Suppressor of cytokine signaling 1 inhibits apoptosis of islet grafts through caspase 3 and apoptosis-inducing factor pathways in rats.

A significant portion of pancreatic islet grafts can be destroyed by apoptosis, failing to engraft in the early period after transplantation. Recently, we observed that overexpression of suppressor of cytokine signaling 1 (SOCS1) in islet grafts achieved an antiapoptotic effect, prolonging graft survival in a rat transplant model. Caspase 3 is the central executioner caspase that is activated by upstream cascades in a caspase-dependent apoptosis pathway. Apoptosis inducing factor (AIF) is a key protein that can be released from mitochondria, translocating to the nucleus in the caspase-independent apoptosis pathway. In this study, we investigated whether these two pathways were involved in cytoprotection afforded by SOCS1 on islet grafts. We used a chimeric adenovirus vector (Ad5F35-SOCS1) to enhance SOCS1 expression in isolated Sprague-Dawley rat islets, which were transplanted into recipients experiencing streptozotocin-induced diabetes. We analyzed the expressions of active (cleaved) caspase 3 and AIF on islets. The Ad5F35-SOCS1-infected islets with higher SOCS1 expression showed decreased levels of active caspase 3 and intranuclear AIF after treatment with tumor necrosis factor-α and cycloheximide in vitro. The diabetic recipients transplanted with Ad5F35-SOCS1-infected islets showed longer periods of normoglycemia versus recipients transplanted with mock-infected islets (P < .05) due to prolonged graft survival. A histological analysis indicated that the Ad5F35-SOCS1-infected islet grafts displayed decreased caspase 3 activation and AIF translocation (to nucleus) in the early posttransplant period. These results demonstrated that the expression of SOCS1 in islet grafts protected them from apoptosis through caspase 3 dependent and AIF caspase-independent-pathways.

Liposome-mediated transfer can improve the efficacy of islet labeling with superparamagnetic iron oxide.

An in vivo method for islet visualization using magnetic resonance imaging (MRI), using superparamagnetic iron oxide (SPIO), has been described. Herein we have developed a protocol that uses cationic liposomes to increase the efficiency of islet cell labeling by SPIO in vitro. Fresh islet cells were incubated in RPMI-1640 medium, to which had been added a range of concentrations of ferucarbotran, a SPIO contrast agent. At each SPIO concentration, duplicate samples were incubated with versus without addition of cationic liposomes. We measured intracellular iron concentration, cell viability, and insulin-release function after labeling. We observed that the amount of iron bound to islet cells increased as the concentration of added SPIO increased. The presence of liposomes increased labeling efficiency at each SPIO concentration. In vitro MRI confirmed the effect of liposomes to improve labeling efficiency of islet cells by SPIO.

Murine dendritic cells modified with CXCL10 gene and tumour cell lysate mediate potent antitumour immune responses in mice.

The aim of our present study was to estimate the effect of a therapeutic vaccine against tumour based on dendritic cells (DC) vaccine modified with tumour cell lysate and chemokine CXCL10 gene. In this study, mouse bone marrow DC were pulsed with tumour cell (RM-1) lysate and then transfected with a plasmid vector expressing CXCL10 cDNA by DOTAP liposome. The protective and therapeutic effects of the DC vaccine in RM-1 tumour model were assessed (divided into CXCL10/Lysate-DC, CXCL10/DC, pcDNA/Lysate-DC, Lysate-DC, pcDNA-DC, DC and PBS). The DC transfected with CXCL10 gene were capable of synthesizing and secreting CXCL10 chemokine. The highest CTL activity against RM-1 cells was induced in mice immunized with DC vaccine that was modified with RM-1 lysate and CXCL10 gene (CXCL10/Lysate-DC) when compared with its counterpart in mice. The CXCL10/Lysate-DC immunized mice also exhibited resistance to tumour challenge most effectively. In the RM-1 tumour model, immunization of CXCL10/Lysate-DC inhibited the tumour growth most significantly when compared with other groups and the survival time of the mice treated with CXCL10/Lysate-DC was greatly extended. These findings provide a potential strategy to improve the efficacy of DC-based tumour vaccine.

NF-kappaB activation for constitutive expression of VCAM-1 and ICAM-1 on B lymphocytes and plasma cells.

Cytokine stimulation can activate NF-kappaB that triggers inducible expression of E-selectin, VCAM-1 (Vascular Cell Adhesion Molecule-1) and ICAM-1 (Intercellular Cell Adhesion Molecule-1) in endothelial cells. In the previous study, we have shown that B lymphocytes and plasma cells can express E-selectin by constitutive activation of NF-kappaB. Here we show that human B lymphocytes and ARH-77 plasma cells expressed VCAM-1 and ICAM-1 in a cytokine dispensable mechanism. NF-kappaB antagonists could inhibit their expressions in ARH-77 cells. The activities of NF-kappaB for VCAM-1 and ICAM-1 promoters prior to cytokine stimulation were detected in ARH-77 cells using electrophoretic mobility shift assays. Again, NF-kappaB antagonists could abrogate these promoter activities. Taken together, our results demonstrate that NF-kappaB activation is the underlying molecular mechanism for constitutive expression of E-selectin, VCAM-1, and ICAM-1 on human B lymphocytes and plasma cells.

B lymphocytes and plasma cells express functional E-selectin by constitutive activation of NF-kappaB.

E-selectin (CD62E), a cell adhesion molecule for most leukocytes, is known to be expressed exclusively on the cytokine-stimulated endothelial cells mainly by inductive activation of NF-kappaB. Using immunohistochemistry and in situ hybridization, we showed that B lymphocytes and plasma cells in the spleens and lymph nodes from nude mice (T-lymphocyte-deficient), but not from SCID mice (T- and B-lymphocyte-deficient), expressed E-selectin prior to cytokine stimulation. The expression of E-selectin was also confirmed on human B lymphocytes isolated from peripheral bloods. The mouse J774A.1 monocytes could adhere to the marginal zones of mouse spleens in an E-selectin Ab inhibitable manner, suggesting the functional activity of the expressed E-selectin. In addition, ARH-77 cells, a cell line derived from human plasma cells, were found to express E-selectin mRNA and protein and to have a NF-kappaB activity for an E-selectin promoter. NF-kappaB antagonists, such as TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone), dexamethasone and a IkappaBalpha mutant plasmid could inhibit both the NF-kappaB activity and the expression of E-selectin. Transfection with an E-selectin promoter-driven reporter gene construct further verified the E-selectin promoter activity in ARH-77 cells. Again, TPCK, dexamethasone, and the IkappaBalpha mutant plasmid could neutralize this activity. These findings suggest that B lymphocytes and plasma cells can express E-selectin, which is functional for monocytic leukocytes, by a mechanism of constitutive activation of NF-kappaB.

[Application of silks as scaffolds for three-dimensional culture of chondrocytes].

OBJECTIVE: To observe the effects of silks on attachment, shape and function of chondrocytes cultured in vitro. METHODS: The silks from silk worm cocoons were digested by trypsin and coated with polylactic acid to from three dimensional scaffolds for rabbit rib chondrocyte culture. The growth and shape of chondrocytes were observed with phase contrast microscopy, scanning electron microscopy. RESULTS: The chondrocytes were adhered to silks slowly after chondrocytes were seeded into silk scaffolds and cells fixed on silks well 1 or 2 days later. Cells began to proliferate after 3 days and multiplicative growth was observed on the 6th day. Microholes of silk scaffolds were filled with chondrocytes 2 weeks later. Scanning electron microscopy showed that there was a lot of extracellular matrix surrounding cells. CONCLUSION: Silks are ideal for attachment, growth and function maintenance of chondrocytes, and silks can be used as scaffolds for chondrocytes in three dimensional culture.

PSMA mimotope isolated from phage displayed peptide library can induce PSMA specific immune response.

Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein expressed predominantly in prostate secretory acinar epithelium and prostate cancer cells as well as in several extraprostatic tissues. Mouse monoclonal antibody 4G5 specific to the extracellular domain of PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library). Three 4G5-reactive phagotopes were identified. Sequence analysis of isolated clones demonstrated that the interaction motif "VDPA/SK" has high homology to 719-725aa on PSMA. Immunohistochemical staining of the prostate cancer sample with the PSMA-mimic phagotope (mimotope) immunized serum antibodies demonstrate that the mimotope isolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo.