The purpose of the study is to establish a colorimetric method of HEC toxin hemolysis test for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). RBCs from normal persons and patients with PNH and non-PNH anemia were treated with HEC toxin secreted by Aeromonas hydrophila J-1 strain and the absorbance at 630 nm was measured to quantitate the extent of hemolysis. The results demonstrated that the RBCs from PNH patients showed resistance to the toxin hemolysis, which was in accord with the percentages of CD59(-) cells, while the RBCs from normal persons and non-PNH anemic patients were nearly totally lysed. It is concluded that the method can be considered as a simple, specific and reliable method for the diagnosis of PNH.
Bacterial Toxins ; toxicity ; Colorimetry ; Flow Cytometry ; Hemoglobinuria, Paroxysmal ; diagnosis ; Hemolysis ; Humans

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.
Amino Acid Substitution ; Animal ; Bacterial Toxins/toxicity* ; Bacterial Toxins/metabolism ; Bacterial Toxins/immunology* ; Bacterial Toxins/genetics ; CHO Cells ; Cyclic AMP/metabolism ; Enterotoxins/toxicity* ; Enterotoxins/metabolism ; Enterotoxins/immunology* ; Enterotoxins/genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli*/metabolism ; Escherichia coli*/genetics ; Female ; Hamsters ; IgA, Secretory/blood ; Ileum/metabolism ; Immunity, Mucosal ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; NAD+ ADP-Ribosyltransferase/metabolism ; Recombinant Proteins/toxicity ; Recombinant Proteins/metabolism ; Recombinant Proteins/immunology ; Recombinant Proteins/chemistry

OBJECTIVETo investigate the association of microcystin (MC) in drinking water with the incidence of colorectal cancer.

METHODSThe study was designed as a retrospective cohort. Eight townships or towns were randomly selected as the study sites in Haining City of Zhejiang Province, China. 408 cases of colon and rectum carcinomas diagnosed from 1977 to 1996 in the study sites were included, and a survey on types of drinking water of these patients was conducted. Samples of different water sources (well, tap, river and pond) were collected separately and microcystin concentrations were determined by indirect competitive ELISA method.

RESULTSThe incidence rate of colorectal cancer was significantly higher in population who drank river and pond water than those who drank well and tap water. Compared to well water, the relative risk (RR) for colorectal cancer was 1.88 (tap), 7.94 (river) and 7.70 (pond) respectively. The positive rate (> 50 pg/mL) of microcystin in samples of well, tap, river and pond water was 0, 0, 36.23% and 17.14% respectively. The concentration of microcystin in river and pond water was significantly higher than that in well and tap water (P < 0.01). Spearman rank correlation analysis showed that in the study sites, the microcystin concentration of river and pond water was positively associated with the incidence of colorectal cancer (rs = 0.881, P < 0.01).

CONCLUSIONSThe types of drinking water are positively associated with the incidence of colorectal cancer in the study sites, and this may be related to microcystin contamination of drinking water. Further biological study is needed to support the possible causative role of mycrocystin in carcinogenesis of colon and rectum.

Bacterial Toxins ; toxicity ; Carcinogens, Environmental ; toxicity ; China ; epidemiology ; Cohort Studies ; Colorectal Neoplasms ; chemically induced ; epidemiology ; Female ; Humans ; Male ; Microcystins ; Peptides, Cyclic ; toxicity ; Retrospective Studies ; Water ; chemistry ; Water Pollutants, Chemical ; toxicity ; Water Supply ; standards

An expression plasmid carrying anthrax protective antigen (PA) gene was constructed, which has an OmpA signal sequence attached to the 5' end of PA gene. The plasmid was transformed into E. coli and induced to express recombinant PA (rPA) . The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange, hydrophobic interaction chromatography, and gel filtration, about 15 mg of 95 % pure rPA was obtained from 1-liter culture. The bioactivity of rPA was proved by in vitro cytotoxicity assay. The polyclonal antiserum from rabbits immunized with rPA could inhibit the action of anthrax lethal toxin in vitro, which suggests that antibodies against rPA can provide high passive protection against anthrax. The results reported here may be helpful to develop a safe and efficacious recombinant PA vaccine against anthrax.
Amino Acid Sequence ; Animals ; Anthrax Vaccines ; immunology ; Antigens, Bacterial ; chemistry ; genetics ; immunology ; toxicity ; Bacterial Toxins ; chemistry ; genetics ; immunology ; toxicity ; Base Sequence ; Mice ; Molecular Sequence Data ; Plasmids ; Rabbits ; Recombinant Proteins ; biosynthesis ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVETo examine the expression of P53, Bcl-2 and Bax proteins in rat liver after exposed to microcystin LR.

METHODSSD rats received microsystin LR by gastric perfusion. The expression of P53, Bax and Bcl-2 in liver was detected by Western blot.

RESULTSThe expression of P53 and Bax in each treatment group increased significantly compared with that in control group(P<0.05), with the exception of 0.1 microg/kg LR exposure group. Moreover, with exposure levels increasing the expression of P53 and Bax increased gradually; while no changes of the expression of Bcl-2 were observed.

CONCLUSIONP53 and Bax may play important roles in microcystin LR induced apoptosis, but Bcl-2 seems not be involved in this process.

Animals ; Apoptosis ; drug effects ; Bacterial Toxins ; toxicity ; Liver ; metabolism ; pathology ; Male ; Marine Toxins ; toxicity ; Microcystins ; Peptides, Cyclic ; toxicity ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; bcl-2-Associated X Protein

The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTx-induced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.
AMP Deaminase ; genetics ; Animals ; Anthrax ; pathology ; Antigens, Bacterial ; toxicity ; Bacterial Toxins ; toxicity ; Base Sequence ; Blotting, Western ; Cell Death ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Cells, Cultured ; Exotoxins ; toxicity ; Macrophages ; cytology ; drug effects ; enzymology ; Mice ; Molecular Sequence Data ; Polymerase Chain Reaction

OBJECTIVETo investigate α-toxin-induced apoptosis of umbilical vein endothelial cells and explore its role in vertical infection of Staphylococcus aureus L-form.

METHODSHUV-EC-C cells exposed to different concentrations (0, 10, 30, 90, and 270 ng/ml) of α-toxin for different time lengths (0, 2, 4, 6, and 8 h) were examined for apoptosis using flow cytometry with Annexin V-PI staining. The levels of tumor necrosis factor-α (TNF-α) and the activities of, caspase-3 and caspase-8 in the cell culture were detected by ELISA and colorimetric method, respectively. α-Toxin-induced cell apoptosis was also analyzed in HUV-EC-C cells treated with a neutralizing antibody of TNF-α or with the inhibitory peptides of caspase-3 (zDEVD-FMK) and caspase-8 (zIETD-fmk).

RESULTSα-Toxin induced apoptosis of HUV-EC-C cells in a dose- and time-dependent manner and caused significantly enhanced expression of TNF-α and the activation of both caspase-3 and caspase-8. Inhibition of TNF-α with its neutralizing antibody and the inhibitory peptides of caspase-3 or -8 all significantly decreased α-toxin-induced cell apoptosis, and the caspase-3 inhibitor completely blocked α-toxin-induced cell apoptosis.

CONCLUSIONα-Toxin-induced apoptosis is partially mediated by the extrinsic cell death pathway of TNF-α and caspase-8 and plays an important role in the vertical infection of S. aureus L-form to affect fetal growth and development.

Apoptosis ; Bacterial Toxins ; toxicity ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; L Forms ; Staphylococcal Infections ; Staphylococcus aureus ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo explore the pathogenesis of the damage to peripheral nerves induced by Campylobacter jejuni exotoxin (CJT).

METHODS(1) Animal models: (1) The CJT was extracted from PEN 19-CJ and injected perineurally and intravenously to Wistar rats. (2) The sera and the supernatants of peripheral blood mononuclear cells (PBMCs), taken from the rats immunized with the CJT, were injected perineurally at sciatic nerves of experimental rats and intravenously, respectively. (2) Histopathologic study of sciatic nerves: the animals were sacrificed and their sciatic nerves were examined for tease fibers, transverse section with toluidine blues staining and electron microscopy. (3) Immunohistochemistry: sections of sciatic nerves of either normal rats or human which were incubated with CJT and the sciatic nerves with pathological changes induced by CJT were obtained for observation of the binding capability of CJT with peripheral nerves by SABC and FITC-immunofluorescence methods, and nucleic acid hybridization techniques for detection of TNF-alpha mRNA expression in pathological sciatic nerves samples.

RESULTS(1) Remarkable peripheral neuropathies with axon degeneration and/or demyelination were found in the nerves induced by both CJT injection perineurally and intravenously. The axon degeneration was more obvious. Pathological changes were identified in 76.8% (2,763/3,600) of teasing fibers after perineural injection, but only 9.6% (230/2,400) of fibers were damaged in control group (P < 0.01). The peak severity of fiber damage was found on the 3rd day after CJT intravenous injection with the incidence of abnormal fibers was 19.5% (390/2,000), and abnormalities of 15.5% (310/2000) on the 14th day. However, no abnormal changes were demonstrated in control group (P < 0.01). So was in the groups injected with anti-CJT sera and the supernatants of PBMCs compared with control (P > 0.05). (2) Binding of CJT to the nerve was found dominant in the sciatic nerves taken from normal rats or human either incubated with CJT or in the pathological sciatic nerves induced by CJT to various degrees. The binding of CJT to all these nerves was determined. (3) After intravenous injection with CJT, no histopathologic change could be found in the other viscera of the rats, with the exception of remarkable pathological change in peripheral nerves.

CONCLUSIONS(1) CJT could remarkably damage the peripheral nerves in rats. Specific pathogenicity of CJT to peripheral nerves was well shown, because no histopathologic abnormalities could be found in the other viscera, such as brain, liver and kidney etc. although there was remarkable pathological change along the peripheral nerve in the animals. (2) No immunological pathogenicity of CJT could be demonstrated in the nerves of rats after immunization with CJT.

Animals ; Antibodies, Anti-Idiotypic ; blood ; Bacterial Toxins ; immunology ; toxicity ; Campylobacter jejuni ; immunology ; Exotoxins ; immunology ; toxicity ; Gene Expression ; drug effects ; Peripheral Nerves ; drug effects ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Sciatic Nerve ; drug effects ; metabolism ; pathology ; Tumor Necrosis Factor-alpha ; genetics

Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an AB5 hexamer structure. LT is a powerful mucosal adjuvant when co-administered with soluble antigens. However, its use in mucosal immunity is inconvenient because of its low yield and depolymerization during long-term storage under normal condition. In this study, we report an efficient expression system and optimized purification and storage strategy of LT. A gene encoding LT was cloned into the vector pET11c and transformed in E. coli BL21(DE3). By growing this strain on modified M9-CAA medium, LT was expressed efficiently. About 46mg/L LT could be purified from the supernatant of bacteria lysate. Using D(+)-Immobilized galactose column, LT could be purified at a wide pH range with various elution buffers. The optimized elution buffers are TEAN (pH 7.3) containing 0.3mol/L galactose and carbonate buffer (pH 10.4) containing 0.3mol/L galactose. After dried by freeze and placed in 4 degrees C, LT dissolved in TEAN (pH 7.3) and carbonate buffer (pH 10.4) were assayed by HPLC. The results indicated that the integrity of AB5 hexamer was kept well. LT could undergo long-term storage under this condition. This was proved to be an optimized strategy of LT storage. The results of GM1 binding assay and toxicity assay showed that the purified recombinant LT has normal biological character.
Animals ; Bacterial Toxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; CHO Cells ; Cell Shape ; drug effects ; Chromatography, High Pressure Liquid ; Cricetinae ; Cricetulus ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Female ; Genetic Vectors ; genetics ; Mice ; Mice, Inbred C57BL ; Toxicity Tests