Conventional dendritic cells 2, the targeted antigen-presenting-cell, induces enhanced type 1 immune responses in mice immunized with CVC1302 in oil formulation.

Multifunctional CD4+ T helper 1 (Th1) cells, producing IFN-γ, TNF-α and IL-2, define a correlate of vaccine-mediated protection against intracellular infection. In our previous study, we found that CVC1302 in oil formulation promoted the differentiation of IFN-γ+/TNF-α+/IL-2+Th1 cells. In order to extend the application of CVC1302 in oil formulation, this study aimed to elucidate the mechanism of action in improving the Th1 immune response. Considering the signals required for the differentiation of CD4+ T cells to Th1 cells, we detected the distribution of innate immune cells and the model antigen OVA-FITC in lymph node (LN), as well as the quantity of cytokines produced by the innate immune cells. The results of these experiments show that, cDC2 and OVA-FITC localized to interfollicular region (IFR) of the draining lymph nodes, inflammatory monocytes localized to both IFR and T cell zone, which mainly infiltrate from the blood. In this inflammatory niche within LN, CD4+ T cells were attracted into IFR by CXCL10, secreted by inflammatory monocytes, then activated by cDC2, secreting IL-12. Above all, CVC1302 in oil formulation, on the one hand, targeted antigen and inflammatory monocytes into the LN IFR in order to attract CD4+ T cells, on the other hand, targeted cDC2 to produce IL-12 in order to promote optimal Th1 differentiation. The new finding will provide a blueprint for application of immunopotentiators in optimal formulations.

Analysis of CVC1302-Mediated Enhancement of Monocyte Recruitment in Inducing Immune Responses.

Monocytes (Mos) are believed to play important roles during the generation of immune response. In our previous study, CVC1302, a complex of PRRs agonists, was demonstrated to recruit Mo into lymph nodes (LNs) in order to present antigen and secret chemokines (CXCL9 and CXCL10), which attracted antigen-specific CD4+ T cells. As it is known that Mos in mice are divided into two main Mo subsets (Ly6C+ Mo and Ly6C- Mo), we aimed to clarify the CVC1302-recruiting Mo subset and functions in the establishment of immunity. In this study, we found that CVC1302 attracted both Ly6C+ Mo and Ly6C- Mo into draining LNs, which infiltrated from different origins, injection muscles and high endothelial venule (HEV), respectively. We also found that the numbers of OVA+ Ly6C+ Mo in the draining LNs were significantly higher compared with OVA+ Ly6C- Mo. However, the levels of CXCL9 and CXCL10 produced by Ly6C- Mo were significantly higher than Ly6C+ Mo, which plays important roles in attracting antigen-specific CD4+ T cells. Under the analysis of their functions in initiating immune responses, we found that the ability of the Ly6C+ monocyte was mainly capturing and presenting antigens, otherwise; the ability of the Ly6C- monocyte was mainly secreting CXCL9 and CXCL10, which attracted antigen-specific CD4+ T cells through CXCR3. These results will provide new insights into the development of new immunopotentiators and vaccines.

Increases in Cellular Immune Responses Due to Positive Effect of CVC1302-Induced Lysosomal Escape in Mice.

This study found a higher percentage of CD8+ T cells in piglets immunized with a CVC1302-adjuvanted inactivated foot-and-mouth disease virus (FMDV) vaccine. We wondered whether the CVC1302-adjuvanted inactivated FMDV vaccine promoted cellular immunity by promoting the antigen cross-presentation efficiency of ovalbumin (OVA) through dendritic cells (DCs), mainly via cytosolic pathways. This was demonstrated by the enhanced levels of lysosomal escape of OVA in the DCs loaded with OVA and CVC1302. The higher levels of ROS and significantly enhanced elevated lysosomal pH levels in the DCs facilitated the lysosomal escape of OVA. Significantly enhanced CTL activity levels was observed in the mice immunized with OVA-CVC1302. Overall, CVC1302 increased the cross-presentation of exogenous antigens and the cross-priming of CD8+ T cells by alkalizing the lysosomal pH and facilitating the lysosomal escape of antigens. These studies shed new light on the development of immunopotentiators to improve cellular immunity induced by vaccines.

Promoting immunity with novel targeting antigen delivery vehicle based on bispecific nanobody.

As the most potent professional antigen presenting cells, dendritic cells (DCs) have been targeted in strategies to enhance vaccination efficacy. To date, targeted delivery has been mainly used for cancer therapy, with few studies focusing on vaccine antigens for animal epidemic diseases. In this study, we selected a series of mouse DC-specific nanobodies from a non-immunized camel. The four candidate nanobodies identified (Nb4, Nb13, Nb17, and Nb25), which showed efficient endocytosis of bone marrow-derived DCs, were evaluated as potential vaccine antigen targeted delivery vehicles. First, green fluorescent protein (GFP) was selected and four corresponding DCNb-GFP fusions were constructed for verification. Nb17-GFP was effective at promoting antibody production, inducing a cellular immune response, and increasing the IL-4 level. Second, foot-and-mouth disease virus (FMDV) and a FMDV-specific nanobody (Nb205) were selected and four bispecific nanobody DCNb-Nb205 fusions were generated to investigate the feasibility of a novel targeting antigen delivery vehicle. The resulting bispecific nanobody, Nb17-Nb205, could not only deliver FMDV particles instead of antigenic peptide, but also induced the production of specific antibodies, a cellular immune response, and IFN-γ and IL-4 levels upon immunization with a single subcutaneous injection. In conclusion, our results demonstrate the potential of bispecific nanobody as a novel and efficient DC-specific antigen delivery vehicle. This highlights the potential to expand targeted delivery to the field of animal epidemic diseases and provides a reference for the general application of nanotechnology in viral diseases.

Multidimensional analysis of job advertisements for medical record information managers.

Objective: The rapid growth of the medical industry has resulted in a tremendous increase in medical record data, which can be utilized for hospital management, aiding in diagnosis and treatment, medical research, and other purposes. For data management and analysis, medical institutions require more qualified medical record information managers. In light of this, we conducted an analysis of the qualifications, abilities, and job emphasis of medical record information managers in order to propose training recommendations. Materials and methods: From online job posting sites, a sample of 241 job advertisements for medical record information management positions posted by Chinese healthcare institutions were collected. We conducted word frequency and keyword co-occurrence analysis to uncover overall demands at the macro level, and job analysis to investigate job-specific disparities at the micro level. Based on content analysis and job analysis, a competency framework was designed for medical record information managers. Results: The most frequent keywords were "code," "job experience," and "coding certification," according to the word frequency analysis. The competency framework for managers of medical record information is comprised of seven domains: essential knowledge, medical knowledge, computer expertise, problem-solving skills, leadership, innovation, and attitude and literacy. One of the fundamental skills required of medical record information managers is coordination and communication. Similarly, knowledge and skill requirements emphasize theoretical knowledge, managerial techniques, performance enhancement, and innovation development. Conclusion: According to organization type and job differences, the most crucial feature of the job duties of medical record information managers is cross-fertilization. The findings can be utilized by various healthcare organizations for strategic talent planning, by the field of education for medical record information managers for qualification and education emphasis adjustment, and by job seekers to enhance their grasp of the profession and self-evaluation.

Enhanced Immune Responses in Mice Induced by the c-di-GMP Adjuvanted Inactivated Vaccine for Pseudorabies Virus.

Cyclic dimeric guanosine monophosphate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice, suggesting potential applications as a vaccine immunopotentiator or therapeutic agent. In this study, we evaluated the efficacy of c-di-GMP as an immunopotentiator for pseudorabies virus (PRV) inactivated vaccine in a murine model. We found that c-di-GMP improved the humoral and cellular immune responses induced by PRV inactivated vaccine and its effects on immunity reached the level comparable to that of a live attenuated vaccine. Furthermore, c-di-GMP enhanced the murine antibody response against the viral glycoprotein gB up to 120 days after immunization. The c-di-GMP-adjuvanted PRV inactivated vaccine induced long-term humoral immunity by promoting a potent T follicular helper cell response, which is known to directly control the magnitude of the germinal center B cell response. Furthermore, the c-di-GMP enhanced the response of bone marrow plasma cells and upregulated the expression of Bcl-2 and Mcl-1, which have been identified as anti-apoptotic regulatory genes of germinal center and memory B cells. Our findings open a new avenue for improving the immune efficacy of PRV inactivated vaccines.

Pattern-Recognition Receptor Agonist-Containing Immunopotentiator CVC1302 Boosts High-Affinity Long-Lasting Humoral Immunity.

Ideally, a vaccine should provide life-long protection following a single administered dose. In our previous study, the immunopotentiator CVC1302, which contains pattern- recognition receptor (PRR) agonists, was demonstrated to prolong the lifetime of the humoral immune response induced by killed foot-and-mouth disease virus (FMDV) vaccine. To elucidate the mechanism by which CVC1302 induces long-term humoral immunity, we used 4-hydroxy-3-nitrophenylacetyl (NP)-OVA as a pattern antigen and administered it to mice along with CVC1302, emulsified together with Marcol 52 mineral oil (NP-CVC1302). From the results of NP-specific antibody levels, we found that CVC1302 could induce not only higher levels of NP-specific antibodies but also high-affinity NP-specific antibody levels. To detect the resulting NP-specific immune cells, samples were taken from the injection sites, draining lymph nodes (LNs), and bone marrow of mice injected with NP-CVC1302. The results of these experiments show that, compared with mice injected with NP alone, those injected with NP-CVC1302 had higher percentages of NP+ antigen-presenting cells (APCs) at the injection sites and draining LNs, higher percentages of follicular helper T cells (TFH), germinal center (GC) B cells, and NP+ plasma-blasts in the draining LNs, as well as higher percentages of NP+ long-lived plasma cells (LLPCs) in the bone marrow. Additionally, we observed that the inclusion of CVC1302 in the immunization prolonged the lifetime of LLPCs in the bone marrow by improving the transcription expression of anti-apoptotic transcription factors such as Mcl-1, Bcl-2, BAFF, BCMA, Bax, and IRF-4. This research provides a blueprint for designing new generations of immunopotentiators.

Retrospective Study on the Influencing Factors and Prediction of Hospitalization Expenses for Chronic Renal Failure in China Based on Random Forest and LASSO Regression.

Aim: With the improvement in people's living standards, the incidence of chronic renal failure (CRF) is increasing annually. The increase in the number of patients with CRF has significantly increased pressure on China's medical budget. Predicting hospitalization expenses for CRF can provide guidance for effective allocation and control of medical costs. The purpose of this study was to use the random forest (RF) method and least absolute shrinkage and selection operator (LASSO) regression to predict personal hospitalization expenses of hospitalized patients with CRF and to evaluate related influencing factors. Methods: The data set was collected from the first page of data of the medical records of three tertiary first-class hospitals for the whole year of 2016. Factors influencing hospitalization expenses for CRF were analyzed. Random forest and least absolute shrinkage and selection operator regression models were used to establish a prediction model for the hospitalization expenses of patients with CRF, and comparisons and evaluations were carried out. Results: For CRF inpatients, statistically significant differences in hospitalization expenses were found for major procedures, medical payment method, hospitalization frequency, length of stay, number of other diagnoses, and number of procedures. The R2 of LASSO regression model and RF regression model are 0.6992 and 0.7946, respectively. The mean absolute error (MAE) and root mean square error (RMSE) of the LASSO regression model were 0.0268 and 0.043, respectively, and the MAE and RMSE of the RF prediction model were 0.0171 and 0.0355, respectively. In the RF model, and the weight of length of stay was the highest (0.730). Conclusions: The hospitalization expenses of patients with CRF are most affected by length of stay. The RF prediction model is superior to the LASSO regression model and can be used to predict the hospitalization expenses of patients with CRF. Health administration departments may consider formulating accurate individualized hospitalization expense reimbursement mechanisms accordingly.

Development of haemagglutination assay for titration of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) was one of the most economically important viral pathogens in all the swine-producing countries and often resulted in tremendous economic losses for the swine industry. As PCV2 could not cause cytopathogenic effects while propagated in infected cells, many complicated experiments should be performed to titrate its virus titer. In this study we developed a simple and effective hemagglutination assay for titration of virus titer of PCV2. To develop the hemagglutination assay, a recombinant bispecific nanobody (BsNb) against PCV2 and chicken red blood cells (cRBCs) was constructed based on two nanobodies (NbPCV11 and NbRBC48) which were selected from the non-immunized nanobody library, respectively. The hemagglutination assay was used to titrate the virus titer of PCV2 propagated in cell culture by simple naked-eye observation within 30 min, with the detection limit of 104.09 tissue culture infective dose 50 (TCID50)/mL, excellent specificity and reproducibility. Therefore, the hemagglutination assay had potential to be a rapid, reliable, cost-effective, user-friendly qualitative and semi-quantitative tool for titration of virus titer of PCV2 during the vaccine manufacturing process.

Development of GEM-PA-nanotrap for purification of foot-and-mouth disease virus.

Vaccination is the primary preventative measure against outbreaks of foot-and-mouth disease (FMD). The efficacy of inactivated FMD vaccines is mainly determined by the integrity of foot-and-mouth disease virus (FMDV) particles (referred to as 146S particles), and impurities in the inactivated vaccines could result in side effects. In this study, we developed an effective affinity purification method for the purification of FMDV from cellular lysates, referred to as GEM-PA-nanotrap. To develop the GEM-PA-nanotrap, a nanobody (Nb205) against FMDV vaccine strain O/MYA98/BY/2010 146S particles was selected from a non-immunized library and fused to a peptidoglycan-binding protein anchor (PA). The PA-Nb205 fusion protein was non-covalently coupled to the surface of Gram-positive enhancer matrix (GEM) particles, which were prepared from the non-living, non-genetically modified, Gram-positive, food-grade Lactococcus lactis bacteria. The GEM-PA-nanotrap was used to purify FMDV from cellular lysates through a simple incubation and centrifugation step. The FMDV recovery rate was more than 99%, the efficiency of nonviral protein removal was about 98.3%, and the purification process had almost no effect on the integrity and immunogenicity of 146S particles. Therefore, the GEM-PA-nanotrap has potential as an effective method for the recovery and purification of FMDV during the vaccine manufacturing process.

In vitro antiviral activity of Griffithsin against porcine epidemic diarrhea virus.

Griffithsin is a lectin with potent antiviral activity against enveloped viruses. The objective of this study was to assess Griffithsin's inhibitory effect on porcine epidemic diarrhea virus (PEDV). The results showed that Griffithsin reduced PEDV infection of Vero cells by approximately 82.8%. Moreover, using time-of-addition assays and RT-qPCR, we found that delayed addition of Griffithsin had a weaker inhibitory effect on PEDV than earlier treatment. The mechanism of Griffithsin's action against PEDV involved both preventing viral attachment to host cells and disrupting cell-to-cell transmission; its dual mode of action distinguished Griffithsin from most other antiviral drugs. In conclusion, Griffithsin was identified as a potent PEDV inhibitor and may represent a candidate drug for preventing PEDV infection.

Novel monoclonal antibody-based immunochromatographic strip for detecting citrinin in fruit from Zhejiang province, China.

Citrinin (CIT) is a hepato-nephrotoxic fungal metabolite produced by the genera Penicillium, Aspergillus and Monascu. There is an increasing demand for rapid and economical methods for detection CIT residues in fruit. In this study, we developed an immunochromatographic strip (ICS) for detection of citrinin (CIT) residues in fruit for the first time. Anti-CIT monoclonal antibody (McAb) 2B9 was prepared, with a binding affinity of 9.39 × 108 L/moL. Conjugates CIT-BSA and McAb 2B9 were used to develop the ICS which could be completed in 5 min, with the detection limit of 50 ng/mL and no cross reactivity with other mycotoxins. Analysis of CIT in 64 fruit samples revealed that data obtained from the ICS test were in good agreement with indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs) and high performance liquid chromatography (HPLC). This result demonstrated that the ICS test could be used as a rapid, reliable, cost-effective and user-friendly qualitative tool for detection of CIT residues on-site.

Characterization of Anti-Citrinin Specific ScFvs Selected from Non-Immunized Mouse Splenocytes by Eukaryotic Ribosome Display.

Single chain variable fragments (scFvs) against citrinin (CIT) were selected from a scFv library constructed from the splenocytes of non-immunized mice by an improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA)/ CIT-BSA and ovalbumin (OVA)/ CIT-OVA were used as the antigens to select specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, expression vector pTIG-TRX carrying specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3) for protein expression. Thirteen positive clones were selected out of which three (designated 23, 68 and 109) showed high binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with carrier proteins. The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA. These specific scFvs offer a potential novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

Fabrication of lactobionic-loaded chitosan microcapsules as potential drug carriers targeting the liver.

This paper demonstrates a general approach for fabrication of lactobionic chitosan microcapsules using layer-by-layer assembly via click chemistry. Chitosan was selectively modified with either azide (CHI-Az) or alkyne (CHI-Alk) groups. The growth of the CHI-Az/CHI-Alk click multilayer was studied experimentally by multilayer assembly on planar supports. Linear buildup of the film was observed. The chitosan click capsules were also analyzed with confocal laser scanning microscopy and transmission electron microscopy. Capsules were found to have regular spherical shapes. In addition, (CHI-Az/CHI-Alk)-coated particles were modified with fluorescein isothiocyanate to ensure that the particles can be easily post-functionalized. Finally, lactobionic acid was conjugated onto the (CHI-Az/CHI-Alk)-coated particles and the lactobionic particles exhibited hepatoma cell (HepG2) targeting behavior.