Phosph-Akt1 expression is associated with a favourable prognosis in pancreatic cancer.

INTRODUCTION: Akt, a serine/threonine protein kinase, mediates growth factor-associated cell survival. In several human cancers, including pancreatic cancer, constitutive activation of Akt (phosphorylated Akt, p-Akt) has been observed and may be associated with chemotherapy and radiotherapy resistance. However, there are contradictory viewpoints in p-Akt in pancreatic cancer on prognosis, and the clinical relevance of p-Akt in pancreatic cancer is not well understood. This study aims to investigate the expressions and relevance of Akt and p-Akt1 in pancreatic cancer tissues and their clinical significance. MATERIALS AND METHODS: The expressions of Akt and p-Akt in 74 surgically resected paraffin-embedded pancreatic ductal adenocarcinoma samples and 10 normal pancreatic tissue samples were examined by immunohistochemistry. The associations of their expression with clinicopathological and survival data were analysed. RESULTS: The positive expression rate of Akt and p-Akt1 were 87.8% and 83.8%, respectively, which were remarkably higher then those in normal pancreatic tissue (P <0.05). There was a positive correlation between the expression of Akt and p-Akt1. High p-Akt1 expression correlated with lower T stage (P = 0.004), while Akt was not associated with any clinicopathologic variables. Kaplan-Meier survival analysis revealed that higher expression of Akt, p-Akt1 were respectively correlated with favourable prognosis (16.0[4.7-27.3] vs 9.3[9.0-9.6] months, P = 0.007, and 23.0[12.2-33.8] vs 11.1[7.5-14.7] months, P = 0.004, respectively). Multivariate analysis identified p-Akt1 as a significant independent favourable prognostic factor (HR=0.421, P = 0.010). CONCLUSIONS: These results suggest that high p-Akt1 expression may be a favourable prognostic factor in pancreatic cancer.

Molecular mutations associated with the in vitro passage of virulent porcine reproductive and respiratory syndrome virus.

Mutants of a highly pathogenic, porcine reproductive, and respiratory syndrome virus (PRRSV), JXA1 strain, were prepared by continuous in vitro passage. Genomic sequence comparisons were made between mutants obtained at different passages and the parental strain JXA1. The mutant strain obtained at passage 80 contained a 12 nucleotide insertion and 108 nucleotide mutations that resulted in 45 amino acid changes. Most of these changes (89%) occurred between passage 10 and 45 and were genetically stable for the next 35-70 passages. A comparison of the mutants, their parental strain, and several American PRRSV strains, identified 13 characteristic amino acid changes. These sites, as well as the distinct 12 nucleotide insertion, represent possible genetic markers for the evaluation of live vaccine applications, particularly for additional studies of the safety and potency of live PRRSV vaccines.

[A synthetic peptide selected by bioinformatics inhibits mouse corneal neovascularization].

OBJECTIVE: To identify the active anti-angiogenic region in the amino acid sequence of human apolipoprotein (a) [apo (a)] kringle V (KV), and to evaluate the role of this synthetic peptide on VEGF-induced angiogenesis of mouse cornea in vivo. METHODS: The characterization of the structure and biological activity of the amino acid sequence of apo (a) KV was analyzed using the bioinformatic methods which included sequence alignment, analysis of antigenicity, surface accessibility and hydrophilicity, and then a peptides was selected. The peptide was synthesized with a high efficiency solid-phase method. Corneal neovascularization was induced with a pellet containing 160 ng vascular endothelial growth factor (VEGF) in a mouse corneal micropocket model. 40 C57BL/6 mice (40 eyes) were divided randomly into 4 groups (10 eyes per group). Four kinds of pellets were made containing 160 ng VEGF plus the dose range of 0.0, 0.5, 1.0 and 1.5 microg synthetic peptide for control group, group A, group B and group C, respectively. Neovascularization was observed biomicroscopically on day 7 after the operation, and the corneas were then examined histologically. RESULTS: The result of bioinformatic analysis showed that the peptide contained a majority of conservative residues and possessed fine properties of antigenicity, surface accessibility and hydrophilicity. The synthetic peptide at the doses of 1.0 microg and 1.5 microg showed significant inhibition of mouse corneal neovascularization induced by VEGF in the parameters of vessel length, clock hours and area compared with the control group on day 7 after the operation (P < 0.01). There was no difference in the two doses (1.0 microg and 1.5 microg peptide) in the inhibition of the neovascularization. The dose of 0.5 microg peptide did not show any significant inhibition of the neovascularization compared with the control group (P > 0.05). CONCLUSIONS: The peptide, selected from the amino acid sequence of apo (a) KV by bioinformatics, appears to inhibit VEGF-induced angiogenesis in a mouse corneal micropocket assay in vivo, therefore, the study suggest that this amino acid sequence may locate at the active anti-angiogenic region of apo (a) KV.

[Subretinal transplantation of human fetal lung fibroblasts expressed ciliary neurotrophic factor gene prevent photoreceptor degeneration in RCS rats].

OBJECTIVE: To investigate the efficacy of subretinal transplantation of CNTF gene transfected fibroblasts for preventing photoreceptor degeneration in RCS. METHODS: The human fetal lung fibroblasts with high level expression of CNTF were established by liposome mediated gene transfer and MTX selection. A 5 microl of cell suspension, containing 1 x 10(5) cells, was injected through pars plana of ciliary body into the subretinal space of the right eye at postnatal 4-5 weeks, the left eye was left without injection or injected with PBS as controls. The both eyes were enucleated for histopathological examinations at 2, 4, 6, 8, 10, 12 and 15 weeks following transplantation. RESULTS: The level of CNTF protein (91,046.15 pg/ml) expressed in the transfected cells was determined by sandwich enzyme-linked immunosorbent assay (ELISA). The four of seven eyes examined by light microscopy and the ten of 14 eyes examined by electro microscopy showed rescue effect. The prolonged photoreceptor survival, reduction of apoptotic cells and debris were observed in transplanted eyes in comparison with untreated or sham-injected eyes. CONCLUSION: This study provides the first indication that transplanted human fibroblasts with high level expression of CNTF are able to rescue photoreceptor degeneration in RCS dystrophic rat retina.

[Exogenous advanced glycosylation end products induce diabetes-like vascular dysfunction in normal rats: a factor for occurrence of diabetic retinopathy].

OBJECTIVE: To disclose the relationship between the deposition of advanced glycosylation end products (AGE) in the retinal vascular tissues and damage of retinal vessels in diabetic retinopathy. METHODS: Sixteen SD rats aged 2 months were divided into 4 groups, with 4 rats in each group. Rats in normal group received no treatment. Diabetes was induced by AGE in the diabetes group. Rat serum albumin (RSA, 40 mg/kg weight) was administered daily to healthy non-diabetic rats through tail veins for 2 weeks (RSA group). AGE-modified RSA was injected to rats in another group at the same route and dosage (AGE-RSA group). The number of pericytes in retinal capillary vessels was counted 2 weeks later. RESULTS: After two weeks continuous AGE treatment, the average amount of pericytes of capillary vessel per 10 microscope visual field (x 100 magnification) in AGE group (4.31 +/- 0.34) was significantly less than that of RSA group (5.80 +/- 0.48) (P < 0.01). Meanwhile, in the AGE-RSA group, AGE were identified in the retinal vascular tissues by immunohistochemical staining. CONCLUSION: Injection of exogenous AGE into healthy rats induces vascular changes resembling those find in the diabetic retinopathy. AGE might be one of the independent pathogenic factors in the occurrence of diabetic retinopathy.

[Inhibitation of tumor angiogenesis, growth and metastasis by blocking VEGF paracrine pathway].

Solid tumors require an adequate vascular supply to grow beyond a certain dimension. It is known that formation of new blood vessels in tumor is mediated by unbalanced expression of angiogenic factors and their inhibitors. Among the former, the vascular endothelial growth factor (VEGF) has been assumed prime candidacy as a major positive physiological effector. To investigate the role of VEGF in angiogenesis associated with development of breast cancer, a sense VEGF and an anti-sense VEGF expression plasmids were constructed, and then were introduced into a human breast carcinoma cell line, MCF-7, expressing middle level of endogenous VEGF. Anti-sense VEGF(121) transfected MCF-7 cells that expressed reduced constitutive levels of VEGF and showed the same growing potential as untransfected MCF-7 cells in vitro, but it showed longer latency, smaller tumor, slower growth and prolonged survival time compared to parental or sense VEGF(165) transfected MCF-7 cells in vivo. Moreover, the tumors derived from anti-sense VEGF(121) transfected MCF-7 cells characterized by minimal vascularization and extensive necrosis. Finally, mice with primary subcutaneous tumors treated with intratumoral administration of anti-sense VEGF, or the plasmid expressing extracellular domain of the Flk-1 VEGF receptor (sFlk-1) followed by electroporation, showed significant tumor suppression. These results suggest that VEGF plays a major angiogenic role in breast cancer and a strategy, which blocks the VEGF paracrine pathway, may provide a means to control tumor growth topically without the risk of systemic antiangiogenesis.

[A novel model for visualization of tumor cell-induced angiogenesis in vivo].

The bladder transitional cell carcinoma cell line, BTT739 from the T739 mouse, was transfected with a plasmid that encoded an enhanced green fluorescence protein (GFP) and the cells stably expressing GFP were selected and subcloned. 1 x 10(3)-1 x 10(4) GFP-labeled BTT739 cells were injected under the skin of ear of T739 mice. On day 2-5 post injection, the most interesting manifestations observed were the chemotaxis-like movement of the tumor cells toward the pre-existing host vasculature, host vessel dilation and tortuosity and increased extravasation. On day 10 or later, the sprout from pre-existing host vasculature was observed. Once angiogenesis was triggered on, the tumor cells grew more rapidly and exhibited a specific growth pattern where tumor cells always associated with or surrounded the vessels. The newly formed microvessels always showed heavy extravasation. Immunohistochemistry staining revealed strong VEGF and VEGFR2 (Flk-1) expression in tumor cells. Angiography using Rhodamin-labeled dextran showed neovascularization with unprecedented clarity. However, the tumor mass, even bigger than 2 mm and being neovascularized, shrunk and then disappear in 3-5 days and left only delicated host vessels and recovered extravasation. The evidence from this observation indicated that angiogenesis induced by tumor cells after implantation into the host begins at very early stage. The micrometastases foci could not form or survive without vigorous and continuous angiogenesis. Furthermore, there was active VEGF paracrine and autocrine expression in tumor and high level VEGF secretion by tumor cells plays an important role in initiating angiogenesis and supporting micrometastases.