OBJECTIVETo explore the important role of the terminal residues containing sialic acid (SA) in Campylobacter jejuni (CJ) lipopolysaccharide (LPS) as the critical antigen to induce nerve damage, and also to identify immunopathological evidence for the hypothesis of molecular mimicry and cross-immunity between CJ LPS and gangliosides.

METHODSA mutant of Pen O:19 CJ with neuB1 gene inactivated and LPS outer core terminal residues losing SA was to be constructed. PCR and RT-PCR were used to confirm the mutant. Capability of CJ LPS binding to cholera toxin B subunit (CTB) was tested. Guinea pigs were systematically immunized with LPS of the wild and the mutant strains, respectively. Titers of anti-LPS and anti-ganglioside GM(1) IgG antibodies in sera of immunized guinea pigs were detected by ELISA. Pathological study for sciatic nerves of both Guinea pigs either immunized systematically or perineural injection with their immunized serum was finished.

RESULTS(1) The mutant of CJ O:19 strain with inactivated neuB1 gene was successfully constructed and lost transcriptional activity of neuB1 gene in the mutant strain was confirmed by PCR and RT-PCR. SA was well demonstrated by both acidic ninhydrin reaction and periodate-resorcinol reaction in the LPS of wild strain but not in the mutant LPS; (2) Compared with the titers before immunization, the titers of anti-GM(1) IgG antibody increased in sera of guinea pigs immunized with LPS of the wild strain. However there were no detectable anti-GM(1) IgG antibody in sera of the animals immunized with mutant LPS and PBS. (3) The incidence of pathological fibers of sciatic nerves in wild CJ LPS group (17.3%) was significantly higher than the mutant CJ LPS group (chi(2) = 125, P < 0.01); the difference between the mutant CJ LPS group and control group was not statistically significant (chi(2) = 1.633, P > 0.05). (4) After perineural injection with immunized serum, the incidence of pathological fibers of sciatic nerves in wild strain group (67.8%) was also significantly higher than the incidence of mutant group (P < 0.01).

CONCLUSIONA mutant of CJ O:19 strain neuB1 gene inactivated and SA component of terminal structure of LPS lost was successfully constructed. And it no longer expressed SA component which is the normal terminal structure of LPS in wild strain. The capability of the wild strain to induce increased titers of anti-GM(1) antibody and immune-mediated nerve damage was simultaneously lost for the mutant strain. It could be a strong immunopathologic evidence to identify the molecular mimicry hypothesis between CJ LPS and ganglioside epitope in nerve on the pathogenesis of CJ related GBS. The terminal residues containing SA should be as the basic GM1-like structure in CJ LPS.

Animals ; Antibodies, Bacterial ; blood ; immunology ; Antigens, Bacterial ; genetics ; immunology ; Campylobacter jejuni ; genetics ; immunology ; G(M1) Ganglioside ; immunology ; Guinea Pigs ; Lipopolysaccharides ; chemistry ; immunology ; Molecular Mimicry ; Mutagenesis ; N-Acetylneuraminic Acid ; chemistry ; immunology ; Peripheral Nervous System Diseases ; immunology ; microbiology

The immunologic reactivity of a lipopolysaccharide (LPS)-protein complex isolated from a potassium thiocyanate extract of a Pasteurella multocida (capsular type A and somatic type 3) strain was evaluated in mice. The LPS-protein complex provided 100% protection in mice against a challenge with the homologous strain. However, when the complex was fractionated into LPS and protein moieties by phenol-water treatment, both components lacked immunogenicity. The complex and extracted components were mitogenic for mouse B lymphocytes with the protein moiety the most active. Although immune serum against the LPS-protein complex protected mice against challenge thereby indicating a role for humoral immunity, the LPS-protein complex of P. multocida was also found to induce cell-mediated immunity. This cell-mediated immunity was demonstrated in mice immunized with the complex by: (1). mitogenic responses of T lymphocytes, (2). induction of delayed type hypersensitivity reaction in the hind footpads, and (3). enhanced resistance to challenge infection with Salmonella enteritidis.
Animals ; Antibodies, Bacterial/blood/immunology ; Bacterial Proteins/chemistry/*immunology ; Chemical Fractionation ; Hypersensitivity, Delayed ; Immune Sera/immunology ; Immunity, Cellular ; Immunization, Passive ; Lipopolysaccharides/chemistry/*immunology ; Lymphocyte Activation ; Mice ; Pasteurella Infections/immunology/*prevention & control ; Pasteurella multocida/*chemistry/immunology ; Salmonella Infections, Animal/immunology/prevention & control ; Salmonella enteritidis/growth & development/immunology ; Spleen/cytology/immunology/microbiology

OBJECTIVETo obtain Chlamydia trachomatis heat shock protein (cHSP) 10 gene from clinical secretion samples.

METHODScHSP10 gene was amplified from 20 cases of clinical secretion samples with positive gold-labelling by specific primers of cHSP10 and identified by sequence analysis.

RESULTScHSP10 full-length gene was amplified from 1 of 20 cases of clinical secretion samples with positive gold-labelling. cHSP10 gene encoding 102 amino acids contains 306 bp, which nuclotide at position 194 changes from T to A, leading to the change of corresponding amino acid.

CONCLUSIONScHSP10 gene may be cloned from clinical secretion samples with positive gold-labelling, which make it possible to further construct expression plasmid of recombinant cHSP10.

Antibodies, Bacterial ; blood ; Bacterial Proteins ; genetics ; Base Sequence ; Chaperonin 10 ; chemistry ; genetics ; immunology ; Chlamydia trachomatis ; chemistry ; immunology ; Cloning, Molecular ; Female ; Humans ; Infertility, Female ; etiology ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction

OBJECTIVETo study the regulating effects of a novel CpG oligodeoxynuleotide and the synergistic effect of chitosan-nanoparticles (CNP) with CpG on immune responses of mice, which were used to develop a novel immunoadjuvant to boost immune response to conventional vaccines.

METHODSA novel CpG ODN containing 11 CpG motifs was synthesized and its bioactivities to stimulate the proliferation of lymphocytes of pig in vitro were detected. Then it was entrapped with CNP prepared in our laboratory by the method of ionic cross linkage, and immunized Kunming mice were co-inoculated with paratyphoid vaccine. The peripheral blood was collected weekly from the tail vein of inoculated mice to detect the contents of IgG, IgA, IgM, and specific antibody against salmonella as well as the levels of interleukin-2 (IL2), IL-4, and IL-6 by SABC-ELISA assay. The numbers of leucocytes, monocytes, granuloytes, and lymphocytes were calculated separately using the routine method. The experimental mice were orally challenged with virulent salmonella 35 days after inoculation.

RESULTSThis CpG ODN could remarkably provoke the proliferation of lymphocytes of pig in vitro in contrast with the control (P < 0.05). Compared with those of the control, immunoglobulins, including IgG, IgA, IgM, and specific antibodies to paratyphoid vaccine, increased significantly in sera from the CpG or CpG-CNP-vaccinated mice (P < 0.05). IL-2, IL-4, and IL-6 increased remarkably in sera from immunized mice (P < 0.05). The leucocytes, monocytes, granuloytes, and lymphocytes of the mice immunized with CpG or CpG-CNP were also increased in number (P < 0.05). After the challenge, these immunity values were elevated in the mice vaccinated with CpG or CpG-CNP. The immunized mice all survived, while the control mice fell ill with evident lesions with diffuse hemorrhage in stomach, small intestine, and peritoneum.

CONCLUSIONSCpG ODN entrapped with CNP is a promising effective immunoadjuvant for vaccination, which promotes humoral and cellular immune responses, enhances immunity and resistance against salmonella by co-administration with paratyphoid vaccine.

Adjuvants, Immunologic ; administration & dosage ; pharmacology ; Animals ; Antibodies, Bacterial ; blood ; Cell Proliferation ; Chitosan ; chemistry ; Drug Therapy, Combination ; Enzyme-Linked Immunosorbent Assay ; Immunoglobulin Isotypes ; blood ; Interleukins ; blood ; Lymphocyte Activation ; drug effects ; Lymphocytes ; cytology ; Mice ; Nanoparticles ; chemistry ; Oligodeoxyribonucleotides ; administration & dosage ; pharmacology ; Paratyphoid Fever ; immunology ; prevention & control ; Salmonella ; physiology ; Salmonella Infections, Animal ; immunology ; prevention & control ; Swine ; immunology ; Typhoid-Paratyphoid Vaccines ; immunology

Diagnosis of scrub typhus is difficult because its symptoms are very similar to other acute febrile illnesses, such as leptospirosis, murine typhus, and other viral hemorrhagic fevers. To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We have developed a chimeric recombinant antigen cr56 and two other recombinant antigens, r21 and kr56, from various serotypes of Orientia tsutsugamushi. They were tested for the detection of antibodies against O. tsutsugamushi in the patient's serum samples using enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. As of conventional immunofluorescence assay (IFA), when the mixture of these three recombinant antigens was used, both sensitivity and specificity of the recombinant antigens were increased up to 98% in IgM and IgG at ELISA and dot blotting. Additionally, both sensitivity and specificity by detection of IgM and IgG antibodies at rapid diagnostic test (RDT), using the mixture of three antigens and gold conjugated antibodies, were 99%. Our results suggest the use of mixture of these recombinant antigen proteins in ELISA or RDT is suitable as a diagnostic test for scrub typhus.
Antibodies, Bacterial/blood/chemistry/immunology ; Antigens, Bacterial/diagnostic use/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Gold/chemistry ; Humans ; Immunoassay ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Orientia tsutsugamushi/immunology/*metabolism ; Recombinant Proteins/biosynthesis/diagnostic use/genetics ; Scrub Typhus/*diagnosis ; Sensitivity and Specificity ; Serotyping

This study is to investigate the effects of paeoniflorin (Pae) on the levels of related serum antibodies and cAMP of splenocytes in rats with adjuvant arthritis. Complete Freund's adjuvant was used to induce AA in rats. The level of circulating immune complexes in serum was determined by PEG6000 assay, and the levels of anti-C II antibody and anti-TB antibody in serum were measured by enzyme-linked immunosorbent assay (ELISA), the level of cAMP in splenocytes was measured by radioimmunoassay, separately. Pae (25, 50, and 100 mg x kg(-1)) and GTW (40 mg x kg(-1)) were given by intragastric administration for 7 days from the 17th day after immunization. Pae (50 and 100 mg x kg(-1)) reduced the levels of circulating immune complexes, anti-C II antibody and anti-TB antibody in serum in rats with adjuvant arthritis. The inhibition ratios of Pae groups to AA model group were dosage-dependent; Pae (12.5, 62.5, and 312.5 mg x L(-1)) decreased the elevated levels of cAMP in splenocytes in vitro. Pae (ig) decreased the levels of related serum antibodies and elevated the level of cAMP in rats with adjuvant arthritis.
Animals ; Antibodies, Bacterial ; blood ; Antigen-Antibody Complex ; blood ; Arthritis, Experimental ; chemically induced ; immunology ; Benzoates ; isolation & purification ; pharmacology ; Bridged-Ring Compounds ; isolation & purification ; pharmacology ; Cyclic AMP ; metabolism ; Dose-Response Relationship, Drug ; Freund's Adjuvant ; Glucosides ; isolation & purification ; pharmacology ; Isoantibodies ; blood ; Monoterpenes ; Mycobacterium tuberculosis ; immunology ; Paeonia ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spleen ; cytology ; metabolism