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Biomedical subjects

R Bos

Publications and source records attributed to R Bos.

At least 55 records · Page 3Linked to original sources
PubMed1997

Monoclonal antibodies against the human mannose receptor that inhibit the binding of tissue-type plasminogen activator.

To study the role of the mannose receptor in cellular uptake and degradation of tissue-type plasminogen activator (t-PA), a set of five monoclonal antibodies (Moab) was generated against the mannose receptor isolated from human placental tissue. All Moab specifically recognised the 175 kDa mannose receptor in a crude placenta extract, as shown in Western blot analysis. By use of immunohistochemistry, we showed that in human placenta only the Hofbauer cells (fetal macrophages) express the mannose receptor. Epitope competition experiments indicated that the Moab bound to at least two different epitopes on the receptor molecule. Moab 14-3, 14-5, and 15-2, which are directed against one of these epitopes, strongly inhibited the interaction between the purified mannose receptor and t-PA. These Moab also inhibited mannose receptor-mediated degradation of t-PA by cultured human macrophages. The low density lipoprotein receptor-related protein (LRP) mediated t-PA degradation was not affected by the Moab. It is concluded that the Moab are useful for studying the expression of the human mannose receptor in Western blot and in immunohistochemistry, and for studying the interactions between the human mannose receptor and the mannose-containing ligand t-PA.

Antibodies, Monoclonal↗
PubMed1997

[Determination of infravesical obstruction in men with benign prostatic hyperplasia using pressure-flow measurement and analysis with the Dx/CLIM software program].

BACKGROUND: Quantification of infravesical obstruction is important in the management of clinical benign prostatic hyperplasia (BPH). OBJECTIVES: The assessment of obstruction degree according to the urethral resistance parameter (URA) and bladder contractility (W) with software Dx/CLIM. METHODS: The study retrospectively analysed a group of 61 patients with BPH in order to assess the degree of obstruction and bladder contractility. Each of the patients underwent pressure/flow studies with Dx/CLIM analysis (URA < 29 cm H2O-unobstructed, Wmax > 12.8 W/m2-normal bladder contractility) and symptom score evaluation (maximum 15 points). In a dose-finding, placebo-controlled study 12 patients received 2 mg of doxazosin (alpha-1 blocker) and 11 patients received placebo during 4 weeks of treatment. RESULTS: 15 patients were unobstructed (25%) out of whom 10 had poor bladder contractility. Obstruction was present in 46 patients (75%) out of whom 18 had impaired contractility. During treatment, the symptom score decreased statistically significantly in both groups (p < 0.05). URA decreased significantly only in the 2 mg group (2 mg group: baseline URA = 53.9, final mean URA = 40.1, p < 0.05; placebo group: baseline URA = 52.9, final mean URA = 56.1). Detrusor contractility was not significantly affected. CONCLUSIONS: Pressure/flow studies with Dx/CLIM analysis are very useful in the selection of BPH patients for treatment modalities and in the assessment of efficacy. Doxazosín decreases urethral resistance effectively. (Tab. 3, Fig. 3, Ref. 12.)

Aged↗
PubMed1996

The onset of inter-human contacts: longitudinal ultrasound observations in early twin pregnancies.

Twin pregnancies provide us with the opportunity to observe first reactions towards touch in utero, and this study illustrates the onset and development of these contacts. Forty women with twin pregnancies volunteered to take part in this study, 25 of whom (five monochorionic, 20 dichorionic pairs) fulfilled the inclusion criteria. Between 8 and 12 weeks' gestation, ultrasound examinations were performed transvaginally, and from 13 weeks onwards transabdominally, at weekly intervals. Ultrasound findings were recorded on videotapes and were analyzed retrospectively. The first contacts producing reactions in the co-twin were defined as primary contacts, which could be slow or fast arm/leg/head/body contacts. Primary contacts followed an action-reaction model, and usually lasted < 3 s. These contacts were initially slow and then became fast. The first reactions of the co-twin towards touch were observed at 65 postmenstrual days. Contacts of longer duration between both bodies including extremities, or contacts initiated by sucking movements towards the co-twin were defined as complex contacts and were observed from 85 and 92 post-menstrual days, respectively. Nearly all contacts occurred significantly earlier in monochorionic compared to dichorionic twins. Female/female pairs seemed to develop complex body contacts earlier than male/male pairs, but for the onset of other contacts we have not yet found significant differences between gender combinations.

Age of Onset↗
PubMed1996

Influence of ionic strength and substratum hydrophobicity on the co-adhesion of oral microbial pairs.

Co-adhesion between oral microbial pairs (i.e. adhesion of a planktonic microorganism to a sessile organism adhering to a substratum surface) has been described as a highly specific interaction, mediated by stereochemical groups on the interacting microbial cell surfaces, and also as a non-specific, critical colloid-chemical interaction. In a colloid-chemical approach, microbial co-adhesion is considered as an interplay between, amongst others, hydrophobic and electrostatic interactions. The aim of this paper was to determine the influence of ionic strength on the co-adhesion of Streptococcus oralis 34 to either Actinomyces naeslundii T14V-J1 or its mutant strain 5951 adhering to glass in a parallel-plate flow chamber. To this end, the ionic strength of the suspension was varied by the addition of KCl. Another aim was to investigate whether substratum hydrophobicity affected the co-adhesion between the organisms by allowing the sessile organisms (in this case the actinomyces) to adhere either to hydrophilic or to hydrophobic, dimethyldichlorosilane (DDS)-coated glass. The kinetics of co-adhesion of S. oralis 34 to the actinomyces decreased with increasing ionic strength, expressed as the ratio, chi, between the local and non-local initial deposition rates of the streptococci in the vicinity of, or far away from, the adhering actinomyces, respectively. In a stationary end-point of co-adhesion, ionic strength appeared not to be a determinant factor for the co-adhesion of S. oralis 34 with A. naeslundii 5951, either when the actinomyces were adhering to hydrophilic glass or to hydrophobic, DDS-coated glass. However, for S. oralis 34 co-adhering in a stationary end-point with A. naeslundii T14V-J1 in the high-ionic-strength (250 mM KCl) suspension, co-adhesion was far less on hydrophobic, DDS-coated glass than on hydrophilic glass. It is possible that the hydrophobic fibrils on A. naeslundii T14V-J1 bearing the lectin responsible for co-adhesion were immobilized in the latter case by adsorption to the hydrophobic substratum, making them less available for interaction with the streptococci.

Actinomyces↗
PubMed1996

Influence of temperature on the co-adhesion of oral microbial pairs in saliva.

Coaggregation (interactions between two planktonic microorganisms) and co-adhesion (interactions between sessile and planktonic microorganisms) are believed to be important factors in the formation of dental plaque by many investigators, although others doubt whether coaggregation and co-adhesion occur in vivo. It is known that coaggregation and co-adhesion generally occur equally well in buffer as in saliva, but the influence of temperature on the co-adhesion of coaggregating oral microbial pairs in saliva is unknown. Therefore, co-adhesion of streptococci suspended in saliva to glass with adhering actinomyces present (1.0 x 10(6) cells cm-2) was studied in a parallel plate flow chamber in the temperature range from 22 degrees C to 40 degrees C. In the range from 22 degrees C up to 35 degrees C both pairs studied, Streptococcus oralis 34 with Actinomyces naeslundii 5951 and Streptococcus oralis J22 with A. naeslundii 5951, displayed similar co-adhesion kinetics and co-adhesion in a stationary end-point, but around and above 37 degrees C co-adhesion almost disappeared. Hence, we conclude that co-adhesion of coaggregating oral microbial pairs in saliva may be critically influenced by temperature, especially around the temperatures prevailing in the oral cavity.

Actinomyces↗
PubMed1996

Co-adhesion of oral microbial pairs under flow in the presence of saliva and lactose.

Co-aggregation (interactions between two suspended micro-organisms) between oral microbial pairs has been studied extensively and is believed to be an important factor in dental plaque formation. However, co-adhesion (interactions between suspended and already-adhering micro-organisms) may well be equally important. The aim of this paper was to determine the influence of saliva and lactose on the co-adhesion of streptococci (S. oralis 34 and S. sanguis PK1889) to actinomyces (A. naeslundii T14V-J1 or 5951) adhering on glass under flow from buffer and saliva in the absence and presence of lactose. The kinetics of co-adhesion as well as co-adhesion in a stationary end-point of co-aggregating and non-co-aggregating pairs was studied in a parallel plate flow chamber by analysis of the spatial arrangement of co-adhering micro-organisms as a function of time. For co-aggregating pairs, initial deposition rates of streptococci in the immediate vicinity of adhering actinomyces (local initial deposition rates) were up to 5 to 10 times higher than the non-local initial deposition rates in buffer and in saliva, respectively. In a stationary end-point of co-adhesion, 5 to 6 times more streptococci co-adhered with the adhering actinomyces than averaged over the entire substratum surface. A non-co-aggregating pair showed only minor preferential (co-)adhesion near the adhering actinomyces. Co-adhesion in buffer was fully lost when lactose was added. However, addition of lactose to saliva did not inhibit co-adhesion, but co-adhesion became more reversible. Detachment of micro-organisms from the substratum due to the passage of an air-liquid interface, as occurs in the oral cavity during eating, drinking, and speaking, was minimal when deposition was carried out from buffer to bare glass. Major detachment of streptococci adhering to the substratum occurred when adhesion was mediated through a salivary conditioning film on the glass, while detachment of adhering actinomyces and streptococci co-adhering with them remained low. It is suggested that, in the development of dental plaque, adhering actinomyces may act as strongholds for other micro-organisms, like streptococci, to adhere.

Actinomyces↗
PubMed1995

Initial microbial adhesion is a determinant for the strength of biofilm adhesion.

This paper presents a hypothesis on the importance of initial microbial adhesion in the overall process of biofilm formation. The hypothesis is based on the realization that dynamic shear conditions exist in many environments, such as in the oral cavity, or on rocks and ship hulls. Recognizing that an entire biofilm is detached during high shear once the bond between the initially adhering organisms and a surface (often constituted through a so-called 'conditioning film') is broken, it becomes clear that research should focus on detachment rather than adhesion. Experiments were done in a parallel plate flow chamber in which attempts were made to detach adhering oral streptococci from glass by applying a high shear caused by the passage of a bubble, giving an air-liquid interface. Detachment of streptococci from bare glass and from an initially adhering actinomycete strain appeared not to occur. However, substantial detachment of adhering streptococci occurred when adhesion was mediated through a salivary conditioning film, presumably because of cohesive failure in the conditioning film.

Actinomyces↗
PubMed1995

A monoclonal antibody with high affinity for a neo-antigenic site in fibrinogen degraded by polymorphonuclear leukocyte-derived elastase.

Elastase, released by stimulated polymorphonuclear leukocytes (PMN), is thought to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD) especially pulmonary emphysema. A test that can detect release of elastase activity from PMN would be valuable to monitor therapy or to identify patients at risk. The authors aimed to isolate and characterize monoclonal antibodies (mAb) with a high affinity for a neo-antigenic determinant in a high-molecular weight degradation product of fibrinogen (Fbg) generated by PMN-derived elastase. Using synthetic peptides, they mimicked the new amino or carboxy terminal sequences of the A alpha-, B beta- and gamma-chains of Fbg that are generated by elastase. These synthetic peptides (A alpha 22-36, A alpha 350-360, B beta 44-55, and gamma 295-305), uni-directionally coupled to a carrier protein, were used to generate mAb specific for elastase-degraded fibrinogen (EDF). mAb that appeared to be specific for a neo-antigenic determinant (neotope) consisting of the new amino terminal amino acid(s) of the Fbg A alpha-chain that is generated by elastase activity were isolated only with the A alpha 22-36 synthetic peptide. One mAb, designated as EF1-4, was further characterized and had an approximately 75-fold higher affinity for EDF, as compared with Fbg, in solution. Using the other peptides, no mAb specific for elastase generated fibrinogen degradation products were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A one-step enzyme immunoassay for total plasminogen activator inhibitor-1 antigen in human plasma.

A one-step enzyme immunoassay (EIA) has been developed for plasminogen activator inhibitor-1 (PAI-1) antigen. The assay is based on polyclonal antibodies, which were found to be slightly more reactive with tissue-type plasminogen activator (t-PA)/PAI-1 complexes and latent PAI-1 than with active PAI-1. To correct this, active PAI-1 is converted to t-PA/PAI-1 complexes. Latent PAI-1 and tPA/PAI-1 complexes were equally reactive. The EIA is specific (PAI-2 and PAI-3 are not detected), precise (CVs range from 1.8% to 11.1%, depending on the PAI-1 concentration), fast (assay time less than 3 h), and easy to perform. It is compatible with the use of Stabilyte plasma. The assay is calibrated against the putative international standard of the National Institute of Biological Standards and Control (NIBSC; lot 87/512). The normal ranges found with this EIA were 13.2-88 ng/ml; one apparently normal donor had a value of 100 ng/ml.

Animals↗
PubMed1995

Assessing health opportunities: a course on multisectoral planning.

Multisectoral planning can yield major benefits for health, especially in the case of water resource development projects. A two-week course, designed to involve mid-level officers from at least six government ministries in the planning and implementation of such projects, has been tested in Ghana, the United Republic of Tanzania, and Zimbabwe.

Curriculum↗
PubMed1994

The role of spontaneous cap domain mutations in haloalkane dehalogenase specificity and evolution.

The first step in the utilization of the xenobiotic chlorinated hydrocarbon 1,2-dichloroethane by Xanthobacter autotrophicus is catalyzed by haloalkane dehalogenase (Dh1A). The enzyme hydrolyses 1-haloalkanes to the corresponding alcohols. This allows the organism to grow also on short-chain (C2-C4) 1-chloro-n-alkanes. We have expressed Dh1A in a strain of Pseudomonas that grows on long-chain alcohols and have selected 12 independent mutants that utilize 1-chlorohexane. Six different mutant enzymes with improved Km or Vmax values with 1-chlorohexane were obtained. The sequences of the mutated dh1A genes showed that several mutants had the same 11-amino acid deletion, two mutants carried a different point mutation, and three mutants had different tandem repeats. All mutations occurred in a region encoding the N-terminal part of the cap domain of Dh1A, and it is concluded that this part of the protein is involved in the evolution of activity toward xenobiotic substrates.

Amino Acid Sequence↗
PubMed1992

One-step purification of tissue-type plasminogen activator using affinity chromatography with a special monoclonal antibody under mild conditions.

We have previously isolated a monoclonal antibody, designated as 1-3-1, specific for tissue-type plasminogen activator (t-PA). We have shown that t-PA dissociates from 1-3-1 in the presence of the lysine analogue 6-aminohexanoic acid (6-AHA). Here we describe a method for the one-step immunoaffinity purification of t-PA from conditioned melanoma cell medium, using 1-3-1 immobilised on Sepharose under mild elution conditions, favourable for t-PA. The yield of t-PA (antigen or total protein) from a 1-3-1-Sepharose column, when eluted using a buffer supplemented with 0.2 M 6-AHA at neutral pH, was as effective as other buffers that involve a strong pH-change, i.e., pH 2-3. However, the enzymatic activity of the t-PA purified with 6-AHA was 25 to 30% higher, as compared with t-PA eluted using a pH change. This resulted in a markedly higher specific activity of t-PA purified with 0.2 M 6-AHA, as compared with t-PA purified using a strong pH-change. The purity of t-PA, purified using the present method, was very high, as determined by gel electrophoresis. An additional advantage of the present procedure is that the mild elution conditions prolong the column life.

Aminocaproic Acid↗
PubMed1992

Characterization of the interaction both in vitro and in vivo of tissue-type plasminogen activator (t-PA) with rat liver cells. Effects of monoclonal antibodies to t-PA.

The interaction of 125I-labelled tissue-type plasminogen activator (125I-t-PA) with freshly isolated rat parenchymal and endothelial liver cells was studied. Binding experiments at 4 degrees C with parenchymal cells and endothelial liver cells indicated the presence of 68,000 and 44,000 high-affinity t-PA-binding sites, with an apparent Kd of 3.5 and 4 nM respectively. Association of 125I-t-PA with parenchymal cells was Ca(2+)-dependent and was not influenced by asialofetuin, a known ligand for the galactose receptor. Association of 125I-t-PA with liver endothelial cells was Ca(2+)-dependent and mannose-specific, since ovalbumin (a mannose-terminated glycoprotein) inhibited the cell association of t-PA. Association of 125I-t-PA with liver endothelial cells was inhibited by anti-(human mannose receptor) antiserum. Anti-(galactose receptor) IgG had no effect on 125I-t-PA association with either cell type. Degradation of 125I-t-PA at 37 degrees C by both cell types was inhibited by chloroquine or NH4Cl, indicating that t-PA is degraded lysosomally. in vitro experiments with three monoclonal antibodies (MAbs) demonstrated that anti-t-PA MAb 1-3-1 specifically decreased association of 125I-t-PA with the endothelial cells, and anti-t-PA Mab 7-8-4 inhibited association with the parenchymal cells. Results of competition experiments in rats in vivo with these antibodies were in agreement with findings in vitro. Both antibodies decreased the liver uptake of 125I-t-PA, while a combination of the two antibodies was even more effective in reducing the liver association of 125I-t-PA and increasing its plasma half-life. We conclude from these data that clearance of t-PA by the liver is regulated by at least two pathways, one on parenchymal cells (not galactose/mannose-mediated) and another on liver endothelial cells (mediated by a mannose receptor). Results with the MAbs imply that two distinct sites on the t-PA molecule are involved in binding to parenchymal cells and liver endothelial cells.

Animals↗
PubMed1992

The potential improvement of thrombolytic therapy by targeting with bispecific monoclonal antibodies: why they are used and how they are made.

The generation of the proteolytic enzyme plasmin from its inactive precursor plasminogen, mediated by so called plasminogen activators, is the essential step in thrombolytic therapy. Plasmin is responsible for the degradation of the insoluble fibrin, the major component of a thrombus, to soluble fibrin degradation products. So far, the use of the more recently developed thrombolytic agents single-chain urokinase-type plasminogen activator (scu-PA) and tissue-type plasminogen activator (t-PA) were disappointing, mainly due to some of their negative properties in vivo, i.e., rapid inhibition and/or hepatic clearance. Besides some background information on the haemostatic balance; t-PA and scu-PA structure; and mechanisms of action, we here review some reported attempts to improve on these agents for thrombolytic therapy following various strategies. One of the more potential strategies, antibody-targeted thrombolytic therapy using bispecific monoclonal antibodies, is discussed somewhat more extensively, as are the several procedures that can be followed for bispecific antibody preparation.

Animals↗
PubMed1992

Enhanced transfection of a bacterial plasmid into hybridoma cells by electroporation: application for the selection of hybrid hybridoma (quadroma) cell lines.

A procedure was investigated to transduce a bacterial plasmid containing a specific drug resistance marker (pSV2-neo), into a hybridoma cell line using electroporation. The effect of several buffers and the form of plasmid DNA (circular or linearized) on the stable transfection frequency were examined. When complete cell culture medium (DMEM) was used as electroporation buffer, we observed a two-fold increase in post-pulse viability and a ten- to thirty-fold increase in the transfection frequency of pSV2-neo, as compared with HEPES buffered 0.15 M sodium chloride. Supplementing DMEM with fetal bovine serum (DMEM + FBS) had some beneficial effect on post-pulse viability of the cells after electroporation, but did not markedly increase stable transfection frequency as compared with DMEM alone. Furthermore, with DMEM + FBS, the intact plasmid was transfected as effectively as linearized PSV2-neo. However, when using HEPES buffered saline, the transfection frequency of pSV2-neo increased two-fold after linearization as compared with intact plasmid. The drug resistance was used successfully as a marker for the selection of hybrid hybridoma (quadroma) cell lines after fusing two different hybridoma cell lines, producing anti-fibrin and anti-plasminogen activator antibodies respectively. The quadroma cells produced bispecific antibodies that are capable of accumulating plasminogen activator on a fibrin surface.

Antibodies, Monoclonal↗
PubMed1992

A one-step enzyme immunoassay for the determination of total tissue-type plasminogen activator (t-PA) antigen in plasma.

A reproducible and sensitive one-step enzyme immunoassay (EIA) was developed to determine total tissue-type plasminogen activator (t-PA) antigen in plasma. The EIA comprises two monoclonal catching antibodies and a polyclonal (goat) tagging antibody conjugated with horseradish peroxidase. There is an equal reactivity towards the several physiological t-PA forms, i.e., single-chain t-PA, two-chain t-PA and t-PA in complex with its naturally occurring inhibitor plasminogen activator inhibitor-type 1 (t-PA/PAI-1 complex). Additionally, the EIA does not discriminate between human melanoma t-PA and recombinant t-PA (Activase). The assay has a lower detection limit of approximately 0.5 ng t-PA per ml plasma, with a time-to-result of only 3.5 h.

Antibodies↗
PubMed1992

DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans.

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants. AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long-chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm. AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk-genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown. The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P. putida chromosome.

Alcohol Dehydrogenase↗