Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.
Adjuvants, Immunologic/administration & dosage* ; Administration, Intranasal ; Animal ; Bacterial Toxins/immunology* ; Bacterial Toxins/genetics ; Bacterial Toxins/administration & dosage ; Enterotoxins/immunology* ; Enterotoxins/genetics ; Enterotoxins/administration & dosage ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli* ; Feces ; Female ; Gastric Mucosa/microbiology ; Gastric Mucosa/immunology* ; Helicobacter pylori ; Human ; IgA, Secretory/immunology* ; IgG/immunology ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; NAD+ ADP-Ribosyltransferase/immunology ; NAD+ ADP-Ribosyltransferase/genetics ; Nasal Mucosa/immunology* ; Point Mutation ; Urease/immunology* ; Urease/administration & dosage ; Vaccination*

Adjuvants, Immunologic ; administration & dosage ; Administration, Intranasal ; Animals ; Antibodies, Viral ; blood ; Bacterial Toxins ; administration & dosage ; Capsid Proteins ; Enterotoxins ; administration & dosage ; Escherichia coli Proteins ; administration & dosage ; Female ; Hemagglutination Inhibition Tests ; Human papillomavirus 16 ; immunology ; Immunization ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomavirus Vaccines ; Vaccines, DNA ; administration & dosage ; immunology ; Viral Vaccines ; administration & dosage ; immunology ; Virion ; immunology

The filtrate of Staphylococcus aureus culture has been used in an ampule form named as staphylococcal enterotoxin C injection for cancer therapy in clinic for ten years in China and proved to be effective. The active constituent of three kinds of injections is claimed to be staphylococcal enterotoxin C2 (SEC2), and the content of SEC2 is used as quality control. However, the correct content of SEC2 was not known and the relative amount of SEC2 was very low because of the complicated components of the filtrate. In this research, we established a proper ELISA system for the detection of SEC2 in staphylococcal enterotoxin C injection, which will improve the quality control of the injection. We produced and identified polyclonal and monoclonal antibodies of SEC2 and established BA-ELISA method based on the method of sandwich ELISA. It was found that the BA-ELISA method had good specificity, sensitivity and reproducibility, and being able to detect SEC2 at concentration from 2 to 20 ng x mL(-1), with an average CV value of 5.08%. The SEC2 content in staphylococcal enterotoxin C injection was calculated. There is some difference between the actual and labeled contents in the injections.
Animals ; Antibodies, Bacterial ; biosynthesis ; immunology ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antineoplastic Agents ; administration & dosage ; analysis ; immunology ; Enterotoxins ; administration & dosage ; analysis ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Hybridomas ; secretion ; Injections ; Mice ; Mice, Inbred BALB C ; Quality Control ; Rabbits ; Staphylococcus aureus ; chemistry

OBJECTIVETo develop an efficient expression, purification system of recombinant Escherichia coli heat-labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization.

METHODSA recombinant, pMMB68-LTB was generated by cloning the LTB cDNA fragment into an expression vector (pMMB68) and transformed it into the host strain marine vibrio VSP60. The relevant target protein was identified using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Sephacryl S-100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60. BALB/c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration. After three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA.

RESULTSrLTB protein was highly expressed in VSP60. After gel filtration with Sephacryl S-100, the purity of rLTB reached 98.1%, the yield rate was about 52%. After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL + rLTB groups were significantly increased, compared with the HEL alone group (P < 0.001).

CONCLUSIONSA set of protocols for large-scale rLTB preparation has been established, which is simple, efficient and applicable. The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co-administered antigen.

Adjuvants, Immunologic ; administration & dosage ; pharmacology ; Administration, Intranasal ; Animals ; Bacterial Toxins ; administration & dosage ; isolation & purification ; pharmacology ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins ; administration & dosage ; isolation & purification ; pharmacology ; Escherichia coli ; Escherichia coli Proteins ; Immunization ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; immunology ; Recombinant Proteins ; administration & dosage ; isolation & purification ; pharmacology

This paper is aimed to investigate the transcription and expression of BCR-ABL-pIRES-SEA fusion gene vaccines in vivo in mice. The reconstructed plasmids (BCR-ABL-pIRES-SEA) which were developed previously in our laboratory were injected into the skeletal muscles of BALB/c mice at 14d intervals for three cycles. The transcription and expression of BCR-ABL and staphylococcal enterotoxin A (SEA) in injection site were detected using RT-PCR and immunohistological methods. The BCR-ABL/SEA mRNA and protein could be identified in the injection site of BCR-ABL-pIRES-SEA vaccinated mice. The reconstructed BCR-ABL-pIRES-SEA plasmids can effectively express gene production in the skeletal muscles of mice and have the common features of DNA vaccine.
Animals ; Enterotoxins ; genetics ; immunology ; metabolism ; Fusion Proteins, bcr-abl ; genetics ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Muscle, Skeletal ; metabolism ; Plasmids ; immunology ; RNA, Messenger ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, DNA ; administration & dosage ; 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

OBJECTIVE: The aim of this study was to determine the expression of GATA-3 and the level of Th1 and Th2 cytokines upon repeated exposure to staphylococcal enterotoxin B(SEB) of different concentrations in the maxillary sinus mucosa of rabbits. METHOD: The rabbits were randomly divided into 2 groups (24 rabbits per group): low-dose SEB group and high-dose SEB group. The low-dose SEB group and high-dose SEB group received daily injections of 0.6 ng of SEB (2 ml) and 60 ng of SEB (2 ml) into the left maxillary sinus of rabbits for 28 days, respectively. Concurrent treatment of the right maxillary sinus with normal saline was used as a control. Six rabbits chosen randomly in two groups were killed on days 3, 7, 14, and 28, and to obtain the sinus mucosa from the two-side maxillary sinuses for measurement. Mucosal levels of IL-2, IL-4, IL-5, and IFN-γ were measured using ABC-ELISA. Tissue expression of GATA-3 were examined using Real-time PCR and immunohistochemistry. RESULT: IFN-γ and IL-2 levels were significantly elevated in the high-dose SEB group compared with the low-dose SEB and control groups on days 7, 14, and 28 (P < 0.05). However, IL-4 and IL-5 levels were markedly enhanced in the low-dose SEB group compared with the high-dose SEB and control groups on days 14 and 28 (P < 0.05). Real-time PCR showed that the expression of GATA-3 mRNA in the low-dose SEB group was markedly enhanced, and immunohistochemical staining illustrated that the number of GATA-3 positive cells was markedly increased in the low-dose SEB group as compared with the high-dose SEB group (P < 0.05). No significant differences were observed in GATA-3 expression between the high-dose SEB and the control groups (P > 0.05). CONCLUSION SEB promoted Th1 cytokines production at high concentrations, and enhanced Th2 cytokines expression and Th2 immune response at low concentrations.
Animals ; Cytokines ; metabolism ; Enterotoxins ; administration & dosage ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Interferon-gamma ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-5 ; metabolism ; Male ; Maxillary Sinus ; drug effects ; metabolism ; Nasal Mucosa ; metabolism ; RNA, Messenger ; metabolism ; Rabbits ; Th1 Cells ; Th2 Cells ; Transcription Factors ; metabolism