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Biomedical subjects

J Verhoef

Publications and source records attributed to J Verhoef.

At least 253 records · Page 14Linked to original sources
PubMed1994

Rapid detection of toxigenic Clostridium difficile in fecal samples by magnetic immuno PCR assay.

Rapid detection of toxigenic Clostridium difficile in fecal samples was accomplished with the magnetic immuno PCR assay (MIPA). Elaborate DNA extraction techniques were unnecessary. First, we generated a mouse monoclonal antibody (MAb) reactive with only C. difficile, Clostridium sordellii, and Clostridium bifermentans. Then, magnetic beads were coated with the MAb, incubated with fecal samples to allow binding with C. difficile, extracted from the stool with a magnet, and processed in the PCR with primers specific for the toxin B gene. After optimizing MIPA by raising the number of PCR cycles from 35 to 40 and adding Chelex 100 to the PCR mixture, we found a sensitivity of 96.7%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94.1% when compared with the culture of cytotoxic C. difficile from fecal samples. MIPA is a rapid, easy, and sensitive PCR method for demonstrating the presence of toxigenic C. difficile in stool samples and avoids the disadvantage of elaborate extraction of DNA from fecal samples.

Antibodies, Monoclonal↗
PubMed1994

Clinical significance of Clostridium difficile and its toxins in faeces of immunocompromised children.

In this study, clinical and laboratory findings were tested for correlation with the presence of Clostridium difficile. The toxigenicity of the isolated strains and the toxins were determined in faecal samples of immunocompromised children admitted to a single room for protective isolation. Using the toxin assay as the gold standard, the culture sensitivity of toxigenic C difficile was 94.1%, the specificity 93.8%, the positive predictive value 62.8%, and the negative predictive value 99.3%. Correction for stools with a positive culture of toxigenic C difficile preceding detection of toxin, resulted in a positive prediction value of 78.4%. A statistically significant association was found between a positive faecal toxin assay and fever, and between a positive culture of toxigenic C difficile and abdominal pain: 42% of the patients with positive toxin assays had fever versus 21% with negative toxin assays, and 66% of the patients with a positive culture for toxigenic C difficile had abdominal pain, versus 22% with negative cultures. Further analysis of the cultures and toxin assays showed no statistically significant association with diarrhoea, fever, white blood cell count, C reactive protein concentrations, or abdominal pain. Based on these findings, it is suggested that immunocompromised children should be treated when toxigenic C difficile is cultured or when toxin is detected in stool samples.

Abdominal Pain↗
PubMed1994

Effect of loracarbef and amoxicillin on the oropharyngeal and intestinal microflora of patients with bronchitis.

The effect on the oropharyngeal and intestinal microflora and the efficacy and tolerance of loracarbef (200 mg b.i.d. for 7 days) versus amoxicillin (500 mg t.i.d. for 7 days) were compared in 80 patients with bronchitis. The oropharyngeal samples of 18% of patients in the amoxicillin group and 5% of patients in the loracarbef group revealed a new Gram-negative species around days 8-10. The presence of other aerobic bacteria than found at baseline in the faeces occurred in 38% of patients treated with amoxicillin compared with 31% in the loracarbef group on days 8-10. After treatment, no loracarbef- or amoxicillin-resistant aerobic Gram-negative bacteria were found in faecal samples in the loracarbef group, while amoxicillin- or loracarbef-resistant (E. coli) strains appeared in 35% of patients receiving amoxicillin (p < 0.001). Treatment success occurred by days 8-10 in all 40 patients receiving loracarbef, compared with 90% in the amoxicillin group, on days 21-28, in 93% and 90% respectively. The results of this study indicate that the use of loracarbef leads to minor changes in the normal oropharyngeal and intestinal microflora compared with amoxicillin, while almost no resistant Gram-negative bacteria emerge after treatment with loracarbef.

Acute Disease↗
PubMed1994

Modulation of lipopolysaccharide binding to human granulocytes.

Using flow cytometry and fluorescein-labelled lipopolysaccharide (LPS) from Salmonella minnesota R595 (FITC-ReLPS), we studied the role of membrane proteins in the recognition of LPS by human polymorphonuclear granulocytes (PMN) in the absence of serum. Treatment of PMN with trypsin, pronase E or proteinase K reduced both the binding of FITC-ReLPS to PMN at 4 degrees and the response of PMN to LPS at 37 degrees, as measured by luminol-enhanced chemiluminescence. Neuraminidase treatment enhanced both activities. Trypsin treatment of PMN after the binding of FITC-ReLPS effectively reduced fluorescence when cells were kept at 4 degrees, while further incubation of FITC-ReLPS-labelled PMN at 37 degrees rendered fluorescence insensible to trypsin. These results indicate a protein structure of the LPS binding site, association of FITC-ReLPS with the cell membrane at 4 degrees and subsequent internalization at 37 degrees. The binding of FITC-ReLPS was not inhibited by the anti-CD14 monoclonal antibody (mAb) 3C10, which recognizes a functional epitope of CD14. Furthermore, binding of FITC-ReLPS was observed to PMN obtained from a patient with paroxysmal nocturnal haemoglobinuria who lacked membrane-bound CD14. Stimulation of PMN with tumour necrosis factor (TNF) or LPS enhanced the binding of FITC-ReLPS at 4 degrees. This was not observed after activation of PMN devoid of granules (cytoplasts), indicating that the binding of LPS at the cell surface is enhanced by mobilization of LPS-binding proteins from intracellular granules. These studies provide evidence that LPS binding and activation of PMN involves protein structures at the cell surface different from CD14, and that granules constitute a pool of LPS-binding proteins that can be translocated to the cell surface upon stimulation.

Antibodies, Monoclonal↗
PubMed1994

In vitro compatibility of a 1.1% amino acid containing peritoneal dialysis fluid with phagocyte function.

The effects of a recently introduced peritoneal dialysis fluid (PDF) containing amino acids (AA) were compared with those of a glucose-based PDF (G-PDF) on viability and function of donor granulocytes (PMNs) in vitro. After 30 min incubation in the PDF, viability, assessed by trypan blue exclusion, and phagocytosis capacity (PC), tested in two assays using a fluorescein and a 3H-labeled Staphylococcus epidermidis strain, were significantly better in AA-PDF than in G-PDF (p < 0.002 in the 3H-assay). Bactericidal activity was not different in the PDFs. If pH of G-PDF was adjusted from 5.2 to neutral, differences in PC disappeared. In AA-PDF, PMN chemiluminescence (CL) response was significantly higher than in G-PDF (p < 0.003). At neutral pH, however, PMNs showed a significant stronger CL-response in 1.36% G-PDF than in AA-PDF (p < 0.05). These data suggest that this AA-PDF has little detrimental effect on phagocyte viability and function. The improved compatibility over G-PDF in in vitro tests seems to be pH dependent. The reduced chemiluminescence response compared to 1.36% G-PDF with neutral pH is possibly due to quenching by (one of the) amino acids and osmolarity.

Amino Acids↗
PubMed1994

Antibacterial peritoneal defence in automated peritoneal dialysis: advantages of tidal over continuous cyclic peritoneal dialysis?

In tidal peritoneal dialysis (TPD) only a part of the infused dialysate is drained with each exchange, leaving a residual volume on top of which fresh fluid is cycled. As the persistent presence of a buffered intraperitoneal reserve volume might favour peritoneal macrophage (PMO) function, PMO obtained from eight patients during a 3-h continuous cyclic peritoneal dialysis (CCPD) or TPD session were studied in a randomized cross-over trial. PMO were studied for uptake of E. coli (complement-dependent) and S. epidermidis (antibody-dependent), as well as for their killing capacity and peak chemiluminescence response. In addition, dialysate was sampled during both treatment sessions and studied for pH, osmolality, and effect on the viability of donor phagocytes and mesothelial cells. TPD-derived PMO were significantly better able to phagocytose E. coli than CCPD-PMO (48 +/- 8 versus 33 +/- 6% uptake, P < 0.05), whereas the other tested functional capacities revealed no significant difference between TPD- and CCPD-PMO. During TPD dialysate pH ranged from 6 to 7 as compared to a pH range from 5 to 7 in CCPD. The presence of a residual dialysate volume resulted in less wash-out of cells and opsonins early in the treatment, and to some extent blunted the noxious effects of fresh dialysis solutions. Overall, however, tidal PD appeared to have no advantage over CCPD regarding preservation of peritoneal defences.

Adult↗
PubMed1993

The sepsis syndrome in a Dutch university hospital. Clinical observations.

BACKGROUND: Most studies of the cause of sepsis syndrome focus on patients hospitalized in intensive care units. In this study, we analyzed the incidence, cause, and outcome of the sepsis syndrome in all hospitalized patients. METHODS: Clinical and microbiologic data were obtained for 382 patients (5.6% of all patients admitted) from whom blood was drawn for culture. RESULTS: The incidence of the sepsis syndrome was 13.6 per 1000 patients admitted (1.06 per 1000 hospital days), while the incidence of septic shock was 4.6 per 1000. The respiratory tract was the predominant infection site. Of all patients with sepsis syndrome, 38% (n = 35) had positive blood cultures. More than half of these cultures (13 [57%]) were caused by gram-positive microorganisms (excluding patients receiving selective decontamination of the digestive tract and those with intravascular device-related bacteremias). The mortality for patients with sepsis syndrome without shock was 28% (17/61), while for patients with septic shock, it was 55% (17/31). Patients with cardiovascular diseases had a significantly (P < .005) greater risk of dying during a sepsis syndrome episode than patients with other predisposing factors. Multivariate analysis of factors influencing outcome identified the development of shock and an immunocompromised state as being significantly associated with outcome in patients with sepsis syndrome. CONCLUSIONS: Patients fulfilling the criteria for the sepsis syndrome are at great risk of developing septic shock or multiple-organ failure and subsequently dying. In our hospital, the majority of bacteremic episodes were associated with gram-positive microorganisms.

Adult↗
PubMed1993

Comparison of immunomagnetic beads coated with protein A, protein G, or goat anti-mouse immunoglobulins. Applications in enzyme immunoassays and immunomagnetic separations.

Immunomagnetic beads were prepared using either protein A (PA) or protein G (PG) coupled to magnetic beads for binding antibodies at their Fc region. The performance of these beads was compared with commercially available beads coated with goat anti-mouse (G alpha M) immunoglobulins. Both the PA- and PG-beads possessed a higher binding capacity than the G alpha M-beads for the monoclonal antibodies tested, although, PA bound weakly with some IgG1 antibodies. PA-beads were compared with G alpha M-beads in a magnetic enzyme immunoassay for the detection of mouse immunoglobulins as an alternative to a conventional capture ELISA. The magnetic enzyme immunoassay was characterized by a detection time of less than 60 min and a linear assay range from 5-10 to 500 ng/ml for G alpha M-beads and 5-10 to 1000 ng/ml for PA-beads. The capture ELISA was linear from 10 to 250 ng/ml. For immunomagnetic separation of Salmonella with immunomagnetic beads, PA-beads were superior to both PG- and G alpha M-beads. For specific isolation of bacteria from heterogeneous suspensions by immunomagnetic separation, PA- and PG-beads are preferable since G alpha M-beads crossreact with bacteria possessing proteins with Fc-binding activity.

Animals↗
PubMed1993

Binding of human immunodeficiency virus type-1 to follicular dendritic cells in vitro is complement dependent.

The authors studied the binding in vitro of HIV-1 virus particles, conjugated to fluorescein isothiocyanate, to follicular dendritic cells (FDC) isolated from human tonsils. Analysis was done using flow cytometry, fluorescence microscopy, and immunogold electron microscopy. The focus of study was on the effect of serum from various origins, including pooled fresh serum and heated serum from control donors and pooled heated serum from HIV-1-infected patients (containing anti-HIV-1 antibodies). In the presence of heated serum, either from controls or from HIV-1-infected patients, the fluorescence signal in flow cytometry was similar to the background value. In the presence of fresh serum, the signal was substantially increased, and an even higher signal was observed in the presence of fresh serum and serum from HIV-1-infected patients. This high fluorescence signal was also found in the presence of serum depleted of complement factor C5, but not with serum deficient in complement factor C3. The binding of HIV-1 virions to FDC in the presence of fresh serum was confirmed by fluorescence microscopy on cytospot preparations. After quenching of the extracellular fluorescence with trypan blue, the fluorescence was reduced to about 30% of the initial value, indicating that most of bound fluorescent virions were present extracellularly. Similar experiments using blood mononuclear cells showed that fluorescent HIV-1 particles after binding to these cells were present intracellularly. This flow cytometry data was confirmed in immunogold electron microscopy demonstrating that most HIV-1 gag p24 or FITC label was present at the outside of FDC and on adherent virus particles. We conclude that HIV-1 virions adhere to FDC in vitro in a complement component C3-dependent way. Anti-HIV-1 antibodies in serum from HIV-1 infected patients enhance binding but, by itself, are unable to mediate binding.

Adhesiveness↗
PubMed1993

A flow cytometric rosetting assay for the analysis of Fc receptors and C3 receptors on HSV-infected cells.

A sensitive and reproducible flow cytometric assay was developed for the analysis of Fc gamma and C3b(i) receptors on HSV-infected cells. The method is based on a rosette technique using fluorochrome-labeled erythrocytes sensitized with IgG or C3b(i). A comparison of flow cytometric and microscopic quantitation demonstrated that the binding of EIgG, EC3b(i) to HSV-infected cells were correlated. Flow cytometric analysis provides the opportunity to study simultaneously the distribution of E per HSV-infected cell and the total binding of E to the whole population of HSV-infected cells. Receptor activity and HSV glycoprotein cell surface expression were shown to be correlated in a linear fashion. The assay could be applied to other Fc gamma R- and C3b(i)R-bearing cells.

Animals↗
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

Infection of epithelial cell line HEp-2 with human immunodeficiency virus type 1 is CD4 dependent.

HEp-2 cells, human epithelial cells derived from a larynx carcinoma, were found to be highly susceptible to infection with HIV-1 stain IIIb and MN, but not to infection with the monotropic strain IIIBa-L or the clinical isolate HIV-1AT. HEp-2 cells infected with HIV-1 IIIb continuously secreted high levels of p24 antigen, while no cytopathic effects were observed. Although no CD4 antigen could be detected on the cells by flow cytometric analysis, CD4 mRNA was detected by reverse transcriptase PCR. Furthermore, infection could be blocked by anti-CD4 monoclonal antibody OKT4a indicating a CD4 mediated viral entry in HEp-2 cells. HEp-2 cells are commonly used in clinical virology for the culture of different viruses from clinical specimens. HEp-2 cells should therefore be handled with caution as they may potentially become infected with HIV.

Antibodies, Monoclonal↗
PubMed1993

Safety and efficacy of lomefloxacin versus norfloxacin in the treatment of complicated urinary tract infections.

Seventy patients with complicated urinary tract infections were treated with either lomefloxacin (400 mg once daily) or norfloxacin (400 mg twice daily) for 10 to 14 days. A total of 19 (86%) of 22 patients treated with lomefloxacin and 20 (74%) of 27 patients treated with norfloxacin were cured, as defined by a negative culture five to nine days after completion of therapy (p = 0.09). After four to six weeks, the figures were 56% for lomefloxacin and 48% for norfloxacin. Adverse effects in the lomefloxacin group were observed on 20 occasions in 12 patients (one stopped treatment) compared to nine occasions in five patients treated with norfloxacin (difference not significant).

Adolescent↗