Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0507296 on August 3, 2007

Published online before print August 3, 2007
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(Journal of Leukocyte Biology. 2007;82:1083-1094.)
© 2007 by Society for Leukocyte Biology

Neuroprotective activities of CD4+CD25+ regulatory T cells in an animal model of Parkinson’s disease

Ashley D. Reynolds*,{dagger}, Rebecca Banerjee*,{dagger}, Jianou Liu*,{dagger}, Howard E. Gendelman*,{dagger},{ddagger},1 and R. Lee Mosley*,{dagger}

* Center for Neurovirology and Neurodegenerative Disorders, Departments of
{dagger} Pharmacology and Experimental Neuroscience and
{ddagger} Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA

1 Correspondence: Center for Neurovirology and Neurodegenerative Disorders, University of Nebraska Medical Center, 985880 Nebraska Medical Center, Omaha, NE 68198-5880, USA. E-mail: hegendel{at}unmc.edu

ABSTRACT

Progressive loss of dopaminergic neurons in the substantia nigra pars compacta and their terminal connections in the striatum are central features in Parkinson’s disease (PD). Emerging evidence supports the notion that microglia neuroinflammatory responses speed neurodegenerative events. We demonstrated previously that this can be slowed by adoptive transfer of T cells from Copolymer-1-immunized mice administered to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) recipients. The cellular basis for this neuroprotective response was the CD4+ T cell population, suggesting involvement of CD4+CD25+ regulatory T cells (Tregs), cells known to suppress immune activation and maintain immune homeostasis and tolerance. We show for the first time that adoptive transfer of CD3-activated Tregs to MPTP-intoxicated mice provides greater than 90% protection of the nigrostriatal system. The response was dose-dependent and paralleled modulation of microglial responses and up-regulation of glial cell-derived neurotrophic factor (CDNF) and TGF-β. Interestingly, that adoptive transfer of effector T cells showed no significant neuroprotective activities. Tregs were found to mediate neuroprotection through suppression of microglial responses to stimuli, including aggregated, nitrated {alpha}-synuclein. Moreover, Treg-mediated suppression was also operative following removal of Tregs from culture prior to stimulation. This neuroprotection was achieved through modulation of microglial oxidative stress and inflammation. As Tregs can be modulated in vivo, these data strongly support the use of such immunomodulatory strategies to treat PD.

Key Words: neurodegeneration • neuroprotection • inflammation • microglia

INTRODUCTION

Parkinson’s disease (PD), second in incidence to Alzheimer’s disease, is a progressive neurodegenerative disorder characterized by the loss of substantia nigra pars compacta (SNpc) dopaminergic neurons and their projections to the caudate-putamen [1 2 3 ]. To date, only symptomatic therapies are available [3 ]. Although disease etiology is incompletely understood, host genetics, environment, age, and inflammatory processes are factors affecting disease onset and progression [4 5 6 7 ]. A large body of evidence links inflammation, defined gliosis, mitochondrial dysfunction, oxidative stress and free radical formation, and altered neurotrophic support as being responsible, in part, for disease-associated nigrostriatal degeneration [8 9 10 ]. The primary mediator of such neuroinflammatory responses is the activated microglia, a cell found in and around degenerating, dopaminergic neurons [11 , 12 ]. A plethora of microglial toxic secretory products are secreted or up-regulated in disease, including reactive oxygen species (ROS), TNF-{alpha}, IFN-{alpha}/β, IL-1β, IL-6, inducible NO synthase (iNOS), and leukotrienes [13 14 15 ]. How microglia are activated and affect disease is not completely known but is thought to be mediated by the release of aggregated and nitrated {alpha}-synuclein (N-{alpha}-syn) during degeneration of dopaminergic neurons in the SNpc, resulting in an inflammatory cytotoxic cascade [16 ] (Ashley D. Reynolds, Jason G. Glanzer, Irena Kadiu, Mary Ricardo-Dukelow, Anathbandhu Chaudhuri, Sanjay K. Garg, Ruma Banerjee, Pawel Ciborowski, Ronald Cerny, Benjamin Gelman, R. Lee Mosley, Mark P. Thomas, Howard E. Gendelman, in revision). Left uncontrolled, this inflammatory cascade affects the tempo of disease.

Previously, immune cells from mice immunized with Copolymer-1 (Cop-1) were demonstrated to attenuate microglial responses and lead to protection against dopaminergic neuronal loss in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD [17 ]. Depletion of T cells abrogated this protection. In recent works, the T cell-dependent response was validated, and CD4+ T cells were found responsible for this neuroprotection [18 ]. The capacity of CD4+ T cells to attenuate microglial responses and protect from MPTP-induced neurodegeneration suggested the possible involvement of T regulatory cells (Tregs).

Based on these observations, we hypothesized that induction of CD4+CD25+ Tregs can serve to modulate immune responses in the brain, perhaps through interactions with the host immune system within and outside the nervous system, resulting in significant neuroprotection in PD model systems. To investigate this possibility, Tregs and naïve CD4+CD25– T cells were isolated by magnetic sorting and activated in vitro with anti-CD3 and IL-2. Activated Tregs and effector T cells (Teffs) were adoptively transferred to MPTP-intoxicated recipients, and neuroinflammation and neuronal survival were evaluated. As few as 3.5 x 106 Tregs were sufficient to reduce the numbers of CD11b immunoreactive microglia and protect dopaminergic neuronal bodies in the SNpc and their striatal termini. These results support the use of therapeutic strategies, which induce Treg responses to attenuate neuroinflammation and inhibit dopaminergic neurodegeneration associated with PD.

MATERIALS AND METHODS

Animals and MPTP intoxication
Male SJL mice (7–8 weeks old, The Jackson Laboratory, Bar Harbor, ME, USA) received four i.p. injections at 2 h intervals of vehicle (PBS, 10 mL/kg bodyweight) or MPTP-HCl (12 or 18 mg MPTP/kg bodyweight of free base in PBS, Sigma-Aldrich, St. Louis, MO, USA). Twelve hours after the last MPTP injection, randomly selected, MPTP-intoxicated mice received adoptive transfers of Tregs or Teffs or no cells (n=5–7 mice per group per time-point). On Days 2 and 7 post-MPTP intoxication, mice were sacrificed, and brains were processed for analyses as described previously [17 ]. All animal procedures were in accordance with National Institutes of Health (Bethesda, MD, USA) guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center (UNMC; Omaha, NE, USA). MPTP handling and safety measures were in accordance with published guidelines [19 ].

Isolation and adoptive transfer of murine CD4+CD25+ and CD4+CD25– T cells
Single cell suspensions were prepared from lymph nodes (axillary, brachial, inguinal, superficial cervical, and mandibular) and spleens. T cells were enriched by negative selection on T cell columns (R&D Systems, Minneapolis, MN, USA). The enriched CD3+ T cells were then incubated with anti-CD8 microbeads (Miltenyi Biotec, Auburn, CA, USA) and subjected to magnetic separation with AutoMACS (Miltenyi Biotec). Nonadherent cells contained enriched CD4+ T cells, which were incubated with PE-labeled anti-CD25 antibody (BD PharMingen, San Diego, CA, USA) and anti-PE microbeads (Miltenyi Biotec). The negatively selected fraction contained enriched CD4+CD25– T cells. The positively selected fraction was subjected to another pass of positive selection to enrich CD4+CD25+ T cells further, and the negatively selected fraction was then further depleted of residual CD25+ cells by negative selection through a deplete program. Samples from cell fractions were labeled with fluorescently labeled antibodies CD3, CD19, CD4, CD8, and CD25 (eBiosciences, San Diego, CA, USA) and analyzed by flow cytometric analysis with a FACSCalibur (BD Biosciences, San Jose, CA, USA). T cells were plated at a density of 1 x 106 cells/ml stimulation media [RPMI medium 1640 (Gibco, Carlsbad, CA, USA), supplemented with 10% FBS, 2 mM L-glutamine, 25 mM HEPES, 1 mM sodium pyruvate, 1x nonessential amino acids, 55 µM 2-ME, 100 units/ml penicillin, 100 µg/ml streptomycin (Mediatech, Herndon, VA, USA)] for 4 days in the presence of mouse recombinant (r)IL-2 (100 U/ml; R&D Systems) and 0.5 µg/ml anti-CD3 (145-2C11, BD PharMingen). Enriched CD4+CD25+ T cells were tested for their capacity to inhibit anti-CD3-induced, proliferative responses. For the assay, CD4+CD25– T cells were cultured for 72 h at 5 x 104 per well with irradiated splenocytes in the presence of 0.5 µg/ml anti-CD3, and numbers of Tregs were serially diluted twofold. Cultures were pulsed with 1 µCi [3H]-methylated thymidine [MP Biomedicals (ICN), Solon, OH, USA], and incorporation was measured 18 h later by β-scintillation spectrometry (Top Count, Packard Instrument Co., Meriden, CT, USA). For adoptive transfers, cells were harvested 4 days after stimulation and resuspended in HBSS. MPTP-intoxicated mice received an i.v. tail injection of 0.5–3.5 x 106 Tregs or Teffs in 0.25 ml HBSS. Samples from cell fractions were labeled with fluorescently labeled antibodies to CD3, CD19, CD4, CD8, and CD25 and analyzed by flow cytometry. A sample of each was also permeabilized and assessed for the transcription factor forkhead box p3 (Foxp3), according to the manufacturer’s protocol. All fluorescently labeled antibodies were purchased from eBiosciences.

RNA isolation and RT-PCR assays
Ventral midbrains were dissected from mice killed by CO2 asphyxiation 24 h and 6 days following adoptive transfers. The samples were snap-frozen on dry ice and stored at -80°C. RNA was prepared from the samples using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) and purified with the RNeasy mini kit (Qiagen Sciences, Valencia, CA, USA) prior to cDNA synthesis. Samples were prepared for RT-PCR and real-time RT-PCR using SYBR Green master mix (Applied Biosystems, Foster City, CA, USA). The PCR reaction for Foxp3 was carried out at 50°C for 32 cycles, for TGF-β at 56°C for 36 cycles, for IL-10 at 50°C for 36 cycles, for IFN-{gamma} at 55°C for 36 cycles, and for β-actin at 45°C for 36 cycles. Signals were analyzed using GelExpert software on Nucleovision (NucleoTech, San Mateo, CA, USA). The real-time reaction was carried out using the ABI Prism program-dissociation protocol. Murine-specific primer pairs: Foxp3, 5'-CATTTGCCAGCAGTGGGTAG-3' and 5'-CAGCTGCCTACAGTGCCCCTAG-3'; Il-10, 5'-CAGTTATTGTCTTCCCGGCTGTA-3' and 5'-CTATGCTGCCTGCTCTTACTGACT-3'; Itgam [membrane-activated complex 1 (Mac-1)], 5'-GCCAATGCAACAGGTGCATAT-3' and 5'-CACACATCGGTGGCTGGTAG-3'; brain-derived neurotrophic factor (Bdnf), 5'-GGAATTCGAGTGATGACCATCCTTTTCCTTAC-3' and 5'-CGGATCCCTATCTTCCCCTTTTAATGGTCAGTG-3'; glial cell line-derived neurotrophic factor (Gdnf), 5'- GAGAGGAATCGGCAGGCTGCAGCTG-3' and 5'-CAGATACATCCACATCGTTTAGCGG-3'; Nos2 (iNOS), 5'-GGCAGCCTGTGAGACCTTTG-3' and 5'-GAAGCGTTTCGGGATCTGAA-3'; glial fibrillary acidic protein (Gfap), 5'-CCCCAGCTGGTTAGAATTGG-3' and 5'-TGGCATTCGTGATGCATAGG-3'; TGF-β (Tgfb), 5'-CT{Gamma}CT{Gamma}CTTTCTCCCTCAAC-3' and 5'-GACTGGCGAGCCTTAGTTTG-3'; IFN-{gamma} (Ifng), 5'-TTTGAGGTCAACAACAACCCACA-3' and 5'-CGCAATCACAGTCTTGGCTA-3'; β-actin (Actb), 5'-TGGAATCCTGTGGCATCCATGAAAC-3' and 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'.

A SYBR Green I detection system was used, and the reactions generated a melting temperature dissociation curve enabling quantification of the PCR products. TNF-{alpha} and GAPDH expression was analyzed using TaqMan gene expression assays. All PCR reagents were obtained from Applied Biosystems. Gene expression was normalized to GAPDH or β-actin and used as an endogenous control.

Immunohistochemistry
For analyses, terminally anesthetized mice were transcardially perfused with saline followed by 4% paraformaldehyde (PFA), their brains removed and fixed in 4% PFA for 24 h, followed by 30% sucrose in PBS for 48 h, flash-frozen, and mounted in OCT medium (Fisher Scientific, Waltham, MA, USA). To assess reactive microglia 48 h after MPTP intoxication, midbrain sections (30 µm), from five to seven mice per treatment group (12 sections per animal), were immunostained for Mac-1 (CD11b, 1:500, Serotec, Raleigh, NC, USA). Cell counts were obtained from ameboid Mac-1+ cells within the SN, and cells per mm2 were calculated. Numbers of Mac-1-positive cells were averaged for each animal, and the mean number of cells per mm2 per animal was estimated. Dopaminergic neuron survival was assessed 7 days following MPTP intoxication. The brains were cryosectioned and immunostained as free-floating tissues for expression of tyrosine hydroxylase (TH; 1:1000, Calbiochem, San Diego, CA, USA) and Nissl substance by thionin staining [20 ], as described previously [17 ]. Total numbers of TH- and Nissl-stained neurons in the SNpc were estimated in a blinded manner by unbiased, stereological analysis with StereoInvestigator software (MicroBrightfield, Williston, VT, USA) using the optical fractionator module [21 ]. Quantitation of striatal TH immunostaining (1:500, Calbiochem) was performed by densitometric analysis as described [20 ]. Measurements of striatal TH density, as a reflection of the dopaminergic innervation, were obtained by digital image analysis (Scion, Frederick, MD, USA).

Confocal microscopy
Midbrain sections (30 µm) from mice of each treatment group were blocked and permeabilized in 10% normal goat serum containing 0.05% saponin in TBS for 1 h and then immunostained with chicken anti-human BDNF or anti-GDNF (1:50, Promega, Madison, WI, USA) overnight at 4°C. Additional primary antibodies used for immunofluorescence staining were anti-TH (1:200, Calbiochem), anti-Mac-1 (1:200, Serotec), and anti-CD4 (1:200, BD PharMingen). Appropriate Alexa Fluor-conjugated secondary antibodies (1:200, Molecular Probes, Carlsbad, CA, USA) were used for fluorescence detection. Images were acquired with a Nikon swept field confocal microscope (Nikon Instruments Inc., Melville, NY, USA).

Microglial isolation and culture
Microglia were prepared from C57BL/6 neonatal mice (1–2 days old, Jackson Laboratory) using techniques described previously [22 ]. Brains were removed and placed in HBSS at 4°C. The tissue was dissociated using a 10-ml plastic pipette and incubated in 0.25% trypsin at 37°C for 30 min. After adding cold, heat-inactivated FBS, the tissue was washed several times with cold HBSS. The tissue was then triturated by pipetting through a series of sterile Pasteur pipettes with reduced bores and filtered through a 40-µm filter. These mixed glial cells were cultured in complete medium containing 2 µg/ml M-CSF (a generous gift from Wyeth Pharmaceuticals, Cambridge, MA, USA). To obtain highly purified microglia, beginning 7 days after harvest, the culture flasks were gently shaken, and the supernatants containing floating microglia were transferred to new flasks, which were incubated for 30 min to allow the microglia to adhere, and loose cells were removed by washing with DMEM. These microglia cells were cultured in complete medium containing M-CSF for 7–14 days and then replated for experiments. Greater than 98% purity was obtained consistently using the methods described above, as determined by CD11b staining.

Determination of cytoxicity and oxidative stress
Cells from the MES23.5 dopaminergic cell line, kindly provided by Dr. Stanley Appel, (Weill Medical College of Cornell University, Houston, Texas, USA) were cultured in 75 cm2 flasks in DMEM/F12 with 15 mM HEPES (Invitrogen Corp.) containing N2 supplement (Invitrogen Corp.), 100 U/ml penicillin, 100 µg/ml streptomycin, and 5% FBS. Cells were grown to 80% confluence and then cocultured in serum-free DMEM/F12 at a density of 1 x 105 cells (1:1 with microglia) on sterile glass coverslips. Assays for viable and dead mammalian cells (Live/Dead Viability/Cytotoxicity Kit, Invitrogen Corp.) were performed, according to the manufacturer’s protocol. Photomicrographs were taken using fluorescence microscopy at 200x magnification. Images were blinded, and a separate investigator performed quantification of live and dead cells. Cell counts were normalized as the percentage of surviving cells from unstimulated culture controls.

Hydrogen peroxide (H2O2) production by microglia, stimulated with N-{alpha}-syn, rTNF-{alpha} (R&D Systems), or LPS (Sigma-Aldrich), plated at 1 x 105 per well in a 96-well fluorometer plate, was measured following removal of media and replacement with Kreb’s ringer buffer (Sigma-Aldrich) containing 0.1 U/ml HRP and 50 µM Amplex red. PMA was added to select wells at 10 µM final concentration. Fluorescence intensity was measured at 563 nm (excitation)/587 nm (emission). Determination of NO was performed using the Greiss reaction according to the manufacturer’s protocol (Invitrogen Corp).

Statistical analyses
All values were expressed as mean ± SEM. Differences among means were analyzed by one-way ANOVA followed by Bonferroni post-hoc testing for pair-wise comparison (SPSS, Inc., Chicago, IL, USA). All effects of treatment were tested at the 95% confidence level. Linear regression analysis was achieved with the Prism software package (GraphPad Software, Inc., San Diego, CA, USA). Differences between paired means for RT-PCR were analyzed with Student’s t-test.

RESULTS

Isolation, activation, and characterization of Tregs and Teffs
Flow cytometric analysis demonstrated that nonactivated isolates of CD4+CD25+ and CD4+CD25– T cells were greater than 96% enriched for those subsets (Fig. 1A ). After activation with anti-CD3, the phenotype of Tregs was unchanged; however, CD25 was up-regulated by greater than 96% of the Teffs. Expression of forkhead/winged-helix transcription factor (Foxp3) distinguishes the Tregs from other effector cell populations and is required for Treg development and function [23 ]. Indeed, Tregs expressed higher levels of Foxp3 transcription factor compared with Teffs. RT-PCR analysis confirmed that Tregs showed increased expression of Foxp3 (34-fold) when compared with Teffs, as well as 1.9- and 3.4-fold increases in levels of TGF-β and IL-10 mRNA, respectively. In contrast, IFN-{gamma} mRNA expression was increased by 3.4-fold in Teffs compared with Tregs (Fig. 1B) . To determine the potential of Tregs to inhibit T cell proliferation, 5 x 104 naïve CD4+CD25– T cells were stimulated with anti-CD3 and cultured with graded amounts of Tregs, which inhibited anti-CD3-induced mitogenesis in a dose-dependent manner, providing maximal suppression (62%) at a 1:1 Treg:Teff ratio (Fig. 1C) . Stimulation of CD4+CD25– T cells induced a robust, proliferative response (43,000±5000 cpm), and activated Tregs alone yielded a response less than 3000 cpm (data not shown).


Figure 1
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  		Figure 1. The molecular properties of CD4+CD25+ Tregs. Tregs and Teffs were purified from lymphocytes of naïve SJL mice and analyzed before and following polyclonal activation. (A) Cells were analyzed for surface expression of the indicated cellular markers in the gated T cell populations by flow cytometry before and after polyclonal activation. (B) cDNA from polyclonal-activated Tregs and Teffs was analyzed by RT-PCR for Foxp3, TGF-β, IL-10, and IFN-{gamma} expression. Data are represented as the relative mRNA expression in reference to β-actin. *, P < 0.05 vs. Teff. (C) Enriched isolates of Tregs were assayed for inhibitory function on the proliferation of Teff responders in the presence of anti-CD3 (0.5 µg/ml) and APC. Mean ± SEM percentage of inhibition for n = 4 wells at each indicated ratio of Treg to a constant number of responding Teffs.

 
Tregs modulate microglial inflammation in MPTP-intoxicated mice
To assess the effects of Tregs and Teffs on MPTP-induced neuroinflammation, 3–5 x 106 anti-CD3-activated Tregs or Teffs were adoptively transferred to MPTP-intoxicated recipients (18 mg MPTP/kg bodyweight) 12 h after the last MPTP injection. MPTP intoxication occurs rapidly with metabolism to the active toxin 1-methyl-4-phenylpyridinium (MPP+) completed within minutes [24 ] and is undetectable after 8 h from the last injection [25 ]. Therefore, T cells can be adoptively transferred without affecting MPTP metabolism and its induced hematopoietic toxicity [26 ]. Throughout these studies, MPTP- and PBS-injected mice, which did not receive T cells, served as intoxicated and nonintoxicated controls, respectively. As the active phase of neuronal death and neuroinflammatory activities peak at ~2 days after the last MPTP injection [21 , 27 ], we analyzed microglial reactions in the ventral midbrain at this time-point (Fig. 2A ). Assessment of Mac-1 (CD11b) antigen expression by immunohistochemistry revealed microglial cells with thin ramifications in PBS controls. In contrast, MPTP- and MPTP/Teff-injected mice exhibited dense Mac-1 immunoreactivity, characterized by larger microglial cells with thicker, retracted ramifications. In MPTP/Treg-injected mice, Mac-1 staining revealed smaller microglia with thinner ramifications in contrast to those seen in MPTP- and MPTP/Teff-injected controls. Enumeration within the SN of Mac-1+ microglia with an activated phenotype showed a significant reduction in the MPTP/Treg group compared with MPTP and MPTP/Teff-injected groups (Fig. 2B) . MPTP intoxication also resulted in a significant increase in Mac-1 mRNA (Fig. 2C) . In contrast, MPTP/Treg-injected mice expressed less Mac-1 mRNA compared with MPTP- and MPTP/Teff-injected animals. Of the microglial secretory factors known to influence secondary degeneration, TNF-{alpha} and iNOS are implicated in stimulating the dopaminergic cell loss observed following MPTP intoxication [21 ]. Expression of TNF-{alpha} and iNOS mRNA was reduced significantly in midbrains of MPTP/Treg-injected mice compared with MPTP- and MPTP/Teff-injected mice. These data indicate that Tregs, but not Teffs, are capable of attenuating MPTP-induced microglial activation to a neurotoxic, inflammatory phenotype.


Figure 2
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  		Figure 2. Tregs attenuate MPTP-induced microglial activation. (A) Photomicrographs of Mac-1 immunostaining within the SN (original scale bars, 25 µm; insets at x1000 original magnification) from PBS, MPTP, MPTP/Treg, and MPTP/Teff groups. (B) Counts of Mac-1+ microglia within the SN. (C) Quantitative RT-PCR results are shown for Mac-1, TNF-{alpha}, iNOS, Foxp3, IL-10, and TGF-β expression from ventral midbrains on Day 2 after MPTP intoxication and normalized to expression of GAPDH mRNA. Values are means ± SEM for four to six mice per group, and P < 0.05 compared with aPBS; bMPTP; or cMPTP/Teff groups.

 
As activated Tregs and Teffs are indistinguishable by cell surface phenotype, Foxp3 expression was assessed as an unbiased, preliminary marker of Tregs entry into the SN (Fig. 2C) . MPTP-intoxicated mice, which received Tregs, had significantly higher expression of Foxp3 in the ventral midbrain by 2 days post-MPTP relative to other groups. In addition, significantly higher levels of IL-10 and TGF-β mRNA expression in MPTP/Treg-injected mice were observed compared with any other MPTP-intoxicated group. In addition, analysis of IFN-{gamma} revealed an increase in mRNA expression in ventral midbrains of MPTP/Teff-injected mice over nonintoxicated mice and mice that received MPTP alone and a 23-fold increase relative to MPTP/Treg-injected mice (data not shown). These data suggest that activated Tregs and Teffs were present in the midbrain of recipient, MPTP-intoxicated mice upon analysis.

Adoptive transfer of Tregs to MPTP-intoxicated mice is neuroprotective
To assess a potential, neuroprotective role for Tregs in the MPTP model of PD, 3.5–5 x 106 in vitro-activated Tregs and Teffs were adoptively transferred to recipient, MPTP-intoxicated mice. Seven days after MPTP intoxication, when no further dopaminergic degeneration is detected [28 ], mice were killed, and sections of the SN were immunostained for TH immunoreactivity. Adoptive transfer of Tregs significantly ameliorated the loss of TH-immunoreactive, dopaminergic neuronal bodies (TH+Nissl+) in the SNpc compared with MPTP-intoxicated mice and MPTP/Teff-injected mice (Fig. 3A ). In three replicate experiments, greater than 90% of the dopaminergic neurons were spared with as few as 3.5 x 106 Tregs (Fig. 3B) . The Teff subset had minimal and insignificant, neuroprotective effects compared with MPTP-intoxicated animals. Moreover, no significant effects on numbers of nondopaminergic neurons (TH–Nissl+) were detected by MPTP intoxication or adoptive transfer of Tregs or Teffs, indicating that the effects of MPTP and neuroprotection were specific to dopaminergic neurons and not associated with loss of TH from viable, neuronal bodies.


Figure 3
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  		Figure 3. Neuroprotection of dopaminergic neuronal bodies in the SN is mediated by Tregs. Unbiased, stereological analysis of TH+Nissl+ and TH–Nissl+ neurons in the SN from mice treated with PBS, MPTP, MPTP/Treg, or MPTP/Teff. (A) Representative results from three replicate experiments transferring 3–5 x 106 Tregs or Teffs to MPTP-intoxicated recipients. Mean ± SEM of five to seven mice per group, and P < 0.001 compared with aPBS, bMPTP, cMPTP/Teff. (B) Combined results of three replicate experiments normalized to percent of surviving neurons. Mean ± SEM of pooled data normalized to PBS controls as percent surviving neurons for 18–20 mice per treatment group. P < 0.0001 compared with aPBS, bMPTP, cMPTP/Teff.

 
Neuroprotection afforded by the transfer of Tregs correlated with the amount of activated T cells transferred. Intoxication with a relatively high concentration of MPTP (18 mg MPTP/kg bodyweight) results in an insurmountable lesion, which may mask potential, neuroprotective effects within the SN as well as the striata; therefore, the concentration of MPTP was titrated and reduced to 12 mg MPTP/kg bodyweight, thereby using a less-acute dose to produce loss of nigral neuronal bodies and striatal termini, which still recapitulate the Parkinsonian lesion (Fig. 4 ). Following MPTP intoxication, increasing numbers of 0.5–3.5 x 106 of Tregs or Teffs were adoptively transferred. Immunohistochemical analysis revealed remarkable losses of TH+ neurons in the SN and striatum from MPTP-intoxicated mice and those receiving 3.5 x 106 Teffs compared with PBS controls; however, neuronal loss was unremarkable in MPTP-intoxicated mice, which received 3.5 x 106 Tregs (Fig. 4A) . Stereological analysis of TH+ neuron counts indicated that 12 mg MPTP/kg bodyweight resulted in a 56% loss of nigral TH+ neurons following MPTP intoxication relative to PBS-injected controls (Fig. 4B) , whereas similar to previous results (Fig. 3) , no effect on TH–Nissl+ neurons was demonstrated (data not shown). Adoptive transfer of Tregs to MPTP-intoxicated mice resulted in significantly greater numbers of surviving TH+ neurons, whereas no significant increase in neuron number was obtained with transfer of Teffs. Moreover, a significant amount of protection (48%) was afforded with as few as 0.5 x 106 Tregs, and transfer of 3.5 x 106 Tregs ameliorated the dopaminergic loss within the SN. In addition, this protection was dose-dependent. Regression analysis demonstrated that TH neuronal survival was strongly correlated with Treg cell number (r=0.83, P<0.0001) and was significantly different (P=0.0001) to the effects of increasing numbers of Teffs on neuronal survival. The density of TH expression in striata was assessed to estimate the extent that Tregs were capable of preserving dopaminergic, axonal fibers following MPTP intoxication. MPTP/kg bodyweight (12 mg) resulted in a 71% reduction in striatal TH density compared with PBS controls (Fig. 4C) . Transfer of Teffs yielded insignificant levels of protection (2–18% over MPTP-intoxicated mice) for the dopaminergic termini. In contrast, adoptive transfer of 0.5–3.5 x 106 Tregs resulted in significant, dose-related protection, and 61% survival of the striatal TH+ termini was afforded by as few as 0.5 x 106 Tregs and near complete protection (85–88%) with as few as 1.0–3.5 x 106 Tregs, respectively. Similar to protection of nigral neurons, regression analysis of the striatal TH density demonstrated a significantly strong correlation with numbers of Tregs transferred (r=0.61, P=0.0008), which was also significantly different (P=0.009) than dose effects afforded by transfer of Teffs.


Figure 4
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  		Figure 4. Treg-mediated dopaminergic neuroprotection in SN and striatum is dose-dependent. (A) Expression of TH by neuronal bodies in SN and terminal fibers in striatum of ventral midbrain from PBS-, MPTP-, MPTP/Treg (0.5–3.5x106)-, or MPTP/Teff (0.5–3.5x106)-treated mice (original scale bars, 25 µm). (B) Unbiased, stereological analysis of TH+ neurons in SN. Values represent means ± SEM for seven mice per group, and P < 0.001 compared with aPBS, bMPTP, or cMPTP/respective number of Teffs. (C) ODs of striatal TH+ fibers. Values represent means ± SEM for seven mice per group, and P < 0.01 compared with aPBS, bMPTP, or cMPTP/respective number of Teffs.

 
Astrocyte GFAP expression was not altered significantly following MPTP intoxication or by adoptive transfer of Treg or Teff to MPTP-treated animals (data not shown). To investigate whether adoptive transfer of Tregs could influence the synthesis of astrocyte-derived, neurotrophic factors, ventral midbrain mRNA expression of BDNF and GDNF was assessed at Days 2 and 7 post-MPTP intoxication. A temporal pattern of expression for these factors was observed in ventral midbrains of MPTP-intoxicated mice treated with 3.5 x 106 Tregs compared with all other treatment groups. BDNF but not GDNF was increased at Day 2 (Fig. 5A , Day 2 GDNF; data not shown), whereas by Day 7, GDNF but not BDNF mRNA was increased significantly in midbrains of MPTP/Treg-injected mice (Day 7 BDNF; data not shown). Confocal microscopy of midbrain slices from Day 2 or 7, stained to detect expression of BDNF or GDNF, respectively, revealed a greater abundance of the neurotrophic factors BDNF and GDNF in the SN of Treg recipients at those respective times compared with all other groups (Fig. 5B) . Colocalization of BDNF from Day 2 brains (Fig. 5C) and GDNF from Day 7 brains (Fig. 5D) revealed that these neurotrophic factors were in part associated with GFAP+ astrocytes and not with Mac-1+ microglia or TH+ neurons. BDNF rarely colocalized with CD4+ T cells. No associations were seen between T cells and GDNF. Taken together, these data suggest that Tregs ameliorate neurodegeneration following MPTP intoxication by suppression of microglial activation and by inducing neurotrophic factors, which support neuronal survival.


Figure 5
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  		Figure 5. Neurotrophic factor expression in MPTP mice after adoptive transfer of Tregs. (A) Quantitative RT-PCR of BDNF and GDNF mRNA expression from ventral midbrains of PBS, MPTP, MPTP/Treg, and MPTP/Teff groups. Values represent ratios of gene expression normalized to GAPDH mRNA and means ± SEM for four to six mice per group, and P < 0.05 compared with aPBS, bMPTP, or cMPTP/Teff. (B) Confocal images of midbrain sections of PBS, MPTP, MPTP/Treg, and MPTP/Teff groups revealed BDNF or GDNF expression within the SN on Days 2 and 7 post-MPTP intoxication, respectively. Colocalization of (C) BDNF (green) or (D) GDNF (green) with (C and D) microglia (Mac-1; red), astrocytes (GFAP; red), T cells (CD4+; red), or dopaminergic neurons (TH+; red) in the ventral midbrain of mice treated with MPTP/Treg on Days 2 (BDNF) and 7 (GDNF) post-MPTP intoxication (original scale bars, 25 µm).

 
Tregs modulate microglia to attenuate ROS production and cytotoxicity in vitro
The deleterious effect of microglial activation on adjacent cells is postulated to account for the majority of neuronal demise. For this reason, suppression of microglial activation by Tregs may be responsible for the profound protection observed in vivo. To investigate specific mechanisms for the ability of Tregs to modulate microglial responses and its effects on neuronal survival, a laboratory model of PD was developed to recapitulate activated microglia and neuronal interactions as they may occur in PD. Mechanisms of microglia activation in PD are unknown; however, interaction with aggregated and N-{alpha}-syn has been implicated [16 ] (A. D. Reynolds et al., in revision). In keeping with this hypothesis, an in vitro model of microglia-mediated cytotoxicity was established using N-{alpha}-syn-activated microglia and MES23.5 cells. We observed a significant 42% loss of MES23.5 cells cocultured for 24 h with microglia, stimulated with 100 nM N-{alpha}-syn compared with control cocultures of MES23.5 with unstimulated microglia (CON) (Fig. 6A ). Coculture of activated microglia with anti-CD3-activated Tregs significantly inhibited MES23.5 cytotoxicity, resulting in a higher percentage of MES23.5 cell survival (95%±13%). In contrast, protection afforded by activated Teffs provided no significant protection (57%±5% survival compared with controls). These data suggested that Tregs could modulate the microglial phenotype to prevent a cytotoxic response. It is interesting that preincubation of naïve microglia with Tregs was also able to inhibit microglia-mediated cytotoxicity of MES23.5 induced by N-{alpha}-syn, as removal of Tregs from cocultures prior to stimulation with N-{alpha}-syn supported MES23.5 cell survival significantly (99%±2%) compared with activated microglia alone or preincubated with Teff (Fig. 6B) . In addition, supernatants of microglia stimulated with N-{alpha}-syn alone or N-{alpha}-syn and cultured in the presence of Teff were significantly cytotoxic to MES23.5 cells, yielding 44% and 30% MES23.5 cell losses, respectively (Fig. 6C) , whereas supernatants from microglia stimulated with N-{alpha}-syn and cultured with Tregs supported significant MES23.5 survival (96%±2%). These data demonstrate the potential of Tregs to suppress cytotoxicity afforded by N-{alpha}-syn-activated microglia and suggest that direct modulation of microglial responses provides one primary mechanism for Treg-mediated neuronal protection.


Figure 6
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  		Figure 6. Mechanisms for Treg suppression of microglial neurotoxic responses. Microglia and MES23.5 cells stimulated with N-{alpha}-syn are cultured with or without Tregs or Teffs; controls (CON) are unstimulated co-cultures of microglia and MES23.5 (A). (B) Microglia and MES23.5 cells were preincubated with Treg or Teff before the addition of N-{alpha}-syn. In both instances, MES23.5 cell survival was measured by live/dead assay. (C) MES23.5 cell survival after culture fluids of N-{alpha}-syn-stimulated microglia were cocultured with Tregs or Teffs, and Tregs or Teffs suppression or induction of ROS on microglial ROS production in response to N-{alpha}-syn (D) celecoxib (CB; E), or TNF-{alpha} (F). Accumulation of NO in conditioned media in response to LPS was also determined from microglia with or without Treg or Teff (G). Values are represented ± SEM (P<0.01 vs. aCon, bstimulus (N-Syn, TNF{alpha} ± PMN, or LPS) cstimulus + corresponding Teff.

 
Oxidative stress, particularly induced by increased levels of superoxide radicals and NO, is known to contribute to neurotoxicity [29 ]. Thus, we hypothesized one mechanism by which Tregs could suppress microglial-induced toxicity through suppression of ROS. We demonstrated previously that N-{alpha}-syn-stimulated microglia afford an inflammatory phenotype and produce significantly detectable levels of ROS [16 ] (A. D. Reynolds et al., in revision). Based on our previous data, showing Treg suppression of microglia-mediated cytotoxicity, we tested whether Tregs could modify this "putative neurotoxic" ROS response. Anti-CD3-activated Tregs or Teffs were cultivated with naïve microglia for 24 h prior to stimulation of microglia with N-{alpha}-syn. After removal of T cells, microglia were stimulated with N-{alpha}-syn for a period of 90 min, and ROS production was measured as a function of H2O2 accumulation. N-{alpha}-syn stimulation of microglia resulted in a 73% increase in H2O2 production over nonstimulated, control microglia (Fig. 6D) . Pretreatment of microglia with Tregs resulted in significant, dose-dependent reductions in the amount of H2O2 produced in comparison with N-{alpha}-syn-stimulated microglial cells and cells pretreated with Teffs (P<0.01). At a 1:1 ratio of microglia:Treg, reduction of the ROS response was comparable with that induced by the known cyclo-oxygenase-2 inhibitor, Celecoxib (Pfizer Pharmaceuticals Co., Cambridge, MA, USA; Fig. 6E ). Significant suppression of the ROS response was also obtained by coculturing Tregs with microglia activated with TNF-{alpha} for 24 h or TNF-{alpha} with PMA (Fig. 6F) or LPS (data not shown). Tregs also suppressed NO production significantly by microglia stimulated with 100 ng LPS (Fig. 6G) . These data support the use of Treg-mediated therapies for the treatment and prevention of microglial inflammatory processes.

DISCUSSION

Our prior works demonstrated that adoptive transfer of immune cells from Cop-1-immunized donors to MPTP-intoxicated recipients attenuated reactive microglial responses and provided nigrostriatal neuronal protection [17 , 30 ]. Although the precise mechanism(s) for this neuroprotection remains undetermined, these works demonstrate the importance of T cells with a regulatory phenotype secreting IL-10 in protection of the nigrostriatal system in this model of PD. These data are also supported by what is known in regards to Cop-1-mediated immunity. Not only does Cop-1 preferentially induce Th2 and Th3 Tregs, which secrete anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β [31 ], but it also promotes the conversion of CD4+CD25– T cells to CD4+CD25+ Tregs through induction of Foxp3 [32 ].

Based on our observation, we postulated that an induction of a Treg response was responsible for the observed, neuroprotective effects in MPTP mice. To test whether a neuroprotective response could be obtained devoid of antigen specificity, isolated Tregs and Teffs were polyclonal-activated with anti-CD3. As only activated lymphocytes enter the CNS of MPTP-intoxicated recipients and accumulate in areas of tissue damage early after cell transfer and at times of peak inflammation [17 ], it is hypothesized that in this model, polyclonal-activated Tregs traffic into the brain and interact with local glial cells. This interaction is thought to alter the reactive microglial phenotype from a toxic to a trophic state and induce astrocyte expression of growth factors to promote neuron survival [33 ] and thereby ameliorate MPTP-induced neurodegeneration [15 , 17 , 34 , 35 ]. This report now demonstrates that adoptive transfer of CD3-activated Tregs indeed attenuated MPTP-induced neuroinflammation, promoted astrocyte-derived BDNF and GDNF expression, and conferred nearly complete neuroprotection.

The neurotoxic effect of MPTP depends on astroglial transformation into the active compound MPP+ once within the brain. MPP+ is then taken up through the dopamine transporter of SNpc TH+ neurons and blocks complex I of the mitochondrial electron transport chain inhibiting synthesis of ATP and eliciting oxidative stress [36 ]. These early effects are thought to account for ~10% of dopaminergic neuronal death [15 , 17 ]. Microglial activation, believed to be a secondary event in models of PD, further impairs survival of TH+ neurons after toxic stimuli. Substantial evidence implicates microglial-induced inflammation in early neurodegenerative processes [14 , 37 38 39 40 ] and in PD [5 , 12 , 41 42 43 ]. The relevance of microglia-associated neurotoxicity in the pathogenesis of PD is substantiated by recent observations that microglial activation to {alpha}-syn, a stimulus relevant to PD, results in an inflammatory phenotype, which was characterized by up-regulation of proinflammatory cytokines including TNF-{alpha} and IL-6, ROS production, and inflammatory factors, which directly affect neuronal death in cellular systems [16 , 44 ] (A. D. Reynolds et al., in revision). Indeed, attenuation of microglial activation in models of PD results in the protection of up to 90% of the neurons destined to die [15 , 45 46 47 48 49 50 ]. The therapeutic implications of these observations were exemplified in recent data, showing administration of daily, nonsteroidal, anti-inflammatory drugs significantly reduced the incidence of PD in a large cohort of health care workers [51 , 52 ].

Ample evidence exists for the role of Tregs as regulators of inflammation in models of graft-versus-host disease [53 ] and autoimmune disorders including arthritis [54 ], diabetes [55 ], colitis [56 , 57 ], autoimmune gastritis [58 ], and experimental autoimmune encephalitis [59 60 61 62 ]. However, with the exception of experimental allergic encephalomyelitis, limited studies of Tregs in models of chronic, neurodegenerative diseases exist. Although the potential for T cell-mediated protection for trauma-induced and degenerative disorders of the CNS has been explored, results have, until this report, been inconclusive [33 , 63 64 65 66 67 68 69 70 71 ]. The importance of Tregs in the pathogenesis of PD is underscored by the observations of a significant diminution of CD4+ T cells and shifts toward proinflammatory immune responses associated with PD [72 , 73 ]. In addition, fluctuations in CD4+CD25+ Tregs in PD combined with their capacity to affect disease outcomes in inflammatory and neurodegenerative models [33 , 53 54 55 56 57 58 59 60 61 , 74 , 75 ] suggested a role for Tregs in PD. Clearly, interest in Tregs has been heightened by the demonstration that Tregs can be harnessed therapeutically to treat autoimmune diseases and facilitate transplantation tolerance or eliminated to potentiate cancer immunotherapy [76 ].

Tregs are phenotypically and functionally distinguished from naïve effector CD4+ T cells by their constitutive expression of CD25+ and Foxp3, their unresponsiveness to anti-CD3 stimulation, and their suppression of Teff proliferation [77 ]. Upon activation, Tregs switch into an active suppressor mode to neutralize or to inactivate other effector cells, such as B cells or T cells, as well as suppressing myeloid and APCs, including microglia [77 78 79 80 ]. Tregs mediate their suppression of proliferative responses through cell–cell contact and/or IL-10- and TGF-β-mediated mechanisms [81 82 83 84 ]. In this report, IL-10 and TGF-β expression was increased in the ventral midbrains of MPTP-intoxicated mice, which received Tregs, and was significantly higher compared with MPTP-intoxicated mice alone or those that received Teffs. This suggests that IL-10 and TGF-β are but two possible mechanisms, whereby Tregs attenuate neuroinflammation in this MPTP model of PD. Indeed, infusion of IL-10 was shown recently to protect against LPS-induced and inflammation-mediated degeneration of neurons in the SN [85 ], possibly by inhibiting microglial activation and proinflammatory cytokine synthesis. Of interest, overexpression of IL-10 has also been shown to induce Treg populations preferentially in vivo [55 ]. Alternatively, as TGF-β inhibits microglial activation and induction of inflammation [86 ] and has been shown to have direct, neuroprotective potential in MPP+-induced, dopaminergic neurodegeneration [87 ], increased Treg-mediated TGF-β production could also have dual effects of attenuating neuroinflammation and direct neuroprotection of neurons. Another mechanism by which T cells can affect the outcome of neurodegenerative processes may be through interactions with astrocytes to promote synthesis of neural growth factors [17 , 33 , 88 ]. In this study, adoptive transfer of activated Tregs, but not Teffs, induced increased expression of astrocyte BDNF and GDNF.

Adoptive transfer of Tregs presents two distinct paradigms by which Tregs could attenuate neuroinflammatory responses and mediate neuronal protection. First, Tregs could act by modulating the peripheral immune system through suppressing peripheral immune cell activation and/or migration to the CNS. It is now known that the peripheral immune system (B cells, T cells, NK cells, and monocytes) is involved in the neurotoxic outcome of MPTP intoxication [89 ] (Eric J. Benner, Rebecca Banerjee, Ashley D. Reynolds, Simon Sherman, Vladimir M. Pisarev, Vladislav Tsiperson, Craig Nemachek, Pawel Ciborowski, Serge Przedborski, R. Lee Mosley, Howard E. Gendelman, in revision) and is likely involved in disease progression of PD patients [12 , 41 42 43 , 72 , 73 ]. Second, Tregs adoptively transferred to MPTP-intoxicated recipients are capable of migrating to the site of injury within the brain and through direct interactions with local glia, attenuate neuroinflammation and as such, affect neuronal protection [33 ]. Previous studies using Cop-1-mediated immunomodulation, as well as those presented herein, suggest the latter is the more likely mechanism. This is evidenced by the presence of T cells from Cop-1-immunized donors in the SN of MPTP-intoxicated recipients as early as 20 h following adoptive transfer [17 ]. Recent investigations using single photon emission-computed tomography and magnetic resonance imaging demonstrated that Tregs were able to traffic to the nigra of MPTP recipients within 24 h post-transfer (our unpublished data). In the present study, midbrains of MPTP-intoxicated mice, which received Tregs, had significantly higher expression of Foxp3, IL-10, and TGF-β mRNA relative to other groups, suggesting the presence of Tregs. Moreover, not only was neuroinflammation greatly attenuated by adoptive transfer of Tregs, but also expression of BDNF and GDNF was increased.

Cross-validation in other model systems of PD coincident with microglial activation is important. To this end, we have developed, in parallel, a cellular model of microglial activation in the presence of nitrated and aggregated {alpha}-syn to recapitulate many of the pathogenic processes, which may occur in PD. Therein, through biological, genomic, and proteomic tests, nitrated and aggregated {alpha}-syn stimulation induced a microglial, neuroinflammatory phenotype consisting of NF-{kappa}B activation, increased inflammatory protein expression, and ROS production, which was linked to neuronal viability [16 ] (A. D. Reynolds et al., in revision). We reasoned that suppression of this response would ameliorate microglial-induced neuronal cell death. Previous studies showed that blockade of a ROS response in microglial prevented dopaminergic cytotoxicity in response to N-{alpha}-syn [44 ]. Our own results suggest that one mechanism by which Tregs may ameliorate neurotoxicity in this cellular model is through suppression of a significant proportion of microglial ROS production in response to a variety of stimuli, including nitrated and aggregated {alpha}-syn, TNF-{alpha}, and LPS. Moreover, ongoing investigations into alterations in the microglial phenotype have uncovered several proteins, which are differentially regulated between N-{alpha}-syn-stimulated microglia in coculture with Tregs compared with N-{alpha}-syn stimulation alone. These proteins were found to be involved in migration, cell–cell interactions, cytoskeletal structure, and redox proteins, including a twofold increase in expression of peroxyredoxins 1 and 4 after just 4 h (our unpublished data). Indeed, peroxyredoxins play an important role in eliminating peroxides and regulating intracellular concentrations of H2O2, further supporting this hypothesis. This model can now be used to decipher specific mechanisms by which Tregs modulate the microglial response and provides novel, therapeutic avenues for the treatment of PD.

The importance of the observations in the present study rests with the idea that they can be readily translated to the clinic. Perhaps of even more importance is the readily understood mechanism of Tregs in human immunology and degenerative disorders outside of the nervous system and their critical role in controlling potentially harmful, inflammatory responses and in maintaining immune homeostasis. Therefore, taken together, these data support the use of immunomodulatory strategies, which induce Treg-mediated, immune responses, affecting neuroinflammation, and as such, inhibit dopaminergic neurodegeneration in PD.

ACKNOWLEDGEMENTS

The work was supported by the Francine and Louis Blumkin Foundation, the Community Neuroscience Pride of Nebraska Research Initiative, the Carol Swarts, M.D., Laboratory of Emerging Neuroscience Research, the Alan Baer Charitable Trust (to H. E. G.), a University of Nebraska Medical Center Graduate Student Excellence Award (to A. D. R.), and NIH grants 1T32 NS07488 (to A. D. R. and H. E. G.), 1R21 NS049264 (to R. L. M.), and 2R37 NS36126, 1 P01 NS043985-01, 5 P01 MH64570-03, and P20 RR15635 (to H. E. G.). The authors thank Chad Laurie, Traci Anderson, David Levy, Irina Usherenko, Dr. Jonathan Kipnis, and Robin Taylor for their scientific advice, critical reading of the manuscripts, and/or technical assistance. The authors have no real or perceived conflict of interest.

Received May 10, 2007; revised July 8, 2007; accepted July 9, 2007.

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