This study aimed to investigate the mechanism by which Shegan Mahuang Decoction(SGMH) and its bitter Chinese herbs(BCHs) regulated the lung-gut axis through the bitter taste receptor 14(TAS2R14)/secretory immunoglobulin A(SIgA)/thymic stromal lymphopoietin(TSLP) to intervene in the epithelial cell barrier of cold asthma rats. Fifty SD rats were randomly divided into the following five groups: normal group, model group, dexamethasone group, SGMH group, and BCHs group. A 10% ovalbumin(OVA) solution was used to sensitize the rats via subcutaneous injection on both sides of the abdomen and groin, combined with 2% OVA atomization and cold(2-4 ℃) stimulation to induce a cold asthma model in rats. The SGMH, BCHs, and dexamethasone groups were given corresponding treatments by gavage and nebulization, while the normal and model groups received normal saline by gavage and nebulization. After the final stimulation, pathological changes in the lung and intestine tissues were observed using hematoxylin-eosin(HE) and periodic acid-Schiff(PAS) staining. Lung function was assessed by measuring the ratio of forced expiratory volume in the first second to forced vital capacity(FEV1/FVC), the ratio of the average flow rate at 25%-75% of forced vital capacity to foned vital capacity(FEV25%-75%/FVC), the peak expiratory flow(PEF), and pulmonary resistance(RL). The levels of IL-4, IL-5, IL-13, and TNF-α in serum, and sIgA in serum, intestinal, and bronchial mucosa were detected by enzyme-linked immunosorbent assay(ELISA). The expression of TAS2R14 protein in lung tissue was detected by Western blot(WB). The content of short-chain fatty acids(SCFAs) in rat feces was determined by gas chromatography-mass spectrometry(GC-MS). The effect of TAS2R14/TSLP on lipopolysaccharide(LPS)-induced inflammation in epithelial cells in the BCHs group was observed, and the expression of TAS2R14 and TSLP in cells was detected by WB. Compared with the normal group, the model group showed reduced water intake, diet, and body weight, increased infiltration of inflammatory cells in the lung and intestinal tissues, goblet cell hyperplasia, significantly decreased FEV1/FVC, FEV25%-75%/FVC, and PEF, and significantly increased RL. Moreover, serum levels of IL-4, IL-5, IL-13, and TNF-α were elevated, and sIgA levels in serum, intestine, and bronchial mucosa were significantly decreased. TAS2R14 expression in lung tissues was inhibited, and the content of acetic acid, propionic acid, and butyric acid in feces was significantly reduced. In the LPS group, TSLP expression increased, and TAS2R14 expression decreased. Compared with the model group, the general condition of rats in the SGMH and BCHs groups improved, with reduced infiltration of inflammatory cells and goblet cell hyperplasia in the lung and intestinal tissues. FEV1/FVC, FEV25%-75%/FVC, and PEF significantly increased, and RL significantly decreased. Serum levels of IL-4, IL-5, IL-13, and TNF-α decreased, while sIgA levels in serum, intestine, and bronchial mucosa significantly increased, and TAS2R14 expression was activated in lung and intestinal tissues. The content of acetic acid, propionic acid, and butyric acid in feces significantly increased. Compared with the model group, the BCHs group and the agonist group showed inhibited TSLP expression and increased TAS2R14 expression. The results showed that both SGMH and BCHs could reduce lung and intestinal inflammatory reactions, improve lung function, and regulate the content of intestinal SCFAs in asthmatic rats. There was no significant difference in TAS2R14 protein expression between the SGMH and BCHs groups, indicating that the clinical efficacy of BCHs may be related to the activation of the bitter receptor TAS2R14 and the regulation of immune inflammatory mediators in lung and intestinal epithelial cells.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Rats, Sprague-Dawley ; Lung/metabolism* ; Asthma/metabolism* ; Cytokines/immunology* ; Male ; Receptors, G-Protein-Coupled/immunology* ; Epithelial Cells/metabolism* ; Thymic Stromal Lymphopoietin ; Immunoglobulin A, Secretory/genetics* ; Humans ; Cold Temperature

OBJECTIVES: To investigate the protective effect of the probiotic bacterium Streptococcus salivarius K12 (K12) against Mycoplasma pneumoniae (Mp) infection in mice. METHODS: Forty male BALB/c mice were randomized into normal control group, K12 treatment group, Mp infection group, and K12 pretreatment prior to Mp infection group. The probiotic K12 was administered daily by gavage for 14 days before Mp infection induced by intranasal instillation of Mp. Three days after Mp infection, the mice were euthanized for analysis of bronchoalveolar lavage fluid (BALF) cell counts and serum levels of secretory immunoglobulin A (sIgA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). RT-qPCR was performed to detect the P1 and community-acquired respiratory distress syndrome ( CARDS ) toxin of Mp in the lung tissues and the mRNA expressions of TNF-α, IL-6, chemokine 1 (CXCL1), matrix metalloproteinase 9 (MMP9), mucin 5ac (MUC5ac), collagen 3a1 (Col3a1), Toll-like receptor 2 (TLR2) and TLR4; the protein expressions of TLR2 and TLR4 in the lung tissue were detected using Western blotting. Pathological changes in the lung tissue and airway remodeling were examined with HE staining and AB/PAS staining. RESULTS: Compared with the Mp-infected mice with PBS treatment, the infected mice with K12 treatment showed significantly lowered mRNA levels of P1 and CARDS in the lung tissue and reduced white blood cell counts in the BALF (P<0.05). In spite of the absence of significant differences in serum levels of inflammatory factors between the two groups, the mRNA expressions of TNF‑α, IL-6, CXCL1, MMP9, MUC5ac and COL3A1 and the mRNA and protein levels of TLR2 and TLR4 in the lung tissues were significantly lower in K12-treated mice, in which AB/PAS staining showed obviously decreased mucus secretion. CONCLUSIONS K12 pretreatment can effectively reduce pulmonary inflammatory responses, improve airway remodeling and alleviate lung injury in Mp-infected mice.
Animals ; Mice ; Pneumonia, Mycoplasma/metabolism* ; Mice, Inbred BALB C ; Toll-Like Receptor 2/metabolism* ; Mycoplasma pneumoniae ; Male ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Lung/microbiology* ; Toll-Like Receptor 4/metabolism* ; Streptococcus salivarius ; Probiotics/administration & dosage* ; Bronchoalveolar Lavage Fluid ; Matrix Metalloproteinase 9/metabolism* ; Mucin 5AC/metabolism* ; Chemokine CXCL1/metabolism* ; Immunoglobulin A, Secretory/metabolism* ; Bacterial Toxins ; Bacterial Proteins

Objective To investigate the effect of salidroside on intestinal mucosal immune status in rats under compound stress of hypoxia and training (HTCS) and the mechanism. Methods SD rats were randomly divided into HTCS model group (model), placebo group (placebo) and salidroside group (salidro). Model group received no intervention, and placebo and salidro group received intraperitoneal injection of normal saline and salidroside, respectively. Then, ileum tissue of rats were collected and the intestinal damage was assayed by HE staining and Chiu scores. Intestinal permeability indices, including serum D-diamine oxidase (DAO), D-lactic acid (DLA) and endotoxin (END) and secretory immunoglobulin A (sIgA) of intestinal tissue were detected by ELISA. T lymphocyte subsets of intestinal tissue were detected by flow cytometry. Expression of tight junction molecules, including ZO-1, Claudin-3, occluding, were detected by PCR and western blot. Activation of TLR4/NF-κB signaling pathway was detected by Western blot analysis. Results Compared with model group and placebo group, salidro group had the decreased intestinal mucosal injury and low Chiu score, and the level of intestinal permeability indices including serum DAO, DLA and END fell off. CD4⁺ T cell percentage, CD4⁺/CD8⁺ ratio and sIgA level were went up, while CD8⁺ T cell percentage was went down. mRNA and the level of protein expressions of ZO-1, claudin-3 and occludin increased, while activation of TLR4/NF-κB signaling pathway was inhibited. Conclusion Salidroside can alleviate the intestinal barrier injury and improve intestinal mucosal immune status of rats under compound stress of hypoxia and training via inhibiting TLR4/NF-κB signalling pathway.
Animals ; Rats ; Rats, Sprague-Dawley ; NF-kappa B ; Toll-Like Receptor 4/genetics* ; Claudin-3 ; Hypoxia ; Immunoglobulin A, Secretory ; Signal Transduction

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.
Animals ; Antibodies ; Bacteria ; Chickens ; Hemagglutination ; HN Protein ; Homologous Recombination ; Immunoglobulin A, Secretory ; Immunoglobulin G ; Lymphocytes ; Methods ; Newcastle disease virus ; Newcastle Disease ; Plasmids ; Poultry ; Salmonella ; Suicide ; Vaccines

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.
Animals ; Cloning, Molecular ; Computational Biology ; Computer Simulation* ; Epitopes ; Epitopes, B-Lymphocyte ; Fusobacterium nucleatum* ; Fusobacterium* ; Hydrophobic and Hydrophilic Interactions ; Immunoglobulin A, Secretory ; Immunoglobulin G ; Membrane Proteins ; Mice ; Periodontitis ; Solubility ; Vaccination

Goose parvovirus (GPV) continues to be a threat to goose farms and has significant economic effects on the production of geese. Current commercially available vaccines only rarely prevent GPV infection. In our study, Lactobacillus (L.) plantarum NC8 was selected as a vector to express the VP2 gene of GPV, and recombinant L. plantarum pSIP409-VP2/NC8 was successfully constructed. The molecular weight of the expressed recombinant protein was approximately 70 kDa. Mice were immunized with a 2 × 109 colony-forming unit/200 µL dose of the recombinant L. plantarum strain, and the ratios and numbers of CD11c⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, and interferon gamma- and tumor necrosis factor alpha-expressing spleen lymphocytes in the pSIP409-VP2/NC8 group were higher than those in the control groups. In addition, we assessed the capacity of L. plantarum SIP409-VP2/NC8 to induce secretory IgA production. We conclude that administered pSIP409-VP2/NC8 leads to relatively extensive cellular responses. This study provides information on GPV infection and offers a clear framework of options available for GPV control strategies.
Agriculture ; Animals ; Geese ; Immunization ; Immunoglobulin A, Secretory ; Interferons ; Lactobacillus plantarum* ; Lactobacillus* ; Lymphocytes ; Mice* ; Molecular Weight ; Parvovirus* ; Spleen ; Tumor Necrosis Factor-alpha ; Vaccines

To construct a novel vaccine candidate against bovine enterotoxigenic Escherichia coli (ETEC), FanC, the major subunit of K99 fimbriae adhesion, was inserted into secretion plasmid pYA3560 containing a β-lactamase secretion system. This was then transformed into Δasd Δcrp Salmonella (S.) Typhimurium and designated as JOL950. Secretion of recombinant fanC fimbrial antigens was confirmed by immunoblot analysis. Groups of mice were inoculated with single or double doses of JOL950. Another group was used as a negative control. Compared to control mice, all immunized mice had significantly higher levels (p < 0.05) of serum immunoglobulin (Ig)G, and secretory IgA against FanC. The IgG2a and IgG1 titer assays revealed that immunization highly induced IgG2a compared to that of IgG1, indicating that T helper-1- related cell-mediated immune responses may be elicited by JOL950. The results show that both systemic and mucosal immunities against selected fimbrial antigens of bovine ETEC expressed by a live attenuated S. Typhimurium strain are prominently produced in mice immunized with JOL950 via an oral route.
Animals ; Enterotoxigenic Escherichia coli* ; Immunization ; Immunoglobulin A, Secretory ; Immunoglobulin G ; Immunoglobulins ; Mice* ; Plasmids ; Salmonella*

The levels of Streptococcus (S.) mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgI/II (PAc) and glucosyltransferase (GTF) for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3-4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A (sIgA), anti-PAc and anti-Glucan binding domain (anti-GLU) were compared to determine the correlation among them. It was found the level of s-IgA against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific sIgA against PAc or GLU between any two groups. No significant correlation was found between such specific sIgA and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of sIgA against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.
Antibodies, Bacterial ; biosynthesis ; Antigens, Bacterial ; chemistry ; immunology ; Bacterial Proteins ; chemistry ; immunology ; Case-Control Studies ; Child, Preschool ; Dental Caries ; immunology ; pathology ; prevention & control ; Epitopes ; chemistry ; immunology ; Female ; Glucosyltransferases ; chemistry ; immunology ; Humans ; Immunoglobulin A, Secretory ; biosynthesis ; Male ; Peptides ; chemistry ; immunology ; Saliva ; chemistry ; microbiology ; Severity of Illness Index ; Streptococcal Vaccines ; biosynthesis ; chemistry ; immunology ; Streptococcus mutans ; chemistry ; immunology ; pathogenicity ; Vaccines, Subunit ; Virulence Factors ; chemistry ; immunology

In the present study, quids from La Cueva de los Muertos Chiquitos (CMC) were subjected to ELISA tests for 2 protozoan parasites, Toxoplasma gondii (n=45) and Trypanosoma cruzi (n=43). The people who occupied CMC, the Loma San Gabriel, lived throughout much of present-day Durango and Zacatecas in Mexico. The known pathoecology of these people puts them into at-risk categories for the transmission of T. gondii and T. cruzi. Human antibodies created in response to these 2 parasites can be detected in modern saliva using ELISA kits intended for use with human serum. For these reasons, quids were reconstituted and subjected to ELISA testing. All test wells yielded negative results. These results could be a factor of improper methods because there is no precedence for this work in the existing literature. The results could equally be a simple matter of parasite absence among those people who occupied CMC. A final consideration is the taphonomy of human antibodies and whether or not ELISA is a sufficient method for recovering antibodies from archaeological contexts. An additional ELISA test targeting secretory IgA (sIgA) was conducted to further examine the failure to detect parasite-induced antibodies from quids. Herein, the methods used for quid preparation and ELISA procedures are described so that they can be further developed by future researchers. The results are discussed in light of the potential future of quid analysis.
Antibodies ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin A, Secretory ; Loma ; Methods ; Mexico ; Parasites ; Saliva ; Toxoplasma ; Trypanosoma cruzi

PURPOSE: Salmonella enterica serovar Gallinarum (SG) ghost vaccine candidate was recently constructed. In this study, we evaluated various prime-boost vaccination strategies using the candidate strain to optimize immunity and protection efficacy against fowl typhoid. MATERIALS AND METHODS: The chickens were divided into five groups designated as group A (non-immunized control), group B (orally primed and boosted), group C (primed orally and boosted intramuscularly), group D (primed and boosted intramuscularly), and group E (primed intramuscularly and boosted orally). The chickens were primed with the SG ghost at 7 days of age and were subsequently boosted at the fifth week of age. Post-immunization, the plasma IgG and intestinal secretory IgA (sIgA) levels, and the SG antigen-specific lymphocyte stimulation were monitored at weekly interval and the birds were subsequently challenged with a virulent SG strain at the third week post-second immunization. RESULTS: Chickens in group D showed an optimized protection with significantly increased plasma IgG, sIgA, and lymphocyte stimulation response compared to all groups. The presence of CD4+ and CD8+ T cells and monocyte/macrophage (M/M) in the spleen, and splenic expression of cytokines such as interferon γ (IFN-γ) and interleukin 6 (IL-6) in the immunized chickens were investigated. The prime immunization induced significantly higher splenic M/M population and mRNA levels of IFN-γ whereas the booster showed increases of splenic CD4+ and CD8+ T-cell population and IL-6 cytokine in mRNA levels. CONCLUSION: Our results indicate that the prime immunization with the SG ghost vaccine induced Th1 type immune response and the booster elicited both Th1- and Th2-related immune responses.
Birds ; Chickens ; Cytokines ; Immunization* ; Immunoglobulin A, Secretory ; Immunoglobulin G ; Interferons ; Interleukin-6 ; Lymphocyte Activation ; Plasma ; RNA, Messenger ; Salmonella enterica ; Salmonella* ; Serogroup ; Spleen ; T-Lymphocytes ; Typhoid Fever ; Vaccination