Fluoxetine Successfully Treats Intracranial Enterovirus E18 Infection in a Patient with CD79a Deficiency Arising from Segmental Uniparental Disomy of Chromosome 19.

The pre BCR complex plays a crucial role in B cell production, and its successful expression marks the B cell differentiation from the pro-B to pre-B. The CD79a and CD79b mutations, encoding Igα and Igß respectively, have been identified as the cause of autosomal recessive agammaglobulinemia (ARA). Here, we present a case of a patient with a homozygous CD79a mutation, exhibiting recurrent respiratory infections, diarrhea, growth and development delay, unique facial abnormalities and microcephaly, as well as neurological symptoms including tethered spinal cord, sacral canal cyst, and chronic enteroviral E18 meningitis. Complete blockade of the early B cell development in the bone marrow of the patient results in the absence of peripheral circulating mature B cells. Whole exome sequencing revealed a Loss of Heterozygosity (LOH) of approximately 19.20Mb containing CD79a on chromosome 19 in the patient. This is the first case of a homozygous CD79a mutation caused by segmental uniparental diploid (UPD). Another key outcome of this study is the effective management of long-term chronic enteroviral meningitis using a combination of intravenous immunoglobulin (IVIG) and fluoxetine. This approach offers compelling evidence of fluoxetine's utility in treating enteroviral meningitis, particularly in immunocompromised patients.

Neutralizing IL-8 potentiates immune checkpoint blockade efficacy for glioma.

Malignant gliomas are largely refractory to immune checkpoint blockade (ICB) therapy. To explore the underlying immune regulators, we examine the microenvironment in glioma and find that tumor-infiltrating T cells are mainly confined to the perivascular cuffs and express high levels of CCR5, CXCR3, and programmed cell death protein 1 (PD-1). Combined analysis of T cell clustering with T cell receptor (TCR) clone expansion shows that potential tumor-killing T cells are mainly categorized into pre-exhausted/exhausted and effector CD8+ T subsets, as well as cytotoxic CD4+ T subsets. Notably, a distinct subpopulation of CD4+ T cells exhibits innate-like features with preferential interleukin-8 (IL-8) expression. With IL-8-humanized mouse strain, we demonstrate that IL-8-producing CD4+ T, myeloid, and tumor cells orchestrate myeloid-derived suppressor cell infiltration and angiogenesis, which results in enhanced tumor growth but reduced ICB efficacy. Antibody-mediated IL-8 blockade or the inhibition of its receptor, CXCR1/2, unleashes anti-PD-1-mediated antitumor immunity. Our findings thus highlight IL-8 as a combinational immunotherapy target for glioma.

Study on the mechanism of Fufang E'jiao Jiang on precancerous lesions of gastric cancer based on network pharmacology and metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang E'jiao Jiang (FEJ) is a prominent traditional Chinese medicine prescription, which consists of Asini Corii Colla (Donkey-hide gelatin prepared by stewing and concentrating from the hide of Equus asinus Linnaeus., ACC), Codonopsis Radix (the dried roots of Codonopsis pilosula (Franch.) Nannf., CR), Ginseng Radix et Rhizoma Rubra (the steamed and dried root of Panax ginseng C.A. Mey., GRR), Crataegi Fructus (the mature fruits of Crataegus pinnatifida Bunge., CF), and Rehmanniae Radix Praeparata (the steamed and sun dried tuber of Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A. Mey., RRP). It is a popularly used prescription for "nourishing Qi and nourishing blood". AIM OF THE STUDY: To explore the potential mechanism of FEJ on precancerous lesion of gastric cancer in rats by combining network pharmacology and metabolomics. METHODS: Traditional Chinese Medicine Systems Pharmacology and Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine were used to identify the ingredients and potential targets of FEJ. GeneCards database was used to define PLGC-associated targets. We built a herb-component-disease-target network and analyzed the protein-protein interaction network. Underlying mechanisms were identified using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. In addition, 40% ethanol, N-methyl-N'-nitro-N-nitroguanidine and irregular eating were used to establish PLGC rats model. We also evaluated the efficacy of FEJ on MNNG-induced PLGC rats by body weight, histopathology, blood routine and cytokine levels, while the predicted pathway was determined by the Western blot. Ultra-performance liquid chromatography-tandem mass spectrometry-based serum non-targeted metabolomics was used to select potential biomarkers and relevant pathways for FEJ in the treatment of PLGC. RESULTS: Network pharmacology showed that FEJ exhibited anti-PLGC effects through regulating ALB, TNF, VEGFA, TP53, AKT1 and other targets, and the potential pathways mainly involved cancer-related, TNF, PI3K-AKT, HIF-1, and other signaling pathways. Animal experiments illustrated that FEJ could suppress inflammation, regulate gastrointestinal hormones, and inhibit the expression of PI3K/AKT/HIF-1α pathway-related proteins. Based on serum non-targeted metabolomics analysis, 12 differential metabolites responding to FEJ treatment were identified, and metabolic pathway analysis showed that the role of FEJ was concentrated in 6 metabolic pathways. CONCLUSION: Based on network pharmacology, animal experiments and metabolomics, we found that FEJ might ameliorate gastric mucosal injury in PLGC rats by regulating gastrointestinal hormones and inhibiting inflammation, and its mechanism of action is related to the inhibition of excessive activation of PI3K/AKT/HIF-1α signaling pathway and regulation of disorders of body energy metabolism. This comprehensive strategy also provided a reasonable way for unveiling the pharmacodynamic mechanisms of multi-components, multi-targets, and multi-pathways in Traditional Chinese Medicine.

WASp Deficiency Selectively Affects the TCR Diversity of Different Memory T Cell Subsets in WAS Chimeric Mice.

Background: The T cell receptor (TCR) diversity is essential for effective T cell immunity. Previous studies showed that TCR diversity in Wiskott-Aldrich Syndrome (WAS) patients was severely impaired, especially in the memory T cell populations. Whether this defect was caused by intrinsic WASp deficiency or extrinsic reasons is still unclear. Methods: We sorted different T cell subsets from the bone marrow chimeric mice model using both magnetic beads and flow cytometry. TCR repertoires of memory T cells, especially CD4+ effector memory T (TEM) cells and CD8+ central memory T (TCM) cells, were analyzed using the UMI quantitative high-throughput sequencing (HTS). Results: An average of 5.51 million sequencing reads of 32 samples was obtained from the Illumina sequencing platform. Bioinformatic analyses showed that compared with wild type (WT), WAS knock out (KO)-CD4+ TEM cells exhibited increased Simpson index and decreased D50 index (P <0.05); The rank abundance curve of KO-CD4+ TEM cells was shorter and steeper than that of WT, and the angle of qD and q in KO-CD4+ TEM cells was lower than that of WT, while these indexes showed few changes between WT and KO chimeric mice in the CD8+TCM population. Therefore, it indicated that the restriction on the TCRVß repertoires is majorly in KO-CD4+ TEM cells but not KO- CD8+ TCM cells. Principal Component Analysis (PCA), a comprehensive parameter for TCRVß diversity, successfully segregated CD4+ TEM cells from WT and KO, but failed in CD8+ TCM cells. Among the total sequences of TRB, the usage of TRBV12.2, TRBV30, TRBV31, TRBV4, TRBD1, TRBD2, TRBJ1.1, and TRBJ1.4 showed a significant difference between WT-CD4+ TEM cells and KO-CD4+ TEM cells (P <0.05), while in CD8+ TCM cells, only the usage of TRBV12.2 and TRBV20 showed a substantial difference between WT and KO (P <0.05). No significant differences in the hydrophobicity and sequence length of TCRVß were found between the WT and KO groups. Conclusion: WASp deficiency selectively affected the TCR diversity of different memory T cell subsets, and it had more impact on the TCRVß diversity of CD4+ TEM cells than CD8+ TCM cells. Moreover, the limitation of TCRVß diversity of CD4+ TEM cells and CD8+ TCM cells in WAS was not severe but intrinsic.

Schwann cell­derived exosomes induce bone marrow­derived mesenchymal stem cells to express Schwann cell markers in vitro.

Following peripheral nerve injury, factors in the local microenvironment can induce the differentiation of bone marrow­derived mesenchymal stem cells (BMSCs) into Schwann cells; however, the specific factors that participate in this process remain unclear. The present study aimed to investigate the role of Schwann cell­derived exosomes in the differentiation of BMSCs into Schwann cells. Exosomes were extracted from Schwann cells or fibroblasts and co­cultured with BMSCs. The morphology, as well as gene and protein expressions of the BMSCs were measured to determine the effect of exosomes on cell differentiation. The levels of Schwann cell­specific markers in BMSCs were significantly increased by Schwann cell­derived exosomes compared with untreated BMSCs; however, fibroblast­derived exosomes did not demonstrate the same effects. In conclusion, Schwann cell­derived exosomes may be involved in the differentiation of BMSCs into Schwann cells, which may provide a novel target for promoting nerve regeneration following injury.

Decreased Circulating MANF in Women with PCOS is Elevated by Metformin Therapy and is Inversely Correlated with Insulin Resistance and Hyperandrogenism.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel neurotrophic factor. Although recent studies have suggested that MANF appeared to be associated with insulin resistance, the results have been inconsistent. The aim of our study was to determine the serum MANF levels in women with PCOS and controls, to investigate their relationship to insulin resistance, and to evaluate circulating MANF changes with metformin intervention in PCOS women. We conducted a series of cross-sectional and interventional studies in 90 newly diagnosed patients with PCOS and 60 age- and gender-matched controls. Oral glucose tolerance test and euglycemic-hyperinsulinemic clamps were performed to assess the glucose tolerance and insulin sensitivity. Forty-three women with PCOS were randomly assigned to six months of oral metformin therapy. Serum MANF levels were significantly lower in women with PCOS than in controls. Serum MANF levels were positively correlated with M-value and negatively correlated with body mass index (BMI), body fat percentage (FAT), homeostatic model assessment of insulin resistance (HOMA-IR), and free androgen index (FAI). Multivariate stepwise regression demonstrated that serum MANF levels were independently associated with M-value and FAI. After six months of metformin treatment, there was a significant increase in serum MANF levels in PCOS women. Serum MANF levels are decreased in women with PCOS, and are reversely related to insulin resistance and hyperandrogenism. Metformin treatment elevates serum MANF levels and alleviates insulin resistance and hyperandrogenism in PCOS women.

E1021K Homozygous Mutation in PIK3CD Leads to Activated PI3K-Delta Syndrome 1.

PURPOSE: Activated PI3Kδ syndrome 1 is a primary immunodeficiency disease, usually caused by heterozygous mutations in PIK3CD. We aimed to identify the cause of homozygous mutation at c.G3061A (p.E1021K) in a patient and the effect of allele dose in this mutation. METHODS: Genomic DNA from the parent-child trio was analyzed by next-generation sequencing. We performed phenotypic analyses in the patient and in Pik3cdE1024K+/+ mice. RESULTS: The patient was a girl harboring a homozygous mutation for p.E1021K in PIK3CD. At the age of 2 months, she began experiencing respiratory tract infections and lymphoproliferation, accompanied by bronchiectasis and extensive atelectasis in the lungs. She suffered from Haemophilus influenzae and Cytomegalovirus infections and experienced restricted growth and development. Whole-exome sequencing showed a region that included PIK3CD, with loss of heterozygosity (LOH) in chromosome 1 of the patient. The patient had not inherited any allele from her father in the LOH region. Copy number variation analysis showed no changes in the patient's father and the patient. Ultra-deep sequencing of genomic DNA from the patient's mother showed that the mutant allele frequency for c.G3061A was 1.64%. Thus, the presence of segmental maternal uniparental disomy and maternal gonosomal mosaicism resulted in the homozygous mutation. Lymphadenopathy, differentiation of activated T cells, and follicular B cells lymphopenia were found to be more prominent in Pik3cdE1024+/+ mice than in Pik3cdE1024+/- mice. CONCLUSION: This report showed the coexistence of uniparental disomy and mosaicism in PIK3CD. Some immunological features were seen to be allele dose-dependent in the presence of p.E1021K mutation.

Exosomes from adipose-derived stem cells alleviate neural injury caused by microglia activation via suppressing NF-kB and MAPK pathway.

Activation of microglia cells play critical role in neuroinflammation after brain injury. Exosomes from adipose-derived stem cells (ADSC) possess immunoregulation effect similar with ADSC. We hypothesized that ADSC derived exosomes (ADSC-exosomes) could inhibit the activation of microglia cells and prevent neuroinflammation. We found that ADSC-exosomes could inhibit the activation of microglia cells by suppressing NF-kB and MAPK pathway. Production of inflammatory factors in lipopolysaccharide-stimulated microglia cells decreased significantly when pretreated with ADSC-exosomes. Furthermore, ADSC-exosomes could decrease the cytotoxicity of activated microglia. These results revealed that ADSC-exosomes might be a promising strategy for the therapy of neural injury.

Role of bone morphogenetic protein-9 in the regulation of glucose and lipid metabolism.

Bone morphogenetic protein (BMP)-9 has been reported to regulate energy balance in vivo. However, the mechanisms underlying BMP9-mediated regulation of energy balance remain incompletely understood. Here, we investigated the role of BMP9 in energy metabolism. In the current study, we found that hepatic BMP9 expression was down-regulated in insulin resistance (IR) mice and in patients who are diabetic. In mice fed a high-fat diet (HFD), the overexpression of hepatic BMP9 improved glucose tolerance and IR. The expression of gluconeogenic genes was down-regulated, whereas the level of insulin signaling molecule phosphorylation was increased in the livers of Adenovirus-BMP9-treated mice and glucosamine-treated hepatocytes. Furthermore, BMP9 overexpression ameliorated triglyceride accumulation and inhibited the expression of lipogenic genes in both human hepatocellular carcinoma HepG2 cells treated with a fatty acid mixture as well as the livers of HFD-fed mice. In hepatocytes isolated from sterol regulatory element-binding protein (SREBP)-1c knockout mice, the effects of BMP9 were ablated. Mechanistically, BMP9 inhibited SREBP-1c expression through the inhibition of liver X receptor response element 1 activity in the SREBP-1c promoter. Taken together, our results show that BMP9 is an important regulator of hepatic glucose and lipid metabolism.-Yang, M., Liang, Z., Yang, M., Jia, Y., Yang, G., He, Y., Li, X., Gu, H. F., Zheng, H., Zhu, Z., Li, L. Role of bone morphogenetic protein-9 in the regulation of glucose and lipid metabolism.

Effective gene delivery of shBMP-9 using polyethyleneimine-based core-shell nanoparticles in an animal model of insulin resistance.

Bone morphogenetic protein (BMP)-9 has been associated with insulin resistance and type 2 diabetes mellitus. However, methods for delivering exogenous BMP-9 genes in vivo are lacking. In this study, we developed a gene delivery system using polyethyleneimine (PEI)-based core-shell nanoparticles (PCNs) as gene delivery carriers, and investigated the effectiveness and safety for delivery of the shBMP-9 gene. PCNs possessed a well-defined core-shell nanostructure with hydrophobic polymer cores and dense PEI shells of uniform particle size and highly positively charged surfaces. In vitro evaluation suggested that PCNs had high loading capacity for exogenous genes and low cytotoxicity toward hepatocytes. The transfection efficiency of PCNs/pENTR-shBMP9 complexes was higher than that of commercial lipofectamine 2000/shBMP9. In vivo studies showed that PCNs/pENTR-shBMP9 transfection led to a significant decrease in hepatic BMP9 expression compared with pENTR-shBMP9 transfection. Under high fat diet (HFD) feeding, PCNs/pENTR-shBMP9 mice exhibited aggravated glucose and insulin tolerance. At a molecular level, PCNs/pENTR-shBMP9 mice displayed elevated PEPCK protein levels and lower levels of InsR and Akt phosphorylation than pENTR-shBMP9 mice. These results suggest that the biological effects of PCNs/pENTR-shBMP9 in vivo are much more effective than those of pENTR-shBMP9. Therefore, the polyethyleneimine (PEI)-based core-shell nanoparticle can be applied as promising nanocarriers for effective and safe gene delivery.

Effects of the tumor suppressor PTEN on biological behaviors of activated pancreatic stellate cells in pancreatic fibrosis.

Pancreatic stellate cells (PSCs), when activated, are characterized by proliferation and collagen synthesis, and contribute to extracellular matrix deposition in pancreatic fibrosis. Concomitantly, fibrosis is linked with the loss of PTEN (phosphatase and tensin homolog) protein in several organs. This study investigated the association between PTEN protein levels and the activated or apoptotic status of PSCs in a rat model of chronic pancreatitis. In addition, the activation status and biological behaviors of culture-activated PSCs were analyzed after lentiviral transfection with wildtype or mutant (G129E) PTEN for upregulation, or PTEN short hairpin RNA for downregulation, of PTEN. In vivo, PTEN levels gradually decreased during pancreatic fibrosis, which positively correlated with apoptosis of activated PSCs, but negatively with PSC activation. In vitro, activated PSCs with wildtype PTEN showed less proliferation, migration, and collagen synthesis compared with control PSCs, and greater numbers were apoptotic; activated PSCs with mutant PTEN showed similar, but weaker, effects. Furthermore, AKT and FAK/ERK signaling was involved in this process. In summary, activated PSCs during pancreatic fibrosis in vivo have lower levels of PTEN. In vitro, PTEN appears to prevent PSCs from further activation and promotes apoptosis through regulation of the AKT and FAK/ERK pathways.

Rapid induction of pancreatic cancer cells to cancer stem cells via heterochromatin modulation.

Mounting evidence supports that CSCs (cancer stem cells) play a vital role in cancer recurrence. Therefore elimination of CSCs is currently considered to be an important therapeutic strategy for complete remission. A major obstacle in CSC research is the obtainment of sufficient numbers of functional CSC populations. Here, we established a method to induce bulk pancreatic cancer cells to CSCs via heterochromatin modulation. Two pancreatic cancer cell lines Panc1 and Bxpc3 were cultured for 4 days in inducing medium (mTeSR containing FBS, B27, MEK inhibitor, GSK3 inhibitor, and VPA), and another 2 days in sphere culture medium (mTeSR supplemented with B27). Then the induced cells were dissociated into single cells and cultured in suspension in sphere culture medium. It was found that the majority of induced cells formed spheres which could grow larger and be passaged serially. Characterization of Panc1 sphere cells demonstrated that the sphere cells expressed increased pancreatic cancer stem cell surface markers and stem cell genes, were more resistant to chemotherapy, and were more tumorigenic in vivo, indicating that the induced sphere cells acquired CSC properties. Thus, the inducing method we developed may be used to obtain a sufficient number of CSCs from cancer cells, and contribute to the research for CSC-targeting therapy.

Myonectin Predicts the Development of Type 2 Diabetes.

Context: Myonectin has been identified as a myokine, expressed predominantly in skeletal muscle. However, its clinical implications are largely unknown. Objective: The aim of this study is to investigate the relationship between myonectin (C1q tumor necrosis factor-α-related protein isoform 15) and type 2 diabetes mellitus (T2DM) in cross-sectional and interventional studies. Design: In a separate study, oral glucose tolerance tests, a 45-minute bout of exercise, lipid infusions, and euglycemic-hyperinsulinemic clamps (EHCs) were performed to investigate the association of myonectin with homeostasis model assessment of insulin resistance (HOMA-IR) and T2DM. Circulating myonectin was measured by enzyme-linked immunosorbent assay. Patients: One hundred four newly diagnosed T2DM (nT2DM), 109 impaired glucose tolerance (IGT), and 128 healthy individuals were recruited for this study. Results: nT2DM and IGT subjects had higher circulating myonectin concentrations than normal subjects (82.3 ± 47.6 and 68.9 ± 46.6 vs. 45.2 ± 23.5 µg/L, P < 0.05 or P < 0.01). It was also found that in nT2DM individuals, circulating myonectin was higher than in IGT subjects. Plasma myonectin correlated positively with waist/hip ratio, percentage of body fat, triglyceride, fasting blood glucose, 2-hour blood glucose after glucose overload, fasting insulin, hemoglobin A1c, and HOMA-IR and negatively with the insulin sensitivity index in all of the study population. Multivariate logistic regression analysis revealed that circulating myonectin levels were significantly correlated with IGT and T2DM. A 45-minute bout of exercise did not change circulating myonectin levels in healthy, young individuals. Circulating myonectin levels were not significantly altered in response to an oral glucose challenge or EHC. In addition, acute elevated free fatty acid levels induced by lipid infusion had no effects on circulating myonectin. Conclusions: These data suggest that myonectin may be a useful marker in predicting the development of prediabetes and diabetes.

[Over-expression of thioredoxin-interacting protein promotes apoptosis of MIN6 cells via activating p38MAPK pathway].

Objective To investigate the effect of thioredoxin interacting protein (TXNIP) over-expression on the apoptosis of MIN6 ß-cells and the mechanism involved. Methods Lentivirus carrying TXNIP gene was used to infect MIN6 ß-cells in logarithmic growth phase, and the infection efficiency was evaluated by fluorescence microscope and Western blotting. Then MIN6 ß-cells were divided into three groups: control group, empty lentivirus vector (LV-GFP) group and TXNIP over-expression (LV-GFP-TXNIP) group. CCK-8 assay was used to detect cell proliferation. AnnexinV-FITC/PI double staining was utilized to measure the apoptosis of MIN6 cells. Western blotting was applied to detect the expressions of TXNIP protein, TRX, Bax, Bcl2, cleaved caspase-3 (c-caspase-3), p38 mitogen-activated protein kinase (p38MAPK), phospho-p38MAPK (p-p38MAPK) proteins in the MIN6 ß-cells before and after treated with p38MAPK inhibitor SP169316. Results At 72 hours after the infection, the infection rate reached (87.10±2.30)% and (92.21±0.54)% in LV-GFP group and LV-GFP-TXNIP group, respectively, suggesting that lentivirus-mediated TXNIP over-expression was desirable. Compared with control group and LV-GFP group, the cell viability markedly decreased, and cell apoptosis, Bax/Bcl2 ratio, expression of c-caspased-3 and p38MAPK phosphorylation significantly increased in LV-GFP-TXNIP group. However, both the Bax/Bcl2 ratio and c-caspase-3 protein expression in LV-GFP-TXNIP group were obviously reduced after treated with p38MAPK inhibitor. Conclusion TXNIP over-expression might promote the apoptosis of MIN6 cells via activating the p38 MAPK pathway.

Circulating CTRP9 levels are increased in patients with newly diagnosed type 2 diabetes and correlated with insulin resistance.

AIMS: C1q/TNF-related protein-9 (CTRP9) is a novel adipokine that has been shown to promote lipid metabolism, enhance insulin sensitivity and protect against cardiovascular disease. However, previous studies in humans have produced controversial results regarding the association between CTRP9 and insulin resistance. The objective of this study was to evaluate the relationships between CTRP9 and insulin resistance in Chinese population. METHODS: Subjects with normal glucose tolerance (NGT, n=108), impaired glucose tolerance (IGT, n=92), and newly diagnosed type 2 diabetes mellitus (nT2DM, n=106) were recruited to determining the circulating CTRP9 and adiponectin levels by enzyme linked immunosorbent assay. Anthropometric and biochemical measurements related to insulin resistance, adiposity and lipid profile were examined for all participants. Oral glucose tolerance test was performed in healthy subjects (17 male and 17 female). RESULTS: Circulating CTRP9 level was significantly higher in both IGT and nT2DM than in individuals with NGT. Overweight/obese subjects had much higher CTRP9 levels than lean individuals, and in all subjects, females also had higher CTRP9 levels than males. In addition, circulating CTRP9 level was positively correlated with markers of obesity and insulin resistance, including body mass index, fasting blood glucose, insulin, HbA1c, homeostasis model assessment of insulin resistance and low-density lipoprotein-cholesterol, while was inversely correlated with high-density lipoprotein-cholesterol and adiponectin. Moreover, hyperglycemia during an oral glucose challenge increased circulating CTRP9 concentrations. CONCLUSIONS: We conclude that CTRP9 was strongly associated with insulin resistance, suggesting that CTRP9 might be important in the development of type 2 diabetes.

Decreased circulating BMP-9 levels in patients with Type 2 diabetes is a signature of insulin resistance.

Bone morphogenetic protein 9 (BMP-9) has been demonstrated to improve glucose homoeostasis in diabetic mice. However, no report has demonstrated the relationship of circulating BMP-9 levels with insulin resistance (IR) or Type 2 diabetes mellitus (T2DM) in humans. The objective of the present study was to investigate the relationship between BMP-9 and IR in cross-sectional and interventional studies. Circulating BMP-9 levels were analysed by ELISA in 280 well-characterized individuals. Two-hour oral glucose tolerance test (OGTT) and euglycaemic-hyperinsulinaemic clamp (EHC) were performed in 20 healthy subjects. Acute IR was induced by lipid infusion for 4 h in 20 healthy volunteers. Real-time (RT)-PCR and Western blotting were used to assess mRNA and protein expression of BMP-9. The effect of a glucagon-like peptide-1 (GLP-1) receptor agonist (PEX168) on circulating BMP-9 was investigated in a 24-week treatment trial. Circulating BMP-9 levels were significantly higher in healthy subjects than in newly diagnosed patients with T2DM. Circulating BMP-9 negatively correlated with HbA1c, fasting blood glucose (FBG), OGTT, the area under the curve for glucose (AUCglucose) and homoeostasis model assessment of insulin resistance (HOMA-IR). Multivariate regression analyses showed that BMP-9 levels were independently associated with non-esterified fatty acid (NEFA) and AUCglucose Both hyperinsulinaemia and lipid infusion decreased circulating BMP-9 levels. BMP-9 mRNA and protein expressions were significantly decreased in muscle and adipose tissues of T2DM patients. In the placebo treated group, BMP-9 levels continued to decline over time, whereas in the PEX 168 treated groups BMP-9 levels remained stable. Our data suggest that BMP-9 is likely to play an important role in IR in humans.

Pregnancy-specific glycoprotein 9 (PSG9), a driver for colorectal cancer, enhances angiogenesis via activation of SMAD4.

PSG9 is a member of the pregnancy-specific glycoprotein (PSG) family and has been shown to contribute to the progression of colorectal cancer (CRC) and cancer-related angiogenesis. Here, we aim to investigate abnormal PSG9 levels in patients with CRC and to emphasize the role of PSG9 in driving tumorigenesis. Serum from 140 patients with CRC and 125 healthy controls as well as 74 paired tumors and adjacent normal tissue were used to determine PSG9 levels. We discovered that PSG9 was significantly increased in serum (P<0.001) and in tumor tissues (P<0.001) from patients with CRC. Interestingly, the increased PSG9 levels correlated with poor survival (P=0.009) and microvessel density (MVD) (P=0.034). The overexpression of PSG9 strongly promoted the proliferation and migration of HCT-116 and HT-29 cells. However, PSG9 depletion inhibited the proliferation of SW-480 cells. Using a human umbilical vein endothelial cell tube-forming assay, we found that PSG9 promoted angiogenesis. The overexpression of PSG9 also increased the growth of tumor xenografts in nude mice. Co-immunoprecipitation experiments revealed that PSG9 was bound to SMAD4. The PSG9/SMAD4 complex recruited cytoplasmic SMAD2/3 to form a complex, which enhanced SMAD4 nuclear retention. The PSG9 and SMAD4 complex activated the expression of multiple angiogenesis-related genes (included IGFBP-3, PDGF-AA, GM-CSF, and VEGFA). Together, our findings illustrate the innovative mechanism by which PSG9 drives the progression of CRC and tumor angiogenesis. This occurs via nuclear translocation of PSG9/SMAD4, which activates angiogenic cytokines. Therefore, our study may provide evidence for novel treatment strategies by targeting PSG9 in antiangiogenic cancer therapy.

Circulating Zinc-α2-glycoprotein levels and Insulin Resistance in Polycystic Ovary Syndrome.

The aim of study was to assess the relationship between zinc-α2-glycoprotein (ZAG) and androgen excess with insulin resistance in polycystic ovary syndrome (PCOS) women. 99 PCOS women and 100 healthy controls were recruited. Euglycemic-hyperinsulinemic clamp (EHC) was preformed to assess their insulin sensitivity. Circulating ZAG was determined with an ELISA kit. In healthy subjects, circulating ZAG levels exhibited a characteristic diurnal rhythm in humans, with a major nocturnal rise occurring between midnight and early morning. Circulating ZAG and M-value were much lower in PCOS women than in the controls. In all population, overweight/obese subjects had significantly lower circulating ZAG levels than lean individuals. Multiple linear regression analysis revealed that only M-value and the area under the curve for glucose were independently related factors to circulating ZAG in PCOS women. Multivariate logistic regression analysis showed that circulating ZAG was significantly associated with PCOS even after controlling for anthropometric variables, blood pressure, lipid profile and hormone levels. The PCOS women with high ZAG had fewer MetS, IGT and polycystic ovaries as compared with the low ZAG PCOS women. Taken together, circulating ZAG levels are reduced in women with PCOS and ZAG may be a cytokine associated with insulin resistance in PCOS women.

Krüppel-like factor 14 increases insulin sensitivity through activation of PI3K/Akt signal pathway.

Genome-wide association studies (GWAS) have shown that Krüppel-like factor 14 (KLF14) is associated with type 2 diabetes mellitus (T2DM). However, no report has demonstrated a relationship between KLF14 and glucose metabolism. The aim of this study was to determine whether KLF14 is associated with glucose metabolism and insulin signaling in vitro. The mRNA and protein expressions of KLF14 were determined by Real-time PCR and Western blotting. Glucose uptake was assessed by 2-[(3)H]-deoxyglucose (2-DG) uptake. Western blotting was used to identify the activation of insulin signaling proteins. KLF14 mRNA and protein in fat and muscle were significantly decreased in HFD-fed mice, db/db mice and T2DM patients. Overexpression of KLF14 enhanced insulin-stimulated glucose uptake and the activation of Akt kinase in Hepa1-6 cells. The phosphorylation of insulin receptor (InsR), insulin receptor substrate-1(IRS-1), glycogen synthase kinase-3ß (GSK-3ß) and Akt also elevated significantly by up-regulation of KLF14. KLF14 overexpression in Hepa1-6 cells prevented the inhibition of glucose uptake and Akt phosphorylation induced by high glucose and/or high insulin, or T2DM serum. However, KLF14's ability to increase glucose uptake and Akt activation was significantly attenuated by LY294002, a PI3-kinase inhibitor. These data suggested that KLF14 could increase insulin sensitivity probably through the PI3K/Akt pathway.

[Effects of JAZF1 overexpression on proinflammatory cytokines in hepatocytes induced by palmitic acid].

OBJECTIVE: To investigate the effects of JAZF1 overexpression on the pro-inflammatory cytokines in hepatic steatosis. METHODS: The model of hepatic steatosis was established by incubating hepatocytes with palmitic acid (PA) at 0, 0.125, 0.25, 0.5 and 1 mM dose and for 0, 6, 12, 24 and 48 hours, after which recombinant adenovirus expressing JAZF1 (Ad-JAZF1) was introduced to up-regulate expression. Triglyceride level was measured by GOD. Cell viability was detected by CCK-8. The mRNA and protein expression of TNF-alpha, MCP-1, IL-8 and JAZF1 was examined by RT-PCR, ELISA, and western blotting. RESULTS: The PA-treated hepatocytes showed dose-dependent significant increases in TNF-alpha, MCP-1 and IL-8 mRNA expression for doses up to 0.25 mM; there were no significant increases for the highest doses of 0.5 and 1 mM. The 0.25 mM PA-treated hepatocytes showed time-dependent significant increases in TNF-alpha, MCP-1 and IL-8 mRNA expressions (FTNF-alpha = 26.51, FMCP-1 = 57.20, FIL-8 = 353.85, P less than 0.01), with the maximum level reached at 12 h and followed by a gradual decrease with longer treatment times. JAZF1 mRNA and protein expression was markedly increased in hepatocytes infected with Ad-JAZF1 (P less than 0.01). However, the AP-treated hepatocytes with JAZF1 overexpression showed down-regulation of TNF-alpha, MCP-1 and IL-8 mRNA expression (decreased by 89.69%, 77.68%, and 83.21%, respectively) and secretion (37%, 37% and 41%, respectively, P less than 0.01). CONCLUSION: Stimulation of hepatocytes by the PA fatty acid in vitro promotes mRNA expression of TNF-alpha, MCP-1 and IL-8, but overexpression of JAZF1 inhibits the PA-induced expression and secretion of these factors.