Salmonella causes a wide variety of diseases ranging from mild diarrhea to severe systemic infections, such as like typhoid fever, in multiple organisms, ranging from mice to humans. A lack of ptsI, which encodes the first component of phosphoenolpyruvate (PEP) : carbohydrate phosphotransferase system (PTS), is known to cause Salmonella Typhimurium attenuation; however, the mechanisms behind this have not yet been elucidated. In this study, a DNA microarray was performed to determine why the virulence of ptsI mutants is attenuated under low-oxygen conditions in which the ptsI expression is enhanced. Of 106 down-regulated genes, the most repressed were pdu and tdc genes, which are required for propanediol utilization and threonine and serine metabolism, respectively. In addition, half the flagellar genes were down-regulated in the ptsI mutant strain. Because pdu genes are induced during infection and Tdc products and flagella-mediated motility are necessary for the invasion of S. Typhimurium, the invasive ability of ptsI mutants was examined. We found that ptsI mutation reduced the ability of S. Typhimurium to invade into host cells, suggesting that reduced expression of the pdu, tdc, and flagellar genes is involved in the attenuation of ptsI mutants.
Animals ; Diarrhea ; Flagella ; Humans ; Metabolism ; Mice ; Microarray Analysis* ; Oligonucleotide Array Sequence Analysis ; Phosphoenolpyruvate ; Salmonella typhimurium* ; Salmonella* ; Serine ; Threonine ; Typhoid Fever ; Virulence

This study is designed to evaluate antioxidant and antigenotoxic activities of corn tassel extracts (CTTs). The major bioactive components of CTTs include flavonoid, saponin and polysaccharide. The antioxidant properties of the three bioactive components of CTTs were investigated by Ferric Reducing Antioxidant Property (FRAP) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assays. The activities of the extracts were determined by assessing the inhibition of mutagenicity of the direct-acting mutagen fenaminosulf, sodium azide, and indirect-acting mutagen 2-aminofluorene using the Ames test (strains TA98 and TA100). The results showed that the extraction rates of flavonoid, saponin, and polysaccharide from the dried corn tassels were 1.67%, 2.41% and 4.76% respectively. DPPH and FRAP assay strongly demonstrated that CTTs had antioxidant properties. CTTs at doses of 625, 1250 and 2500 μg per plate reduced 2-aminofluorene mutagenicity by 12.52%, 28.76% and 36.49% in Salmonella typhimurium TA98 strain assay respectively and by 10.98%, 25.27% and 37.83%, at the same doses in Salmonella typhimurium TA100 assay system, respectively. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay showed that the different concentrations of CTTs inhibited the proliferation of MGC80-3 cells in a dose-dependent manner (P<0.01). It is concluded that these integrated approaches to antioxidant and antigenotoxicity assessment may be useful to study corn tassel as a natural herbal material.
Antimutagenic Agents ; pharmacology ; Antioxidants ; pharmacology ; Biphenyl Compounds ; antagonists & inhibitors ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Flavonoids ; pharmacology ; Fluorenes ; pharmacology ; Free Radical Scavengers ; pharmacology ; Humans ; Inflorescence ; chemistry ; Mutagens ; pharmacology ; Picrates ; antagonists & inhibitors ; metabolism ; Plant Extracts ; pharmacology ; Polysaccharides ; pharmacology ; Salmonella typhimurium ; drug effects ; genetics ; Saponins ; pharmacology ; Zea mays ; chemistry

Th-2-biased immune responses are known to play a key role in the pathogenesis of atopic dermatitis. In particular, the macrophage-derived chemokine CCL22 is directly implicated in Th-2-associated skin inflammatory reactions, and its levels are significantly elevated in serum and are correlated with disease severity in atopic dermatitis. In this study, we tested the development of genetic therapeutic options to treat atopic dermatitis using bacteria expressing miRNA. We constructed a recombinant strain of Salmonella typhimurium expressing CCL22 miRNA (ST-miRCCL22) for the in vivo knockdown of CCL22. The CCL22 gene was downregulated with CCL22 miRNA in activated lymphocytes. In mice with a cutaneous disease similar to atopic dermatitis, interleukin-4 was inhibited and interferon-gamma was induced after treatments with ST-miRCCL22. Furthermore, CCL22 levels were suppressed in the atopic mice treated with ST-miRCCL22. These results suggest that ST-miRCCL22 may be an effective genetic agent for treating atopic dermatitis.
Animals ; Cell Line ; *Chemokine CCL22/genetics ; Cytokines/blood ; Dermatitis, Atopic/pathology ; Disease Models, Animal ; Female ; Gene Expression Regulation/drug effects ; Gene Silencing ; Immunoglobulin E/blood ; Mice ; *MicroRNAs/genetics/pharmacology ; *Organisms, Genetically Modified/genetics ; *Salmonella typhimurium/genetics/metabolism ; Skin/*drug effects/pathology

OBJECTIVETo explore the effects of Huoxiang Zhengqi liquid (HXZQ) on enteric mucosal immune responses in mice with Bacillus dysenteriae and Salmonella typhimurium induced diarrhea (BSD).

METHODMice were randomly divided into four groups with 10 mice in each group: control group (control), BSD group, Huoxiang Zhengqi liquid treated BSD groups at high dosage and low dosage (HXZQ high, HXZQ low). HXZQ was administrated from the day of diarrhea induction at dosage of 5.21 g kg(-1) and 0.52 g kg (-1) respectively. Peyer's patch and periphery lymphocytes were prepared for flow cytometry, and level of TNF-alpha in periphery and enteric tissue homogenate were determined with ELISA. Student's t-test was used for statistics.

RESULTMice in BSD group started showing continuous diarrhea at the day of induction till the fourth day when the mice were sacrificed. Diarrhea in the mice of HXZQ high and low groups lasted for 36 and 54 h respectively. There were more CD4+ and CD8+ cells in periphery, less CD4+ cells in peyer's patch in BSD mice comparing to normal mice. In peyer's patch, there were more CD8+ cells in mice in HXZQ high and low groups and more CD4+ in mice in HXZQ high group. Higher level TNF-alpha in periphery and intestinal tissue homogenate in BSD group were observed. Mice in HXZQ high group showed the decreased level TNF-alpha in periphery and enteric tissue homogenate.

CONCLUSIONThe immune regulation on peyer's patch CD4+ and CD8+ cells and suppression on TNF-alpha level in enteric homogenate might partially explain the effect of HXZQ on improvement of BSD.

Animals ; CD4-CD8 Ratio ; Colon ; immunology ; metabolism ; pathology ; Diarrhea ; immunology ; metabolism ; microbiology ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Dysentery, Bacillary ; immunology ; metabolism ; microbiology ; Immunity, Mucosal ; drug effects ; Intestinal Mucosa ; immunology ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Peyer's Patches ; drug effects ; immunology ; pathology ; Plants, Medicinal ; chemistry ; Random Allocation ; Salmonella Infections ; immunology ; metabolism ; microbiology ; Salmonella typhimurium ; immunology ; Shigella dysenteriae ; immunology ; T-Lymphocyte Subsets ; drug effects ; immunology ; pathology ; Tumor Necrosis Factor-alpha ; blood ; metabolism

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3 CD4 T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/ pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.
Animals ; Hepacivirus ; genetics ; immunology ; Mice ; Plasmids ; genetics ; Salmonella typhimurium ; genetics ; metabolism ; Vaccines, DNA ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Hepatitis Vaccines ; genetics ; immunology

Non-typhoidal Salmonella (NTS) is an important commensal microorganism. The purpose of this study was to determine the epidemiological relation between NTS isolates from livestock and NTS isolates from human by analyzing antimicrobial susceptibilities and performing molecular typing. We determined the serotypes of 36 human clinical isolates and 64 livestock isolates, performed antimicrobial susceptibility testing against 8 antibiotics, and determined the molecular types of isolated NTS spp. by pulsed field gel electrophoresis (PFGE). In human isolates, S. enteritidis was the most common serotype (17 isolates; 47.2%) and S. typhimurium the second most (8 isolates; 22.2%). In livestock isolates, S. typhimurium was the most common serotype (15 isolates; 23.44%), and S. enteritidis was the second most (14 isolates; 21.88%). Ampicillin and tetracycline resistance were 50% (32/64 isolates) each among broiler-chicken NTS isolates. No human or livestock NTS isolates showed resistance to ciprofloxacin, TMP-SMX, or ceftriaxone. However, 19.4% (7/36) and 46.8% (30/64) of the human and livestock NTS isolates were resistant to nalidixic acid (MIC > or =16 mg/mL), respectively. The presence of the three identical PFGE molecular types from human and broiler-chicken NTS isolates suggests the possibility of transmission from livestock to humans.
Adult ; Animals ; Chickens ; Cluster Analysis ; Drug Resistance, Bacterial ; Female ; Humans ; Korea ; Male ; Nalidixic Acid/pharmacology ; Salmonella Infections/epidemiology/metabolism/*microbiology ; Salmonella Infections, Animal/epidemiology/metabolism/*microbiology ; Salmonella enteritidis/metabolism ; Salmonella typhimurium/*metabolism ; Serotyping

OBJECTIVETo investgate the metabolism of terephthalic acid (TPA) in rats and its mechanism. Methods Metabolism was evaluated by incubating sodium terephthalate (NaTPA) with rat normal liver microsomes, or with microsomes pretreated by phenobarbital sodium, or with 3-methycholanthrene, or with diet control following a NADPH-generating system. The determination was performed by high performance liquid chromatography (HPLC), and the mutagenic activation was analyzed by umu tester strain Salmonella typhimurium NM2009. Expression of CYP4B1 mRNA was detected by RT-PCR. Results The amount of NaTPA (12.5-200 micromol x L(-1)) detected by HPLC did not decrease in microsomes induced by NADPH-generating system. Incubation of TPA (0.025-0.1 mmol x L(-1)) with induced or noninduced liver microsomes in an NM2009 umu response system did not show any mutagenic activation. TPA exposure increased the expression of CYP4B 1 mRNA in rat liver, kidney, and bladder.

CONCLUSIONLack of metabolism of TPA in liver and negative genotoxic data from NM2009 study are consistent with other previous short-term tests, suggesting that the carcinogenesis in TPA feeding animals is not directly interfered with TPA itself and/or its metabolites.

Animals ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Genes, Bacterial ; genetics ; Kidney ; enzymology ; Liver ; enzymology ; Male ; Microsomes, Liver ; drug effects ; enzymology ; Mutagenicity Tests ; Phthalic Acids ; pharmacokinetics ; toxicity ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Salmonella typhimurium ; genetics ; Urinary Bladder ; enzymology ; beta-Galactosidase ; metabolism

OBJECTIVETo evaluate the feasibility of oral cytokine gene therapy against tumor using live attenuated Salmonella as a vector.

METHODSA live attenuated AraA- autotrophic mutant of Salmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1 and pCMVmIL-12 administered orally in BALB/c and C57BL/6 mice. After 6 weeks, the mice were challenged with 4T1 or Lewis tumor cells, respectively, and flow cytometry and confocal microscopy were used to detect the expression of green fluorescence protein (GFP) in the tissues. PCR and ELISA were performed to detect the integration and expression of mIL-12 gene, and the survival time of the mice was also recorded.

RESULTSGFP expression and mIL-12 gene integration could be detected in the liver, spleen, intestinal, kidney and tumor tissues of the mice. The serum level of mIFN-gamma, mIL-12 increased significantly in mice with oral mIL-12 administration (P<0.05), which resulted in the survival prolongation of the mice as compared with the control mice (P<0.05).

CONCLUSIONOral gene therapy using live attenuated Salmonella can be potentially a simple, effective and above all, safe means for tumor treatment.

Administration, Oral ; Animals ; Carcinoma, Lewis Lung ; therapy ; Flow Cytometry ; Genetic Therapy ; methods ; Genetic Vectors ; administration & dosage ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Interleukin-12 ; genetics ; metabolism ; Mammary Neoplasms, Animal ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Microscopy, Confocal ; Neoplasms, Experimental ; therapy ; Salmonella typhimurium ; genetics ; immunology ; metabolism ; Vaccines, Attenuated ; immunology

OBJECTIVETo increase the immune effect of gene vaccine, T7 RNA polymerase was used to establish a system of cytoplasmic expression.

METHODS(1) The plasmid pT7 EMCVP1, including T7 promoter sequence, 5'-untranslated sequence of encephalomyocarditis (EMC) virus, VP1 sequence of coxsackievirus B3 (CVB3), was cotransfected with the plasmid pAR 3132, which codes for the T7 RNA polymerase, into HeLa cells and murine peritoneal macrophages. (2) The plasmid pT7 EMCVP1 and pAR 3132 were respectively transformed into the attenuated Salmonella typhimurium SL 7207. The two kinds of transformed bacteria were coinfected into murine peritoneal macrophages.

RESULTS(1) The target antigen VP1 in the cytoplasm was about 2-4-fold higher than that of pcDNA3 VP1 singly transfected. (2) After the murine peritoneal macrophages were coinfected by two kinds of transformed bacteria, the target antigen VP1 could also be detected.

CONCLUSIONThe pT7 EMCVP1 and pAR 3132 could be expressed in the cytoplasm of HeLa cells and murine peritoneal macrophages and the amount of the antigen VP1 increased remarkably as compared with that of pcDNA3 VP1 singly transfected.

Animals ; Bacteriophage T7 ; genetics ; Capsid Proteins ; genetics ; metabolism ; Cytoplasm ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enterovirus B, Human ; genetics ; Gene Expression ; HeLa Cells ; Humans ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Salmonella typhimurium ; genetics ; Transfection

OBJECTIVETo prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacter pylori (H. pylori) B subunit (UreB) and to determine whether it could be used as an oral vaccine against H. pylori infection.

METHODSUsing genomic DNA of H. pylori Sydney strain (SSI) as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LB5000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups (including body weight, gastric inflammation, etc.) were also collected.

RESULTSThe sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-gamma and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed.

CONCLUSIONThe attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; administration & dosage ; immunology ; Female ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Helicobacter Infections ; immunology ; prevention & control ; Helicobacter pylori ; enzymology ; genetics ; immunology ; Immunoglobulin G ; blood ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Mice ; Mice, Inbred BALB C ; Salmonella typhimurium ; genetics ; immunology ; metabolism ; Urease ; genetics ; immunology ; metabolism ; Vaccines, Attenuated ; genetics ; immunology ; Weight Loss