MicroRNA-146a Deficiency Protects against Listeria monocytogenes Infection by Modulating the Gut Microbiota.

The gut microbiota and microRNAs play important roles in the defense against infection. However, the role of miR-146a in L. monocytogenes infection and gut microbiota remains unclear. We tried to determine whether miR-146a controlled L. monocytogenes infection by regulating the gut microbiota. Wild-type and miR-146a-deficient mice or macrophages were used to characterize the impact of miR-146a on animal survival, cell death, bacterial clearance, and gut microbiota following L. monocytogenes challenge. We found that L. monocytogenes infection induced miR-146a expression both in vitro and in vivo. When compared to wild-type mice, miR-146a-deficient mice were more resistant to L. monocytogenes infection. MiR-146a deficiency in macrophages resulted in reduced invasion and intracellular survival of L. monocytogenes. High-throughput sequencing of 16S rRNA revealed that the gut microbiota composition differed between miR-146a-deficient and wild-type mice. Relative to wild-type mice, miR-146a-deficient mice had decreased levels of the Proteobacteria phylum, Prevotellaceae family, and Parasutterella genus, and significantly increased short-chain fatty acid producing bacteria, including the genera Alistipes, Blautia, Coprococcus_1, and Ruminococcus_1. Wild-type mice co-housed with miR-146a-deficient mice had increased resistance to L. monocytogenes, indicating that miR-146a deficiency guides the gut microbiota to alleviate infection. Together, these results suggest that miR-146a deficiency protects against L. monocytogenes infection by regulating the gut microbiota.

Cytosolic double-stranded DNA induces nonnecroptotic programmed cell death in trophoblasts via IFI16.

The mechanisms underlying the immune defense by trophoblasts against pathogens remain ill defined. We demonstrated that placental cell death was increased upon in vivo exposure to Listeria monocytogenes. The death of infected cells is an important host innate defense mechanism. Meanwhile, double-stranded DNA (dsDNA) derived from intracellular bacteria or dsDNA viruses is emerging as a potent pathogen-associated molecular pattern recognized by host cells. We sought to characterize trophoblast death in response to cytosolic dsDNA challenge. Our results showed that dsDNA induced caspase-dependent and -independent cell death in human trophoblasts. However, necroptosis, a cell death pathway independent of caspase, could not be induced by dsDNA treatment, even in the presence of exogenously expressed RIPK3. L. monocytogenes-derived genomic DNA triggered a similar cell death pattern. Moreover, the cell death in response to dsDNA was IFI16 dependent. These data suggest that cytosolic dsDNA induces nonnecroptotic cell death in trophoblasts via IFI16, and this could contribute to placental barrier against infection.

Identification and characterization of Th cell epitopes in MrkD adhesin of Klebsiella pneumoniae.

In this study, we identified the Th epitopes in MrkD of Klebsiella pneumoniae, an excellent vaccine candidate antigen. By using the RANKPEP prediction algorithm, we have identified and characterized three Th epitopes within the MrkD antigen, which can be recognized by CD4+ T cells from BALB/c (H-2(d)) mice. They were M(221-235), M(175-189), and M(264-278). These epitopes have important value for studying the immune response of K. pneumoniae infection and for designing effective vaccine against K. pneumoniae.

DNA Sensor IFI204 Contributes to Host Defense Against Staphylococcus aureus Infection in Mice.

Interferon-inducible protein (IFI204) (p204, the murine homolog of human IFI16) is known as a cytosolic DNA sensor to recognize DNA viruses and intracellular bacteria. However, little is known about its role during extracellular bacterial infection. Here we show that IFI204 is required for host defense against the infection of Staphylococcus aureus, an extracellular bacterial pathogen. IFI204 deficiency results in decreased survival, increased bacterial loads, severe organs damage, and decreased recruitment of neutrophils and macrophages. Production of several inflammatory cytokines/chemokines including IFN-ß and KC is markedly decreased, as well as the related STING-IRF3 and NF-κB pathways are impaired. However, exogenous administration of recombinant KC or IFN-ß is unable to rescue the susceptibility of IFI204-deficient mice, suggesting that other mechanisms rather than KC and IFN-ß account for IFI204-mediated host defense. IFI204 deficiency leads to a defect in extracellular bacterial killing in macrophages and neutrophils, although bacterial engulf, and intracellular killing activity are normal. Moreover, the defect of bactericidal activity is mediated by decreased extracellular trap formation in the absence of IFI204. Adoptively transferred WT bone marrow cells significantly protect WT and IFI204-deficient recipients against Staphylococcus infection compared with transferred IFI204-deficient bone marrow cells. Hence, this study suggests that IFI204 is essential for the host defense against Staphylococcus infection.

A corn straw-based diet increases release of inflammatory cytokines in peripheral blood mononuclear cells of dairy cows.

Recent studies have shown that diet can affect the body's immunity. Roughage of dairy cows consists of a variety of plant materials which make different contributions to health. This study investigated the effect of different roughages on the immunity of dairy cows. Serum, peripheral blood mononuclear cells (PBMCs), and milk samples were collected from 20 multiparous mid-lactation cows fed mixed forage (MF)- or corn straw (CS)-based diets. Expression profile analysis was used to detect the differentially expressed genes (DEGs) from PBMCs. The results showed that milk protein in the MF group increased to 3.22 g/100 ml, while that of the CS group milk was 2.96 g/100 ml; by RNA sequencing, it was found that 1615 genes were differentially expressed between the CS group and the MF group among the 24 027 analyzed probes. Gene ontology (GO) and pathway analysis of DEGs suggested that these genes (especially genes coding cytokines, chemokine and its receptors) are involved in the immune response. Results were confirmed at the protein level via detecting the levels of interleukin-2 (IL-2), IL-6, IL-10, IL-12, leptin (LEP), interferon-γ (IFN-γ), transforming growth factor-ß1 (TGF-ß1), and tumor necrosis factor-α (TNF-α) in peripheral blood by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis. Our data supported the conclusions that the protein content in milk of the MF group was higher than that of the CS group, the CS-based diets induced more release of cytokines than the MF-based diets in dairy cows' PBMCs, and milk protein content may be affected by cytokines.

Non-Hematopoietic MLKL Protects Against Salmonella Mucosal Infection by Enhancing Inflammasome Activation.

The intestinal mucosal barrier is critical for host defense against pathogens infection. Here, we demonstrate that the mixed lineage kinase-like protein (MLKL), a necroptosis effector, promotes intestinal epithelial barrier function by enhancing inflammasome activation. MLKL-/- mice were more susceptible to Salmonella infection compared with wild-type counterparts, with higher mortality rates, increased body weight loss, exacerbated intestinal inflammation, more bacterial colonization, and severe epithelial barrier disruption. MLKL deficiency promoted early epithelial colonization of Salmonella prior to developing apparent intestinal pathology. Active MLKL was predominantly expressed in crypt epithelial cells, and experiments using bone marrow chimeras found that the protective effects of MLKL were dependent on its expression in non-hematopoietic cells. Intestinal mucosa of MLKL-/- mice had impaired caspase-1 and gasdermin D cleavages and decreased interleukin (IL)-18 release. Moreover, administration of exogenous recombinant IL-18 rescued the phenotype of increased bacterial colonization in MLKL-/- mice. Thus, our results uncover the role of MLKL in enhancing inflammasome activation in intestinal epithelial cells to inhibit early bacterial colonization.

Comparison of effectiveness of whole viral, N and N199 proteins by ELISA for the rapid diagnosis of severe acute respiratory syndrome coronavirus.

BACKGROUND: Although severe acute respiratory syndrome (SARS) has been controlled, the subsequently emerging sporadic cases in 2004 emphasize the necessity of developing a rapid diagnostic method, which would be of great help in clinical diagnosis and also wild host screening. This study aims to establish an effective and rapid serological tool for the diagnosis of SARS-CoV by comparison among whole viral, N and N199 proteins by ELISA. METHODS: SARS-CoV N and N199 (a truncated nucleocapsid gene) genes were cloned, expressed, identified by Western blotting, and applied in screening of human and swine samples. Sera of SARS convalescent-phase patients, normal human sera, sera of patients with other respiratory diseases, and swine sera were screened by ELISA, with whole SARS-CoV F69, N and N199 proteins as antigens. RESULTS: The sensitivity and specificity of N and N199 proteins in human sera diagnosis were approximate (P = 0.743), which was higher than whole viral protein but the difference was not significant (P = 0.234). The N199 protein proved to be more specific in swine sera screening than whole viral and N protein (P < 0.001). CONCLUSION: N199 protein is feasible in both clinical diagnosis and SARS-CoV reservoir screening.

An anti-Propionibacterium acnes antibody shows heterologous resistance to an Actinobacillus pleuropneumoniae infection independent of neutrophils in mice.

Porcine contagious pleuropneumonia is a highly fatal respiratory disease that is caused by Actinobacillus pleuropneumoniae (APP) and results in tremendous economic losses for the pig breeding industry worldwide. Previous studies have demonstrated that Propionibacterium acnes (PA) could effectively prevent APP infection in mice and pigs. The humoral immune response played a primary role during this process and anti-PA antibody could mediate macrophages to kill the bacteria. However, the role of neutrophils in this process is currently unknown. In this study, mice were injected with cyclophosphamide to deplete neutrophils and then passively immunized with anti-PA serum or negative serum. Mice were subsequently challenged with APP serotype 1. The results showed that the mice exhibited less bacterial colonization, less lung damage, and a high survival rate, which were immunized with the anti-PA antibody whether neutrophils were depleted or not. Worse still, the presence of neutrophils increased the damage to the mice after challenge. These results suggest that the activity of the anti-PA antibody against APP infection was independent of neutrophils. These findings have important significance for understanding the mechanisms of humoral immunity conferred by heterologous immunization and lay a good foundation for preventing APP infection.

Genipin inhibits NLRP3 and NLRC4 inflammasome activation via autophagy suppression.

Inflammasomes are cytoplasmic, multiprotein complexes that trigger caspase-1 activation and IL-1ß maturation in response to diverse stimuli. Although inflammasomes play important roles in host defense against microbial infection, overactive inflammasomes are deleterious and lead to various autoinflammatory diseases. In the current study, we demonstrated that genipin inhibits the induction of IL-1ß production and caspase-1 activation by NLRP3 and NLRC4 inflammasomes. Furthermore, genipin specifically prevented NLRP3-mediated, but not NLRC4-mediated, ASC oligomerization. Notably, genipin inhibited autophagy, leading to NLRP3 and NLRC4 inflammasome inhibition. UCP2-ROS signaling may be involved in inflammasome suppression by genipin. In vivo, we showed that genipin inhibited NLRP3-dependent IL-1ß production and neutrophil flux in LPS- and alum-induced murine peritonitis. Additionally, genipin provided protection against flagellin-induced lung inflammation by reducing IL-1ß production and neutrophil recruitment. Collectively, our results revealed a novel role in inhibition of inflammatory diseases for genipin that has been used as therapeutics for centuries in herb medicine.

Staphylococcus aureus infection of intestinal epithelial cells induces human umbilical cord-derived mesenchymal stem cell migration.

There is a growing interest in umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) for cellular therapy in regenerative medicine. To aid in tissue repair, MSCs are recruited to sites of inflammation induced by a bacterial infection. The primary objective of this study was to explore the mechanisms of MSC recruitment to intestinal epithelial cells infected with Staphylococcus aureus. First, we isolated and characterized the UCB-derived MSCs used in our experiments. Next, we determined the ability of S. aureus infected intestinal epithelial cells to induce migration of UCB-derived MSCs. Expression analysis of cytokines secreted by infected epithelial cells indicated that MSC migration occurred predominately via a nuclear factor-kappa B (NF-κB)-dependent signaling pathway. Altogether, our data provide the first evidence for a role of S. aureus infection in MSC migration and reveal the function of UCB-derived MSCs in intestinal pathophysiology.

Identification and characterization of antigenic epitope of Staphylococcus aureus ClfA adhesin.

Staphylococcus aureus ClfA adhesin is a protective antigen that induces partial immunity against S. aureus infection in mice. To identify the antigenic epitope of ClfA, a monoclonal antibody (mAb) D01 against the recombinant protein was produced by the hybridoma technique. The mAb was used to immunoscreen a random phage-displayed peptide library as the immunogen. After three rounds of biopanning, 41 positive clones were identified. Sixteen phage clones were sequenced and their amino acids were deduced. One mimotope (SKVGIDKRRGTA) showed good match with ClfA adhesin at 383-394 aa and the serum of mice induced by the phage clone clearly recognized ClfA adhesin.

Different transcriptional profiles of RAW264.7 infected with Mycobacterium tuberculosis H37Rv and BCG identified via deep sequencing.

BACKGROUND: The Mycobacterium tuberculosis H37Rv and BCG effects on the host cell transcriptional profile consider a main research point. In the present study the transcriptome profiling analysis of RAW264.7 either infected with Mycobacterium tuberculosis H37Rv or BCG have been reported using Solexa/Illumina digital gene expression (DGE). RESULTS: The DGE analysis showed 1,917 different expressed genes between the BCG and H37Rv group. In addition, approximately 5% of the transcripts appeared to be predicted genes that have never been described before. KEGG Orthology (KO) annotations showed more than 71% of these transcripts are possibly involved in approximately 210 known metabolic or signaling pathways. The gene of the 28 pathways about pathogen recognition receptors and Mycobacterium tuberculosis interaction with macrophages were analyzed using the CLUSTER 3.0 available, the Tree View tool and Gene Orthology (GO). Some genes were randomly selected to confirm their altered expression levels by quantitative real-time PCR (qRT-PCR). CONCLUSION: The present study used DGE from pathogen recognition receptors and Mycobacterium tuberculosis interaction with macrophages to understand the interplay between Mycobacterium tuberculosis and RAW264.7. Meanwhile find some important host protein which was affected by Mycobacterium tuberculosis to provide evidence for the further improvement of the present efficacy of existing Mycobacterium tuberculosis therapy and vaccine.

[Comparison of plaque-forming characteristics and morphogenetic changes of Newcastle disease virus isolated from chickens and geese on host cells].

The plaque-forming characteristics of Newcastle disease viruses of chickens and geese source were compared on various cells. The result showed that there were obvious differences of plaque formation between F48E9 and NA-1 on Vero cells, chicken embryo fibroblast cells (CEF) and goose embryo fibroblast cells (GEF). The plaque-forming ability of NA-1 was higher than F48E9 on GEF, but lower than F48E9 on CEF. On Vero cells, the plaque-forming ability of NA-1 was slightly stronger than F48E9. It demonstrated that the plaque-forming characteristics were consistent with host tropism of virus. The morphogenesis of F48E9 and NA-1 on Vero cells was observed with transmission electron microscope. There were different replication processes between F48E9 and NA-1 on cells at different stages. NA-1 had stronger adaptability to host than F48E9 according to budding processes and envelope integrity.

Klebsiella pneumoniae MrkD adhesin-mediated immunity to respiratory infection and mapping the antigenic epitope by phage display library.

In this study, the K. pneumoniae MrkD adhesin was identified an immunodominant antigen which correlated with protection against infection by K. pneumoniae. The mouse monoclonal antibodies (mAbs) against MrkD adhesin were produced by the hybridoma technique using recombinant MrkD-GST as the immunogen and were immunoscreened against phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 36 phage clones were randomly selected and their specificity to mAb was verified by sandwich and competitive inhibition ELISA. Sixteen phage clones were sequenced and their amino acids were deduced. One mimotope (QKTLAKSTYMSA) showed good match with MrkD adhesion at 148-159 aa and the serum of mice induced by the phage clone clearly recognized MrkD adhesion.

[Salmonella choleraesuis C500 delivering DNA immunization against classical swine fever virus].

Classical Swine Fever Virus (CSFV) E2 protein eukaryotic expression plasmid pVAXE2 was constructed. The plasmid pVAXE2 was transformed into Salmonella choleraesuis C500 (S. C500) attenuated vaccine strain by electroporation to generate Salmonella choleraesuis engineering strain S. C500/pVAXE2. The characterization of S. C500/pVAXE2 in morphology, growth, biochemistry and serology indicated that it retained the same properties as its original strain S. C500 with exception of kanamycin resistance originated from the plasmid pVAXE2. The plasmid stable in the bacteria after 15 passages. Kunming mice and rabbits were vaccinated three times at two weeks interval with S. C500/pVAXE2 in oral and intramuscular routes at the dosage of 1 x 10(8) CFU for mice and 2 x 10(9) CFU for rabbits each time. The specific antibody response against CSFV and Salmonella choleraesuis was detected by ELISA. Two weeks after the third boost the immunized rabbits were challenged with 20 ID50 of hog cholera lapinized virus (HCLV), followed by a virulent strain of Salmonella choleraesuis two week later than HCLV challenge. The results showed that all immunized mice and rabbits produced significant antibodies against CSFV and Salmonella choleraesuis, and the immunized rabbits demonstrated the effective protection against the challenge of HCLV and virulent Salmonella choleraesuis. These results indicated the potential of developing multiplex swine DNA vaccine by using this bacteria as the vector.

Preparation and development of equine hyperimmune globulin F(ab')2 against severe acute respiratory syndrome coronavirus.

AIM: The resurgence of severe acute respiratory syndrome (SARS) is still a threat because the causative agent remaining in animal reservoirs is not fully understood, and sporadic cases continue to be reported. Developing high titers of anti-SARS hyperimmune globulin to provide an alternative pathway for emergent future prevention and treatment of SARS. METHODS: SARS coronavirus (CoV)F69 (AY313906) and Z2-Y3 (AY394989) were isolated and identified from 2 different Cantonese onset SARS patients. Immunogen was prepared from SARS-CoV F69 strain. Six health horses were immunized 4 times and serum was collected periodically to measure the profile of specific IgG and neutralizing antibodies using indirect enzyme-linked immunosorbent assay and a microneutralization test. Sera were collected in large amounts at the peak, where IgG was precipitated using ammonium sulphate and subsequently digested with pepsin. The product was then purified using anion-exchange chromatography to obtain F(ab')2 fragments. RESULTS: The specific IgG and neutralizing antibody titers peaked at approximately week 7 after the first immunization, with a maximum value of 1:14210. The sera collected at the peak were then purified. Fragment of approximately 15 g F(ab')2 was obtained from 1litre antiserum and the purity was above 90% with the titer of 1:5120, which could neutralize the other strain (SARS-CoV Z2-Y3) as well. CONCLUSION: This research provides a viable strategy for the prevention and treatment of SARS coronavirus infection with equine hyperimmune globulin, with the purpose of combating any resurgence of SARS.

[Construction and Identification of DNA vaccine containing chimeric gene gag-gp120 of HIV-1].

AIM: To construct DNA vaccine expression plasmid containing the chimeric gene gag-gp120 of HIV-1. METHODS: The recombinant eukaryotic expression vector pVAXGE was constructed via inserting the chimeric gene gag-gp120 into the vector pVAX1. Hela cells had been transfected by recombinant plasmid via liposome. After 72 h, the transfected cells was detected by RT-PCR and analyzed by Dot-ELISA. RESULTS: The transcript products of target gene could be amplified from the cells transfected by recombinant plasmid. Dot-ELISA detection showed that the target gene was expressed in Hela cells. CONCLUSION: The DNA vaccine plasmid expressing chimeric gene gag-gp120 was successfully constructed, which lays the foundation for preparing DNA vaccine against HIV-1.

[Construction of recombinant fowlpox virus coexpressing gp120 of Chinese HIV-1 strain and IL-18 and its immunogenicity in mice].

To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.