Expression and characterization of a soluble VEGF receptor 2 protein

Objective: To clone and express a truncated, soluble vascular endothelial growth factor receptor 2 (sVEGFR2) possessing the combined-functional domains 1-3 and 5 in eukaryotic cells and to test the inhibitory effects of full length VEGFR2 in vivo. Results: pCMV6-trunctated-rVegfr2 (6100 bp) was successfully cloned. The transfection experiments showed that either pCMV6-truncated-rat-Vegfr2 (pCMV6-truncated-rVegfr2) or pCMV6-rVegfr2 inhibited the expression of intracellular green fluorescent protein, which is usually used as an exogenous transfected reporter gene to determine the transfected efficiency. An analysis of the transfected cells revealed that the amount of full-length VEGFR2 protein in the pCMV6-truncated-rVegfr2 transfected cells was 20% lower than that in the negative control (non-transfected HEK 293 cells). The differences in test results between the transfected and negative control groups were greatest from 24-30 h after transfection; this period was therefore chosen as optimal for collecting culture supernatants. This analysis was highly sensitive for detecting the amount of sVEGFR2 protein expressed and secreted by the cells, and the sVEGFR2 protein content was found to increase by approximately 26% in the transfected cells compared to that in the negative control cells (68.2% ± 1.7% vs. 41.9% ± 2.9%, P = 0.000) and by 18% compared to the negative control cells (68.2% ± 1.7% vs. 50.0% ± 0.5%, P = 0.003). Propidium iodide and Hoechst staining indicated no significant change in the number of HEK293 cells undergoing apoptosis 6 days after pCMV6-trucated-Vegfr2 transfection, compared to the negative control. Soluble VEGFR2 produced by pCMV6-truncated-rVegfr2 inhibited full-length VEGFR2 protein expression in the cell membrane. Conclusions: This study employed a eukaryotic system to express sVEGFR2. The use of transient transfection technology greatly improved transfect efficiency. Recombinant sVEGFR2 inhibited the effect of endogenous full-length VEGFR2 but was not cytotoxic

Intestinal IgA plasma cells of the B1 lineage are IL-5 dependent

Two lineages of B cells, designated B1 and B2 cells, have been identified based upon their origins, anatomical distribution, cell surface markers, antibody repertoire and self-replenishing potential. B1 cells are maintained by self-renewal of cells resident in the peritoneal cavity (PerC) and they utilize a limited repertoire of germline V-region genes, mostly directed against ubiquitous bacterial antigens such as phosphoryl choline (PC). B2 cells are replenished from bone marrow precursors and use a larger repertoire of immunoglobulin V-region genes. Whereas most immunoglobulin A (IgA) plasma cells in the intestine derive from B2 lineage precursors in the Peyer's patch, a subpopulation of Per C-derived B1 cells populate the intestinal lamina propria where they mature into IgA plasma cells. In previous in vivo studies we have shown that whereas IgA+ B2 cells are interleukin (IL)-6 dependent, B1 cells are IL-6 independent. In view of the in vitro evidence that IL-5 is also involved in IgA expression, in the studies reported here we have used IL-5-deficient mice to evaluate the role of IL-5 in vivo in IgA expression in the gut. The results demonstrate that although total IgA cell numbers are only marginally depressed in IL-5- deficient mice, there is a marked selective depletion of IgA+ cells of the BI lineage in the gut and a corresponding depression in the capacity of these mice to mount an intestinal response to a B1 antigen (PC) but not to a B2 antigen (oralbumin; OVA), reflecting intact B2-derived IgA cell function but a defect in the B1 cell contribution to IgA responses in IL-5 deficient mice. Collectively these data demonstrate differential cytokine regulation of subsets of IgA+ cells in the gut in that IgA+ cells of the B2 lineage are IL-6 dependent but IL-5 independent, but B1-derived IgA+ cells are IL-5 dependent and IL-6 independent.</p

Amelioration of Liver Injury by Continuously Targeted Intervention against TNFRp55 in Rats with Acute-on-Chronic Liver Failure

<div><p>Background</p><p>Acute-on-chronic liver failure (ACLF) is an acute deterioration of established liver disease. Blocking the TNF (tumor necrosis factor)/TNFR (tumor necrosis factor receptor) 1 pathway may reduce hepatocyte apoptosis/necrosis, and subsequently decrease mortality during development of ACLF. We demonstrated that a long-acting TNF antagonist (soluble TNF receptor: IgG Fc [sTNFR:IgG-Fc]) prevented/reduced development of acute liver failure by blocking the TNF/TNFR1 (TNFRp55) pathway. However, it is still unclear if sTNFR:IgG-Fc can inhibit hepatocyte damage during development of ACLF.</p><p>Methodology</p><p>Chronic liver disease (liver fibrosis/cirrhosis) was induced in Wistar rats by repeatedly challenging with human serum albumin (HSA), and confirmed by histopathology. ACLF was induced with D-galactosamine (D-GalN)/lipopolysaccharide (LPS) i.p. in the rats with chronic liver disease. Serum and liver were collected for biochemical, pathological and molecular biological examinations.</p><p>Principal Findings</p><p>Reduced mortality was observed in sTNFR:IgG-Fc treated ACLF rats, consistent with reduced interleukin (IL)-6 levels in serum and liver, as well as reduced hepatic caspase-3 activity, compared to that of mock treated group. Reduced hepatic damage was confirmed with histopathology in the sTNFR:IgG-Fc treated group, which is consistent with reduced Bcl-2 and Bax, at mRNA and protein levels, but increased hepatocyte proliferation (PCNA). This is also supported by the findings that caspase-3 production was up-regulated significantly in ACLF group compared to the mock treated group. Moreover, up-regulated caspase-3 was inhibited following sTNFR:IgG-Fc treatment. Finally, there was up-regulation of hepatic IL-22R in sTNFR:IgG-Fc treated ACLF rats.</p><p>Conclusions</p><p>sTNFR:IgG-Fc improved survival rate during development of ACLF <i>via</i> ameliorating liver injury with a potential therapeutic value.</p></div

The levels of IL-6 in the serum (Figure 3A) and the liver (Figure 3B), IL-22 in the liver (Figure 3C) and IL-22R in the liver (Figure 3D) were detected.

<p>X-axis represents different treatments [the control (n = 6) (white bar), the ACLF liver (n = 8) (black bar), and sTNFR:IgG-Fc treatment (n = 6) (grey bar)]. Y-axis represents the levels of respective cytokine. (** P<0.01, ***P<0.001).</p

Survival rate of the animals treated with ACLF induction (n = 8) (black square), ACLF with sTNFR:IgG-Fc treatment (n = 8) (grey triangle) and control (n = 6) (white circle) with significant difference (p<0.05) (Figure 1A).

<p>Histopathological scores (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g001" target="_blank">Figure 1B</a>). No obvious lesion was detected in the control (n = 6) (white bar), high level of lesion was observed in the ACLF (n = 8) (black bar), and significant reduced lesion was observed in the ACLF+sTNFR:IgG-Fc treatment (n = 8) (grey bar). The corresponding pictures are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g002" target="_blank">Figure 2</a> A–C. ALT level was detected in all three groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g001" target="_blank">Figure 1C</a>). X-axis represents different treatments, Y-axis represents ALT in international units in log scale (IU). As expected, there was constitutive level of ALT in the control (n = 6) (white bar), and ALT was substantially induced in the ACLF liver (n = 8) (black bar), whereas the induced ALT can be inhibited significantly by sTNFR:IgG-Fc treatment (n = 6) (grey bar). Prothrombin time (PT) was determined in the three groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g001" target="_blank">Figure 1D</a>). X-axis represents different treatments, Y-axis represents PT level. As expected, there was constitutive level of PT in the control (n = 6) (white bar), and PT was substantially induced in the ACLF liver (n = 8) (black bar), whereas the induced PT can be inhibited significantly by sTNFR:IgG-Fc treatment (n = 6) (grey bar). (** P<0.01, ***P<0.001).</p

Histopathology of the liver from the control (n = 6) (Figure 2A), ACLF liver (n = 8) (Figure 2B) and ACLF+sTNFR:IgG-Fc treatment (n = 8) (Figure 2 C).

<p>Immunohistochemistry of caspase-3 production in the liver with different treatments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g002" target="_blank">Figure 2D</a>, control), ACLF (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g002" target="_blank">Figure 2E</a>) and ACLF+sTNFR:IgG-Fc treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g002" target="_blank">Figure 2F</a>). Caspase-3 activity detected by the kit (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g002" target="_blank">Figure 2G</a>) in the liver from the different treatments. Quantification of immunohistochemistry of caspase-3⁺ cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g002" target="_blank">Figure 2H</a>) in the liver with different treatments. Y axis represents the relative mRNA expression of Bcl-2, Bax or PCNA compared to that of housekeeping gene GAPDH (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g002" target="_blank">Figure 2</a> I–K). The data are the average with SEM of all the samples in each group. X-axis represents different treatments [the control (n = 6) (white bar), the ACLF liver (n = 8) (black bar), and sTNFR:IgG-Fc treatment (n = 6) (grey bar)]. Y-axis represents levels of caspase-3 activity, caspase-3⁺ cells, or respective gene expression. (** P<0.01, ***P<0.001).</p

Protein levels of Bax, Bcl-2 and PCNA were detected by Western blot (Figure 4A) and quantified individually Bax (Figure 4B), Bcl-2 (Figure 4C) and PCNA (Figure 4D).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068757#pone-0068757-g004" target="_blank">Figure 4B–D</a>: X axis represents the relative gray values of Western blot bands of Bcl-2, Bax or PCNA compared to that of house keeping protein beta-actin.</p

Histopathological score for liver.

<p>Histopathological score for liver.</p