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1.
J Gabelsberger B Knapp S Bauersachs UI Enz BU von Specht H Domdey 《Canadian Metallurgical Quarterly》1997,(98):302-314
Recently a hybrid protein containing parts of the outer membrane proteins OprF (aa 190-342) and OprI (aa 21-83) from Pseudomonas aeruginosa fused to the glutathione-S-transferase was shown to protect mice against a 975-fold 50% lethal dose of P. aeruginosa. To omit the use of the GST-protein, the hybrid protein OprF-OprI was expressed in E. coli using distinct modifications which have not to be eliminated after its expression. Using different signal peptides, the yield of the hybrid protein OprF-OprI in E. coli could be increased to 30% of the total cell protein, however, only a very small amount of the hybrid preprotein was processed and could be isolated from the periplasm of the host. A construct containing an N-terminal extension of 11 amino acids from the original OprF gene gave rise to a significantly higher expression in the cytoplasm. Purification was facilitated by the addition of a five histidine tag at the C-terminus. An even higher expression was obtained by a construct in which a six histidine tag was attached to the N-terminus of the hybrid protein. The N-terminal extended OprF-OprI as well as the N-terminal his-tagged OprF-OprI hybrid antigens were purified by immobilized-metal affinity chromatography under native and denaturing conditions and can now be tested for protectivity against P. aeruginosa in animal model systems. 相似文献
2.
BU von Specht HC Lücking B Blum A Schmitt KD Hungerer H Domdey 《Canadian Metallurgical Quarterly》1997,(98):326-337
After expression in Escherichia coli and purification by Ni++ chelate-affinity chromatography, the outer membrane protein I (OprI) of Pseudomonas aeruginosa was tested in experimental animals for its safety and pyrogenicity. Four groups of 7 adult human volunteers were then vaccinated 3 times at four-weekly intervals with either 500 micrograms, 200 micrograms, 50 micrograms or 20 micrograms of OprI adsorbed onto aluminum hydroxide. The vaccinations were well tolerated and without systemic side effects, but a significant rise of antibody titers against OprI was measured in the serum of those who had received the 500 micrograms, 200 micrograms or 50 micrograms doses. Raised antibody titers against OprI were still present 30 weeks after the final vaccination. It was possible to demonstrate binding of the complement component C1q to the elicited antibodies, and this confirms their ability to promote antibody-mediated complement-dependent opsonization. 相似文献
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ER van der Voort P van der Ley J van der Biezen S George O Tunnela H van Dijken B Kuipers J Poolman 《Canadian Metallurgical Quarterly》1996,64(7):2745-2751
A set of isogenic strains was constructed from the meningococcal reference strain H44/76 (B:15:P1.7,16) which differed only in their outer membrane protein (OMP) compositions. First, three isogenic strains lacking the expression of either class 3 (PorB) or class 4 (RmpM) OMP or both were obtained. Second, three isogenic class 1 OMP loop-deficient strains of H44/76 lacking the predicted loop 1 or 4 or both of class 1 OMP (PorA) were obtained. Third, three isogenic class 1 OMP strains which differed by point mutations in the predicted loop 4 of subtype P1.16 were constructed. Strains were constructed through transformation with gene constructs made in Escherichia coli and their homologous recombination into the meningococcal chromosome. This study describes the contribution of one of the six class 1 OMPs, PorA P1.7,16, in the development of bactericidal antibodies after a single immunization of adult volunteers with 50 or 100 micrograms of protein within a hexavalent PorA outer membrane vesicle vaccine. PorA-, PorB-, and RpmM-deficient isogenic strains were used to define the human immune response against PorA. The loop-deficient isogenic strains were used to define the contribution of loops 1 and 4 of PorA in the development of bactericidal anti-PorA antibodies. The isogenic strains carrying a point mutation in loop 4 were used to study the cross-reactivity of the induced bactericidal antibodies against target strains showing microheterogeneity. The results indicate that a single immunization with the hexavalent PorA vaccine induced a dose-dependent bactericidal immune response, which is directed mainly against PorA. The epitope specificity of antibodies is directed mostly against loop 1, although loop 4 and as-yet-unidentified epitopes of PorA P1.7,16 are also involved. 相似文献
6.
L Martínez-Martínez A Pascual MC Conejo L Picabea EJ Perea 《Canadian Metallurgical Quarterly》1999,43(2):397-399
The activity of imipenem against Pseudomonas aeruginosa HUS-3 decreased by 16 times in the presence of substances eluted from siliconized latex urinary catheters (SLUCs). SLUCs did not inactivate imipenem or increase beta-lactamase activity. The outer membrane of P. aeruginosa HUS-3 grown in the presence of eluate lacked an OprD-like protein and expressed a new 50-kDa protein. The decreased activity of imipenem against P. aeruginosa in the presence of SLUCs is related to the loss of an OprD-like protein and the expression of a new outer membrane protein. 相似文献
7.
When purified without the use of ionic detergents, both OmpA and OprF proteins contained nearly 20% alpha-helical structures, which disappeared completely upon the addition of sodium dodecyl sulfate. This result suggests that the proteins fold in a similar manner, with an N-terminal, membrane-spanning beta-barrel domain and a C-terminal, globular, periplasmic domain. 相似文献
8.
SJ Cryz A Lang A Rüdeberg J Wedgwood JU Que E Fürer U Schaad 《Canadian Metallurgical Quarterly》1997,(98):345-349
Healthy, non-colonized cystic fibrosis (CF) patients (N = 26) were immunized with an octavalent Pseudomonas aeruginosa O-polysaccharide-toxin A conjugate vaccine. Vaccination was well tolerated and induced anti-lipopolysaccharide (LPS) antibodies of a high affinity capable of promoting the opsonophagocytic killing of P. aeruginosa by human peripheral lymphocytes. In contrast, anti-LPS antibodies acquired after natural infection possessed a very low affinity and were non-opsonic. To determine if immunization could prevent or delay infections due to P. aeruginosa, the infection rate among immunized patients was compared retrospectively to age and gender-matched controls. After 6 years of clinical follow-up, 15/20 (75%) of control and 8/23 (35%) of immunized subjects were classified as infected (p = 0.022). The persistence of high-affinity antibodies among immunized patients correlated with a significantly lower rate of infection after 4-6 years of observation. Infection of immunized patients was correlated with a dramatic decline in total antibody titer between year 2 and 3 of follow-up. Smooth, typeable strains of P. aeruginosa predominated among immunized patients. In contrast, rough, nontypeable strains were most frequently isolated from nonimmunized patients. Mucoid P. aeruginosa strains were isolated from 6 nonimmunized patients versus only I immunized subject. 相似文献
9.
The phenotypic characteristics of three Serpulina pilosicoli strains isolated from humans with diarrhoea (WesB, Kar, Hrm7) and two porcine S. pilosicoli strains isolated from pigs with intestinal spirochaetosis (1648, 3295), were compared with the type strain of the species P43/6/78T (T = type strain) and other intestinal spirochaetes within the genus Serpulina. All S. pilosicoli strains had a characteristic ultrastructural appearance, displayed similar growth rates, hydrolysed hippurate, lacked beta-glucosidase activity, utilised D-ribose as a growth substrate, and had similar sensitivities to rifampicin and spiramycin. The only consistent phenotypic characteristic that differentiated human strains from porcine strains of S. pilosicoli was that the human strains all utilised the pentose sugar D-xylose. These distinguishing phenotypic traits appear useful for identifying S. pilosicoli. 相似文献
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Pseudomonas aeruginosa is not generally considered a cause of infectious diarrhoea. However, it was the predominant organism isolated from the faeces of 23 unrelated, hospital outpatients investigated in the course of a year for persistent (> 1 week duration) diarrhoea. To investigate the possible aetiological role of P. aeruginosa, these patient histories were reviewed and a selection of their faecal isolates were investigated in vitro (n > or = 10) and in vivo (n = 2) for virulence. The patients had a mean age of 60 years, were receiving antibiotics and/or had an underlying illness. Extensive microbiological investigations identified no other potential or recognized enteropathogen in the faeces of 20 of these patients. More than 40% of the isolates tested were able to adhere to HEp-2 cells and exhibited twitching motility (type IV pili), properties indicative of their ability to colonize the human intestine. Cytotoxic activity was demonstrated in bacterium-free cell supernatants of over 80% of isolates; supernatants of four isolates tested in infant mice were weakly enterotoxigenic. Two isolates intragastrically inoculated into clindamycin pre-treated rats established persistent infections and induced signs and symptoms of enteritis. Overall these findings suggest that P. aeruginosa can cause diarrhoea particularly in immunodeficient individuals. 相似文献
11.
HS El-Zaim AK Chopra JW Peterson ML Vasil JP Heggers 《Canadian Metallurgical Quarterly》1998,66(11):5551-5554
Pseudomonas aeruginosa is an opportunistic pathogen that causes serious and sometimes fatal infections in the compromised host, especially in patients with major trauma or thermal injuries. Exotoxin A (ETA) is the major and most lethal virulence factor produced by this ubiquitous microorganism. In a recent study (H. S. Elzaim, A. K. Chopra, J. W. Peterson, R. Goodheart, and J. P. Heggers, Infect. Immun. 66:2170-2179, 1998), we identified two major epitopes, one within the translocation domain (amino acid [aa] residues 289 to 333) of ETA and another within the enzymatic domain (aa 610 to 638), by using a panel of antipeptide antibodies. Synthetic peptides representing these two epitopes induced ETA-specific antibodies which were able to abrogate the cytotoxic activity of ETA, as measured by incorporation of [3H]leucine into 3T3 fibroblasts. In the present study, these antibodies were tested for the ability to provide protection against ETA and infection with a toxin-producing strain of P. aeruginosa in a mouse model. Antibodies to either of the synthetic peptides conferred protection against ETA. Also, when used for immunization, both peptides induced active immunity to ETA in mice. Antibodies to the peptide representing a region within the enzymatic domain of ETA, in combination with the antibiotic amikacin, enhanced the survival of mice infected with a toxin-producing strain of P. aeruginosa. Thus, antipeptide antibodies specific for ETA might be paired with antibiotic treatment for passive immunization of patients suffering from P. aeruginosa infection. 相似文献
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The major outer membrane protein (MOMP) of Chlamydia species shares several biochemical properties with classical porin proteins. Secondary structure analysis by circular dichroism now reveals that MOMP purified from Chlamydia psittaci has a predominantly beta-sheet content (62%), which is also typical of bacterial porins. Can MOMP form functional ion channels? To directly test the "porin channel" hypothesis at the molecular level, the MOMP was reconstituted into planar lipid bilayers, where it gave rise to multibarreled channels, probably trimers, which were modified by an anti-MOMP monoclonal antibody. These observations are consistent with the well-characterized homo-oligomeric nature of MOMP previously revealed by biochemical analysis and with the triple-barreled behavior of other porins. MOMP channels were weakly anion selective (PCl/PK approximately 2) and permeable to ATP. They may therefore be a route by which Chlamydia can take advantage of host nucleoside triphosphates and explain why some anti-MOMP antibodies neutralize infection. These findings have broad implications on the search for an effective chlamydial vaccine to control the significant human and animal diseases caused by these organisms. 相似文献
13.
S Tamura A Yajima E Hatori S Tamura H Asanuma Y Suzuki C Aizawa T Kurata 《Canadian Metallurgical Quarterly》1997,15(16):1784-1790
In previous papers, we have shown that Escherichia coli heat-labile enterotoxin B subunit, supplemented with a trace amount of the holotoxin (LTB*) could be used as a potent adjuvant for a nasal influenza HA (haemagglutinin) vaccine in humans. The present study was designed to determine whether the effectiveness of a combined LTB*-HA vaccine could be limited by preexisting immunity to LTB and how many times the adjuvant-combined vaccine could be administered intranasally without reducing its protective efficacy in BALB/c, C3H and B10 mice. The magnitude of both nasal and serum Ab responses to HA vaccine was correlated with the degree of protection against virus infection. Higher doses of LTB*-combined vaccine were required for inducing high enough levels of anti-HA Ab responses to provide complete protection in low responder mice. Repeated pretreatments with LTB* alone (more than six times), which provided high levels of preexisting Abs to LTB, inhibited the induction of anti-HA Ab responses and reduced the protective efficacy of the adjuvant-combined vaccine. However, the LTB*-combined vaccine could be given repeatedly (about ten times) to mice without reducing the effectiveness of the adjuvant-combined vaccine. These results suggest that the LTB*-combined nasal influenza vaccine can be given to humans once every few years when an epidemic of influenza may occur. 相似文献
14.
OBJECTIVE: To describe an immunocompromised patient (without AIDS) with nosocomial infectious diarrhea caused by Pseudomonas aeruginosa. Oral ciprofloxacin therapy proved to be effective. CASE SUMMARY: An 80-year-old woman with type II diabetes mellitus and hypertension developed progressive renal insufficiency, was hospitalized because of uremia, and underwent hemodialysis. When the patient developed hematochezia, Duke's C sigmoid colon cancer was detected and successfully resected. She received broad-spectrum antibiotics in the perioperative period. The patient then developed profuse diarrhea associated with abdominal cramping, a low-grade fever, prostration, and headache. The patient then started to received vancomycin 500 mg po qid empirically. Four days later, the diarrhea continued unabated, the Clostridium difficile titer was negative, and the vancomycin therapy was stopped. However, the stool culture was positive for heavy growth of P. aeruginosa sensitive to ciprofloxacin. The patient then began to receive ciprofloxacin 500 mg po bid. Within 3 days the diarrhea stopped. Oral ciprofloxacin therapy was continued for 10 days and the patient remained free of symptoms with formed stools thereafter. DISCUSSION: Diarrhea following the use of broad-spectrum antibiotics implicates pseudomembranous colitis as the cause. The patient did not respond to oral vancomycin therapy and had a negative stool assay for C. difficile toxin. This patient was believed to have Pseudomonas enteritis, which was confirmed by 2 positive stool cultures. The administration of oral ciprofloxacin therapy stopped her diarrhea with a rapid resolution of symptoms. CONCLUSIONS: P. aeruginosa as a cause of infectious diarrhea is unusual. When it occurs, it usually represents a nosocomial infection in an immunocompromised host. This report illustrates that oral ciprofloxacin therapy is effective for Pseudomonas enteritis, with rapid resolution of symptoms. 相似文献
15.
Moraxella (Branhamella) catarrhalis is an important human respiratory tract pathogen. Outer membrane protein (OMP) CD is highly conserved among strains and has characteristics that indicate it may be an effective vaccine antigen. This study investigated the effect of immunization with OMP CD on pulmonary clearance following intratracheal challenge of mice with M. catarrhalis. Two routes of immunization were studied: mucosal immunization (intra-Peyer's patch followed by intratracheal boost) and intramuscular immunization with OMP CD. Both resulted in enhanced pulmonary clearance of M. catarrhalis compared with sham-immunized controls. Immunization with OMP CD induced specific antibodies in serum and bronchoalveolar lavage fluid and induced a specific lymphocyte proliferative response in T cells from mesenteric lymph nodes from mice mucosally immunized with OMP CD. On the basis of these results, OMP CD should undergo continued testing to determine whether it will induce a protective immune response in humans. 相似文献
16.
GQ Zhang A Hotta T Ho T Yamaguchi H Fukushi K Hirai 《Canadian Metallurgical Quarterly》1998,42(6):423-428
The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera. 相似文献
17.
Using a granulocytopenic murine model, we evaluated the efficacy of cefoperazone plus sulbactam against disseminated infection due to isolates of beta-lactamase-producing, cefoperazone-resistant (MIC, > or = 50 micrograms/ml) Escherichia coli and Pseudomonas aeruginosa. Both isolates were susceptible in vitro to cefoperazone plus sulbactam (MIC, < or = 6.3 micrograms/ml). Mice rendered granulocytopenic with cyclophosphamide were divided into three groups: group A--infected, untreated mice (controls); group B--infected, cefoperazone-treated mice (700 mg/kg of body weight); and group C--infected, cefoperazone-plus-sulbactam-treated mice (700 mg plus 350 mg). In the E. coli experiment, survival rates in groups A, B, and C were 25, 46, and 73%, respectively. In the experiment with P. aeruginosa, survival rates in groups A, B, and C were 0, 10, and 50%, respectively (P < 0.001). Highly significant differences also were noted for colony counts in the blood, liver, and spleen of group C mice versus group A or B mice in both experiments. Thus, cefoperazone plus sulbactam appears to be a promising combination for the treatment of infections due to certain cefoperazone-resistant gram-negative bacilli, including P. aeruginosa. 相似文献
18.
In 1994, the Malm?-Klaipeda twinning programme was approved by the World Federation of Hemophilia. One of the first steps in the collaboration has been to set up a registry of the haemophilia patients in the Klaipeda area. In order to collect important clinical data the patients have been examined jointly by experts on haemophilia from the two centres. Seventeen out of 25 patients with severe haemophilia known at the Klaipeda centre were examined and compared to a matched cohort of patients from the Malm? centre. The main differences between the cohorts were that home treatment was not available to the Klaipeda patients, they received less treatment in general, had higher joint scores and more frequent bleeds. The pattern of transmission of blood-borne virus was very similar, with a high prevalence of hepatitis C antibodies. We conclude that the twinning programme between Malm? and Klaipeda has resulted in several achievements, including training of staff and a necessary inventory of the patients. This should not only form a suitable platform for the future development of haemophilia care in Lithuania, but could also serve as an example for liaisons between other haemophilia centres. 相似文献
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A crude outer membrane preparation from Francisella tularensis Live Vaccine Strain (LVS) was used to immunize mice. Immunized mice were completely protected from a F. tularensis challenge. We evaluated the role of two major outer membrane antigens in the induction of protective immunity, namely lipopolysaccharide and an outer membrane protein FopA. We presented FopA to the immune system using an aromatic amino acid-dependent Salmonella typhimurium as a vector. Although mice mounted an immune response to cloned FopA no significant protection was induced. However, LPS immunized mice were completely protected. We conclude that LPS is a major protective antigen whereas FopA has a limited or no role in the induction of protective immunity. 相似文献
20.
CR Zanetti MT de Franco RC Vass?o CA Pereira OA Pereira 《Canadian Metallurgical Quarterly》1998,143(9):1745-1756
This report shows that the SMB vaccine currently used in Brazil for human immunisation provides different degrees of protection in mice, depending on the rabies virus strain used as challenge. Using the NIH and Habel potency tests to evaluate the protective activity of rabies vaccine, we observed that vaccinated mice showed a higher resistance to a challenge with a fixed rabies virus (CVS-Challenge Virus Strain). The vaccine potency using the Habel or NIH tests was respectively > 6.4 (log 10) and 1.0 (Relative Potency-RP) when the fixed rabies virus was used for challenge, and from 2.9 to 4.3 (log 10) or 0.13 to 0.8 (RP) when different wild rabies viruses were used for challenge. The presence of virus neutralising antibodies (VNA) could not explain the differences of susceptibility after vaccination, since sera of vaccinated animals had similar VNA levels against both fixed and wild strains before virus challenge (respectively, 5.6 +/- 0.24 and 5.0 +/- 0.25 IU/ml of VNA against the fixed rabies virus and the 566-M strain of wild rabies virus in sera of mice vaccinated with 0.2 units of vaccine). Only cell-mediated immunity parameters correlated with the protection induced by vaccination. The IFN gamma titers found in sera and brain tissues of animals challenged with CVS strain were higher (from 36.7 +/- 5.7 to 293.3 +/- 46.2 IU/ml) than those found in mice challenged with 566-M virus strain (from 16.7 +/- 5.8 to 36.7 +/- 5.8). The proliferation index of spleen cells obtained with CVS stimulation reached a maximal value of 15.1 +/- 0.7 while spleen cells from vaccinated mice stimulated with 566-M virus failed to proliferate. The implications of these data in human protection by vaccination are discussed. 相似文献