Intermedin alleviates diabetic vascular calcification by inhibiting GLUT1 through activation of the cAMP/PKA signaling pathway.

BACKGROUND AND AIMS: Vascular calcification (VC) is regarded as an independent risk factor for cardiovascular events in type 2 diabetic patients. Glucose transporter 1 (GLUT1) involves VC. Intermedin/Adrenomedullin-2 (IMD/ADM2) is a cardiovascular protective peptide that can inhibit multiple disease-associated VC. However, the role and mechanism of IMD in diabetic VC remain unclear. Here, we investigated whether IMD inhibits diabetic VC by inhibiting GLUT1. METHODS AND RESULTS: It was found that plasma IMD concentration was significantly decreased in type 2 diabetic patients and in fructose-induced diabetic rats compared with that in controls. Plasma IMD content was inversely correlated with fasting blood glucose level and VC severity. IMD alleviated VC in fructose-induced diabetic rats. Deficiency of Adm2 aggravated and Adm2 overexpression attenuated VC in high-fat diet-induced diabetic mice. In vitro, IMD mitigated high glucose-induced calcification of vascular smooth muscle cells (VSMCs). Mechanistically, IMD reduced advanced glycation end products (AGEs) content and the level of receptor for AGEs (RAGE). IMD decreased glucose transporter 1 (GLUT1) levels. The inhibitory effect of IMD on RAGE protein level was blocked by GLUT1 knockdown. GLUT1 knockdown abolished the effect of IMD on alleviating VSMC calcification. IMD receptor antagonist IMD17-47 and cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) inhibitor H89 abolished the inhibitory effects of IMD on GLUT1 and VSMC calcification. CONCLUSIONS: These findings revealed that IMD exerted its anti-calcification effect by inhibiting GLUT1, providing a novel therapeutic target for diabetic VC.

Analysis of Bacterial Biofilm Formation in Patients with Malignancy Undergoing Double J Stent Indwelling and Its Influencing Factors.

OBJECTIVE: To analyze the bacterial biofilm (BF) formation in patients with malignancy undergoing double J stent indwelling and its influencing factors. METHODS: A total of 167 patients with malignant tumors who received double J stent indwelling in the hospital from January 2018 to January 2021 were included in the study. The urine and double J stent samples were collected for bacterial identification and observed for BF formation on the surface of the urinary catheter under a scanning electron microscope (SEM). Univariate and multivariate logistic regression analyses were used to analyze the influencing factors of BF. RESULTS: The BF formation rate was 34.73% (58/167). The BF formation rate of positive specimens cultured in urine and double J stent was significantly higher than that of negative ones (P<0.05). Staphylococcus was the main BF bacteria in double J stent and urine culture specimens, followed by Enterococcus, Pseudomonas, Enterobacter, and Acinetobacter. Compared with the non-BF group, the number of viable bacteria in the double J stent and urine and the catheterization time in the BF group rose markedly (P<0.05). Advanced age, chemotherapy, anemia, indwelling time ≥90d, and urinary tract infection were risk factors for BF formation in patients with malignancy undergoing double J stent indwelling (P<0.05). CONCLUSION: There is a high rate of BF formation in patients with malignancy undergoing double J stent indwelling, with Staphylococcus as the dominant species. Treatment requires enhanced urinary catheter management and nutritional status to inhibit BF formation and lower the rate of urinary catheter-related infections.

Intermedin1-53 attenuates atherosclerotic plaque vulnerability by inhibiting CHOP-mediated apoptosis and inflammasome in macrophages.

Atherosclerotic plaque vulnerability and rupture increase the risk of acute coronary syndromes. Advanced lesion macrophage apoptosis plays important role in the rupture of atherosclerotic plaque, and endoplasmic reticulum stress (ERS) has been proved to be a key mechanism of macrophage apoptosis. Intermedin (IMD) is a regulator of ERS. Here, we investigated whether IMD enhances atherosclerotic plaque stability by inhibiting ERS-CHOP-mediated apoptosis and subsequent inflammasome in macrophages. We studied the effects of IMD on features of plaque vulnerability in hyperlipemia apolipoprotein E-deficient (ApoE-/-) mice. Six-week IMD1-53 infusion significantly reduced atherosclerotic lesion size. Of note, IMD1-53 lowered lesion macrophage content and necrotic core size and increased fibrous cap thickness and vascular smooth muscle cells (VSMCs) content thus reducing overall plaque vulnerability. Immunohistochemical analysis indicated that IMD1-53 administration prevented ERS activation in aortic lesions of ApoE-/- mice, which was further confirmed in oxidized low-density lipoproteins (ox-LDL) induced macrophages. Similar to IMD, taurine (Tau), a non-selective ERS inhibitor significantly reduced atherosclerotic lesion size and plaque vulnerability. Moreover, C/EBP-homologous protein (CHOP), a pro-apoptosis transcription factor involved in ERS, was significantly increased in advanced lesion macrophages, and deficiency of CHOP stabilized atherosclerotic plaques in AopE-/- mice. IMD1-53 decreased CHOP level and apoptosis in vivo and in macrophages treated with ox-LDL. In addition, IMD1-53 infusion ameliorated NLRP3 inflammasome and subsequent proinflammatory cytokines in vivo and in vitro. IMD may attenuate the progression of atherosclerotic lesions and plaque vulnerability by inhibiting ERS-CHOP-mediated macrophage apoptosis, and subsequent NLRP3 triggered inflammation. The inhibitory effect of IMD on ERS-induced macrophages apoptosis was probably mediated by blocking CHOP activation.

Inhibition of Notch1-mediated inflammation by intermedin protects against abdominal aortic aneurysm via PI3K/Akt signaling pathway.

The Notch1-mediated inflammatory response participates in the development of abdominal aortic aneurysm (AAA). The vascular endogenous bioactive peptide intermedin (IMD) plays an important role in maintaining vascular homeostasis. However, whether IMD inhibits AAA by inhibiting Notch1-mediated inflammation is unclear. In this study, we found Notch intracellular domain (NICD) and hes1 expression were higher in AAA patients' aortas than in healthy controls. In angiotensin II (AngII)-induced AAA mouse model, IMD treatment significantly reduced AAA incidence and maximal aortic diameter. IMD inhibited AngII-enlarged aortas and -degraded elastic lamina, reduced NICD, hes1 and inflammatory factors expression, decreased infiltration of CD68 positive macrophages and the NOD-like receptor family pyrin domain containing 3 protein level. IMD inhibited lipopolysaccharide-induced macrophage migration in vitro and regulated macrophage polarization. Moreover, IMD overexpression significantly reduced CaCl2-induced AAA incidence and down-regulated NICD and hes1 expression. However, IMD deficiency showed opposite results. Mechanically, IMD treatment significantly decreased cleavage enzyme-a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) level. Pre-incubation with IMD17-47 (IMD receptors blocking peptide) and the phosphatidylinositol 3-kinase/protein kinase b (PI3K/Akt) inhibitor LY294002 reversed ADAM10 level. In conclusion, exogenous and endogenous IMD could inhibit the development of AAA by inhibiting Notch1 signaling-mediated inflammation via reducing ADAM10 through IMD receptor and PI3K/Akt pathway.

STAT3 Promotes Schistosome-Induced Liver Injury by Inflammation, Oxidative Stress, Proliferation, and Apoptosis Signal Pathway.

Schistosomiasis is a parasitic helminth disease that can cause organ lesions leading to health damage. During a schistosome infection, schistosome eggs can flow into the liver along the portal vein. Numerous inflammatory cells gather around the eggs, causing granulomas and fibrosis in the liver. In this process, many molecules are involved in the initiation and regulation of the fibrous scar formation. However, the precise molecular mechanisms responsible for the progression of granuloma formation and fibrosis initiation caused by schistosome infection have not been extensively studied. In this study, C57BL/6 wild-type mice and Stat3flox/flox Alb-Cre mice were infected with cercariae of Schistosoma japonicum Liver injury, effector molecule levels, and RNA transcriptome resequencing of liver tissue were detected at 4, 5, and 6 weeks postinfection. We investigated the role of STAT3 (signal transducer and activator of transcription 3) in Schistosoma-induced liver injury in mice. After 6 weeks postinfection, there was obvious liver fibrosis. A sustained pathological process (inflammation, oxidative stress, proliferation, and apoptosis) occurred in S. japonicum-induced liver fibrosis initiation. Meanwhile, we observed activation of the STAT3 pathway in hepatic injury during S. japonicum infection by RNA transcriptome resequencing. Liver deficiency of phospho-STAT3 alleviated infection-induced liver dysfunction, hepatic granuloma formation, and fibrosis initiation. It also promoted STAT3-dependent apoptosis and reduced liver inflammation, oxidative stress, and proliferation. Our results suggest that STAT3 signal pathway and its mediating inflammation, oxidative stress, proliferation, and apoptosis are involved in S. japonicum-induced liver injury and may be a new potential guideline for the treatment of schistosomiasis.

Intermedin alleviates pathological cardiac remodeling by upregulating klotho.

Cardiac remodeling is accompanied by cardiac hypertrophy, fibrosis, dysfunction, and eventually leading to heart failure. Intermedin (IMD), as a paracrine/autocrine peptide, has a protective effect in cardiovascular diseases. In this study, we elucidated the role and the underlying mechanism of IMD in pathological remodeling. Pathological remodeling mouse models were induced by abdominal aorta constriction for 4 weeks or angiotensin II (Ang II) infusion for 2 weeks in wildtype, IMD-overexpression, IMD-knockout and klotho-knockdown mice. Western blot, real-time PCR, histological staining, echocardiography and hemodynamics were used to detect the role of IMD in cardiac remodeling. Cardiac hypertrophy, fibrosis and dysfunction were significantly aggravated in IMD-knockout mice versus wildtype mice, and the expression of klotho was downregulated. Conversely, cardiac remodeling was alleviated in IMD-overexpression mice, and the expression of klotho was upregulated. Hypertension induced by Ang II infusion rather than abdominal aorta constriction was mitigated by IMD. However, the cardioprotective effect of IMD was blocked in klotho-knockdown mice. Similar results were found in cultured neonatal rat cardiomyocytes, which was pretreated with IMD before Ang II stimulation. Mechanistically, IMD inhibited the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the activity of calcineurin to protect against cardiac hypertrophy through upregulating klotho in vivo and in vitro. Furthermore, peroxisome proliferator-activated receptor γ (PPARγ) might mediate IMD upregulating klotho. In conclusion, pathological remodeling may be alleviated by endogenous IMD, which inhibits the expression of calcineurin and p-CaMKII by upregulating klotho via the PPARγ pathway. It suggested that IMD might be a therapeutic target for heart disease.

Intermedin1-53 attenuates aging-associated vascular calcification in rats by upregulating sirtuin 1.

Vascular calcification is a common phenomenon in older adults. Intermedin (IMD) is a cardiovascular bioactive peptide inhibiting vascular calcification. In this study, we aimed to investigate whether IMD1-53 attenuates aging-associated vascular calcification. Vascular calcification was induced by vitamin D3 plus nicotine (VDN) in young and old rats. The calcification in aortas was more severe in old rats treated with VDN than young control rats, and IMD expression was lower. Exogenous administration of IMD1-53 significantly inhibited the calcium deposition in aortas and the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) in VDN-treated old rats. Moreover, levels of aging-related p16, p21 and ß-galactosidase were all greatly decreased by IMD1-53. These results were further confirmed in rat and human VSMCs in vitro. In addition, IMD-deficient mouse VSMCs showed senescence features coinciding with osteogenic transition as compared with wild-type mouse VSMCs. Mechanistically, IMD1-53 significantly increased the expression of the anti-aging factor sirtuin 1 (sirt1); the inhibitory effects of IMD1-53 on calcification and senescence were blocked by sirt1 knockdown. Furthermore, preincubation with inhibitors of PI3K, AMPK or PKA efficiently blunted the upregulatory effect of IMD1-53 on sirt1. Consequently, IMD1-53 could attenuate aging-associated vascular calcification by upregulating sirt1 via activating PI3K/Akt, AMPK and cAMP/PKA signaling.

Linking the low-density lipoprotein receptor-binding segment enables the therapeutic 5-YHEDA peptide to cross the blood-brain barrier and scavenge excess iron and radicals in the brain of senescent mice.

INTRODUCTION: Iron accumulates in the brain during aging, which catalyzes radical formation, causing neuronal impairment, and is thus considered a pathogenic factor in Alzheimer's disease (AD). To scavenge excess iron-catalyzed radicals and thereby protect the brain and decrease the incidence of AD, we synthesized a soluble pro-iron 5-YHEDA peptide. However, the blood-brain barrier (BBB) blocks large drug molecules from entering the brain and thus strongly reduces their therapeutic effects. However, alternative receptor- or transporter-mediated approaches are possible. METHODS: A low-density lipoprotein receptor (LDLR)-binding segment of Apolipoprotein B-100 was linked to the 5-YHEDA peptide (bs-5-YHEDA) and intracardially injected into senescent (SN) mice that displayed symptoms of cognitive impairment similar to those of people with AD. RESULTS: We successfully delivered 5-YHEDA across the BBB into the brains of the SN mice via vascular epithelium LDLR-mediated endocytosis. The data showed that excess brain iron and radical-induced neuronal necrosis were reduced after the bs-5-YHEDA treatment, together with cognitive amelioration in the SN mouse, and that the senescence-associated ferritin and transferrin increase, anemia and inflammation reversed without kidney or liver injury. DISCUSSION: bs-5-YHEDA may be a mild and safe iron remover that can cross the BBB and enter the brain to relieve excessive iron- and radical-induced cognitive disorders.

Intermedin1-53 protects against cardiac hypertrophy by inhibiting endoplasmic reticulum stress via activating AMP-activated protein kinase.

OBJECTIVE: Intermedin (IMD), a novel member of the calcitonin/calcitonin gene-related peptide family, is involved in maintaining circulatory homeostasis and is a protective factor of heart and vessel. Here, we investigated the effects of IMD on cardiac hypertrophy in vivo and in vitro and explored the mechanisms involved. METHODS AND RESULTS: IMD1-53 (100 ng/kg/h) was systemically administered to rats with cardiac hypertrophy induced by abdominal aortic constriction (AAC) by a mini-osmotic pump the next day after surgery continuously for 4 weeks. The AAC-treated rats before IMD infusion showed increased IMD content and expression of its receptors in the hearts. In-vivo administration of IMD1-53 greatly attenuated the cardiac hypertrophy as shown by heart weight to body weight ratio (HW/BW), haemodynamics, echocardiography, histological analyses and expression of hypertrophic markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) induced by AAC. IMD1-53 treatment significantly reduced the myocardial protein expression of endoplasmic reticulum stress (ERS) markers such as glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP) and caspase-12, whereas the protein level of phosphorylated AMP-activated protein kinase (p-AMPK) was upregulated with IMD1-53 treatment, which was further confirmed in cultured cardiomyocytes. Concurrently, cardiomyocyte apoptosis in vivo and in vitro was ameliorated by IMD1-53 treatment. The inhibitory effects of IMD1-53 on ERS and apoptosis were eliminated on pretreatment with compound C, an AMPK inhibitor. CONCLUSION: IMD1-53 could exert its cardioprotective effect on cardiac hypertrophy by inhibiting myocardial ERS and apoptosis, possibly via activation of AMPK signalling.

Peroxisome proliferator-activated receptor γ ligands retard cultured vascular smooth muscle cells calcification induced by high glucose.

Peroxisome proliferator-activated receptor γ (PPARγ) and its ligands have profound effects on glucose homeostasis, cardiovascular diseases, and bone metabolism. To explore the pathophysiological roles of PPARγ in diabetes with concomitant vascular calcification, we investigated changes in PPARγ expression and the effect of the PPARγ ligands troglitazone and rosiglitazone on vascular smooth muscle cell (VSMC) calcification induced by high glucose (HG, 25 mmol/L). Compared with low glucose, HG-induced VSMC calcification, and PPARγ mRNA, protein level was decreased. Troglitazone and rosiglitazone treatment markedly attenuated the VSMC calcification, whereas PPARγ antagonist GW9662 abolished the effect of rosiglitazone on calcification. Pretreatment of VSMCs with rosiglitazone, but not troglitazone, restored the loss of lineage marker expression: the protein levels of α-actin and SM-22α were increased 52 % (P < 0.05) and 53.1% (P < 0.01), respectively, as compared with HG alone. Troglitazone and rosiglitazone reversed the change in bone-related protein expression induced by HG: decreased the mRNA levels of osteocalcin, bone morphogenetic protein 2 (BMP2), and core binding factor α 1 (Cbfα-1) by 26.9% (P > 0.05), 50.0 % (P < 0.01), and 24.4% (P < 0.05), and 48.4% (P < 0.05), 41.4% (P < 0.01) and 56.2% (P < 0.05), respectively, and increased that of matrix Gla protein (MGP) 84.2% (P < 0.01) and 70.0%, respectively (P < 0.05), as compared with HG alone. GW9662 abolished the effect of rosiglitazone on Cbfα-1 and MGP expression. PPARγ ligands can inhibit VSMCs calcification induced by high glucose.

Tissue-specific distribution of fatty acids, polychlorinated biphenyls and polybrominated diphenyl ethers in fish from Taihu Lake, China, and the benefit-risk assessment of their co-ingestion.

The fish tissues from four species collected from Taihu Lake, China, were analyzed including dorsal, ventral, and tail muscles, heart, liver, and kidney. The highest and lowest concentrations of fatty acids were respectively observed in livers and muscles. There were significant intraspecies and interspecies differences in the compositions of most fatty acids among muscle, heart, liver, and kidney. All the tissues were generally beneficial for consumption considering fatty acids. People mainly consume the muscle. Hence, the benefits from two polyunsaturated fatty acids, i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and risks from PCBs and PBDEs via fish consumption were evaluated by calculating the benefit-risk quotient (BFQ) for the intake of fish muscle containing EPA+DHA vs. PCBs or PBDEs. The BFQ values considering carcinogenic and noncarcinogenic effects for PCBs were ∼3000 and 10 times higher than those of PBDEs via fish consumption to achieve the recommended EPA+DHA intake of 250 mg d(-1), respectively. The results also suggested that the risk consuming the dorsal muscle was generally lower than the ventral and tail muscles.

Polycyclic aromatic hydrocarbons and organochlorine pesticides in fish from Taihu Lake: their levels, sources, and biomagnification.

The investigation of biomagnification of polycyclic aromatic hydrocarbons (PAHs) and endosulfan, an organochlorine pesticide (OCP) and a new persistent organic pollutant, has been limited in freshwater food chains. The objective of the present study was to investigate the levels with focus on the sources and biomagnification of PAHs and OCPs in fish from Taihu Lake, China. In 193 samples of 24 species investigated, the concentrations ranged from 289 to 9 500 ng/g lipid weight (lw) for PAHs, and from 121 to 904 ng/g lw for OCPs, indicating that the fish in the lake was moderately contaminated. The PAHs mainly originated from both unburned petroleum and combustion of fossil fuels, and the OCPs from aged residues. It was unlikely that most of the PAHs and OCPs were biodiluted through the food chain because their trophic magnification factors were higher than one nevertheless the P-values >0.05. Aldrin, dieldrin, p,p'-DDE, p,p'-DDD, and endosulfan sulfate were significantly biomagnified through the food chain.

[Level, distribution, and source identification of polychlorinated naphthalenes in surface agricultural soils from an electronic waste recycling area].

The concentration of 46 polychlorinated naphthalenes (PCNs) in the agricultural soils from Luqiao was analyzed by GC-NCI-MS. The objectives of this study were to investigate the contents, spatial distribution and sources of PCNs. The total concentrations of PCNs (sigma PCNs) in soil samples were in the range of 0.062 to 2.92 ng x g(-1), with a mean of 0.630 ng x g(-1). Tetra-CNs and penta-CNs were the predominant homologues in most of the samples, accounting for 18.4% - 88.8% and 8.40% - 53.1%, with average values of 46.7% and 30.7%, respectively, followed by tri-CNs, accounting for 0 - 47.3%, with a mean of 10.6%. Cluster analysis and combustion marker analysis showed that the sampling sites were mainly polluted by Halowax 1014 and Halowax 1013, also possibly polluted by PCBs mixtures and e-waste combustion process. Compared to other studies, the PCNs concentration in this study was at a medium level.

Diepoxybutane induces the formation of DNA-DNA rather than DNA-protein cross-links, and single-strand breaks and alkali-labile sites in human hepatocyte L02 cells.

1,3-Butadiene (BD) is an air pollutant and a known carcinogen. 1,2,3,4-Diepoxybutane (DEB), one of the major in vivo metabolites of BD, is considered the ultimate culprit of BD mutagenicity/carcinogenicity. DEB is a bifunctional alkylating agent, being capable of inducing the formation of monoalkylated DNA adducts and DNA cross-links, including DNA-DNA and DNA-protein cross-links (DPC). In the present study, we investigated DEB-caused DNA cross-links and breaks in human hepatocyte L02 cells using comet assay. With alkaline comet assay, it was observed that DNA migration increased with the increase of DEB concentration at lower concentrations (10-200µM); however, at higher concentrations (200-1000µM), DNA migration decreased with the increase of DEB concentration. This result indicated the presence of cross-links at >200µM, which was confirmed by the co-treatment experiments using the second genotoxic agents, tert-butyl hydroperoxide and methyl methanesulfonate. At 200µM, which appeared as a threshold, the DNA migration-retarding effect of cross-links was just observable by the co-treatment experiments. At <200µM, the effect of cross-links was too weak to be detected. The DEB-induced cross-links were determined to be DNA-DNA ones rather than DPC through incubating the liberated DNA with proteinase K prior to unwinding and electrophoresis. However, at the highest DEB concentration tested (1000µM), a small proportion of DPC could be formed. In addition, the experiments using neutral and weakly alkaline comet assays showed that DEB did not cause double-strand breaks, but did induce single-strand breaks (SSB) and alkali-labile sites (ALS). Since SSB and ALS are repaired more rapidly than cross-links, the results suggested that DNA-DNA cross-links, rather than DPC, were probably responsible for mutagenicity/carcinogenicity of DEB.

[Levels, distribution and possible sources of polybrominated diphenyl ethers in river sediments from an electronic waste recycling area].

The concentrations of 41 polybrominated diphenyl ethers (PBDEs) in the river sediments from Luqiao were analyzed by GC-NCI-MS. The objectives of this study were to understand the contents, spatial distribution and sources. The Sigma40 PBDEs (excluding BDE209) concentrations in sediments sampled were in the range of 0.177 to 161 ng x g(-1), with a mean of 22.5 ng x g(-1), and the concentrations of BDE209 were from 0.36 to 958 ng x g(-1), with a mean of 148 ng x g(-1). Deca-BDE was the most predominant in 9 PBDE homologues in most of samples, accounting for 38.4%-96.0%, with an average of 74.4% nona-BDEs and hepta-BDEs, accounting for 3.3%-25.8% and 0.01%-14.1%, respectively. Significant correlations were observed among all of PBDEs congeners, which suggested these PBDEs might have the similar sources. The homologue composition of PBDEs and hierarchical cluster analysis (HCA) showed that most of sampling sites were mainly polluted by deca-BDE formulation, and others polluted by deca-BDE and octa-BDE formulations. Compared to other studies from different countries and regions, the PBDEs concentrations in the present study were at a medium-to-low level. But it should be concerned that a few of sampling sites were highly contaminated by point sources.

[Glycosyl-phosphatidylinositol-anchored interleukin-2 expressed on tumor-derived exosomes induces anti-tumor immune response].

OBJECTIVE: To prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2. METHODS: To construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2. RESULTS: The pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70, intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P<0.05). CONCLUSIONS: GPI-IL-2 gene-modified tumor cells can make the exosomes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder transitional cell tumors.

[Preparation of renal cancer vaccine of IL-12-anchored exosomes and its antitumor effect in vitro].

OBJECTIVE: To prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro. METHODS: A mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay. RESULTS: Mammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro. CONCLUSION: A vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.

[Residues of organochlorine pesticides in urban soil of Shanghai].

The 55 soil samples were collected from Shanghai urban areas in March 2007. The residues and distribution characteristics of organochlorine pesticides (OCPs) in the soil samples were investigated with gas chromatography. The results showed that HCHs, DDTs, and HCB were in the ranges of nd-38.58 microg x kg(-1), 1.81-79.61 microg x kg(-1) and 0.16-40.25 microg x kg(-1), respectively. The total OCPs concentrations in urban soil of Shanghai ranged from 3.12 microg x kg(-1) to 91.07 microg x kg(-1) with a mean of 22.33 microg x kg(-1), and the p,p'-DDE took over 60% of the total OCPs. The main contaminated areas were distributed in the park and greenbelts. The composition of OCPs indicated that OCPs in soil samples mainly came from historical application. Compared to the reference data, the pollution burden in soil of Shanghai was lower than those in other areas of China and in German, Argentina and Poland.

Interleukin-12-anchored exosomes increase cytotoxicity of T lymphocytes by reversing the JAK/STAT pathway impaired by tumor-derived exosomes.

Tumor-derived exosomes express tumor antigens, leading to their promising utility as tumor vaccines, but they also can suppress T-cell signaling molecules and reduce cytotoxic effects. We investigated whether interleukin-12 (IL-12)-anchored exosomes (EXO/IL-12) reverse tumor exosome-mediated inhibition of T-cell activation and cytotoxicity was associated with inhibition of JAK3 and p-STAT5. A co-expression plasmid of pBudCE4.1/IL-12A/ IL-12B-GPI was constructed. EXO/IL-12 was identified by transmission electron microscopy and Western blotting, which induced proliferation and cytotoxicity of T-cells and were analyzed by CFSE-based flow cytometry. Expression of JAK2, JAK3 and p-STAT5 was detected by Western blotting. Our results showed that EXO/IL-12 was much more efficient in induction of the proliferation, release of IFN-gamma and cytotoxic effect of T lymphocytes than conventional exosomes in vitro. Exosomes inhibited the expression of JAK3 and phosphorylation of STAT5 in high doses in T-cells, but not JAK2, while EXO/IL-12 had much less attenuated reduction of the expression of p-STAT5. The enhanced cytotoxic effects of T lymphocytes might partly depend on EXO/IL-12 reversing the suppressed expression of p-Stat5 by Jak2/Stat5 pathway. These findings might provide an alternative approach for developing exosomes into tumor vaccines.

[Indoor deposition flux and congener profiles of polybrominated diphenyl ethers (PBDEs) in Shanghai, China].

Indoor dry deposition of eight homes and offices in the urban area of Shanghai, China were sampled with clean glass plate during July to August of 2008 to study the indoor deposition flux and congener profiles of polybrominated diphenyl ethers (PBDEs). 16 PBDEs congeners which including BDE-17, -28, -71, 47, -66, -77, -100, -99, -85, -118, -154, -153, -138, -183, -190 and BDE-209 were measured by GC-MS with negative chemical ionization (NCI) in selected ion monitoring (SIM) mode. The particulate deposition flux of PBDEs in homes and offices were (10.9 +/- 8.2) and (14.2 +/- 11.9) ng x (m2 x d)(-1) respectively. Deca-BDE (BDE-209) was the major compound, accounting for 88.2% -99.2% of the quantified PBDEs. The particulate deposition flux in the offices [(3.1 +/- 2.0) mg x (m2 x d)(-1)] was relatively lower than that of homes, but the concentration of PBDEs in the particles [(3361.6 +/- 1987.4) ng x g(-1)] was significantly higher than that of homes [(1169.1 +/- 647.1) ng x g(-1)]. The concentration of PBDEs in the indoor dry deposition of Shanghai ranked in the middle level comparing with other cities around the world. The indoor deposition flux of PBDEs was mainly correlated with the flux of particle deposition and the usage of electrical and electronic products, but not the interior decoration and the amount of furniture.