RESUMO
PURPOSE: Infection with Shiga-like toxin (SLT)-producing Escherichia coli, an emerging human pathogen found particularly in young children under 5 years of age, causes a spectrum of illnesses with high morbidity and mortality, ranging from diarrhea to hemorrhagic colitis and hemolytic uremic syndrome. Host mediators play an important role in the pathogenesis of SLT-I toxicity. The experiments described here were designed to investigate the effect of SLT-I on TNF- alpha production and to understand the effect of TNF-alpha on GB3 expression. We also further examine the relationship between the Gb3 level and the differential susceptibility of cells to the cytotoxic action of SLT-I. METHODS: The effect of purified SLT-1 from E. coli O157 : H7 (ATCC 43890) on tumor necrosis factor-alpha (TNF- alpha) production in Raw264.7 cells was investigated. Many mediators regulate endothelial cell membrane expression of the glycolipid globotriaosyleramide (Gb3), which serves as the toxin receptor, suggesting that the host response to the toxin or other bacterial products may contribute to pathogenesis by regulating target cell sensitivity to the toxins. Therefore, the relationships between Gb3 expression and cytotoxicity against SLT-I on three types of cells were evaluated. RESULTS: Detectable levels of TNF-alpha were produced as early as six hours after induction and continued to increase during 48 hours by SLT-I. It was also found that Vero cells and dendritic cells (DC2.4 cells) expressed high levels of Gb3, 83% and 68%, respectively, and that Raw264.7 cells had a low level of Gb3 (29%) and appeared refractory to cytotoxicity against SLT-I. Vero cells and DC2.4 cells expressing high levels of Gb3 were highly susceptible to SLT-I. Furthermore, macrophages showed a resistance to SLT-I cytotoxicity, despite the fact that Gb3 expression was enhanced. CONCLUSION: These results strongly suggest that the expression of Gb3 is necessary but not sufficient to confer sensitivity of macrophages to SLT-I and further underpin the important role of SLT-I and its Gb3 receptors in the pathogenesis of E. coli O157 infection.
Assuntos
Criança , Humanos , Colite , Células Dendríticas , Diarreia , Células Endoteliais , Escherichia coli , Síndrome Hemolítico-Urêmica , Macrófagos , Membranas , Mortalidade , Toxina Shiga I , Fator de Necrose Tumoral alfa , Células VeroRESUMO
Escherichia coli (E. coli) O157:H7 is an important cause of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). LPS-based vaccines need improvement since the anti-LPS antibodies raised by the vaccines are bactericidal and release toxin that may precipitate the development of HUS. Despite huge efforts, the treatment of infection with E. coli O157 has been difficult because antibiotics do not change the course of the enteritis caused by E. coli O157 and may increase the incidence of HUS through the release of Shiga-like toxin (Stx)-I. For this aim we tried the conjugate of E. coli O157 OSP bound to the nontoxic B subunit of Stx-I B as a vaccine that can induce both serum IgG anti-LPS antibody with bactericidal activity and neutralizing antibody to Stx-I. Mice were immunized s.c. with OSP-Stx-I B conjugate. Anti-sera were analyzed for the anti-LPS antibody, anti-Stx-I B antibody, complement-dependent bactericidal activity, and Stx-I neutralization activity. Mice injected with the bivalent conjugate elicited both bactericidal antibodies to E. coli O157 and neutralization antibodies to Stx-1. We also analyzed the distributional, functional changes of T lymphocytes in vitro and in vivo. After the injection of Stx-I, splenocytes showed a decrease in proliferation when stimulated with phytohemagglutinin (PHA) or LPS, and the number of CD4+ and CD8+ T cells also decreased. Interleukin (IL)-2, IL-4 and IFN-gamma productions by CD3+ T cells were slightly increased in the Stx-I injected mice.
Assuntos
Animais , Camundongos , Antibacterianos , Anticorpos , Anticorpos Neutralizantes , Colite , Enterite , Escherichia coli O157 , Escherichia coli , Escherichia , Síndrome Hemolítico-Urêmica , Imunoglobulina G , Imunotoxinas , Incidência , Interleucina-4 , Interleucinas , Antígenos O , Linfócitos T , VacinasRESUMO
BACKGROUND: Salvia miltiorrhiza (SM), a traditional oriental medicine, has been reported to have anti-tumor properties, but its exact mechanism remains to be elucidated. In this study, we investigated several of the molecular events that occur in human breast carcinoma MCF-7 cells and human pulmonary adenocarcinoma A549 cells. METHODS: For this purpose, we evaluated the growth-inhibitory effect of SM in association with the expressions of p53, p21, cyclin D1, and pRb, which are known to be involved in cell cycle arrest. The extent of thymidine incorporation was also examined to assess G1/S phase cell cycle arrest in both cells by 3H-thymidine incorporation. RESULTS: Our results show that SM inhibits the growth and the proliferation of MCF-7 and A549 cells. Furthermore, we also observed increased expression of p21 via a p53-dependent pathway in both cell lines after treating with SM. In addition, treatment with SM for 24 hours caused the suppression of hyperphosphorylated retinoblastoma protein (pRb) expression and the dephosphorylation of pRb. CONCLUSION: These findings suggest that the growth inhibitory and the anti-proliferation effects of SM on MCF-7 cells and A549 cells are mediated via the decreased expression and dephosphorylation of pRB by p21 up-regulation in a p53-dependent manner. To the best of our knowledge, this study is the first to report upon the molecular mechanisms involved in SM-induced tumor cell growth inhibition.
Assuntos
Humanos , Adenocarcinoma , Neoplasias da Mama , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Ciclina D1 , Células MCF-7 , Medicina Tradicional do Leste Asiático , Proteína do Retinoblastoma , Salvia miltiorrhiza , Salvia , Timidina , Regulação para CimaRESUMO
No Abstract Available.
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Vibrio parahaemolyticus , Vibrio , Fatores de Virulência , VirulênciaRESUMO
No Abstract Available.
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Resistência a Meticilina , Meticilina , Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus , StaphylococcusRESUMO
No Abstract Available.
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Vibrio parahaemolyticus , Vibrio , Fatores de Virulência , VirulênciaRESUMO
No Abstract Available.
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Resistência a Meticilina , Meticilina , Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus , StaphylococcusRESUMO
BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) are becoming increasingly responsible for the outbreaks of nosocomial infections around the world. Because MRSA are often resistant to antimicrobial agents and because of the current necessity to use non-beta-lactam antimicrobial therapy, infections with these organisms are difficult to treat. The purpose of this study was to explain how the genetic background contributes to the phenotypic expression of MRSA and to enable immediate and proper identification of MRSA in nosocomial infections. METHODS: A total of 240 staphylococci strains were tested for epidemiological research. The molecular genetic methods, such as dot blot hybridization, polymerase chain reaction, and southern blot hybridization, were compared in terms of the immediate and proper identification of the pathogens. The arrangement of dru sequence, the repeat unit of genes, was clarified particularly through mecA probe to compare and distinguish the isolated MRSA strains. RESULTS: By the dot blot hybridization, the mecA gene was detected in 120 MRSA strains and in 8 of 50 methicillin-suscpetible S. aureus (MSSA) in Group II (resistance to methicillin is induced in vitro). Among isolates included in Group I (resistance to methicillin is not induced in vitro) mecA genes were not detected. By PCR, the amplified DNA band of 533 bp was confirmed in 120 MRSA but not in MSSA. By the southern blot hybridization, the signal of the amplified electrophoresis band in PCR was similarly observed. The amplified band of mec related hypervariable region was observed in all methicilin resistant (MR) staphylococci but not in MSSA. The size of PCR products was between 194 bp and 1,353 bp. mec-related HVR was classified into seven genotypes. The nucleotide sequence of genotype E was similar to that of mec related HVR and eight units were directly repeated. CONCLUSION: Both methods allowed rapid detection of mecA gene. Further differentiation of MRSA and other staphylococci strains was possible by PCR detection of polymorphism of HVR sequence which would be useful in tracing back the source of resistant clones.
Assuntos
Anti-Infecciosos , Sequência de Bases , Southern Blotting , Células Clonais , Infecção Hospitalar , Surtos de Doenças , DNA , Eletroforese , Genótipo , Meticilina , Resistência a Meticilina , Staphylococcus aureus Resistente à Meticilina , Biologia Molecular , Reação em Cadeia da PolimeraseRESUMO
A total of 100 Vibrio spp. strains were examined for production of various extracellular enzyme and for plasmid content plasmid were subjected to digestion with restriction enzymes. Most of them produced extracellular enzyme more than one, especially V. parahaemolyticus and V. cholerae non-01 strains were showed production of various extracellular enzymes. About the 55% Vibrio spp. have the plasmid more than one, but a lot of Vibrio spp. (about 45%) did not possess any plasmid. Most of these plasmid various derivatives ranged from 2.4 kb-23 kb, especially two strains of V. mimicus and one strain of V. furnissii carried one high-molecular weight plasmid (molecular weight ranging between 70 kb-100 kb). Results of restriction analysis for plasmid of this three strains were by no means the rule. For detection of tdh and ctx gene, the virulence factor involved in the pathogenesis, we carried out the TDH, CT assay, PCR amplification, and hybridization. A total 11 strains were produced TDH, involved in 4 strains of V. parahaemolyticus and 1 strain of V. cholerae non-01 from clinical isolates and 6 strains of environmental isolates. Nine strains of 11 strains, involved in 4 strains of V. parahaemolyticus and 1 strain of V. cholerae non-01 from clinical isolates and 4 strains of V. parahaemolyticus from environmental isolates, could be successfully amplified in 400 bp by PCR, no amplification products were obtained from TDH-negative strains. The PCR results were consistent with DNA hybridization. In the experiments of ctx gene detection, in all, 3 strains of V. cholerae non-01 from clinical isolate and 1 strains of V. cholerae non-01 from environmental isolate were observed CT- positive. These CT-producing strains amplified in 302 bp by PCR for the detection of ctx gene. All CT-producing strains hybridized with digoxigenin-labeled DNA probe, while CT-negative strains did not hybridize. Also hybridization tests results for detection of ctx gene consistent with PCR.
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Cólera , Digestão , DNA , Plasmídeos , Reação em Cadeia da Polimerase , Vibrio , Fatores de Virulência , VirulênciaRESUMO
Cholera enterotoxin (CT) is a major virulence determinant of Vibrio cholerae 01. CI' is known to be the major virulence factor of Vibrio cholerae 01 and in accordance with the recent report showing which V. cholerae non-01 has ctx gene, we performed the molecular genetic study for the detection of ctx gene related to the production of CT at the subject Vibrio spp. except for V. cholerae non-01 and V. cholerae non-01 stock cultured in the laboratory of microbiology, College of Medicine, Pusan National University and the Vibrio spp. isolated from the marine products of Pusan General Fish Market and the sea water, and then its results are as follows: 1. PCR for the detection of ctx gene at the subject of V. cholerae 01:61H-151 having the ctx gene of which the denaturation is 1 rninute at 95'C, annealing to 1min, 30 sec at 60'C, the extension to be 1min. 30 sec at 72'C and 30 or 40 cycles. ctx gene was detected from 4 strains of V. cholera non-01 derived from the environment isolates. 2. Adjusting the quantity of chromosomal DNA used as template DNA to be from 0.1 pg to 1 ng, in order to know the PCR conditions for the effective search of ctx gene, and the detection limit of the system was 10 pg of chromosomal DNA. 3. The broth culture was used for template DNA, ctx gene of 302 bp was detected from 4 V. cholerae non-01, as in the case of chromosomal DNA, and the cell number was possible to be detected to 3 * 10.4. We attempted the confirmation of ctx gene through Southern blot hybridization, labeling with P and then it was confirmed only from 4 V. cholerae non-01 as like PCR results. 5. As the result of the sensitivity of PCR and Southern blot hybridization, it was shown to be possible which 10 pg was detected in case of chromosomal DNA and in case of cultured broth, the cell number was detected until 10 at PCR and Southern blot hybridization, and thus it was examed which its sensitivity was same.
Assuntos
Southern Blotting , Contagem de Células , Toxina da Cólera , Cólera , DNA , Enterotoxinas , Limite de Detecção , Biologia Molecular , Óperon , Reação em Cadeia da Polimerase , Água do Mar , Vibrio cholerae , Vibrio , VirulênciaRESUMO
Fecal isolates of Escherichia coli which were collected from diarrheal patients and HUS patient in Pusan National University Hospital between 1990 and 1996, were serotyped and analyzed for plasmid DNA profile, biotype, HEp-2 cell adherence ability, reactivity to eae probe and for production of verotoxins (VT). In order to ease the diagnosis of EHEC infection, a LPS- based solid phase enzyme linked immunosorbent assay was utilized to detect serological diagnosis of EHEC infection. The following results were obtained. Among 150 EPEC isolates and HUS patient's stool, 7 EHEC were found. The 7 EHEC belonged to 5 different serotypes 0157:H7, 0143:H-, 0166:H-, 0128:H2, 026:H-, and 0111:H 21 previously associated with human haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Biochemical cheracteristic analysis indicated 7 strains were biotype 1 and was found to have siderophilins suggesting their advantagous growth in vivo. For plasmid profiles, all strains had 60 MDa plasmid and several smaller sizes of plasmids. Three strains of Escherichia coli serotype 0157:H7, 0128:H2, and 026:H- showed one pattern of adherence in the HEp-2 cell assay namely, localized adherence and were positive for eae probe when tested by colony blot hybridization assay. PCR using specific primers for VT1, VT2 was tested, and all 7 strains carried VT1 gene only. PCR products of 130-bp (VT1) and 346-bp (VT2) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC. The serum obtained from HUS patient of enterohemorrhagic E. coli were analyzed for rises in titer of intibody to somatic 02, 026, 0111, 0128, 0143, 0145, 0157, and 0165. Although response to the somatic 0 correlated significantly with response to the 026 rises of antibody titer to somatic 0 in acute stage of disease and anti-VT had not so many relation to that of VT. These results suggest that ELISA can be used to detect somatic 0 in serum and it is a useful method to diagnose the infection caused by EHEC rapidly.
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Humanos , Anticorpos , Colite , Diagnóstico , DNA , Escherichia coli Êntero-Hemorrágica , Escherichia coli Enteropatogênica , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Reação em Cadeia da Polimerase Multiplex , Plasmídeos , Reação em Cadeia da Polimerase , Toxinas Shiga , TransferrinaRESUMO
BACKGROUND: Bacterial extracellular protease has been implicated as an important virulence factor in several infections. In Vibrio sp., protease involvement has been demonstrated by the decreased virulence of protease-deficient mutants. We characterized the protease from V. parahemolyticus bc-7 with purification. METHODS: 100 Vibrio isolates from sea water and sea products were studied. The bacteria grown in Brain Heart Infusion broth were screened for protease production by incubating on tryptic soy agar plate containing gelatin and subsequently flooding the plate with 12.5% HgCl2-1M HCl. A clear zone indicated protease activity. Optimal conditions for protease production were determined by pH, temperature and NaCl concentration. Protease was purified from culture filtrates and its biological analysis was carried out. RESULTS: Out of Vibrio isolates studied, V. parahemolyticus bc-7 produced protease in the highest titer. Maximal yields of the protease was obtained when the bacteria were grown in Brain Heart Infusion broth with initial pH of 8.0, 3% NaCl at 30degrees C. Production of the protease was optimal during the late exponential phase. Homogeneity of the purified protease was demonstrated by revealing a 65kDa band on SDS-PAGE. The protease was stable at 50degrees C and 70% of the activity was lost by heating at 60degrees C for 30 min. The protease was stable at a pH between 5~10, but under pH 4.0, the activity was lost completely. CONCLUSION: General and serological studies are warranted to clarify the diffences and further characterize the protease as a virulence factor in Vibrio sp.