BACKGROUND: Burn wounds lack normal barriers that protect against pathogenic bacteria, and burn patients are easily colonized and infected by Staphylococcus aureus. Toxic shock syndrome (TSS) is a rare but fatal disease caused by S. aureus. A lack of detectable antibodies to TSS toxin-1 (TSST-1) in serum indicates susceptibility to TSS. METHODS: A total of 207 patients (169 men and 38 women; median age, 42.5 yr) admitted to a burn center in Korea were enrolled in this study. The serum antibody titer to TSST-1 was measured by sandwich ELISA. S. aureus isolates from the patients' nasal swab culture were tested for TSST-1 toxin production by PCR-based detection of the TSST-1 toxin gene. RESULTS: One hundred seventy-four (84.1%) patients showed positive results for antibody against TSST-1. All patients aged > or =61 yr (n=28) and <26 months (n=7) were positive for the anti-TSST-1 antibody. S. aureus was isolated from 70 patients (33.8%), and 58.6% of the isolates were methicillin resistant. Seventeen patients were colonized with TSST-1-producing S. aureus. The antibody positivity in these 17 carriers was 88.2%, and the positivity in the non-carriers was 83.7%. CONCLUSIONS: Most burn patients had antibody to TSST-1, and nasal colonization with TSST-1-producing S. aureus was associated with positive titers of anti-TSST-1 antibody. Additionally, patients with negative titers of anti-TSST-1 antibody might be susceptible to TSS.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Bacterial/*blood ; Bacterial Toxins/genetics/immunology/*metabolism ; Burns/blood/*immunology/*microbiology/pathology ; Child ; Child, Preschool ; Enterotoxins/genetics/immunology/*metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Infant ; Male ; Middle Aged ; Nasal Cavity/microbiology ; Polymerase Chain Reaction ; Prevalence ; Staphylococcal Infections/epidemiology ; Staphylococcus aureus/isolation & purification/*metabolism ; Superantigens/genetics/immunology/*metabolism ; Young Adult

OBJECTIVETo explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro.

METHODSAfter BMDCs stimulated by E.coli LLO/OVA, their Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) receptor signalling pathway were examined by superarray hybridization; and the priming effect of the vaccine activated BMDCs on CD4(+)T and CD8(+)T was determined by [3H]thymidine uptake and ELISA, the tumor cytotoxic effect of activated CD8(+)T cells was determined by cytotoxic assay.

RESULTSAfter BMDCs were activated by E. coli LLO/OVA via TLR4, NOD1 receptor and NF-κB signalling pathway, the expression of their surface molecules including MHC class I, MHC class II, CD40, CD80 and CD86 significantly up-regulated; the secretion of IL-12 and IFN-γ increased also. The mature BMDCs stimulated the allergic CD4(+)T and CD8(+)T cells proliferation and their IL-2 and IFN-γ secretion, and the activated CD8(+)T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.

CONCLUSIONE.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.

Animals ; Antigens, Neoplasm ; genetics ; pharmacology ; Bacterial Toxins ; genetics ; pharmacology ; Cancer Vaccines ; genetics ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; immunology ; Coculture Techniques ; Cytokines ; immunology ; secretion ; Dendritic Cells ; cytology ; drug effects ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Female ; Flow Cytometry ; Heat-Shock Proteins ; genetics ; pharmacology ; Hemolysin Proteins ; genetics ; pharmacology ; Immunity, Innate ; drug effects ; Mice ; Mice, Inbred C57BL ; Nod1 Signaling Adaptor Protein ; genetics ; physiology ; Ovalbumin ; genetics ; pharmacology ; Recombinant Fusion Proteins ; genetics ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Toll-Like Receptor 4 ; genetics ; physiology

In order to study the pathogenesis of Listeria monocytogenes (LM), we cloned listeriolysin gene into prokaryotic expression vector PET21a. The expression vector was transformed into Escherichia coli BL21 for expression of listeriolysin O (LLO). LLO-His tag fusion protein was purified with a Ni-NTA affinity column and was used as an immunogen to vaccinate BALB/C mice. Hybridomas were developed by fusing mouse myeloma cells Sp2/0 and splenocytes from the immunized mice and screened with purified LLO. Three hybridomas secreting antibodies against listeriolysin O were obtained and named anti-LLO1, anti-LLO2 and anti-LLO3, respectively. Western blotting analysis showed that all of them could specifically bind to the LLO secreted by the LM. The titers of anti-LLO monoclonal antibodies in the supernatants of three hybridomas cultures were 1:3.6 x 10(4), 1:6.4 x 10(4) and 1:1.6 x 10(4), respectively, and the titers of ascites from the hybridoma-injected mice were 1:2 x 10(7), 1:2 x 10(7) and 1:1 x 10(7), respectively, based on ELISA test. The isotypes of the monoclonal antibodies were determined to be IgG1. The dissociation constants (Kd) of these three monoclonal antibodies were determined to be 6.18 x 10(-11), 7.50 x 10(-11) and 6.27 x 10(-11) respectively. These data and reagents will be of great assistance to elucidate the pathogenesis of Listeriosis.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Bacterial Toxins ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Heat-Shock Proteins ; biosynthesis ; genetics ; immunology ; Hemolysin Proteins ; biosynthesis ; genetics ; immunology ; Listeria monocytogenes ; genetics ; pathogenicity ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

LTB gene fragment was amplified by PCR from plasmid pMDTLT, and a recombinant plasmid pETLTBVP1 was constructed by inserting LTB gene fragment into VP1 gene expression plasmid pETVP1 constructed previously. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The recombinant protein existed in the inclusion body and its molecular weight was about 39 kD proved by SDS-PAGE analysis. Western blotting showed that the fusion protein could be reacted with both anti-FMDV and anti-cholera toxin serum demonstrating the immunoactivity of the fusion protein. Strong immune responses can be induced in mice inoculated with the fusion protein intraperitoneally, and the serum antibody level is higher than that of commercial foot-and-mouth disease vaccines.
Animals ; Antibodies, Viral ; blood ; Bacterial Toxins ; genetics ; immunology ; metabolism ; Capsid Proteins ; genetics ; immunology ; metabolism ; Enterotoxins ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; Female ; Gene Fusion ; genetics ; Mice ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

BACKGROUND: Enzyme immunoassay (EIA) capable of detecting both toxin A and toxin B is strongly recommended for the diagnosis of Clostridium difficile associated disease. Therefore, we evaluated two different EIAs for the detection of C. difficile toxin A/B. METHODS: For a total of 228 stool specimens we performed bacteriologic cultures for C. difficile and examined for toxin A and toxin B using enzyme linked fluorescent immunoassay (ELFA; VIDAS CDAB, Bio-Merieux sa, France) and ELISA (C.DIFFICILE TOX A/B II, TECHLAB, USA). We also performed PCR assays for toxin A and B genes in 117 C. difficile isolates that grew from the stool cultures and compared the results with those obtained with the two different EIAs. RESULTS: The concordance rate between ELFA and ELISA was 85.5% (195/228). Using the culture and PCR results as the standard, the sensitivity/specificity of the ELFA and ELISA were 65.0%/72.1% and 71.8%/70.3%, and for positive/negative predictive values were 78.4%/69.6% and 71.8%/70.3%, respectively (P value >0.05). No differences were observed between the results of ELFA and ELISA with toxin A- toxin B+ strains of C. difficile. CONCLUSIONS: The sensitivity of the ELISA was slightly higher than that of ELFA for toxin A and toxin B, but the specificity and positive predictive value of the ELFA were rather higher than those of the ELISA, although no statistical differences were observed. A bacteriologic culture and PCR assay for toxin genes are recommended in case the both EIAs are negative.
Bacterial Proteins/*analysis/genetics/immunology ; Bacterial Toxins/*analysis/genetics/immunology ; Clostridium difficile/genetics/isolation & purification/*metabolism ; Enterotoxins/*analysis/genetics/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Feces/microbiology ; Fluorescent Dyes/chemistry ; Humans ; Reagent Kits, Diagnostic

It has been well established that bacterial superantigens lead to the induction and aggravation of chronic inflammatory skin diseases. We investigated the clinical significance of serum specific immunoglobulin E (lgE) to the staphylococcal superantigens staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), and toxic shock syndrome toxin (TSST)-1 in patients with chronic urticaria (CU), focusing on the differences in these prevalences between aspirin-intolerant CU (AICU) and aspirin-tolerant CU (ATCU) patients. Aspirin sensitivity was confirmed by oral aspirin provocation test. There were 66 patients AICU and 117 patients ATCU in the study. Serum IgE antibodies specific for SEA, SEB, and TSST-1 were measured by the ImmunoCAP test and the patients were compared with 93 normal controls (NC). The prevalences of serum specific IgE to staphylococcal superantigens were significantly higher in CU than in NC patients (IgE to SEA, 13.7% vs. 5.4%; IgE to SEB, 12.0% vs. 4.3%; IgE to TSST-1, 18.0% vs. 6.5%; p<0.05, respectively). The patients with specific IgE to SEA, SEB, and TSST-1 had higher serum total IgE levels and higher rates of atopy. Significant associations were noted between the prevalence of specific IgE to SEA and SEB and the HLA DQB1*0609 and DRB1*1302 alleles in the AICU group. We confirmed that a sub-population of patients with CU possesses serum IgE antibodies to SEA, SEB, and TSST- 1. Particularly, the IgE immune response to TSST-1 is associated with aspirin sensitivity in CU patients.
Adolescent ; Adult ; Aged ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Antigens/*chemistry ; Aspirin/pharmacology ; Bacterial Toxins/metabolism ; Chronic Disease ; Enterotoxins/metabolism ; Female ; Humans ; Immunoglobulin E/*chemistry/metabolism ; Male ; Middle Aged ; Phenotype ; Staphylococcus/*genetics/immunology ; Superantigens/metabolism ; Urticaria/*immunology

In order to amplify alpha toxin gene of Clostridium septicum HeB01 strain, one pair of primers was designed according to the GenBank sequence, and a 1323bp alpha toxin gene fragment was obstained by PCR. Sequence analysis indicated that the homology of the nucleotid sequence of HeB01 strain to those other reference strains was more than 99.5% . The expression plasmid pQE30-alpha was constructed by inserting alpha toxin gene into the prokaryotic expression vector pQE30. The plasmid expressed when the recombinant strain M15(pQE30-alpha) was induced by IPTG. The specific 48 kD protein was detected SDS-PAGE and the immunogenicity of the expressed alpha toxin was confirmed by Western blot and ELISA. The expressed alpha toxin was transformed into alpha toxoid vaccine by adding 0.3% formaldehyde into alpha toxin. The protective immune response was proved after the mice was immunized with alpha toxoid vaccine. The results showed that the recombinanted strain M15 (pQE30-alpha) could be as a candidate of alpha toxoid vaccine to provide protective immune response against clostridium septicum infection.
Animals ; Antibodies, Bacterial ; blood ; immunology ; Bacterial Toxins ; genetics ; immunology ; metabolism ; Blotting, Western ; Cloning, Molecular ; Clostridium Infections ; immunology ; microbiology ; prevention & control ; Clostridium septicum ; genetics ; immunology ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Gene Expression Regulation, Bacterial ; Mice ; Polymerase Chain Reaction ; Recombinant Proteins ; administration & dosage ; immunology ; metabolism ; Toxoids ; immunology ; Vaccination

A completely synthetic gene encoding the He domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287 bp, 429 aa, -50 kD) was constructed with oligonucleotides. After expressed in Escherichia coli, soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36%-53% of soluble total proteins) and the purified yield was more than 30 mg/L by one-step purification. Then, the purified AHc was used to vaccinate Balb/c mice, which developed a strong and specific immune response as expected following administration of AHe protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble, stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab, but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against botulinum toxin serotype A.
Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; genetics ; immunology ; Botulinum Toxins, Type A ; biosynthesis ; genetics ; immunology ; Botulism ; immunology ; prevention & control ; Clostridium botulinum type A ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; T-Lymphocytes ; immunology ; Vaccines, DNA ; genetics ; immunology

Staphylococcus aureus may perform an crucial function in atopic dermatitis (AD), via the secretion of superantigens, including staphylococcal enterotoxins (SE) A or B, and toxic shock syndrome toxin-1 (TSST-1). Dysregulated cytokine production by keratinocytes (KCs) upon exposure to staphylococcal superantigens (SsAgs) may be principally involved in the pathophysiology of AD. We hypothesized that lesional KCs from AD may react differently to SsAgs compared to nonlesional skin or normal skin from nonatopics. We conducted a comparison of HLA-DR or CD1a expression in lesional skin as opposed to that in nonlesional or normal skin by immunohistochemistry (IHC). We also compared, using ELISA, the levels of IL-1alpha, IL-1beta, and TNF-alpha secreted by cultured KCs from lesional, nonlesional, and normal skin, after the addition of SEA, SEB and TSST-1. IHC revealed that both HLA-DR and CD1a expression increased significantly in the epidermis of lesional skin versus nonlesional or normal skin in quite a similar manner. IL-1alpha, IL-1beta, and TNF-alpha secretion was also significantly elevated in the cultured KCs from lesional skin after the addition of SsAgs. Our results indicated that KCs from lesional skin appear to react differently to SsAgs and increased proinflammatory cytokine production in response to SsAgs may contribute to the pathogenesis of AD.
Tumor Necrosis Factor-alpha/biosynthesis/genetics ; *Superantigens/administration & dosage/immunology ; Staphylococcus aureus/*immunology/pathogenicity ; Male ; Keratinocytes/immunology/*microbiology ; Interleukin-1/biosynthesis/genetics ; Inflammation Mediators/metabolism ; Humans ; HLA-DR Antigens/metabolism ; Enterotoxins/administration & dosage/immunology ; Dermatitis, Atopic/etiology/immunology/*microbiology ; DNA, Complementary/genetics ; Case-Control Studies ; Base Sequence ; Bacterial Toxins/administration & dosage/immunology ; Antigens, CD1/metabolism ; Adult

ApxI is one of the most important virulence factors of Actinobacillus pleuropneumoniae (APP). To study the immunogenicity of the ApxI, the complete coding sequence (3146bp) and its 5'-terminal 1140 bp fragment of the apxIA gene were separately cloned into the prokaryotic expression vector pET-28a, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The expression products, rApxIA and rApxIAN, were present in a form of inclusion bodies and showed the same immunological reactivity as natural ApxI (nApxI) in Western-blot analysis. BALB/c mice were intraperitoneally immunized with the rApxIA, rApxIAN and nApxI respectively. The serum antibody levels of the rApxIAN immunized mice were significantly lower than those immunized with rApxIA or nApxI in an ApxI-specific ELISA, but serum neutralization test demonstrated that immunized mice with rApxIAN, rApxIA and nApxI could generate similar levels of antibodies neutralizing the hemolytic activity of the natural ApxI. The rApxIAN was able to elicite 80% protection rate against APP serovar 1 and 100% against serovar 2 when challenged at a dose of one LD50 after 2 weeks of boost immunization.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; Animals ; Antibodies ; blood ; Bacterial Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Cytotoxins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hemolysin Proteins ; genetics ; immunology ; Inclusion Bodies ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Peptides ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology