A Novel Strategy of US3 Codon De-Optimization for Construction of an Attenuated Pseudorabies Virus against High Virulent Chinese Pseudorabies Virus Variant

In this study, we applied bacterial artificial chromosome (BAC) technology with PRVΔTK/gE/gI as the base material to replace the first, central, and terminal segments of the US3 gene with codon-deoptimized fragments via two-step Red-mediated recombination in E. coli GS1783 cells. The three constructed BACs were co-transfected with gI and part of gE fragments carrying homologous sequences (gI+gE’), respectively, in swine testicular cells. These three recombinant viruses with US3 codon de-optimization ((PRVΔTK&gE-US3deop−1, PRVΔTK&gE-US3deop−2, and PRVΔTK&gE-US3deop−3) were obtained and purified. These three recombinant viruses exhibited similar growth kinetics to the parental AH02LA strain, stably retained the deletion of TK and gE gene fragments, and stably inherited the recoded US3. Mice were inoculated intraperitoneally with the three recombinant viruses or control virus PRVΔTK&gEAH02 at a 107.0 TCID50 dose. Mice immunized with PRVΔTK&gE-US3deop−1 did not develop clinical signs and had a decreased virus load and attenuated pathological changes in the lungs and brain compared to the control group. Moreover, immunized mice were challenged with 100 LD50 of the AH02LA strain, and PRVΔTK&gE-US3deop−1 provided similar protection to that of the control virus PRVΔTK&gEAH02. Finally, PRVΔTK&gE-US3deop−1 was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 106.0 TCID50. Immunized piglets showed only slight temperature reactions and mild clinical signs. However, high levels of seroneutralizing antibody were produced at 14 and 21 days post-immunization. In addition, the immunization of PRVΔTK&gE-US3deop−1 at a dose of 105.0 TCID50 provided complete clinical protection and prevented virus shedding in piglets challenged by 106.5 TCID50 of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRVΔTK&gE-US3deop−1 displays great potential as a vaccine candidate

Secondary infection of Fasciola gigantica in buffaloes shows a similar pattern of serum cytokine secretion as in primary infection

BackgroundAs a natural host of Fasciola gigantica, buffalo is widely infected by F. gigantica. Its impact on buffalo production has caused great losses to the husbandry sector, and repeat infection is non-negligible. In buffaloes experimentally infected with F. gigantica, primary and secondary infection have yielded the same rate of fluke recovery, indicating a high susceptibility of buffalo to F. gigantica, which contributes to the high infection rate. Determining the immunological mechanism of susceptibility will deepen the understanding of the interaction between F. gigantica and buffalo. Here, we explored the immune response of buffaloes against primary and secondary F. gigantica infection, with a focus on cytokines’ dynamics explored through serum cytokine detection.MethodsBuffaloes were assigned to three groups: group A (noninfected, n = 4), group B (primary infection, n = 3), and group C (secondary infection, n = 3). Group B was infected via oral gavage with 250 viable F. gigantica metacercariae, and group C was infected twice with 250 metacercariae at an interval of 4 weeks. The second infection of group C was performed simultaneously with that of group B. Whole blood samples were collected pre-infection (0 weeks) and at 1–6, 10, and 12  weeks after that. The serum levels of seven cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-13, TGF-β, and IL-17) were simultaneously determined using ELISA and further analyzed.ResultsIn the present study, no significant changes in Th1-type cytokines production were detected in early infection, both in primary and secondary infections, while the Th2-type response was strongly induced. A comparison of primary and secondary infection showed no significant difference in the cytokine secretion, which may indicate that the re-infection at 4 weeks after primary infection could not induce a robust adaptive immune response. The full extent of interaction between buffalo and F. gigantica in re-infection requires further study

AMPK Phosphorylates and Inhibits SREBP Activity to Attenuate Hepatic Steatosis and Atherosclerosis in Diet-Induced Insulin-Resistant Mice

SummaryAMPK has emerged as a critical mechanism for salutary effects of polyphenols on lipid metabolic disorders in type 1 and type 2 diabetes. Here we demonstrate that AMPK interacts with and directly phosphorylates sterol regulatory element binding proteins (SREBP-1c and -2). Ser372 phosphorylation of SREBP-1c by AMPK is necessary for inhibition of proteolytic processing and transcriptional activity of SREBP-1c in response to polyphenols and metformin. AMPK stimulates Ser372 phosphorylation, suppresses SREBP-1c cleavage and nuclear translocation, and represses SREBP-1c target gene expression in hepatocytes exposed to high glucose, leading to reduced lipogenesis and lipid accumulation. Hepatic activation of AMPK by the synthetic polyphenol S17834 protects against hepatic steatosis, hyperlipidemia, and accelerated atherosclerosis in diet-induced insulin-resistant LDL receptor-deficient mice in part through phosphorylation of SREBP-1c Ser372 and suppression of SREBP-1c- and -2-dependent lipogenesis. AMPK-dependent phosphorylation of SREBP may offer therapeutic strategies to combat insulin resistance, dyslipidemia, and atherosclerosis

LKB1/AMPK and PKA Control ABCB11 Trafficking and Polarization in Hepatocytes.

Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation

Impact of Visual Field Testing on Intraocular Pressure Change Trends in Healthy People and Glaucoma Patients

Purpose. To compare the impact of visual field (VF) testing on intraocular pressure (IOP) change trends between healthy subjects and glaucoma patients. Methods. We recruited healthy volunteer subjects who did not have previous ocular diseases and open-angle glaucoma patients who were medically controlled well. IOP in both eyes of each participant was measured by using a noncontact tonometer at five time points: before, immediately after (0 minute), and 10, 30, and 60 minutes after the standard automated perimetry. Repeated measures ANOVA was used to analyze the effect of VF testing on IOP change trends in healthy and glaucoma eyes. Results. Forty healthy subjects (80 eyes) and 31 open-angle glaucoma patients (62 eyes) were included for the study. The baseline IOP of healthy and glaucoma eyes was 16.11 ± 3.01 mmHg and 15.78 ± 3.57 mmHg, respectively. After the VF testing, the IOP in healthy eyes was decreased by 1.5% at 0 minute, 6.5% at 10 minutes (P<0.001), 6.6% at 30 minutes (P<0.001), and 7.0% at 1 hour (P<0.001), indicating that this reduction was sustained for at least 1 hour. However, the IOP in glaucoma eyes was increased by 12.7% at 0 minute (P<0.001) and, then, returned towards initial values 1 hour after the VF testing. Conclusions. IOP change trends after VF field testing between healthy subjects and glaucoma patients were quite different. VF testing led to a mild and relatively sustained IOP decrease in healthy subjects, whereas IOP in open-angle glaucoma patients tended to significantly increase immediately after VF testing and, then, returned to pretest values after 1 hour. These findings indicate that the factors of VF testing should be considered in the clinical IOP measurements

Dysregulated CXCL12 expression in osteoblasts promotes B-lymphocytes preferentially homing to the bone marrow in MRL/lpr mice

AbstractObjective: Todetect the abnormal distribution of B-lymphocytes between peripheral and bone marrow (BM) compartments and explore the mechanism of abnormal chemotaxis of B-lymphocytes in lupus subjects. Methods: The proportions of CXC chemokine receptor (CXCR)4+ B cells and CFDA-labeled MRL/lpr-derived B cells were detected by flow cytometry. The levels of CXC chemokine ligand (CXCL)12in peripheral blood (PB)were measured by ELISA. The migrated B cells to osteoblasts (OBs) was measured by transwell migration assay. The relative spatial position of B cells, OBs and CXCL12 was presented by Immunofluorescence assay. Results: Firstly, we found that the percentage of CXCR4+ B cells was lower in PB and higher in the BM from both MRL/lpr mice and patientswith Systemic lupus erythematosus (SLE). Secondly, OBs from MRL/lpr mice produced more CXCL12 than that from C57BL/6 mice. Besides, MRL/lpr-derived OBs demonstrated more potent chemotactic ability toward B-lymphocytes than control OBs by vitro an vivo. Additionally, more B-lymphocytes were found to co-localize with OBs within the periosteal zone of bone in MRL/lpr mice. Lastly, the percentages of CXCR4+B cells were found to be negatively correlated with serum Immunoglobulin (Ig) G concentration, moreover, BM CXCL12 levels were found to be positively correlated with SLE disease activity index Score and negatively correlated with serum Complement3 (C3) concentration. Conclusions: our results indicated that there is a shifted distribution of B-lymphocytes between BM and peripheral compartments in both SLE patients and MRL/lpr mice. Besides, the up-regulated levels of CXCL12 in OBs was indicated to contribute to the enhanced chemotactic migration and anchorage of B-lymphocytes to OBs