A unique B cell epitope-based particulate vaccine shows effective suppression of hepatitis B surface antigen in mice.

OBJECTIVE: This study aimed to develop a novel therapeutic vaccine based on a unique B cell epitope and investigate its therapeutic potential against chronic hepatitis B (CHB) in animal models. METHODS: A series of peptides and carrier proteins were evaluated in HBV-tolerant mice to obtain an optimised therapeutic molecule. The immunogenicity, therapeutic efficacy and mechanism of the candidate were investigated systematically. RESULTS: Among the HBsAg-aa119-125-containing peptides evaluated in this study, HBsAg-aa113-135 (SEQ13) exhibited the most striking therapeutic effects. A novel immunoenhanced virus-like particle carrier (CR-T3) derived from the roundleaf bat HBV core antigen (RBHBcAg) was created and used to display SEQ13, forming candidate molecule CR-T3-SEQ13. Multiple copies of SEQ13 displayed on the surface of this particulate antigen promote the induction of a potent anti-HBs antibody response in mice, rabbits and cynomolgus monkeys. Sera and purified polyclonal IgG from the immunised animals neutralised HBV infection in vitro and mediated efficient HBV/hepatitis B virus surface antigen (HBsAg) clearance in the mice. CR-T3-SEQ13-based vaccination induced long-term suppression of HBsAg and HBV DNA in HBV transgenic mice and eradicated the virus completely in hydrodynamic-based HBV carrier mice. The suppressive effects on HBsAg were strongly correlated with the anti-HBs level after vaccination, suggesting that the main mechanism of CR-T3-SEQ13 vaccination therapy was the induction of a SEQ13-specific antibody response that mediated HBV/HBsAg clearance. CONCLUSIONS: The novel particulate protein CR-T3-SEQ13 suppressed HBsAg effectively through induction of a humoural immune response in HBV-tolerant mice. This B cell epitope-based therapeutic vaccine may provide a novel immunotherapeutic agent against chronic HBV infection in humans.

Transcriptomic and Metabolomic Analyses Provide Insights into the Formation of the Peach-like Aroma of Fragaria nilgerrensis Schlecht. Fruits.

Fragaria nilgerrensis Schlecht. is a wild diploid strawberry species. The intense peach-like aroma of its fruits makes F. nilgerrensis an excellent resource for strawberry breeding programs aimed at enhancing flavors. However, the formation of the peach-like aroma of strawberry fruits has not been comprehensively characterized. In this study, fruit metabolome and transcriptome datasets for F. nilgerrensis (HA; peach-like aroma) and its interspecific hybrids PA (peach-like aroma) and NA (no peach-like aroma; control) were compared. In total, 150 differentially accumulated metabolites were detected. The K-means analysis revealed that esters/lactones, including acetic acid, octyl ester, δ-octalactone, and δ-decalactone, were more abundant in HA and PA than in NA. These metabolites may be important for the formation of the peach-like aroma of F. nilgerrensis fruits. The significantly enriched gene ontology terms assigned to the differentially expressed genes (DEGs) were fatty acid metabolic process and fatty acid biosynthetic process. Twenty-seven DEGs were predicted to be associated with ester and lactone biosynthesis, including AAT, LOX, AOS, FAD, AIM1, EH, FAH, ADH, and cytochrome P450 subfamily genes. Thirty-five transcription factor genes were predicted to be associated with aroma formation, including bHLH, MYB, bZIP, NAC, AP2, GATA, and TCPfamily members. Moreover, we identified differentially expressed FAD, AOS, and cytochrome P450 family genes and NAC, MYB, and AP2 transcription factor genes that were correlated with δ-octalactone and δ-decalactone. These findings provide key insights into the formation of the peach-like aroma of F. nilgerrensis fruits, with implications for the increased use of wild strawberry resources.

Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells.

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

[The expression of substance P in airway mucosa of guinea pigs with repetitive esophageal stimulation by hydrochloric acid].

OBJECTIVE: To observe the expression of substance P (SP) in the airway mucosa of guinea pigs with repetitive esophageal stimulation by hydrochloric acid (HCL). METHODS: Twenty adult guinea pigs were randomly divided into 2 groups (n = 10 each): (1) The HCL model group: On the day of experimentation, guinea pigs were maintained under ketamine anesthesia. A 5F catheter was inserted orally into the lumen of the middle and lower esophagus. The esophagus of each animal was perfused with HCl-P for 20 min/d for 14 d. (2) The PBS control group: The esophagus of each animal was perfused with PBS instead. The bronchial responsiveness to Ach given intravenously with increasing doses (3.125, 6.25, 12.5, 25, 50, 100 microg/kg) was measured after the last perfusion. The left lung was isolated for pathological examination. Lung sections were stained with hematoxylin and eosin, and other sections were prepared for immunohistochemistry using monoclonal antibodies against SP. RESULTS: In response to increasing doses of ACh, all guinea pigs showed dose-dependent increases in R(L). However, when the dose of ACh was increased to 25 microg/kg, the airway responsiveness increased significantly in the HCl-P model animals compared with the PBS control group (t values = 43.057, 51.410, 57.359 respectively, all P<0.01). The mean gray values of SP decreased significantly in the tracheal epithelia and the distal airway walls of the model group compared with the PBS control group (t values = 3.44, 2.16 respectively, all P<0.01). CONCLUSION: There was airway neurogenic inflammation in guinea pigs with repetitive esophageal stimulation by HCL, which maybe closely related to the pathogenesis of gastro-esophageal reflux disease.

Anti-inflammatory effects of ethanol extract from Kummerowia striata (Thunb.) Schindl on lps-stimulated RAW 264.7 cell.

Kummerowia striata (Thunb.) Schindl has long been used as a fork herb in inflammation-related therapy. This study was undertaken to determine the anti-inflammatory effect of the plant. High performance liquid chromatography (HPLC) was used for evaluating the extract. While dexamethasone (DM) was used as a positive control, the effects of ethanol extract on the production of IL-1beta, IL-6, NO, COX-2 and TNF-alpha, the expression of iNOS mRNA, TNF-alpha mRNA, COX-2 mRNA, protein production of COX-2 and HO-1, NF-kappaB and I-kappaB of LPS-stimulated RAW 264.7 cells were studied by sandwich ELISA, real-time PCR, Western blot analysis and immunocytochemistry assay respectively. The results showed that K. striata (Thunb.) Schindl had a good anti-inflammatory effect on LPS-stimulated RAW264.7 cell. On one hand, it could significantly inhibit the production of IL-1beta, IL-6, NO, TNF-alpha, COX-2 in LPS-stimulated cell than that of single LPS stimulated cell (p < 0.01 or p < 0.05). On the other hand, it could increase the production of IL-10 and HO-1 than that of single LPS intervention cell (p < 0.01 or p < 0.05). Furthermore, the extract also could inhibit the production of NF-kappaB and I-kappaB compared to single LPS stimulated cell. In a word, it suggested that the anti-inflammatory actions of K. striata (Thunb.) Schindl ethanol extract might be due to the down-regulation of IL-1beta, IL-6, NO, TNF-alpha and COX-2 via the suppression of NF-kappaB activation and conversation of I-kappaB production, and another pathway was up regulating the production of IL-10 and HO-1.

[The impact of exercise rehabilitation on left ventricular remodeling and systolic function in acute myocardial infarction patients].

OBJECTIVE: To investigate the influence of rehabilitation on left ventricular remodeling and systolic function in acute myocardial infarction patients. METHODS: Patients meeting the inclusion criteria with uneventful clinical course after a first myocardial infarction were randomly assigned to a 3-month exercise training period (exercise group, n = 35) or a control group (n = 29). Before randomization, all patients underwent NT-proBNP test, 2-dimensional echocardiography, and submaximal exercise test. RESULTS: (1) At baseline, there were no significant differences in NT-proBNP, left ventricular end-diastolic diameter (LVDd) and left ventricular ejection fraction (LVEF) between the exercise and control groups. After 3 months, a significant decrease in NT-proBNP was observed only in the exercise group [from (845.29 +/- 93.48) ng/L to (335.64 +/- 246.14) ng/L, P < 0.05], but not in the control group [from (1091.62 +/- 101.78) ng/L to (1099.83 +/- 168.75) ng/L, P > 0.05) and there was significant difference in NT-proBNP level between the two groups after 3 months (P < 0.01). The LVDd increased in the control group [from (50.9 +/- 5.8) to (52.6 +/- 5.4) mm, P < 0.05] but not in the exercise group [from (50.7 +/- 4.5) to (50.3 +/- 3.9) mm, P > 0.05] and there was significant difference in LVDd between the two groupsafter 3 months (P < 0.05). Conversely, LVEF improved in the exercise group [from (55.0 +/- 8.6)% to (60.0 +/- 8.0)%, P < 0.05] but not in the control group (P > 0.05) and there was significant difference in LVEF between the two groups after 3 months (P < 0.05). (2) NT-proBNP was inversely correlated with LVEF. CONCLUSIONS: Rehabilitation exercise training under instructions based on family condition in the early and recovery stage of AMI can lower the NT-proBNP level, improve ejection fraction, and prevent the increase of left ventricular end-diastolic diameter. Therefore, it may reduce unfavorable remodeling response and improve cardiac systolic function hereafter.

Effects of Contrast-Enhanced Ultrasonography in Monitoring Hepatic Microcirculation After Rat Liver Ischemia-Reperfusion Injury.

OBJECTIVES: Our objective was to evaluate the effects of contrast-enhanced ultrasonography in monitoring microcirculation after rat liver ischemia-reperfusion injury. MATERIALS AND METHODS: Male Wistar rats (n = 36) were divided into sham-operated and ischemia-reperfusion groups. Rats in the ischemia-reperfusion groups underwent normothermic liver ischemia for 15 minutes followed by 1, 6, or 24 hours of reperfusion. At different time points, contrast-enhanced ultrasonography was performed to determine peak intensity in monitoring hepatic microcirculation. In addition, serum levels of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor α, and interleukin 1ß levels were measured. Histopathologic changes were also observed. RESULTS: One hour after reperfusion, peak intensity values decreased, and serum levels of alanine aminotransferase, tumor necrosis factor α, and interleukin 1ß increased significantly in the ischemia-reperfusion group compared with the sham-operated group. Histology results showed mild injury. Six hours after reperfusion, peak intensity values decreased continuously, serum levels of alanine aminotransferase, tumor necrosis factor α, and interleukin 1ß decreased, and aspartate aminotransferase levels increased. Histology results showed severe injury compared with 1 hour after reperfusion. Twenty-four hours after reperfusion, peak intensity values increased, alanine aminotransferase and aspartate aminotransferase levels decreased, and histology results showed moderate injury compared with 6 hours after reperfusion. Peak intensity values were negatively correlated to alanine aminotransferase (P < .05; γ = -0.38) and aspartate aminotransferase (P < .01; γ = -0.78) levels. CONCLUSIONS: Microcirculation dysfunction after liver ischemia-reperfusion injury can be monitored by contrast-enhanced ultrasonography. The perfusion of contrast agents negatively correlates to the severity of injuries.

Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation.

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

Epcam, CD44, and CD49f distinguish sphere-forming human prostate basal cells from a subpopulation with predominant tubule initiation capability.

BACKGROUND: Human prostate basal cells expressing alpha-6 integrin (CD49f(Hi)) and/or CD44 form prostaspheres in vitro. This functional trait is often correlated with stem/progenitor (S/P) activity, including the ability to self-renew and induce differentiated tubules in vivo. Antigenic profiles that distinguish tubule-initiating prostate stem cells (SCs) from progenitor cells (PCs) and mature luminal cells (LCs) with less regenerative potential are unknown. METHODOLOGY/PRINCIPLE FINDINGS: Prostasphere assays and RT-PCR analysis was performed following FACS separation of total benign prostate cells based upon combinations of Epcam, CD44, and/or CD49f expression. Epithelial cell fractions were isolated, including Epcam(+)CD44(+) and Epcam+CD44+CD49f(Hi) basal cells that formed abundant spheres. When non-sphere-forming Epcam(+)CD44(-) cells were fractionated based upon CD49f expression, a distinct subpopulation (Epcam(+)CD44(-)CD49f(Hi)) was identified that possessed a basal profile similar to Epcam(+)CD44(+)CD49f(Hi) sphere-forming cells (p63(+)AR(Lo)PSA(-)). Evaluation of tubule induction capability of fractionated cells was performed, in vivo, via a fully humanized prostate tissue regeneration assay. Non-sphere-forming Epcam(+)CD44(-) cells induced significantly more prostate tubular structures than Epcam(+)CD44(+) sphere-forming cells. Further fractionation based upon CD49f co-expression identified Epcam(+)CD44(-)CD49f(Hi) (non-sphere-forming) basal cells with significantly increased tubule induction activity compared to Epcam(+)CD44(-)CD49f(Lo) (true) luminal cells. CONCLUSIONS/SIGNIFICANCE: Our data delineates antigenic profiles that functionally distinguish human prostate epithelial subpopulations, including putative SCs that display superior tubule initiation capability and induce differentiated ductal/acini structures, sphere-forming PCs with relatively decreased tubule initiation activity, and terminally differentiated LCs that lack both sphere-forming and tubule-initiation activity. The results clearly demonstrate that sphere-forming ability is not predictive of tubule-initiation activity. The subpopulations identified are of interest because they may play distinct roles as cells of origin in the development of prostatic diseases, including cancer.