24 results on '"Wielinga PR"'
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2. Food Safety: at the center of a One Health approach for combating zoonoses.
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Wielinga PR and Schlundt J
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- Animals, Drug Resistance, Bacterial, Food Microbiology, Humans, Food Safety, Global Health, Zoonoses prevention & control
- Abstract
Food Safety is at the center of One Health. Many, if not most, of all important zoonoses relate in some way to animals in the food production chain. Therefore, the food becomes an important vehicle for many, but not all, of these zoonotic pathogens. One of the major issues in food safety over the latest decennia has been the lack of cross-sectoral collaboration across the food production chain. Major food safety events have been significantly affected by the lack of collaboration between the animal health, the food control, and the human health sector. Examples range from BSE and E. coli outbreaks over dioxin crises to intentional melamine contamination. One Health formulates clearly both the need for and the benefit of cross-sectoral collaboration. In this chapter, we will focus on the human health risk related to zoonotic microorganisms present both in food animals and food from these animals, and typically transmitted to humans through food. We focus on these issues because they are very important in relation to the human disease burden, but also because this is the area where some experience of cross-sectoral collaboration already exist. Food related zoonoses can be separated in three major classes: parasites, bacteria, and viruses. While parasites often relate to very specific animal hosts and contribute significantly to the human disease burden, virus have often been related to major, well-published global outbreaks, e.g. SARS and avian- and swine-influenza. The bacterial zoonoses on the other hand often result in sporadic, but very wide-spread disease cases, resulting in a major disease burden in all countries, e.g. Salmonella and Campylobacter. Next to these traditional zoonotic problems, the use of antimicrobials in (food) animals has also caused the emergence of antimicrobial resistant (AMR) zoonotic bacteria. It is important to realize the difference in the nature of disease epidemiology, as well as, in society's reaction to these diseases in different socio-economic settings. Some diseases have global epidemic-or pandemic-potential, resulting in dramatic action from international organizations and national agricultural-and health authorities in most countries, for instance as was the case with avian influenza. Other diseases relate to the industrialized food production chain and have been-in some settings-dealt with efficiently through farm-to-fork preventive action in the animal sector, e.g. Salmonella. Finally, an important group of zoonotic diseases are 'neglected diseases' in poor settings, while they have been basically eradicated in affluent economies through vaccination and culling policies in the animal sector, e.g. Brucella.
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- 2013
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3. Evaluation of DNA extraction methods for Bacillus anthracis spores spiked to food and feed matrices at biosafety level 3 conditions.
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Wielinga PR, de Heer L, de Groot A, Hamidjaja RA, Bruggeman G, Jordan K, and van Rotterdam BJ
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- Animals, Bacillus anthracis growth & development, Bacillus anthracis isolation & purification, Bacillus thuringiensis genetics, Bacillus thuringiensis growth & development, Bacillus thuringiensis isolation & purification, DNA, Bacterial analysis, DNA, Bacterial chemistry, DNA, Bacterial genetics, Flour microbiology, Milk microbiology, Sequence Analysis, DNA, Soy Foods microbiology, Spores, Bacterial chemistry, Spores, Bacterial genetics, Spores, Bacterial isolation & purification, Triticum microbiology, Zea mays microbiology, Bacillus anthracis genetics, Bacterial Typing Techniques methods, Food Microbiology
- Abstract
The DNA extraction efficiency from milk, whey, soy, corn gluten meal, wheat powders and heat-treated corn grain that were spiked with Bacillus anthracis and Bacillus thuringiensis spores was determined. Two steps were critical: lysis of the spores and binding of the free DNA to the DNA binding magnetic beads in the presence of the interfering powders. For the guanidine-thiocyanate based Nuclisens lysis buffer from Biomerieux we found that between 15 and 30% of the spores survived the lysis step. As most lysis buffers in DNA/RNA extraction kits are guanidine based it is likely that other lysis buffers will show a similar partial lysis of the Bacillus spores. Our results show that soybean flour and wheat flour inhibited the DNA extraction process strongest, leading to unreliable DNA extractions when using too much of the matrix. For corn gluten meal, heat-treated corn grain and milk powders, DNA extraction efficiencies in the presence of 100mg and 10mg of powder resulted in 70%-95% reduced DNA recoveries. The inhibition was, however, reliable and intermediate compared to the inhibition by soy and wheat. Whey powder had the lowest inhibitory effect on DNA-extraction efficiency and recoveries of 70-100% could be reached when using 10mg of powder. The results show that reducing the amount of matrix leads to better DNA-extraction efficiencies, particularly for strongly inhibiting powders such as soy and wheat. Based on these results, a standard protocol to directly isolate DNA from micro-organisms present in complex matrixes such as food and feed powders was designed., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
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4. Detection of Coxiella burnetii in complex matrices by using multiplex quantitative PCR during a major Q fever outbreak in The Netherlands.
- Author
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de Bruin A, de Groot A, de Heer L, Bok J, Wielinga PR, Hamans M, van Rotterdam BJ, and Janse I
- Subjects
- Animals, Animals, Domestic, Bacillus thuringiensis genetics, Bacteriological Techniques standards, Coxiella burnetii genetics, Female, Goats, Multiplex Polymerase Chain Reaction standards, Netherlands epidemiology, Q Fever epidemiology, Q Fever microbiology, Real-Time Polymerase Chain Reaction standards, Sensitivity and Specificity, Sheep, Vagina microbiology, Bacteriological Techniques methods, Coxiella burnetii isolation & purification, Disease Outbreaks, Environmental Microbiology, Multiplex Polymerase Chain Reaction methods, Q Fever veterinary, Real-Time Polymerase Chain Reaction methods
- Abstract
Q fever, caused by Coxiella burnetii, is a zoonosis with a worldwide distribution. A large rural area in the southeast of the Netherlands was heavily affected by Q fever between 2007 and 2009. This initiated the development of a robust and internally controlled multiplex quantitative PCR (qPCR) assay for the detection of C. burnetii DNA in veterinary and environmental matrices on suspected Q fever-affected farms. The qPCR detects three C. burnetii targets (icd, com1, and IS1111) and one Bacillus thuringiensis internal control target (cry1b). Bacillus thuringiensis spores were added to samples to control both DNA extraction and PCR amplification. The performance of the qPCR assay was investigated and showed a high efficiency; a limit of detection of 13.0, 10.6, and 10.4 copies per reaction for the targets icd, com1, and IS1111, respectively; and no cross-reactivity with the nontarget organisms tested. Screening for C. burnetii DNA on 29 suspected Q fever-affected farms during the Q fever epidemic in 2008 showed that swabs from dust-accumulating surfaces contained higher levels of C. burnetii DNA than vaginal swabs from goats or sheep. PCR inhibition by coextracted substances was observed in some environmental samples, and 10- or 100-fold dilutions of samples were sufficient to obtain interpretable signals for both the C. burnetii targets and the internal control. The inclusion of an internal control target and three C. burnetii targets in one multiplex qPCR assay showed that complex veterinary and environmental matrices can be screened reliably for the presence of C. burnetii DNA during an outbreak.
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- 2011
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5. A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types.
- Author
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Wielinga PR, Hamidjaja RA, Agren J, Knutsson R, Segerman B, Fricker M, Ehling-Schulz M, de Groot A, Burton J, Brooks T, Janse I, and van Rotterdam B
- Subjects
- Bacillus anthracis isolation & purification, Bacillus anthracis pathogenicity, Bacillus cereus classification, Bacillus cereus genetics, Bacillus thuringiensis classification, Bacillus thuringiensis genetics, DNA Primers, Virulence genetics, Bacillus anthracis classification, Polymerase Chain Reaction methods
- Abstract
Bacillus anthracis is closely related to the endospore forming bacteria Bacillus cereus and Bacillus thuringiensis. For accurate detection of the life threatening pathogen B. anthracis, it is essential to distinguish between these three species. Here we present a novel multiplex real-time PCR for simultaneous specific identification of B. anthracis and discrimination of different B. anthracis virulence types. Specific B. anthracis markers were selected by whole genome comparison and different sets of primers and probes with optimal characteristic for multiplex detection of the B. anthracis chromosome, the B. anthracis pXO1 and pXO2 plasmids and an internal control (IC) were designed. The primer sets were evaluated using a panel of B. anthracis strains and exclusivity was tested using genetically closely related B. cereus strains. The robustness of final primer design was evaluated by laboratories in three different countries using five different real-time PCR thermocyclers. Testing of a panel of more than 20 anthrax strains originating from different locations around the globe, including the recent Swedish anthrax outbreak strain, showed that all strains were detected correctly., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2011
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6. An innovative molecular detection tool for tracking and tracing Clostridium botulinum types A, B, E, F and other botulinum neurotoxin producing Clostridia based on the GeneDisc cycler.
- Author
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Fach P, Fenicia L, Knutsson R, Wielinga PR, Anniballi F, Delibato E, Auricchio B, Woudstra C, Agren J, Segerman B, de Medici D, and van Rotterdam BJ
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- Animals, Botulinum Toxins, Type A genetics, Clostridium botulinum genetics, Clostridium botulinum type A genetics, Clostridium botulinum type A isolation & purification, Clostridium botulinum type B genetics, Clostridium botulinum type B isolation & purification, Clostridium botulinum type E genetics, Clostridium botulinum type E isolation & purification, Clostridium botulinum type F genetics, Clostridium botulinum type F isolation & purification, Feces microbiology, Mice, Botulinum Toxins genetics, Clostridium botulinum isolation & purification, Food Microbiology methods, Oligonucleotide Array Sequence Analysis methods, Polymerase Chain Reaction
- Abstract
Rapid and specific detection of botulinum neurotoxin (BoNT) producing Clostridia is a priority for public health authorities, in case of both natural and intentional botulism outbreaks. This study reports on the evaluation of a detection system based on the GeneDisc Cycler designed for simultaneously testing the bont/A, bont/B, bont/E and bont/F genes encoding for the botulinum neurotoxins types A, B, E and F. BoNT-producing Clostridia (n = 102) and non-BoNT-producing bacteria (n = 52) isolated from clinical, food and environmental samples were tested using this macro-array and results were compared to the reference lethality test on mice. The bont genes were correctly detected in all C. botulinum type A, B, E and F strains available, as well as in toxigenic C. baratii type F and toxigenic C. butyricum type E. No cross reactivity was observed with non human-toxigenic bacteria, C. botulinum types C, D and G. The identification of the bont genotype using the macro-array was correlated to toxino-typing of the BoNTs as determined by the mouse bioassay. An "evaluation trial" of the GeneDisc array performed blind in four European laboratories with 77 BoNT-producing Clostridia as well as 10 food and clinical samples showed that the developed macro-array is specific and reliable for identifying BoNT/A-, BoNT/B-, BoNT/E- and BoNT/F-producing clostridial strains and for screening naturally contaminated food and fecal samples. The test is robust, has a low detection limit (c.a. 5 to 50 genome copies in the PCR reaction microwell) and is promising for monitoring BoNT-producing Clostridia in different kinds of samples including food and clinical samples., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2011
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7. Towards an international standard for detection and typing botulinum neurotoxin-producing Clostridia types A, B, E and F in food, feed and environmental samples: a European ring trial study to evaluate a real-time PCR assay.
- Author
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Fenicia L, Fach P, van Rotterdam BJ, Anniballi F, Segerman B, Auricchio B, Delibato E, Hamidjaja RA, Wielinga PR, Woudstra C, Agren J, De Medici D, and Knutsson R
- Subjects
- Animal Feed microbiology, Animals, Botulinum Toxins genetics, Botulism microbiology, Clostridium botulinum type A classification, Clostridium botulinum type A genetics, Clostridium botulinum type A isolation & purification, Clostridium botulinum type B classification, Clostridium botulinum type B genetics, Clostridium botulinum type B isolation & purification, Clostridium botulinum type E classification, Clostridium botulinum type E genetics, Clostridium botulinum type E isolation & purification, Clostridium botulinum type F classification, Clostridium botulinum type F genetics, Clostridium botulinum type F isolation & purification, Environmental Microbiology, Europe, Food Microbiology methods, Food Microbiology standards, Humans, Mice, Molecular Typing standards, Polymerase Chain Reaction standards, Sensitivity and Specificity, Clostridium botulinum classification, Molecular Typing methods, Polymerase Chain Reaction methods
- Abstract
A real-time PCR method for detection and typing of BoNT-producing Clostridia types A, B, E, and F was developed on the framework of the European Research Project "Biotracer". A primary evaluation was carried out using 104 strains and 17 clinical and food samples linked to botulism cases. Results showed 100% relative accuracy, 100% relative sensitivity, 100% relative specificity, and 100% selectivity (inclusivity on 73 strains and exclusivity on 31 strains) of the real-time PCR against the reference cultural method combined with the standard mouse bioassay. Furthermore, a ring trial study performed at four different European laboratories in Italy, France, the Netherlands, and Sweden was carried out using 47 strains, and 30 clinical and food samples linked to botulism cases. Results showed a concordance of 95.7% among the four laboratories. The reproducibility generated a relative standard deviation in the range of 2.18% to 13.61%. Considering the high level of agreement achieved between the laboratories, this real-time PCR is a suitable method for rapid detection and typing of BoNT-producing Clostridia in clinical, food and environmental samples and thus support the use of it as an international standard method., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
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8. Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry flea-borne Rickettsia species.
- Author
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Sprong H, Wielinga PR, Fonville M, Reusken C, Brandenburg AH, Borgsteede F, Gaasenbeek C, and van der Giessen JW
- Abstract
Background: Hard ticks have been identified as important vectors of rickettsiae causing the spotted fever syndrome. Tick-borne rickettsiae are considered to be emerging, but only limited data are available about their presence in Western Europe, their natural life cycle and their reservoir hosts. Ixodes ricinus, the most prevalent tick species, were collected and tested from different vegetation types and from potential reservoir hosts. In one biotope area, the annual and seasonal variability of rickettsiae infections of the different tick stages were determined for 9 years., Results: The DNA of the human pathogen R. conorii as well as R. helvetica, R. sp. IRS and R. bellii-like were found. Unexpectedly, the DNA of the highly pathogenic R. typhi and R. prowazekii and 4 other uncharacterized Rickettsia spp. related to the typhus group were also detected in I. ricinus. The presence of R. helvetica in fleas isolated from small rodents supported our hypothesis that cross-infection can occur under natural conditions, since R. typhi/prowazekii and R. helvetica as well as their vectors share rodents as reservoir hosts. In one biotope, the infection rate with R. helvetica was ~66% for 9 years, and was comparable between larvae, nymphs, and adults. Larvae caught by flagging generally have not yet taken a blood meal from a vertebrate host. The simplest explanation for the comparable prevalence of R. helvetica between the defined tick stages is, that R. helvetica is vertically transmitted through the next generation with high efficiency. The DNA of R. helvetica was also present in whole blood from mice, deer and wild boar., Conclusion: Besides R. helvetica, unexpected rickettsiae are found in I. ricinus ticks. We propose that I. ricinus is a major reservoir host for R. helvetica, and that vertebrate hosts play important roles in the further geographical dispersion of rickettsiae.
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- 2009
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9. Persistent detection of Babesia EU1 and Babesia microti in Ixodes ricinus in the Netherlands during a 5-year surveillance: 2003-2007.
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Wielinga PR, Fonville M, Sprong H, Gaasenbeek C, Borgsteede F, and van der Giessen JW
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- Animals, Babesiosis parasitology, DNA, Protozoan genetics, Female, Humans, Male, Netherlands epidemiology, RNA, Ribosomal, 18S genetics, Sequence Analysis, DNA, Babesia genetics, Babesia microti genetics, Babesiosis epidemiology, Ixodes parasitology
- Abstract
We report the finding of Babesia EU1 and Babesia microti in Ixodes ricinus ticks in the Netherlands. During 5 years of surveillance between 2003 and 2007, 1488 ticks were collected in a dune forest area near the North Sea and were screened for Babesia infections. In 17 ticks, DNA of the protozoan parasite genus Babesia was detected using a Babesia-specific 18S rRNA polymerase chain reaction. Further, reverse line blot analysis and DNA sequence analysis showed that 13 of these ticks carried Babesia EU1, two ticks carried B. microti, and one tick carried B. divergens. This study shows that the human pathogenic species Babesia EU1 and B. microti can complete their life cycle in the Netherlands.
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- 2009
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10. [Small risk of developing Lyme borreliosis following a tick bite on Ameland: research in a general practice].
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Jacobs JJ, Noordhoek GT, Brouwers JM, Wielinga PR, Jacobs JP, and Brandenburg AH
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- Animals, Bites and Stings epidemiology, DNA, Bacterial analysis, Humans, Lyme Disease pathology, Lyme Disease transmission, Prospective Studies, Risk Assessment, Risk Factors, Time Factors, Borrelia burgdorferi isolation & purification, Ixodes microbiology, Lyme Disease epidemiology, Tick Infestations epidemiology
- Abstract
Objective: To investigate the percentage of ticks infected with Borrelia burgdorferi on the Dutch North Sea island of Ameland, and the risk of developing Lyme disease following tick bite on the island., Design: Prospective, observational., Method: Ticks were collected from patients who visited a general practitioner and were tested for the DNA of B. burgdorferi. After 6 months the patients were interviewed by phone using a standardised questionnaire., Results: From 2004-2006, 216 ticks were collected from 167 persons. Most ticks were removed within 24 hours. In 44 ticks (20.4%) B. burgdorferi DNA was detected. Follow up information was available on 146 persons, 41 (28.1%) of whom had been bitten by a Borrelia-positive tick. None of the persons developed a typical erythema migrans. From the 13 persons (9%) reporting a non-specific redness of the skin (diameter less than 5 cm) at the site of the tick bite, 5 had been bitten by a positive tick and 8 by a negative tick. One patient bitten by a positive tick reported systemic symptoms related to Lyme borreliosis, namely fatigue, perspiration and joint ache, without local redness., Conclusion: The probability of developing Lyme borreliosis was low even though a relatively large percentage of the ticks collected were positive for B. burgdorferi. This is probably connected to the fact that in the majority of cases the tick had been removed within 24 hours.
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- 2008
11. Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands.
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Wielinga PR, de Vries A, van der Goot TH, Mank T, Mars MH, Kortbeek LM, and van der Giessen JW
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- Adolescent, Adult, Animals, Base Sequence, Cattle, Cattle Diseases transmission, Child, Child, Preschool, Cryptosporidiosis transmission, DNA Primers, DNA, Protozoan genetics, Disease Reservoirs, Genetic Markers, Genotype, Humans, Infant, Infant, Newborn, Molecular Epidemiology, Molecular Sequence Data, Netherlands, Sequence Analysis, DNA, Species Specificity, Zoonoses, Cattle Diseases parasitology, Cryptosporidiosis parasitology, Cryptosporidiosis veterinary, Cryptosporidium genetics
- Abstract
The protozoan parasite Cryptosporidium is found world-wide and can cause disease in both humans and animals. To study the zoonotic potential of Cryptosporidium in The Netherlands we isolated this parasite from the faeces of infected humans and cattle and genotyped those isolates for several different markers. The overall genotyping results showed: for humans isolates, 70% Cryptosporidium hominis, 19% Cryptosporidium parvum, 10% a combination of C. hominis and C. parvum, and 1% Cryptosporidium felis; and for cattle isolates 100% C. parvum. Analysis of the genetic variants detected for the HSP70, ML1 and GP60 markers showed: for human isolates, one C. hominis and two C. parvum variants (C. parvum and C. parvum NL) for HSP70, one C. hominis and five C. parvum variants (C1, C2, C3, and C2 NL1 and C2 NL2) for ML1, four C. hominis (mainly IbA10G2) and four C. parvum variants (mainly IIaA15G2R1) for GP60; and the cattle isolates only C. parvum (not C. parvum NL1) for HSP70, C1 and C2 for ML1, and 17 different IIa sub-types (mainly IIaA15G2R1) for GP60. Molecular epidemiological analysis of the human data showed a C. hominis peak in autumn. The majority (80%) of the human cases were children aged between 0 and 9 years and >70% of these were caused by C. hominis. Patients >25 years of age were infected mainly with C. parvum. We conclude that C. hominis IbA10G2 is found at high frequencies in autumn in humans and not in cattle. The high prevalence of C. parvum IIaA15G2R1 in both humans and cattle indicates that cattle may be a reservoir for this sub-type in The Netherlands.
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- 2008
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12. cGMP transport by vesicles from human and mouse erythrocytes.
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de Wolf CJ, Yamaguchi H, van der Heijden I, Wielinga PR, Hundscheid SL, Ono N, Scheffer GL, de Haas M, Schuetz JD, Wijnholds J, and Borst P
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- ATP Binding Cassette Transporter, Subfamily G, Member 2, ATP-Binding Cassette Transporters chemistry, ATP-Binding Cassette Transporters genetics, Alprostadil metabolism, Animals, Biological Transport, Dose-Response Relationship, Drug, Erythrocyte Membrane metabolism, Erythrocytes metabolism, Humans, Mice, Mice, Knockout, Multidrug Resistance-Associated Proteins genetics, Neoplasm Proteins genetics, Cyclic GMP metabolism, Erythrocytes cytology
- Abstract
cGMP secretion from cells can be mediated by ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCC11. Indirect evidence suggests that ABCC4 and ABCC5 contribute to cGMP transport by erythrocytes. We have re-investigated the issue using erythrocytes from wild-type and transporter knockout mice. Murine wild-type erythrocyte vesicles transported cGMP with an apparent Km that was 100-fold higher than their human counterparts, the apparent Vmax being similar. Whereas cGMP transport into human vesicles was efficiently inhibited by the ABCC4-specific substrate prostaglandin E1, cGMP transport into mouse vesicles was inhibited equally by Abcg2 and Abcc4 inhibitors/substrates. Similarly, cGMP transport into vesicles from Abcc4-/- and Abcg2-/- mice was 42% and 51% of that into wild-type mouse vesicles, respectively, whereas cGMP transport into vesicles from Abcc4(-/-)/Abcg2(-/-) mice was near background. The knockout mice were used to show that Abcg2-mediated cGMP transport occurred with lower affinity but higher Vmax than Abcc4-mediated transport. Involvement of Abcg2 in cGMP transport by Abcc4-/- erythrocyte vesicles was supported by higher transport at pH 5.5 than at pH 7.4, a characteristic of Abcg2-mediated transport. The relative contribution of ABCC4/Abcc4 and ABCG2/Abcg2 in cGMP transport was confirmed with a new inhibitor of ABCC4 transport, the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride.
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- 2007
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13. Longitudinal analysis of tick densities and Borrelia, Anaplasma, and Ehrlichia infections of Ixodes ricinus ticks in different habitat areas in The Netherlands.
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Wielinga PR, Gaasenbeek C, Fonville M, de Boer A, de Vries A, Dimmers W, Akkerhuis Op Jagers G, Schouls LM, Borgsteede F, and van der Giessen JW
- Subjects
- Anaplasma genetics, Anaplasma isolation & purification, Animals, Borrelia genetics, Borrelia isolation & purification, DNA, Bacterial analysis, Ecosystem, Ehrlichia genetics, Ehrlichia isolation & purification, Female, Male, Netherlands, Polymerase Chain Reaction, Population Density, Prevalence, Arachnid Vectors microbiology, Arachnid Vectors physiology, Ixodes growth & development, Ixodes microbiology
- Abstract
From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m2). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m2. The highest tick density was found in the dune area (139 to 551/100 m2). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.
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- 2006
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14. Lyme borreliosis in the Netherlands: strong increase in GP consultations and hospital admissions in past 10 years.
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Hofhuis A, van der Giessen JW, Borgsteede FH, Wielinga PR, Notermans DW, and van Pelt W
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- Animals, Causality, Comorbidity, Erythema Chronicum Migrans epidemiology, Humans, Incidence, Netherlands epidemiology, Population Surveillance, Risk Assessment methods, Risk Factors, Bites and Stings epidemiology, Disease Outbreaks statistics & numerical data, Family Practice statistics & numerical data, Hospitalization statistics & numerical data, Lyme Disease epidemiology, Referral and Consultation statistics & numerical data, Ticks
- Published
- 2006
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15. Mice lacking Mrp3 (Abcc3) have normal bile salt transport, but altered hepatic transport of endogenous glucuronides.
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Zelcer N, van de Wetering K, de Waart R, Scheffer GL, Marschall HU, Wielinga PR, Kuil A, Kunne C, Smith A, van der Valk M, Wijnholds J, Elferink RO, and Borst P
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- Animals, Bile Ducts physiopathology, Bilirubin analogs & derivatives, Bilirubin blood, Biological Transport genetics, Biological Transport physiology, Cholic Acids metabolism, Deoxycholic Acid metabolism, Ileum metabolism, Immunoblotting, Immunohistochemistry, Ligation, Liver chemistry, Male, Mice, Mice, Inbred Strains, Multidrug Resistance-Associated Proteins analysis, Multidrug Resistance-Associated Proteins genetics, Taurocholic Acid metabolism, Bile Acids and Salts metabolism, Glucuronides metabolism, Liver metabolism, Multidrug Resistance-Associated Proteins deficiency, Multidrug Resistance-Associated Proteins physiology
- Abstract
Background/aim: Multidrug Resistance Protein 3 (MRP3) transports bile salts and glucuronide conjugates in vitro and is postulated to protect the liver in cholestasis. Whether the absence of Mrp3 affects these processes in vivo is tested., Methods: Mrp3-deficient mice were generated and the contribution of Mrp3 to bile salt and glucuronide conjugate transport was tested in (1): an Ussing-chamber set-up with ileal explants (2), the liver during bile-duct ligation (3), liver perfusion experiments, and (4) in vitro vesicular uptake experiments., Results: The Mrp3((-/-)) mice show no overt phenotype. No differences between WT and Mrp3-deficient mice were found in the trans-ileal transport of taurocholate. After bile-duct ligation, there were no differences in histological liver damage and serum bile salt levels between Mrp3((-/-)) and WT mice, but Mrp3-deficient mice had lower serum bilirubin glucuronide concentrations. Glucuronide conjugates of hyocholate and hyodeoxycholate are substrates of MRP3 in vitro and in livers that lack Mrp3, there is reduced sinusoidal secretion of hyodeoxycholate-glucuronide after perfusion with hyodeoxycholate., Conclusions: Mrp3 does not have a major role in bile salt physiology, but is involved in the transport of glucuronidated compounds, which could include glucuronidated bile salts in humans.
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- 2006
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16. Mice lacking multidrug resistance protein 3 show altered morphine pharmacokinetics and morphine-6-glucuronide antinociception.
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Zelcer N, van de Wetering K, Hillebrand M, Sarton E, Kuil A, Wielinga PR, Tephly T, Dahan A, Beijnen JH, and Borst P
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- ATP Binding Cassette Transporter, Subfamily B deficiency, Animals, Bile metabolism, Cell Line, Glucuronosyltransferase, Humans, Liver metabolism, Mice, Mice, Knockout, Morphine Derivatives blood, Morphine Derivatives pharmacokinetics, Morphine Derivatives pharmacology, Pain Measurement drug effects, Protein Transport, Spodoptera, Tissue Distribution, Transport Vesicles metabolism, ATP Binding Cassette Transporter, Subfamily B metabolism, ATP-Binding Cassette Transporters metabolism, Morphine pharmacokinetics, Morphine Derivatives metabolism
- Abstract
Glucuronidation is a major detoxification pathway for endogenous and exogenous compounds in mammals that results in the intracellular formation of polar metabolites, requiring specialized transporters to cross biological membranes. By using morphine as a model aglycone, we demonstrate that multidrug resistance protein 3 (MRP3/ABCC3), a protein present in the basolateral membrane of polarized cells, transports morphine-3-glucuronide (M3G) and morphine-6-glucuronide in vitro. Mrp3(-/-) mice are unable to excrete M3G from the liver into the bloodstream, the major hepatic elimination route for this drug. This results in increased levels of M3G in liver and bile, a 50-fold reduction in the plasma levels of M3G, and in a major shift in the main disposition route for morphine and M3G, predominantly via the urine in WT mice but via the feces in Mrp3(-/-) mice. The pharamacokinetics of injected morphine-glucuronides are altered as well in the absence of Mrp3, and this results in a decreased antinociceptive potency of injected morphine-6-glucuronide.
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- 2005
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17. Online fluorescent method to assess BCRP/ABCG2 activity in suspension cells.
- Author
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Hooijberg JH, Peters GJ, Kaspers GJ, Wielinga PR, Veerman AJ, Pieters R, and Jansen G
- Subjects
- ATP Binding Cassette Transporter, Subfamily G, Member 2, ATP-Binding Cassette Transporters chemistry, Benzimidazoles pharmacology, Biological Transport, Cell Line, Tumor, Cells, Cultured, Fluorescent Dyes pharmacology, Humans, Kinetics, Neoplasm Proteins chemistry, Radiation-Sensitizing Agents pharmacology, Software, Time Factors, ATP-Binding Cassette Transporters metabolism, Neoplasm Proteins metabolism, Spectrometry, Fluorescence methods
- Abstract
An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells. To this end, a 2-compartment system consisting of a transwell cup and a cuvette was used. In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment. 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells. This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143.
- Published
- 2004
- Full Text
- View/download PDF
18. Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells.
- Author
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Wielinga PR, van der Heijden I, Reid G, Beijnen JH, Wijnholds J, and Borst P
- Subjects
- 1-Methyl-3-isobutylxanthine pharmacology, Blotting, Western, Cell Line, Cyclic AMP metabolism, Cyclic GMP metabolism, Dose-Response Relationship, Drug, Glutathione metabolism, Humans, Multidrug Resistance-Associated Proteins chemistry, Nitric Oxide metabolism, Nitric Oxide Donors pharmacology, Nitroprusside pharmacology, Phosphodiesterase Inhibitors pharmacology, Ribosomal Proteins chemistry, Time Factors, Biological Transport, Multidrug Resistance-Associated Proteins metabolism, Ribosomal Proteins metabolism
- Abstract
Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.
- Published
- 2003
- Full Text
- View/download PDF
19. Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells.
- Author
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Wielinga PR, Reid G, Challa EE, van der Heijden I, van Deemter L, de Haas M, Mol C, Kuil AJ, Groeneveld E, Schuetz JD, Brouwer C, De Abreu RA, Wijnholds J, Beijnen JH, and Borst P
- Subjects
- Biological Transport, Cells, Cultured, Chromatography, High Pressure Liquid, Drug Interactions, Humans, Kidney cytology, Kidney embryology, Kinetics, Mercaptopurine pharmacology, Methylthioinosine pharmacology, Multidrug Resistance-Associated Proteins biosynthesis, Ribosomal Proteins biosynthesis, Thioguanine pharmacology, Transfection, Mercaptopurine metabolism, Multidrug Resistance-Associated Proteins metabolism, Ribosomal Proteins metabolism, Thioguanine metabolism
- Abstract
Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.
- Published
- 2002
- Full Text
- View/download PDF
20. Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export.
- Author
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Evers R, de Haas M, Sparidans R, Beijnen J, Wielinga PR, Lankelma J, and Borst P
- Subjects
- Anion Transport Proteins, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Benzbromarone pharmacology, Biological Transport, Active drug effects, Dose-Response Relationship, Drug, Humans, Indomethacin pharmacology, Probenecid pharmacology, Tumor Cells, Cultured, Antineoplastic Agents, Phytogenic metabolism, Carrier Proteins metabolism, Glutathione metabolism, Sulfinpyrazone metabolism, Uricosuric Agents metabolism, Vinblastine metabolism
- Abstract
The multidrug resistance proteins MRP1 and MRP2 are members of the same subfamily of ATP-binding cassette transporters. Besides organic molecules conjugated to negatively charged ligands, these proteins also transport cytotoxic drugs for which no negatively charged conjugates are known to exist. In polarized MDCKII cells, MRP1 routes to the lateral plasma membrane, and MRP2 to the apical plasma membrane. In these cells MRP1 transports daunorubicin, and MRP2 vinblastine; both transporters export reduced glutathione (GSH) into the medium. We demonstrate that glutathione transport in MDCKII-MRP1 cells is inhibited by the inhibitors of organic anion transporters sulfinpyrazone, indomethacin, probenecid and benzbromarone. In MDCKII-MRP2 cells, GSH export is stimulated by low concentrations of sulfinpyrazone or indomethacin, whereas export is inhibited down to control levels at high concentrations. We find that unmodified sulfinpyrazone is a substrate for MRP2, also at concentrations where GSH export is inhibited. We also show that GSH export in MDCKII-MRP2 cells increases in the presence of vinblastine, and that the stoichiometry between drug and GSH exported is between two and three. Our data indicate that transport of sulfinpyrazone and vinblastine is associated with GSH export. However, at high sulfinpyrazone concentrations this compound is transported without GSH. Models of MRP action are discussed that could explain these results.
- Published
- 2000
- Full Text
- View/download PDF
21. The relative importance of passive and P-glycoprotein mediated anthracycline efflux from multidrug-resistant cells.
- Author
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Wielinga PR, Westerhoff HV, and Lankelma J
- Subjects
- Biological Transport, Biological Transport, Active drug effects, Cell Compartmentation, Cell Line, Cell Membrane Permeability drug effects, Daunorubicin pharmacokinetics, Digitonin pharmacology, Doxorubicin pharmacokinetics, Drug Resistance, Multiple, Epirubicin pharmacokinetics, Humans, Idarubicin pharmacokinetics, Kinetics, Verapamil pharmacology, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Antibiotics, Antineoplastic pharmacokinetics
- Abstract
For the four anthracyclines idarubicin, daunorubicin, epirubicin and doxorubicin the passive and active efflux rates in intact multidrug resistant cells were compared. Although highly similar structurally, these anti-tumor agents differ in lipophilicity and membrane permeability (k). The method we used was based on the continuous measurement of the cellular efflux and determination of the ratio (RVp) of transport rates just before and just after inhibition of the active transport with verapamil (Vp). Hence, RVp - 1 should reflect the active transport rate relative to the passive transport rate. If cells were single, well-stirred compartments, RVp - 1 should equal Vmax/(k.Km), where Vmax is the maximal pumping rate and Km is the Michaelis constant. However, using the plasma membrane permeabilizing agent digitonin, we found an effective intracellular anthracycline store. Particularly, when the efflux was fast, e.g. with idarubicin or in intensively pumping cells, the intracellular transport began to control the cellular efflux. Under these conditions, k underestimated the true plasma membrane permeability (k0) and RVp - 1 underestimated Vmax/(k.Km). Based on the effects of digitonin on the efflux rates in pumping and nonpumping cells, we developed an index (RVp,corrected - 1) which should equal Vmax/(k0. Km). The term Vmax/(k0.Km) varied substantially between the drugs. It appears that differences in lipophilicity between the drugs do not affect passive efflux and pumping equally. This demonstrates that passive permeation plays a substantial and independent role in determining the drug resistance for these anthracyclines. The methods developed here enable dissection of this role from that of drug pumping and intracellular subcompartmentation.
- Published
- 2000
- Full Text
- View/download PDF
22. In vitro transepithelial drug transport by on-line measurement: cellular control of paracellular and transcellular transport.
- Author
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Wielinga PR, de Waal E, Westerhoff HV, and Lankelma J
- Subjects
- Algorithms, Animals, Biological Transport, Active, Cattle, Cell Line, Cells, Cultured, Chemical Phenomena, Chemistry, Physical, Cyclosporins chemistry, Daunorubicin chemistry, Daunorubicin pharmacokinetics, Dextrans, Dogs, Fluorescein-5-isothiocyanate analogs & derivatives, Genes, MDR genetics, Humans, Idarubicin chemistry, Idarubicin pharmacokinetics, Models, Biological, Online Systems, Tight Junctions metabolism, Epithelial Cells metabolism
- Abstract
Studies on transcellular transport across epithelial cell layers are performed mostly by discontinuous sampling of the transported compound. This has several drawbacks, e.g., it gives disturbances in volume, it limits the time-resolution, and is often laborious. In this report we introduce a method to measure transepithelial transport of fluorescent compounds continuously. The time-resolution is at the (sub)minute scale, allowing the measurement of the change in transport rate before and after transport modulation. We will describe how we used the method to measure transcellular and paracellular transport. For highly membrane-impermeable compounds, the paracellular transport and the regulation of the tight junctions was studied in wild-type and MDR1 cDNA transfected epithelial canine kidney cells (MDCKII). The effect of the multidrug transporter P-glycoprotein (Pgp) on the transepithelial transport was studied. Addition of the Pgp inhibitor SDZ PSC 833 showed a modulation of the idarubicin (IDA) and daunorubicin (DNR) transport, which was larger during transport from the basolateral to the apical side than in the reverse direction. By modeling the transepithelial transport, we found that in these cells Pgp had more effect on the basolateral to apical transport than vice versa, which can be attributed to a relatively large passive permeation coefficient for the cellular basolateral plasma membrane.
- Published
- 1999
- Full Text
- View/download PDF
23. A method for studying plasma membrane transport with intact cells using computerized fluorometry.
- Author
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Wielinga PR, Heijn M, Westerhoff HV, and Lankelma J
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Biological Transport, Computers, Fluorescent Dyes, Kinetics, Mathematics, Rhodamine 123 metabolism, Cell Membrane metabolism, Fluorometry methods
- Abstract
A new method is presented for measuring rapid efflux of fluorescent compounds from monolayer cells. Cells grown on a glass coverslip were loaded with a fluorescent substrate. Thereafter, the coverslip was installed outside the light path in a stirred and thermostated cuvette of a fluorometer. The efflux was recorded by measuring the changes of fluorescence in the extracellular medium. The method was used to study the kinetics of active and passive plasma membrane transport of the P-glycoprotein substrates rhodamine 123 and daunorubicin. The method has advantages over other methods: (1) no radioactively labeled substrate is needed, (2) fluorescence of the transported substrate is not compromised by the cells, (3) changes in the extracellular concentration of the substrate can be monitored continuously and therefore a substantial improvement of the kinetic resolution is obtained, and (4) the measurement setup is relatively simple and a standard fluorometer can be used. From the efflux data, cellular transport parameters could be calculated, such as passive permeation coefficients and active transport rates., (Copyright 1998 Academic Press.)
- Published
- 1998
- Full Text
- View/download PDF
24. P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells.
- Author
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Wielinga PR, Heijn M, Broxterman HJ, and Lankelma J
- Subjects
- Daunorubicin pharmacokinetics, Dose-Response Relationship, Drug, Drug Resistance, Multiple, Etoposide pharmacokinetics, Fluoresceins pharmacokinetics, Humans, Hydrogen-Ion Concentration, KB Cells, Protein Kinase C physiology, ATP Binding Cassette Transporter, Subfamily B, Member 1 physiology, Antineoplastic Agents pharmacokinetics, Tetradecanoylphorbol Acetate pharmacology
- Abstract
The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-overexpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4alpha-phorbol, 12,13-didecanoate (4alphaPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.
- Published
- 1997
- Full Text
- View/download PDF
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