PBS-injected Control Group Research Articles

Sortilin Inhibition Protects Neurons From Degeneration in the Diabetic Retina.

To investigate the level and localization of the multifunctional receptor sortilin in the diabetic retina, as well as the effect of sortilin inhibition on retinal neurodegeneration in experimental diabetes. The localization of sortilin and colocalization with the p75 neurotrophin receptor (p75NTR) and Müller cell (MC) markers were determined using immunofluorescence on retinal sections from human patients with diabetes and streptozotocin-induced diabetic C57BL/6J male mice. In the diabetic mice, levels were further quantified using Western blot and quantitative PCR. Therapeutic studies were performed on diabetic mice using intravitreally injected anti-sortilin antibodies. Neuroprotection was evaluated in vivo by optical coherence tomography and by quantification of retinal ganglion cells (RGCs) in flat mounts. Increased levels of sortilin were observed in human and murine diabetic retinas compared with nondiabetic control retinas. Sortilin was highly localized to retinal MCs, and, notably, colocalization with p75NTR was only seen in diabetic retinas. A remarkable protective effect of sortilin inhibition on inner retinal cells was observed in diabetic mice. At eight weeks after diabetes induction, inner retinal thickness was reduced by 9.7% (-12.7%, -6.6%; P < 0.0001; n = 11-12) in the PBS-injected control group compared with the anti-sortilin injected group. Similarly, the count of RGCs was reduced by 20.5% (-30.8%, -10.2%; P = 0.0009) in the PBS-injected control group compared with the anti-sortilin-injected group. Sortilin is upregulated in the diabetic retina, and sortilin inhibition effectively protects against neuronal loss. Thus sortilin emerges as a novel pharmacological target in diabetic retinal neurodegeneration-an important early event in the pathogenesis of diabetic retinopathy.

Tryptophan and Kynurenine Enhances the Stemness and Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro and In Vivo.

Aging tissues present a progressive decline in homeostasis and regenerative capacities, which has been associated with degenerative changes in tissue-specific stem cells and stem cell niches. We hypothesized that amino acids could regulate the stem cell phenotype and differentiation ability of human bone marrow-derived mesenchymal stromal cells (hBMSCs). Thus, we performed a screening of 22 standard amino acids and found that D-tryptophan (10 μM) increased the number of cells positive for the early stem cell marker SSEA-4, and the gene expression levels of OCT-4, NANOG, and SOX-2 in hBMSCs. Comparison between D- and L-tryptophan isomers showed that the latter presents a stronger effect in inducing the mRNA levels of Oct-4 and Nanog, and in increasing the osteogenic differentiation of hBMSCs. On the other hand, L-tryptophan suppressed adipogenesis. The migration and colony-forming ability of hBMSCs were also enhanced by L-tryptophan treatment. In vivo experiments delivering L-tryptophan (50 mg/kg/day) by intraperitoneal injections for three weeks confirmed that L-tryptophan significantly increased the percentage of cells positive for SSEA-4, mRNA levels of Nanog and Oct-4, and the migration and colony-forming ability of mouse BMSCs. L-kynurenine, a major metabolite of L-tryptophan, also induced similar effects of L-tryptophan in enhancing stemness and osteogenic differentiation of BMSCs in vitro and in vivo, possibly indicating the involvement of the kynurenine pathway as the downstream signaling of L-tryptophan. Finally, since BMSCs migrate to the wound healing site to promote bone healing, surgical defects of 1 mm in diameter were created in mouse femur to evaluate bone formation after two weeks of L-tryptophan or L-kynurenine injection. Both L-tryptophan and L-kynurenine accelerated bone healing compared to the PBS-injected control group. In summary, L-tryptophan enhanced the stemness and osteoblastic differentiation of BMSCs and may be used as an essential factor to maintain the stem cell properties and accelerate bone healing and/or prevent bone loss.

Abstract 252: Adhesion GPCR, Latrophilin-2 Induces Cardiac Differentiation Through CDK5, Contributing to Repair After Myocardial Infarction

There has been an increasing demand for cardiomyocyte (CMC) for research into cardiovascular disease and the toxicology of drug metabolites. We identified a G protein-coupled receptor, latrophilin-2 (Lphn2), as a functional marker for cardiomyogenic lineage cells during both in vitro differentiation from ESCs/iPSCs and in vivo heart development. Lphn2 was selectively expressed on CPCs and CMCs during the differentiation of mouse and human PSCs, and cell sorting with an anti-Lphn2 antibody promoted the isolation of populations highly enriched in CPCs and CMCs. Lphn2 knock-down iPSCs did not express cardiac genes. Heterozygous Lphn2 -KO mice ( Lphn2 +/-) were alive and fertile. Homozygous Lphn2 -/- mice were embryonically lethal and showed underdevelopment of the ventricular myocardium. Interestingly, the hearts of Lphn2 -/- embryos revealed the disrupted conotruncal septation of ventricles at E13.5. Lphn2 induces the expression of the central transcription factors Gata4, Nkx2.5, and Tbx5 in mouse heart development. The amino acid sequence of Lphn2 is highly conserved across humans and mice (95.8% identical). For the purpose of cardiac regeneration, we transplanted PSC-derived Lphn2+ cells into the infarcted heart. Transplantation of Lphn2+ cells significantly reduced the fibrosis area and length compared with those in the PBS-injected control group and Lphn2- cells. Echocardiography revealed significantly small LV dimensions at the systole and diastole, and the LV systolic function was higher in the Lphn2+ cell group than in the control and Lphn2- cell groups. To investigate the molecular mechanism underlying the induction of cardiac differentiation by Lphn2, we used the Phospho Explorer Antibody Array, which encompasses nearly all known signaling pathways. Lphn2-dependent phosphorylation was strongest for CDK5 at Tyr15. We identified CDK5, Src, and P38MAPK as key downstream molecules of Lphn2. Our findings provide a valuable tool for identifying CPCs and CMCs differentiated from PSCs, as well as revealing novel insights into cardiac development. Furthermore, the results of this study could be of potential use in regenerative cell therapy for the restoration of cardiac muscle. Funding Source: This study was supported by "Strategic Center of Cell and Bio Therapy for Heart, Diabetes &amp; Cancer" (HI17C2085) through the Korea Health Industry Development Institute, funded by the Ministry of Health &amp; Welfare, Republic of Korea.

A comparison of novel inactivation methods for production of a vaccine against Streptococcus agalactiae in Nile tilapia Oreochromis niloticus

Disease prevention and control are crucial for the development of intensive commercial aquaculture. For some fish species, several vaccines are available to contain the effects of pathogens. However, in Nile tilapia there are just a few vaccines available. The current study compared the efficacy of three inactivated vaccines against Streptococcus agalactiae; in Nile tilapia. Formaldehyde, hydrogen peroxide and pH manipulation were used to inactivate the bacteria. Nile tilapia fingerlings were distributed into six experimental groups in duplicate: T1: naïve group; T2: sham group (PBS-injected, non-challenged); T3: PBS-injected control group (PBS-injected, challenge-infected); T4: vaccinated with hydrogen peroxide-inactivated vaccine (vaccinated, challenge-infected); T5: vaccinated with formaldehyde-inactivated vaccine (vaccinated, challenge-infected); T6: vaccinated by pH manipulation (vaccinated, challenge-infected). Twenty-eight days after vaccination, groups T3, T4, T5 and T6 were challenged intraperitoneally with 0.1 mL of the bacteria and monitored for 21 days. Additionally, replicates of non-challenged fish were used for determination of total serum protein and anti-Streptococcus agalactiae IgM antibodies. The percentage of survival of the groups T1, T2, T3, T4, T5 and T6 were 100%, 100%, 62.5%, 80%, 75% and 85%, respectively. The group vaccinated by pH manipulation presented slight higher survival when compared to the control group (P > .05). However, there were no differences in survival between vaccinated groups. The relative percentage survival (RPS) in groups T4, T5 and T6 were 46.6%, 33.3% and 60%, respectively. No differences were observed for total serum proteins and anti-Streptococcus. agalactiae IgM antibodies. The results observed in this study indicate equivalent moderate vaccine efficacy between alternative inactivated (hydrogen peroxide and pH), and conventional formaldehyde vaccines, with slight higher values for the pH manipulated vaccine, which suggests that future studies should be performed for each alternative method in order to improve their performance.

A 3D nanofibrous hydrogel and collagen sponge scaffold promotes locomotor functional recovery, spinal repair, and neuronal regeneration after complete transection of the spinal cord in adult rats

Central nervous system neurons in adult mammals display limited regeneration after injury, and functional recovery is poor following complete transection (>4 mm gap) of a rat spinal cord. A novel combination scaffold composed of 3D nanofibrous hydrogel PuraMatrix and a honeycomb collagen sponge was used to promote spinal repair and locomotor functional recovery following complete transection of the spinal cord in rats. We transplanted this scaffold into 5 mm spinal cord gaps and assessed spinal repair and functional recovery using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. The BBB score of the scaffold-transplanted group was significantly higher than that of the PBS-injected control group from 24 d to 4 months after the operation (P < 0.001–0.01), reaching 6.0 ± 0.75 (mean ± SEM) in the transplant and 0.70 ± 0.46 in the control groups. Neuronal regeneration and spinal repair were examined histologically using Pan Neuronal Marker, glial fibrillary acidic protein, growth-associated protein 43, and DAPI. The scaffolds were well integrated into the spinal cords, filling the 5 mm gaps with higher numbers of regenerated and migrated neurons, astrocytes, and other cells than in the control group. Mature and immature neurons and astrocytes in the scaffolds became colocalized and aligned longitudinally over >2 mm, suggesting their differentiation, maturation, and function. The spinal cord NF200 content of the transplant group, analyzed by western blot, was more than twice that of the control group, supporting the histological results. Transplantation of this novel scaffold promoted functional recovery, spinal repair, and neuronal regeneration.

Effects of Prunella vulgaris labiatae extract on specific and non-specific immune responses in tilapia (Oreochromis niloticus).

We examined the effects of Prunella vulgaris Labiatae (P. vulgaris L.) on specific and non-specific immune responses of Nile tilapia, Oreochromis niloticus. The optimal concentration without toxicity of P. vulgaris was determined to 30-40 μg/ml in vitro and 120 μg/100 g of fish in vivo. P. vulgaris significantly elicited an antibody titer compared to FCA or β-glucan. β-glucan plus P. vulgaris group synergistically enhanced antibody production. No significant difference in antibody production was observed between P. vulgaris and P. vulgaris plus β-glucan group. A respiratory burst activity of head kidney (HK) leucocytes of tilapia administered with 300 or 500 μg P. vulgaris was significantly (p < 0.05) enhanced compared with the PBS-injected control group and FCA-treated group. Maximum increase in the NBT reduction value was observed in 500 μg P. vulgaris group but no significant difference was found between 300 and 500 μg P. vulgaris group. The level of serum lysozyme activity was significantly (p < 0.05) higher in the 300 and 500 μg P. vulgaris than 100 μg P. vulgaris and FCA group. The phagocytic activities of HK leucocytes from tilapia administered with 300 and 500 μg P. vulgaris were significantly (p < 0.05) higher than 100 μg P. vulgaris and the control group. P. vulgaris was revealed with a good immunoadjuvant evoking the specific and non-specific immune responses of tilapia.

MHC genes determine fetal susceptibility in a rat model of congenital heart block

ObjectiveCongenital heart block (CHB) develops in fetuses of anti-Ro52 antibody positive women. A recurrence rate of 12–25%, despite the persistance of maternal autoantibodies indicates that there are additional, yet unidentified,...

Blocking the secretion of saliva by silencing the HlYkt6 gene in the tick Haemaphysalis longicornis

Soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) have been identified as the key components of the protein complexes that facilitate vesicle traffic, of which Ykt6 (from Saccharomyces cerevisiae, v-SNARE) is proved to be a multifunctional protein in the membrane fusion. In the present study, a tick homologue of Ykt6 (HlYkt6, predicted 22.6 kDa), was isolated from the ixodid tick Haemaphysalis longicornis. RT-PCR and Western blot analysis indicated that the gene and the encoded protein were expressed ubiquitously in different tissues of the partially fed adult tick. Silencing of the HlYkt6 gene resulted in a significant decrease of the engorged body weight (82.9 ± 26.8 mg vs. 232.17 ± 59.1 mg in the PBS-injected control group and 178.7 ± 57.0 mg in the GFP dsRNA-injected control group) and high mortality of replete ticks (100% in tested group vs. 4.8% in the PBS and 20.4% in GFP dsRNA-injected control groups). Disruption of HlYkt6 mRNA led to the suppression of saliva secretion, and a lower anticoagulant activity of the released liquid from the glands (APTT time: 25.25 ± 1.50 s) than that of the control groups (39.25 ± 0.50 s in the PBS-treated group and 40.0 ± 1.41 s in the GFP dsRNA-treated group). These results suggest the vital role of the HlYkt6 protein in the exocytosis of saliva proteins, the feeding and survival of ticks.

Role of Mycobacterium vaccae in the protection induced by first generation Leishmania vaccine against murine model of leishmaniasis

Various Leishmania antigens showed to induce protection when used with IL-12 as an adjuvant in an animal model of leishmaniasis. Limitations in using IL-12 justify searching for an appropriate adjuvant to accelerate induction of a Th1-type immune response and protection. In this study, the role of Mycobacterium vaccae as an adjuvant mixed with either autoclaved Leishmania major (ALM) or freeze-thawed-killed L. major (KLM) in increasing protection in susceptible and resistant mice was studied. Nineteen groups of BALB/c and 19 groups of C57BL/6 mice, ten mice per group, were immunized three times in 45 days interval with different doses of either KLM or ALM alone or mixed with either BCG or different doses of M. vaccae. Immunized groups of mice and PBS-injected control group were challenged with 2 x 10(6) promastigotes of L. major at the base of the tail. The evolution of the lesion was monitored, and the size of the lesion was measured and recorded weekly. Anti-Leishmania total IgG Ab was titrated before and after challenge. The results showed that immunization of either susceptible or resistant mice with KLM or ALM mixed with low dose of M. vaccae increased protection defined by significantly smaller ulcer size in immunized mice compared with the PBS-injected control group.

861. Neonatal Gene Therapy with a Lentiviral Vector Results in Therapeutic Levels of α-Galactosidase A Correction in Fabry Mice

Fabry diseaseis a lysosomal storage disease caused by a systemic deficiency of α-galactosidase A (α-gal A) activity. Humans affected with Fabry disease respond to enzyme replacement therapy (ERT), however frequent infusions are required, strong antibody responses develop, and the long-term benefits in key organs are unknown or inadequate. Life-long correction of inherited or acquired disorders by a single non-invasive treatment is the ultimate goal of most gene therapy research. One of the strategies toward this aim is neonatal gene transfer. In the case of lysosomal storage disorders such as Fabry disease, this approach could prevent the disease progression at a very early stage before irreversible organ damage occurs. Lentiviral vectors (LVs) are well suited to this application because of their ability to confer stable, long-term expression of transgenes in a broad host range. LVs are also known to be refractory to inactivation by complement in human serum. Here we explore use of single intravenous injection of a VSV-g pseudotyped LV system to deliver the α-gal A cDNA in order to correct Fabry disease. We have synthesized an HIV-1-based LV that encodes the human α\-gal A cDNA and a cell surface marker human CD25 (huCD25) in a bicistronic format. In in vitro studies we first established efficient transduction of Fabry patient fibroblasts and Fabry mouse primary bone marrow stromal cells. High levels of α-gal A activity (up to 69-fold above normal) were detected in cell lysates and secreted into the culture media. In an in vivo study, neonatal Fabry mice were injected intravenously through the temporal vein with 100 μgl of concentrated LV (1 × 107 functional viral particles/ml determined by huCD25 expression in HeLa cells) at 2 to 3 days after birth. The survival rate was 92.3% (12/13) in the treated group, 83.3% (5/6) in the PBS injected control group. No hematological abnormalities or huCD25 expression on ononuclear cells was observed in peripheral blood. In plasma from Fabry mice injected once with the therapeutic LV, we observed that α-gal A activity rose to ~110.0% of normal levels (35.7 ± 34.7%, n = 12) 6 weeks after injection. The persistence of enzyme activity in our mice is encouraging since a previous report of ERT in Fabry mice showed rapid clearance of this enzyme (T1/2 = ~5min) in plasma. Numerous studies are underway to gauge long-term corrective effects mediated by this simple vector delivery method in Fabry mice. Organ α-gal A activity and Gb3 clearance in clinically relevant organs will be assessed. Anti-human α-gal A antibody levels in plasma will be quantified throughout the experiment. Since it is important to know the systemic biodistribution of vector for pre-clinical safety and efficacy assessments, we will further investigate the distribution of transduced cells by tracking the co-expressed cell surface marker huCD25. To our knowledge, this study is the first report of using neonatal LV-mediated gene therapy to achieve long-term correction in any disease model. These results suggest that this facile LV delivery system may offer significant long-term therapeutic advantages for a variety of gene therapy applications.

Mixture of N-carbamoyl- l-glutamate plus l-arginine can protect rats with liver cirrhosis from acute ammonia intoxication

Background/Aims : We earlier reported that N-carbamoyl- l-glutamate (CG) plus l-arginine (Arg) protected normal and 70% hepatectomized rats from intoxication by a lethal or sub-lethal dose of ammonium acetate, respectively. In the present study, the protective effect of these compounds on cirrhotic rats was assessed. Methods : CG plus Arg were administered prior to the injection of a sub-lethal dose of ammonium acetate into dimethylnitrosamine-induced cirrhotic rats. Control rats were given phosphate-buffered saline (PBS) instead of the mixture. The behavior of the rats was monitored until the time of sacrifice. Blood ammonia level, blood urea nitrogen (BUN) and liver carbamoylphosphate synthetase I (CPS I) activity were determined. Results : Pretreatment of rats with the mixture of CG plus Arg could significantly lower the blood ammonia level ( P<0.05), increase the activity of CPS I ( P<0.05), improve abnormal behavior associated with ammonia intoxication ( P<0.05), and increase BUN ( P<0.05), as compared with the PBS-injected control group. There were significantly close correlations between (1) the increase of CPS I activity; (2) the improvement of abnormal behavior; (3) the increase of BUN; and (4) the decrease of the blood ammonia level. Conclusions : A mixture of CG plus Arg could protect rats with liver cirrhosis from acute ammonia intoxication.

HIGH INFLAMMATION AND MILD DEMYELINATION IN THE PERIPHERAL NERVOUS SYSTEM INDUCED BY AN INTRANEURAL INJECTION OF RR INTERLEUKIN-4

To examine the direct effects of IL-4 on peripheral nervous system (PNS) we injected recombinant rat IL-4 (rrIL-4) into the sciatic nerve of normal adult Lewis rats. Histopathological and immunohistochemical observations revealed that 1 day after injection, a large number of macrophages and MHC class II-positive cells appeared within both the perineurium and endoneurium. Only few CD4+and CD8+T cells existed in the endoneurium. From day 4 to day 7, we observed a gradual decline of inflammation, but the number of infiltrates in rrIL-4 injected nerves was significantly higher compared with sterile PBS-injected control group. On the contrary, demyelination affected significantly fewer nerve fibres in the rrIL-4-injected nerves compared with control group on day 7. Intraneural injection of rrIL-4 results in high grade inflammation and mild demyelination in the PNS.

N-Carbamoyl-l-Glutamate Plusl-Arginine Can Protect Ammonia Intoxication in Rats with Reduced Functional Liver Mass

We previously reported the protective effect of N-carbamoyl-L-glutamate plus L-arginine in rats given a lethal dose (LD99.9) of ammonium acetate (Kim, S.et al.,Proc. Natl. Acad. Sci. 69, 3530-3533, 1971; ref.1). The present study was undertaken to find out whether the same compounds could also be effective even after the functional mass of the liver was significantly reduced. Thus, the protective effect of these compounds in 70% partial hepatectomized rats following the injection of sublethal dose of ammonium acetate was assessed. The mixture could significantly decrease blood ammonia level compared with PBS-injected control group. In addition, abnormal behaviors observed in the control rats were significantly improved. The protective effect on the behavioral change seemed to be closely related with their effect on blood ammonia level, showing a strong correlation between the blood ammonia level and the behavioral score. The findings provide a rational basis for the clinical use of N-carbamoyl-L-glutamate plus L-arginine in the prevention and treatment of hyperammonemia encountered in liver diseases.