, whereas the risk of implant-associated infection, particularly given the rise of antimicrobial resistance, has compelled the development of implants with innovative antimicrobial strategies in which a small amount of bactericidal agent can effectively kill a wide range of bacteria. To induce antibacterial property, the surface of Grade-5 bone plate titanium implants used in clinical applications was modified using direct current (DC) sputter coating followed by thermal annealing. The 15 nm silver film-coated implants were thermally annealed in the furnace for 15 min at 750 °C. The modified implant surface's antibacterial efficacy againstEscherichia coli(E. coli),Staphylococcus aureus(S. aureus),Salmonella typhi, andMethicillin-resistant staphylococcus aureusbacteria has been assessed using a colony-forming assay. On the modified implant surface, the growth ofE. coliandS. aureusbacteria is reduced by 99.72%, while highly drug-resistant bacteria are inhibited by 96.59%. The MTT assay was used to assess the cytotoxicity of the modified bone-implant surface against NIH3T3 mouse fibroblast cells. The modified bone-implant surface promoted fibroblast growth and demonstrated good cytocompatibility. Furthermore, the mechanical properties of the implant were not harmed by this novel surface modification method. This method is simple and provides new insight into surface modification of commercial metallic implants to have effective antibacterial properties against various classes of bacteria.

FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury.

BACKGROUND: Neutrophils are traditionally viewed as first responders but have a short onset of action in response to traumatic brain injury (TBI). However, the heterogeneity, multifunctionality, and time-dependent modulation of brain damage and outcome mediated by neutrophils after TBI remain poorly understood. METHODS: Using the combined single-cell transcriptomics, metabolomics, and proteomics analysis from TBI patients and the TBI mouse model, we investigate a novel neutrophil phenotype and its associated effects on TBI outcome by neurological deficit scoring and behavioral tests. We also characterized the underlying mechanisms both in vitro and in vivo through molecular simulations, signaling detections, gene expression regulation assessments [including dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays], primary cultures or co-cultures of neutrophils and oligodendrocytes, intracellular iron, and lipid hydroperoxide concentration measurements, as well as forkhead box protein O1 (FOXO1) conditional knockout mice. RESULTS: We identified that high expression of the FOXO1 protein was induced in neutrophils after TBI both in TBI patients and the TBI mouse model. Infiltration of these FOXO1high neutrophils in the brain was detected not only in the acute phase but also in the chronic phase post-TBI, aggravating acute brain inflammatory damage and promoting late TBI-induced depression. In the acute stage, FOXO1 upregulated cytoplasmic Versican (VCAN) to interact with the apoptosis regulator B-cell lymphoma-2 (BCL-2)-associated X protein (BAX), suppressing the mitochondrial translocation of BAX, which mediated the antiapoptotic effect companied with enhancing interleukin-6 (IL-6) production of FOXO1high neutrophils. In the chronic stage, the "FOXO1-transferrin receptor (TFRC)" mechanism contributes to FOXO1high neutrophil ferroptosis, disturbing the iron homeostasis of oligodendrocytes and inducing a reduction in myelin basic protein, which contributes to the progression of late depression after TBI. CONCLUSIONS: FOXO1high neutrophils represent a novel neutrophil phenotype that emerges in response to acute and chronic TBI, which provides insight into the heterogeneity, reprogramming activity, and versatility of neutrophils in TBI.

The podoplanin-CLEC-2 interaction promotes platelet-mediated melanoma pulmonary metastasis.

BACKGROUND: Podoplanin (PDPN) expressed on tumour cells interacts with platelet C-type lectin-like receptor 2 (CLEC-2). This study aimed to investigate the role of the PDPN-platelet CLEC-2 interaction in melanoma pulmonary metastasis. METHODS: Murine melanoma B16-F0 cells, which have two populations that express podoplanin, were sorted by FACS with anti-podoplanin staining to obtain purified PDPN + and PDPN- B16-F0 cells. C57BL/6J mice transplanted with CLEC-2-deficient bone marrow cells were used for in vivo experiments. RESULTS: The in vivo data showed that the number of metastatic lung nodules in WT mice injected with PDPN + cells was significantly higher than that in WT mice injected with PDPN- cells and in WT or CLEC-2 KO mice injected with PDPN- cells. In addition, our results revealed that the platelet Syk-dependent signalling pathway contributed to platelet aggregation and melanoma metastasis. CONCLUSIONS: Our study indicates that the PDPN-CLEC-2 interaction promotes experimental pulmonary metastasis in a mouse melanoma model. Tumour cell-induced platelet aggregation mediated by the interaction between PDPN and CLEC-2 is a key factor in melanoma pulmonary metastasis.

Sialylation on vesicular integrin ß1 determined endocytic entry of small extracellular vesicles into recipient cells.

BACKGROUND: Small extracellular vesicles (sEV) are closely associated with the development and metastasis of many types of mammalian cancer. Glycoconjugates are highly expressed on sEV and play important roles in sEV biogenesis and their interaction with other cells. However, the study on vesicular glycoconjugates are far behind proteins and nucleic acids. Especially, the functions of sialic acids which are the terminal components of glycoconjugates, are poorly understood in sEV. METHODS: Sialic acid levels on sEV from plasma and bladder cancer cells were determined by ELISA and lectin blotting. Effects of sialylation on sEV uptake were determined by flow cytometry. Vesicular glycoproteins bearing sialic acids responsible for sEV uptake was identified by proteomics and density gradient centrifugation, and their site-specific sialylation functions were assayed by N-glycosylation site mutation. Effects of integrin ß1 bearing sialic acids on the pro-metastatic function of sEV in vivo were explored using Balb/c nu/nu mice. RESULTS: (1) Increased sialic acid levels were observed in sEV from malignant bladder cancer cells. (2) Elimination of sialic acids on sEV impaired sEV uptake by recipient cells. (3) Vesicular integrin ß1 bearing sialic acids was identified to play a key role in sEV uptake. (4) Desialylation of the hybrid domain of vesicular integrin ß1 inhibited its binding to matrix fibronectin, and reduced sEV entry into recipient cells. (5) Sialylation on integrin ß1 affected pro-metastatic function of sEV in Balb/c nu/nu mice. CONCLUSIONS: Taken together, our findings indicate important functional roles of sialic acids in sEV uptake and reprogramming plasticity of surrounding normal epithelial cells.

CYLD alleviates NLRP3 inflammasome-mediated pyroptosis in osteoporosis by deubiquitinating WNK1.

BACKGROUND: Osteoporosis (OP) is the result of bone mass reduction and bone structure disorder. Bone marrow mesenchymal stem cells (BMSCs) are the main source of osteogenic precursor cells involved in adult bone remodeling. The involvement of the deubiquitinating enzyme CYLD in OP has recently been discovered. However, the detailed role and mechanism of CYLD remain unknown. METHODS: The OP mouse model was established by performing ovariectomy (OVX) on mice. Hematoxylin and eosin staining, Masson and Immunohistochemical staining were used to assess pathologic changes. Real-time quantitative PCR, Western blot, and immunofluorescence were employed to assess the expression levels of CYLD, WNK1, NLRP3 and osteogenesis-related molecules. The binding relationship between CYLD and WNK1 was validated through a co-immunoprecipitation assay. The osteogenic capacity of BMSCs was determined using Alkaline phosphatase (ALP) and alizarin red staining (ARS). Protein ubiquitination was evaluated by a ubiquitination assay. RESULTS: The levels of both CYLD and WNK1 were decreased in bone tissues and BMSCs of OVX mice. Overexpression of CYLD or WNK1 induced osteogenic differentiation in BMSCs. Additionally, NLRP3 inflammation was activated in OVX mice, but its activation was attenuated upon overexpression of CYLD or WNK1. CYLD was observed to reduce the ubiquitination of WNK1, thereby enhancing its protein stability and leading to the inactivation of NLRP3 inflammation. However, the protective effects of CYLD on osteogenic differentiation and NLRP3 inflammation inactivation were diminished upon silencing of WNK1. CONCLUSION: CYLD mitigates NLRP3 inflammasome-triggered pyroptosis in osteoporosis through its deubiquitination of WNK1.

MicroRNA-21a-5p inhibition alleviates systemic sclerosis by targeting STAT3 signaling.

BACKGROUND: MicroRNA (miRNA)-21-5p participates in various biological processes, including cancer and autoimmune diseases. However, its role in the development of fibrosis in the in vivo model of systemic sclerosis (SSc) has not been reported. This study investigated the effects of miRNA-21a-5p overexpression and inhibition on SSc fibrosis using a bleomycin-induced SSc mouse model. METHODS: A murine SSc model was induced by subcutaneously injecting 100 µg bleomycin dissolved in 0.9% NaCl into C57BL/6 mice daily for 5 weeks. On days 14, 21, and 28 from the start of bleomycin injection, 100 µg pre-miRNA-21a-5p or anti-miRNA-21a-5p in 1 mL saline was hydrodynamically injected into the mice. Fibrosis analysis was conducted in lung and skin tissues of SSc mice using hematoxylin and eosin as well as Masson's trichrome staining. Immunohistochemistry was used to examine the expression of inflammatory cytokines, phosphorylated signal transducer and activator of transcription-3 (STAT3) at Y705 or S727, and phosphatase and tensin homologue deleted on chromosome-10 (PTEN) in skin tissues of SSc mice. RESULTS: MiRNA-21a-5p overexpression promoted lung fibrosis in bleomycin-induced SSc mice, inducing infiltration of cells expressing TNF-α, IL-1ß, IL-6, or IL-17, along with STAT3 phosphorylated cells in the lesional skin. Conversely, anti-miRNA-21a-5p injection improved fibrosis in the lung and skin tissues of SSc mice, reducing the infiltration of cells secreting inflammatory cytokines in the skin tissue. In particular, it decreased STAT3-phosphorylated cell infiltration at Y705 and increased the infiltration of PTEN-expressing cells in the skin tissue of SSc mice. CONCLUSION: MiRNA-21a-5p promotes fibrosis in an in vivo murine SSc model, suggesting that its inhibition may be a therapeutic strategy for improving fibrosis in SSc.

NF-κB subunits RelA and c-Rel selectively control CD4+ T cell function in multiple sclerosis and cancer.

The outcome of cancer and autoimmunity is often dictated by the effector functions of CD4+ conventional T cells (Tconv). Although activation of the NF-κB signaling pathway has long been implicated in Tconv biology, the cell-autonomous roles of the separate NF-κB transcription-factor subunits are unknown. Here, we dissected the contributions of the canonical NF-κB subunits RelA and c-Rel to Tconv function. RelA, rather than c-Rel, regulated Tconv activation and cytokine production at steady-state and was required for polarization toward the TH17 lineage in vitro. Accordingly, RelA-deficient mice were fully protected against neuroinflammation in a model of multiple sclerosis due to defective transition to a pathogenic TH17 gene-expression program. Conversely, Tconv-restricted ablation of c-Rel impaired their function in the microenvironment of transplanted tumors, resulting in enhanced cancer burden. Moreover, Tconv required c-Rel for the response to PD-1-blockade therapy. Our data reveal distinct roles for canonical NF-κB subunits in different disease contexts, paving the way for subunit-targeted immunotherapies.