Tuberculosis infection in children visiting friends and relatives in countries with high incidence of tuberculosis: A study protocol.

INTRODUCTION: Tuberculosis (TB) is a global infectious disease. In low-incidence countries, paediatric TB affects mostly immigrant children and children of immigrants. We hypothesize that these children are at risk of exposure to Mycobacterium tuberculosis when they travel to the country of origin of their parents to visit friends and relatives (VFR). In this study, we aim to estimate the incidence rate and risk factors associated to latent tuberculosis infection (LTBI) and TB in VFR children. METHODS AND ANALYSIS: A prospective study will be carried out in collaboration with 21 primary health care centres (PCC) and 5 hospitals in Catalonia, Spain. The study participants are children under 15 years of age, either immigrant themselves or born to immigrant parents, who travel to countries with high incidence of TB (≥ 40 cases/100,000 inhabitants). A sample size of 492 children was calculated. Participants will be recruited before traveling, either during a visit to a travel clinic or to their PCC, where a questionnaire including sociodemographic, epidemiological and clinical data will be completed, and a tuberculin skin test (TST) will be performed and read after 48 to 72 hours; patients with a positive TST at baseline will be excluded. A visit will be scheduled eight to twelve-weeks after their return to perform a TST and a QuantiFERON-TB Gold Plus test. The incidence rate of LTBI will be estimated per individual/month and person/year per country visited, and also by age-group. ETHICS AND DISSEMINATION: The study protocol was approved by the Clinical Research Ethics Committee of the Hospital Universitari Mútua Terrassa (code 02/16) and the Clinical Research Ethics Committee of the Fundació Institut Universitari per a la Recerca a l'Atenció Primària de Salut Jordi Gol i Gurina (code P16/094). Articles will be published in indexed scientific journals. TRIAL REGISTRATION: Clinical-Trials.gov: NCT04236765.

In vitro evaluation of 225 Ac-DOTA-substance P for targeted alpha therapy of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant form of brain tumors with dismal prognosis despite treatment by surgery combined with radiotherapy and chemotherapy. The neuropeptide Substance P (SP) is the physiological ligand of the neurokinin-1 receptor, which is highly expressed in glioblastoma cells. Thus, SP represents a potential ligand for targeted alpha therapy. In this study, a protocol for the synthesis of SP labeled with the alpha emitter 225 Ac was developed and binding affinity properties were determined. The effects of 225 Ac-DOTA-SP were investigated on human glioblastoma cell lines (T98G, U87MG, U138MG) as well as GBM stem cells. A significant dose-dependent reduction in cell viability was detected up to 6 days after treatment. Also, colony-forming capacity was inhibited at the lower doses tested. In comparison, treatment with the conventional agent temozolomide showed higher cell viability and colony-forming capacity. 225 Ac-DOTA-SP treatment caused induction of late apoptosis pathways. Cells were arrested to G2/M-phase upon treatment. Increasing doses and treatment time caused additional S-phase arrest. Similar results were obtained using human glioblastoma stem cells, known to show radioresistance. Our data suggest that 225 Ac-DOTA-SP is a promising compound for treatment of GBM.

Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells.

Decitabine (5-aza-2'-deoxycytidine) is a DNA methyltransferase inhibitor and an archetypal epigenetic drug for the therapy of myeloid leukemias. The mode of action of decitabine strictly depends on the incorporation of the drug into DNA. However, DNA incorporation and ensuing genotoxic effects of decitabine have not yet been investigated in human cancer cell lines or in models related to the approved indication of the drug. Here we describe a robust assay for the quantitative determination of decitabine incorporation rates into DNA from human cancer cells. Using a panel of human myeloid leukemia cell lines we show appreciable amounts of decitabine incorporation that closely correlated with cellular drug uptake. Decitabine incorporation was also detectable in primary cells from myeloid leukemia patients, indicating that the assay is suitable for biomarker analyses to predict drug responses in patients. Finally, we also used next-generation sequencing to comprehensively analyze the effects of decitabine incorporation on the DNA sequence level. Interestingly, this approach failed to reveal significant changes in the rates of point mutations and genome rearrangements in myeloid leukemia cell lines. These results indicate that standard rates of decitabine incorporation are not genotoxic in myeloid leukemia cells.

DNA methyltransferase inhibition reverses epigenetically embedded phenotypes in lung cancer preferentially affecting polycomb target genes.

PURPOSE: Cancer cell phenotypes are partially determined by epigenetic specifications, such as DNA methylation. Metastasis development is a late event in cancerogenesis and might be associated with epigenetic alterations. EXPERIMENTAL DESIGN: An in vivo selection approach was used to generate highly aggressive non-small cell lung cancer (NSCLC) cell lines (A549 and HTB56) followed by genome-wide DNA methylation analysis. Furthermore, the therapeutic effects of the epigenetic agent azacytidine on DNA methylation patterns and the in vivo phenotypes were explored. RESULTS: Widespread changes of DNA methylation were observed during development of highly aggressive cell lines. Up to 2.5% of the CpG-rich region was differentially methylated as identified by reduced representation bisulfite sequencing compared with the less aggressive parental cell lines. DNA methyltransferase inhibition by azacytidine reversed the prometastatic phenotype; this was highly associated with the preferential loss of DNA methylation at sites that were hypermethylated during the in vivo selection. Of note, polycomb (PRC2) binding sites were particularly affected by DNA methylation changes after azacytidine exposure that persisted over time. CONCLUSIONS: We could show that metastatic capability of NSCLC is closely associated with DNA methylome alterations. Because inhibition of DNA methyltransferase reversed metastasis-prone phenotype, epigenetic modulation seems to be a potential therapeutic approach to prevent metastasis formation.

Cardiopulmonary exercise testing in healthy children and adolescents at moderately high altitude / Prueba de esfuerzo cardiopulmonar a niños y adolescentes sanos en altitud moderadamente elevada

Objective: Cardiopulmonary exercise testing is a tool that helps clinicians to establish diagnosis and calculate risk stratification in adults. However, the utility of this test among children with congenital heart disease has not been fully explored. The goal of this study was to describe reference values for cardiopulmonary performance of healthy children. Methods: This study included 103 apparently healthy children (aged from 4 to 18 years; 61 boys), who underwent cardiopulmonary test using a treadmill protocol. All tests took place at 2240m above sea level (Mexico City). Results: Exercise time was 11 ± 4 min. There were no complications. Peak oxygen uptake correlated closely with height in both genders (girls r = 0.84; boys r = 0.84, p < 0.001). A multivariable linear regression model showed that body surface area, exercise time, gender and heart rate reserve were significant predictors of peak oxygen uptake (R² =0.815, p<0.001). Peak oxygen uptake was strongly associated with age even among children younger than thirteen years (r = 0.74, p <0.001). Conclusion: This study provides physiological values for the major cardiopulmonary variables obtained from exercise testing using a treadmill among healthy children. Cardiopulmonary exercise test can be safely and effectively performed in young children even as young as 4 years old. Variables including age, gender and height are strongly associated with exercise time, peak heart rate and peak oxygen uptake. Regression equations for predicting peak heart rate and peak oxygen uptake are presented as reference values that allow researchers to compare children with heart disease versus those who are healthy.

Objetivo: La prueba de esfuerzo cardiopulmonar es una herramienta que ayuda a los médicos a establecer el diagnóstico y estratificar el riesgo en adultos. Sin embargo, su utilidad en los niños no se ha explorado a fondo. El objetivo fue describir los valores de esta prueba en niños sanos en altitud moderadamente alta. Métodos: Se realizaron pruebas de esfuerzo cardiopulmonar a 103 niños sanos (4 a 18 años, 61 varones) mediante tapiz rodante y a 2240m sobre el nivel del mar (Ciudad de México). Resultados: El tiempo de ejercicio fue de 11 ± 4 min, sin complicaciones. El consumo de oxígeno pico se correlacionó estrechamente con la talla en ambos géneros (niñas r = 0.84; niños r = 0.84, p < 0.001). El modelo multivariado que incluyó superficie corporal, tiempo de ejercicio, género y la frecuencia cardíaca de reserva fue un fuerte predictor del consumo de oxígeno pico (R²=0.815, p<0.001). Conclusión: Las pruebas de esfuerzo cardiopulmonar mediante tapiz rodante se pueden realizar con seguridad y eficacia en niños, incluso de 4 años de edad. Variables como la edad, el género y la talla están fuertemente asociados con el tiempo de ejercicio, la frecuencia cardiaca máxima y el de oxígeno pico. Las ecuaciones de regresión obtenidas para calcular la frecuencia cardíaca máxima y el consumo de oxígeno pico pueden ayudar, tanto a clínicos como a investigadores, a comparar el comportamiento de niños con cardiopatías frente a los que no las tienen.

The role of human equilibrative nucleoside transporter 1 on the cellular transport of the DNA methyltransferase inhibitors 5-azacytidine and CP-4200 in human leukemia cells.

The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹4C]5-azacytidine demonstrated Na⁺-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances.

Cardiopulmonary exercise testing in healthy children and adolescents at moderately high altitude.

OBJECTIVE: Cardiopulmonary exercise testing is a tool that helps clinicians to establish diagnosis and calculate risk stratification in adults. However, the utility of this test among children with congenital heart disease has not been fully explored. The goal of this study was to describe reference values for cardiopulmonary performance of healthy children. METHODS: This study included 103 apparently healthy children (aged from 4 to 18 years; 61 boys), who underwent cardiopulmonary test using a treadmill protocol. All tests took place at 2240m above sea level (Mexico City). RESULTS: Exercise time was 11±4min. There were no complications. Peak oxygen uptake correlated closely with height in both genders (girls r=0.84; boys r=0.84, p<0.001). A multivariable linear regression model showed that body surface area, exercise time, gender and heart rate reserve were significant predictors of peak oxygen uptake (R(2)=0.815, p<0.001). Peak oxygen uptake was strongly associated with age even among children younger than thirteen years (r=0.74, p<0.001). CONCLUSION: This study provides physiological values for the major cardiopulmonary variables obtained from exercise testing using a treadmill among healthy children. Cardiopulmonary exercise test can be safely and effectively performed in young children even as young as 4 years old. Variables including age, gender and height are strongly associated with exercise time, peak heart rate and peak oxygen uptake. Regression equations for predicting peak heart rate and peak oxygen uptake are presented as reference values that allow researchers to compare children with heart disease versus those who are healthy.

[Cardiac rehabilitation in congenital heart disease]. / Rehabilitación cardiaca en cardiopatías congénitas.

Pediatric Cardiology is a medical subspecialty that emerged in a systematic manner during the beginning of the 20th century. Throughout time, with the use of several methods we have been able to establish a series of diagnosis, offer surgical treatments and currently we evaluate and analyze the results of such proceedings. In the cardiac rehabilitation programs, children and adolescents are taught to identify the safety limits of their hearts, being able to relate them to their daily effort activities, providing them with a better quality of life and where they learn to live with the limitations that their illness implies.

Rehabilitación cardiaca en cardiopatías congénitas / Cardiac rehabilitation in congenital heart disease

La cardiología pediátrica es una subespecialidad que surgió de manera sistemática, al inicio del siglo XX. A lo largo del tiempo y a través de diversos métodos se han establecido diagnósticos, se ha ofrecido tratamiento farmacológico, intervencionista y quirúrgico y actualmente, se evalúan y analizan los resultados de dichos procedimientos. A través de los programas de rehabilitación cardiaca, se le enseña a conocer los límites seguros de su corazón en actividades de la vida diaria, brindando a los pequeños una mejor calidad de vida donde aprenderán a vivir con las limitaciones que la enfermedad trae consigo.

Pediatric Cardiology is a medical subspecialty that emerged in a systematic manner during the beginning of the 20th century. Throughout time, with the use of several methods we have been able to establish a series of diagnosis, offer surgical treatments and currently we evaluate and analyze the results of such proceedings. In the cardiac rehabilitation programs, children and adolescents are taught to identify the safety limits of their hearts, being able to relate them to their daily effort activities, providing them with a better quality of life and where they learn to live with the limitations that their illness implies.

Proton pump inhibitors inhibit metformin uptake by organic cation transporters (OCTs).

Metformin, an oral insulin-sensitizing drug, is actively transported into cells by organic cation transporters (OCT) 1, 2, and 3 (encoded by SLC22A1, SLC22A2, or SLC22A3), which are tissue specifically expressed at significant levels in various organs such as liver, muscle, and kidney. Because metformin does not undergo hepatic metabolism, drug-drug interaction by inhibition of OCT transporters may be important. So far, comprehensive data on the interaction of proton pump inhibitors (PPIs) with OCTs are missing although PPIs are frequently used in metformin-treated patients. Using in silico modeling and computational analyses, we derived pharmacophore models indicating that PPIs (i.e. omeprazole, pantoprazole, lansoprazole, rabeprazole, and tenatoprazole) are potent OCT inhibitors. We then established stably transfected cell lines expressing the human uptake transporters OCT1, OCT2, or OCT3 and tested whether these PPIs inhibit OCT-mediated metformin uptake in vitro. All tested PPIs significantly inhibited metformin uptake by OCT1, OCT2, and OCT3 in a concentration-dependent manner. Half-maximal inhibitory concentration values (IC(50)) were in the low micromolar range (3-36 µM) and thereby in the range of IC(50) values of other potent OCT drug inhibitors. Finally, we tested whether the PPIs are also transported by OCTs, but did not identify PPIs as OCT substrates. In conclusion, PPIs are potent inhibitors of the OCT-mediated metformin transport in vitro. Further studies are needed to elucidate the clinical relevance of this drug-drug interaction with potential consequences on metformin disposition and/or efficacy.

Delivery of 5-azacytidine to human cancer cells by elaidic acid esterification increases therapeutic drug efficacy.

Azacytidine is an established nucleoside drug that is well known for its ability to modulate epigenetic gene regulation by inhibition of DNA methylation. Despite recent advances in the clinical development of azacytidine, the use of the drug is limited by its low bioavailability and dependency on variably expressed nucleoside transporters for cellular uptake. We show here that CP-4200, an elaidic acid derivative of azacytidine, has strong epigenetic modulatory potency in human cancer cell lines, as evidenced by efficient depletion of DNA methyltransferase protein, genome-wide DNA demethylation, and robust reactivation of epigenetically silenced tumor suppressor genes. Importantly, however, the cellular uptake of CP-4200 was substantially less dependent on the nucleoside transporters that are known to be involved in azacytidine uptake. In agreement with this notion, CP-4200 showed a significantly higher antitumoral activity than azacytidine in an orthotopic mouse tumor model for acute lymphocytic leukemia. Together, these data represent a detailed characterization of the CP-4200 mode of action and suggest that elaidic acid modification improves the therapeutic efficacy of azacytidine.

Vectorial transport of nucleoside analogs from the apical to the basolateral membrane in double-transfected cells expressing the human concentrative nucleoside transporter hCNT3 and the export pump ABCC4.

The identification of the transport proteins responsible for the uptake and the efflux of nucleosides and their metabolites enables the characterization of their vectorial transport and a better understanding of their absorption, distribution, and elimination. Human concentrative nucleoside transporters (hCNTs/SLC28A) are known to mediate the transport of natural nucleosides and some nucleoside analogs into cells in a sodium-dependent and unidirectional manner. On the other hand, several human multidrug resistance proteins [human ATP-binding cassette transporter, subfamily C (ABCC)] cause resistance against nucleoside analogs and mediate transport of phosphorylated nucleoside derivatives out of the cells in an ATP-dependent manner. For the integrated analysis of uptake and efflux of these compounds, we established a double-transfected Madin-Darby canine kidney (MDCK) II cell line stably expressing the human uptake transporter hCNT3 in the apical membrane and the human efflux pump ABCC4 in the basolateral membrane. The direction of transport was from the apical to the basolateral compartment, which is in line with the unidirectional transport and the localization of both recombinant proteins in the MDCKII cells. Recombinant hCNT3 mediated the transport of several known nucleoside substrates, and we identified 5-azacytidine as a new substrate for hCNT3. It is of interest that coexpression of both transporters was confirmed in pancreatic adenocarcinomas, which represent an important clinical indication for the therapeutic use of nucleoside analogs. Thus, our results establish a novel cell system for studies on the vectorial transport of nucleosides and their analogs from the apical to the basolateral compartment. The results contribute to a better understanding of the cellular transport characteristics of nucleoside drugs.

Intraindividual comparison between gadopentetate dimeglumine and gadobutrol for magnetic resonance perfusion in normal brain and intracranial tumors at 3 Tesla.

BACKGROUND: In vitro studies have shown that the 3-Tesla (T) magnetic resonance (MR) characteristics of high- and standard-molar gadolinium-based contrast agents differ. Such differences may indicate that high-molar (1.0 M) agents offer advantages for perfusion-weighted imaging (PWI) at 3T, as has been previously reported at 1.5 T. PURPOSE: To investigate possible intraindividual differences of high- versus low-molar contrast agents on PWI at 3T in patients with intracranial space-occupying lesions. MATERIAL AND METHODS: Six patients with intraaxial and five patients with extraaxial tumors underwent two MR examinations at 3T, separated by at least 48 hours. On each occasion, an exogenous contrast-based, T2*-weighted, gradient-recalled echo-planar imaging (EPI) technique was used to determine the intracranial perfusion characteristics using one of two intravenous contrast agents: either 5 ml of 1.0 M gadobutrol or 10 ml of 0.5 M gadopentetate dimeglumine. The primary PWI outcome measure was region-of-interest maximal signal change (C(max)). RESULTS: The difference in C(max) for gray and white matter (Delta C(max)) was significantly higher for gadobutrol compared to gadopentetate dimeglumine (P<0.01). The ratio of C(max) between gray and white matter (rC(max) = C(maxGray)/C(maxWhite)) was also significantly higher (median 24.6%, range 13.7-36.5%) for gadobutrol (P<0.01). The ratio of C(max) between the whole tumor and whole normal side hemisphere was higher in five out of the six intraaxial tumor cases. A significantly higher ratio (Delta C(max)/C(max)) in the difference between C(max) of gray and white matter (from hemisphere without brain lesion) compared to C(max) for the hemisphere containing the neoplasm (hemisphere with brain lesion) was demonstrated for gadobutrol in intraaxial tumors (P<0.05). CONCLUSION: Higher-concentration 1.0 M gadobutrol can offer advantages over standard 0.5 M gadopentetate dimeglumine, particularly with respect to delineation between gray and white matter and for the demarcation of highly vascularized tumor tissue on brain PWI performed at 3T.

Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation.

The DNA methyltransferase inhibitors 5-azacytidine (5-azaCyd) and 5-aza-2'-deoxycytidine have found increasing use for the treatment of myeloid leukemias and solid tumors. Both nucleoside analogues must be transported into cells and phosphorylated before they can be incorporated into DNA and inactivate DNA methyltransferases. The members of the human equilibrative and concentrative nucleoside transporter families mediate transport of natural nucleosides and some nucleoside analogues into cells. However, the molecular identity of the transport proteins responsible for mediating the uptake of 5-azanucleosides has remained unknown. To this end, we have generated a stably transfected Madin-Darby canine kidney strain II cell line expressing recombinant hCNT1. An antiserum directed against hCNT1 specifically detected the protein in the apical membrane of hCNT1-expressing Madin-Darby canine kidney cells. Using [14C]5-azaCyd, we show here that hCNT1 mediated the Na+-dependent uptake of this drug with a Km value of 63 micromol/L. Na+-dependent transport of radiolabeled cytidine, uridine, and 5-fluoro-5'-deoxyuridine further showed the functionality of the transporter. hCNT1-expressing cells were significantly more sensitive to 5-azaCyd, and drug-dependent covalent trapping of DNA methyltransferase 1 was substantially more pronounced. Importantly, these results correlated with a significant sensitization of hCNT1-expressing cells toward the demethylating effects of 5-azaCyd and 5-aza-2'-deoxycytidine. In conclusion, our study identifies 5-azaCyd as a novel substrate for hCNT1 and provides direct evidence that hCNT1 is involved in the DNA-demethylating effects of this drug.

ATP-dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4).

The proinflammatory mediators leukotriene (LT) B(4) and LTC(4) must be transported out of cells before they can interact with LT receptors. Previously, we identified the multidrug resistance protein ABCC1 (MRP1) as an efflux pump for LTC(4). However, the molecular basis for the efflux of LTB(4) was unknown. Here, we demonstrate that human ABCC4 mediates the ATP-dependent efflux of LTB(4) in the presence of reduced glutathione (GSH), whereby the latter can be replaced by S-methyl GSH. Transport studies were performed with inside-out membrane vesicles from V79 fibroblasts and Sf9 insect cells that contained recombinant ABCC4, with vesicles from human platelets and myelomonocytic U937 cells, which were rich in endogenous ABCC4, but ABCC1 was below detectability. Moreover, human polymorphonuclear leukocytes contained ABCC4. K(m) values for LTB(4) were 5.2 muM with vesicles from fibroblasts and 5.6 muM with vesicles from platelets. ABCC4, with its broad substrate specificity, also functioned as an ATP-dependent efflux pump for LTC(4) with a K(m) of 0.13 muM in vesicles from fibroblasts and 0.32 muM in vesicles from platelets. However, GSH was not required for the transport of this glutathionylated leukotriene. The transport of LTC(4) by ABCC4 explains its release from platelets during transcellular synthesis. ATP-dependent transport of LTB(4) and LTC(4) by ABCC4 was inhibited by several organic anions, including S-decyl GSH, sulindac sulfide, and by the LTD(4) receptor antagonists montelukast and 3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-((3-dimethyl-amino-3-oxopropyl)-thio)-methyl)thio)propanoic acid (MK571). Thus, as an efflux pump for the proinflammatory mediators LTB(4) and LTC(4), ABCC4 may represent a novel target for anti-inflammatory therapies.

Data-based mathematical modeling of vectorial transport across double-transfected polarized cells.

Vectorial transport of endogenous small molecules, toxins, and drugs across polarized epithelial cells contributes to their half-life in the organism and to detoxification. To study vectorial transport in a quantitative manner, an in vitro model was used that includes polarized MDCKII cells stably expressing the recombinant human uptake transporter OATP1B3 in their basolateral membrane and the recombinant ATP-driven efflux pump ABCC2 in their apical membrane. These double-transfected cells enabled mathematical modeling of the vectorial transport of the anionic prototype substance bromosulfophthalein (BSP) that has frequently been used to examine hepatobiliary transport. Time-dependent analyses of (3)H-labeled BSP in the basolateral, intracellular, and apical compartments of cells cultured on filter membranes and efflux experiments in cells preloaded with BSP were performed. A mathematical model was fitted to the experimental data. Data-based modeling was optimized by including endogenous transport processes in addition to the recombinant transport proteins. The predominant contributions to the overall vectorial transport of BSP were mediated by OATP1B3 (44%) and ABCC2 (28%). Model comparison predicted a previously unrecognized endogenous basolateral efflux process as a negative contribution to total vectorial transport, amounting to 19%, which is in line with the detection of the basolateral efflux pump Abcc4 in MDCKII cells. Rate-determining steps in the vectorial transport were identified by calculating control coefficients. Data-based mathematical modeling of vectorial transport of BSP as a model substance resulted in a quantitative description of this process and its components. The same systems biology approach may be applied to other cellular systems and to different substances.

Gadofluorine m uptake in stem cells as a new magnetic resonance imaging tracking method: an in vitro and in vivo study.

OBJECTIVES: Cell tracking using ultrasmall iron particles is well established in magnetic resonance imaging (MRI). However, in experimental models, intrinsic iron signals derived from erythrocytes mask the labeled cells. Therefore, we evaluated Gadofluorine M with other gadolinium chelates for a T1-weighted positive enhancement for cell tracking in vitro. In addition, Gadofluorine M was tested in vivo. MATERIAL AND METHODS: Gadofluorine M and other gadolinium chelates were used to label stem cells with and without uptake-mediating agents in vitro and in vivo using a 1.5 T MRI. In addition, histology and molecular modeling was investigated. RESULTS: Gadofluorine M revealed comparable properties to an uptake mediating agent in molecular modeling. Without an uptake-mediating agent Gadofluorine M-labeled cells were detected as a T1-weighted positive contrast in vitro and in vivo. Histology confirmed a 100% success rate for intracellular labeling. CONCLUSION: This study describes a novel contrast agent with the capability of intracellular accumulation without an uptake mediator providing a T1-positive MRI signal at 1.5 T and may be suitable for cell tracking in animal models with intraparenchymal hemorrhages such as stroke or malignant tumors.

Substrate specificity of human ABCC4 (MRP4)-mediated cotransport of bile acids and reduced glutathione.

The multidrug resistance protein ABCC4 (MRP4), a member of the ATP-binding cassette superfamily, mediates ATP-dependent unidirectional efflux of organic anions out of cells. Previous studies showed that human ABCC4 is localized to the sinusoidal membrane of hepatocytes and mediates, among other substrates, the cotransport of reduced glutathione (GSH) with bile acids. In the present study, using inside-out membrane vesicles, we demonstrated that human ABCC4 in the presence of physiological concentrations of GSH has a high affinity for the taurine and glycine conjugates of the common natural bile acids as well as the unconjugated bile acid cholate. Chenodeoxycholyltaurine and chenodeoxycholylglycine were the GSH cosubstrates with the highest affinities for ABCC4, with K(m) values of 3.6 and 5.9 microM, respectively. Ursodeoxycholyltaurine and ursodeoxycholylglycine were cotransported together with GSH by ABCC4 with K(m) values of 7.8 and 12.5 microM, respectively, but no transport of ursodeoxycholate and deoxycholate was observed. The simultaneous transport of labeled GSH and cholyltaurine or cholylglycine was demonstrated in double-labeled cotransport experiments with a bile acid-to-GSH ratio of approximately 1:22. K(m) values of the bile acids for ABCC4 were in a range similar to those reported for the canalicular bile salt export pump ABCB11. Under physiological conditions, the sinusoidal ABCC4 may compete with canalicular ABCB11 for bile acids and thereby play a key role in determining the hepatocyte concentration of bile acids. In cholestatic conditions, ABCC4 may become a key pathway for efflux of bile acids from hepatocytes into blood.

Prostanoid transport by multidrug resistance protein 4 (MRP4/ABCC4) localized in tissues of the human urogenital tract.

PURPOSE: The seminal vesicles are the major source of prostaglandins in seminal fluid. For prostanoid action on cell surfaces they must be released from synthesizing cells. MRP4/ABCC4 (multidrug resistance protein 4 adenosine triphosphate-binding cassette, subfamily C, member 4) is an adenosine triphosphate dependent export pump for organic anions that may mediate prostanoid transport across the plasma membranes. Therefore, we analyzed whether MRP4 is expressed in the seminal vesicles and other tissues of the human urogenital tract, whether MRP4 and prostanoid synthesizing enzymes are co-expressed in the same cell type and whether MRP4 functions as a prostanoid export pump. MATERIALS AND METHODS: The expression and localization of MRP4 and prostanoid synthesizing enzymes were investigated in several tissues of the male human urogenital tract by immunoblot and immunofluorescence analyses. Prostanoid transport was measured into inside-out membrane vesicles from cells expressing recombinant human MRP4. RESULTS: MRP4 and prostanoid synthesizing enzymes were co-expressed in the epithelial cells of human seminal vesicles. Moreover, MRP4 was localized in the plasma membrane of epithelial cells of the ureter, in the basolateral membrane of glandular epithelial cells of the prostate, and in smooth muscle cells of the bladder and corpus cavernosum. Transport studies established MRP4 as an efflux pump for prostaglandin E2 (Michaelis constant [Km] 3.5 muM), thromboxane B2 (Km 9.9 muM) and prostaglandin F2alpha (Km 12.6 muM). CONCLUSIONS: The co-expression of prostanoid synthesizing enzymes and MRP4 in epithelial cells of the human seminal vesicles and the function of MRP4 as a prostanoid efflux pump indicate that MRP4 mediates prostanoid transport from these cells, which are the main prostanoid synthesizing cells in the male urogenital tract.

Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane.

The liver is the major source of reduced glutathione (GSH) in blood plasma. The transport protein mediating the efflux of GSH across the basolateral membrane of human hepatocytes has not been identified so far. In this study we have localized the multidrug resistance protein 4 (MRP4; ABCC4) to the basolateral membrane of human, rat, and mouse hepatocytes and human hepatoma HepG2 cells. Recombinant human MRP4, expressed in V79 hamster fibroblasts and studied in membrane vesicles, mediated ATP-dependent cotransport of GSH or S-methyl-glutathione together with cholyltaurine, cholylglycine, or cholate. Several monoanionic bile salts and the quinoline derivative MK571 were potent inhibitors of this unidirectional transport. The K(m) values were 2.7 mmol/L for GSH and 1.2 mmol/L for the nonreducing S-methyl-glutathione in the presence of 5 micromol/L cholyltaurine, and 3.8 micromol/L for cholyltaurine in the presence of 5 mmol/L S-methyl-glutathione. Transport of bile salts by MRP4 was negligible in the absence of ATP or without S-methyl-glutathione. These findings identify a novel pathway for the efflux of GSH across the basolateral hepatocyte membrane into blood where it may serve as an antioxidant and as a source of cysteine for other organs. Moreover, MRP4-mediated bile salt transport across the basolateral membrane may function as an overflow pathway during impaired bile salt secretion across the canalicular membrane into bile. In conclusion, MRP4 can mediate the efflux of GSH from hepatocytes into blood by cotransport with monoanionic bile salts.