Potential of CD34 in the Regulation of Symmetrical and Asymmetrical Divisions by - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:56 |只看该作者 |倒序浏览 |打印

作者：Tabitha E. Bullock, Baiping Wen, Stephen B. Marley, Myrtle Y. Gordon作者单位：Department of Haematology, Imperial College Faculty of Medicine, Hammersmith Campus, London, United Kingdom









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      【摘要】9 _: D* ~1 g! k0 Q/ L7 t5 M
   The control of symmetric and asymmetric division in the hematopoietic stem/progenitor cell population is critically important for the regulation of blood cell production. Asymmetric divisions depend on cell polarization, which may be conferred by location and/or interaction with neighboring cells. In this study, we sought evidence for polarization in CD34  cells, which interact by binding to one another. In these cells, surface molecules became redistributed by mechanisms that included transport by lipid rafts, and the interacting cells were able to communicate via gap junctions. These changes were accompanied by modulation of cell cycle regulating proteins (p16Ink4a, p27kip1, cyclins D, and the retinoblastoma pathway proteins) and a reduction in progenitor cell proliferation in vitro. These results are consistent with an increase in asymmetric cell division kinetics. Accordingly, we found that interaction between CD34  cells influenced the plane of cell division in a way that suggests unequal sharing of Notch-1 between daughter cell progeny. We conclude that interaction between CD34  cells may coordinate cell function and participate in the control of hematopoietic stem/progenitor cell division kinetics.$ H' U- e2 l# b+ w. ~/ Z

Disclosure of potential conflicts of interest is found at the end of this article. * d: G  |7 s! Y
      【关键词】 Adhesion molecules Stem cell asymmetry Self-renewal Hematopoietic stem cells CD
   INTRODUCTION

Throughout life, human hematopoietic cell production is sustained by the proliferative activity of the stem cell pool. During development, stem cells originate in the aorta-gonad-mesonephros region and then migrate and multiply in order to populate the fetal liver and, ultimately, the adult bone marrow. In the normal steady-state, stem cell numbers are maintained at a constant level but may be required to increase, for example, after treatment with myelotoxic chemotherapy. It is thought that stem cell numbers are regulated by the balance between symmetric and asymmetric cell divisions . Asymmetric divisions are associated with homeostasis, whereas symmetrical divisions are required for stem cell recovery from depletion. This is because asymmetrical divisions do not allow for changes in stem cell numbers, each division producing one new stem cell and one cell destined for differentiation. In contrast, symmetrical divisions permit expansion in stem cell number by altering the proportions of stem cells that produce two new stem cells versus those that produce two differentiating cells. These considerations imply either that the probabilities of symmetric divisions producing stem cells and differentiated cells are flexible or that asymmetric and symmetric divisions coexist in variable proportions in hematopoiesis.

Asymmetric divisions are a property of polarized cells. Polarity may be conferred by location and interaction with surrounding cells, as has been demonstrated by the spatial relationship between stem cells and their niches in Drosophila and in mammalian hematopoiesis. Another mechanism that may confer asymmetric cell division is the unequal partitioning of cell fate determinants as a result of orientation of the mitotic spindle and the plane of cell division, which ensures the unequal distribution of cellular constituents between the daughter cells .
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Hematopoietic stem and progenitor cells are generally perceived as round, nonpolarized cells. However, CD34  cells aggregate together when exposed to the class II anti-CD34 antibody, QBEND10, but not antibodies belonging to class III . Gap junctions are specialized structures that form direct channels between the cytoplasms of adjacent cells. These hydrophilic pores allow ions and molecules of up to 1 kDa, including second messengers such as cyclic-AMP and Ca2 , to pass through, whereas proteins and nucleic acids cannot. By coupling the metabolic activities of adjacent cells, gap junctions facilitate coordinated responses by groups of cells to environmental stimuli.
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In this study, we sought evidence for the polarization of cell division in aggregated CD34  cells and CD34  cells found in doublets within developing myeloid colonies in vitro, together with the effect of this interaction on hematopoietic progenitor cell kinetics. Our results suggest that aggregation between CD34  cells may offer a potential setting for the formation of a complex multimolecular structure facilitating cell polarization and communication that may relate to the regulation of cell proliferation kinetics. We show that a multimolecular complex consisting of cell adhesion and signaling molecules forms at the contact point between aggregated CD34  cells and that the cells are highly polarized with regard to proteins that are inside and outside the complex. Also, we found that the association between CD34  cells resulted in orientation of the centrosomes, indicating mitotic spindle orientation, and evidence that intercellular communication occurred via gap junctions. Therefore, aggregated CD34  cells may be considered as a functional coordinated unit that may facilitate adaptive responses to environmental changes.7 y/ J$ w" D9 N1 E
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MATERIALS AND METHODS; c* C8 B* Q3 ^8 P+ Z
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Cells for Study1 A% k1 H0 Y  J# Q" f

Peripheral blood progenitor cells were obtained by leukapheresis following mobilization with granulocyte colony-stimulating factor from donors for transplantation. The samples used were in excess of clinical requirements and were provided with informed consent and Research Ethics Committee approval.

The samples were diluted in Hanks' balanced salt solution (HBSS; Gibco, Grand Island, NY, http://www.invitrogen.com) layered over Lymphoprep (Axis-Shield, Oslo, Norway, http://www.axis-shield.com) and centrifuged for 30 minutes at 1,800 rpm. Mononuclear cells were harvested from the interface and washed twice in HBSS. CD34  cells were isolated using MiniMACS technology according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com).
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Cell Aggregation
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CD34  cells were suspended at a concentration of 1 x 106/ml in alpha medium (Gibco) supplemented with 15% fetal calf serum (FCS). Aliquots of 100 µl were transferred to the wells of 96-well microtitre plates and then treated with 2 µl of the anti-CD34 monoclonal antibody QBEND10 (a kind gift from Dr. L. Healy) or isotype control antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) for 3 hours at 37¡ãC in 5% CO2 in air. Aliquots of 30 µl were then air-dried onto Teflon-coated multiwell slides (BDH, Lutterworth, U.K.), fixed in ethanol, stained with Romanowsky dyes, and scored for aggregation by light microscopy. At least 500 cells per well were scored to give the percentage of cells in aggregates versus the percentage of single cells. The score for cells exposed to the isotype control antibody was subtracted from this value to give the specific aggregation index. Myeloid colonies were grown in standard culture conditions, harvested, and examined for the presence of CD34  cell doublets .' {! t; O/ @# R1 o) B' c! _& u
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Immunostaining; r" E4 O' L2 z3 J0 m7 A; q
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After aggregation, cells were gently resuspended, and aliquots of 30 µl were then air-dried onto Teflon-coated multiwell slides. The cells were fixed in 3% paraformaldehyde for 10 minutes or acetone for 1 minute, rehydrated for 10 minutes in phosphate-buffered saline (PBS), and then blocked for 1 hour with 3% bovine serum albumin (PAA Laboratories, Linz, Austria, http://www.paa.at). The cells were covered with 100 µg/ml primary antibody or isotype control for 1 hour at room temperature in the dark. After four 5-minute washes in PBS, cells were incubated with 0.5% or 1% fluorochrome-conjugated secondary antibody. For dual staining experiments, the above steps were repeated for the second antibody. All slides were mounted in VECTASHIELD containing 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Immunostained cells were examined using a Zeiss Axiovert 100 fluorescent microscope with SmartCapture imaging software or a Zeiss Meta 512 confocal microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) using the argon laser to view fluorescein isothiocyanate (FITC).1 ~9 S1 }! d$ ?. Q' A

Western Blotting- f: t8 G! c6 o( X5 U

Cell lysates and Kaleidoscopic prestained markers (Bio-Rad, Hercules, CA, http://www.bio-rad.com) were electrophoresed on polyacrylamide gels at 100 V and transferred to 0.45-µm polyvinylidene difluoride membrane (Millipore, Billerica, MA, http://www.millipore.com) by semidry transfer (Bio-Rad). Membranes were blocked for 1 hour at room temperature in 5% milk (Marvel, Dublin, Ireland) made up with TBS containing 0.5% Tween (BDH). Primary antibody (Santa Cruz) was applied to the membranes at 1/500 milk/Tris-buffered saline (TBS)-Tween and left at 4¡ãC overnight. After four 5-minute washes in TBS-Tween, the relevant secondary horseradish peroxidase conjugate was diluted 1/1,000 in milk/TBS-Tween and applied to the membrane for 1 hour at room temperature with shaking. The membranes were then washed and visualized using an ECL chemiluminescent visualization kit (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com).

Lipid Rafts1 U" ~3 T- D# F+ N( y1 [
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Lipid rafts were isolated from aggregated and nonaggregated CD34  cells by sucrose density gradient ultracentrifugation. Cells were washed once, resuspended in 0.4 ml of ice-cold MBS-BL  (50 mM 2-(N-morpholino)ethanesulfonic acid, 150 mM NaCl, 1 mM phenymethyl sulfonylfluoride, 1 mM sodium vanadate, 1 µg/ml protease inhibitor cocktail, 1% Triton X-100 ), and kept on ice for 30 minutes. Cells were then lysed after the addition of 0.4 ml of 80% sucrose and dispensed into a 5-ml ultracentrifuge tube. The lysate was gently overlaid with 2.7 ml of 30% sucrose followed by 1.5 ml of 5% sucrose. The tubes were placed into an SW55Ti rotor and centrifuged for 24 hours at 4¡ãC. Fractions of 0.5 ml were than removed from the top of the gradient. The pellet was resuspended in 0.5 ml of 1x sample buffer, diluted to 1x with an equal volume of PBS, and boiled for 3 minutes. The fractions were analyzed by Western blotting.7 a1 J+ z& L9 s4 q

To stain lipid rafts, FITC-labeled cholera toxin-B (100 µl of 10 µg/ml; Sigma) was added to slides of aggregated and nonaggregated cells followed by incubation on ice for 30 minutes in the dark. After one wash, slides were mounted in VECTASHIELD.

To deplete cells of cholesterol, they were incubated with 10 mM methyl-ß-cyclodextrin (MBCD; Sigma) in RPMI for 15 minutes and washed three times in PBS. Treated cells were also incubated with cholesterol (Sigma) for 15 minutes at 37¡ãC to reconstitute cellular cholesterol and reverse the effects of MBCD treatment.
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Gap Junctions
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CD34  cells were split into two groups. One was stained with 500 nM calcein (Sigma) for 30 minutes at 37¡ãC and the other with 4 x 10¨C6 M PKH26 (Sigma). The cells were washed, mixed 50:50, and aggregated by exposure to QBEND10. Then, cells were mounted on slides in VECTASHIELD and analyzed using a fluorescent microscope to document dye transfer between cells. To inhibit gap junction communication, 0.2% carbenoxalone (Sigma) was added to the cells and incubated for 18 hours.

Small Interfering RNA-Mediated Knockdown of Lymphocyte Function-Associated Antigen-1

Cells were seeded at a concentration of 2 x 105/ml in 400 µl of X-vivo medium (BioWhittaker, Walkersville, MD, http://www.cambrex.com) in 12-well plates. For each well to be transfected, 7 µl of Opti-MEM (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) was mixed with 3 µl of Oligofectamine (Invitrogen) and left at room temperature for 10 minutes. In a separate stock tube, lymphocyte function-associated antigen-1 (LFA-1) small interfering RNA (siRNA) (Santa Cruz) was diluted by adding 3 µl of 10 µM stock solution to 87 µl of Opti-MEM for each well and mixed with the diluted Oligofectamine. After 20 minutes at room temperature, 100 µl of the mixture was added to each well and incubated overnight at 37¡ãC in 5% CO2 in air. The next day, an additional 100 µl of siRNA/Oligofectamine was added to each well. Four hours later, 60 µl of FCS was added to each well, and the plate was incubated at 37¡ãC in 5% CO2 in air. At the relevant time point, 30-ml aliquots of cells were removed and examined by immunofluorescence and Western blotting and tested in the aggregation assay.
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Colony Replating Assay
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Colony-forming units-granulocyte-macrophage were cultured in standard assays for 7 days, then 120 individual colonies per group were plated into wells in 96-well microtitre plates. After an additional 7 days, secondary colonies were scored and the proliferation index was calculated .
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Statistical Analysis

Results were analyzed using the Mann-Whitney U test or chi-squared test for statistical significance.' |! S& w8 G1 l8 Z8 U' \# a

RESULTS* H8 I7 l' U$ B
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Homotypic Aggregation Between CD34  Cells Involves a Multimolecular Adhesion Complex and Localization of Notch-1
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CD34 is diffusely distributed in nonaggregated cells but adopts a ring-like configuration at the intercellular binding site when the cells are induced to aggregate by exposure to QBEND10 monoclonal antibody (Fig. 1A¨C1C). In addition to CD34, CD44, LFA-1, and F actin, but not intercellular adhesion molecule (ICAM)-1, were found to be concentrated at the contact point between CD34  cells induced to aggregate by treatment with QBEND10 (Fig. 1D¨C1K). Although CD44 and LFA-1 were concentrated with CD34, antibodies to CD44 and LFA-1 did not cause CD34  cell aggregation (data not shown). In spite of the exclusion of ICAM-1, downregulation of LFA-1 using siRNA, which was confirmed by immunofluorescence and Western blotting (data not shown), reduced the ability of the cells to aggregate (Fig. 1L, 1M). CD44 also colocalizes with CD34 in antibody-treated cells (Fig. 2A¨C2C), whereas CD164 locates to the opposite pole and is excluded from the adhesion site (Fig. 2D, 2F). However, unlike CD34, CD44 and CD164 are polarized without exposure to QBEND10 (Fig. 2G, 2H). This result indicates that CD34  cell polarization may be both constitutive and induced. Interestingly, Notch-1 exhibits a patchy distribution in untreated cells (Fig. 2I) but was concentrated in the same cell hemisphere as CD34 in 67% of aggregated CD34  cells (Fig. 2J¨C2L).

Figure 1. Redistribution of surface proteins on QBEND10-treated CD34  cells. (A): Diffuse distribution of CD34 on untreated cells. (B): Polarization of CD34 between treated cells. (C): Confocal microscopy of the ring configuration of CD34 on treated cells. (D): Polarization of CD34 on treated cells. (E): Polarization of CD44 on treated cells. (F): Merge of (D) and (E). (G): Polarization of CD34 on treated cells. (H): Polarization of actin on treated cells. (I): Merge of (G) and (H). (J): Diffuse distribution of LFA-1 on untreated cells. (K): Polarization of LFA-1 on treated cells. (L): siRNA knockdown of LFA-1 evaluated by Western blotting. (M): Influence of siRNA knockdown of LFA-1 on aggregation induced by QBEND10. siRNA = LFA-1-specific siRNA; MM = mismatch siRNA control. * p
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Figure 2. Polarization and relative localizations of CD34, CD44, CD164, and Notch. (A): Polarization of CD34 on treated cells. (B): Polarization of CD44 on treated cells. (C): Merge of (A) and (B). (D): Polarization of CD34 on treated cells. (E): Polarization of CD164 on treated cells. (F): Merge of (D) and (E). (G): Polarization of CD44 on untreated cells. (H): Polarization of CD164 on untreated cells. (I): Diffuse distribution of Notch on untreated cells. (J): Polarization of CD34 on treated cells. (K): Polarization of Notch on treated cells. Notch-1 polarization was scored on treated cells; at least 100 cells were scored per experiment, n = 3. (L): Merge of (J) and (K).

Lipid Rafts Participate in Aggregation and Complex Formation Between CD34  Cells
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LFA-1 and CD44 are lipid-raft-associated proteins , reduced aggregation of CD34  cells incubated with QBEND10 (Fig. 3C). Incubation of MBCD-treated cells with cholesterol restored aggregation to control levels (p = NS). Sucrose density gradient ultracentrifugation was used to confirm the presence of lipid rafts in aggregated and nonaggregated CD34  cells. Following lysis with Brij 35, lyn (a lipid raft marker), CD34, LFA-1, CD44, and CD164 were found within the lipid raft fraction (Fig. 3D). Thus, lipid-raft-mediated transfer may be involved in the relocalization of molecules that are excluded from, as well as those that are included in, the intercellular complex., S* ^3 @6 m3 J. m, Z2 I9 ]' b: l6 S

Figure 3. Role of lipid rafts in the polarization of QBEND10-treated CD34  cells. Distribution of lipid rafts in (A) untreated cells and (B) treated cells. (C): Influence of methyl-ß-cyclodextrin (MBCD) on QBEND10-induced aggregation. Black columns represent cells pretreated with 10 mM MBCD; white columns are untreated cells. The time following addition of QBEND10 is on the x-axis, * p + ^1 f* V8 u6 h4 {2 G& l
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Evidence for Communication via Gap Junctions Between Aggregated CD34  Cells7 M* L) W" C/ K" h  G9 \
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Since connexin-43 has been found in lipid rafts , we next sought evidence for gap junction-mediated communication between aggregated CD34  cells. Figure 4A shows that connexin-43 is detectable in CD34  cells by Western blotting. CD34  cells were stained with the membrane dye PKH26 (red fluorescence) or calcein, a green fluorescent dye that is cleaved to a membrane-impermeable form by cellular esterases and can only pass between cells through gap junctions (Fig. 4B, 4C). PKH26- and calcein-stained cells were mixed 50:50 and treated with QBEND10 to induce aggregation. Gap junction-mediated dye transfer, detected by the appearance of orange/yellow cells in the mixture (Fig. 4D), was expressed as the percentage of the number of potential calcein recipients (i.e., the number of PKH26-stained cells that were present in the aggregates) that turned orange/yellow. Figure 4E shows that 40% of the possible recipients received calcein via gap junctions. Figure 4E also shows that carbenoxalone, an inhibitor of gap junction transport, reduced the transfer of calcein to 10% of the control levels. Carbenoxolone was not toxic to the cells, as assessed by trypan blue dye exclusion, and did not influence the ability of the cells to aggregate when they were treated with QBEND10 (data not shown).
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Figure 4. Evidence for gap junction-mediated communication between aggregated CD34  cells. (A): Detection of connexin-43 in CD34  cells by Western blotting. (B): Calcein-stained cell. (C): PKH26-stained cell. (D): PKH26-stained recipient of calcein via gap junction-mediated transfer. (E): Effect of carbenoxalone on gap junction-mediated transfer; white column = untreated control, black column = carbenoxalone-treated cells. At least 500 cells were scored per experiment, n = 5, * p
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QBEND10-Induced CD34  Cell Aggregation Modulates the Cell Cycle Machinery

The cell cycle machinery is central to the regulation of cell proliferation kinetics . Therefore, we tested the effect of QBEND10 pretreatment on the ability of clonogenic CD34  cells to proliferate in culture. As may have been anticipated, the treatment reduced proliferation significantly (Fig. 5B). Antibodies to class I and class III CD34 epitopes had no effect on proliferation (data not shown), indicating that the reduction was due to aggregation itself rather than engagement of CD34 alone.7 Z, \; N2 P. q/ X

Figure 5. Influence of QBEND10-mediated polarization and aggregation on cell cycle proteins and progenitor proliferation. (A): Western blotting analysis. The x-axis shows the number of hours treated with QBEND10 prior to cell lysis. (B): Progenitor proliferation index. The black bar represents untreated cells and the white bar represents cells exposed to QBEND10 for 3 hours before culture, n = 5. Abbreviation: pRb, retinoblastoma protein.
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Orientation of Cell Division
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A reduction in proliferation (i.e., output of secondary colony-forming cells) could be due to increased asymmetric cell divisions . Accordingly, we investigated whether aggregation between CD34  cells might influence the plane of cell division. We used pericentrin to stain the centrosomes in interphase cells and scored their positions relative to the binding site between aggregated CD34  cells. The centrosomes were nonrandomly distributed, since in 89.6% (199/122) of the cells they were in the cell hemisphere opposite the contact point. The distributions of the centrosomes and CD164 were similar, and comparing their localizations revealed close spatial association between them in 83.1% of the cells (Fig. 6A¨C6C). Similarly, when we examined cell doublets harvested from 6-day-old cultured myeloid colonies, 89.2% (173/194) of single centrosomes were located in the hemisphere opposite the contact point. Moreover, a close association between CD164 and the centrosome was seen in 95.9% of the 98 CD34  cells harvested from myeloid colonies and scored.

Figure 6. Proposed influence of polarization on the symmetry/asymmetry of cell division by CD34  cells. (A): Polarization of CD164 in treated cells. (B): Localization of the centrosome in treated cells. (C): Merge of (A) and (B). (D): Model relating distribution of CD34 and Notch to the plane of cell division in CD34  cells. (E): Scheme used for scoring potential planes of division, defined by the positions of CD34 and the centrosome. We examined 149 doublets in total, 16 of which contained one cell with two centrosomes, n = 3. The chi-squared test confirmed a significant difference in the frequency of each plane of division cell from the expected values, p
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The position of the centrosome in nondividing cells can indicate incipient mitotic spindle orientation, since the interphase centrosome divides, and the resultant two centrosomes may occupy opposite poles of the cell* S8 ?! Q! @* w9 y' G1 Q6 q

DISCUSSION& {8 s/ Z) a2 A2 f, q: x

Asymmetric and symmetric divisions are required for the steady-state maintenance and expansion of hematopoiesis, respectively, and the balance between the two must be tightly controlled. Although the mechanisms of this control are not well understood, certain common themes are emerging from studies in Drosophila and other systems. These themes include roles for cell interaction, communication, and polarization .1 e2 J' g! b' \( n) K# T6 d* Z- t/ M

In aggregated CD34  cells, the CD34 adopted a ring-like configuration in a manner that is reminiscent of the supramolecular activation cluster (SMAC) seen in the formation of the "immunological synapse" between T cells and antigen-presenting cells
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Notch is a key regulator of cell fate, and Notch-1 is expressed by hematopoietic stem cells , the observation that it is relocated when CD34  cells are induced to aggregate is persuasive evidence that it may be involved in CD34  cell regulation.
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The mechanism underlying the dynamic reorganization of molecules in the cell membrane to result in the configuration seen in aggregated cells is likely to be part of the overall regulatory system. Lipid raft-mediated transport is one candidate for this, since it has been shown to participate in the relocalization of molecules in polarized T cells . Accordingly, an accumulation of lipid rafts stained with cholera toxin was seen at the contact point between aggregated CD34  cells. Unexpectedly, CD164, which is excluded from the CD34 contact zone, as well as CD34, LFA-1, and CD44 were found in the lipid raft fraction, demonstrating that rafts are heterogeneous and may be responsible for transporting particular molecules to specific locations on the cell surface. The importance of lipid rafts for aggregation of CD34  cells was demonstrated by treating the cells with methyl-ß-cyclodextrin to extract cholesterol and disrupt lipid raft integrity.0 |8 y$ X* S! B4 m% a, C

The close association between aggregated CD34  cells provides suitable conditions for intercellular communication, which is known to be important for coordinating cellular responses in many contexts. In particular, cell-cell contacts may determine the symmetry/asymmetry of cell division . The dye transfer studies shown in Figure 4 demonstrate clearly that gap junction communication occurs between aggregated CD34  cells and the experiment using carbenoxalone shows that it can be regulated./ ~9 w) v; ]; I1 k2 Q# I- x$ `

Activated Notch modulates stem and progenitor cell renewal, which is associated with upregulation of the cyclin-dependent kinase inhibitors p27kip1 and p21cip1 , these aggregation-mediated effects on the cell cycle machinery were associated with a reduction in the proliferative index of progenitor cells after aggregation.
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The results so far do not provide any information about symmetry versus asymmetry of cell division. This is because symmetry/asymmetry depends on the plane of cell division, which is determined by the orientation of the mitotic spindle. Centrosomes are mitotic spindle organizers, and their position can be used to predict mitotic spindle orientation , thereby allowing a range of symmetrical and asymmetrical outcomes of CD34  cell division. Importantly, the same arrangement was observed in the aggregated CD34  cell model and the in vitro colony model, thereby showing that the results are not an artifact resulting from exposure to QBEND10.
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We acknowledge that the results reported here are related to hematopoietic progenitor cells and may not apply to the hematopoietic stem cell population. Stem cells, including hematopoietic stem cells, are frequently considered in the context of the stem cell niche. In this situation they may, like Drosophila germline stem cells, be restricted to asymmetric cell division to preserve the stem cell pool .8 h( h; m$ j0 ~8 p) C/ ?0 A8 @( g
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST2 m% Q, R) y1 T+ J# P- l) }( R
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The authors indicate no potential conflicts of interest.0 }% T! D( ?3 m0 \+ D
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