Novel strategy for expression and characterization of rabies virus glycoprotein.

Rabies is a fatal zoonosis which could affect all mammals. Glycoprotein (G protein) from the rabies virus plays an important role in the binding of virus to target cells. However, expression of the G protein with native conformation has been a great challenge for many years. In this study, we solved this problem by replacing the original signal peptide of rabies virus G protein with the one from the heavy chain of human IgG. The expression levels of recombinant G protein dramatically increased from a few µg/L to 50 mg/L in the culture supernatants. The identity of the recombinant G protein was confirmed by western blotting using both 6XHis mAb 6E2 and rabies G protein mAb 7G3. The correct conformation of the recombinant G protein was shown by using rabies virus neutralizing antibodies. In addition, the recombinant G protein had immune-reactivities with mice sera raised against rabies vaccines and vice versa. Taken together, our data suggested that by replacing the signal peptide, the expression level of the G protein with native conformation could be significantly improved. This would help the development of a rabies subunit vaccine, structural studies of rabies G protein, elucidation of the signal pathway of RABV infection.

Down-regulation of miR-301a suppresses pro-inflammatory cytokines in Toll-like receptor-triggered macrophages.

In many types of tumours, especially pancreatic adenocarcinoma, miR-301a is over-expressed. This over-expression results in negative regulation of the target gene of miR-301a, the nuclear factor-κB (NF-κB) repressing factor (NKRF), increasing the activation of NF-κB and production of NF-κB-responsive pro-inflammatory cytokines such as interleukin-8, interferon-ß, nitric oxide synthase 2A and cytochrome oxidase subunit 2 (COX-2). However, in immune cells, mechanisms that regulate miR-301a have not been reported. Similar to tumour cells, Toll-like receptor (TLR) -activated macrophages produce NF-κB-responsive pro-inflammatory cytokines. Therefore, it is of considerable interest to determine whether miR-301a regulates the secretion of cytokines by immune cells. In the present study, we demonstrate that the expression of miR-301a was decreased in TLR-triggered macrophages. Through targeting NKRF, miR-301a affected the activity of NF-κB and the expression of pro-inflammatory genes downstream of NF-κB such as COX-2, prostaglandin E2 and interleukin-6. In addition, when lipopolysaccharide-treated macrophages were simultaneously stimulated with trichostatin A, an inhibitor of histone deacetylases, the expression of miR-301a increased, whereas NKRF and pro-inflammatory cytokine expression decreased. However, further investigation revealed that there was no correlation between the induction of miR-301a and the inhibitory effect of trichostatin A on lipopolysaccharide-induced gene expression in macrophages. In summary, our study indicates a new mechanism by which miR-301a regulates inflammatory cytokine expression in macrophages, which may clarify the regulatory role of microRNAs in immune-mediated inflammatory responses.

RNF19a inhibits antiviral immune response to RNA viruses through degradation of TBK1.

TANK-binding kinase 1 (TBK1) plays a pivotal role in antiviral innate immunity. TBK1 mediates the activation of interferon regulatory factor (IRF) 3, leading to the induction of type I IFNs (IFN-α/ß) and of NF-κB signal transduction following viral infections. TBK1 must be tightly regulated to effectively control viral infections and maintain immune homeostasis. Here, we found that E3 ubiquitin ligase RNF19a mediated K48-linked ubiquitination and proteasomal degradation of TBK1. Specifically, the silence of RNF19a enhanced the production of type I interferons and suppressed RNA viral replication. Our results uncover that RNF19a acts as a negative mediator in the RIG-I signaling pathway to attenuate antiviral immune responses and suggest RNF19a as a potential therapy target in clinical infectious and inflammatory diseases.

Evaluation of Rabies Immunoglobulin Administration Status in China: a Retrospective, Cross-Sectional Study at a Tertiary Hospital in Beijing.

This retrospective cross-sectional single-center study included patients with category III exposure to rabies virus between January and December 2019. Exposure characteristics and clinical data were compared and statistically analyzed between groups who were willing and unwilling to receive the rabies immunoglobin (RIG) injection, and the determinants of its administration were identified by stepwise multivariate logistic regression analyses. In total, 1,757 patients with category III exposure were enrolled: 845 men (48.1%) and 912 women (51.9%; median age: 28 [9-50] years). Among them, 1,297 (73.8%) received the RIG injection (median age: 28 [8-50] years) and 460 (26.2%) refused to receive the injection (median age: 25 [15-48] years). Patients aged 16-25 years (odds ratio [OR] = 3.006, 95% confidence interval [CI] = 1.957-4.619), 26-45 years (OR = 2.940, 95% CI = 2.011-4.298), 46-55 years (OR = 3.647, 95% CI = 2.233-5.959), and above 56 years (OR = 6.660, 95% CI = 4.009-11.062); those with injuries caused by cats (OR = 1.937, 95% CI = 1.476-2.542); and people with scratch (OR = 3.319, 95% CI = 2.510-4.390) and minor (OR = 35.281, 95% CI = 18.524-64.198), and moderate (OR = 12.711, 95% CI = 7.221-22.375) injuries were more likely to refuse injection. The RIG administration level in the settings studied herein was found to be insufficient. Educational and awareness programs on rabies prevention, especially those targeted at people not injured by dogs, people with minor injuries, and the elderly should be considered.

Evaluation of Pilot-Scale Radio Frequency Heating Uniformity for Beef Sausage Pasteurization Process.

Radio frequency (RF) heating has the advantages of a much faster heating rate as well as the great potential for sterilization of food compared to traditional thermal sterilization. A new kettle was designed for sterilization experiments applying RF energy (27.12 MHz, 6 kW). In this research, beef sausages were pasteurized by RF heating alone, the dielectric properties (DPs) of which were determined, and heating uniformity and heating rate were evaluated under different conditions. The results indicate that the DPs of samples were significantly influenced (p < 0.01) by the temperature and frequency. The electrode gap, sample height and NaCl content had significant effects (p < 0.01) on the heating uniformity when using RF energy alone. The best heating uniformity was obtained under an electrode gap of 180 mm, a sample height of 80 mm and NaCl content of 3%. The cold points and hot spots were located at the edge of the upper section and geometric center of the sample, respectively. This study reveals the great potential in solid food for pasteurization using RF energy alone. Future studies should focus on sterilization applying RF energy and SW simultaneously using the newly designed kettle.

Implantation of bFGF-treated islet progenitor cells ameliorates streptozotocin-induced diabetes in rats.

AIM: To examine whether implantation of islet preparation-derived proliferating islet cells (PIC) could ameliorate diabetes in rats. METHODS: PIC were expanded from rat islet preparation by supplementation of basic fibroblast growth factor (bFGF) and implanted into rats with streptozotocin (STZ)-induced diabetes through the portal vein. Body weight and blood glucose levels were measured. Serum insulin levels were measured by radioimmunoassay. The presence of insulin-positive cells was determined by hematoxylin and immunohistochemical staining. RESULTS: Cultured islet cells (CIC) were demonstrated to dedifferentiate in vitro, and the apoptosis ratios reached more than 50% by the 15th day post-isolation. PIC cells treated with bFGF (20 ng/mL) continued growing within 30 days after isolation, and no apoptotic cells were detected. Implantation of PIC into diabetic rats was capable of ameliorating diabetes, in terms of the restoration of euglycemia, weight gain, improved glucose response and elevated serum insulin levels for up to 130 days. Livers derived from PIC-implanted rats were examined for insulin expression and single insulin-positive cells. In addition, most islets of PIC-implanted STZ-induced diabetic rats were intact at 130 days post-transplantation and comparable to those of normal rats. CONCLUSION: Implantation of bFGF-treated proliferating islet cells is a promising cellular therapeutic approach for diabetes.

Rabies virus glycoprotein serology ELISA for measurement of neutralizing antibodies in sera of vaccinated human subjects.

BACKGROUND: Vaccination provides protection against infection by inducing VNAs mainly against RABV surface GP. The measurement of VNAs to RABV is commonly used to assess the level of immunity in humans and animals after vaccination. A VNA titer of  ≥ 0.5 IU/mL of sera indicates adequate response to vaccination. Here, we report the development and validation of a RABV GP serology ELISA kit for semi-quantitative measurement of VNA titers in sera of vaccinated human subjects. METHODS: Using a recombinant RABV GP expressed in mammalian cells as the capture antigen, the ELISA method was established using HuMAb NM57 reference initially and HRIG reference subsequently. The limit of detection (LOD), linear range, reproducibility, and precision of the method were examined. Specificity and sensitivity were established to assess the diagnostic accuracy. RESULTS: RABV GP for ELISA plate coating and optimal dilution of human serum sample was 1 µg/mL and 1:20, respectively. Multiple assays were carried out by different technicians at different laboratories for assay standardization. Using the HRIG reference, the LOD was found to be 0.02-0.06 IU/mL and the linear range was 0.2-10.0 IU/ mL. The inter-assay CVs were in the range of 6.60-10.79%, indicating the reproducibility. None of the 12 known negative human sera, tested positive by ELISA, highlighting the specificity. A total of 415 unknown positive human sera were double-blind tested by the RFFIT and ELISA. The VNA titer cut-off value of ELISA was set at 1.5 IU/mL to ensure no false-positive. The diagnostic specificity and sensitivity were 100% and 91.1%, respectively. CONCLUSIONS: The validation data characterize this ELISA as a suitable method for semi-quantitative measurement of VNA titers in human serum samples to assess vaccination status. The ELISA kit can offer simplicity, speed, low cost and high throughput, making it a practical tool for monitoring the immune response following vaccination.

RhoB induces the production of proinflammatory cytokines in TLR-triggered macrophages.

Toll-like receptors (TLRs) are the primary sensors detecting conserved molecular patterns on microorganisms, thus acting as important components of innate immunity against invading pathogens. Many positive and negative regulators of TLR-triggered signaling have been identified. The Rho GTPase RhoB plays a key role in cell migration, division and polarity; however, the function and regulatory mechanisms of RhoB in TLR ligand-triggered innate immune responses remain to be investigated. Here, we report that the expression of RhoB is induced by TLR agonists (lipopolysaccharide (LPS), CpG, poly(I:C)) in macrophages. Knockdown of RhoB expression markedly decreased TLR ligand-induced activation of mitogen activated protein kinases and nuclear factor-κB (NF-κB), and the production of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1ß in macrophages stimulated with TLR ligands. Furthermore, we demonstrated that RhoB interacts with major histocompatibility complex class II (MHCII) α chain, but not ß chain, in endosomes of macrophages. Knockdown of MHCII expression greatly reduced the interaction of RhoB with Btk, and attenuated the induction of NF-κB and interferon ß activity by RhoB upon LPS stimulation. These findings suggest that RhoB is a positive physiological regulator of TLRs signaling via binding to MHCII in macrophages, and therefore RhoB may be a potential therapeutic target in inflammatory diseases.