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Profiles of multidrug resistance protein-1 in the peripheral blood mononuclear cells of patients with refractory epilepsy.

Ban JJ, Jung KH, Chu K, Lee ST, Jeon D, Park KI, Moon HJ, Kim H, Kim S, Lee SK, Roh JK - PLoS ONE (2012)

Bottom Line: The MDR1 conformational change level was significantly higher in the high-medication-use group than the low-use group (p = 0.028).Basal MDR1 (OR = 1.16 [95% CI: 1.060-1.268]) and conformational change level (OR = 1.11 [95% CI: 1.02-1.20]) were independent predictors for seizure frequency and number of medications, respectively.The MDR1 profile of PBMNCs is associated with seizure frequency and medication conditions in patients with epilepsy.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Laboratory for Neurotherapeutics, Seoul National University Hospital, Seoul, South Korea.

ABSTRACT

Background: About one third of patients with epilepsy become refractory to therapy despite receiving adequate medical treatment, possibly from multidrug resistance. P-glycoprotein, encoded by multidrug resistance protein-1 (MDR1) gene, at the blood brain barrier is considered as a major factor mediating drug efflux and contributing to resistance. Given that peripheral blood mononuclear cells (PBMNCs) express MDR1, we investigated a MDR1 status of PBMNCs in various subsets of epilepsy patients and demonstrated their association with clinical characteristics.

Methodology/principal findings: Clinical and MDR1 data were collected from 140 patients with epilepsy, 30 healthy volunteers, and 20 control patients taking anti-epileptic drugs. PBMNCs were isolated, and basal MDR1 levels and MDR1 conformational change levels were measured by flow cytometry. MDR1 profiles were analyzed according to various clinical parameters, including seizure frequency and number of medications used in epilepsy patients. Epilepsy patients had a higher basal MDR1 level than non-epilepsy groups (p<0.01). Among epilepsy patients, there is a tendency for higher seizure frequency group to have higher basal MDR1 level (p = 0.059). The MDR1 conformational change level was significantly higher in the high-medication-use group than the low-use group (p = 0.028). Basal MDR1 (OR = 1.16 [95% CI: 1.060-1.268]) and conformational change level (OR = 1.11 [95% CI: 1.02-1.20]) were independent predictors for seizure frequency and number of medications, respectively.

Conclusions/significance: The MDR1 profile of PBMNCs is associated with seizure frequency and medication conditions in patients with epilepsy.

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Related in: MedlinePlus

Analysis of MDR1 profiles of PBMNCs with the flow cytometry.(A) PBMNCs were stained with MDR1 antibody and propidium iodide (PI) and examined forward scatter (FSC) and side scatter (SSC) and leukocytes were gated (R1) to exclude aggregates and debris. (B) In R1 gated population, PI positive dead cells were excluded and PI negative live cells were gated (lined box). (C) In gated cells, the MDR1-positive population was assessed using the baseline determined from a negative control of IgG2a background staining (dotted box). (D) Representative histogram figures show the basal MDR1 level (left) and conformational change level (right).
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pone-0036985-g001: Analysis of MDR1 profiles of PBMNCs with the flow cytometry.(A) PBMNCs were stained with MDR1 antibody and propidium iodide (PI) and examined forward scatter (FSC) and side scatter (SSC) and leukocytes were gated (R1) to exclude aggregates and debris. (B) In R1 gated population, PI positive dead cells were excluded and PI negative live cells were gated (lined box). (C) In gated cells, the MDR1-positive population was assessed using the baseline determined from a negative control of IgG2a background staining (dotted box). (D) Representative histogram figures show the basal MDR1 level (left) and conformational change level (right).

Mentions: Materials for the flow cytometry staining including MDR1 antibody (UIC2 antibody), dimethyl sulfoxide (DMSO), vinblastine, working solutions, and binding buffers were all included in the MDR1 shift assay kit (Chemicon International, Temecula, CA, USA). Peripheral blood samples (5cc) were obtained from all enrolled subjects. The heparinized blood samples were added to Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 400 ×g for 30 min to separate the PBMNCs. Isolated cells were counted and centrifuged at 200 ×g for 5 min. The supernatant was removed, and the pellets were resuspended to 1×106 cells/mL in warmed UIC2 binding buffer (1% bovine serum albumin in PBS). Basal MDR1 level assay procedure is as follows: cells were divided into two groups and treated with 5 µL of DMSO respectively. The tubes were incubated at 37°C for 10 min; 25 µL of the kit’s IgG2a antibody working solution was added to one group and 25 µL of UIC2 antibody working solution was added to the other group. The tubes were incubated at 37°C for another 15 min and washed three times with cold UIC2 binding buffer at 200×g for 10 min at 4°C. The cells were resuspended in 250 µL ice-cold secondary antibody working solution and incubated for 15 min at 4°C in the dark. After being washed three times, the cells were resuspended in 0.5 mL ice-cold propidium iodide staining buffer. Samples were then maintained on ice until analysis with a flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA, USA), using FL2 channel for indirect UIC2 staining and FL3 channel for propidium iodide to exclude dead cells. To filter out cell debris, forward scatter and side scatter of the cells were measured and lymphocytes and monocytes regions were gated (Fig. 1A). After gating cell populations, propidium iodide negative cells were gated to wipe out dead cells (Fig. 1B). Using this cell population, baseline for gating positive population was confirmed using IgG2a isotype. UIC2 positive populations were gated using this baseline and positive cell number was counted using FL2 channel (Fig. 1C). Positive level was calculated as a percentage of UIC2 positive cell number in whole live cell population. For MDR1 conformational change level assay, we used 5 µL of 22 mM vinblastine instead of DMSO and rest of the process is identical to basal MDR1 level. UIC2 antibody reacts with membrane bound MDR1 and their reactivity is increased when conformational change of MDR1 occurs. The basal MDR1 level reflects the number of cells expressing high level of MDR1 and the conformational change level reflects all functional MDR1 level in PBMNCs [22], [23].


Profiles of multidrug resistance protein-1 in the peripheral blood mononuclear cells of patients with refractory epilepsy.

Ban JJ, Jung KH, Chu K, Lee ST, Jeon D, Park KI, Moon HJ, Kim H, Kim S, Lee SK, Roh JK - PLoS ONE (2012)

Analysis of MDR1 profiles of PBMNCs with the flow cytometry.(A) PBMNCs were stained with MDR1 antibody and propidium iodide (PI) and examined forward scatter (FSC) and side scatter (SSC) and leukocytes were gated (R1) to exclude aggregates and debris. (B) In R1 gated population, PI positive dead cells were excluded and PI negative live cells were gated (lined box). (C) In gated cells, the MDR1-positive population was assessed using the baseline determined from a negative control of IgG2a background staining (dotted box). (D) Representative histogram figures show the basal MDR1 level (left) and conformational change level (right).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351424&req=5

pone-0036985-g001: Analysis of MDR1 profiles of PBMNCs with the flow cytometry.(A) PBMNCs were stained with MDR1 antibody and propidium iodide (PI) and examined forward scatter (FSC) and side scatter (SSC) and leukocytes were gated (R1) to exclude aggregates and debris. (B) In R1 gated population, PI positive dead cells were excluded and PI negative live cells were gated (lined box). (C) In gated cells, the MDR1-positive population was assessed using the baseline determined from a negative control of IgG2a background staining (dotted box). (D) Representative histogram figures show the basal MDR1 level (left) and conformational change level (right).
Mentions: Materials for the flow cytometry staining including MDR1 antibody (UIC2 antibody), dimethyl sulfoxide (DMSO), vinblastine, working solutions, and binding buffers were all included in the MDR1 shift assay kit (Chemicon International, Temecula, CA, USA). Peripheral blood samples (5cc) were obtained from all enrolled subjects. The heparinized blood samples were added to Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 400 ×g for 30 min to separate the PBMNCs. Isolated cells were counted and centrifuged at 200 ×g for 5 min. The supernatant was removed, and the pellets were resuspended to 1×106 cells/mL in warmed UIC2 binding buffer (1% bovine serum albumin in PBS). Basal MDR1 level assay procedure is as follows: cells were divided into two groups and treated with 5 µL of DMSO respectively. The tubes were incubated at 37°C for 10 min; 25 µL of the kit’s IgG2a antibody working solution was added to one group and 25 µL of UIC2 antibody working solution was added to the other group. The tubes were incubated at 37°C for another 15 min and washed three times with cold UIC2 binding buffer at 200×g for 10 min at 4°C. The cells were resuspended in 250 µL ice-cold secondary antibody working solution and incubated for 15 min at 4°C in the dark. After being washed three times, the cells were resuspended in 0.5 mL ice-cold propidium iodide staining buffer. Samples were then maintained on ice until analysis with a flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA, USA), using FL2 channel for indirect UIC2 staining and FL3 channel for propidium iodide to exclude dead cells. To filter out cell debris, forward scatter and side scatter of the cells were measured and lymphocytes and monocytes regions were gated (Fig. 1A). After gating cell populations, propidium iodide negative cells were gated to wipe out dead cells (Fig. 1B). Using this cell population, baseline for gating positive population was confirmed using IgG2a isotype. UIC2 positive populations were gated using this baseline and positive cell number was counted using FL2 channel (Fig. 1C). Positive level was calculated as a percentage of UIC2 positive cell number in whole live cell population. For MDR1 conformational change level assay, we used 5 µL of 22 mM vinblastine instead of DMSO and rest of the process is identical to basal MDR1 level. UIC2 antibody reacts with membrane bound MDR1 and their reactivity is increased when conformational change of MDR1 occurs. The basal MDR1 level reflects the number of cells expressing high level of MDR1 and the conformational change level reflects all functional MDR1 level in PBMNCs [22], [23].

Bottom Line: The MDR1 conformational change level was significantly higher in the high-medication-use group than the low-use group (p = 0.028).Basal MDR1 (OR = 1.16 [95% CI: 1.060-1.268]) and conformational change level (OR = 1.11 [95% CI: 1.02-1.20]) were independent predictors for seizure frequency and number of medications, respectively.The MDR1 profile of PBMNCs is associated with seizure frequency and medication conditions in patients with epilepsy.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Laboratory for Neurotherapeutics, Seoul National University Hospital, Seoul, South Korea.

ABSTRACT

Background: About one third of patients with epilepsy become refractory to therapy despite receiving adequate medical treatment, possibly from multidrug resistance. P-glycoprotein, encoded by multidrug resistance protein-1 (MDR1) gene, at the blood brain barrier is considered as a major factor mediating drug efflux and contributing to resistance. Given that peripheral blood mononuclear cells (PBMNCs) express MDR1, we investigated a MDR1 status of PBMNCs in various subsets of epilepsy patients and demonstrated their association with clinical characteristics.

Methodology/principal findings: Clinical and MDR1 data were collected from 140 patients with epilepsy, 30 healthy volunteers, and 20 control patients taking anti-epileptic drugs. PBMNCs were isolated, and basal MDR1 levels and MDR1 conformational change levels were measured by flow cytometry. MDR1 profiles were analyzed according to various clinical parameters, including seizure frequency and number of medications used in epilepsy patients. Epilepsy patients had a higher basal MDR1 level than non-epilepsy groups (p<0.01). Among epilepsy patients, there is a tendency for higher seizure frequency group to have higher basal MDR1 level (p = 0.059). The MDR1 conformational change level was significantly higher in the high-medication-use group than the low-use group (p = 0.028). Basal MDR1 (OR = 1.16 [95% CI: 1.060-1.268]) and conformational change level (OR = 1.11 [95% CI: 1.02-1.20]) were independent predictors for seizure frequency and number of medications, respectively.

Conclusions/significance: The MDR1 profile of PBMNCs is associated with seizure frequency and medication conditions in patients with epilepsy.

Show MeSH
Related in: MedlinePlus