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Biomedical subjects

J Verhoef

Publications and source records attributed to J Verhoef.

At least 217 records · Page 12Linked to original sources
PubMed1995

Opsonic activities of surfactant proteins A and D in phagocytosis of gram-negative bacteria by alveolar macrophages.

Surfactant proteins A and D (SP-A, SP-D) can interact with lipopolysaccharide (LPS) and stimulate alveolar macrophages. The opsonic activities of SP-A and SP-D for bacteria with different types of LPS and alveolar macrophages were investigated. In flow cytometric studies with fluorescein-labeled rough (J5) and smooth (O111) Escherichia coli and rat alveolar macrophages, SP-A enhanced binding of J5 but not O111 bacteria to macrophages. Most importantly, SP-A enhanced ingestion of J5 bacteria by alveolar macrophages and subsequent bacterial killing. Immunoelectron microscopy demonstrated that J5 bacteria, the interface between the bacterium and the outer membrane of the alveolar macrophage, and ingested bacteria were heavily labeled with SP-A. In contrast, SP-D did not mediate phagocytosis. SP-A acted as an opsonin in the phagocytosis of rough LPS-containing bacteria by alveolar macrophages, emphasizing the possible role for SP-A in the alveolar defense system.

Animals↗
PubMed1995

Anti-polysaccharide immunoglobulin isotype levels and opsonic activity of antisera: relationships with protection against Streptococcus pneumoniae infection in mice.

Relationships between in vitro parameters (opsonic activity and anti-pneumococcal polysaccharide [PS] antibody subclasses) and in vivo mouse protection were established by logistic regression analysis. Data were from 158 mice challenged with pneumococci after vaccination with synthetic oligosaccharide- and PS-protein conjugates in combination with the adjuvant Quil A. The hypothesis that serum opsonic activity has predictive value for protection against pneumococcal infection was tested. Serum opsonic activity was well correlated with protection (chi 2 = 35.5, P < 0.001), although a stronger correlation was observed for anti-PS IgM and IgG. The combined use of IgG and opsonic activity as predictor variables yielded the best fitting model for predicting protection (chi 2 = 74.1, P < 0.001). When opsonic activity data were added to models that included various antibody isotypes, the statistical significance of the models was enhanced. Thus, the opsonic activity of antisera induced by pneumococcal vaccines can predict mouse protection.

Adjuvants, Immunologic↗
PubMed1995

Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis.

The purpose of this study was to investigate complement resistance in Branhamella (Moraxella) catarrhalis isolated from healthy schoolchildren or sputum-producing adult patients. Two techniques were used: a serum bactericidal assay as the gold standard and an easier 'culture and spot' test. Children (age 4-13; n = 303) and patients (n = 1047) showed high colonization/infection rates with B. catarrhalis (31% and 19%, respectively). Complement resistance or intermediate sensitivity occurred frequently in patient isolates (62% and 27%, respectively) and less often in children (33% and 8.5%, respectively; P << 0.0001). In young children (age 4-5 years), the proportion of complement-resistant strains was around 50%. Complement resistance in B. catarrhalis is associated with illness and may hence be considered a virulence factor.

Adolescent↗
PubMed1995

Assessment of complement-mediated killing of Moraxella (Branhamella) catarrhalis isolates by a simple method.

Recently, we showed that complement resistance is an important virulence factor of Moraxella (Branhamella) catarrhalis. Our study used a serum bactericidal assay to determine complement resistance in M. catarrhalis. Although the serum bactericidal assay is considered the "gold standard" for determining complement resistance, it is laborious and time-consuming and therefore not well suited for large-scale studies. Using a large number (n = 324) of M. catarrhalis isolates obtained from the sputa of patients with lower respiratory tract infections (n = 200) and young carriers (n = 124), we assessed the value of a simple "culture-and-spot" test as an alternative to the serum bactericidal assay. For both groups of isolates, the degree of concordance between the two tests used was very significant (P < 0.0001). The agreement between the two assays was estimated to be "excellent beyond chance" (as determined by Cohen's kappa test). The culture-and-spot assay is a valuable alternative to the serum bactericidal assay, not only for screening purposes as shown here but also for studying the mechanism of complement resistance in M. catarrhalis at the molecular level.

Bacteriological Techniques↗
PubMed1995

Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human peripheral blood mononuclear cells by flow cytometry.

Monocytes may represent an important defense mechanism in disseminated cryptococcosis. We have developed a flow cytometric method to study the interaction of Cryptococcus neoformans with monocytes. For phagocytosis, C. neoformans was labelled with fluorescein isothiocynate (FITC). Monocytes were identified on the flow cytometer by labelling with anti-CD14-R-phycoerythrin. Discrimination between attached cells (association) and internalized cells (uptake) was made by quenching FITC-labelled C. neoformans with trypan blue. Only internalized cells kept their FITC fluorescence after quenching. For comparison under the microscope, specific staining of the cell wall of C. neoformans with Uvitex was used. Internalized C. neoformans cells were not stained, as Uvitex was occluded from phagocytes. To assay killing, C. neoformans was labelled with 0.2 mM 2'-7(1)-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethylester. After phagocytosis of labelled cells by monocytes, blood cells were lysed with 25 mM deoxycholate. Viable yeast cells retained the fluorescence, but nonviable cells lost it. Quantitative counts of viable cells on Sabouraud dextrose agar were performed for comparison. The change in the relative fluorescence of green within the monocyte region was used to quantitate association, uptake, and killing of C. neoformans by monocytes on the flow cytometer. The flow cytometry methods showed that 18% +/- 2%, 35% +/- 14%, 50% +/- 11%, 51% +/- 6% of monocytes had become associated with C. neoformans after 0, 30, 60, and 120 min, respectively. After 2 h of phagocytosis time, 30% of C. neoformans-associated monocytes had taken up the cells, and killing rates of 23% +/- 17%, 22% +/- 9%, and 40% +/- 13% were obtained with effector-to-target cell ratios of 1:1, 10:1, and 50:1, respectively. Results with the flow cytometry methods compared favorably with those by the conventional methods used, but the flow cytometry methods are simpler, rapid, more reproducible, and objective.

Blood Bactericidal Activity↗
PubMed1995

Synthetic peptides representing T-cell epitopes act as carriers in pneumococcal polysaccharide conjugate vaccines.

Improvement of antibody responses to polysaccharides through their linkage to proteins is thought to be mediated by protein-specific T helper (Th) cells. To investigate whether the carrier protein of a conjugate could be substituted by a Th epitope, Streptococcus pneumoniae type 17F polysaccharide (PS) was bromoacetylated and coupled to different peptides via their carboxy-terminal cysteines. Two peptides, one from the mycobacterial 65-kDa heat shock protein (hsp65) and the other from influenza virus hemagglutinin, are well-known Th epitopes. Two other peptides were selected from the pneumolysin sequence by Th epitope prediction methods; one of them was synthesized with cysteine either at the carboxy or the amino terminus. Three conjugates consistently elicited in mice anti-PS immunoglobulin M (IgM) and IgG responses that were not observed upon immunization with derivatized PS without peptide. The same conjugates induced no anti-PS antibody responses in athymic (nu/nu) mice, whereas clear responses were elicited in euthymic (nu/+) controls, demonstrating the thymus-dependent character of these conjugates. Only the three conjugates inducing anti-PS responses were capable of eliciting antipeptide antibodies. One of the immunogenic conjugates was studied in more detail. It induced significant protection and an anti-PS IgG response comprising all subclasses. On the basis of these results and proliferation studies with peptide and conjugate-primed cells, it is concluded that linkage of Th epitopes to PS in the right orientation enhances its immunogenicity in a thymus-dependent manner. Future possibilities for using peptides as carriers for inducing antibody responses to poorly immunogenic saccharide antigens are discussed.

Adjuvants, Immunologic↗
PubMed1995

Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria.2+ fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid.

Bacteria↗
PubMed1995

Phenotypic characterization of epidemic versus sporadic strains of methicillin-resistant Staphylococcus aureus.

Forty strains of methicillin-resistant Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behavior into two subgroups, sporadic MRSA (SMRSA) and epidemic MRSA (EMRSA) strains. The strains were examined for binding of 125I-labelled fibronectin, vitronectin, collagen, Fc fragments of immunoglobulin G, and fibrinogen. A significant difference between EMRSA and SMRSA strains was found for binding of 125I-labelled fibrinogen and for Fc fragments of immunoglobulin G, (P < 0.05). No significant difference in the binding of 125I-labelled fibronectin and collagen was found between EMRSA and SMRSA strains. The binding of 125I-labelled vitronectin to MRSA strains was found to be aspecific. Capsular serotypes of the strains were determined with monoclonal antibodies against capsular types 5 and 8. Strains could be divided into the following four groups: types 5, 8, and 5/8 and nontypeable. More nontypeable strains were found in the EMRSA group (66.6%). Significantly more EMRSA strains (79%) than SMRSA strains (44%) produced alpha-toxin (P < 0.025). Logistic regression analysis using a combination of the parameters 125I-labelled immunoglobulin G binding, capsular type, and alpha-toxin production predicted the epidemic character with a sensitivity of 83% and a specificity of 75%.

Bacterial Toxins↗
PubMed1995

Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines.

Although pneumococcal conjugate vaccines are close to being licensed, a more profound knowledge of the virulence factors responsible for the morbidity and mortality caused by Streptococcus pneumoniae is necessary. This review deals with the major structures of pneumococci involved in the pathogenesis of pneumococcal disease and their interference with the defense mechanisms of the host. It is well known that protection against S. pneumoniae is the result of phagocytosis of invading pathogens. For this process, complement and anticapsular polysaccharide antibodies are required. Besides, relatively recent experimental data suggest that protection is also mediated by the removal of disintegrating pneumococci and their degradation products (cell wall, pneumolysin). These structures seem to be major contributors to illness and death caused by pneumococci. An effective conjugate vaccine should therefore preferably include the capsular polysaccharide and at least one of these inflammatory factors.

Adult↗
PubMed1994

Airborne gram-negative bacteria and endotoxin in sick building syndrome. A study in Dutch governmental office buildings.

METHODS: During a survey of building-related symptoms among 1355 employees working in 19 governmental office buildings in the Netherlands, we measured physical and chemical factors, airborne microorganisms, and endotoxin. Mechanically ventilated buildings were grouped as "healthy" or "sick" based on symptom prevalence (mean symptom prevalence < 15% or > or = 15%). We compared physical, chemical, and microbiological characteristics between mechanically ventilated and naturally ventilated buildings. RESULTS: Gram-negative rods were found in higher numbers in the "sick" buildings. The concentration of airborne endotoxin was six to seven times higher in the "sick" buildings than in the "healthy" buildings (254 vs 46 ng/m3); it was 35 ng/m3 in naturally ventilated buildings. No differences in physical characteristics of the buildings were found. Volatile organic compounds and carbon dioxide were present at higher concentrations in naturally ventilated buildings. CONCLUSION: Airborne microbial contamination, in particular with gram-negative rods and perhaps with endotoxin, may have a role in the causation of sick building syndrome.

Adolescent↗
PubMed1994

Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides.

Surfactant protein A (SP-A) enhances the phagocytosis of opsonized and non-opsonized bacteria by alveolar macrophages, but it is not known with which component of the bacterial surface it associates. We investigated the interaction of SP-A with lipopolysaccharides (LPS), which are important biologically active constituents of the outer membranes of Gram-negative bacteria. Flow cytometry was used to study the binding of fluorescein isothiocyanate-labelled SP-A either to LPS of various chain lengths coupled to magnetic beads or to Gram-negative bacteria. The binding of SP-A to LPS-coated beads was saturable, both time- and concentration-dependent, and required both Ca2+ and Na+. SP-A bound to the lipid A moiety of LPS and to LPS from either the Re-mutant of Salmonella minnesota or the J5-mutant of Escherichia coli. In contrast, it did not bind to O111 LPS of E. coli, suggesting that SP-A binds only to rough LPS. The binding of SP-A to LPS was not affected by mannan and heparin or by deglycosylation of the SP-A, indicating that the carbohydrate-binding domain and the carbohydrate moiety of SP-A are not involved in its interaction with LPS. We also observed saturable and concentration-dependent binding of SP-A to the live J5 mutant of whole E. coli, but not to its O111 mutant. In addition, Re LPS aggregated in the presence of SP-A, Ca2+ and Na+. We conclude that SP-A associates with LPS via the lipid A moiety of rough LPS and may be involved in the anti-bacterial defences of the lung.

Bronchoalveolar Lavage Fluid↗
PubMed1994

Clinical efficacy and morbidity associated with continuous cyclic compared with continuous ambulatory peritoneal dialysis.

OBJECTIVE: To assess the clinical efficacy and morbidity of continuous cyclic peritoneal dialysis compared with continuous ambulatory peritoneal dialysis with a Y-connector as renal replacement therapy. DESIGN: Prospective, randomized study. SETTING: University hospital. PATIENTS: All new patients with end-stage renal failure consecutively entering the dialysis program from January 1988 through July 1991 were randomly assigned to receive continuous ambulatory peritoneal dialysis with a Y-connector or continuous cyclic peritoneal dialysis and were followed prospectively. MEASUREMENTS: Patient and technique survival, dialysis adequacy, and (infectious) morbidity. RESULTS: Forty-one patients (median age, 56 years; range, 18 to 86 years) started continuous ambulatory peritoneal dialysis with a Y-connector (follow-up, 688 patient-months), and 41 patients (median age, 54 years; range 21 to 76 years) started continuous cyclic peritoneal dialysis (follow-up, 723 patient-months). The two groups showed no significant differences in adequacy of dialysis (as assessed by blood pressure control and laboratory and neurologic variables) and patient or technique survival. Renal transplant was the primary reason for discontinuing the assigned dialysis technique in both groups. The average number of hospitalizations per patient-year was 1.0 using continuous ambulatory peritoneal dialysis with a Y-connector and 0.6 per patient-year using continuous cyclic peritoneal dialysis (P = 0.02), with a mean duration of 10.8 and 9.6 days per admission, respectively (not significant). Peritonitis occurred significantly less often in those receiving continuous cyclic peritoneal dialysis (0.94 compared with 0.51 episodes per patient-year; P = 0.03). No difference in causative pathogens was observed. Exit site infection rate was 0.38 episodes per patient-year in both groups. CONCLUSION: In an unselected patient group, continuous cyclic peritoneal dialysis was accompanied by significantly lower rates of peritonitis and dialysis-related hospital admission, whereas it was as effective as continuous ambulatory peritoneal dialysis with a Y-connector for patient and technique survival.

Adolescent↗
PubMed1994

Role for oxygen radicals in self-sustained HIV-1 replication in monocyte-derived macrophages: enhanced HIV-1 replication by N-acetyl-L-cysteine.

N-acetyl-L-cysteine (NAC) has been proposed as a therapeutic agent for AIDS patients because it reduces human immunodeficiency virus type 1 (HIV-1) replication in stimulated T cells. However, NAC and glutathione enhanced acute HIV-1 replication in monocyte-derived macrophages. Buthionine sulfoximine did not affect NAC-mediated enhanced HIV-1 replication, indicating that the NAC-mediated effects are glutathione-independent. Superoxide dismutase and the hydroxyl radical scavengers dimethylthiourea and thiourea, but not urea, inhibited acute HIV-1 replication in macrophages. NAC reduced ferricytochrome c and increased dose-dependently Fe(III)-citrate and Fe(III)-EDTA-catalyzed hydroxyl radical formation in a system using glucose and glucose oxidase. Dimethylthiourea and thiourea, but not urea and superoxide dismutase, dose-dependently inhibited NAC-mediated enhancement of HIV-1 replication. These data suggest that oxygen radicals play an important role in self-sustained HIV-1 replication in macrophages and that oxygen radical scavengers other than NAC should be considered as therapeutic agents for AIDS patients.

Acetylcysteine↗
PubMed1994

Hypocalcemic effect of salmon calcitonin following single and repeated nasal and intravenous administration in young rabbits.

The effect of the polypeptide salmon calcitonin (sCT) on serum calcium concentrations following intranasal and intravenous administration was studied in young rabbits. A small, hypocalcemic effect was observed after nasal administration of sCT without additives, indicating that the nasal sCT absorption was low. The absorption could be improved by addition of an absorption-enhancing adjuvant to the nasal preparation. The absorption, however, was still far from complete as was apparent from the much stronger effect of intravenously injected sCT. When a number of sCT doses were given during a 10-week period, the hypocalcemic effect per sCT dose in the young rabbits decreased after intravenous and, although less pronounced, after nasal administration. The decreased response to sCT is probably not related to the induction of neutralizing antibodies or desensitization of sCT receptors, but is more likely associated with the age-dependent level of bone activity.

Absorption↗