Folate Deficiency and/or the Genetic Variant Mthfr677C >T Can Drive Hepatic Fibrosis or Steatosis in Mice, in a Sex-Specific Manner.

SCOPE: Disturbances in one-carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr677C >T variant on NAFLD. METHODS AND RESULTS: This study uses the new Mthfr677C >T mouse model for the human MTHFR677C >T variant. Mthfr677CC and Mthfr677TT mice were fed control diet (CD) or folate-deficient (FD) diets for 4 months. FD and Mthfr677TT alter choline/methyl metabolites in liver and/or plasma (decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr677TT, provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex-based differences in methylation potential, betaine, sphingomyelin, and trimethylamine-N-oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females. CONCLUSION: This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr677TT, whereas Mthfr677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD-related disorders.

UTX inhibition suppresses proliferation and promotes apoptosis in patient-derived glioblastoma stem cells by modulating periostin expression.

Glioblastoma stem cells (GSCs) exert a crucial influence on glioblastoma (GBM) development, progression, resistance to therapy, and recurrence, making them an attractive target for drug discovery. UTX, a histone H3K27 demethylase, participates in regulating multiple cancer types. However, its functional role in GSCs remains insufficiently explored. This study aims to investigate the role and regulatory mechanism of UTX on GSCs. Analysis of TCGA data revealed heightened UTX expression in glioma, inversely correlating with overall survival. Inhibiting UTX suppressed GBM cell growth and induced apoptosis. Subsequently, we cultured primary GSCs from three patients, observing that UTX inhibition suppressed cell proliferation and induced apoptosis. RNA-seq was performed to analyze the gene expression changes after silencing UTX in GSCs. The results indicated that UTX-mediated genes were strongly correlated with GBM progression and regulatory tumor microenvironment. The transwell co-cultured experiment showed that silencing UTX in the transwell chamber GSCs inhibited the well plate cell proliferation. Protein-protein interaction analysis revealed that periostin (POSTN) played a role in the UTX-mediated transcriptional regulatory network. Replenishing POSTN reversed the effects of UTX inhibition on GSC proliferation and apoptosis. Our study demonstrated that UTX inhibition hindered POSTN expression by enhancing the H3K27me2/3 level, eventually resulting in inhibiting proliferation and promoting apoptosis of patient-derived GSCs. Our findings may provide a novel and effective strategy for the treatment of GBM.

CCN3/NOV Regulates Proliferation and Neuronal Differentiation in Mouse Hippocampal Neural Stem Cells via the Activation of the Notch/PTEN/AKT Pathway.

Neural stem cells (NSCs) persist in the subgranular zone (SGZ) throughout the lifespan and hold immense potential for the repair and regeneration of the central nervous system, including hippocampal-related diseases. Several studies have demonstrated that cellular communication network protein 3 (CCN3) regulates multiple types of stem cells. However, the role of CCN3 in NSCs remains unknown. In this study, we identified CCN3 expression in mouse hippocampal NSCs and observed that supplementing CCN3 improved cell viability in a concentration-dependent manner. Additionally, in vivo results showed that the injection of CCN3 in the dentate gyrus (DG) increased Ki-67- and SOX2-positive cells while decreasing neuron-specific class III beta-tubulin (Tuj1) and doublecortin (DCX)-positive cells. Consistently with the in vivo results, supplementing CCN3 in the medium increased the number of BrdU and Ki-67 cells and the proliferation index but decreased the number of Tuj1 and DCX cells. Conversely, both the in vivo and in vitro knockdown of the Ccn3 gene in NSCs had opposite effects. Further investigations revealed that CCN3 promoted cleaved Notch1 (NICD) expression, leading to the suppression of PTEN expression and eventual promotion of AKT activation. In contrast, Ccn3 knockdown inhibited the activation of the Notch/PTEN/AKT pathway. Finally, the effects of changes in CCN3 protein expression on NSC proliferation and differentiation were eliminated by FLI-06 (a Notch inhibitor) and VO-OH (a PTEN inhibitor). Our findings imply that while promoting proliferation, CCN3 inhibits the neuronal differentiation of mouse hippocampal NSCs and that the Notch/PTEN/AKT pathway may be a potential intracellular target of CCN3. Our findings may help develop strategies to enhance the intrinsic potential for brain regeneration after injuries, particularly stem cell treatment for hippocampal-related diseases.

ISL1 Promotes Human Glioblastoma-Derived Stem Cells' Self-Renewal by Activation of Sonic Hedgehog/GLI1 Function.

Glioblastoma (GBM), the most aggressive primary heterogeneous primary brain tumor, is a glioma subtype that originates from the glial cells of the central nervous system. Glioblastoma stem cells (GSCs), situated at the top of the hierarchy, initiate and maintain the tumor and are largely accountable for GBM resistance to the mainstay treatment and recurrence. The LIM homeobox transcription factor islet 1 (ISL1) induces tumorigenicity in various tumors; however, its function in GSCs has been less reported. We aimed to generate GSCs from surgical specimens of human GBM and investigate the effect of ISL1 knockdown on GSCs. We established patient-derived GSCs, determined cancer stem cell marker expression, and immunostained GSCs to assess cell viability and apoptosis. We demonstrated that ISL1 deletion decreased the GSC viability and proliferation, and upregulated apoptosis. Moreover, we performed enzyme-linked immunosorbent assay and western blotting and found that ISL1 knockdown affected the expression of sonic hedgehog (SHH) and its downstream regulator GLI1, and further validated these results by supplementing the cells with recombinant SHH. Our results suggested that ISL1 played a critical role in regulating GBM growth and that an ISL1/SHH/GLI1 pathway was required for the maintenance of GBM progression and malignancy. The regulation of GSC growth through ISL1 might be a mechanism of interest for future therapeutic studies.

Glypican 4 Regulates Aß Internalization in Neural Stem Cells Partly via Low-Density Lipoprotein Receptor-Related Protein 1.

Neural stem cell (NSC) damage has been reported in patients with Alzheimer's disease. Intracellular Aß plays a vital role in NSC damage. Heparan sulfate proteoglycans are potent mediators of Aß enrichment in the brain. We hypothesized the heparan sulfate proteoglycan glypican 4 (Gpc4) regulates Aß internalization by NSCs. We evaluated Gpc4 expression in NSCs from P0-P2 generations using immunofluorescence. Adenovirus and lentivirus were used to regulate Gpc4 expression in NSCs and APP/PS1 mice, respectively. Co-immunoprecipitation was used to determine the relationship between Gpc4, Aß, and low-density lipoprotein receptor-related protein 1 (LRP1). Intracellular Aß concentrations were detected using enzyme-linked immunosorbent assay and immunofluorescence. The role of Gpc4/LRP1 on toxic/physical Aß-induced effects was evaluated using the JC-1 kit, terminal deoxynucleotidyl transferase dUPT nick end labeling, and western blotting. Gpc4 was stably expressed in NSCs, neurons, and astrocytes. Gpc4 was upregulated by Aß in NSCs and regulated Aß internalization. Gpc4 attenuation reduced Aß uptake; Gpc4 overexpression increased Aß uptake. Gpc4 regulated Aß internalization through LRP1 and contributed to Aß internalization and toxic/physical concentrations of Aß-induced mitochondrial membrane potential and cell apoptosis, partly via LRP1. Therefore, Gpc4 is a key regulator of Aß enrichment in NSCs. Inhibiting Gpc4 rescued the Aß-induced toxic effect and attenuated the nontoxic Aß enrichment into intracellular toxic concentrations. Gpc4 contributed to Aß internalization and toxic/physical concentrations of Aß-induced mitochondrial membrane potential damage and cell apoptosis, partly via LRP1. These findings suggest a potential role of Gpc4 in treating Alzheimer's disease at an early stage, by targeting NSCs.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Altered Methyl Metabolism and in Sex-Specific Placental Transcription Changes.

SCOPE: Many pregnant women have higher folic acid (FA) intake due to food fortification and increased vitamin use. It is reported that diets containing five-fold higher FA than recommended for mice (5xFASD) during pregnancy resulted in methylenetetrahydrofolate reductase (MTHFR) deficiency and altered choline/methyl metabolism, with neurobehavioral abnormalities in newborns. The goal is to determine whether these changes have their origins in the placenta during embryonic development. METHODS AND RESULTS: Female mice are fed control diet or 5xFASD for a month before mating and maintained on these diets until embryonic day 17.5. 5xFASD led to pseudo-MTHFR deficiency in maternal liver and altered choline/methyl metabolites in maternal plasma (increased methyltetrahydrofolate and decreased betaine). Methylation potential (S-adenosylmethionine:S-adenosylhomocysteine ratio) and glycerophosphocholine are decreased in placenta and embryonic liver. Folic acid supplemented diet results in sex-specific transcriptome profiles in placenta, with validation of dietary expression changes of 29 genes involved in angiogenesis, receptor biology or neurodevelopment, and altered methylation of the serotonin receptor 2A gene. CONCLUSION: Moderate increases in folate intake during pregnancy result in placental metabolic and gene expression changes, particularly in angiogenesis, which may contribute to abnormal behavior in pups. These results are relevant for determining a safe upper limit for folate intake during pregnancy.

Activation of metabotropic glutamate receptor 4 regulates proliferation and neural differentiation in neural stem/progenitor cells of the rat subventricular zone and increases phosphatase and tensin homolog protein expression.

Neural stem/progenitor cells (NSPCs) persist in the mammalian subventricular zone throughout life, where they can be activated in response to physiological and pathophysiological stimuli. A recent study indicates metabotropic glutamate receptor 4 (mGluR4) is involved in regulating NSPCs behaviors. Therefore, defining mGluR4 function in NSPCs is necessary for determining novel strategies to enhance the intrinsic potential for brain regeneration after injuries. In this study, mGluR4 was functionally expressed in SVZ-derived NSPCs from male Sprague-Dawley rats, in which the cyclic adenosine monophosphate concentration was reduced after treatment with the mGluR4-specific agonist VU0155041. Additionally, lateral ventricle injection of VU0155041 significantly decreased 5-bromo-2'-deoxyuridine (BrdU)+ and Ki67+ cells, while increased Doublecortin (DCX)/BrdU double-positive cells in SVZ. In cultured NSPCs, mGluR4 activation decreased the ratio of BrdU+ cells, G2/M-phase cells, and inhibited Cyclin D1 expression, whereas it increased neuron-specific class III ß-tubulin (Tuj1) expression and the number of Tuj1, DCX, and PSA-NCAM-positive cells. However, pharmacological blocking mGluR4 with the antagonist MSOP or knockdown of mGluR4 abolished the effects of VU0155041 on NSPCs proliferation and neuronal differentiation. Further investigation demonstrated that VU0155041 treatment down-regulated AKT phosphorylation and up-regulated expression of the phosphatase and tensin homolog protein (PTEN) in NSPCs culture. Moreover VU0155041-induced proliferating inhibition and neuronal differentiating amplification in NSPCs were significantly hampered by VO-OHpic, a PTEN inhibitor. We conclude that activation of mGluR4 in SVZ-derived NSPCs suppresses proliferation and enhances their neuronal differentiation, and regulation of PTEN may be involved as a potential intracellular target of mGluR4 signal. Cover Image for this issue: https://doi.org/10.1111/jnc.15052.

Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8+ T Cell Apoptosis.

Increasing evidence demonstrates that interleukin-10 (IL-10), known as an immunosuppressive cytokine, induces antitumor effects depending on CD8+ T cells. However, it remains elusive how immunosuppressive effects of IL-10 contribute to CD8+ T cell-mediated antitumor immunity. We generated Cetuximab-based IL-10 fusion protein (CmAb-(IL10)2) to prolong its half-life and allow tumor-targeted delivery of IL-10. Our results demonstrated potent antitumor effects of CmAb-(IL10)2 with reduced toxicity. Moreover, we revealed a mechanism of CmAb-(IL10)2 preventing dendritic cell (DC)-mediated CD8+ tumor-infiltrating lymphocyte apoptosis through regulating IFN-γ production. When combined with immune checkpoint blockade, CmAb-(IL10)2 significantly improves antitumor effects in mice with advanced tumors. Our findings reveal a DC-regulating role of IL-10 to potentiate CD8+ T cell-mediated antitumor immunity and provide a potential strategy to improve cancer immunotherapy.

Activation of type 4 metabotropic glutamate receptor promotes cell apoptosis and inhibits proliferation in bladder cancer.

Bladder cancer, the second most common genitourinary malignancy, severely endangers the human health. Rising evidence suggests that metabotropic glutamate receptors (mGluRs) are involve in tumor progression. In this study, we observed that metabotropic glutamate receptor 4 (mGluR4) was functionally expressed in normal and cancerous bladder cells and its expression was positively correlated with high bladder cancer grading. We further confirmed that the activation of mGluR4 by VU0155041, an mGluR4-specific agonist, decreased cyclic adenosine monophosphate (cAMP) concentration and cell viability, promoted apoptosis and inhibited proliferation in bladder cancer cells, whereas MSOP (group III mGluR antagonist) or mGluR4 knockdown eliminated the effects of mGluR4 activity. Western blotting revealed the decreased cyclin D1 expression, increased procaspase-8/9/3 cleavage, and unbalanced Bcl-2/Bax expression in bladder cancer cell lines after mGluR4 activation, and likewise MSOP and mGluR4 knockdown abrogated the actions of mGluR4 activity. In vivo study showed that mGluR4 activation significantly inhibited tumor growth of bladder cancer via suppressing proliferation and promoting apoptosis. Furthermore, upregulation of phosphatase and tensin homolog (PTEN) and inhibition of Akt phosphorylation were also observed after mGluR4 activation. Similar with VU0155041, the Akt-specific inhibitor markedly promoted apoptosis and inhibited proliferation. Nevertheless, the PTEN-specific inhibitor significantly abolished the mGluR4 activation-induced cell apoptosis and proliferative inhibition in bladder cancer cell lines. These results indicate that mGluR4 can regulate the switch between survival and death via the cAMP/PTEN/AKT signaling pathway in bladder cancer cells. Our findings suggest that mGluR4 has diagnostic and prognostic potential for bladder cancer, and the development of mGluR4 agonist may be a promising strategy for bladder cancer treatment.

Dual Targeting of Innate and Adaptive Checkpoints on Tumor Cells Limits Immune Evasion.

CD47 on tumor cells protects from phagocytosis, while PD-L1 dampens T cell-mediated tumor killing. However, whether and how CD47 and PD-L1 coordinate is poorly understood. We reveal that CD47 and PD-L1 on tumor cells coordinately suppress innate and adaptive sensing to evade immune control. Targeted blockade of both CD47 and PD-L1 on tumor cells with a bispecific anti-PD-L1-SIRPα showed significantly enhanced tumor targeting and therapeutic efficacy versus monotherapy. Mechanistically, systemic delivery of the dual-targeting heterodimer significantly increased DNA sensing, DC cross-presentation, and anti-tumor T cell response. In addition, chemotherapy that increases "eat me" signaling further synergizes with the bispecific reagent for better tumor control. Our data indicate that tumor cells evolve to utilize both innate and adaptive checkpoints to evade anti-tumor immune responses and that tumor cell-specific dual-targeting of both checkpoints represents an improved strategy for tumor immunotherapy.

Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most lethal glioma variant in the adult brain and among the deadliest of human cancers. Increasing evidence has shown that metabotropic glutamate receptor subtype 4 (mGluR4) expression may play roles in regulating the growth of neural stem cells as well as several cancer cell lines. Here, we investigated the effects of mGluR4 on the growth and apoptosis of the LN229 GBM cell line. Involvement of Gli-1, one of the key transcription factors in the sonic Hedgehog (SHH) signaling pathway, was further explored. In this study, mGluR4 was activated using selective agonist VU0155041; and gene-targeted siRNAs were used to generate loss of function of mGluR4 and Gli-1 in LN229 cells. The results demonstrated that LN229 cells expressed mGluR4 and the agonist VU0155041 decreased cell viability in a dose- and time-dependent manner. Activation of mGluR4 inhibited cyclin D1 expression, activated pro-caspase-8/9/3, and disrupted the balance of Bcl-2/Bax expression, which indicated cell cycle arrest and apoptosis of LN229 cells, respectively. Furthermore, Gli-1 expression was reduced by mGluR4 activation in LN229 cells, and downregulation of Gli-1 expression by gene-targeted siRNA resulted in both inhibition of cell proliferation and promotion of apoptosis. Moreover, VU0155041 treatment substantially blocked SHH-induced cyclin D1 expression and cell proliferation, while increasing TUNEL-positive cells and the activation of apoptosis-related proteins. We concluded that activation of mGluR4 expressed in LN229 cells could inhibit GBM cell growth by decreasing cell proliferation and promoting apoptosis. Further suppression of intracellular Gli-1 expression might be involved in the action of mGluR4 on cancer cells. Our study suggested a novel role of mGluR4, which might serve as a potential drug target for control of GBM cell growth.

Rational selection of antibacterial drugs and postoperative nursing for gynecologic and obstetric surgery patients.

Infection after gynecologic surgery is very common and frequent. If the control is not good, it will lead to serious consequences. Therefore, it is necessary to use antibiotics in the period of obstetrics and gynecology. This study will explore the use of antimicrobial agents in gynecologic and obstetric surgery, thus standardizing the use of antibiotics in the process of obstetrics and gynecology. Through the analysis of the use of antibacterials, we can see that the highest utilization rate of 5 kinds of antibacterial drugs followed by Cefaclor Sustained Release Tablets (65.7%), metronidazole (32.5%), cefathiamidine (26.8%), enoxacin (22.5%) and cefoperazone tazobactam sodium (11.8%). At the same time, the hospital should improve the consciousness of rational drug use and strengthen the administration of antibacterials in the operative period of obstetrics and gynecology. The application of antibiotics in the operative period of the department of obstetrics and gynecology can improve the current situation of its irrational use. Nursing work must take strict aseptic operation to prevent cross infection. At the same time, we should strengthen the observation of the effect of medication, monitor the body temperature and blood pressure, and identify the side effects of drugs.

BMP­6 protects retinal pigment epithelial cells from oxidative stress­induced injury by inhibiting the MAPK signaling pathways.

Worldwide, neovascular age­related macular degeneration (nAMD) is one of the most common causes of blindness in the elderly. In particular, degeneration of retinal pigment epithelial (RPE) cells represents the main pathological process in the development of nAMD, and oxidative stress serves a major role. The present study aimed to investigate the association between bone morphogenetic protein 6 (BMP­6) and nAMD. BMP­6 concentration was significantly reduced in patients with wet nAMD compared with in the control group. Furthermore, the present study investigated the protective effects of BMP­6 on RPE cells following oxidative stress­induced injury. Cell Counting Kit­8 assay and terminal deoxynucleotidyl transferase dUTP nick­end labeling staining demonstrated that BMP­6 increased RPE cell viability, which was decreased following treatment with hydrogen peroxide (H2O2), and reduced H2O2­induced apoptosis. In addition, western blotting revealed that BMP­6 reversed the decrease in pro­caspase­3 levels and the dysregulation of the B­cell lymphoma 2 (Bcl­2)/Bcl­2­associated X protein (Bax) balance caused by H2O2. In addition, alterations in c­Jun N­terminal protein kinase (JNK) and p38 mitogen­activated protein kinase (MAPK) expression were examined, and pretreatment with BMP­6 was demonstrated to reduce H2O2­induced activation of JNK and p38 MAPK. Conversely, the effects of BMP­6 were attenuated by its inhibitor noggin. In conclusion, the present study demonstrated that BMP­6 may protect RPE cells from oxidative stress injury to a certain extent, which may be associated with alterations in the MAPK signaling pathway. However, the specific mechanism of action underlying this effect requires further investigation. Overall, the present study laid a foundation for exploring novel nAMD treatment methods.

[The effect of Toll-like receptor 4 in nicotine suppressing the osteogenic potential of periodontal ligament stem cells].

Objective To explore the impact of nicotine on proliferation and osteogenic capability of periodontal ligament stem cells (PDLSCs), and the role of Toll-like receptor 4 (TLR4) in nicotine, suppressing the osteogenic capability of PDLSCs. Methods PDLSCs were cultured in vitro, and the flow cytometer was used to identify the surface antigen markers of PDLSCs. WST-1 was used to detect the proliferation ability of PDLSCs, which were stimulated by different concentrations of nicotine. Alizarin red staining was used to observe the formation of mineralized nodules after PDLSCs stimulation with different concentrations of nicotine. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the change in osteogenic potential of PDLSCs stimulated by nicotine, after TAK-242, and with the inhibitor of TLR4. Results PDLSCs expressed mesenchymal stem cell-associated markers CD90 and CD105. When the concentration of nicotine was 10⁻4 mol·L⁻¹, the PDLSC proliferation could be suppressed after 3 d compared with the control group (P<0.05). The amount of mineralized nodules reduced after osteogenic differentiation at 21 d by alizarin red staining. RT-PCR and Western blot showed the expression levels of alkaline phosphatase (ALP), and osteocalcin (OCN), and the Runt-related transcription factor-2 (Runx-2) were lower than in the control group when nicotine suppressed the PDLSCs (P<0.05). This effect was attenuated after TAK-242 was added. Conclusion Nicotine suppresses the proliferation and osteogenic capability of PDLSCs, which may be regulated by TLR4.

Converting Lymphoma Cells into Potent Antigen-Presenting Cells for Interferon-Induced Tumor Regression.

Anti-hCD20 is a therapeutic mAb that is clinically used to treat B-cell lymphoma. Some lymphomas are resistant to anti-hCD20; others relapse after treatment with anti-hCD20. Using a syngeneic immunocompetent mouse model, we observed that targeting lymphoma with interferon-α (IFNα) abolished resistance of B-cell lymphoma to anti-CD20 while limiting interferon (IFN)-associated systemic toxicity in the host. Control of tumors by a fusion of anti-CD20 and IFNα (anti-CD20-IFNα) depended on existing tumor-infiltrating CD8+ T cells. Although lymphomas were resistant to IFN-directed killing, IFN-exposed tumor cells became the dominant antigen-presenting cells (APC) for the reactivation of tumor-infiltrating CD8+ T cells that then controlled those lymphomas. Anti-CD20-IFNα also abolished checkpoint blockade resistance in advanced B-cell lymphoma. Our findings indicate that anti-CD20-IFNα eradicates B-cell lymphoma by employing tumor cells as APCs to reactivate tumor-infiltrating CD8+ T cells and synergizing with anti-PD-L1 treatment. Cancer Immunol Res; 5(7); 560-70. ©2017 AACR.

CTLA-4 Limits Anti-CD20-Mediated Tumor Regression.

PURPOSE: The inhibition of tumor growth by anti-CD20 antibody (Ab) treatment is mediated by Ab- and complement-dependent cytotoxicity in xenograft tumor models. In addition, anti-CD20 therapy for B-cell lymphoma can result in intrinsic and extrinsic tumor resistance to further Ab treatment. However, adaptive immune response-related resistance has not been well studied in anti-CD20-mediated tumor control, and adaptive immunity has long been underestimated. The purpose of this study was to explore whether T cells are involved in mediating the effects of anti-CD20 therapy and what factors contribute to adaptive immune response-related resistance. EXPERIMENTAL DESIGN: Using a syngeneic mouse B-cell lymphoma model, we investigated the role of CD8+ T cells in anti-CD20-mediated tumor regression. Furthermore, we revealed how the tumor-specific T-cell response was initiated by anti-CD20. Finally, we studied adaptive immune response-related resistance in advanced B-cell lymphoma. RESULTS: CD8+ T cells played an essential role in anti-CD20-mediated tumor regression. Mechanistically, anti-CD20 therapy promoted dendritic cell (DC)-mediated cross-presentation. Importantly, macrophages were also necessary for the increase in the tumor-specific CTL response after anti-CD20 treatment, via the production of type I IFN to activate DC function. Furthermore, adaptive resistance is gradually developed through the CTLA-4 pathway in Treg cells in larger lymphomas. Further blockade of CTLA-4 can synergize with anti-CD20 treatment in antitumor activities. CONCLUSIONS: The therapeutic function of anti-CD20 depends on tumor-specific CD8+ T-cell responses initiated by anti-CD20 through macrophages and DCs. CTLA-4 blockade can synergize with anti-CD20 to overcome adaptive immune response-related resistance in advanced B-cell lymphoma. Clin Cancer Res; 23(1); 193-203. ©2016 AACR.

A fully human monoclonal antibody targeting PD-L1 with potent anti-tumor activity.

BACKGROUND: Programmed cell death ligand-1 (PD-L1) with its receptor PD-1 pathway is overactivated in many tumors. Inhibiting the interaction of PD-L1 and PD-1 is an attractive strategy to restore tumor-specific T cell immunity for tumor therapy. METHODS: A fully human anti-PD-L1 monoclonal antibody (mAb) B60-55 was identified by yeast surface display. The affinity, specificity, activity, and efficacy of mAb B60-55 were investigated in vitro or in vivo. RESULTS: mAb B60-55 (purity >99%) could bind to PD-L1 that is expressed on HEK293 cells with a dissociation constant of 0.2 nM, and specifically bind to human or cynomolgus macaque PD-L1 without a cross-reaction with murine PD-L1. Moreover, mAb B60-55 is an antagonistic antibody, which can block PD-L1 binding to its receptors, including PD-1 (PDCD1) and B7.1 (CD80). In vitro assays demonstrated the ability of mAb B60-55 to enhance T cell responses and cytokine production in the mixed lymphocyte reaction. In vivo studies showed that administration of mAb B60-55 exhibited a potent antitumor activity toward tumor cell carcinoma xenograft, with a mean half-life of 177.9h in cynomolgus monkeys. CONCLUSION: mAb B60-55 is a potential candidate for clinical development in cancer treatment.

Ameliorative effects of tannic acid on carbon tetrachloride-induced liver fibrosis in vivo and in vitro.

We investigated the ameliorative effects and potential mechanisms of tannic acid (TA) in carbon tetrachloride (CCl4)-intoxicated mice and hepatic stellate cells (HSCs). Liver fibrosis was observed in CCl4 (800 ml/kg)-induced mice, and high viability was observed in CCl4 (10 mM)-intoxicated HSCs. Pre-treatment of mice with TA (25 or 50 g/kg/day) significantly ameliorated hepatic morphology and coefficient values and reduced the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), the concentrations of malondialdehyde (MDA) and serum levels of endothelin-1 (ET-1). In addition, TA increased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and endothelial nitric oxide synthase (eNOS) and the serum level of NO. Moreover, TA reduced the expression of angiotensin II receptor-1 (ATR-1), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), caspase-3, c-fos, c-jun, the ratio of Bax/bcl-2, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TA increased matrix metal proteinase-9 (MMP-9), matrix metalloproteinase-1 (MMP-1). Furthermore, TA (0.01 µM, 0.1 µM or 1 µM) decreased the TIMP-1/MMP-1 ratio and reduced the viability of HSCs. These results indicated that TA exerts significant liver-protective effects in mice with CCl4-induced liver fibrosis. The potential mechanism may rely on the inhibition of collagen accumulation, oxidative stress, inflammation and apoptosis.

Inhibitory effects of subcutaneous tumors in nude mice mediated by low-frequency ultrasound and microbubbles.

The aim of the present study was to investigate the sonication effects of 21-kHz ultrasound (US) with microbubbles (MBs) on the subcutaneous prostate tumors of nude mice. In total, 15 tumor-bearing nude mice were divided into three groups: The control group, the low-frequency US group and the US+MB group. The MBs used were from US contrast agent SonoVue. The parameters of the US were as follows: 21 kHz, 26 mW/cm2 and a 40% duty cycle (2 sec on, 3 sec off) for 3 min, once every other day for 2 weeks. Color Doppler flow imaging, hematoxylin and eosin (HE) staining, immunoblotting and transmission electron microscopy (TEM) were used to evaluate the results. Following 2 weeks of treatment, the blood flow signal disappeared in the US+MB group only, and the tumor size was smaller when compared with the control and US groups. For the immunoblotting, the intensity of cyclooxygenase-2 and vascular endothelial growth factor in the US+MB group was lower compared with the other two groups. Tumor necrosis was present and the nucleus disappeared upon HE staining in the US+MB group. Upon TEM analysis, increased cytoplasmic vacuolation and dilatation of the perinuclear cisternae of the tumor cells were found in the US+MB group. In the control and US groups, the tumors had intact vascular endothelia and vessel lumens. However, lumen occlusion of the vessels was observed in the US+MB group. In conclusion, 21-kHz low-intensity US with MBs may result in vessel occlusion and growth inhibitory effects in the subcutaneous tumors of nude mice.

Hepatitis B virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and M2-like macrophages.

The mechanisms of chronic HBV infection and immunopathogenesis are poorly understood due to a lack of a robust small animal model. Here we report the development of a humanized mouse model with both human immune system and human liver cells by reconstituting the immunodeficient A2/NSG (NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice with human HLA-A2 transgene) with human hematopoietic stem cells and liver progenitor cells (A2/NSG-hu HSC/Hep mice). The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75% of HBV infected mice established persistent infection for at least 4 months. We detected human immune responses, albeit impaired in the liver, chronic liver inflammation and liver fibrosis in infected animals. An HBV neutralizing antibody efficiently inhibited HBV infection and associated liver diseases in humanized mice. In addition, we found that the HBV mediated liver disease was associated with high level of infiltrated human macrophages with M2-like activation phenotype. Importantly, similar M2-like macrophage accumulation was confirmed in chronic hepatitis B patients with liver diseases. Furthermore, gene expression analysis showed that induction of M2-like macrophage in the liver is associated with accelerated liver fibrosis and necrosis in patients with acute HBV-induced liver failure. Lastly, we demonstrate that HBV promotes M2-like activation in both M1 and M2 macrophages in cell culture studies. Our study demonstrates that the A2/NSG-hu HSC/Hep mouse model is valuable in studying HBV infection, human immune responses and associated liver diseases. Furthermore, results from this study suggest a critical role for macrophage polarization in hepatitis B virus-induced immune impairment and liver pathology.