Development of novel DNA vaccine for VEGF in murine cancer model

We developed DNA vaccine for vascular endothelial growth factor (VEGF), which may provide the therapeutic option instead of anti-VEGF antibody, bevacizumab. Plasmid containing VEGF mini-gene was constructed in the insertion of B-cell epitope of Hepatitis B core protein [HBc-VEGF], which was an epitope carrier. High titer of anti-VEGF antibody was observed in BALB/c mice which were intramuscularly immunized with HBc-VEGF by electropolator. In mice inoculated with colon 26 cells, tumor volume and microvessel density was decreased in HBc-VEGF with a significant prolonged survival. Co-treatment of purified IgG from immunized mice with HBc-VEGF showed in vitro neutralizing activity for VEGF-induced ERK phosphorylation and tube formation in cultured endothelial cells. Furthermore, intravitreally injection of this purified IgG reduced the neovessel formation in the mouse oxygen-induced retinopathy and laser-induced choroidal neovascularization models. These results first provided that DNA vaccine against VEGF possessed the anti-angiogenic effect, leading to prolonged survival in mouse cancer model.Y

Acute neuropharmacological effects of atomoxetine on inhibitory control in ADHD children: A fNIRS study

The object of the current study is to explore the neural substrate for effects of atomoxetine (ATX) on inhibitory control in school-aged children with attention deficit hyperactivity disorder (ADHD) using functional near-infrared spectroscopy (fNIRS). We monitored the oxy-hemoglobin signal changes of sixteen ADHD children (6–14 years old) performing a go/no-go task before and 1.5 h after ATX or placebo administration, in a randomized, double-blind, placebo-controlled, crossover design. Sixteen age- and gender-matched normal controls without ATX administration were also monitored. In the control subjects, the go/no-go task recruited the right inferior and middle prefrontal gyri (IFG/MFG), and this activation was absent in pre-medicated ADHD children. The reduction of right IFG/MFG activation was acutely normalized after ATX administration but not placebo administration in ADHD children. These results are reminiscent of the neuropharmacological effects of methylphenidate to up-regulate reduced right IFG/MFG function in ADHD children during inhibitory tasks. As with methylphenidate, activation in the IFG/MFG could serve as an objective neuro-functional biomarker to indicate the effects of ATX on inhibitory control in ADHD children. This promising technique will enhance early clinical diagnosis and treatment of ADHD in children, especially in those with a hyperactivity/impulsivity phenotype

Effect of Ang II vaccine on SHR.

<p>A) Experimental protocol is shown. Ang II-KLH (Ang II-KLH group, n = 5) or saline (saline group, n = 5) were injected on days 0, 14, and 21 in twenty-four-week old male SHR. The anti-Ang II antibody titer and systolic BP were measured on days 0, 14, and 28. The Ang II-KLH group rats were immunized with 5 µg Ang II-KLH with CFA on day 0 and with 5 µg Ang II-KLH with IFA on day 14 and 21. B) Anti-Ang II antibody titers in the sera of immunized rats on days 0, 14, 28, and that of saline injected rats on day 28 were examined. *<i>P</i><0.01 vs. day 0. C) Systolic BP on days 0, 14, and 28 were shown. The data are expressed as the mean systolic BP ± standard error (SE) of the mean. *<i>P</i><0.05. vs. Saline.</p

Neutralizing antibody production induced by Ang II vaccine.

<p>(A) The antibody titers in the sera of mice immunized with saline, Ang II, KLH or Ang II-KLH (dose: 25 ng/mouse) with or without Freund’s adjuvant (FA) are shown. †<i>P</i><0.001 vs. saline. ELISA was performed using the sera of mice on day 42. (B) The serum antibody titers elicited by various doses (10, 25, 100, 300, 1000 ng/mouse) of Ang II-KLH with Freund’s adjuvant (FA). *<i>P</i><0.05 vs. 10 ng. The titer is expressed as the dilution of serum giving half-maximal binding (optical density: OD 50%) ± SE of the mean. (C) Western blot using anti-phosphorylated ERK (P-ERK) and anti-ERK antibodies. The total protein was extracted from HASMCs treated with 10⁻⁷ M Ang II that was previously incubated with 1 % serum. (D) Promoter activity of <i>c-fos</i> was measured using luciferase activity. HASMCs were transfected with <i>c-fos</i> luciferase reporter gene and stimulated by pre-incubated Ang II (10⁻⁷ M) with 1 % serum of the control or immunized mice for 24 hours. The results from 3 samples are expressed as the mean of the ratio to no stimulation ± SE of the mean. ‘‘Control serum’’ indicates serum from mice immunized by KLH (1 mg, with adjuvant), and ‘‘Immunized serum’’ indicates serum from mice immunized by Ang II-KLH (1,000 ng, with adjuvant). All serum was obtained on day 42. †<i>P</i><0.01.</p

T cell activation by Ang II-KLH, KLH or Ang II.

<p>(A) T cell proliferation was determined by analyzing [³H]-thymidine incorporation. Splenocytes from mice on day 42 were stimulated for 36 hours with Ang II-KLH, KLH, Ang II or angiotensinogen (AGT) at a concentration of 10 µg/ml. The stimulation index is expressed as the ratio of stimulation to no stimulation. The data are expressed as the mean stimulation index ± the standard error of the mean per 10⁶ splenocytes. *<i>P</i><0.001 vs. no stimulation. (B) A representative photograph from the ELISPOT assay. The ELISPOT assay detected splenocytes that produced IL-4 and/or IFN-γ? Splenocytes from mice on day 42 were stimulated for 48 hours with 10 µg/ml Ang II-KLH, KLH, Ang II or angiotensinogen (AGT). (C) The quantification of spots in the ELISPOT assay. The data are expressed as the mean number of spots ± SEM per 10⁶ splenocytes. *<i>P</i><0.001 vs. saline. (a,c) The results are from 6 control mice (saline) and 6 experimental mice (1,000 ng Ang II-KLH with adjuvant).</p

Effect of Ang II vaccine on cardiac remodeling induced by Ang II.

<p>(A) The mouse heart weights are shown. The results are expressed as the mean heart weight per body weight ± the standard error of the mean. (B) The cardiac tissue was stained with Masson trichrome and analyzed for cardiac fibrosis. Representative photographs are shown. The scale bar represents 20 µm. The results are expressed as the mean of the fibrotic area ± SE of the mean. (C) The mRNA levels of collagen type I (collagen I), collagen type III (collagen III), atrial natriuretic factor (ANF) and β-myosin heavy chain (β-MHC) were evaluated using RT-PCR. The results are expressed as the ratio of gene expression in the experimental mice to gene expression in the control mice ± SE of the mean. Control mice were immunized with KLH (1 mg, with adjuvant) and experimental mice were immunized with Ang II-KLH (1,000 ng, with adjuvant). All groups contained 6 to 8 mice on day 56. *<i>P</i><0.05.</p

Histochemical analysis of kidney and heart after vaccination.

<p>(A and B) Kidney (upper panel) or heart with coronary artery (lower panel) was stained with H&E in control (saline) or immunized mice (1,000 ng Ang II-KLH with adjuvant) . The scale bar represents 100 µm in the upper panel or 20 µm in the lower panel. The results were examined (A) at day 42 after vaccination and (B) after Ang II infusion (day 56 after vaccination).</p

Effect of Ang II vaccine on Ang II-induced hypertension.

<p>(A) Systolic BP at steady state and under Ang II infusion on day 49. The control mice were immunized with 1 mg KLH. The experimental mice were immunized with 100ng or 1,000 ng Ang II-KLH. All groups contained 6 to 8 mice. The data are expressed as the mean systolic BP ± standard error (SE) of the mean. †<i>P</i><0.01 *<i>P</i><0.05. (B) The correlation between systolic BP under Ang II infusion and the anti-Ang II antibody titers in the sera of immunized mice. The titer is expressed as the dilution of serum giving half-maximal binding (optical density: OD 50%).</p

Conceptual schematic of the experiment.

<p>(A)Immunization step (Ang II-KLH: an antigen) (Step1) The antigen presenting cells (APCs) phagocytose the Ang II-KLH conjugate and present a T cell epitope of KLH to T cells through the major histocompatibility complex (MHC). T cells recognize it through T cell receptors and become activated. (Step2) B cells specific to Ang II (pentagons) differentiate to plasmacytes and proliferate with the help of activated T cells. Then, B cells produce anti-Ang II antibody. (B)Post-Immunization step (response to Ang II) (Step1) The APCs do not present the T cell epitope of Ang II to T cells. Therefore, T cells do not recognize it and are not activated by Ang II. (Step2) B cells specific to Ang II (pentagons) differentiate and proliferate in response to Ang II. Therefore, Ang II does not stimulate the production of anti-AngII antibody.</p

Antibody production induced by Ang II vaccine.

<p>(A) Anti-Ang II antibody titers in the sera of immunized mice on days 28, 42, 70, and 98 were examined. The results are from 6 immunized mice (100 ng Ang II-KLH with adjuvant). †<i>P</i><0.01 vs. day 28, *<i>P</i><0.01 vs. day 42. (B) Anti-Ang II antibody titer of immunized mice pre-Ang II infusion (day 42) and post-Ang II infusion (day 56). The results are from 8 immunized mice (1,000 ng Ang II-KLH with adjuvant). The titer is expressed as the dilution of serum giving half-maximal binding (optical density: OD 50%) ± SE of the mean. *<i>P</i><0.01 vs. day 42. (C) Anti-Ang II, anti-Ang I, and anti-angiotensinogen (AGT) antibody titers in the sera of immunized mice (1,000??ng Ang II-KLH with adjuvant). The titer is expressed as the dilution of serum exhibiting half-maximal binding (optical density: OD 50%) ± SE of the mean. All groups contained 6 mice. “N.D.” indicates “not detected” †<i>P</i><0.001 vs. AGT, *<i>P</i><0.001 vs. Ang I. (D) The binding ability of the antibodies to native Ang II was examined by competitive ELISA. The 1000-fold diluted serum was pre-incubated with various doses of recombinant Ang II and evaluated for binding to plates coated with Ang II-BSA by means of the optical density (OD) at 450 nm. †<i>P</i><0.001 vs. 0.01 µmol/L.</p