An alternative to blunt-end ligation for cloning DNA fragments with incompatible ends.
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Animals↗
Biomedical subjects
R Díaz
Publications and source records attributed to R Díaz.
Effect of composition and method of preparation of liposomes on their stability and interaction with murine monocytes infected with Brucella abortus.
The success of the use of liposomes as drug carriers depends on both their formulation and the method of preparation. We have carried out a series of in vitro studies using different formulations and preparation methods, with the aim of obtaining a type of liposome which is efficient in the treatment of brucellosis. On the basis of results obtained in studies of stability at 37 degrees C in the presence of serum lipoproteins and of the activation of phagocytic cells and antibiotic transport to the interior of monocytes infected with Brucella abortus, we conclude that the most suitable vesicles are positively charged, stable plurilamellar vesicles (phosphatidylcholine, 30% cholesterol, and 10% stearylamine). Gentamicin incorporated into these cationic liposomes completely eliminated all of the intracellular Brucella organisms (4.6 logs), while free gentamicin was capable of reducing the number of intracellular bacteria by only 0.3 log.
Animals↗
Outer membrane differences between pathogenic and environmental Yersinia enterocolitica biogroups probed with hydrophobic permeants and polycationic peptides.
Sensitivities to polycationic peptides and EDTA were compared in Yersinia enterocolitica pathogenic and environmental biogroups. As shown by changes in permeability to the fluorescent hydrophobic probe N-phenylnaphthylamine (NPN), the outer membranes (OMs) of pathogenic and environmental strains grown at 26 degrees C in standard broth were more resistant to poly-L-lysine, poly-L-ornithine, melittin, cecropin P1, polymyxin B, and EDTA than Escherichia coli OMs. At 37 degrees C, OMs of pathogenic biogroups were resistant to EDTA and polycations and OMs of environmental strains were resistant to EDTA whereas E. coli OMs were sensitive to both EDTA and polycations. Similar results were found when testing deoxycholate sensitivity after polycation exposure or when isogenic pairs with or without virulence plasmid pYV were compared. With bacteria grown without Ca++ available, OM permeability to NPN was drastically increased in pathogenic but not in environmental strains or E. coli. Under these conditions, OMs of pYV+ and pYV- cells showed small differences in NPN permeability but differences in polycation sensitivity could not be detected by fluorimetry. O:1,6 (environmental type) lipopolysaccharide (LPS), but not O:3 or O:8 LPS, was markedly rough at 37 degrees C, and this could explain the differences in polycation sensitivity. LPSs from serotypes O:3 and O:8 grown at 37 degrees C were more permeable to NPN than O:1,6 LPS, and O:8 LPS was resistant to polycation-induced permeabilization. These data suggest that LPSs relate to some but not all the OM differences described. It is hypothesized that the different OM properties of environmental and pathogenic biogroups reflect the adaptation of the latter biogroups to pathogenicity.
Bacterial Outer Membrane Proteins↗
Characterization of Brucella abortus and Brucella melitensis native haptens as outer membrane O-type polysaccharides independent from the smooth lipopolysaccharide.
Brucella native haptens (NHs) extracted with hot water from smooth (S)-type B. abortus and B. melitensis were purified to high levels of serological activity and compared with the polysaccharide obtained by acid hydrolysis (PS) of the S lipopolysaccharide (S-LPS). By 13C nuclear magnetic resonance analysis, NHs showed the spectrum of a homopolymer of alpha-1,2- or alpha-1,2- plus alpha-1,3-linked 4-formamido-4,6-dideoxy-D-mannose (N-formylperosamine) previously reported for the LPS O chain. However, while PS contained up to 0.6% 3-deoxy-D-manno-2-octulosonate, this LPS-core marker was absent from NH. High performance liquid chromatography and thin-layer chromatography showed heterogeneity in NH purified from whole cells but not in PS. By immunoprecipitation, polysaccharides indistinguishable from NH were demonstrated in extracts obtained with phenol-water, saline at 60 degrees C, and ether-water treatments, and none of these treatments caused S-LPS hydrolysis detectable with antibodies to the O chain and lipid A. Two lines of evidence showed that NH was in the cell surface. First, NH became biotinylated when B. abortus live cells were labelled with biotin-hydrazide, and the examination of cell fractions and electron microscopy sections with streptavidin-peroxidase and streptavidin-coloidal gold, respectively, showed that labelling was extrinsic. Moreover, whereas only traces of NH were found in cytosols, the amount of NH was enriched in cell envelopes and in the outer membrane blebs spontaneously released by brucellae during growth. Interactions between NH and S-LPS were observed in crude cell extracts, and such interactions could be reconstituted by using purified NH and LPS. The results demonstrate that NH is not a hydrolytic product of S-LPS and suggest a model in which LPS-independent O-type polysaccharides (NH) are intertwined with the O chain in the outer membrane of S-type brucellae.
Brucella abortus↗
[Utility of the Stamp method].
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Bacteremia↗
[Acute kidney failure in patients with and without sepsis: prognosis and clinical course].
The purpose of this prospective study was to determine whether the course and prognosis of acute renal failure (ARF) in patients with and without sepsis are different. Two hundred fifty-two (8%) of 3086 consecutive patients admitted to a medical-surgical intensive care unit (ICU) developed ARE. One hundred forty-nine (59%) were septic and 103 (41%) were non-septic. No differences were found between groups regarding the incidence of oliguria, hyperkalemia, hypercatabolism, gastrointestinal bleeding, duration of oligria and renal deficit, severity of axotemia, dialysis requirements and duration of stay in the hospital. There were statistically significant differences between septic and non septic patients with respect of hyponatremia (67.8 vs 54.4%, p < 0.04), respiratory failure (68 vs 54%, p < 0.04), and thrombocytopenia (64 vs 48%, p < 0.02). Mortality in septic patients was higher than in non-septics (56 vs 42.7%, p < 0.009). Factors associated with increased mortality in ARF septic patients were respiratory failure, metabolic acidosis and oliguria while in the non-septics they were hepatic dysfunction, hyperkalemia, respiratory failure and infection acquired during the course of renal failure. We conclude that ARF developing in septic patients has a higher mortality than that of non-septic patients, whereas the incidence of hypercatabolism and oliguria was not different between both groups.
Acute Kidney Injury↗
Penicillium purpurogenum produces several xylanases: purification and properties of two of the enzymes.
The fungus Penicillium purpurogenum produces several extracellular xylanases. The two major forms (xylanases A and B) have been purified and characterized. After ammonium sulfate precipitation and chromatography in Bio-Gel P 100, xylanase A was further purified by means of DEAE-cellulose, hydroxylapatite and CM-Sephadex, and xylanase B by DEAE-cellulose and CM-Sephadex. Both xylanases showed apparent homogeneity in SDS-polyacrylamide gel electrophoresis. Xylanase A (33 kDa) has an isoelectric point of 8.6, while xylanase B (23 kDa) is isoelectric at pH 5.9. Antisera against both enzymes do not cross-react. The amino terminal sequences of xylanases A and B show no homology. The results obtained suggest that the enzymes are produced by separate genes and they may perform different functions in xylan degradation.
Amino Acid Sequence↗
Comparative activity of azithromycin and doxycycline against Brucella spp. infection in mice.
The activities of a short therapeutic regimen with azithromycin and the classic treatment doxycycline with streptomycin were compared and evaluated in mice infected with Brucella melitensis. In a chronic model, starting therapy 31 days after challenge, azithromycin (10 days, 50 mg/kg/day) significantly reduced the infection (2.9 logs, day 48 post-infection). The effectiveness of doxycycline (21 days, 50 mg/kg/12 hourly) was greater than azithromycin (4.1 logs of reduction, day 48 post-infection), and when doxycycline was administered for a period of 45 days, all the animals were bacteriologically cured from day 78. The combination with streptomycin (14 days, 10 mg/kg/day) did not improve the effect of any of the regimens. In an acute model infection, treatments with doxycycline or doxycycline-streptomycin, for a period of 3 days, starting 1 day after lethal challenge, were able to protect all the mice. In contrast, only 50% of the mice treated with azithromycin survived the challenge. In conclusion, although a short oral treatment with azithromycin was able to reduce the infection significantly, it was not able to cure the animals as effectively as the classic regimen with doxycycline administered for a longer period of time.
Acute Disease↗
Antibiotic treatment induces an increase of the specific antibody levels in Brucella melitensis infected mice.
The effects of doxycycline (DOX) and streptomycin (SM) treatment of Brucella melitensis infected mice on humoral immune response were examined. In female BALB/c mice, DOX was administered at a dose of 50 mg/kg/12 h, for 21 or 45 consecutive days, alone or combined with SM (10 mg/kg/12 h) for 14 days. All treatments induced a significant increase in specific IgG levels (ELISA and CIEP) against LPS and cytosolic antigens of Brucella during treatment. This was not related with therapeutic failure or relapse since all treatments induced a significant reduction in the degree of infection.
Animals↗
Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats.
Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.
Animals↗
Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep.
A total of 291 unvaccinated sheep from Brucella melitenesis-infected flocks were examined for delayed-type hypersensitivity (DTH) responses with Brucellergene commercial allergen and with cold saline extract and cytosol from rough B. melitensis 115, and their sera were tested in the rose bengal test (RBT), complement fixation test (CFT), and enzyme-linked immunosorbent assay (ELISA) with lipopolysaccharide. DTH reactions were maximal after 72 h, with no intensity differences among allergens, inoculation sites (eyelid and tail), and doses tested. There were no differences in the results recorded by visual inspection and palpation of inoculation sites, by measuring skin thickness with a caliper, or by microscopic examination of samples taken at necropsy. Six days after DTH testing, energy was observed in 100% of the animals, and 100% reactivity was recovered only after 24 days. All animals were necropsied, and thorough bacteriological searches were performed. The sensitivities found with the 140 animals from which B. melitensis was isolated were ELISA, 100%; DTH, 97.1%; RBT, 92.1%; and CFT, 88.6%. Those results put into question the value of RBT and CFT as screening and confirmatory tests for sheep brucellosis and at least indicate that their standardization should be modified. For 151 tested sheep from which B. melitensis was not isolated, the percentages of positive animals were ELISA, 100%; DTH, 94.0%; RBT, 57.6%; and CFT, 53.6%. All tests were negative for 100 tested sheep from Brucella-free flocks. The different results of bacteriological and immunological tests suggest the usefulness of developing indirect tests able to distinguish truly infected animals from those that have developed an immunological response.
Allergens↗
[Permanent venous access through a +portacath reservoir in patients receiving prolonged chemotherapy].
A significant number of oncology patients need intravenous chemotherapy. Peripheral venous access is generally difficult, frequently requiring central venous access. This study reviews the Author's experience with permanent venous portals. A total of 66 patients records, from January 1992 to April 1994, were reviewed. The central venous portals remained in place an average of 272 days. An average of 8 courses of chemotherapy were given through each port. There was no significant difference in the rate of complications secondary to the portal site. The Authors conclude that the placement of permanent venous portals is a safe and reliable chronic venous access in patients with cancer who require intravenous therapy. The rate of serious complication was 3% which is relatively low for these immunosuppressed patients. All venous portals, except one (98.5%) remained functional.
Antineoplastic Agents↗
[Evaluation of an ELISA for the serologic diagnosis of tuberculosis].
The use of an ELISA method for the serological diagnosis of tuberculosis was assessed through the study of the presence of circulating IgG antibodies to PPD in 220 serum samples. An 82% sensibility was determined in 50 serum samples from patients with pulmonary tuberculosis, and a specificity of 95.33% in 150 serum samples from apparently healthy subjects. 20 serum samples from patients with disorders other than tuberculosis were included in the study to determine possible cross reactions.
Antibodies, Bacterial↗
Brucella melitensis Rev-1 vaccine as a cause of human brucellosis.
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Accidents, Occupational↗
Brucella group 3 outer membrane proteins contain a heat-modifiable protein.
Brucella melitensis and B. ovis outer membrane blebs contained a protein displaying a temperature-dependent molecular mass upshift from 25 kDa to 30 kDa. A fraction of the protein tightly bound to LPS did not show the molecular mass upshift which was also blocked by exposure of the protein to Zwittergent 314. The B. melitensis heat-modifiable protein and Escherichia coli OmpA shared antigenic determinants. These data indicate that the Brucella group 3 outer membrane proteins belonged to the OmpA family of proteins.
Bacterial Outer Membrane Proteins↗
Evaluation of whole cell and subcellular vaccines against Brucella ovis in rams.
Five antigen preparations from Brucella ovis strain REO 198 were incorporated with the pluronic polymer L-121 and muramyl dipeptide and tested as vaccines against B. ovis infection of rams. The antigenic preparations were: (1) a fraction enriched in outer membrane proteins and rough lipopolysaccharide (hot saline extract, HS); (2) the proteins from HS substantially free of lipopolysaccharide; (3) outer membrane blebs; (4) outer membrane-peptidoglycan complexes extracted with detergent; (5) killed whole cells. The experimental vaccines were compared with two standard vaccines, rough Brucella abortus 45/20 whole killed cells in an oil based adjuvant, and live Brucella melitensis Rev 1. Immunizations with non-living vaccines were performed on two occasions, 18 weeks apart. The rams were challenged with a virulent strain of B. ovis 31 weeks after the second vaccination and slaughtered 15 weeks thereafter. Rates of infection in groups vaccinated with Rev 1 (33%), and HS (40%) were significantly lower (P < 0.005 and P < 0.025, respectively) than that in the non-vaccinated control group (87%). Strain 45/20 was the only other vaccine that conferred a significant level of protection (50%) (P < 0.05). The organ distribution of the infection and the level of colonization of infected organs did not differ significantly between infected animals in the various vaccine groups and those in the unvaccinated control group. No statistically significant relationship was detected between the magnitude of the antibody responses to the HS extract, to outer membrane proteins, or to the rough lipopolysaccharide, and freedom from infection. The results indicate that the HS extract of B. ovis may represent a useful alternative to B. melitensis Rev 1 or B. abortus 45/20 as a vaccine against B. ovis.
Animals↗
The Myf fibrillae of Yersinia enterocolitica.
The Myf antigen produced by Yersinia enterocolitica appeared as a proteic polymer composed of 21 kDa subunits. By transposon mutagenesis we isolated Myf-defective mutants. Those allowed us to clone and sequence a 4.4 kb chromosomal locus involved in Myf production. This region was found to contain three genes that we called myfA, myfB and myfC. Genes myfB and myfC encode an assembly machine related to those involved in the synthesis of many fimbriae: MyfB, the putative chaperone, possesses the consensus residues of the PapD family and myfC encodes a putative outer-membrane protein. MyfA, the major subunit, was found to be 44% identical to the pH 6 antigen of Y. pestis. Myf is thus the Y. enterocolitica counterpart of this antigen, but it is by far not so well conserved as the other virulence determinants such as the Yops, suggesting that Myf and pH 6 antigen do not necessarily play the same role in Y. enterocolitica and Y. pestis. The study of the prevalence of myfA in various species of Yersinia revealed that, like the yst enterotoxin gene, its presence is restricted to the pathogenic serotypes of Y. enterocolitica. By immunogold labelling, Myf appeared as a layer of extracellular material extending locally 2 microns from the bacterial surface, indicative of a fibrillar structure.
Amino Acid Sequence↗
Comparative analysis of Brucella serotype A and M and Yersinia enterocolitica O:9 polysaccharides for serological diagnosis of brucellosis in cattle, sheep, and goats.
Hapten polysaccharides of Brucella smooth M and A serotypes were prepared from Brucella sp. and Yersinia enterocolitica O:9 by previously described hydrolytic (O chain) or nonhydrolytic (native hapten [NH]) procedures. The purified polysaccharides differed only in the presence (O chain) or absence (NH) of lipopolysaccharide core sugars. The polysaccharides were compared by reverse radial immunodiffusion for the diagnosis of brucellosis in cattle (Brucella abortus biotype 1 [A serotype] and Brucella melitensis biotype 3 [AM serotype]), sheep (B. melitensis biotypes 1 [M serotype] and 3), and goats (B. melitensis biotype 1). The reverse radial immunodiffusion test with the NH from B. melitensis 16 M (serotype M) showed the highest sensitivity (89.6 to 97.3%), regardless of the host species and the serotype of the infecting Brucella sp. Y. enterocolitica O:9 NH (A serotype) was useful for diagnosing disease in cattle infected with B. abortus biotype 1, but not in cattle infected with B. melitensis biotype 3, sheep, or goats. The different results obtained with the serotype M and A polysaccharides and the sera from animals infected with M, A, and AM serotypes of Brucella spp. showed that in naturally infected animals, a large proportion of the antibodies are directed to or react with a previously defined common epitope(s) (J. T. Douglas and D. A. Palmer, J. Clin. Microbiol. 26:1353-1356, 1988) different from the A or M epitopes. By using the radial immunodiffusion test with B. melitensis 16M NH, it was possible to differentiate infected from vaccinated cattle, sheep, and goats with a sensitivity and specificity similar to that of the complement fixation test.
Animals↗