The effect of milrinone on mortality in adult patients who underwent CABG surgery: a systematic review of randomized clinical trials with a meta-analysis and trial sequential analysis.

BACKGROUND: As an inodilator, milrinone is commonly used for patients who undergo coronary artery bypass graft (CABG) surgery because of its effectiveness in decreasing the cardiac index and mitral regurgitation. The aim of this study was to perform a systematic review and meta-analysis of existing studies from the past 20 years to evaluate the impact of milrinone on mortality in patients who undergo CABG surgery. METHODS: We performed a systematic literature search on the application of milrinone in patients who underwent CABG surgery in studies published between 1997 and 2017 in BioMed Central, PubMed, EMBASE, and the Cochrane Central Register. The included studies evaluated milrinone groups compared to groups receiving either placebo or standard treatment and further compared the systemic administration. RESULTS: The network meta-analysis included 723 patients from 16 randomized clinical trials. Overall, there was no significant difference in mortality between the milrinone group and the placebo/standard care group when patients underwent CABG surgery. In addition, 9 trials (with 440 randomized patients), 4 trials (with 212 randomized patients), and 10 trials (with 470 randomized patients) reported that the occurrence of myocardial infarction (MI), myocardial ischemia, and arrhythmia was lower in the milrinone group than in the placebo/standard care group. Between the milrinone treatment and placebo/standard care groups, the occurrence of myocardial infarction, myocardial ischemia, and arrhythmia was significantly different. However, the occurrence of stroke and renal failure, the duration of inotropic support (h), the need for an intra-aortic balloon pump (IABP), and mechanical ventilation (h) between these two groups showed no differences. CONCLUSIONS: Based on the current results, compared with placebo, milrinone might be unable to decrease mortality in adult CABG surgical patients but can significantly ameliorate the occurrence of MI, myocardial ischemia, and arrhythmia. These results provide evidence for the further clinical application of milrinone and of therapeutic strategies for CABG surgery. However, along with milrinone application in clinical use, sufficient data from randomized clinical trials need to be collected, and the potential benefits and adverse effects should be analyzed and reevaluated.

N-glycosylation-mutated HCV envelope glycoprotein complex enhances antigen-presenting activity and cellular and neutralizing antibody responses.

BACKGROUND: The development of an efficient vaccine and broadly cross-neutralizing antibodies of hepatitis C virus (HCV) remains a priority. The heavily glycosylated viral envelope glycoprotein E1E2 complex is a candidate vaccine antigen. Bacteria-derived unmethylated CpG DNA, a potent stimulator of immune cells, is important for vaccine research. METHODS: Here, the immunogenicities of wild type (WT) E1E2, five N-glycosylation site mutated E1E2 glycoproteins, and five CpG-coupled E1E2 N-glycosylation mutated glycoproteins were analyzed in BALB/c mice by DNA vaccination using in vivo electroporation. RESULTS: The E1E2 protein expression levels were examined and shown to be unaffected by these N-glycosylation mutations. We found that a CpG-coupled E1-N209D-E2-N430D DNA vaccine (named CpG-E1E2-M4) induced the highest cellular immune response compared to the WT E1E2, CpG-E1E2, and other mutants. Furthermore, the CpG-E1E2-M4 anti-serum effectively neutralized the infection of cell-cultured HCV (HCVcc, genotype 2a)- and HCV pseudo particles (HCVpp, genotypes 1 to 7) to Huh-7.5.1 hepatocytes. Additionally, CpG-E1E2-M4 enhanced the Interleukin-12 (IL-12) production and antigen-presenting activity of CD11c(+) dendritic cells (DCs) by inducing CD4(+) Th1 polarization and the production of perforin and granzyme B (GrB) in CD8(+) T cells. CONCLUSIONS: As our knowledge this is the first study revealing that the naturally poor immunogenicity of E1E2 can be enhanced by the deletion of N-glycans combined with the addition of immune activator CpG by DNA vaccination. GENERAL SIGNIFICANCE: Deletion of N-glycans can enhance viral immunogenicity. The selected CpG-E1E2-M4 mutant is a novel potential HCV DNA vaccine that elicits enhanced CD4(+) Th1 and CD8(+) T cell responses and neutralizing antibody production against HCV infection. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Cycloastragenol mediates activation and proliferation suppression in concanavalin A-induced mouse lymphocyte pan-activation model.

CONTEXT: Cycloastragenol (CAG) is a molecule isolated from various species in the genus Astragalus. Although the regulatory activity of Astragalus on immune system has been investigated, the effect of CAG on activated lymphocytes is poorly understood. OBJECTIVE: We aimed to biologically address the possible anti-inflammation potential of CAG on concanavalin A (Con A)-mediated mouse lymphocyte pan-activation model. MATERIALS AND METHODS: Mouse lymphocytes were obtained from spleens and subjected to Con A for 24 h. Herein, the cells were treated with different concentrations of CAG. Cell viability was assayed by MTT. Pretreated by CAG and stimulated by Con A, the expression of CD69 and CD25, Th1/Th2/Th17 cytokines, cell cycle, proliferation and intracellular Ca2+ concentration ([Ca2+]i) were analyzed by flow cytometry. RESULTS: The results declared that CAG significantly downregulated both CD69 and CD25 expressed on Con A activated CD3 + T cells' surface, as well as inhibiting proliferation of activated lymphocytes. In addition, CAG blocked the Con A-induced mitogenesis, exhibiting lymphocyte G0/G1-phase cell-cycle arrest with significant reduction of cells in S and G2/M phases. Meanwhile, pretreated by CAG, a significant decline in [Ca2+]i was observed. Furthermore, CAG significantly inhibited the production of Th1 cytokines IFN-γ, TNF, IL-2, Th2 cytokines IL-4, IL-6, IL-10 and Th17 cytokine IL-17 A on Con A-activated lymphocytes. CONCLUSION: Our results reinforce that CAG has important anti-inflammatory activity by inhibiting lymphocytes activation, proliferation and cytokines expression, and shows, that this effect may be related to reduction of overall intracellular Ca2+ overload.

Ficolin-2 inhibits hepatitis C virus infection, whereas apolipoprotein E3 mediates viral immune escape.

Human ficolin-2 (L-ficolin/p35) is a lectin-complement pathway activator that is present in normal human plasma and is associated with infectious diseases; however, little is known regarding the roles and mechanisms of ficolin-2 during chronic hepatitis C virus (HCV) infection. In this study, we found that ficolin-2 inhibits the entry of HCV at an early stage of viral infection, regardless of the viral genotype. Ficolin-2 neutralized and inhibited the initial attachment and infection of HCV by binding to the HCV envelope surface glycoproteins E1 and E2, blocking HCV attachment to low-density lipoprotein receptor (LDLR) and scavenger receptor B1, and weakly interfering with CD81 receptor attachment. However, no interference with claudin-1 and occludin receptor attachment was observed. The C-terminal fibrinogen domain (201-313 aa) of ficolin-2 was identified as the critical binding region for the HCV-E1-E2 N-glycans, playing a critical role in the anti-HCV activity. More importantly, we found that apolipoprotein E (ApoE)3, which is enriched in the low-density fractions of HCV RNA-containing particles, promotes HCV infection and inhibits ficolin-2-mediated antiviral activity. ApoE3, but not ApoE2 and ApoE4, blocked the interaction between ficolin-2 and HCV-E2. Our data suggest that the HCV entry inhibitor ficolin-2 is a novel and promising antiviral innate immune molecule, whereas ApoE3 blocks the effect of ficolin-2 and mediates an immune escape mechanism during chronic HCV infection. HCV may be neutralized using compounds directed against the lipoprotein moiety of the viral particle, and ApoE3 may be a new target to combat HCV infection.

Decidual natural killer cells dysfunction is caused by IDO downregulation in dMDSCs with Toxoplasma gondii infection.

Myeloid-derived suppressor cells (MDSCs) play a crucial role in maintaining maternal-fetal tolerance by expressing some immune-suppressive molecules, such as indoleamine 2,3-dioxygenase (IDO). Toxoplasma gondii (T. gondii) infection can break the immune microenvironment of maternal-fetal interface, resulting in adverse pregnancy outcomes. However, whether T. gondii affects IDO expression in dMDSCs and the molecular mechanism of its effect are still unclear. Here we show, the mRNA level of IDO is increased but the protein level decreased in infected dMDSCs. Mechanistically, the upregulation of transcriptional levels of IDO in dMDSCs is regulated through STAT3/p52-RelB pathway and the decrease of IDO expression is due to its degradation caused by increased SOCS3 after T. gondii infection. In vivo, the adverse pregnancy outcomes of IDO-/- infected mice are more severe than those of wide-type infected mice and obviously improved after exogenous kynurenine treatment. Also, the reduction of IDO in dMDSCs induced by T. gondii infection results in the downregulation of TGF-ß and IL-10 expression in dNK cells regulated through Kyn/AhR/SP1 signal pathway, eventually leading to the dysfunction of dNK cells and contributing the occurrence of adverse pregnancy outcomes. This study reveals a novel molecular mechanism in adverse pregnancy outcome induced by T. gondii infection.

Single-cell transcriptomic analysis of decidual immune cell landscape in the occurrence of adverse pregnancy outcomes induced by Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can lead to adverse pregnancy outcomes, particularly in early pregnancy. Previous studies have illustrated the landscape of decidual immune cells. However, the landscape of decidual immune cells in the maternal-fetal microenvironment during T. gondii infection remains unknown. METHODS: In this study, we employed single-cell RNA sequencing to analyze the changes in human decidual immune cells following T. gondii infection. The results of scRNA-seq were further validated with flow cytometry, reverse transcription-polymerase chain reaction, western blot, and immunofluorescence staining. RESULTS: Our results showed that the proportion of 17 decidual immune cell clusters and the expression levels of 21 genes were changed after T. gondii infection. Differential gene analysis demonstrated that T. gondii infection induced the differential expression of 279, 312, and 380 genes in decidual NK cells (dNK), decidual macrophages (dMφ), and decidual T cells (dT), respectively. Our results revealed for the first time that several previously unknown molecules in decidual immune cells changed following infection. This result revealed that the function of maternal-fetal immune tolerance declined, whereas the killing ability of decidual immune cells enhanced, eventually contributing to the occurrence of adverse pregnancy outcomes. CONCLUSIONS: This study provides valuable resource for uncovering several novel molecules that play an important role in the occurrence of abnormal pregnancy outcomes induced by T. gondii infection.

The effect of Toxoplasma gondii infection on galectin-9 expression in decidual macrophages contributing to dysfunction of decidual NK cells during pregnancy.

BACKGROUND: Toxoplasma gondii infection causes adverse pregnancy outcomes by affecting the expression of immunotolerant molecules in decidual immune cells. Galectin-9 (Gal-9) is widely expressed in decidual macrophages (dMφ) and is crucial for maintaining normal pregnancy by interacting with the immunomodulatory protein T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3). However, the effects of T. gondii infection on Gal-9 expression in dMφ, and the impact of altered Gal-9 expression levels on the maternal-fetal tolerance function of decidual natural killer (dNK) cells, are still unknown. METHODS: Pregnancy outcomes of T. gondii-infected C57BL/6 and Lgals9-/- pregnant mice models were recorded. Expression of Gal-9, c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), and Forkhead box protein O1 (FOXO1) was detected by western blotting, flow cytometry or immunofluorescence. The binding of FOXO1 to the promoter of Lgals9 was determined by chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR). The expression of extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), cAMP-response element binding protein (CREB), phosphorylated CREB (p-CREB), T-box expressed in T cells (T-bet), interleukin 10 (IL-10), and interferon gamma (IFN-γ) in dNK cells was assayed by western blotting. RESULTS: Toxoplasma gondii infection increased the expression of p-JNK and FOXO1 in dMφ, resulting in a reduction in Gal-9 due to the elevated binding of FOXO1 with Lgals9 promoter. Downregulation of Gal-9 enhanced the phosphorylation of ERK, inhibited the expression of p-CREB and IL-10, and promoted the expression of T-bet and IFN-γ in dNK cells. In the mice model, knockout of Lgals9 aggravated adverse pregnancy outcomes caused by T. gondii infection during pregnancy. CONCLUSIONS: Toxoplasma gondii infection suppressed Gal-9 expression in dMφ by activating the JNK/FOXO1 signaling pathway, and reduction of Gal-9 contributed to dysfunction of dNK via Gal-9/Tim-3 interaction. This study provides new insights for the molecular mechanisms of the adverse pregnancy outcomes caused by T. gondii.

Telocinobufagin, a PLK1 suppressor that inhibits tumor growth and metastasis by modulating CDC25c and CTCF in HNSCC cells.

BACKGROUND: The high metastasis and mortality rates of head and neck squamous cell carcinoma (HNSCC) urgently require new treatment targets and drugs. A steroidal component of ChanSu, telocinobufagin (TBG), was verified to have anti-cancer effects in various tumors, but its activity and mechanism in anti-HNSCC were still unknown. PURPOSE: This study tried to demonstrate the anti-tumor effect of TBG on HNSCC and verify its potential mechanism. METHODS: The effect of TBG on cell proliferation and metastasis were performed and the TBG changed genes were detected by RNA-seq analysis in HNSCC cells. The GSEA and PPI analysis were used to identify the pathways targeted for TBG-regulated genes. Meanwhile, the mechanism of TBG on anti-proliferative and anti-metastasis were investigated in vitro and in vivo. RESULTS: The in vitro and in vivo experiments confirmed that TBG has favorable anti-tumor effects by induced G2/M phase arrest and suppressed metastasis in HNSCC cells. Further RNA-seq analysis demonstrated the genes regulated by TBG were enriched at the G2/M checkpoint and PLK1 signaling pathway. Then, the bioinformatic analysis of clinical data found that high expressed PLK1 were closely associated with poor overall survival in HNSCC patients. Furthermore, PLK1 directly and indirectly modulated G2/M phase and metastasis (by regulated CTCF) in HNSCC cells, simultaneously. TBG significantly inhibited the protein levels of PLK1 in both phosphorylated and non-phosphorylated forms and then, in one way, inactivated PLK1 failed to activate G2/M phase-related proteins (including CDK1, CDC25c, and cyclin B1). In another way, be inhibited PLK1 unable promote the nuclear translocation of CTCF and thus suppressed HNSC cell metastasis. In contrast, the anti-proliferative and anti-metastasis effects of TBG on HNSCC cell were vanished when cells high-expressed PLK1. CONCLUSION: The present study verified that PLK1 mediated TBG induced anti-tumor effect by modulated G2/M phase and metastasis in HNSCC cells.

Hernandezine acts as a CDK4 suppressor inhibiting tumor growth by the CDK4/PKM2/NRF2 axis in colon cancer.

BACKGROUND: The cyclin-dependent kinase 4 (CDK4) interacts with its canonical and non-canonical substrates modulating the cell cycle in tumor cells. However, the potential substrates and the beyond-cell-cycle-regulated functions of CDK4 in colon cancer (CC) are still unknown. Hernandezine (HER) is previously verified to induce G0/G1 phase arrest and autophagic cell death in human cancer cells, which implies that HER might target G0/G1 phase-related proteins, including CDK4. PURPOSE: The present study tried to investigate the glycolytic metabolism and oxidative stress functions of CDK4 in colon cancer. Furthermore, the inhibitory effects and potential binding sites of HER on CDK4, as well as its anti-tumor activity were investigated in CC cells. METHODS: The mass spectrometry assay was performed to identify potential endogenous substrates of CDK4 and the correlation between glycolytic metabolic rate and CDK4 level in COAD patient tissues. Meanwhile, after inhibiting the activity or the expression of CDK4, the binding capacity of CDK4 to PKM2 and NRF2 and the latter two protein distributions in cytoplasm and nucleus were detected in CC cells. In vitro, the regulatory effects of the CDK4-PKM2-NRF2 axis on glycolysis and oxidative stress were performed by ECAR, OCR, and ROS assay. The inhibitory effect of HER on CDK4 activity was explored in CC cells and the potential binding sites were predicted and testified in vitro. Furthermore, tumor growth inhibition of HER by suppressing the CDK4-PKM2-NRF2 axis was also investigated in vitro and in vivo. RESULTS: PKM2 and NRF2 were identified as endogenous substrates of CDK4 and, high-expressed CDK4 was associated with low-level glycolysis in COAD. In vitro, inactivated CDK4 facilitated CDK4-PKM2-NRF2 complex formation which resulted in 1) inhibited PKM2 activity and retarded the glycolytic rate; 2) cytoplasm-detained NRF2 failed to transcript anti-oxidative gene expressions and induced oxidant stress. Additionally, as a CDK4 inhibitor, HER developed triple anti-tumor effects including induced G0/G1 phase arrest, suppressed glycolysis, and disrupted the anti-oxidative capacity of CC cells. CONCLUSION: The results first time revealed that CDK4 modulated glycolytic and anti-oxidative capacity of CC cells via bound to its endogenous substrates, PKM2 and NRF2. Additionally, 140Asp145Asn amino acid sites of CDK4 were potential targets of HER. HER exerts anti-tumor activity by inhibited the activity of CDK4, promoted the CDK4-PKM2-NRF2 complex formation in the CC cells.

LILRB4 regulates the function of decidual MDSCs via the SHP-2/STAT6 pathway during Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii infection can cause adverse pregnancy outcomes, such as recurrent abortion, fetal growth restriction and infants with malformations, among others. Decidual myeloid-derived suppressor cells (dMDSCs) are a novel immunosuppressive cell type at the fetal-maternal interface which play an important role in sustaining normal pregnancy that is related to their high expression of the inhibitory molecule leukocyte immunoglobulin-like receptor B4 (LILRB4). It has been reported that the expression of LILRB4 is downregulated on decidual macrophages after T. gondii infection, but it remains unknown whether T. gondii infection can induce dMDSC dysfunction resulting from the change in LILRB4 expression. METHODS: LILRB4-deficient (LILRB4-/-) pregnant mice infected with T. gondii with associated adverse pregnancy outcomes, and anti-LILRB4 neutralized antibodies-treated infected human dMDSCs were used in vivo and in vitro experiments, respectively. The aim was to investigate the effect of LILRB4 expression on dMDSC dysfunction induced by T. gondii infection. RESULTS: Toxoplasma gondii infection was observed to reduce STAT3 phosphorylation, resulting in decreased LILRB4 expression on dMDSCs. The levels of the main functional molecules (arginase-1 [Arg-1], interleukin-10 [IL-10]) and main signaling molecules (phosphorylated Src-homology 2 domain-containing protein tyrosine phosphatase [p-SHP2], phosphorylated signal transducer and activator of transcription 6 [p-STAT6]) in dMDSCs were all significantly reduced in human and mouse dMDSCs due to the decrease of LILRB4 expression induced by T. gondii infection. SHP-2 was found to directly bind to STAT6 and STAT6 to bind to the promoter of the Arg-1 and IL-10 genes during T. gondii infection. CONCLUSIONS: The downregulation of LILRB4 expression on dMDSCs induced by T. gondii infection could regulate the expression of Arg-1 and IL-10 via the SHP-2/STAT6 pathway, resulting in the dysfunction of dMDSCs, which might contribute to adverse outcomes during pregnancy by T. gondii infection.

Procyanidin C1 inhibits tumor growth and metastasis in colon cancer via modulating miR-501-3p/HIGD1A axis.

INTRODUCTION: Although colon (COAD) and rectal adenocarcinoma (READ) combined to refer to colorectal cancer (CRC), substantial clinical evidence urged that CRC should be treated as two different cancers due to compared with READ, COAD showed higher morbidity and worse 5-year survival. OBJECTIVES: This study has tried to screen for the crucial gene that caused the worse prognosis and investigate its mechanism for mediating tumor growth and metastases in COAD. Meanwhile, the potential anti-COAD compound implicated in this mechanism was identified and testified from 1,855 food-borne chemical kits. This study aims to bring a new perspective to the development of new anti-COAD drugs and personalized medicine for patients with COAD. METHODS AND RESULTS: The survival-related hub genes in COAD and READ were screened out from The Cancer Genome Atlas (TCGA) database and the results showed that HIGD1A, lower expressed in COAD than in READ, was associated with poor prognosis in COAD patients, but not in READ. Over-expressed HIGD1A suppressed CRC cell proliferation, invasion, and migration in vitro and in vivo. Meanwhile, the different expressed microRNA profiles between COAD and READ showed that miR-501-3p was highly expressed in COAD and inhibited HIGD1A expression by targeting 3'UTR of HIGD1A. MiR-501-3p mimics promoted cell proliferation and metastasis in CRC cells. In addition, Procyanidin C1 (PCC1), a kind of natural polyphenol has been verified as a potential miR-501-3p inhibitor. In vitro and in vivo, PCC1 promoted HIGD1A expression by suppressing miR-501-3p and resulted in inhibited tumor growth and metastasis. CONCLUSION: The present study verified that miR-501-3p/HIGD1A axis mediated tumor growth and metastasis in COAD. PCC1, a flavonoid that riched in food exerts anti-COAD effects by inhibiting miR-501-3p and results in the latter losing the ability to suppress HIGD1A expression. Subsequently, unfettered HIGD1A inhibited tumor growth and metastasis in COAD.

Shen-Qi-Jiang-Tang granule ameliorates diabetic nephropathy via modulating tumor necrosis factor signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shen-Qi-Jiang-Tang granule (SQJTG), a classic traditional Chinese medicine (TCM) prescription, has been widely used in clinical for diabetes, especially type Ⅱ diabetes. Previous anti-diabetic studies stumbled across that SQJTG has a potential kidney protective effect on diabetic nephropathy (DN). However, the protective mechanism of SQJTG on DN still needs to be explored. AIM OF THE STUDY: The purpose of the present study was to explore the therapeutic effect of SQJTG on DN through both bioinformatics analysis and in vivo experiments. METHODS AND MATERIALS: The TCMIP database was used for screening potential compounds and targets of SQJTG, and the GeneCards, OMIM, DrugBank, and TTD databases were used for collecting DN-related genes. Then protein-protein interaction analysis for the common targets of SQJTG and DN was performed by the STRING database. Meanwhile, KEGG and GO were carried out using the Metascape and DAVID databases. In vivo experiments, to testify the potential kidney protective effects of SQJTG, STZ-induced DN mice with different dosages of SQJTG treatment were collected and the renal tissues were detected by H&E, PAS, Masson and TUNEL staining. Immunohistochemistry and immunoblotting were used to assess the proteins' expressions. Flow cytometry and ELISA assay were used to detect the levels of pro-inflammatory cytokines. RESULTS: Among the 338 compounds ascertained by SQJTG, there were 789 related targets as well. Moreover, 1,221 DN-related targets were predicted and 20 core targets were screened by the PPI analyses. According to GO and KEGG pathway analysis, SQJTG may affect DN via the TNF pathway. For the in vivo experiments, renal histomorphological examinations demonstrated that SQJTG treatment significantly ameliorated STZ-induced kidney damage and had a dosage dependence. Meanwhile, mice with DN were found to have dramatic increases in IL-1, TNF-α, IL-6, and IL-12, but markedly decreased after administration of SQJTG. In addition, the protein levels of TNF signaling molecules, like p-P65, p-JNK, and p-p38, showed significantly elevated in kidney tissues of DN mice and attenuated after SQJTG treatment. CONCLUSIONS: SQJTG exerts a kidney protective effect in DN mice via modulating TNF signaling pathways, and it has promising applications for the treatment of DN.

Knockdown of core 1 beta 1, 3-galactosyltransferase prolongs skin allograft survival with induction of galectin-1 secretion and suppression of CD8+ T cells: T synthase knockdown effects on galectin-1 and CD8+ T cells.

Core 1 beta 1,3-galactosyltransferase also known as T-antigen-synthase or T-synthase is a key enzyme for the synthesis of the common core 1 O-glycan structure (T-antigen). Although T-synthase is known to be important in human immune-related diseases, the effects of T-synthase and T-antigen on host immune responses remain poorly defined. In this study, a T-synthase-specific short hairpin RNA (shRNA) was transfected into murine colon carcinoma CT26 cells or mouse muscle tissues via intramuscular electroporation to assess the effects of T-synthase on T cells and cytokines. T-synthase knockdown significantly induced galectin-1 secretion both in vivo and in vitro and strongly enhanced Th2 cytokine (IL-10 and IL-4) production in vivo. Further, the increased production of galectin-1 induced by T-synthase knockdown promoted CD8(+) T-cell apoptosis, which, when combined with the increased production of CD4(+) T cell-derived Th2 cytokines prolonged the survival of skin allografts in mice. Our data suggest core 1 beta 1,3-galactosyltransferase-shRNA could serve not only as a useful tool in organ transplantation but also as a powerful tool for investigating O-glycans and glycoprotein synthesis and function.

A novel 2B4 receptor leads to worse pregnancy outcomes by facilitating TNF-α and IFN-γ production in dNK cells during Toxoplasma gondii infection.

BACKGROUND: Infections are a major threat to human reproductive health because they can induce pregnancy failure, including recurrent abortion, stillbirth, and preterm birth. Toxoplasma gondii (T. gondii) infection can result in adverse pregnancy outcomes by affecting certain immune molecules and cytokines. However, the detailed mechanisms behind T. gondii-induced pregnancy failure are poorly understood. METHODS: Toxoplasma gondii-infected wild-type (WT) pregnant mice and 2B4 knockout (2B4-/-) pregnant mice were established for in vivo study. Human decidual natural killer (dNK) cells were cultured for in vitro study. Abnormal pregnancy outcomes were observed, and the expression of 2B4, functional molecules (CD69, CD107a, tumor necrosis factor alpha [TNF-α], interferon gamma [IFN-γ]), and signaling molecules (SHP-2, Fyn, p-ERK, p-P38) in dNK cells were detected by flow cytometry, Western blot, reverse transcriptase polymerase chain reaction (RT-PCR), and/or immunofluorescence. The direct interactions (2B4 interacts with SHP-2 and Fyn; SHP-2 interacts with p-P38 and 2B4; Fyn interacts with p-ERK and 2B4) were verified by co-immunoprecipitation (co-IP) in NK-92 cells. RESULTS: Here, results showed that 2B4 was significantly downregulated after T. gondii infection. Subsequently, infected 2B4-/- pregnant mice displayed worse pregnancy outcomes compared with infected WT pregnant mice. Also, increased TNF-α and IFN-γ expression and elevated dNK cell cytotoxicity were found in 2B4-/- pregnant mice during T. gondii infection. In contrast, reduced TNF-α and IFN-γ expression and decreased human dNK cell activity were found following 2B4 activation during T. gondii infection. Interestingly, results showed that 2B4 binds to adaptor SHP-2 or Fyn, which then triggers different signaling pathways to regulate TNF-α and IFN-γ expression in dNK cells during T. gondii infection. Further, SHP-2 binds 2B4 and p-P38 directly after 2B4 activation, which generates an inhibitory signal for TNF-α and IFN-γ in NK-92 cells. In addition, Fyn can bind to 2B4 and p-ERK after activation of 2B4, thereby inhibiting TNF-α and IFN-γ expression in NK-92 cells following T. gondii infection. CONCLUSIONS: These data suggest that 2B4 may be a novel danger-signaling molecule that is implicated in pregnancy failure during T. gondii infection. Unraveling the mechanism by which 2B4 regulates dNK cell activity will provide novel insights to aid our understanding of T. gondii-induced adverse pregnancy outcomes.

Tim-3 downregulation by Toxoplasma gondii infection contributes to decidual dendritic cell dysfunction.

BACKGROUND: Women in early pregnancy infected by Toxoplasma gondii may have severe adverse pregnancy outcomes, such as spontaneous abortion and fetal malformation. The inhibitory molecule T cell immunoglobulin and mucin domain 3 (Tim-3) is highly expressed on decidual dendritic cells (dDCs) and plays an important role in maintaining immune tolerance. However, whether T. gondii infection can cause dDC dysfunction by influencing the expression of Tim-3 and further participate in adverse pregnancy outcomes is still unclear. METHODS: An abnormal pregnancy model in Tim-3-deficient mice and primary human dDCs treated with Tim-3 neutralizing antibodies were used to examine the effect of Tim-3 expression on dDC dysfunction after T. gondii infection. RESULTS: Following T. gondii infection, the expression of Tim-3 on dDCs was downregulated, those of the pro-inflammatory functional molecules CD80, CD86, MHC-II, tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) were increased, while those of the tolerant molecules indoleamine 2,3-dioxygenase (IDO) and interleukin-10 (IL-10) were significantly reduced. Tim-3 downregulation by T. gondii infection was closely associated with an increase in proinflammatory molecules and a decrease in tolerant molecules, which further resulted in dDC dysfunction. Moreover, the changes in Tim-3 induced by T. gondii infection further reduced the secretion of the cytokine IL-10 via the SRC-signal transducer and activator of transcription 3 (STAT3) pathway, which ultimately contributed to abnormal pregnancy outcomes. CONCLUSIONS: Toxoplasma gondii infection can significantly downregulate the expression of Tim-3 and cause the aberrant expression of functional molecules in dDCs. This leads to dDC dysfunction, which can ultimately contribute to abnormal pregnancy outcomes. Further, the expression of the anti-inflammatory molecule IL-10 was significantly decreased by Tim-3 downregulation, which was mediated by the SRC-STAT3 signaling pathway in dDCs after T. gondii infection.

Effect of B7-H4 downregulation induced by Toxoplasma gondii infection on dysfunction of decidual macrophages contributes to adverse pregnancy outcomes.

BACKGROUND: Toxoplasma gondii infection during pregnancy can lead to fetal defect(s) or congenital complications. The inhibitory molecule B7-H4 expressed on decidual macrophages (dMφ) plays an important role in maternal-fetal tolerance. However, the effect of B7-H4 on the function of dMφ during T. gondii infection remains unclear. METHODS: Changes in B7-H4 expression on dMφ after T. gondii infection were explored both in vivo and in vitro. B7-H4-/- pregnant mice (pregnant mice with B7-H4 gene knockout) and purified primary human dMφ treated with B7-H4 neutralizing antibody were used to explore the role of B7-H4 signaling on regulating the membrane molecules, synthesis of arginine metabolic enzymes and cytokine production by dMφ with T. gondii infection. Also, adoptive transfer of dMφ from wild-type (WT) pregnant mice or B7-H4-/- pregnant mice to infected B7-H4-/- pregnant mice was used to examine the effect of B7-H4 on adverse pregnancy outcomes induced by T. gondii infection. RESULTS: The results illustrated that B7-H4-/- pregnant mice infected by T. gondii had poorer pregnancy outcomes than their wild-type counterparts. The expression of B7-H4 on dMφ significantly decreased after T. gondii infection, which resulted in the polarization of dMφ from the M2 toward the M1 phenotype by changing the expression of membrane molecules (CD80, CD86, CD163, CD206), synthesis of arginine metabolic enzymes (Arg-1, iNOS) and production of cytokines (IL-10, TNF-α) production. Also, we found that the B7-H4 downregulation after T. gondii infection increased iNOS and TNF-α expression mediated through the JAK2/STAT1 signaling pathway. In addition, adoptive transfer of dMφ from a WT pregnant mouse donor rather than from a B7-H4-/- pregnant mouse donor was able to improve adverse pregnancy outcomes induced by T. gondii infection. CONCLUSIONS: The results demonstrated that the downregulation of B7-H4 induced by T. gondii infection led to the dysfunction of decidual macrophages and contributed to abnormal pregnancy outcomes. Moreover, adoptive transfer of B7-H4+ dMφ could improve adverse pregnancy outcomes induced by T. gondii infection.

Gut microbiota dysbiosis as an inflammaging condition that regulates obesity-related retinopathy and nephropathy.

Diabetes-specific microvascular disease is a leading cause of blindness, renal failure and nerve damage. Epidemiological data demonstrated that the high morbidity of T2DM occurs as a result of obesity and gradually develops into serious complications. To date, the mechanisms that underlie this observation are still ill-defined. In view of the effect of obesity on the gut microflora, Leprdb/db mice underwent antibiotic treatment and microbiota transplants to modify the gut microbiome to investigate whether microbes are involved in the development of diabetic nephropathy (DN) and/or diabetic retinopathy (DR). The mouse feces were collected for bacterial 16S ribosomal RNA gene sequencing. Cytokines including TNF-α, TGF-ß1, IFN-γ, IL-1ß, IL-6, IL-17A, IL-10, and VEGFA were detected by enzyme-linked immunosorbent assay (ELISA), flow cytometry, real-time PCR and immunofluorescent assay. Eyes and kidney were collected for histopathological assay. Intestinal permeability was also detected using Evans Blue. The results showed that obesity influenced metabolic variables (including fast/fed glucose, insulin, and triglyceride), retinopathy and nephropathy, and the gut microbiota. Obesity mainly reduced the ratio of Bacteroidetes/Firmicutes and influenced relative abundance of Proteobacteria, Actinobacteria, and Spirochetes. Obesity also increased intestinal permeability, metabolic endotoxemia, cytokines, and VEGFA. Microbiota transplants confirm that obesity aggravates retinopathy and nephropathy through the gut microbiota. These findings suggest that obesity exacerbates retinopathy and nephropathy by inducing gut microbiota dysbiosis, which further enhanced intestinal permeability and chronic low-grade inflammation.

MicroRNA-31/184 is involved in transforming growth factor-ß-induced apoptosis in A549 human alveolar adenocarcinoma cells.

AIMS: TGF-ß-induced alveolar epithelial cells apoptosis were involved in idiopathic pulmonary fibrosis (IPF). This study aimed to explore potential targets and mechanisms of IPF. MAIN METHODS: mRNA and microRNA arrays were used to analyze differentially expressed genes and miRNAs. Several essential targets of TGF-ß-SMADs and TGF-ß-PI3K-AKT pathways were detected. KEY FINDINGS: miR-31 and miR-184 expression levels were positively correlated with smad6 and smad2/akt expression levels in IPF patients. TGF-ß could induce miR-31 and suppress miR-184 levels in A549 cells. miR-31 was confirmed to bind to the smad6-3'UTR and functionally suppress its expression. Down-regulated SMAD6 enhanced SMAD2/SMAD4 dimer formation and translocation due to its failure to prevent SMAD2 phosphorylation. In contrast, anti-fibrotic functions of miR-184 were abolished due to TGF-ß directly suppressing miR-184 levels in A549 cells. When A549 was stimulated by TGF-ß combined with or without miR-31 inhibitor/miR-184 mimic, it was showed that depleted miR-31 and/or increased miR-184 significantly ameliorated TGF-ß-induced viability of A549 cells, as well as inhibited the expression of profibrotic factors, MMP7 and RUNX2. SIGNIFICANCE: Inhibiting miR-31 and/or promoting miR-184 protect against TGF-ß-induced fibrogenesis by respectively repressing the TGF-ß-SMAD2 and TGF-ß-PI3K-AKT signaling pathways, implying that miR-31/184 are potential targets and suggesting a new management strategy for IPF.

Discovery of Aryl Formyl Piperidine Derivatives as Potent, Reversible, and Selective Monoacylglycerol Lipase Inhibitors.

Most of the current monoacylglycerol lipase (MAGL) inhibitors function by an irreversible mechanism of action, causing a series of side effects. Herein, starting from irreversible inhibitors, 25 compounds were synthesized and evaluated in vitro for MAGL inhibition, among which, compound 36 showed the most potent inhibitory activity (IC50 = 15 nM). Crucially, docking studies demonstrated that the m-chlorine-substituted aniline fragment occupied a hydrophobic subpocket enclosed by side chains of Val191, Tyr194, Val270, and Lys273, which creatively identify a new key anchoring point for the development of new MAGL inhibitors. Furthermore, in vivo evaluation innovatively revealed that this reversible inhibitor 36 significantly ameliorated depressive-like behaviors induced by reserpine. To the best of our knowledge, this is the first time that reversible inhibitors of MAGL were developed to support MAGL as a potential therapeutic target for depression.

Application quantitative proteomics approach to identify differentially expressed proteins associated with cardiac protection mediated by cycloastragenol in acute myocardial infarction rats.

Acute myocardial infarction (AMI) is an acute heart disease. Cycloastragenol, as a natural product, inhibits inflammation and protects cardiomyocytes. Cycloastragenol (Y006) modulates inflammation in AMI is not known. To explore the function of Cycloastragenol in AMI, this study investigated the effect of Y006 and its mechanisms both in vitro and in vivo. Y006 influences the concentration of 11 proteins, as shown by a proteomics analysis, immunohistochemistry and western blotting. Among these 11 proteins, Erk1/2, PLCG1, IKBKG, and ZEB1 are related to inflammatory regulation. BAX, COX2, and GSK3ß are involved in modulating cardiomyocyte apoptosis, and RhoA and DSC2 are directly associated with myocardial function. However, the functions of ARHGAP17 and Rit2 in heart are less well established. Additionally, Y006 suppressed TNF-α, IFN-γ and IL-17 production in PBMCs (peripheral blood monocytes) from patients with acute myocardial infarction and enhanced IL-10 and IL-4 expression. Similar results were obtained in a rat model of AMI by flow cytometry detection and ELISA. Our findings indicate that Y006 protects rats from AMI through direct or indirect inhibition of inflammation and cardiomyocyte apoptosis. However, the specific mechanism of Y006's protective function requires further study. Nonetheless, this research revealed a novel aspect for the treatment of myocardial infarction. SIGNIFICANCE: In the present study, we undertook the first proteomic evaluation of Cycloastragenol (Y006) function in acute myocardial infarction (AMI). Y006 significantly improved myocardial function in vivo by regulating multiple molecular expressions. Hypoxia is a direct reason for AMI. And our data support a role of Y006 in gene expression, cell apoptosis under hypoxia. The conclusions of this research assist to explain the potential molecular mechanism in Cycloastragenol treating AMI and supply a new method for ameliorating AMI.