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Heart failure is associated with sympatho-βAR (β-adrenoceptor) activation and cardiac fibrosis. Gal-3 (galectin-3) and K Ca 3.1 channels that are upregulated in diverse cells of diseased heart are implicated in mediating myocardial inflammation and fibrosis. It remains unclear whether Gal-3 interacts with K Ca 3.1 leading to cardiac fibrosis in the setting of βAR activation. We tested the effect of K Ca 3.1 blocker TRAM-34 on cardiac fibrosis and inflammation in cardiac-restricted β 2 -TG (β 2 AR overexpressed transgenic) mice and determined K Ca 3.1 expression in β 2 -TG×Gal-3 −/− mouse hearts. Mechanisms of K Ca 3.1 in mediating Gal-3 induced fibroblast activation were studied ex vivo. Expression of Gal-3 and K Ca 3.1 was elevated in β 2 -TG hearts. Gal-3 gene deletion in β 2 -TG mice decreased K Ca 3.1 expression in inflammatory cells but not in fibroblasts. Treatment of β 2 -TG mice with TRAM-34 for 1 or 2 months significantly ameliorated cardiac inflammation and fibrosis and reduced Gal-3 level. In cultured fibroblasts, Gal-3 upregulated K Ca 3.1 expression and channel currents with enhanced membrane potential and Ca 2+ entry through TRPV4 (transient receptor potential V4) and TRPC6 (transient receptor potential C6) channels leading to fibroblast activation. In conclusion, βAR stimulation promotes Gal-3 production that upregulates K Ca 3.1 channels in noncardiomyocyte cells and activates K Ca 3.1 channels in fibroblasts leading to hyperpolarization of membrane potential and Ca 2+ entry via TRP channels. Gal-3–K Ca 3.1 signaling mobilizes diverse cells facilitating regional inflammation and fibroblast activation and hence myocardial fibrosis.

The opening of endothelial small-conductance calcium-activated potassium channels (K

Heart failure is associated with sympatho-βAR (β-adrenoceptor) activation and cardiac fibrosis. Gal-3 (galectin-3) and K

Chronic islet inflammation is associated with development of type 2 diabetes mellitus (T2DM). Intermediate-conductance calcium-activated K

Background Cardiac fibrosis is a core pathological process associated with heart failure. The recruitment and differentiation of primitive fibroblast precursor cells of bone marrow origin play a critical role in pathological interstitial cardiac fibrosis. The K C a 3.1 channels are expressed in both ventricular fibroblasts and circulating mononuclear cells in rats and are upregulated by angiotensin II . We hypothesized that K C a 3.1 channels mediate the inflammatory microenvironment in the heart, promoting the infiltrated bone marrow–derived circulating mononuclear cells to differentiate into myofibroblasts, leading to myocardial fibrosis. Methods and Results We established a cardiac fibrosis model in rats by infusing angiotensin II to evaluate the impact of the specific K C a 3.1 channel blocker TRAM ‐34 on cardiac fibrosis. At the same time, mouse CD 4 + T cells and rat circulating mononuclear cells were separated to investigate the underlying mechanism of the TRAM ‐34 anti–cardiac fibrosis effect. TRAM ‐34 significantly attenuated cardiac fibrosis and the inflammatory reaction and reduced the number of fibroblast precursor cells and myofibroblasts. Inhibition of K C a 3.1 channels suppressed angiotensin II –stimulated expression and secretion of interleukin‐4 and interleukin‐13 in CD 4 + T cells and interleukin‐4– or interleukin‐13–induced differentiation of monocytes into fibrocytes. Conclusions K C a 3.1 channels facilitate myocardial inflammation and the differentiation of bone marrow‐derived monocytes into myofibroblasts in cardiac fibrosis caused by angiotensin II infusion.

Background Cardiac fibrosis is a core pathological process associated with heart failure. The recruitment and differentiation of primitive fibroblast precursor cells of bone marrow origin play a critical role in pathological interstitial cardiac fibrosis. The KC a3.1 channels are expressed in both ventricular fibroblasts and circulating mononuclear cells in rats and are upregulated by angiotensin II. We hypothesized that KC a3.1 channels mediate the inflammatory microenvironment in the heart, promoting the infiltrated bone marrow–derived circulating mononuclear cells to differentiate into myofibroblasts, leading to myocardial fibrosis. Methods and Results We established a cardiac fibrosis model in rats by infusing angiotensin II to evaluate the impact of the specific KC a3.1 channel blocker TRAM‐34 on cardiac fibrosis. At the same time, mouse CD4+ T cells and rat circulating mononuclear cells were separated to investigate the underlying mechanism of the TRAM‐34 anti–cardiac fibrosis effect. TRAM‐34 significantly attenuated cardiac fibrosis and the inflammatory reaction and reduced the number of fibroblast precursor cells and myofibroblasts. Inhibition of KC a3.1 channels suppressed angiotensin II–stimulated expression and secretion of interleukin‐4 and interleukin‐13 in CD4+ T cells and interleukin‐4– or interleukin‐13–induced differentiation of monocytes into fibrocytes. Conclusions KC a3.1 channels facilitate myocardial inflammation and the differentiation of bone marrow‐derived monocytes into myofibroblasts in cardiac fibrosis caused by angiotensin II infusion.

The opening of endothelial small-conductance calcium-activated potassium channels (KCa2.3) is essential for endothelium-dependent hyperpolarization (EDH), which predominantly occurs in small resistance arteries. Adenosine monophosphate-activated protein kinase (AMPK), an important metabolic regulator, has been implicated in regulating endothelial nitric oxide synthase activity. However, it was unclear whether AMPK regulated endothelial KCa2.3-mediated EDH-type vasodilation. Using bioinformatics analysis and myograph system, we investigated the regulation by AMPK of KCa2.3 in human umbilical vein endothelial cells (HUVECs) or mouse second-order mesenteric resistance arteries. In HUVECs, AMPK activation either by activators (AICAR, A769662 and MK-8722) or expression of the constitutively active form of AMPK significantly upregulated KCa2.3 expression. Such effects were abolished by AMPK inhibitor (compound C) or AMPK α1-/α2-siRNA, extracellular-signal-regulated-kinase 5 (ERK5) inhibitor (ERK5-IN-1), and specific siRNA to myocyte-enhancer factor 2 (MEF2) or kruppel-like factor 2/4 (KLF2/4). KCa2.3 expression was significantly reduced in mesenteric resistance arteries in AMPKα2 knockout mice when compared with littermate control mice. Furthermore, in high-fat diet fed mice, 2-week treatment with AICAR restored endothelial KCa2.3 expression in mesenteric resistance arteries with improved endothelial dysfunction. Our results demonstrate that activation of AMPK upregulates KCa2.3 channel expression through the ERK5-MEF2-KLF2/4 signaling pathway in vascular endothelium, which contributes to benefits through KCa2.3-mediated EDH-type vasodilation in mesenteric resistance arteries.

The endothelial-to-mesenchymal transition (EndoMT) is a cellular process featuring decreased expression of endothelial marker genes but increased expression of mesenchymal marker genes. The EndoMT is involved in endothelial dysfunction and the pathogenesis of atherosclerosis. To investigate the dynamic expression of EndoMT genes in vascular endothelial cells under atheroprotective pulsatile shear stress (PS) and atheroprone oscillatory shear stress (OS), we analyzed RNA sequencing data from multitimepoint shear-stress experiments. This unbiased analysis involving next-generation sequencing confirmed that PS and OS had an opposite effect in regulating EndoMT genes. Further experimental validations with H2O2 and gain- and loss-of-function approaches indicated that reactive oxygen species are involved in OS-induced EndoMT, whereas AMP-activated protein kinase and sirtuin-1 could inhibit OS-induced EndoMT. Furthermore, compared with PS, OS increased the DNA methylation of the promoter regions of von Willebrand factor, CD31, and cadherin 5 genes but decreased that of cadherin 2, fibroblast-specific protein 1, and vimentin. The translational implication of the present study builds on the ability of the antidiabetic drug metformin and cholesterol-lowering drug atorvastatin to suppress the EndoMT in cultured endothelial cells and in mouse aortas. NEW & NOTEWORTHY Our RNA sequencing data provided a genome-wide and unbiased view of the shear stress regulation of the endothelial-to-mesenchymal transition (EndoMT) in the endothelium. Furthermore, epigenetic regulation (e.g., DNA methylation) is a key mechanism involved in shear stress-regulated EndoMT. The translational implication of this study is that cardiovascular medications such as statins and metformin have similar beneficial effects as that of atheroprotective flow by mitigating EndoMT.

To improve the existing human papillomavirus type 16 (HPV16) virus-like particle (VLP) preparation, the Drosophila inducible/secreted expression system, a highly efficient, economical method, was used to produce HPV16 VLPs. Drosophila Schneider-2 cells were cotransfected with pMT/BiP/V5-His expression vector containing the target gene encoding HPV16L1 protein without nucleus localization sequence and the selection vector pCoHygro plasmids at the ratio of 4:1. The stabled hygromycin-resistant cell line was obtained 1 month later, and the protein expression was induced by copper sulfate. The molecular mass of expressed HPV16L1 protein was 66 kDa, as revealed by SDS-PAGE, and confirmed by Western blot analysis. The yield of HPV16L1 protein was 0.554 mg per 1x10(7) cells. The characteristics of HPV16L1 protein were further analyzed by mouse erythrocyte hemagglutination assay, hemagglutination inhibition assay, and transmission electron microscopy. Results showed that the truncated protein was as biologically active as natural HPVL1 protein, inducing murine erythrocyte agglutination and VLP formation. These findings indicate that the Drosophila inducible/secreted expression system is promising as a convenient and economical method for the preparation of HPV16 VLP vaccine.

To generate type specific and conformation dependent monoclonal antibodies against human papillomavirus 16 major capsid L1 protein (HPV16 L1).HPV16 L1 protein was expressed by modified insect-baculovirus expression system and purified by ProBond(TM); purification system in nature conditions. BALB/c mice were immunized with the purified HPV16 L1 protein. Hybridoma technique was used to prepare the hybridoma cell lines. Positive colonies were screened by ELISA. The non-denaturing electrophoresis and Western blot methods were used for detecting the conformation dependent antibodies. Immunocytochemistry was used to confirm the type specific of the antibodies. The sub-class, isotype and titer were also identified respectively.One hybridoma cell line named 2E3 was obtained which can constantly secret type-specific and conformation-dependent HPV16 L1 mAb. The titers of the antibody are 1:800 in the cell culture medium and 1:12 800 in ascetic fluid. The sub-class and isotype were IgG1kappa. The antibody could selectively recognize non-denaturing HPV16 L1 protein and there was no cross-reaction with HPV18, HPV58 and HPV11 L1 protein.One cell line which secretes type specific and conformation dependent HPV16 L1 monoclonal antibody is successfully generated. This monoclonal antibody can be used in study HPV16 L1 protein epitope.

To analyze retrospectively the quantity and activation status of the tumor infiltrating cytotoxic lymphocytes in breast cancer and the draining lymph nodes, and its relation to the clinical pathological significance.Seventy-four breast cancer samples with their corresponding axillary lymph nodes were histologically typed and staged. Cytotxic lymphocytes were analyzed by immunohistochemistry with the monoclonal antibodies against CD8, CD56, granzyme B and perforin.The number of infiltrating CD8(+) T cells in the cancerous interstitial tissue were much higher than that in the tumor parenchyma. Compared with the metastatic tumor samples, the CD8(+) T cells were more intensive in the primary tumors (35.7 +/- 16.0 vs. 23.7 +/- 9.6). The tumor infiltrating CD8(+) T cells of patients with 5 years survivals were more than that of the dead cases in this follow-up series death (32.9 +/- 14.1 vs. 20.1 +/- 9.9). There was no significant difference of activated tumor infiltrating cytotoxic T cell analyzed by using the activation marker granzyme B(+) and there was also no significant correlation between the intensity of CD8(+), CD56(+) cells and the clinicopathological stages. However, percentages of the activated cytotoxic lymphocytes in Stage I groups were significantly higher than those in stage III and IV. Moreover, the number of perforin(+) cells was significantly less than that of granzyme B(+) cells, particularly in the cancerous tissue, indicating a dysfunctional status of tumor infiltrating cytotoxic lymphocytes.Activated cytotoxic lymphocytes may play a significant role against the tumor progression and is associated with a favorable prognosis to some extent. However, a putative dysfunctional status of cytotoxic lymphocytes at tumor site may compromise the host immunity against cancer.

The targeting of tumor cells by cytotoxic T lymphocytes is a promising strategy for biotherapy, but T cells require 2 signals via the T-cell receptor - CD3 complex and CD28 molecules for activation. To bridge the gap between cytotoxic T lymphocytes and tumor cells, our objective in this study was to describe the construction and the cell surface-anchored expression of a fusion protein, anti-CD3 scFv-B7.1, derived from inserting a fusion gene encoding anti-CD3 scFv and the extra-cellular domain of B7.1 fused by the splicing by overlap extension method into a mammalian expression vector, pDisplay. Transfection of the recombinant vector by electroporation into HeLa cells resulted in the production of protein migrating at approximately 57 kDa under reducing conditions. The expressed fusion protein could bind to T lymphocytes and induce strong T-cell activation. Meanwhile, a potent cytotoxicity was induced in the mixed culture of T-cell-modified tumor cells in a 96 h methyl-thiazolyl-diphenyl tetrazolium bromide assay. Our results indicate that this bifunctional protein, through activating T lymphocytes to lyse homologous human carcinomas, may be of potential value for T-cell-based immunotherapeutical treatment protocols in vivo.

To observe the distribution of immunocompetent cells (ICCs) in tumor's local draining lymph nodes (LDLN) at different stages of disease including non-metastasis, micro-metastasis and late metastasis.71 LDLN from 22 breast cancer, 28 LDLN from 7 gastric carcinoma were analysed by using catalyzed signal amplification (CSA) immunohistochemical staining. Monoclonal antibodies (mAbs) to perforin, granzyme B, CD8, CD56, CD68, S-100, CD134 and CD25 were used to detect the amount and functional change of ICCs.Paracortical hyperplasia and sinus histocytosis was mainly observed in tumor's LDLN. As it was reported, the density of S100(+) dendritic cells (DCs) was decreased as the draining lymph nodes became tumor-containing from the status of tumor free (P0.05), and the morphology of DCs turned to be inactivated. As the lymph nodes were invaded by tumors, the densities of CD134(+) lymphocytes and CD25(+) cells were significantly increased (P0.01). Furthermore, there was a significant trend of decreasing in the number of activated cytotoxic T lymphocytes (granzyme B(+) cells) in LDLN as tumor progressed from non/micro-metastasis, early metastasis to advanced stage metastasis (P0.01). There was no obvious difference in distribution of ICC between non/micro-metastasis sentinel lymph nodes and non-sentinel lymph nodes.The dynamic change of the ICCs in LDLN implied that immune response of tumor bearing host tends to be inhibited with the progress of tumors.

To evaluate the immunopotentiation of novel adjuvant SWZY on the cancer vaccine of poorly immunogenic melanoma.C57BL/6 mice immunized with inactivated D5 melanoma cells were divided into 6 groups: the control without adjuvants, with FCA, FCA+IL-2+GM-CSF, FIA+IL-2+GM-CSF, FIA+SWZY, and FIA+SWZY+IL-2+GM-CSF. Three days after completion of immunization, DTH response, specific killing activity of splenocytes and the level of IFN-gamma and IL-10 in serum and splenocytes' culture supernatant were assayed in half of the mice in each group. The rest were subject to live D5 tumor cell challenge. Three weeks after tumor inoculation, the same immunological parameters were measured.DTH response, splenocytes' killing activity of all the experimental groups were markedly higher than those in the control group (P0.05), but decreased as the tumor grew larger. The level of IFN-gamma in serum or splenocyte culture supernatant of each experimental group was significantly higher than that of the control (P0.05) before tumor formation, whereas IL-10 level was lower than that of the control (P0.05). After tumor formation, the level of IFN-gamma in all groups decreased while that of IL-10 increased in general, but the levels of IFN-gamma and IL-10 in group FCA and FIA+SWZY didn't change very much.All kinds of adjuvants can enhance the cell-mediated immune response against poorly immunogenic tumor to some extent. The novel adjuvant SWZY can strengthen immunoresponses similar to FCA. With few harmful side-effect, SWZY might be a promising adjuvant for cancer vaccine.

To conduct a proteomic analysis of human colorectal carcinoma cell lines LS174T and SW480 by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).The total proteins of human colorectal carcinoma cell lines LS174T and SW480 were separated with 2-DE using immobilized pH gradient strips and analyzed by MALDI-TOF-MS to obtain peptide mass fingerprints (PMFs). Proteins were identified by using Mascot software to search protein databases.Good resolution 2-DE maps were obtained. Some proteins including immunoglobulin heavy chain variable region (IgVH) and co-stimulatory molecule B7-1 were identified. IgVH and B7-1 were confirmed by electrospray ionization tandem spectrometry (ESI-MS/MS) and immunocytochemistry.There are IgVH and B7-1 expressions in human colorectal carcinoma cell lines LS174T and SW480. Results obtained will help to elucidate the mechanisms of tumor immune escape.

Using Non-small cell lung cancer (NSCLC) as subject, to explore the characteristics of immune response in immunological microenvironment at the tumor site and its effect on anti-tumor immunity.Using in situ hybridization (ISH), the expressions of IL-2, INF-gamma, IL-12 (p40), IL-18, IL-4, IL-10, TGF-beta1, IL-1, IL-3, IL-8, GM-CSF, TNF-alpha and TGF-alpha mRNAs in lymphocytes and tumor cells from five fresh pleural effusion samples and 18 tumor tissue samples of NSCLC patients were detected by in situ hybridization with digoxin end-labeled oligonucleotide probe, using 5 tuberculous pleurisy patients as control.In pleural effusion and tumor tissue of NSCLC patients, the expression levels of IL-4, IL-10, TGF-alpha, and TGF-beta1 mRNAs were significantly higher than those of IL-2, IL-12, IL-18 and INF-gamma mRNAs. In contrast, the analysis of tuberculous pleural effusion samples revealed lower levels of above-mentioned cytokine mRNA. There was no significant difference among expression levels of these cytokine mRNAs.The expressions of type II cytokine mRNAs and immunosuppressive cytokine mRNAs in pleural effusion and tumor tissue of NSCLC patients occupied a dominant position, suggesting that the immunological microenvironment about tumor be in an immunosuppressive state. The present study has contributed to a better understanding the mechanisms of tumor escape and provides important experimental basis for working out the scheme for an effective immunomodulatory treatment of NSCLC patients.

To conduct the proteomic analysis of human colorectal carcinoma cell line, SW480 by using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDI-TOFMS).The total proteins of human colorectal carcinoma cell line, SW480 were separated with 2-DE by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysis software, Image Master 2D Elite. Nineteen distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis of the tryptic digest peptides mixture was performed by using MALDI-TOF MS. Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF analysis were used to search NCBI, SWISS-PROT and MSDB databases by using Mascot software.PMF maps of all spots were obtained by MALDI-TOF MS and thirteen proteins were preliminarily identified.The methods of analysis and identification of protein spots of tumor cells in 2-DE gel with silver staining by MALDI-TOF MS derived PMF have been established. Protein expression profile of SW480 has been obtained. It is demonstrated that a combination of proteomics and cell culture is a useful approach to comprehend the process of colon carcinogenesis.

To express anti-CD3 scFv in Hela cells and investigate its biological activity.DNA fragment encoding anti-CD3 scFv was inserted into eukaryotic expression vector pDisplay. The recombinant expression vector was sequenced and then transfected into Hela cells by electroporation method. The expression of anti-CD3 scFv was identified by in situ hybridization. In-vitro T lymphocyte activation was then detected by (3)H-TdR incoporation method. Anti-CD3 scFv gene-transfected Hela cells were co-cultured with T cells and cytotoxicity was measured by MTT colorimetry.Anti-CD3 scFv gene was correctly inserted into pDisplay and expressed in Hela cells. The secreted anti-CD3 scFv was able to activate T lymphocytes in the presence of anti-CD28 mAb. Cytotoxicity could be observed when anti-CD3 scFv gene-transfected Hela cells were mixed and co-cultured with T lymphocytes.Anti-CD3 scFv expressed by Hela cells can activate T lymphocytes.

To construct an eukaryotic expression plasmid pcDNA3.1/hIL-18 and express it in mammalian cells.cDNA encoding mature hIL-18 was cleavaged by enzyme digestion from mesomeric clone vector pGEM-T/hIL-18 and inserted into an eukaryotic expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pcDNA3.1/hIL-18. Then the constructed plasmid was transfected into COS-7 and Rlc310 cells by liposome-mediated gene transfer method. hIL-18 expressed in transfected COS-7 and Rlc310 cells was detected by immunohistochemical staining and level of hIL-18 mRNA in transfected Rlc310 cells was assayed by RT-PCR.A recombinant eukaryotic expression plasmid pcDNA3.1/hIL-18 was successfully constructed and expressed transiently in COS-7 cells and stably in Rlc310 cells.The construction and expression of pcDNA3.1/hIL-18 have been achieved successfully, which lays a foundation for further research on anti-tumor effect of IL-18.

To explore the immunoregulatory functions of Escherichia coli heat-labil enterotoxin B subunit(LTB) and its possible mechanism.The rLTB in engineering bacteria VSP60 was purified by Sephacryl S-100 gel filtration chromatography. BALB/c mice were immunized nasally with hen egg lysozyme(HEL) alone or in combination with various doses of LTB. After three times of immunization, specific anti-HEL IgG and IgA levels in serum and small intestinal secretory fluid were determined by ELISA. The effects of LTB on alloantigen- and ConA- mediated lymphocyte proliferation reactions were measured by (3)H-TdR incorporation assay.Very weak serum anti-HEL IgG response and no IgA response were detected in mice immunized with HEL alone. However, levels of both serum anti-HEL IgG, IgA and intestinal secretory fluid IgA in mice immunized with various doses of HEL+LTB were higher than those in mice immunized with HEL alone(P0.001). In-vitro, LTB could obviously inhibit the lymphocyte proliferation induced by mitogen and alloantigen.The effects of LTB protein on immnune system may be bi-directional. LTB is not only a powerful mucocal immunoadjuvant in-vivo, but also a potent immunosuppressive agent In-vitro.

The hscIL-12 gene(no signal sequence), amplified by PCR, was subcloned into an expressing vector pMT/Bip/V5. The recombinant plasmid was transferedinto Drosophila cell line S2. The secreted hscIL-12 fusion protein was identified by Western blot, and the bioactivity of hscIL-12 was investigated by lymphocyte proliferation assay. The results showed hscIL-12 expressed in S2 cells could stimulate lymphocyte proliferation in dose-dependent manner. hscIL-12 has bioactivity identical to natural interleukin 12. These findings suggest that hscIL-12 is promising in theoretical reseach and in clinical use.

AIM: To observe the status of tumor-associated B7 molecule mRNA expression in human colorectal cancer tissue by in situ hybridization. METHODS: The mRNA expression patterns of cancer-associated B7-1,B7H1,B7H2,ICOS in 22 specimens of human colorectal cancer tissue were monitored by in situ hybridization (ISH) with digoxin-labeled oligonucleotide probes. RESULTS: B7-1,B7H1,B7H2,ICOS mRNA were detected in both cancer cells and tumor infiltrating lymphocytes (TIL). The mRNA expression level of these molecules in tumor cells was higher than that in TIL (0.76 ± 0.54-1.62 ± 0.82 vs 0.38 ± 0.19-0.65 ± 0.33, P