ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 17, NUMBER 4, 2008

Cell Transplantation, Vol. 16, pp. 363-372, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Neuroinflammation and Peripheral Immune Infiltration in Parkinson's Disease: An Autoimmune Hypothesis

Angela J. Monahan, Michael Warren, and Paul M. Carvey

Department of Pharmacology, Rush University Medical Center, Chicago, IL, USA

Despite decades of research and the development of a large group of animal models, our understanding of the mechanisms responsible for the progressive loss of dopamine neurons in Parkinson's disease (PD) is unknown. So-called neuroprotective studies demonstrate that a vast group of molecules readily attenuate the dopamine (DA) neuron loss produced by DA neurotoxin insult. Despite these successes, these neuroprotective strategies have been surprisingly ineffective in patients. This may reflect the fact that the initial pathogenic event and the subsequent disease progression is a consequence of different mechanisms. As we began to think about this disconnect, we discovered that animals exposed to DA neurotoxins exhibited blood-brain barrier (BBB) dysfunction. If the BBB in PD patients is disrupted, then the barrier that normally segregates peripheral vascular factors from brain parenchyma is no longer present. Immune cells could then enter brain and produce a self-perpetuating (progressive) degenerative process. In this review, we propose that peripheral immunity contributes to the degenerative process of PD and may be responsible for the progressive nature of the disease. This hypothesis is supported by a broad and diverse literature that is just beginning to come together to suggest that PD is, in part, an autoimmune disease. In order to understand this hypothesis, the reader must question the conventional wisdom that the BBB is intact in PD, the brain is an immune privileged area, and that pathogenic insult and disease progression may reflect different mechanisms.

Key words: T cells; B cells; Inflammation; Multiple sclerosis; Animal models; Disease progression

Address correspondence to Angela J. Monahan, 1735 West Harrison St. (Suite 406), Rush University Medical Center, Chicago, IL 60612, USA. Tel: 312-563-2563; Fax: 312-563-3552; E-mail: Angela_Monahan@rush.edu

Cell Transplantation, Vol. 16, pp. 373-381, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Intraputamenal Infusion of Exogenous Neurturin Protein Restores Motor and Dopaminergic Function in the Globus Pallidus of MPTP-Lesioned Rhesus Monkeys

R. Grondin,1,2 Z. Zhang,1,2 Y. Ai,1,2 F. Ding,1,3 A. A. Walton,1 S. P. Surgener,1 G. A. Gerhardt,1,2 and D. M. Gash1,2

1Department of Anatomy & Neurobiology, University of Kentucky Medical Center, Lexington, KY, USA
2Morris K. Udall Parkinson's Disease Research Center of Excellence, University of Kentucky Medical Center, Lexington, KY, USA
3Department of Neurosurgery, Shandong Provincial Hospital, Shandong University, Jinan, Shandong, PR China

The neurorestorative effects of exogenous neurturin (NTN) delivered directly into the putamen via multiport catheters were studied in 10 MPTP-lesioned rhesus monkeys expressing stable parkinsonism. The parkinsonian animals were blindly assigned to receive coded solutions containing either vehicle (n = 5) or NTN (n = 5, 30 mg/day). Both solutions were coinfused with heparin using convection-enhanced delivery for 3 months. The NTN recipients showed a significant and sustained behavioral improvement in their parkinsonian features during the treatment period, an effect not seen in the vehicle-treated animals. At study termination, locomotor activity levels were increased by 50% in the NTN versus vehicle recipients. Also, DOPAC levels were significantly increased by 150% ipsilateral (right) to NTN infusion in the globus pallidus, while HVA levels were elevated bilaterally in the NTN-treated animals by 10% on the left and 67% on the right hemisphere. No significant changes in DA function were seen in the putamen. Volumetric analysis of putamenal NTN labeling showed between-subject variation, with tissue distribution ranging from 214 to 744 mm3, approximately equivalent to 27-93% of area coverage. Our results support the concept that intraparenchymal delivery of NTN protein may be effective for the treatment of PD. More studies are needed to determine strategies that would enhance tissue distribution of exogenous NTN protein, which could contribute to optimize its trophic effects in the parkinsonian brain.

Key words: Parkinson's disease; MPTP; Neurturin; Putamen; Neuroregeneration

Address correspondence to Dr. Richard Grondin, Ph.D., University of Kentucky Medical Center, Department of Anatomy and Neurobiology, Room 313, Whitney-Hendrickson Building, Lexington, KY 40536-0098, USA. Tel: 859-323-8925; Fax: 859-257-3625; E-mail: rcgron0@uky.edu

Cell Transplantation, Vol. 16, pp. 383-395, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

GDNF-Secreting Human Neural Progenitor Cells Increase Tyrosine Hydroxylase and VMAT2 Expression in MPTP-Treated Cynomolgus Monkeys

Marina E. Emborg,1,2 Allison D. Ebert,3 Jeff Moirano,1,3 Sun Peng,1 Masatoshi Suzuki,3 Elizabeth Capowski,3 Valerie Joers,1 Ben Z. Roitberg,1,5 Patrick Aebischer,6 and Clive N. Svendsen1,2,3

1Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI, USA
2Department of Medical Physics, University of Wisconsin, Madison, WI, USA
3Waisman Center, University of Wisconsin, Madison, WI, USA
4Department of Anatomy, University of Wisconsin, Madison, WI, USA
5Department of Neurosurgery, University of Illinois, Chicago, IL, USA
6Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

Human neural progenitor cells (hNPCs) have been proposed as a potential source of cells for ex vivo gene therapy. In this pilot study, three 5-year-old female cynomolgus monkeys received a single intracarotid infusion of MPTP, followed 1 week later by MRI-guided stereotaxic intrastriatal and intranigral injections of male hNPCs transgenic for GDNF. Immunosupression with oral cyclosporine (30-40 mg/kg) began 48 h before hNPC transplants and continued throughout the study. We monitored the animals using a clinical rating scale (CRS). Three months postsurgery, we euthanized the animals by transcardiac perfusion, then retrieved and processed their brains for morphological analysis. Our findings include the following. 1) hNPCs survived and produced GDNF in all animals 3 months postsurgery. 2) hNPCs remained in the areas of injection as observed by GDNF immunostaining and in situ hybridization for the human Y chromosome. 3) A "halo" of GDNF expression was observed diffusing from the center of the graft out into the surrounding area. 4) We observed increased TH- and VMAT2-positive fibers in areas of GDNF delivery in two of the three animals. The two animals with TH- and VMAT2-positive fibers also showed reductions in their CRS scores. 5) Some GFAP-positive perivascular cuffing was found in transplanted areas. 6) General blood chemistry and necropsies did not reveal any abnormalities. Therefore, we conclude that hNPCs releasing GDNF may be a possible alternative for intracerebral trophic factor delivery in Parkinson's disease.

Key words: Neuroprotection; Parkinson's disease; Dopamine; Trophic factors; Stem cells

Address correspondence to Marina E. Emborg, 1220 Capitol Court, Madison, WI 53715, USA. Tel: 608-262-9714; Fax: (608) 263-3524; E-mail: emborg@primate.wisc.edu

Cell Transplantation, Vol. 16, pp. 397-408, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Contrasting In Vivo Effects of Two Peptide-Based Amyloid-b Protein Aggregation Inhibitors in a Transgenic Mouse Model of Amyloid Deposition

Qingyou Li,1 Marcia Gordon,1 Marcus A. Etienne,2 Robert P. Hammer,2 and Dave Morgan1

1Alzheimer's Research Laboratory, Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
2Department of Chemistry, Louisiana State University, Baton Rouge, LA, USA

Previous studies have shown that 17,19,21-tri-N-methyl-Ab16-22 peptide (Ab16-22m), and a peptide analogue containing a,a-disubsituted amino acids (aaAA) in the hydrophobic core domain of Ab, termed AMY-1, effectively inhibited full-length Ab aggregation in vitro. To investigate the amyloid-modifying effects of these agents in vivo, we injected these compounds into the hippocampus of 13-month-old amyloid precursor protein (APP) transgenic mice, a model of amyloid deposition. After 7 days, brain tissues were stained for immunohistochemistry to detect total Ab and thioflavine-S (THIO-S) to measure Acompact plaques. Both diffuse Ab deposits and compact amyloid plaques were significantly increased when injecting 0.3 nmol Ab16-22m compared to the PBS vehicle. The amyloid aggregation-modifying peptide AMY-1 showed a slight reduction of Ab deposition in the injection area at a dose of 0.3 nmol, but neuronal toxicity, measured by Fluoro-Jade and Nissl stains, appeared when higher doses (3 nmol) were tested. Our data indicate that, unlike observations reported in vitro, the Ab16-22m increased deposition of Ab in the brain of APP transgenic mice in vivo. Possible explanations for this outcome include unique influences of the brain environment and/or modification of Ab production or clearance by the administered agent. The AMY-1 peptide showed a trend for reducing Ab deposits, but led to toxicity at higher doses. These data emphasize the need for evaluating potential Ab aggregation inhibitors with in vivo models of amyloid deposition before assuming they will have benefit in treating Alzheimer's disease patients.

Key words: Alzheimer's disease; Amyloid-b peptide; Neurotoxicity; Transgenic mice

Address correspondence to Dave Morgan, Ph.D., Alzheimer Research Laboratory, Department of Molecular of Pharmacology and Physiology, MDC 8, University of South Florida, College of Medicine, Tampa, FL 33612-4799, USA. Tel: 1-813-974-3949; Fax: 1-813-974-3079; E-mail: dmorgan@health.usf.edu

Cell Transplantation, Vol. 16, pp. 409-415, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Biocompatibility of PEG-Based Hydrogels in Primate Brain

K. B. Bjugstad,1 D. E. Redmond, Jr.,2 K. J. Lampe,3 D. S. Kern,1 J. R. Sladek, Jr.,1 and M. J. Mahoney3

1Department Pediatrics, University Colorado Denver and Health Sciences Center, Aurora, CO, USA
2Departments Psychiatry and Neurosurgery, Yale University, New Haven, CT, USA
3Department Chemical and Biological Engineering, University Colorado, Boulder, CO, USA

Degradable polymers have been used successfully in a wide variety of peripheral applications from tissue regeneration to drug delivery. These polymers induce little inflammatory response and appear to be well accepted by the host environment. Their use in the brain, for neural tissue reconstruction or drug delivery, also could be advantageous in treating neurodegenerative disorders. Because the brain has a unique immune response, a polymer that is compatible in the body may not be so in the brain. In the present study, polyethylene glycol (PEG)-based hydrogels were implanted into the striatum and cerebral cortex of nonhuman primates. Four months after implantation, brains were processed to evaluate the extent of astrogliosis and scaring, the presence of microglia/macrophages, and the extent of T-cell infiltration. Hydrogels with 20% w/v PEG implanted into the brain stimulated a slight increase in astrocytic and microglial/macrophage presence, as indicated by a small increase in glial fibrillary acidic protein (GFAP) and CD68 staining intensity. This increase was not substantially different from that found in the sham-implanted hemispheres of the brain. Staining for CD3+ T cells indicated no presence of peripheral T-cell infiltration. No gliotic scarring was seen in any implanted hemisphere. The combination of low density of GFAP-positive cells and CD68-positive cells, the absence of T cells, and the lack of gliotic scarring suggest that this level of immune response is not indicative of immunorejection and that the PEG-based hydrogel has potential to be used in the primate brain for local drug delivery or neural tissue regeneration.

Key words: Hydrogel; Primate; Biocompatibility; Polyethylene glycol

Address correspondence to K. B. Bjugstad, Department of Pediatrics, Mail Stop F8313, University of Colorado Denver and Health Sciences Center, P.O. Box 6511, Aurora, CO 80045, USA. Tel: 303-724-3041; E-mail: kimberly.bjugstad@uchsc.edu

Cell Transplantation, Vol. 16, pp. 417-425, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Pharmacological MRI (phMRI) Monitoring of Treatment in Hemiparkinsonian Rhesus Monkeys

Liming Luan,1,2* Feng Ding,1,2* Yi Ai,1,3 Anders Andersen,3,4 Peter Hardy,4 Eric Forman,1 Greg A. Gerhardt,1,3 Don M. Gash,1,3 Richard Grondin,1,3 and Zhiming Zhang1,3,4

1Department of Anatomy & Neurobiology, College of Medicine, University of Kentucky, Lexington, KY, USA
2Department of Neurosurgery, Shandong Provincial Hospital, Shandong University, Jinan, Shandong, PR China
3Morris K. Udall Parkinson's Disease Research Center of Excellence, College of Medicine, University of Kentucky, Lexington, KY, USA
4Magnetic Resonance Imaging and Spectroscopy Center, College of Medicine, University of Kentucky, Lexington, KY, USA

There is a great need for the development of noninvasive, highly sensitive, and widely available imaging methods that can potentially be used to longitudinally monitor treatment of Parkinson's disease (PD). Here we report the monitoring of GDNF-induced functional changes of the basal ganglia in hemiparkinsonian monkeys via pharmacological MRI measuring the blood oxygenation level-dependent (BOLD) response to a direct dopamine agonist (apomorphine, APO). After testing BOLD responsiveness to APO in their normal state, two additional scans were taken with the same dose of APO stimulation after induced parkinsonism. Then all animals were chronically treated with GDNF for 18 weeks by a programmable pump and catheter system. The catheter was surgically implanted into the right putamen and connected to the pump via flexible polyurethane tubing. phMRI scans were taken at both 6 and 18 weeks while they received 22.5 mg of GDNF per day. In addition, behavioral changes were monitored throughout the entire study. The primary finding of this study was that APO-evoked activations in the DA denervated putamen were attenuated by the chronic intraputamenal infusion of GDNF accompanied by improvements of parkinsonian features, movement speed, and APO-induced rotation compared to data collected before the chronic GDNF treatment. The results suggest that phMRI methods in combination with administration of a selective DA agonist may be useful for monitoring neurorestorative therapies in PD patients in the future.

Key words: Apomorphine; GDNF; phMRI; Rhesus monkey; Parkinson's disease; MPTP; Pump; Catheter; Putamen

Address correspondence to Zhiming Zhang, M.D., Department of Anatomy & Neurobiology, University of Kentucky College of Medicine, 48 Whitney-Hendrickson Bldg., Lexington, KY 40536-0098, USA. Tel: (859) 257-6032; Fax: (859) 323-3625; E-mail: zzhan01@uky.edu

*These authors contributed equally to this study.

Cell Transplantation, Vol. 16, pp. 427-444, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Embryonic Substantia Nigra Grafts Show Directional Outgrowth to Cografted Striatal Grafts and Potential for Pathway Reconstruction in Nonhuman Primate

J. R. Sladek, Jr.,1 K. B. Bjugstad,1 T. J. Collier,2 E. A. Bundock,3 B. C. Blanchard,1 J. D. Elsworth,4 R. H. Roth,4 and D. E. Redmond, Jr.5

1Department of Pediatrics, The University of Colorado School of Medicine, Aurora, CO, USA
2Department of Neurology, University of Cincinnati School of Medicine, Cincinnati, OH, USA
3Department of Pathology, University of Vermont School of Medicine, Burlington, VT, USA
4Department of Pharmacology, Yale University School of Medicine, New Haven, CT, USA
5Departments of Psychiatry and Neurosurgery, Yale University School of Medicine, New Haven, CT, USA

Transplantation of embryonic dopamine (DA) neurons has been tested as a therapy for Parkinson's disease. Most studies placed DA neurons into the striatum instead of the substantia nigra (SN). Reconstruction of this DA pathway could serve to establish a more favorable environment for control of DA release by grafted neurons. To test this we used cografts of striatum to stimulate growth of DA axons from embryonic SN that was implanted adjacent to the host SN in African green monkeys. Embryonic striatum was implanted at one of three progressive distances rostral to the SN. Immunohistochemical analysis revealed DA neuron survival and neuritic outgrowth from the SN grafts at 12-36 weeks after grafting. Each animal showed survival of substantial numbers of DA neurons. Most fibers that exited SN grafts coursed rostrally. Striatal grafts showed evidence of target-directed outgrowth and contained dense patterns of DA axons that could be traced from their origin in the SN grafts. A polarity existed for DA neurites that exited the grafts; that is, those seen caudal to the grafts did not appear to be organized into a directional outflow while those on the rostral side were arranged in linear profiles coursing toward the striatal grafts. Some TH fibers that reached the striatal grafts appeared to arise from the residual DA neurons of the SN. These findings suggest that grafted DA neurons can extend neurites toward a desired target over several millimeters through the brain stem and caudal diencephalon of the monkey brain, which favors the prospect of circuit reconstruction from grafted neurons placed into appropriate locations in their neural circuitry. Further study will assess the degree to which this approach can be used to restore motor balance in the nonhuman primate following neural transplantation.

Key words: Dopaminergic neurons; Tract reconstruction; Cografts; Directed outgrowth; Parkinsonism

Address correspondence to John Sladek, Ph.D., Department of Pediatrics, University of Colorado School of Medicine, Neuroscience Mail Stop 8315, P.O Box 6511, Aurora CO 80045, USA. Tel: (303) 724-0629; E-mail: john.sladek@uchsc.edu

Cell Transplantation, Vol. 16, pp. 445-455, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Sustained Analgesic Peptide Secretion and Cell Labeling Using a Novel Genetic Modification

Shyam Gajavelli,1 Daniel A. Castellanos,1 Orion Furmanski,1 Paul C. Schiller,2 and Jacqueline Sagen1

1The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA
2Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center, Miami, FL, USA

Cell-based therapy for neuropathic pain could provide analgesics to local pain modulatory regions in a sustained, renewable fashion. In order to provide enhanced analgesic efficacy, transplantable cells may be engineered to produce complementary or increased levels of analgesic peptides. In addition, genetic labeling of modified cells is desirable for identification and tracking, but it should be retained intracellularly as desired analgesic peptides are secreted. Usually constructs encode proteins destined for either extra- or intracellular compartments, as these pathways do not cross. However, interactions between intracellular destinations provide a window of opportunity to overcome this limitation. In this report, we have explored this approach using a potential supplementary analgesic peptide, [Ser1]-histogranin (SHG), the stable synthetic derivative of a naturally occurring peptide with N-methyl D-aspartate (NMDA) antagonistic properties. A synthetic SHG gene was combined with (i) nerve growth factor-b (NGF-b) amino-terminal signal peptide to enable secretion, and (ii) a fluorescent cellular label (mRFP) with intervening cathepsin L cleavage site, and subcloned into a lentiviral vector. In addition, an endoplasmic retention signal, KDEL, was added to enable retrieval of mRFP. Using immunocytochemistry and confocal microscopic profile analysis, cells transduced by such lentiviruses were shown to synthesize a single SHG-mRFP polypeptide that was processed, targeted to expected subcellular destinations in several cell types. Dot blot and Western analysis revealed stable transduction and long-term secretion of SHG from PC12 cells in vitro. Transplantation of such cells provided modest analgesia in a rodent pain model consistent with low levels of SHG peptide in the cerebrospinal fluid (CSF). These results suggest that it is possible to deliver proteins with different final destinations from a single construct, such as pharmacologically active peptide for secretion and intracellular label for identifying transplantable cells.

Key words: Pain; Transplantation; Lentiviral; NMDA antagonist; PC12 cells; Histogranin

Address correspondence to Shyam Gajavelli, Ph.D., The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, 1095 NW 14th Terrace (R-48), Miami, FL 33136, USA. Tel: (305) 243-6038; Fax: (305) 243-3923; E-mail: SGajavel@med.miami.edu