Dendritic cells (DCs) are potent antigen-presenting cells and generated from diverse sources including monocytes which are known to be the sites for human cytomegalovirus (HCMV) latency. HCMV has been known to suppress or evade from immune functions involving monocytes and DC. Thus, it was attempted to investigate the effect of HCMV infection on the differentiation of DCs from monocytes. Monocytes were prepared from peripheral blood mononuclear cells and they expressed high levels of CD14, CD11a, CD11b, CD11c and HLA molecules, while they did not express lymphocyte-specific CD molecules. The surface expressions of CD molecules and HLA in immature DCs (imDCs) differentiated from HCMV-infected monocytes differed from those in uninfected imDCs. Specifically, the expressions of CD11a, CD11b, CD11c, CD40 and HLA-DR were decreased compared to uninfected imDCs, while CD80 expression was increased in imDCs differentiated from HCMV-infected monocytes. DCs were allowed to mature (mDCs) by treating imDCs with interferon gamma and LPS. When HCMV-infected imDCs were differentiated to mDCs, the expression of mDC-specific CD83 as well as HLA molecules were decreased. Thus, our results suggest that HCMV inhibits phenotytpic differentiation of DCs.
Antigen-Presenting Cells ; Cytomegalovirus* ; Dendritic Cells* ; HLA-DR Antigens ; Humans* ; Interferons ; Monocytes

Invasive aspergillosis is the most common Aspergillus fumigatus infection in immunocompromised patients. Although the treatment of the invasive aspergillosis has been mostly relied on antifungal agents, there still exists the need for more effective therapy. To develop cellular immunotherapy specific for Aspergillus fumigatus, we generated specific T cells using dendritic cells (DCs) pulsed with an Aspergillus fumigatus derived recombinant protein in vitro and examined their functions. The f16. p2+3 region containing the conserved region of Asp f16 gene was cloned from Aspergillus fumigatus and the recombinant protein was produced in E. coli. IFN-gamma secretion from the T cells stimulated with recombinant f16. p2+3 (rf16. p2+3) was measured by enzyme linked immunospot (ELISPOT) assay and the cytolytic activity of the stimulated T cells by 51Cr release assay. The number of IFN-gamma secreting cells were significantly increased in the peripheral blood mononuclear cells (PBMCs) stimulated with the rf16. p2+3 pulsed DCs (31+/-12 spots/10(4) cells), compared to that of PBMCs directly stimulated with rf16. p2+3 (83+/-15 spots/10(6) cells). IFN-gamma ELISPOT assay using purified CD4+ or CD8+ as responder cells showed that CD4+ T cells (43 spots/10(4) cells) mainly produced IFN-gamma compared with CD8+ T cells (7 spots/10(4) cells). Furthermore, helper T cells specific for rf16. p2+3 could be efficiently generated by the stimulation with DCs for two weeks. However, cytotoxic T lymphocyte activity was not induced. Our results suggest that the rf16. p2+3 protein could be used for the generation of helper T cells in vitro.
Antifungal Agents ; Aspergillosis ; Aspergillus fumigatus* ; Aspergillus* ; Clone Cells ; Dendritic Cells* ; Enzyme-Linked Immunospot Assay ; Immunocompromised Host ; Immunotherapy ; Lymphocytes ; T-Lymphocytes* ; T-Lymphocytes, Helper-Inducer ; Viperidae*

Recently we have shown that a bacterial flagellin, Vibrio vulnifiucs FlaB (Vv-FlaB), has a strong adjuvant activity to induce protective immune response. In order to investigate the adjuvanticity of Vv-FlaB, we prepared highly purified recombinant protein by using an intein fusion protein purification system. However, in the process of the purification, we unexpectedly encountered a contamination with a 70 kDa protein. We proved the 70 kDa protein as the heat shock protein 70 (HSP70) by Western blotting. Unfortunately, it was reported that the HSP70 has a strong adjuvanticity. In this study we investigated the role of contaminating HSP70 on the Vv-FlaB-mediated adjuvanticity. We separated Vv-FlaB and HSP70 by using a high performance protein purification chromatography and compared adjuvant activities of Vv-FlaB, HSP70 and Vv-FlaB/HSP70 mixture. Using an intranasal immunization mouse model, we observed that co-administration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific antibody (Ig) responses. However contaminating doses of HSP70 did not affect the adjuvanticity of Vv-FlaB and furthermore HSP70 alone did not enhance TT-specific Ig response and protective immunity against lethal challenge with tetanus toxin. These results show that the HSP70 contaminating Vv-FlaB preparations did not affect the adjuvanticity of Vv-FlaB.
Animals ; Blotting, Western ; Chromatography ; Flagellin* ; Heat-Shock Proteins* ; Hot Temperature* ; HSP70 Heat-Shock Proteins* ; Immunization ; Inteins ; Mice ; Staphylococcal Protein A ; Tetanus Toxin ; Tetanus Toxoid ; Vibrio vulnificus ; Vibrio*

Salmonellosis is one of the most common food born diseases in Korea. However, it takes more than 8 days and many expensive antiserums are used for the identification of Salmonella serovars since the microorganism easily undergoes phase variation. According to the data that 65.5% of Salmonella isolates in 2000~2004 year had monophasic flagella, we have developed a rapid serological identification method using a hin gene-specific polymerase chain reaction (PCR) for monophasic Salmonella isolates that does not require the time-consuming phase conversion experiments. Using our new method, 'hin specific PCR-based serological test', we could identify serovars of monophasic Salmonella in 4 days. For the purpose of rapid identification of salmonella serovars collected from outbreaks and sporadic cases, hin specific PCR-based serological tests will be a fast and efficient method.
Disease Outbreaks ; Flagella ; Korea ; Polymerase Chain Reaction* ; Salmonella Infections ; Salmonella* ; Serologic Tests

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates slowly in the host cytoplasm. To investigate the changes of antigen expression during six days of in vitro growth, the reactivity of various monoclonal antibodies (MAbs) was examined in time course. Using immunofluorescence staining, some antigens were shown to be differentially expressed in contrast to 56-kDa protein that was produced at a constant level throughout culture. Three MAbs (NT1, M716B and M716G) revealed antigens, appearing only at 3 days after infection. MAb NT19 recognized an antigen that appeared mainly at the late stage of infection whereas two MAbs (M686-13 and M686-20) demonstrated antigens, being expressed at the early infection stage and showing distinct morphology under immunofluorescence staining. In addition, when cells were infected and then treated with chloramphenicol, granular cytoplasmic bodies were detected and the bacterial development was inhibited. These results show that O. tsutsugamushi changes the antigenic expression to adapt to and replicate in host cells.
Antibodies, Monoclonal* ; Chloramphenicol ; Cytoplasm ; Fluorescent Antibody Technique ; Orientia tsutsugamushi* ; Scrub Typhus

Persistence is dormant phenotypic variants of regular cells that are tolerant to antibiotics. The persistent cells did not acquire antibiotic resistance genetically, being produced in response to antibiotic stress. Because of dormant phenotypic variants due to little or no cell-wall synthesis, translation, or topoisomerase activity, persistent cells show antibiotic tolerance. Recently, such persistent cells have been reported in many bacterial pathogens and are known to play significant roles in clinical settings, particularly in chronic diseases such as cystic fibrosis. Therefore, development of anti-persister drug and appropriate antibiotic treatment are required to eliminate the persisters and to prevent the development of antibiotic resistance. Screening of genes related to persister formation would lead to new drugs to combat persisters during infection. By reviewing recent publications, we summarize phenomenon of survival and tolerance in persistent cells.
Anti-Bacterial Agents ; Chronic Disease ; Cystic Fibrosis ; Drug Resistance, Microbial ; Mass Screening

Vast array of microbes colonize to each anatomical environment of human body. Culture based methods are important in investigating the microbial structure, but they are extremely biased in their evaluation of microbial diversity by selecting particular population of microbiota. Recent advance in molecular technology has allowed sophisticated analysis of complex human microbiota by culture-independent methods. Here, we will discuss features of tools for human microbiota studies including Roche-454 and Illumina platform. We will also briefly discuss features of some strategies that are commonly applied to these platforms including 16S rRNA targeting and shotgun sequencing. New platforms such as PacBio and Oxford Nanopore are also introduced.
Bias (Epidemiology) ; Colon ; Human Body ; Humans ; Metagenome ; Nanopores

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) play important role in immune responses by regulating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), type I interferon and inflammasomes. Nod-like receptor pyrin domain-containing protein 6 (NLRP6) is vital for protection of mice from colitis and colorectal tumorigenesis. However, the role of NLRP6 in microbial infection remains unclear. NLRP6 was recently reported as negative regulator of inflammatory signaling and demonstrated that how it hinders clearance of bacterial pathogens.
Animals ; B-Lymphocytes ; Cell Transformation, Neoplastic ; Colitis ; Cytoskeletal Proteins ; Immunity, Innate ; Inflammasomes ; Interferon Type I ; Listeria monocytogenes ; Mice

We have previously observed that a sequence in coat protein (CP) ORF of Turnip yellow mosaic virus (TYMV) is required for efficient replication of the virus. The sequence was predicted to take a stem-loop structure, thus termed SL2. While examining various SL2 mutants, we observed that all the modifications resulting in extension of translation beyond the CP ORF significantly suppressed subgenomic RNA accumulation. The genomic RNA level, in contrast, was not affected. Introduction of an in-frame stop codon in the CP ORF of these constructs restored the level of subgenomic RNA. Overall, the results suggest that the read-through makes the subgenomic RNA unstable.
Animals ; Brassica napus ; Codon, Terminator ; Ecthyma, Contagious ; RNA ; Tymovirus ; Viruses

15-deoxy-delta12,14 prostaglandin J2 (15d-PGJ2) may hold promise in treatment of the pathologies associated with human immunodeficiency virus (HIV) infection of the central nervous system. However, its precise role and neuroprotective mechanism in the hippocampus remain poorly understood. In the present study, rat hippocampal slices were stimulated with HIV-1 Tat protein to investigate the protective role of 15d-PGJ2 on the hippocampal cytotoxicity. Full-length HIV-1 Tat protein (Tat1-86), but neither its Tat32-62 nor Tat30-86 fragment, significantly induced cytotoxicity in the hippocampus, the brain region most commonly damaged in HIV-associated dementia. This Tat-induced cytotoxicity was associated with inactivation of MEK/extracellular signal-regulated kinase (ERK) signaling pathway. In contrast, Tat1-86 did not alter Wnt signaling pathway necessary for cell survival. Pretreatment of slices with 15d-PGJ2 markedly reduced Tat-driven cytotxicity. Interestingly, this reduction was accompanied by suppression of ERK inactivation in response to Tat. Moreover, the inhibition of the MEK/ERK pathway with SL327 enhanced the Tat-induced cytotoxicity, confirming the ERK-dependent mechanism of Tat-driven cytotoxicity. Collectively, these data demonstrate that the protective action of 15d-PGJ2 against the hippocampal cytotoxicity upon Tat stimulation is exerted through suppression of Tat-mediated ERK1/2 inactivation.
Animals ; Brain ; Cell Survival ; Central Nervous System ; Dementia ; Gene Products, tat ; Hippocampus ; HIV ; HIV-1 ; Phosphotransferases ; Prostaglandin D2 ; Rats ; Wnt Signaling Pathway