Blockade of PI3K/AKT signaling pathway by Astragaloside IV attenuates ulcerative colitis via improving the intestinal epithelial barrier.

BACKGROUND: The specific pathogenesis of UC is still unclear, but it has been clear that defects in intestinal barrier function play an important role in it. There is a temporary lack of specific drugs for clinical treatment. Astragaloside IV (AS-IV) is one of the main active ingredients extracted from Astragalus root and is a common Chinese herbal medicine for the treatment of gastrointestinal diseases. This study aimed to determine whether AS-IV has therapeutic value for DSS or LPS-induced intestinal epithelial barrier dysfunction in vivo and in vitro and its potential molecular mechanisms. METHODS: The intestinal tissues from UC patients and colitis mice were collected, intestinal inflammation was observed by colonoscopy, and mucosal barrier function was measured by immunofluorescence staining. PI3K/AKT signaling pathway activator YS-49 and inhibitor LY-29 were administered to colitic mice to uncover the effect of this pathway on gut mucosal barrier modulation. Then, network pharmacology was used to screen Astragaloside IV (AS-IV), a core active component of the traditional Chinese medicine Astragalus membranaceus. The potential of AS-IV for intestinal barrier function repairment and UC treatment through blockade of the PI3K/AKT pathway was further confirmed by histopathological staining, FITC-dextran, transmission electron microscopy, ELISA, immunofluorescence, qRT-PCR, and western blotting. Finally, 16 S rRNA sequencing was performed to uncover whether AS-IV can ameliorate UC by regulating gut microbiota homeostasis. RESULTS: Mucosal barrier function was significantly damaged in UC patients and murine colitis, and the activated PI3K/AKT signaling pathway was extensively involved. Both in vivo and vitro showed that the AS-IV-treated group significantly relieved inflammation and improved intestinal epithelial permeability by inhibiting the activation of the PI3K/AKT signaling pathway. In addition, microbiome data found that gut microbiota participates in AS-IV-mediated intestinal barrier recovery as well. CONCLUSIONS: Our study highlights that AS-IV exerts a protective effect on the integrality of the mucosal barrier in UC based on the PI3K/AKT pathway, and AS-IV may serve as a novel AKT inhibitor to provide a potential therapy for UC.

Deficiency of microRNA-10b promotes DSS-induced inflammatory response via impairing intestinal barrier function.

Inflammatory bowel disease (IBD) is a non-specific inflammatory disease of the intestine with the pathogenesis to be largely unknown. We found that microRNA (miR)-10b knock-out mice displayed mild IBD symptoms, suggesting that miR-10b may be involved in the onset and development of IBD. This study focuses on elucidating the role of miR-10b in IBD. The colitis model was induced by feeding the mice with 2.5% dextran sodium sulfate (DSS), and the expression levels of miR-10b in colon tissue and blood samples were examined. The severity of colitis was assessed by disease activity index, colon length, histopathological damage, intestinal permeability and ELISA. Then, after transfection of Caco-2 cells with miR-10b mimic and inhibitor, qRT-PCR was used to detect the expression levels of intestinal barrier related genes in colon tissues and cells. miR-10b levels were significantly reduced in mice with DSS-induced acute colitis. Compared with wild-type (WT) mice, miR-10b knockout mice were more sensitive to DSS-induced colitis characterized by increased inflammatory cell infiltration and more severe disruption of colonic barrier function. In addition, by inhibiting miR-10b and thus increasing intestinal barrier gene expression in Caco-2 cells, we found that miR-10b suppressed inflammatory responses and enhanced intestinal barrier function both in vivo and in vitro. miR-10b inhibits the inflammatory response in DSS-induced acute colitis mice in vivo and enhances intestinal barrier function in vitro, suggesting that miR-10b plays a key role in the developmental process of IBD. Thus, miR-10b may be expected to be a new target for the treatment of IBD.

ΗΙF1α, EGR1 and SP1 co-regulate the erythropoietin receptor expression under hypoxia: an essential role in the growth of non-small cell lung cancer cells.

BACKGROUND: Overexpression of erythropoietin (EPO) and EPO receptor (EPO-R) is associated with poor prognosis in non-small-cell lung carcinoma (NSCLC). Hypoxia, a potent EPO inducer, is a major stimulating factor in the growth of solid tumors. However, how EPO-R expression is regulated under hypoxia is largely unknown. METHODS: The role of EPO-R in NSCLC cell proliferation was assessed by RNA interference in vitro. Luciferase reporter assays were performed to map the promoter elements involved in the EPO-R mRNA transcription. Nuclear co-immunoprecipitation and chromatin immunoprecipitation were performed to assess the interaction among transcription factors HIF1α, SP1, and EGR1 in the regulation of EPO-R under hypoxia. The expression of key EPO-R transcription factors in clinical specimens were determined by immunohistochemistry. RESULTS: Hypoxia induced a dosage and time dependent EPO-R mRNA expression in NSCLC cells. Knockdown of EPO-R reduced NSCLC cell growth under hypoxia (P < 0.05). Mechanistically, a SP1-EGR1 overlapped DNA binding sequence was essential to the hypoxia induced EPO-R transcription. In the early phase of hypoxia, HIF1α interacted with EGR1 that negatively regulated EPO-R. With the exit of EGR1 in late phase, HIF1α positively regulated EPO-R expression through additive interaction with SP1. In clinical NSCLC specimen, SP1 was positively while EGR1 was negatively associated with active EPO-R expression (P < 0.05). CONCLUSIONS: HIF1α, SP1 and EGR1 mediated EPO-R expression played an essential role in hypoxia-induced NSCLC cell proliferation. Our study presents a novel mechanism of EPO-R regulation in the tumor cells, which may provide information support for NSCLC diagnosis and treatment.

Synergistic tumoricidal effect of combined hPD-L1 vaccine and HER2 gene vaccine.

Immunotherapy is gathering momentum as a kind of important therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only in a small subset of patients. Combination therapies targeting multiple pathways often can augment an immune response to improve survival further. Here, the tumoricidal effects of the dual hPD-L1(human programmed cell death ligand 1) vaccination/HER2(human epidermal growth factor receptor 2) gene vaccination immunotherapy against the established HER2-expressed cancers were observed. Animals treated with combination therapy using hPD-L1 vaccine and HER2 gene vaccine had significantly improved survival in a mammary carcinoma model. We observed an increase in tumor growth inhibition following treatment. The percentage of the tumor-free mice (%) was much higher in the combined PD-L1/HER2 group. Furthermore, under the tumor-burden condition, hPD-L1 vaccine enhanced humoral immunity of HER2 gene vaccine. And the combination treatment increased the IFN-γ-producing effector T cells. Additionally, splenocytes from the combined PD-L1/HER2 group immunized mice possessed higher CTL activity. Notably, vaccination with combination therapy induced a significant decrease in the percentage of CD4+CD25+ Treg cells. Collectively, these data demonstrate that PD-L1/HER2 gene vaccine combination therapy synergistically generates marked tumoricidal effects against established HER2-expressing cancers.

Acetylation and Amination Protect Angiotensin 1-7 from Physiological Hydrolyzation and Therefore Increases Its Antitumor Effects on Lung Cancer.

The recently reported inhibitory effects of angiotensin 1-7 (Ang-(1-7)) on various cancers indicate its potential use as a therapeutic agent for primary and metastatic cancers. However, its extremely short half-life in the circulation greatly compromises its potential applications. Here, we reported an Ang-(1-7) analogue peptide with the amino and carboxy termini protected by acetylation and amination. The in vitro and in vivo degradation of the resulting analogue, Ang-AA, were determined using high-performance liquid chromatography (HPLC). At the same time, small RNA interference and competition studies were performed to evaluate the specific capacity of Ang-AA to bind to the cell surface Mas receptor. Cell Counting Kit-8 (CCK8), wound-healing, and Boyden chamber assays were performed to investigate the inhibitory effects of Ang-AA on A549 cells. Finally, the synergistic inhibitory effects of Ang-AA and paclitaxel (PTX) on A549 xenografts in mice were observed using animal imaging systems and survival observations. The toxicity of Ang-AA in mice was evaluated. Our results showed that acetylation and amination significantly inhibited the hydrolyzation of Ang-(1-7) in vitro and in vivo. The half-life of Ang-(1-7) in rats was prolonged from 2.4 ± 0.6 min to 238.7 ± 61.3 min ( p < 0.001). The specific binding of Ang-AA to the Mas receptor was well preserved, and Ang-AA exerted significantly greater inhibitory effects on the proliferation, migration, and invasion of A549 cells than Ang-(1-7). The combination of Ang-AA and PTX exhibited a significantly greater synergistic inhibitory effect on A549 xenografts than the combination of Ang-(1-7) and PTX. Ang-AA did not display obvious toxicity in mice. Our findings indicate acetylation and amination is a simple and effective method for producing Ang-(1-7) as a bioactive peptide.

miR-10a inhibits dendritic cell activation and Th1/Th17 cell immune responses in IBD.

OBJECTIVE: Although both innate and adaptive responses to microbiota have been implicated in the pathogenesis of IBD, it is still largely unknown how they are regulated during intestinal inflammation. In this report, we investigated the role of microRNA (miR)-10a, a small, non-coding RNA, in the regulation of innate and adaptive responses to microbiota in IBD. METHODS: miR-10a expression was analysed in the inflamed mucosa of IBD patients treated with or without antitumour necrosis factor (anti-TNF) monoclonal antibodies (mAb) (infliximab) by qRT-PCR. Human monocyte-derived dendritic cells (DC) and IBD CD4+ T cells were transfected with miR-10a precursor to define their effect on the function of DC and CD4+ T cells. RESULTS: The expression of miR-10a was markedly decreased, while NOD2 and interleukin (IL)-12/IL-23p40 were significantly increased, in the inflamed mucosa of IBD patients compared with those in healthy controls. Commensal bacteria, TNF and interferon-γ inhibited human DC miR-10a expression in vitro. Anti-TNF mAb treatment significantly promoted miR-10a expression, whereas it markedly inhibited NOD2 and IL-12/IL-23p40 in the inflamed mucosa. We further identified NOD2, in addition to IL-12/IL-23p40, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited IL-12/IL-23p40 and NOD2 in DC. Moreover, miR-10a was found to markedly suppress IBD T helper (Th)1 and Th17 cell responses. CONCLUSIONS: Our data indicate that miR-10a is decreased in the inflamed mucosa of IBD and downregulates mucosal inflammatory response through inhibition of IL-12/IL-23p40 and NOD2 expression, and blockade of Th1/Th17 cell immune responses. Thus, miR-10a could play a role in the pathogenesis and progression of IBD.

FOXP3 suppresses breast cancer metastasis through downregulation of CD44.

Forkhead box protein 3 (FOXP3) plays an important role in breast cancer as an X-linked tumor suppressor gene. However, the biological functions and significance of FOXP3 in breast cancer metastasis remain unclear. Here, we find that, clinically, nuclear FOXP3 expression is inversely correlated with breast cancer metastasis. Moreover, we demonstrate that FOXP3 significantly inhibits adhesion, invasion and metastasis of breast cancer cells in vivo and in vitro. In addition, the adhesion molecule CD44 is found to be suppressed by FOXP3 through transcriptome sequence analysis (RNA-seq). A luciferase reporter assay, chromatin immunoprecipitation and electrophoretic mobility shift assay identify CD44 as a direct target of FOXP3. The expression of CD44 is downregulated by FOXP3 in breast cancer cells. Importantly, anti-CD44 antibody reverses the FOXP3 siRNA-induced effects on the breast cancer cells in vitro and FOXP3 expression level in the nucleus of breast cancer cells is inversely correlated with CD44 expression level in clinic breast cancer tissues. Taken together, the results from the present study suggest that FOXP3 is a suppressor of breast cancer metastasis. FOXP3 directly binds to the promoter of CD44 and inhibits its protein expression, thereby suppressing adhesion and invasion of human breast cancer cells. This finding highlights the therapeutic potential of FOXP3-CD44 signaling to inhibit breast cancer metastasis.

Downregulation of microRNA-107 in intestinal CD11c(+) myeloid cells in response to microbiota and proinflammatory cytokines increases IL-23p19 expression.

Commensal flora plays an important role in the development of the mucosal immune system and in maintaining intestinal homeostasis. However, the mechanisms involved in regulation of host-microbiota interaction are still not completely understood. In this study, we examined how microbiota and intestinal inflammatory conditions regulate host microRNA expression and observed lower microRNA-107 (miR-107) expression in the inflamed intestines of colitic mice, compared with that in normal control mice. miR-107 was predominantly reduced in epithelial cells and CD11c(+) myeloid cells including dendritic cells and macrophages in the inflamed intestines. We demonstrate that IL-6, IFN-γ, and TNF-α downregulated, whereas TGF-ß promoted, miR-107 expression. In addition, miR-107 expression was higher in the intestines of germ-free mice than in mice housed under specific pathogen-free conditions, and the presence of microbiota downregulated miR-107 expression in DCs and macrophages in a MyD88- and NF-κB-dependent manner. We determined that the ectopic expression of miR-107 specifically repressed the expression of IL-23p19, a key molecule in innate immune responses to commensal bacteria. We concluded that regulation of miR-107 by intestinal microbiota and proinflammatory cytokine serve as an important pathway for maintaining intestinal homeostasis.

UXT is a novel regulatory factor of regulatory T cells associated with Foxp3.

Regulatory T (Treg) cells are a constitutively immunosuppressive subtype of T cells that contribute to the maintenance of immunological self-tolerance and immune homeostasis. However, the molecular mechanisms involved in the regulation of Treg cells remain unclear. In the present study, we identified ubiquitously expressed transcript (UXT) to be a novel regulator of human Treg-cell function. In cultured human Treg cells, UXT associates with Foxp3 in the nucleus by interacting with the proline-rich domain in the N-terminus of Foxp3. Knockdown of UXT expression in Treg cells results in a less-suppressive phenotype, demonstrating that UXT is an important regulator of the suppressive actions of Treg cells. Depletion of UXT affects the localization stability of Foxp3 protein in the nucleus and downregulates the expression of Foxp3-related genes. Overall, our results show that UXT is a cofactor of Foxp3 and an important player in Treg-cell function.

Expression, purification and characterization of galectin-1 in Escherichia coli.

As a member of beta-galactoside-binding proteins family, Galectin-1 (Gal-1) contains a single carbohydrate recognition domain, by means of which it can bind glycans both as a monomer and as a homodimer. Gal-1 is implicated in modulating cell-cell and cell-matrix interactions and may act as an autocrine negative growth factor that regulates cell proliferation. Besides, it can also suppress TH1 and TH17 cells by regulating dendritic cell differentiation or suppress inflammation via IL-10 and IL-27. In the present study, Gal-1 monomer and concatemer (Gal-1②), which can resemble Gal-1 homodimer, were expressed in Escherichia coli and their bioactivities were analyzed. The results of this indicate that both Gal-1 and Gal-1② were expressed in E. coli in soluble forms with a purity of over 95% after purifying with ion-exchange chromatography. Clearly, both Gal-1 and Gal-1② can effectively promote erythrocyte agglutination in hemagglutinating activity assays and inhibit Jurkat cell proliferation in MTT assays. All these data demonstrate that bacterially-expressed Gal-1 and Gal-1② have activities similar to those of wild type human Gal-1 whereas the bioactivity of concatemer Gal-1② was stronger than those of the bacterially-expressed and wild type human Gal.

Lymphocyte oxidative stress/genotoxic effects are related to serum IgG and IgA levels in coke oven workers.

We investigated oxidative stress/genotoxic effects levels, immunoglobulin levels, polycyclic aromatic hydrocarbons (PAHs) levels exposed in 126 coke oven workers and in 78 control subjects, and evaluated the association between oxidative stress/genotoxic effects levels and immunoglobulin levels. Significant differences were observed in biomarkers, including 1-hydroxypyrene levels, employment time, percentages of alcohol drinkers, MDA, 8-OHdG levels, CTL levels and CTM, MN, CA frequency, and IgG, IgA levels between the control and exposed groups. Slightly higher 1-OHP levels in smoking users were observed. For the dose-response relationship of IgG, IgA, IgM, and IgE by 1-OHP, each one percentage increase in urinary 1-OHP generates a 0.109%, 0.472%, 0.051%, and 0.067% decrease in control group and generates a 0.312%, 0.538%, 0.062%, and 0.071% decrease in exposed group, respectively. Except for age, alcohol and smoking status, IgM, and IgE, a significant correlation in urinary 1-OHP and other biomarkers in the total population was observed. Additionally, a significant negative correlation in genotoxic/oxidative damage biomarkers of MDA, 8-OH-dG, CTL levels, and immunoglobins of IgG and IgA levels, especially in coke oven workers, was found. These data suggest that oxidative stress/DNA damage induced by PAHs may play a role in toxic responses for PAHs in immunological functions.

Akkermansia muciniphila isolated from forest musk deer ameliorates diarrhea in mice via modification of gut microbiota.

BACKGROUND: The forest musk deer, a rare fauna species found in China, is famous for its musk secretion which is used in selected Traditional Chinese medicines. However, over-hunting has led to musk deer becoming an endangered species, and their survival is also greatly challenged by various high incidence and high mortality respiratory and intestinal diseases such as septic pneumonia and enteritis. Accumulating evidence has demonstrated that Akkermannia muciniphila (AKK) is a promising probiotic, and we wondered whether AKK could be used as a food additive in animal breeding programmes to help prevent intestinal diseases. METHODS: We isolated one AKK strain from musk deer feces (AKK-D) using an improved enrichment medium combined with real-time PCR. After confirmation by 16S rRNA gene sequencing, a series of in vitro tests was conducted to evaluate the probiotic effects of AKK-D by assessing its reproductive capability, simulated gastrointestinal fluid tolerance, acid and bile salt resistance, self-aggregation ability, hydrophobicity, antibiotic sensitivity, hemolysis, harmful metabolite production, biofilm formation ability, and bacterial adhesion to gastrointestinal mucosa. RESULTS: The AKK-D strain has a probiotic function similar to that of the standard strain in humans (AKK-H). An in vivo study found that AKK-D significantly ameliorated symptoms in the enterotoxigenic Escherichia coli (ETEC)-induced murine diarrhea model. AKK-D improved organ damage, inhibited inflammatory responses, and improved intestinal barrier permeability. Additionally, AKK-D promoted the reconstitution and maintenance of the homeostasis of gut microflora, as indicated by the fact that AKK-D-treated mice showed a decrease in Bacteroidetes and an increase in the proportion of other beneficial bacteria like Muribaculaceae, Muribaculum, and unclassified f_Lachnospiaceae compared with the diarrhea model mice. CONCLUSION: Taken together, our data show that this novel AKK-D strain might be a potential probiotic for use in musk deer breeding, although further extensive systematic research is still needed.

miR-99b-targeted mTOR induction contributes to irradiation resistance in pancreatic cancer.

BACKGROUND: Radiation exerts direct antitumor effects and is widely used in clinics, but the efficacy is severely compromised by tumor resistance. Therefore uncovering the mechanism of radioresistance might promote the development of new strategies to overcome radioresistance by manipulating activity of the key molecules. METHODS: Immunohistochemistry were used to find whether mTOR were over-activated in radioresistant patients' biopsies. Then Western blot, real-time PCR and transfection were used to find whether radiotherapy regulates the expression and activity of mTOR by modulating its targeting microRNA in human pancreatic cancer cell lines PANC-1, Capan-2 and BxPC-3. Finally efficacy of radiation combined with mTOR dual inhibitor AZD8055 was assessed in vitro and in vivo. RESULTS: Ionizing radiation promoted mTOR expression and activation in pancreatic cancer cells through reducing miR-99b expression, which negatively regulated mTOR. Novel mTOR inhibitor, AZD8055 (10 nM, 100 nM, 500 nM) synergistically promoted radiation (0-10 Gy) induced cell growth inhibition and apoptosis. In human pancreatic cancer xenografts, fractionated radiation combined with AZD8055 treatment further increased the anti-tumor effect, the tumor volume was shrinked to 278 mm3 after combination treatment for 3 weeks compared with single radiation (678 mm3) or AZD8055 (708 mm3) treatment (P < 0.01). CONCLUSIONS: Our data provide a rationale for overcoming radio-resistance by combined with mTOR inhibitor AZD8055 in pancreatic cancer therapy.

TNFα induced FOXP3-NFκB interaction dampens the tumor suppressor role of FOXP3 in gastric cancer cells.

Controversial roles of FOXP3 in different cancers have been reported previously, while its role in gastric cancer is largely unknown. Here we found that FOXP3 is unexpectedly upregulated in some gastric cancer cells. To test whether increased FOXP3 remains the tumor suppressor role in gastric cancer as seen in other cancers, we test its function in cell proliferation both at basal and TNFα mimicked inflammatory condition. Compared with the proliferation inhibitory role observed in basal condition, FOXP3 is insufficient to inhibit the cell proliferation under TNFα treatment. Molecularly, we found that TNFα induced an interaction between FOXP3 and p65, which in turn drive the FOXP3 away from the promoter of the well known target p21. Our data here suggest that although FOXP3 is upregulated in gastric cancer, its tumor suppressor role has been dampened due to the inflammation environment.

Mimotope vaccination for epitope-specific induction of anti-VEGF antibodies.

BACKGROUND: Tumor angiogenesis is critical for tumor growth, infiltration and metastasis. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and targeting it is important in reducing angiogenesis. Bevacizumab (Avastin), a monoclonal antibody that reacts directly against VEGF, has been demonstrated to be an effective treatment for various cancers such as rectal cancer, colon carcinoma, and non-small cell lung cancer, etc. RESULTS: In the current study, we used the phage display technique to generate mimotopes that complemented the screening Avastin antibody (Ab). The candidate mimotopes of VEGF were isolated from a 12-mer peptide library. The phage displaying peptide DHTLYTPYHTHP (designated as 12P) exhibited high affinity to Avastin. The chemically synthesized 12P was conjugated to keyhole limpet hemocyanin (KLH) by glutaraldehyde (GA) to form vaccine KLH-12 peptide (KLH-12P). This epitope vaccine significantly induced humoral immunity in mice. The blood serum from KLH-12P-immunized mice associated with VEGF and blocked its binding to VEGFR, thus inhibiting vascular endothelial cell proliferation and migration. CONCLUSIONS: Our data indicate that the isolated mimotope 12P reported here could potentially elicit specific antibodies against VEGF and result in the induction of anti-angiogenesis responses.

High level expression, purification and characterization of recombinant CCR5 as a vaccine candidate against HIV.

Cysteine-cysteine chemokine receptor type 5 (CCR5) is an important co-receptor for human immunodeficiency virus (HIV) infection and CCR5 neutralizing agents have proven efficient in patients suffering from HIV infection. Here, we expressed and purified various CCR5 vaccines named rCCR5, PADRE-rCCR5, GST-C1 and GST-C2 composed of different epitopes of CCR5. Results showed that vaccines containing multiple epitopes (rCCR5 and PADRE-rCCR5) induced stronger immune responses than single-epitope ones (GST-C1 and GST-C2). In addition, the elicited antibodies can specifically bind CCR5(+) U937 but not CCR5(-) Wish cells. These results demonstrate that the CCR5 vaccines are useful for further research, especially for the in vitro preclinical evaluation of their potential as biological CCR5 neutralizing agents.

Microbiota downregulates dendritic cell expression of miR-10a, which targets IL-12/IL-23p40.

Commensal flora plays important roles in the regulation of the gene expression involved in many intestinal functions and the maintenance of immune homeostasis, as well as in the pathogenesis of inflammatory bowel diseases. The microRNAs (miRNAs), a class of small, noncoding RNAs, act as key regulators in many biological processes. The miRNAs are highly conserved among species and appear to play important roles in both innate and adaptive immunity, as they can control the differentiation of various immune cells, as well as their functions. However, it is still largely unknown how microbiota regulates miRNA expression, thereby contributing to intestinal homeostasis and pathogenesis of inflammatory bowel disease. In our current study, we found that microbiota negatively regulated intestinal miR-10a expression, because the intestines, as well as intestinal epithelial cells and dendritic cells of specific pathogen-free mice, expressed much lower levels of miR-10a compared with those in germ-free mice. Commensal bacteria downregulated dendritic cell miR-10a expression via TLR-TLR ligand interactions through a MyD88-dependent pathway. We identified IL-12/IL-23p40, a key molecule for innate immune responses to commensal bacteria, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited, whereas the miR-10a inhibitor promoted, the expression of IL-12/IL-23p40 in dendritic cells. Mice with colitis expressing higher levels of IL-12/IL-23p40 exhibited lower levels of intestinal miR-10a compared with control mice. Collectively, our data demonstrated that microbiota negatively regulates host miR-10a expression, which may contribute to the maintenance of intestinal homeostasis by targeting IL-12/IL-23p40 expression.

Histopathological changes of the dural myeloid cells and lymphatic vessels in a mouse model of sepsis-associated encephalopathy.

As a common diffuse encephalopathy caused by sepsis, sepsis-associated encephalopathy (SAE) is closely associated with increased mortality, severe cognition dysfunction and increased cost of health care in patients of sepsis. Accumulating evidence suggests that the dura mater, the outermost meninges of the central nervous system (CNS), plays an important role in CNS immunity, especially with the discovery of meningeal lymphatic vessels (mLVs), as well as a plentiful array of resident or infiltrating immune cells harbored in the dura. Although these findings have significantly enhanced our understanding of the immune function of dura under both steady-state and pathological condition of CNS, whether and how the immune cells and mLVs within dura response to SAE still remains largely unexplored. Here, we established lipopolysaccharide (LPS) intraperitoneal injection-induced SAE model and examined the dural resident immune cells and mLVs. We analysed the histological change in dura by performing hematoxylin and eosin (H&E) and immunofluorescence staining. Results showed that systemic exposure to LPS induced neutrophils recruitment, exudation and gathering around the dural blood vessels. Moreover, resident macrophage altered its shape as well as location, and downregulated major histocompatibility (MHC) class II expression following LPS injection. We also found that LPS exposure induced dorsal meningeal lymphangiogenesis. Together, these findings collectively demonstrated that LPS-induced SAE can stimulate immune cells and mLVs within dura and provided more information about the immune response of the dura in sepsis.

Engineered Extracellular Vesicle-Delivered CRISPR/Cas9 for Radiotherapy Sensitization of Glioblastoma.

Radiotherapy is a mainstay of glioblastoma (GBM) treatment; however, the development of therapeutic resistance has hampered the efficacy of radiotherapy, suggesting that additional treatment strategies are needed. Here, an in vivo loss-of-function genome-wide CRISPR screen was carried out in orthotopic tumors in mice subjected to radiation treatment to identify synthetic lethal genes associated with radiotherapy. Using functional screening and transcriptome analyses, glutathione synthetase (GSS) was found to be a potential regulator of radioresistance through ferroptosis. High GSS levels were closely related to poor prognosis and relapse in patients with glioma. Mechanistic studies demonstrated that GSS was associated with the suppression of radiotherapy-induced ferroptosis in glioma cells. The depletion of GSS resulted in the disruption of glutathione (GSH) synthesis, thereby causing the inactivation of GPX4 and iron accumulation, thus enhancing the induction of ferroptosis upon radiotherapy treatment. Moreover, to overcome the obstacles to broad therapeutic translation of CRISPR editing, we report a previously unidentified genome editing delivery system, in which Cas9 protein/sgRNA complex was loaded into Angiopep-2 (Ang) and the trans-activator of the transcription (TAT) peptide dual-modified extracellular vesicle (EV), which not only targeted the blood-brain barrier (BBB) and GBM but also permeated the BBB and penetrated the tumor. Our encapsulating EVs showed encouraging signs of GBM tissue targeting, which resulted in high GSS gene editing efficiency in GBM (up to 67.2%) with negligible off-target gene editing. These results demonstrate that a combination of unbiased genetic screens, and CRISPR-Cas9-based gene therapy is feasible for identifying potential synthetic lethal genes and, by extension, therapeutic targets.

Phase II multicenter, randomized, double-blind study of recombinant mutated human tumor necrosis factor-α in combination with chemotherapies in cancer patients.

We previously prepared a tumor necrosis factor (TNF)-α mutant (rmhTNF-α) that showed higher antitumor activity and lower systemic toxicity compared with native TNF-α. The safety profile and the pharmacokinetic characteristics of rmhTNF-α were suited for clinical use according to biological Investigational New Drug application, a standard guideline for new drug investigation in China. Here, we evaluate the activity and safety of rmhTNF-α combined with chemotherapies in head/neck, lung, colorectal, stomach, and renal cancer patients. Ninety-five eligible patients received i.m. rmhTNF-α treatment combined with standard chemotherapies. Another 95 patients were treated with standard chemotherapies. After two treatment cycles, one patient achieved a complete response and 24 patients had partial response, yielding an overall response rate (complete response + partial response) of 27.47% in the rmhTNF-α plus chemotherapy cohort. The chemotherapy alone group acquired only a 11.39% response rate (P < 0.05). When compared between different cancers, a 48.89% response rate was detected in the 45 lung cancer patients of the combination cohort. The most common grade 1-2 adverse events of rmhTNF-α were drug-related fever, allergy, flu-like symptoms, and myalgia. No significant difference was found in grade 3-4 toxicities between the two cohorts. Based on the results of this research, rmhTNF-α can significantly enhance the effectiveness of chemotherapy. An extended phase III trial of rmhTNF-α combined with standard chemotherapy is warranted for evaluating its antitumor activity and toxicity in a larger cohort of tumor patients. The studies in this paper were registered with the State Food and Drug Administration of China (No. 2003S00692).