Reciprocal interaction between macrophages and T cells stimulates IFN-γ and MCP-1 production in Ang II-induced cardiac inflammation and fibrosis.

BACKGROUND: The inflammatory response plays a critical role in hypertension-induced cardiac remodeling. We aimed to study how interaction among inflammatory cells causes inflammatory responses in the process of hypertensive cardiac fibrosis. METHODOLOGY/PRINCIPAL FINDINGS: Infusion of angiotensin II (Ang II, 1500 ng/kg/min) in mice rapidly induced the expression of interferon γ (IFN-γ) and leukocytes infiltration into the heart. To determine the role of IFN-γ on cardiac inflammation and remodeling, both wild-type (WT) and IFN-γ-knockout (KO) mice were infused Ang II for 7 days, and were found an equal blood pressure increase. However, knockout of IFN-γ prevented Ang II-induced: 1) infiltration of macrophages and T cells into cardiac tissue; 2) expression of tumor necrosis factor α and monocyte chemoattractant protein 1 (MCP-1), and 3) cardiac fibrosis, including the expression of α-smooth muscle actin and collagen I (all p<0.05). Cultured T cells or macrophages alone expressed very low level of IFN-γ, however, co-culture of T cells and macrophages increased IFN-γ expression by 19.8±0.95 folds (vs. WT macrophage, p<0.001) and 20.9 ± 2.09 folds (vs. WT T cells, p<0.001). In vitro co-culture studies using T cells and macrophages from WT or IFN-γ KO mice demonstrated that T cells were primary source for IFN-γ production. Co-culture of WT macrophages with WT T cells, but not with IFN-γ-knockout T cells, increased IFN-γ production (p<0.01). Moreover, IFN-γ produced by T cells amplified MCP-1 expression in macrophages and stimulated macrophage migration. CONCLUSIONS/SIGNIFICANCE: Reciprocal interaction between macrophages and T cells in heart stimulates IFN-γ expression, leading to increased MCP-1 expression in macrophages, which results a forward-feed recruitment of macrophages, thus contributing to Ang II-induced cardiac inflammation and fibrosis.

[A multi-gene DNA vaccine encoding non-fusion membrane-anchoring antigen against Schistosoma japonicum].

OBJECTIVE: To construct DNA vaccine (pIRES-Sj97-Sj14-Sj26) and study its immunogenicity and protective immunity against Schistosoma japonicum. METHODS: The plasmid pIRES-Sj97-Sj14-Sj26 containing fatty binding protein (Sj14), GST (Sj26) and paramyosin (Sj97) was constructed and expressed on the membrane. RT-PCR was used to detect the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, and IFA for detecting the expression of trans-membrane Sj14, Sj26 and Sj97. Sixty BALB/c mice were randomly divided into 3 groups. Mice in each group respectively received normal saline, pIRES blank vector and pIRES-Sj97-Sj14-Sj26 by intramuscular injection. Two weeks after the 3rd immunization, 10 mice from each group were sacrificed and total IgG in serum and the level of IFN-y were detected by ELISA and lymphocyte stimulating index (SI) by MTt. FCM was used to analyze the subgroups of splenocytes. The level of NO secreted by peritoneal macrophages was determined by nitrate reductase approaches. The left 10 mice in each group were challenged with (40 +/- 1) cercariae of S. japonicum by abdominal skin penetration. Forty-five days after challenge, mice were sacrificed, and numbers of recovered worms and hepatic eggs were counted. RESULTS: RT-PCR showed the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA. IFA proved the expression of Sj26, Sj14 and Sj97 protein. Level of total IgG in the vaccination group, saline group and pIRES blank vector group was (5.62 +/- 0.64), (1.22 +/- 0.20) and (1.48 +/- 0.36) mg/ml respectively, showing a statistical significance (P < 0.01, P < 0.05). The NO level in macrophages was (321.19 +/- 18.03), (184.12 +/- 11.05) and (213.51 +/- 15.93) nmol/ml in the 3 groups respectively (P < 0.05), and the lymphocyte stimulating index in the 3 groups was (2.25 +/- 0.29), (1.18 +/- 0.07) and(1.22 +/- 0.09) respectively (P < 0.01). The INF-gamma level was higher in the vaccination group than others (P < 0.01). The percentage of CD4+ and CD8+ T cells increased considerably (P < 0.01). The vaccination group showed a worm reduction rate of 39.9% (P < 0.01) and an egg reduction rate of 43.9% (P < 0.01). CONCLUSION: The vaccine candidate pIRES-Sj97-Sj14-Sj26 induces an immune protection in BALB/c mice against Schistosoma japonicum.

[Construction of a secreting prokaryotic shuttle expressing plasmid pBCG-sP-HSP65 and its expression in Mycobacterial smegmatis].

To construct the secretive prokaryotic shuttle expression plasmid pBCG-SP-HSP65, the signal peptide sequence of antigen 85B amplified from Bacillus Calmette-guérin (BCG) genome by PCR and the whole HSP65 DNA sequence of human M. tuberculosis obtained from the plasmid pCMV-MTHSP65 by PCR were cloned into the plasmid pBCG-2100 under the control of the promoter of Heat Shock Protein 70 (HSP70) from human M. tuberculosis. Recombinants were electroporated into Mycobacterial smegmatis and induced by heating. Results of the induced expression were detected by SDS-PAGE and the biological activity of the expressed protein was tested by Western-blot analysis. Results showed pBCG-SP-MTHSP65 was constructed successfully and confirmed by restriction endonuclease analysis, PCR detection and DNA sequencing analysis. After it was electroporated into Mycobacterial smegmatis and induced by heating, the percentage of expressed 65kD protein in Mycobacterial smegmatis detected by SDS-PAGE was 20% in total bacterial protein. But the percentage of expressed 65kD protein in recombibinant Mycobacterial smegmatis was up to 34.46% in total bacterial protein and 68.56% in the total protein of cell lysate supernants, Which demonstrated the recombinant HSP65 gene could express in recombinant with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive proteins could specially combine with antibody against human M. tuberculosis HSP65. Orally, pBCG-SP-HSP65 was successfully constructed; HSP65 gene could express in Mycobacterial smegmatis with high efficiency via it. And the expressed proteins possess the biological activity. So it provids experimental evidence for the application of the recombinant Mycobacterial smegmatis and the development of the vaccine against tuberculosis.

Bombesin stimulates nuclear factor kappa B activation and expression of proangiogenic factors in prostate cancer cells.

The majority of deaths from prostate cancer occur in patients with androgen-insensitive metastatic disease. An important early event in the development of the metastatic phenotype is the induction of genes that promote angiogenesis, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), which are released from tumor cells into their microenvironment. Coincident with progression from prostatic carcinoma in situ to metastatic disease is an increase in the number of tumor cells exhibiting neuroendocrine (NE) differentiation. NE cells express a variety of peptide hormones, including the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP), and its cognate receptor, GRP-R. Although there is a strong positive correlation between the degree of NE differentiation and the metastatic potential of prostate cancers, a mechanistic link between increased expression of peptide hormone receptors, such as GRP-R, and proangiogenic gene expression has not been established. Here we report that BBS stimulates nuclear factor kappa B (NF kappa B) activation and proangiogenic gene expression in the androgen-insensitive prostate cancer cells lines, PC-3 and DU-145. In PC-3 cells, BBS stimulation of GRP-R resulted in the up-regulation of IL-8 and VEGF expression through a NF kappa B-dependent pathway. We show that BBS treatment induced inhibitor of NF kappa B degradation, NF kappa B translocation to the cell nucleus, increased NF kappa B binding to its DNA consensus sequence, and increased IL-8 and VEGF mRNA expression and protein secretion. Treatment with the proteasome inhibitor, MG-132, blocked BBS-stimulated NF kappa B DNA binding, and IL-8 and VEGF expression and secretion. Finally, media collected from PC-3 cell cultures, after BBS treatment, stimulated an NF kappa B-dependent migration of human umbilical vascular endothelial cells in vitro. Together, our data demonstrate a role for BBS and GRP-R in the NF kappa B-dependent up-regulation of proangiogenic gene expression, and suggest a possible molecular mechanism linking NE differentiation and the increased metastatic potential of androgen-insensitive prostate cancers.

Gastrin stimulates cyclooxygenase-2 expression in intestinal epithelial cells through multiple signaling pathways. Evidence for involvement of ERK5 kinase and transactivation of the epidermal growth factor receptor.

Gastrin is a hormone produced by G-cells in the normal gastric antrum. However, colorectal carcinoma cells may aberrantly produce gastrin and exhibit increased expression of cholecystokinin B (CCK-B)/gastrin receptors. Gastrin is trophic for the normal gastric oxyntic mucosa and exerts a growth-promoting action on gastrointestinal malignancy. Thus, gastrin may act as an autocrine/paracrine or endocrine factor in the initiation and progression of colorectal carcinoma. The molecular mechanisms involved have not been elucidated. Hypergastrinemia induced by Helicobacter pylori infection is associated with increased cyclooxygenase-2 (COX-2) expression in gastric and colorectal tissues, suggesting the possibility that gastrin up-regulates COX-2 expression in these tissues; this has not been confirmed. We report here that gastrin significantly increases the expression of COX-2 mRNA and protein, the activity of the COX-2 promoter, and the release of prostaglandin E(2) from a rat intestinal epithelial cell line transfected with the CCK-B receptor. These actions were dependent upon the activation of multiple MAPK signal pathways, including ERK5 kinase; transactivation of the epidermal growth factor receptor; and the increased expression and activities of transcription factors ELK-1, activating transcription factor-2, c-Fos, c-Jun, activator protein-1, and myocyte enhancer factor-2. Thus, our findings identify the signaling pathways coupling the CCK-B receptor with up-regulation of COX-2 expression. This effect may contribute to this hormone-dependent gastrointestinal carcinogenesis, especially in the colon.

Protection of HLE B-3 cells against hydrogen peroxide- and naphthalene-induced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2.

PURPOSE: To investigate the physiological role of two major alpha-class glutathione S-transferases (GSTs), hGSTA1-1 and hGSTA2-2 in protection against oxidative stress and lipid peroxidation (LPO) in human lens epithelial (HLE B-3) cells. METHODS: Total GSTs were purified from HLE B-3 cells by glutathione (GSH)-affinity chromatography and characterized by Western blot analysis, isoelectric focusing, and kinetic studies. The relative contributions of the alpha-class GSTs and the Se-dependent glutathione peroxidase (GPx)-1 in GSH-dependent reduction of phospholipid hydroperoxide (PL-OOH) were quantitated through immunoprecipitation studies using separately the specific polyclonal antibodies against human alpha-class GSTs and GPx-1. HLE B-3 cell membranes were prepared, peroxidized, and used to examine whether hGSTA1-1 and hGSTA2-2 catalyzes the reduction of membrane PL-OOH in situ using the microiodometric and spectrophotometric assays. The protective effects of the alpha-class GSTs against H2O2- and naphthalene-induced LPO and apoptosis were examined by transfecting HLE B-3 cells with cDNAs of hGSTA1 and hGSTA2. RESULTS. HLE B-3 cells expressed only the alpha and pi class GSTs. The Michaelis-Menten constant (k(m)) and turnover number (k(cat)) of purified total GSTs toward phosphatidylcholine hydroperoxide (PC-OOH) were found to be 30 +/- 4 microM and 1.95 +/- 0.26 seconds, respectively. The alpha-class GSTs accounted for approximately 65% of the total GPx activity of HLE B-3 cells toward PC-OOH. Our results demonstrate for the first time that hGSTA1-1 and hGSTA2-2 effectively catalyzed GSH-dependent reduction of membrane PL-OOH in situ in HLE B-3 cells. Transfection with hGSTA1 or hGSTA2 protected these cells from H2O2- and naphthalene-induced LPO and attenuated H2O2- and naphthalene-induced apoptosis through inhibiting caspase 3 activation. CONCLUSIONS: These results demonstrate that the alpha-class GSTs hGSTA1-1 and hGSTA2-2 play a major role as antioxidant enzymes and are the main determinants of the levels of LPO caused by oxidative stress in human lens epithelial cells.