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R Torensma

Publications and source records attributed to R Torensma.

At least 37 records · Page 2Linked to original sources
PubMed1993

Human granulocytes express a 55-kDa lipopolysaccharide-binding protein on the cell surface that is identical to the bactericidal/permeability-increasing protein.

Several LPS-binding proteins have been identified on the surface of human granulocytes (polymorphonuclear leukocyte (PMN)). We describe a plasma-membrane associated ca. 55-kDa LPS-binding protein of human PMN that is indistinguishable from the bactericidal/permeability-increasing protein (BPI). To detect LPS-binding proteins on the cell surface, PMN were biotinylated before detergent solubilization and incubation with LPS-coated beads. Several biotinylated proteins bound to LPS-coated beads but not to uncoated beads and were characterized after elution with detergent by SDS-PAGE and western blotting using streptavidin-horseradish peroxidase. The spectrum of biotinylated proteins binding to and eluting from LPS-coated beads increased as the number of beads incubated with PMN lysate increased. However, at all concentrations of beads a 55-kDa protein was a dominant component of the eluate. Binding of the 55-kDa protein to LPS-coated beads was inhibited by lipid A, and both homologous and heterologous LPS, but not by peptidoglycan. Similar amounts of biotinylated 55-kDa LPS-binding protein were detected on PMN from patients with paroxysmal nocturnal hemoglobinuria who lacked membrane bound CD14, a known ca. 55-kDa plasma membrane-associated LPS-binding protein, indicating that the recovered biotinylated protein is not CD14. Several pieces of evidence, however, do indicate that the 55-kDa surface protein is BPI: 1) flow cytometry of PMN after labeling with rabbit anti-BPI serum and FITC-labeled goat anti-rabbit IgG revealed immunoreactive surface molecules on resting PMN and, in increased amounts, on PMN stimulated with FMLP or TNF; 2) This antiserum specifically and quantitatively inhibited binding of the biotinylated 55-kDa species to LPS-coated beads; 3) both BPI and the 55-kDa protein migrated as a doublet during SDS-PAGE and were both converted to single migrated species after N-glycosidase F treatment; 4) chemical cleavage of the biotinylated protein and native BPI with N-chlorosuccinimide yielded the same fragments. Thus, we have positively identified BPI as a LPS-binding protein on the surface of PMN. The role of this potent antibacterial, endotoxin neutralizing protein on the surface of PMN remains to be established.

Acute-Phase Proteins↗
PubMed1993

Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.

Antibodies, Monoclonal↗
PubMed1993

Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay.

Rapid detection of salmonellae in chicken meat was accomplished by using the magnetic immuno-polymerase chain reaction assay (MIPA). A direct polymerase chain reaction assay performed with chicken meat spiked with Salmonella typhimurium resulted in poor sensitivity (approximately 10(7) CFU/g of meat). The use of immunoseparation with a Salmonella serogroup B-specific monoclonal antibody improved the sensitivity, but enrichment was required for the detection of low levels of contamination. Enrichment for 6 h in either buffered peptone water, lactose broth containing tergitol-7, or selenite-cystine broth resulted in the detection of an initial inoculum of 100 CFU per g of meat. Enrichment of the salmonellae present on 25 g of spiked chicken meat for 24 h in either buffered peptone water or selenite-cystine broth before detection by the MIPA yielded a detection limit of approximately 0.1 CFU/g of meat. A detection limit of approximately 1 CFU/g of meat was obtained when the spiked meat was stored at -20 degrees C before enrichment for 24 h and analysis with the MIPA. Although the MIPA was developed for S. typhimurium, a MIPA in which a panel of six monoclonal antibodies specific for Salmonella serogroups A through E was used detected the presence of 0.1 CFU of Salmonella enteritidis per g of chicken meat. These data indicate that the method is applicable to other commonly isolated serotypes.

Animals↗
PubMed1993

Monoclonal antibodies that react with live Listeria spp.

Seven monoclonal antibodies (MAbs) against Listeria spp. that were reactive with live Listeria spp. were developed. Two of these MAbs (55-8 and 55-37) were members of the immunoglobulin M class, and all other MAbs were members of the immunoglobulin G class. MAb 55-23 reacted with 148 of 157 strains tested. MAb 34-51 reacted with serotype 1/2a, 1/2b, and 1/2c strains and exhibited a scattered reaction pattern with strains belonging to other serotypes. MAb 55-44 reacted with all of the strains belonging to serotype 4b tested. MAb 55-4 reacted with all of the serotype 1/2a isolates tested, although reactivity with other isolates also was observed. The other MAbs exhibited scattered reaction patterns. No correlation of reactivity pattern with serotype was found. Marked differences were observed between the reactivities of MAbs as determined by a magnetic immunoluminescence assay and a whole-cell enzyme-linked immunosorbent assay. Only MAb 55-23 exhibited minor reactivity with three Streptococcus spp. isolates, while no reactivity was observed with six Bacillus spp. strains, one Escherichia coli strain, and one Citrobacter sp. strain. In Western blots (immunoblots) MAbs 55-23, 55-44, and 34-9 exhibited reactivity; all other MAbs were negative in this assay.

Animals↗
PubMed1993

Escherichia coli in bacteremia: O-acetylated K1 strains appear to be more virulent than non-O-acetylated K1 strains.

A total of 174 blood isolates of Escherichia coli, collected during a 5-year period at the University Hospital Utrecht, were serotyped with rabbit sera against 171 O antigens and 73 capsule (K) antigens. The four most prevalent O-antigen serotypes were O6 (n = 22), O18 (n = 19), O1 (n = 19), and O2 (n = 15). Thirty-one strains were not typeable with any of the O-antigen-typing sera. Of the 148 strains that were subjected to K-antigen serotyping, 34 strains lacked a K antigen and 41 were not typeable with the K-antigen-specific antisera used in the study. K1 was by far the most frequently found K-antigen serotype; this was followed by K2, K53, K5, K13, K7, K(A)28, and K15. Strains possessing a K1 antigen were further classified as either O-acetyl-positive (n = 12) or O-acetyl-negative (n = 21) strains. Retrospective analysis of patients infected with different E. coli isolates--nonencapsulated (n = 23), O-acetylated K1 (n = 12), and non-O-acetylated K1 (n = 21)--revealed clinical differences. More patients suffered from sepsis (94% versus 74%), and a higher rate of mortality was found in the group infected with K1 isolates (18 versus 9%) than in the group infected with nonencapsulated isolates. More patients with severe sepsis (25 versus 10%) and a higher mortality (33 versus 10%) were found in the group infected with O-acetylated K1 isolates than in the group infected with non-O-acetylated isolated. Also, the hospitalization of these patients was prolonged. Thus, O-acetylated E. coli K1 strains seem to be more virulent than non-O-acetylated K1 strains.

Acetylation↗
PubMed1993

Comparison of typing methods for Clostridium difficile isolates.

A simple discriminative typing method for Clostridium difficile has been developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and restriction enzyme analysis are relatively simple techniques but are difficult to evaluate, especially the restriction enzyme analysis. Immunoblotting and restriction fragment length polymorphism typing facilitate simple discrimination of patterns.

Bacterial Typing Techniques↗
PubMed1992

Monoclonal antibodies that detect live salmonellae.

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.

Animals↗
PubMed1992

The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples.

Direct polymerase chain reaction (PCR)-based detection with fecal specimens is hampered by inhibitory compounds, such as bilirubin and bile salts. These fecal compounds showed significant inhibition of PCR at low concentrations (10 to 50 micrograms/ml). For direct PCR analysis, fecal samples must be diluted 500-fold to overcome inhibition. Therefore, the magnetic immuno PCR assay (MIPA), which combines immunomagnetic separation by using specific monoclonal antibodies and PCR, was used to directly detect salmonellae in feces from humans. Immunomagnetically extracted stool samples needed to be diluted only 10-fold when 1 microgram of T4 gene 32 protein was added to the PCR. The MIPA sensitivity obtained was 10(5) CFU/ml of feces. A panel of monoclonal antibodies specific for Salmonella serogroups A to E was used to extract salmonellae from clinical samples. MIPA detection of salmonellae occurred with 11 out of 14 stool samples stored at 4 degrees C for 2 months. MIPA detection of salmonellae in stool samples is a promising, fast method for detection and identification.

Antibodies, Monoclonal↗
PubMed1991

Evaluation of the magnetic immuno PCR assay for rapid detection of Salmonella.

A new technique, the Magnetic Immuno PCR Assay (MIPA), has been developed for the detection of Salmonella. The assay utilizes magnetic particles coated with monoclonal antibodies against Salmonella to extract these bacteria from the sample. Trapped bacteria are lysed, and the supernatant, which contains bacterial DNA, is then subjected to the polymerase chain reaction (PCR) using primers from the Salmonella typhimurium origin of DNA replication to amplify a 163 bp region. The specificity of the primer set was tested in the PCR; amplification occurred with all 25 Salmonella strains tested but not with 19 other species of Enterobacteriaceae tested. A sensitivity of 100 cfu Salmonella typhimurium was achieved for the MIPA by visualization of the amplified products by ethidiumbromide stained agarose gel electrophoresis. A ten-fold higher sensitivity was obtained by Southern blotting of the amplified products. The presence of 10(7) cfu Escherichia coli did not interfere with these detection levels. The MIPA thus specifically detected 100 cfu of Salmonella within 5 h and may be potentially useful for rapid detection of Salmonella in clinical specimens and food.

Base Sequence↗
PubMed1991

Immunization of mice with antibiotic-treated Escherichia coli results in enhanced protection against challenge with homologous and heterologous bacteria.

The murine immune response to Escherichia coli exposed to subminimal inhibitory concentrations of four antibiotics was investigated. Groups of mice were injected for 8 weeks with formalin-killed bacteria and subsequently challenged with 10 x LD50 of viable E. coli. Mice receiving saline only (controls) died within 24 h. The mortality of mice immunized with ciprofloxacin-treated E. coli was significantly lower than that of mice immunized with E. coli untreated or treated with other antibiotics. Sera from mice immunized with ciprofloxacin-treated bacteria showed better bacteriostatic capacity and enhanced production of antibodies that bound to homologous and heterologous lipopolysaccharide isolated from several smooth and rough gram-negative strains. The better protection observed in mice immunized with ciprofloxacin-treated E. coli was probably due to an enhanced production of antibodies to epitopes on lipopolysaccharide that became better exposed and so more accessible after treatment with ciprofloxacin.

Animals↗
PubMed1991

Characterisation and functional aspects of monoclonal antibodies specific for surface proteins of coagulase-negative staphylococci.

Mouse monoclonal antibodies (MAbs) raised against whole cells of Staphylococcus epidermidis strain 354 were characterised morphologically and functionally. Nine MAbs showed strong reactivity with coagulase-negative staphylococci (CNS). Only two MAbs were specific for CNS; both belonged to the IgG1 subclass, and one, MAb 36.4, reacted only with the strain used for immunisation. In immunoblotting, both CNS-specific MAbs 36.3 and 36.4 reacted strongly with cell-wall protein bands of 220 Kda of S.epidermidis strain 354 and weak reactivity was observed with a 110-Kda band. MAb 36.3 reacted also with 220-230 Kda bands of two other S.epidermidis strains (291 and ATCC 35984) and a 160-180 Kda band of S.epidermidis strain 354. Only MAb 36.4 promoted phagocytosis of strain 354 by polymorphonuclear leucocytes (PMNL) and monocytes, whereas MAb 36.3 and the other MAbs lacked this activity. Opsonisation of S. epidermidis with MAb 36.4 in the presence of complement enhanced uptake by PMNL, but not by monocytes. Furthermore, S.epidermidis strain 354 opsonised with MAb 36.4 induced chemiluminescence of PMNL. Immuno-gold electronmicroscopy with both MAbs 36.3 and 36.4 demonstrated a homogeneous distribution of gold particles on the surface as well as close to the surface of S.epidermidis.

Animals↗
PubMed1991

Nontoxigenic strains of Clostridium difficile lack the genes for both toxin A and toxin B.

A total of 39 toxigenic and 20 nontoxigenic strains of Clostridium difficile were tested for the presence of either toxin A or toxin B by the polymerase chain reaction (PCR). All toxigenic strains produced cytotoxin as assayed by using highly sensitive fetal lung fibroblasts and were positive for toxin A as well as toxin B in the PCR assay. All nontoxigenic strains failed to produce toxin and were negative in the PCR assay. This study shows that nontoxigenic strains of Clostridium difficile lack the toxin A as well as the toxin B gene.

Bacterial Proteins↗
PubMed1991

Monoclonal antibodies specific for the phase-variant O-acetylated K1 capsule of Escherichia coli.

Two monoclonal antibodies, one each of the immunoglobulin M and G2b types, were produced from mouse spleen cells. These monoclonal antibodies only reacted with approximately 50% of the Escherichia coli K1 strains and not against group B meningococci. No reaction was observed after the strains were boiled. E. coli K1 strains that reacted with the monoclonal antibodies could become nonreactive after subculture. Based on these findings, we conclude that the monoclonal antibodies react with the O-acetylated K1 capsule.

Acetylation↗
PubMed1990

Binding of rough lipopolysaccharides (LPS) to human leukocytes. Inhibition by anti-LPS monoclonal antibody.

The binding of rough LPS (ReLPS from Salmonella minnesota R595) to human peripheral blood polymorphonuclear leukocytes (PMN), monocytes, and lymphocytes was examined by using fluorescein-labeled LPS and flow cytometry. At 4 degrees C, FITC-ReLPS bound rapidly in a concentration- and time-dependent way to PMN, monocytes, and lymphocytes. Because mononuclear cells showed both binding and nonbinding cell populations, FITC-ReLPS was used in conjunction with specific phycoerythrin-labeled mAb to identify these cell subpopulations. In contrast to T lymphocytes and NK cells, all monocytes and B lymphocytes efficiently bound FITC-ReLPS. PMN and monocytes showed two to three times more cell-associated FITC-ReLPS when cells were incubated at 37 degrees C compared with incubation at 4 degrees C. Binding of FITC-ReLPS to lymphocytes was similar for both 4 degrees C and 37 degrees C incubation conditions. In contrast to 4 degrees C, at 37 degrees C cell-associated LPS reflects surface-bound as well as internalized LPS, as demonstrated with fluorescence quenching of extracellular FITC-ReLPS by trypan blue. At 4 degrees C, binding of FITC-ReLPS was inhibited by polymyxin B. In addition, purified IgM mAb directed against hydrophobic acyl residues of ReLPS showed more than 95% inhibition of ReLPS binding to leukocytes, indicating the ability of specific mAb to prevent LPS-cell interactions necessary to exert biologic effects. The use of mAb, directed against different parts of the LPS molecule, provides an alternative method for LPS binding-inhibition studies.

Antibodies, Monoclonal↗