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Toll-like receptor 2 (TLR2) signaling is a pivotal component of immune system activation, and it is closely linked to the lipidation of bacterial proteins. This lipidation is guided by bacterial signal peptides (SPs), which ensure the precise targeting and membrane anchoring of these proteins. The lipidation process is essential for TLR2 recognition and the activation of robust immune responses, positioning lipidated bacterial proteins as potent immunomodulators and adjuvants for vaccines against bacterial-, viral-, and cancer-related antigens. The structural diversity and cleavage pathways of bacterial SPs are critical in determining lipidation efficiency and protein localization, influencing their immunogenic potential. Recent advances in bioinformatics have significantly improved the prediction of SP structures and cleavage sites, facilitating the rational design of recombinant lipoproteins optimized for immune activation. Moreover, the use of SP-containing lipobox motifs, as adjuvants to lipidate heterologous proteins, has expanded the potential of vaccines targeting a broad range of pathogens. However, challenges persist in expressing lipidated proteins, particularly within heterologous systems. These challenges can be addressed by optimizing expression systems, such as engineering E. coli strains for enhanced lipidation. Thus, lipoprotein signal peptides (SPs) demonstrate remarkable versatility as adjuvants in vaccine development, diagnostics, and immune therapeutics, highlighting their essential role in advancing immune-based strategies to combat diverse pathogens.

Abstract Background Flagellin elicits potent immune response and may serve as a vaccine adjuvant. We previously reported that the N-terminus of flagellin (residues 1–99, nFliC) is sufficient for vaccine efficacy enhancement against Pasteurella multocida challenge in chickens. In this study, we futher tested the adjuvancy of nFliC in a subunit vaccine against the pig pathogen Actinobacillus pleuropneumoniae in a mice model. For vaccine formulation, the antigen ApxIIPF (the pore-forming region of the exotoxin ApxII) was combined with nFliC, either through genetic fusion or simple admixture. Results Immune analysis showed that nFliC, introduced through genetic fusion or admixture, enhanced both humoral (antibody levels) and cellular (T cell response and cytokine production) immunity. In a challenge test, nFliC increased vaccine protective efficacy to 60–80%, vs. 20% for the antigen-only group. Further analysis showed that, even without a supplemental adjuvant such as mineral salt or oil emulsion, genetically linked nFliC still provided significant immune enhancement. Conclusions We conclude that nFliC is a versatile and potent adjuvant for vaccine formulation.

Vaccinations can serve as an important preventive measure against the porcine epidemic diarrhea (PED) virus that currently threatens the swine industry. This study focuses on the development of a fusion protein vaccine, FliC99-mCOE, which combines the N-terminus of flagellin (FliC99) with a modified core neutralizing epitope (mCOE) of PEDV. In silico immunoinformatic analysis confirmed the construct’s non-toxic, non-allergenic, and highly antigenic nature. Molecular docking and molecular dynamics (MD) simulations demonstrated FliC99-mCOE’s strong binding to the TLR-5 immunological receptor. Repeated exposure simulations and immunological simulations suggested enhanced cell-mediated immunity. Both FliC99-mCOE and an inactivated PEDV vaccine were produced and tested in mice. The results from cell proliferation, ELISA, and neutralization assays indicated that FliC99-mCOE effectively stimulated cellular immunity and neutralized PEDV. We conclude that the FliC99-mCOE fusion protein may serve as a promising vaccine candidate against PEDV.

ABSTRACT: Recombinant Pasterurella multocida lipoprotein E (PlpE) has been shown to protect against fowl cholera. This study aimed to determine if the signal sequence may contribute to the antigenicity and protective efficacy of recombinant PlpE. A small antigenic domain of PlpE (termed truncated PlpE, tPlpE) was constructed with (SP-tPlpE) or without (tPlpE) the signal sequence and evaluated in vitro and in vivo. In vitro, the HEK-Bule hTLR2 Cells were used to evaluate the activation of NF-kB in the test associated with the stimulation of the SP-tPlpE and tPlpE proteins. When chickens were immunized, compared to the tPlpE vaccine group, the SP-tPlpE group showed higher antibody levels and enhanced CD4+ T cell response. In a challenge test, the SP-tPlpE group showed a survival rate of 87.5% (n = 8), compared to 25% for the tPlpE group. It is confirmed that the inclusion of the native signal sequence enhanced protective efficacy against fowl cholera and may act as a vaccine adjuvant. The short SP-tPlpE construct is amenable to further vaccine engineering and has potential to be developed as a fowl cholera vaccine.

Abstract. Desmoplakin (DSP), encoded by the DSP gene, is the main desmosome component and is abundant in the myocardial tissue. There are three DSP isoforms that assume the role of supporting structural stability through intercellular adhesion. It has been found that DSP regulates the transcription of adipogenic and fibrogenic genes, and maintains appropriate electrical conductivity by regulating gap junctions and ion channels. DSP is essential for normal myocardial development and the maintenance of its structural functions. Studies have suggested that DSP gene mutations are associated with a variety of hereditary cardiomyopathy, such as arrhythmia cardiomyopathy, dilated cardiomyopathy (DCM), left ventricular noncompaction, and is also closely associated with the Carvajal syndrome, Naxos disease, and erythro-keratodermia-cardiomyopathy syndrome with skin and heart damage. The structure and function of DSP, as well as the clinical manifestations of DSP-related cardiomyopathy were reviewed in this article.

Some antituberculosis agents may cause hypothyroidism, and thyroid hormones play a vital role in Mycobacterium tuberculosis infection. However, the relationship between tuberculosis (TB) and hypothyroidism has not been clearly established. Therefore, this retrospective, longitudinal cohort study aimed to investigate the association between these two diseases using the 2000–2017 data from the Taiwan's National Health Insurance Research Database. The hypothyroidism and TB cohorts were matched with the control group in a 1:4 ratio. Adjusted hazard ratios (aHRs) were assessed using Cox proportional hazards regression analysis in each cohort. In total, 3,976 individuals with hypothyroidism and 35 120 individuals with TB were included in this study. The risk of developing TB in patients with hypothyroidism was 2.91 times higher than that in those without hypothyroidism (95% confidence interval [CI], 1.50–3.65). The subgroup of thyroxine replacement therapy (TRT) had a 2.40 times higher risk (95% CI, 1.26–3.01), whereas the subgroup of non-TRT had a 3.62 times higher risk of developing TB than those without hypothyroidism (95% CI, 2.19–4.84). On the other hand, the risk of developing hypothyroidism in patients with TB was 2.01 times higher than that in those without TB (95% CI, 1.41–2.38). Our findings provide evidence that TB and hypothyroidism are interrelated. Thus, clinicians and public health authorities should monitor the association between these two diseases to reduce the relevant disease burden.

Abstract Background Bovine ephemeral fever virus (BEFV) causes fever and muscle stiffness in cattle, resulting in negative economic impact for cattle and dairy farms. During the manufacturing process of inactivated vaccine for virus control, it is important to determine the virus titer, but traditional methods such as plaque assay and TCID50 assay require days of waiting time. We sought to develop a quick dot blot assay for BEFV titering. Results Three different kinds of BEFV antigens were prepared to raise primary antibodies for BEFV detection in dot blot assays: 1) purified BEFV particles, 2) Escherichia coli (E. coli)-expressed BEFV G1 region, and 3) E. coli-expressed BEFV N protein. Results showed that antibodies raised against purified BEFV particles can detect BEFV particles, but it also showed a high background level from the proteins of BHK-21 cells. Antibodies raised against E.coli-expressed BEFV G1 region could not detect BEFV particles in dot blot assays. Finally, antibodies raised against E.coli-expressed BEFV N protein detected BEFV particles with a high signal-to-noise ratio in dot blot assays. Conclusions E.coli-expressed N protein is a suitable antigen for the production of antiserum that can detect BEFV particles with a high signal-to-noise ratio. A dot blot assay kit using this antiserum can be developed as a quick and economical way for BEFV titering.

Flagellin activates the immune system through Toll-like receptor 5 (TLR5) and can work as an adjuvant for subunit vaccines. In this study, we tested the adjuvancy of two different N-terminal fragments of flagellin, (1) FliC99, residues 1–99, and (2) FliC176, residues 1–176, to incorporate larger areas of the hotspot region for potentially higher levels of TLR5 activation and immune response. A truncated version of the VP2 protein (name tVP2, residues 199–356) of the Infectious bursal disease virus (IBDV) was genetically linked to the flagellin constructs, and the immune response was evaluated in chickens. Results showed that both chimeric antigen–adjuvant constructs increased humoral (total IgG titers), cellular and cytokine immune response (IL-4, IFN-γ). The resulting antibody also successfully neutralized IBDV. We conclude that the N-terminus of flagellin can act as an immune activator to enhance vaccine efficacy.

Abstract. Objective:. Physiologic cardiac pacing is a novel technique which has been largely popularized in recent decades. His bundle pacing (HBP) has been long considered the most physiologic pacing method; however, with the widespread implementation of this method, its disadvantages have become apparent. In this context, left bundle branch pacing (LBBP)—directly engaged in the His-Purkinje system—has been foreseen as the best pacing method to mimic physiologic activation patterns. This review aimed to summarize recent approaches to physiologic cardiac pacing. Data sources:. This review included fully peer reviewed publications up to July 2018, found in the PubMed database using the keywords “His bundle branch pacing,” “right ventricular pacing,” and “physiologic pacing.” Study selection:. All selected articles were in English, with no restriction on study design. Results:. The HBP has been studied worldwide, and is currently considered the most physiologic pacing method. However, it has disadvantages, such as high pacing threshold, unsatisfactory sensing and long procedure times, among others. Although LBBP is theoretically superior to HBP, the clinical relevance of this difference remains under debate, as few large randomized clinical trials with LBBP have been published. Conclusions:. Although HBP indeed appears to be the most physiologic pacing method, it has certain shortcomings, such as high pacing threshold, difficult implantation due to specific anatomic features, and others. Further studies are required to clarify the clinical significance of LBBP.

Poloxamer-188 (P188) is a nonionic triblock linear copolymer that can be used as a pharmaceutical excipient because of its amphiphilic nature. This study investigated whether P188 can act as an adjuvant to improve the immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) subunit vaccine. BALB/c mice were vaccinated twice with the RBD antigen alone or in combination with P188 or MF59 (a commercial adjuvant for comparison purposes). The resulting humoral and cellular immunity were assessed. Results showed that P188 helped elicit higher neutralizing activity than MF59 after vaccination. P188 induced significant humoral immune response, along with type 1 T helper (Th1) and type 2 T helper (Th2) cellular immune response when compared with MF59 due to repressing p38MAPK phosphorylation. Furthermore, P188 did not result in adverse effects such as fibrosis of liver or kidney after vaccination. In conclusion, P188 is a novel adjuvant that may be used for safe and effective immune enhancement of the SARS-CoV-2 RBD antigen.

The feedback strategy, or controlled exposure of pig herd to the porcine epidemic diarrhea virus (PEDV), significantly decreased losses during a severe outbreak in late 2013 in Taiwan. However, some pig farms still suffered from recurrent outbreaks. To evaluate the association between antibody titers and clinical manifestations, sera and colostra were analyzed from one pig farm that employed the feedback strategy. Furthermore, spike (S) gene full sequences from six positive samples of two farms with and without using feedback were compared to investigate the evolution of PEDV variants circulating in pig herds. The results in this study showed that high PEDV antibody titers do not correlate with the high rate of protection from PEDV infection. In addition, repeated feedback generated the emergence of PEDV variants with unique substitutions of N537S and Y561H in the COE domain and S769F in the SS6 epitopes. These mutations indicated the pathogenetic evolution of PEDV strains existing in the cycle of the feedback method. A very strict biosecurity practice to block the routes of pathogen transfer should be followed to achieve successful control of PEDV infections in pig herds.

In this study, we sought to develop a subunit vaccine against the increasingly prevalent Duck hepatitis A virus serotype 3 (DHAV-3). The VP1 protein of DHAV-3 and a truncated version containing the C-terminal region of VP1, termed VP1-C, were expressed recombinantly in Escherichia coli as vaccine antigens. For enhanced immune response, a truncated version of flagellin, nFliC, was included as vaccine adjuvant. Ducklings were vaccinated once for immune response analysis and challenge test. Results showed that VP1-C elicited a higher level of virus-specific antibody response and neutralization titer than VP1. The addition of nFliC further enhanced the antibody response. In terms of cellular immune response, the VP1-C + nFliC vaccine elicited the highest level of T cell proliferation among the vaccine formulations tested. Examination of the cytokine expression profile showed that peripheral blood mononuclear cells from the VP1-C + nFliC vaccine group expressed the highest levels of pro-inflammatory (IL-6) and TH-1 type (IL-12 and IFN-γ) cytokines. Finally, in a DHAV-3 challenge test, the VP1-C + nFliC vaccine provided a 75% protection rate (n = 8), in contrast to 25% for the VP1 vaccine. In conclusion, E. coli-expressed VP1-C has been shown to be a promising antigen when combined with nFliC and may be further developed as a single-dose subunit vaccine against DHAV-3.

Abstract Background Inactivated and subunit bovine viral diarrhea virus (BVDV) vaccines have shown limited protective efficacy. This study aimed to evaluate the effectiveness of a vaccine containing both inactivated BVDV (iBVDV) and baculovirus-expressed recombinant E2 (rE2), an important BVDV antigen with strongly neutralizing epitopes. Results Four groups of goats were immunized twice with one of four vaccine preparations: 1) iBVDV+rE2, 2) rE2, 3) iBVDV, and 4) saline, and challenged with BVDV. For goats vaccinated with the iBVDV+rE2 vaccine, no viremia was observed after challenge, and clinical signs, pyrexia, and leukopenia were reduced compared to the saline group. In contrast, for goats vaccinated with either iBVDV or rE2 alone, viremia was still detectable. Conclusion The combination of iBVDV and rE2 elicited stronger protective immune responses against BVDV than iBVDV or rE2 alone.

Impaired wound healing is an ongoing issue that cancer patients undergoing chemotherapy or radiotherapy face. Our previous study regarding lung-cancer-associated pleural fluid (LCPF) demonstrated its propensity to promote endothelial proliferation, migration, and angiogenesis, which are crucial features during cutaneous wound healing. Therefore, the current study aimed to investigate the effect of pleural fluid on cutaneous wound closure in vitro and in vivo using HaCaT keratinocytes and a full-thickness skin wound model, respectively. Both heart-failure-associated pleural fluid (HFPF) and LCPF were sequentially centrifuged and filtered to obtain a cell-free status. Treatment with HFPF and LCPF homogeneously induced HaCaT proliferation with cell cycle progression, migration, and MMP2 upregulation. Western blotting revealed increased PI3K/Akt phosphorylation and VEGFR2/VEGFA expression in HaCaT cells. When treated with the PI3K inhibitor, LCPF-induced keratinocyte proliferation was attenuated with decreased pS6 levels. By applying the VEGFR2 inhibitor, LCPF-induced keratinocyte proliferation was ameliorated by pS6 and MMP2 downregulation. The effect of LCPF-induced cell junction rearrangement was disrupted by co-treatment with a VEGFR2 inhibitor. Compared with a 0.9% saline dressing, LCPF significantly accelerated wound closure and re-epithelization when used as a dressing material in a full-thickness wound model. Histological analysis revealed increased neo-epidermis thickness and dermis collagen synthesis in the LCPF-treated group. Furthermore, LCPF treatment activated basal keratinocytes at the wound edge with the upregulation of Ki-67, VEGFA, and MMP2. Our preliminaries provided the benefit of wet dressing with pleural fluid to improve cutaneous wound closure through enhanced re-epithelization and disclosed future autologous application in cancer wound treatment.

Malignant-associated pleural fluid (MAPF) represented an unsolved problem in advanced lung cancer. Our previous work characterized increased pleural angiogenesis in lung adenocarcinoma and the propensity of MAPF on endothelial angiogenesis. This study investigated the combined efficacy of the tyrosine kinase inhibitor (gefitinib) and bevacizumab in opposing MAPF-induced angiogenesis. In lung adenocarcinoma patients with malignant pleural effusion (MPE), Kaplan–Meier analysis revealed the benefit of cotreatment with target therapy and bevacizumab. Increased EGFR expression was observed in the pleural microvessels of patients with lung adenocarcinoma both with and without mutations in EGFR. MAPF was obtained from lung adenocarcinoma patients both wild-type and mutant EGFRs. Total and phosphorylated EGFR were upregulated in HUVEC cultured with MAPF. Treatment with gefitinib as an EGFR inhibitor suppressed MAPF-induced endothelial migration and partially attenuated endothelial proliferation in both wild-type and mutant EGFR lung adenocarcinoma. Cotreatment with gefitinib and bevacizumab produced better inhibition of MAPF-induced endothelial angiogenesis than gefitinib alone in the mutant EGFR subgroup. Protein analysis of MAPF-derived exosomes revealed abundant EGFR and p-EGFR components that implied possible transfer to endothelial cells. Concluding Kaplan–Meier analysis and in vitro studies, the results indicated that the addition of bevacizumab on gefitinib treatment could suppress MAPF-induced angiogenesis in lung adenocarcinoma patients.

Flagellin from bacteria elicits a proinflammatory immune response and may act as a vaccine adjuvant. In this study, we evaluated the adjuvant effect of the N-terminus of flagellin (residues 1–99) when linked to an antigen (a truncated, conserved domain of lipoprotein E of Pasteurella multocida). Immunization of chickens with the antigen-adjuvant chimeric protein showed that the N-terminus of flagellin accelerated the antibody response and enhanced the cellular immunity (CD8+ T cell expansion). Stimulation of peripheral blood mononuclear cells from vaccinated chickens showed both TH1 (IFN-γ and IL-12) and TH2 (IL-4)-type cytokine gene expressions. In a challenge test, the N-terminus of flagellin increased the survival rate to 75%, compared to 25% in the antigen-only group. In conclusion, our study found that the N-terminus of flagellin can increase the immune response and enhance vaccine protection.

In this study, we propose nondestructive testing methods and combined forecasting models-based stress wave and impedance measurements to obtain accurate internal defects information for wooden building components. Internal defects data for major wooden components of an ancient building in China and reverse laboratory test data on matching tree species indicated various degrees of damage on the pavilion wood structure surface and internal defects in certain pillars. The stress wave method enabled rapid acquisition of two-dimensional plots of test sections; however, the results revealed that the area of stress wave detection was greater than the actual defect area. Moreover, the impedance meter was able to determine the defect position and type in a single path, and the actual defect area was proportional to the absolute error of the drilling resistance. By distributing the errors from the two nondestructive testing methods on the basis of a Shapley value algorithm, we determined the weights of stress wave and impedance meter data in the forecasting models and established combined forecasting models that showed greater accuracy with a mean relative error of less than 6%. This method can improve the prediction accuracy of internal defects in ancient buildings and provide effective data support for practical engineering repair and reinforcement schemes.

Actinobacillus pleuropneuminiae is the major cause of invasive respiratory disease in swine. To develop an efficient subunit vaccine against pathogens, we designed a dual antigen in a single vaccine that increased the potential for an immune response. We present a dual antigen vaccine (pore-forming domain and nutritional immunity) that induces humoral and cell-mediated T-cell responses, antibodies and TH1 and TH2 cells. Mice were immunized with the dual antigen and individual antigen and then subjected to immune response analysis. A significant antibody response was observed for the dual antigen group. For the cellular immune response, CD4+ and CD8+ T-cell expansion, proinflammatory cytokine (IL-1β, IL-6) and TH2-type cytokine (IL-4, IL-10) gene expression was observed for the dual antigen. Finally, in a challenge test with A. pleuropneumoniae serotype1, the dual antigen, individual antigen and PBS vaccine conferred 100, 40 and 0 % protection. In conclusion, the dual antigen presents critical antigens and increases the efficacy of the vaccine. [ABSTRACT FROM AUTHOR]

Background: Streptococcus suis (S. suis) causes arthritis, meningitis, septicemia, and sudden death in pigs and is also an zoonotic agent for humans. The present study demonstrated that immunization with recombinant Sao-L (surface antigen one-L, rSao-L) protein from a strain of S. suis serotype 2 in pigs was able to increase cross-serotype protection against S. suis serotype 1 and 2 challenge. Since weaning piglets are more susceptible to S. suis infections due to the stresses associated with weaning, prepartum immunization in sows may convey passive immunity to piglets and provide protection. Results: Pregnant sows were immunized with a vaccine containing inactivated S. suis serotype 2 plus rSao as the antigens. Blood samples were collected from their piglets after birth for analysis of antigen-specific antibody titers and levels of various cytokines. Results demonstrated that the titers of S. suis and rSao-specific antibodies were significantly (p < 0.05) higher in the vaccinated piglets in comparison with that of piglets in the control group. The serum levels of interferon (IFN)-β, interleukin (IL)-4, IL-6, and IL-12 were significantly (p < 0.05) increased in piglets born from vaccinated sows when compared to piglets from unvaccinated sows. In addition, piglets were challenged by heterologous and homologous S. suis. All piglets from unvaccinated sows developed severe symptoms of bacteremia, fever, anorexia, depression, and arthritis. On the other hand, piglets from vaccinated sows had significantly (p < 0.05) reduced clinical symptoms and lesion score (by 75 and 81%). Conclusions: Our results revealed that immunizing pregnant sows with the vaccine containing inactivated S. suis bacterin plus rSao as the antigens is able to enhance passive immunity against heterologous and homologous S. suis challenge in their piglets. [ABSTRACT FROM AUTHOR]