Localized donor cells in brain of a Hunter disease patient after cord blood

stem cell transplantation

Ken Araya
a
, Norio Sakai
a
, Ikuko Mohri
a,b
, Kuriko Kagitani-Shimono
a
, Takeshi Okinaga
a
, Yoshiko Hashii
a
,
Hideaki Ohta
a
, Itsuko Nakamichi
c
, Katsuyuki Aozasa
c
, Masako Taniike
a,b,
*
, Keiichi Ozono
a
a
Department of Pediatrics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
b
Department of Molecular Research Center for Childrens Mental Development, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
c
Department of Histopathology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
Article history:
Received 21 May 2009
Accepted 21 May 2009
Available online 24 May 2009
Keywords:
Neuropathology
Immunohistochemistry
Hunter disease
Mucopolysaccharidosis II
Cord blood stem cell transplantation
Iduronate-2-sulfatase
a b s t r a c t
The efcacy of hematopoietic stem cell transplantation (HSCT) for Hunter disease (deciency of
iduronate-2-sulfatase, IDS) remains unclear. We treated a 6-year-old male suffering from a severe type
of Hunter disease with cord blood stem cell transplantation (CBSCT); however, he died at 10 months
post-therapy due to a laryngeal post-transplantation lymphoproliferative disorder. During the follow-
up period after CBSCT, his hyperactivity, estimated mental age, and brain MR ndings had not improved.
We assessed the efcacy of CBSCT by biochemical and pathological analyses of the autopsied tissues.
There were many distended cells with accumulated substrate in the brain, but not in the liver. IDS
enzyme activity in the cerebrum remained very low, although that in the liver reached about 40% of
the normal control level. However, a variable number of tandem repeats analyses demonstrated a weak
donor-derived band not only in the liver but also in the cerebrum. Furthermore, IDS-immunoreactivity in
the liver was recognized broadly not only in Kupffer cells but also in hepatocytes. On the other hand, IDS-
immunoreactivity was recognized exclusively in CD68-positive microglia/monocytes in the patients
brain; whereas that in the normal brain was also detected in neurons and oligodendrocytes. These
donor-derived IDS-positive cells were predominantly localized in perivascular spaces and some of them
were evidently present in the brain parenchyma. The efcacy of CBSCT was judged to be insufcient for
the brain at 10 months post-therapy. However, the pathological detection of donor-derived cells in the
brain parenchyma suggests the potential of HSCT for treatment of neurological symptoms in Hunter dis-
ease. This is the rst neuropathological report documenting the distribution of donor-derived cells in the
brain after CBSCT into a Hunter disease patient.
2009 Elsevier Inc. All rights reserved.
1. Introduction
Hunter disease [1], mucopolysaccharidosis (MPS) II (MIM
+309900), is an X-linked recessive lysosomal storage disorder
(LSD) that arises due to a deciency of iduronate-2-sulfatase
(IDS: EC 3.1.6.13), and leads to the accumulation of glycosamino-
glycan (GAG) in various organs including the central nervous sys-
tem (CNS). In the CNS, distended cells with large clear vacuoles
are observed, and dilatation of the perivascular (VirchowRobin)
space is conspicuous within the cerebral white matter. The neuro-
nal storage materials are positive by periodic acid Schiff (PAS)
staining [2]. A considerable degree of neuronal loss and gliosis is
common [3]. The accumulated substrate with membranous cyto-
plasmic bodies or Zebra bodies is observed in electron micrographs
[4,5]. In the liver, hepatocytes and Kupffer cells are also swollen
with a vacuolated cytoplasm, and accumulated substrate in them
is positive by colloidal iron staining [6,7].
The patients present with multiple progressive symptoms such
as coarse facial features, skeletal deformities, hepatosplenomegaly,
respiratory tract infection, cardiovascular disorders, and various
degrees of CNS involvement. There is a wide spectrum from mild
to severe types, which are classied on clinical grounds because
IDS activity appears equally decient in both types. Although the
mild type is characterized by preservation of intelligence, the se-
vere type, with onset usually occurring between 2 and 4 years of
age, is characterized by mental retardation and death before adult-
hood mainly due to obstructive airway disease and cardiac failure
[8].
There have been no established treatments for MPS II; however,
2 therapeutic possibilities have been pursued, that is, hematopoi-
etic stem cell transplantation (HSCT) and enzyme replacement
therapy (ERT). Although the enzyme administered intravenously
by ERT is not expected to cross the bloodbrain barrier (BBB) [9],
1096-7192/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymgme.2009.05.006
* Corresponding author. Address: Department of Pediatrics, Osaka University
Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
E-mail address: masako@ped.med.osaka-u.ac.jp (M. Taniike).
Molecular Genetics and Metabolism 98 (2009) 255263
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism
j our nal homepage: www. el sevi er . com/ l ocat e/ ymgme
HSCT is considered to provide a constant source of enzyme replace-
ment through the engrafted donor cells, which are not impeded by
the BBB and can thus deliver the enzyme to the CNS [10]. Several
in vitro studies using cultured cells [11] and in vivo ones using
model animals [1214] have conrmed the biochemical, patholog-
ical or functional improvement; therefore, many LSD patients have
been treated with HSCT over the last 25 years [15]. However, only a
few studies have reported about the pathological evaluation of the
human CNS after HSCT [16,17].
Here we document a clinicopathological evaluation of the ef-
cacy of umbilical cord blood stem cell transplantation (CBSCT) on
the neuropathology made by detecting the accumulated substrate
and donor-derived cells in the autopsied tissue of a 6-year-old
male with a severe type of MPS II, involving moderate mental
retardation and brain atrophy, 10 months after CBSCT. Since sex-
matched transplantation had been performed, the strategy of the
detection of donor cells by the use of uorescence in situ hybridiza-
tion for sex chromosomes [18] was not available. Therefore, the
detection of donor cells was performed by immunohistochemical
demonstration of IDS-positive cells, assays of IDS activity, and var-
iable number of tandem repeats (VNTR) analysis.
2. Patient, materials, and methods
2.1. Patient and case history
The patient was born by normal delivery at 40 weeks gestation.
His developmental milestones were normal by the age of
18 months; however, a delay in his language and motor skills
was rst recognized at the age of 2 years. He was suspected of suf-
fering from MPS II because of the developmental delay, the charac-
teristic facial features, and the characteristic X-ray ndings of
skeletal deformities. At the age of 3 years 8 months, the diagnosis
of MPS II was conrmed based on the low activity of IDS in his lym-
phocytes (0.5 nmol/4 h/mg vs. adult control range of 58.4
114 nmol/4 h/mg, measured by SRL Inc., Tokyo, Japan). Genetic
analysis revealed that he harbored a nonsense mutation, W267X,
in exon 6 of his IDS gene on Xq28.1.
At the age of 5 years 11 months, CBSCT was performed by
using cells from an unrelated male donor who had completely
matched HLA phenotypes but 3 loci mismatched HLA genotypes
(donor/recipient: A0201/0206, DR0406/0410, DR1406/1402), after
obtaining informed consent from his parents. The conditioning
regimen for CBSCT consisted of busulfan, cyclophosphamide,
and udarabine. He received short-term methotrexate (15 mg/
m
2
on day 1, 10 mg/m
2
on days 3, 6, and 11) and tacrolimus of
which whole blood concentration was maintained at a trough le-
vel of 515 ng/ml. The detailed protocol was described by Toki-
masa et al. (as Patient 5) [19]. He had not experienced chronic
graft-versus-host-disease (GVHD) but only grade I acute GVHD
requiring no steroid therapy. His bone marrow was conrmed
to be completely of the donor type by VNTR analysis 1 month,
6 months, and 9 months after CBSCT (Supplemental Fig. 1). How-
ever, at the age of 6 years 7 months, he developed severe upper-
airway obstruction necessitating intensive care. The biopsy of his
swollen larynx yielded the diagnosis of EpsteinBarr virus-unas-
sociated post-transplantation lymphoproliferative disorder, and
his condition deteriorated despite 6 courses of rituximab admin-
istration. He eventually died at the age of 6 years 9 months, that
is, 10 months post-CBSCT.
At the age of 5 years 11 months, just before CBSCT, he was
hyperactive and had an estimated mental age of 28 months. His li-
ver was palpable 6 cm below the right costal margin. By the age of
6 years 6 months, 7 months post-CBSCT, his hyperactivity and
mental retardation had not changed signicantly (estimated men-
tal age of 30 months), although his hepatomegaly had improved
(palpable 3 cm below the right costal margin).
2.2. Materials and post-mortem brain tissue processing
We performed a general autopsy of the patient after having
obtained written informed consent from his parents. The post-
morteminterval (PMI) was 3 h. At the autopsy, small blocks of liver
and brain samples were snap-frozen or xed for the electron
microscopical investigation. After 10 months of formalin-xation,
blocks from cerebrum, hippocampus, and cerebellum were cut
out and used for the study. For comparative analysis, formalin-
xed brain blocks (13 months of xation) from a 6-year-5-
month-old boy who had died in our hospital were used as a
non-MPS II control, with informed consent. The formalin-xed
brain blocks were embedded in Tissue-Tek Optimal Cutting Tem-
perature compound (Sakura Finetek Inc., Torrance, CA, USA) after
immersion for 6 days in 0.1 M phosphate buffer (PB) in which
the concentration of saccharose was increased from 5% to 30%,
by 5% per day. Thereafter, they were quickly frozen and sectioned
at 5 lm by a cryostat (JUNG CM 1800, Leica Microsystems GmbH,
Wetzlar, Germany) and mounted onto the aminosilane-coated
microscope slides (Matsunami Glass Ind. Ltd., Osaka, Japan).
In addition, we obtained unxed-frozen cerebrumsamples from
an 11-year-old untreated MPS II patient and a 12-year-old non-
MPS II control, whose PMI were 15 h and 18.5 h, respectively, from
the NICHD Brain and Tissue Bank for Developmental Disorders at
the University of Maryland, Baltimore, MD, USA. The unxed-fro-
zen cerebral samples were sectioned at 5 lm, mounted onto ami-
nosilane-coated microscope slides, and xed by immersion in 20%
formaldehyde solution for 5 min at room temperature. Further-
more, we obtained three unxed-frozen liver samples from
non-MPS II controls (27 years old) from Dr. Masahiro Nakayama,
Osaka Medical Center and Research Institute for Maternal and
Child Health, Osaka, Japan.
This study was approved by the Institutional Review Boards of
Osaka University Graduate School of Medicine.
2.3. Methods
2.3.1. Colloidal iron staining study
After having been rinsed in 12% acetic acid, 5 lm-thick unxed-
frozen liver sections were placed in a solution of 0.25% ferric chlo-
ride and 12% acetic acid for 1 h. They were then rinsed again in 12%
acetic acid, immersed in a mixture of 2.5% potassium ferrocyanide
and 2.5% hydrochloric acid, and counterstained with 0.1% nuclear
fast red and 5% aluminum sulfate. They were observed under a
Microscope BX40 (Olympus Co., Tokyo, Japan) equipped with a
high-sensitivity cooled CCD color camera VB-7010 (Keyence Co.,
Osaka, Japan).
2.3.2 PAS staining studies
The brain sections were oxidized with 0.5% orthoperiodic acid,
and placed in Lillies Cold Schiffs Reagent (0.5% basic fuchsin,
100 mM hydrochloric acid, and 0.5% acid sodium sulte). They
were then rinsed in 0.5% sodium bisulte and 50 mM hydrochloric
acid, counterstained with Mayers hematoxylin (Wako Pure Chem-
ical Industries Ltd., Osaka, Japan), and observed under the Micro-
scope BX40.
2.3.3. Electron microscope (EM) studies
The cerebral and cerebellar samples were xed with 1% parafor-
maldehyde and 3% glutaraldehyde in 0.1 M PB, postxed in 1%
osmium tetroxide, stained with 1% uranyl acetate, and embedded
inEpon812(TAABLaboratories Equipment Ltd., Berkshire, UK). After
256 K. Araya et al. / Molecular Genetics and Metabolism 98 (2009) 255263
observation of semi-thin sections, ultra-thin sections were stained
withleadcitrate andobservedunder a TransmissionElectronMicro-
scope H-7650 (Hitachi High-Technologies Co., Tokyo, Japan).
2.3.4. Enzyme assays
Each unxed tissue sample was homogenized mechanically in
ice-cold deionized water. After having been frozen and thawed,
the homogenate was centrifuged at 1000g for 10 min. After the
determination of protein concentration by the Lowry method
[20], each supernatant was used for enzyme assays. The enzyme
activity of IDS was measured by the method described by Voznyi
et al. [21], and those activities of b-galactosidase (EC 3.2.1.23)
and b-hexosaminidase (EC 3.2.1.52) were measured as described
by Hindman and Cotlier [22].
2.3.5. VNTR analysis
Deoxyribonucleic acid (DNA) samples (0.2 lg), extracted from
each blood or tissue sample, were amplied with 35 cycles of poly-
merase chain reaction in a reaction mixture containing 50 ll of
200 lM deoxynucleotide triphosphate mixture, 2.5 U of AmpliTaq
Gold DNA Polymerase (Applied Biosystems Inc., Foster City, CA,
USA), 10 mM Trishydrochloric acid (pH 8.3), 50 mM potassium
chloride, 1.5 mM magnesium chloride, and an 1 lM concentration
of each primer for amplication of a polymorphic DNA sequence
(pMCT118) on chromosome 1p (D1S80) [23,24]. Each cycle con-
sisted of 1 min at 95 C for denaturation, 1 min at 65 C for anneal-
ing, and 2 min at 70 C for extension. After amplication,
polymorphic bands were detected by agarose gel electrophoresis.
2.3.6. Immunoblot analysis
After solubilization in lysis buffer (50 mM Tris, 150 mM NaCl,
1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, 2 lg/ml aprotinin,
2 lg/ml leupeptin, 2 lg/ml pepstatin A, 0.5 mM phenylmethylsul-
fonyl uoride, and 1 mM dithiothreitol), 40 ng and 0.4 ng recombi-
nant human IDS (Elaprase, Shire Human Genetic Therapies Inc.,
Cambridge, MA, USA) and 40 ng recombinant human a-glucosidase
(Myozyme, Genzyme Co., Cambridge, MA, USA) as a negative con-
trol were separated by sodium dodecyl sulfatepolyacrylamide
gel electrophoresis and transferred electrophoretically to a nitro-
cellulose membrane (Trans-Blot Transfer Medium, Bio-Rad Labora-
tories Inc., Hercules, CA, USA). The membrane was incubated
overnight at 4 C with goat antibody against IDS (0.1 lg/ml,
AF2449, R&D Systems Inc., Minneapolis, MN, USA) after treatment
with 5% skim milk/Tris-buffered saline for blocking nonspecic
reactions. The immune complexes were detected with peroxi-
dase-labeled horse anti-goat IgG (1:10,000, Vector Laboratories
Inc., Burlingame, CA, USA) and SuperSignal West Dura Extended
Duration Substrate (Pierce Chemical Co., Rockford, IL, USA).
2.3.7. Immunohistochemical studies
Brain sections were incubated for 30 min in 0.3% hydrogen per-
oxide/methanol for quenching endogenous peroxidase activity,
and then pretreated with 1% bovine serum albumin (BSA)/phos-
phate buffered saline (PBS) for blocking nonspecic reactions. They
were next incubated overnight at 4 C with the goat antibody
against IDS (10 lg/ml) in 1% BSA/PBS, followed by biotinylated rab-
bit anti-goat IgG (1:100, Vector Laboratories Inc.) for 2 h at room
temperature. The reaction products were visualized by using a Vec-
stain ABC Peroxidase Kit (Vector Laboratories Inc.) and diam-
inobenzidine, and counterstained with Mayers hematoxylin.
They were observed under the Microscope BX40.
For the double immunouorescence study, after the endoge-
nous peroxidase activity had been quenched, brain sections were
incubated for 20 min in 0.1% Sudan black B/70% ethanol for block-
ing autouorescence. After pretreatment with TNB blocking buffer
(PerkinElmer Inc., Waltham, MA, USA), they were incubated
overnight at 4 C with the goat antibody against IDS (2 lg/ml)
and primary antibody against Lamp2 (1:500; made in mouse,
Developmental Studies Hybridoma Bank at the Univ. of Iowa, Iowa,
IA, USA), NSE (1:50; made in rabbit, AB951, Chemicon International
Inc., Temecula, CA, USA), transferrin (1:50; made in rabbit, A0061,
Dako A/S, Glostrup, Denmark), S-100 (1:50; made in rabbit, Z0311,
Dako A/S) or CD68 (1:50; made in mouse, M0876, Dako A/S) in 1%
BSA/PBS, followed by the peroxidase-labeled horse anti-goat IgG
(1:100) and biotinylated horse anti-mouse/rabbit IgG (1:100, Vec-
tor Laboratories Inc.) for 2 h at room temperature. They were sub-
sequently incubated in TSA Plus uorescence reagent working
solution (1:50, PerkinElmer Inc.) for 5 min, followed by Texas Red
streptavidin (1:100, Vector Laboratories Inc.) for 2 h at room tem-
perature, and mounted by using Vectashield mounting medium
with DAPI (Vector Laboratories Inc.). Fluorescence was observed
under a laser scanning confocal microscope LSM 510 META (Carl
Zeiss MicroImaging GmbH, Jena, Germany).
Frozen liver sections were xed by immersion in 4% paraformal-
dehyde solution for 10 min at room temperature. After quenching
endogenous peroxidase activity, they were incubated for 30 min in
0.1% Triton X-100/the TNB blocking buffer with Avidin D solution
(in Avidin/Biotin Blocking Kit, Vector Laboratories Inc.). They were
next incubated overnight at 4 C with the goat antibody against IDS
(2 lg/ml) in 1% BSA/PBS with Biotin solution (in the Avidin/Biotin
Blocking Kit) and subsequently with the peroxidase-labeled horse
anti-goat IgG (1:100) for 30 min at room temperature. As a nega-
tive control, the sections reacted with IDS antibody which had
been preabsorbed overnight at 4 C with an excess amount
(20 lg/ml) of the recombinant human IDS. They were subse-
quently incubated in Biotinyl TSA reagent working solution (1:50,
PerkinElmer Inc.) for 10 min, followed by Streptavidin horseradish
peroxidase (1:100, provided in the TSA kit) for 30 min at room tem-
perature. After having been visualized by use of diaminobenzidine,
the reaction products were counterstained and observed as de-
scribed above.
A double immunouorescence study using the antibody against
IDS (2 lg/ml) and CD68 (1:50) was also performed as described
above.
3. Results
3.1. Brain MR imaging before and after CBSCT
The patients brain MR images just before CBSCT showed evi-
dence of brain atrophy including widening of the cortical sulci
and ventricles (Fig. 1A and B). The uid-attenuated inversion
recovery image also revealed enlarged VirchowRobin spaces
(Fig. 1B, arrowheads) and an abnormally high intensity of the
white matter (Fig. 1B, arrows). At 7 months post-CBSCT, calculation
using the software for structural image evaluation of atrophy (SIE-
NA [25]; a part of Functional MRI of the Brain Software Library, FSL
[26]) estimated the rate of brain volume loss to be 9.9% for
7 months, indicating the progression of brain atrophy (Fig. 1C).
This reduced volume may have been due to not only the procedure
of HSCT [27] but also the natural course of MPS II. These ndings
indicate that the patient had the CNS abnormalities of MPS II be-
fore CBSCT and that the brain atrophy was progressive after CBSCT.
3.2. Detection of accumulated substrate
At autopsy, the crown-heel length was 116 cm and the body
weight was 25.4 kg. The brain was 935 g in weight and showed
marked dilatation of the lateral ventricles and widening of the
cortical sulci. Histologically, the neurons in the cerebral cortex
seemed to be reduced in number with reactive gliosis and
K. Araya et al. / Molecular Genetics and Metabolism 98 (2009) 255263 257
showed moderate cytoplasmic ballooning (Fig. 2A, arrows). On
the other hand, the liver weight (1645 g) had increased (standard
liver weight calculated with reference to the body surface area
was 650 g [28]); and the edge of the organ was dull. However,
hepatocytes and Kupffer cells did not appear to be swollen with
vacuolar changes (Fig. 2B), and most of them were not stainable
with colloidal iron (Fig. 2C). The CNS showed many PAS-positive
cells in the MPS II post-CBSCT (Fig. 2D and E, arrows), whereas no
PAS-positive neurons were observed in the non-MPS II control
(Fig. 2F and G). Toluidine blue-stained cerebellar Purkinje cells
were also ballooned (Fig. 3A, asterisks), and membranous cyto-
plasmic bodies were observed in these cells on electron micro-
graphs (Fig. 3B). In addition, many phagocytic cells with
enlarged intracytoplasmic vacuoles in the VirchowRobin spaces
were observed in the toluidine blue-stained sections (Fig. 3C, ar-
rows) and on electron micrographs (Fig. 3D).
These results indicate that the characteristic abnormal ndings
of MPS II and the accumulated substrate had persisted in the CNS
of the patient at 10 months post-CBSCT, although the characteristic
pathology was not observed in the liver.
3.3. IDS enzyme activity and VNTR analysis
After conrming that the IDS enzyme activity of normal bro-
blasts (88.7 nmol/4 h/mg) was in the normal range (31
110 nmol/4 h/mg [21]) by our assay, we measured IDS activity in
cerebrum and liver of the normal control, the patient (MPS II
post-CBSCT), and the untreated MPS II (Table 1). IDS enzyme activ-
ity in the cerebrum of the patient, as well as in that of the un-
treated MPS II, was only about 1% of that of the normal control.
On the other hand, in the liver of the patient, it was about 40% of
that of the normal control.
The analysis of the VNTR locus D1S80 revealed that the recipi-
ent blood (pre-CBSCT) showed a single band (about 600 bp;
Fig. 4, black arrow) and the donor cord blood had two bands due
Fig. 1. MR images of our MPS II patient just before CBSCT (A and B) and at 7 months
post-CBSCT (C). A and C, T1-weighted image; B, uid-attenuated inversion recovery
(FLAIR) image. The lower gure in B is an enlarged view of the square outlined in
the upper gure in B. Just before CBSCT, the whole brain is atrophic, and the
cortical sulci and ventricles are enlarged (A and B). The enlargement of Virchow-
Robin spaces (B, arrowheads) and abnormally high intensity of the white matter (B,
arrows) are evident. At 7 months post-CBSCT, evidence of the progression of brain
atrophy is observed (C).
Fig. 2. (AC) Hematoxylin and eosin (HE) staining (A and B) and colloidal iron (CI)
staining (C) of MPS II post-CBSCT. A, cerebrum; B and C, liver. Neurons (A, arrows),
but not hepatocytes (B), show cytoplasmic ballooning. Hepatocytes are not
stainable with colloidal iron (C). DG, PAS staining of MPS II post-CBSCT (D and
E) and non-MPS II control (F and G). D and F, cerebrum and E and G, cerebellum.
Many PAS-positive cells (D and E; arrows) are observed in the MPS II post-CBSCT,
whereas no PAS-positive neurons are observed in the non-MPS II control. P,
cerebellar Purkinje cells.
Fig. 3. Toluidine blue-stained semi-thin sections (A and C) and corresponding
electron micrographs (B and D) of MPS II post-CBSCT. In the cerebellum, Purkinje
cells are ballooned (A, asterisks) with membranous cytoplasmic bodies (B). In the
VirchowRobin space, many enlarged cells lled with vacuoles are observed (C,
arrows; D). V, blood vessel; N, nucleus of a vacuolated cell.
258 K. Araya et al. / Molecular Genetics and Metabolism 98 (2009) 255263
to its heterozygosity. Therefore, the presence of donor-derived
cells was able to be conrmed by their lower band (about
450 bp; Fig. 4, white arrow). In addition to the concrete recipi-
ent-derived band which was detected in both liver and cerebrum
of the patient after CBSCT, the weak donor-derived bands were de-
tected in the liver and the cerebrum (Fig. 4, white arrowhead) of
the patient at 10 months post-CBSCT, indicating that some donor
cells had migrated into the liver and the cerebrum. The lower
intensity of the donor-derived band in the cerebrum than in the li-
ver suggested fewer donor-derived cells residing in the cerebrum.
3.4. Immunohistochemical study on IDS
Before performing immunohistochemical studies on IDS, we
investigated the validity of the antibody. According to immunoblot
analysis of IDS, the antibody reacted with recombinant human IDS
having the predicted size of 76 kDa but not with recombinant hu-
man a-glucosidase as a negative control (Fig. 5A, arrow). Although
by IDS immunoblot analysis the antibody did not react with nor-
mal human broblasts, probably because of low level of IDS in
them (data not shown), immunocytochemical study revealed
IDS-immunoreactivity in normal control broblasts (Supplemental
method and Supplemental Fig. 2). According to immunohisto-
chemical studies on unxed-frozen cerebrum samples, IDS-immu-
noreactivity was positive in the non-MPS II control (Fig. 5B,
arrows) but negative in the untreated MPS II (Fig. 5C). Furthermore,
immunoreactivity of IDS (Fig. 5D, green arrows) and that of Lamp2
(Fig. 5E, red arrows), a lysosomal marker, were co-localized in the
cerebrum of the non-MPS II control (Fig. 5F, yellow arrows). These
results conrmed the validity of the antibody against IDS used in
this study.
3.4.1. Distribution of IDS-positive cells in the liver
The routine avidinbiotin complex procedure used in the
immunohistochemistry for the CNS did not work in the normal
liver (data not shown), perhaps because the IDS enzyme activity
there was only about one-tenth of that in the normal cerebrum
(Table 1). Therefore, the Tyramide Signal Amplication system
was applied for the study of the liver.
IDS-immunoreactivity was detected in many cells of the MPS II
post-CBSCT (Fig. 6A) as well as in the non-MPS II control (data not
shown). Prior incubation of IDS antibody with excess antigen com-
pletely abolished the immunoreactivity, conrming the validity of
the staining (Fig. 6B). In the double immunouorescence staining
for IDS and CD68 (Fig. 6CE), IDS-immunoreactivity was detected
in many cells (Fig. 6C, green arrows), not only in CD68-positive
Kupffer cells (Fig. 6D, red arrows; and 6E, yellow arrows) but also
in hepatocytes (Fig. 6E, green arrows) in the liver of the MPS II at
10 months post-CBSCT. These results indicate that there were
many IDS-positive cells were widely distributed not only among
Kupffer cells but also among hepatocytes in the liver at 10 months
after CBSCT.
Table 1
Enzyme activities in cerebrum and liver.
IDS
a
b-Hex
b
b-Gal
b
Cerebrum
Control
c
131.5 1141 71
Patient
c
1.4 2135 44
Untreated MPS II
c
0.5 1931 19
Liver
Control (n = 3) 14.019.7 11441197 214270
Patient
c
6.3 1303 233
Fibroblast
Control 88.7 Not done Not done
a
nmol/4 h/mg.
b
nmol/h/mg.
c
Average of duplicate measurements from different pieces of the tissue.
Fig. 4. VNTR analysis of MPS II post-CBSCT. The donor cord blood-derived band
(about 450 bp, white arrow) can be distinguished from that of the recipient (pre-
CBSCT). The brain of the patient after CBSCT, as well as the liver, seems to have the
donor-derived band (white arrowhead) in addition to the recipient-derived band
(about 600 bp, black arrow).
Fig. 5. (A) Immunoblot analysis of IDS. The antibody against IDS reacts specically
with recombinant human IDS having the predicted size of 76 kDa (arrow). GAA
indicates recombinant human a-glucosidase, used as a negative control. (B and C)
Immunohistochemical study on IDS in the cerebrum of non-MPS II control (B) and
untreated MPS II (C). IDS-immunoreactivity is positive in the non-MPS II control (B,
arrows) but negative in the untreated MPS II (C). (DF) Double immunouorescence
for IDS and Lamp2, a lysosomal marker, in the cerebrum of the non-MPS II control.
Immunoreactivity of IDS (D, green arrows) and that of Lamp2 (E, red arrows) are co-
localized (F, yellow arrows). Blue uorescence in F is from DAPI.
Fig. 6. Immunohistochemical studies on IDS in the liver of MPS II post-CBSCT. (A
and B) IDS-immunoreactivity is extensively detected in many cells (A); however, it
disappears completely when the antibody is preabsorbed with the excess
recombinant human IDS (B). (CE) Double immunouorescence for IDS and CD68.
IDS-immunoreactivity is detected in many cells (C, green arrows), not only in CD68-
positive Kupffer cells (D, red arrows; and E, yellow arrows) but also hepatocytes (E,
green arrows). The blue uorescence is from DAPI.
K. Araya et al. / Molecular Genetics and Metabolism 98 (2009) 255263 259
3.4.2. Distribution of IDS-positive cells in the CNS
IDS-immunoreactivity of the antibody was more evident in the
formalin-xed frozen sections of the non-MPSII control (Fig. 7A)
than in the unxed-frozen sections (Fig. 5B), but the antibody
was not reactive with the parafn sections (data not shown).
Therefore, we used the formalin-xed frozen CNS samples in the
following studies.
In the immunohistochemical study on IDS of the non-MPS II
control and MPS II post-CBSCT, many IDS-immunoreactive large
cells, presumably neurons, were found in the non-MPS II control
(Fig. 7A and B); whereas only a few IDS-immunoreactive small
cells, reminiscent of microglia/monocytes, were found in the MPS
II post-CBSCT (Fig. 7CF). Most IDS-positive small cells were local-
ized in perivascular spaces in the MPS II post-CBSCT (Fig. 7CE).
Although many of them seemed to be in the distended Virchow-
Robin spaces (Fig. 7C, area between red dashed lines), some of
them were evidently found in the brain parenchyma (Fig. 7E, ar-
rows). Also, a very small minority of them were present in the
parenchyma where no blood vessels were found in the
neighborhood (Fig. 7F). On the other hand, no IDS-immunoreactive
neurons were found in the MPS II post-CBSCT.
We next performed double immunouorescence staining for
IDS and Lamp2, NSE, transferrin, S-100 or CD68. IDS-immunoreac-
tivity in the cerebrum of the non-MPS II control was observed in
NSE-positive neurons (Fig. 8AC) and transferrin-positive oligo-
dendrocytes (Fig. 8DF) but not in S-100-positive astrocytes
(Fig. 8GI). Although CD68-positive microglia/monocytes were
only few in number, they were also immunoreactive for IDS
(Fig. 8JL). On the other hand, IDS-immunoreactivity in the
cerebrum of the MPS II post-CBSCT was co-localized with that of
Lamp2 (Fig. 8MO) and found exclusively in CD68-positive cells
(Fig. 8PR) but neither in NSE-, transferrin- nor S-100-positive cells
(data not shown). A cell count showed that about 5.7% (17/297) of
CD68-positive microglia/monocytes had IDS-immunoreactivity.
Fig. 7. Immunohistochemical studies on IDS in the CNS of non-MPS II control (A
and B) and MPS II post-CBSCT (CF). A, C, and E, cerebrum; B and D, cerebellum; F,
hippocampus. Insets represent higher magnication of the rectangle outlined in the
corresponding gures, focusing on IDS-positive cells. The non-MPS II control has
many IDS-positive neurons (A and B), whereas the MPS II post-CBSCT has only a few
IDS-positive small cells predominantly localized in the perivascular space (CE).
Many of IDS-positive cells in the MPS II post-CBSCT seemed to be in the distended
VirchowRobin space (C, area between red dashed lines); however, some of them
have migrated into the brain parenchyma nearby the blood vessel (E, arrows). A
very small number of them are present in the parenchyma where no blood vessels
are found in the neighborhood (F). V, blood vessel; VR, VirchowRobin space, and
arrowhead: an enlarged cell with many vacuoles in the VirchowRobin space (see
also Fig. 3C).
Fig. 8. Double immunouorescence in the cerebrum for IDS and NSE (AC),
transferrin (DF), S-100 (GI) or CD68 (JL) of the non-MPS II control; and for IDS
and Lamp2 (MO) or CD68 (PR) of MPS II post-CBSCT. Green arrows indicate IDS-
positive cells; and red ones, NSE-, transferrin-, S-100-, CD68- or Lamp2-positive
cells. Yellow ones indicate double-positive cells. The blue uorescence is from DAPI.
IDS-immunoreactivity in the non-MPS II control is observed in NSE-, transferrin-,
and CD68-positive cells but not in S-100-positive cells. On the other hand, IDS-
immunoreactivity in the MPS II post-CBSCT is co-localized with that of Lamp2, and
observed exclusively in CD68-positive cells.
260 K. Araya et al. / Molecular Genetics and Metabolism 98 (2009) 255263
4. Discussion
In this study, we evaluated the CNS pathology of a 6-year-old
MPS II male who died at 10 months post-CBSCT. There were many
distended cells with accumulated substrate in the CNS, like in un-
treated cases previously reported [35]; and IDS enzyme activity
there remained very low. However, IDS-immunoreactivity was
found in a few microglia/monocytes predominantly localized in
the perivascular spaces. Furthermore, as far as we know, this study
also identied the IDS-positive cells in the normal brain for the rst
time. The fact that oligodendrocytes expressed IDS in the non-MPS
II control, but not in the MPS II patient, may partly be the reason for
the white matter lesions seen in MPS II [4,29], since impairment of
oligodendrocytes due to IDS deciency may lead to the secondary
dysmyelination.
The antibody we used was produced in goats immunized
with recombinant human IDS (2449-SU, R&D Systems Inc.),
which contains whole mature protein (Ser 26Pro 550); there-
fore, there might be some residual recipient IDS detected by
the antibody in vitro. However, this was not the case in situ be-
cause nonsense-mediated mRNA decay generally degrades
mRNAs that terminate translation more than 5055 nucleotides
upstream of a splicing-generated exonexon junction such as
in our patient [30]. In addition, Chang et al. reported in an
expression study in vitro of the W267X mutation of the IDS gene
that the truncated IDS proteins produced seemed to be trapped
in the endoplasmic reticulum, not in the lysosomes, of transfec-
ted COS-7 [31]; therefore, the trapped proteins were probably
degraded by proteasomes [32]. Furthermore, even if there were
the residual recipient IDS proteins despite of the conditions de-
scribed above, it is very unlikely that they would exist exclu-
sively in microglia/monocytes predominantly localized in the
perivascular spaces. These lines of evidence indicate that the
IDS-immunoreactive cells in our MPS II post-CBSCT patient are
judged to be donor-derived microglia/monocytes that had pene-
trated the blood vessels.
The precise physiological events after HSCT still remain to be
determined. Especially, the efciency of microglia replenishment
by hematopoietic stem cells has remained controversial [33]. In a
study on B6/129 F2 mice after bone marrow transplantation
(BMT), Kennedy and Abkowitz found that donor microglia repre-
sented only 30% of the total microglia at 12 months and that the
donor cells were predominantly seen at perivascular and leptome-
ningeal, but not parenchymal, sites; although 89% of the splenic
monocytes/macrophages were of donor origin by 1 month [34].
Furthermore, in the same report they also showed that the engraft-
ment rate of Kupffer cells in the liver was higher than that of
microglia in the CNS at 6 months after BMT (36% vs. 23%) and that
the engraftment rate increased at 12 months after BMT (52% vs.
30%). Cogle et al. demonstrated that transgender microglia, con-
taining a Y chromosome, made up 12% of all microglia and that
transgender neurons and astrocytes were present in the hippocam-
pus of female patients up to 6 years after HSCT [18]. On the other
hand, microglia progenitor recruitment from the circulation was
not found in denervation or CNS neurodegenerative disease of chi-
meric animals obtained by parabiosis without brain conditioning
procedures such as irradiation [35]. The discrepancy about the
microglia progenitor recruitment in the CNS might reect the dif-
ference in neurological disorders, the age, and the conditioning
regimen for HSCT. In our study, IDS-immunoreactivity was found
in about 5.7% of the microglia/monocytes predominantly localized
in perivascular spaces of the patient at 10 months post-CBSCT. Our
result is largely compatible with the above-mentioned study by
Kennedy et al. The engraftment rate might have been higher if
the patient had lived longer. On the other hand, we detected no
IDS-immunoreactivity in neurons, oligodendrocytes or astrocytes
of the patient.
HSCT has been reported to be much more effective in the liver
thanintheCNS[16,34,36]. Resnicket al. foundthat liver biopsyspec-
imens fromMPS patients who had achieved metabolic correction by
BMTwere not stainable withcolloidal iron[37], althoughthose from
untreatedcases had hepatocellular dilatationwithrarefactionof the
cytoplasm, which gave positive staining with colloidal iron [38]. In
addition, ina studyperformingBMTonmodel mice of Hurler disease
(MPS I: MIM +607014), Zheng et al. reported the disappearance of
the storage vacuoles in both Kupffer cells and hepatocytes, and the
broad distribution of a-L-iduronidase (EC 3.2.1.76), a decient en-
zyme of MPS I, with sparse distribution of the bone-marrowderived
cells in the liver [14]. In our case, the hepatomegaly was clinically
improved at 7 months post-CBSCT, and hepatocytes and Kupffer
cells did not appear to be swollen with apparent intracytoplasmic
colloidal iron-positive substrate at 10 months post-CBSCT. Also, a
higher intensity of the donor-derived band was detected in the liver
than in the cerebrum by VNTR analysis. Furthermore, the liver
showed about 40% of the normal IDS enzyme activity, and many
IDS-immunoreactive Kupffer cells andhepatocytes. Althoughevalu-
ation of the hepatic pathology of the patient before CBSCT had been
not performed, it is very unlikely that residual IDS activity was pres-
ent in a tissue-specic way such that the residual IDS activity was
preserved only in the liver. Therefore, IDS-immunoreactivity in the
liver after CBSCT was judged to be donor-derived. Our result and
those of previous studies suggest that donor-derivedmigratingcells,
such as Kupffer cells, secreted IDS enzyme that was taken up by
neighboring recipient hepatocytes and Kupffer cells sufciently to
correct GAG metabolism in the liver of the MPS II patient at
10 months post-CBSCT.
There are some studies documenting that accumulated sub-
strate persists in the peripheral nerves of MPS II patients for up
to 2 years after HSCT [36,39]. With regard to other inherited met-
abolic diseases, Will et al. reported that pronounced cytoplasmic
vacuolation was seen in the brain of a 7-year-old boy with a-man-
nosidosis (MIM #248500) at 18 weeks post-BMT [16]. Although
the precise evaluation of peripheral nerves was impossible, our
data seem to be compatible with their study showing that the
accumulated substrate in neural tissues had not diminished less
than 2 years after HSCT.
There are many clinical studies and reviews that have concluded
that HSCT does not signicantly alter the natural history of severe
cases of MPS II [4044]. Guffon et al. reported that 4 MPS II children,
whose development or intelligence quotient at BMT had been from
65 to 70, had deteriorated after BMT [44]. On the other hand, in a
study of 54 MPS I children, many patients could achieve a favorable
long-term outcome after successful BMT [45]. The reason for the
neurologically poorer outcome in MPS II than in MPS I is not clear;
however, there may be two explanations for the different efcacy.
First of all, it may be because the diagnosis and treatment are gener-
allydelayedmoreinchildrenwithMPSII due totheslower onset and
progressionthaninthose withMPSI. Indeed, Escolar et al. concluded
that CBSCT in newborns with infantile Krabbes disease (MIM
#245200), but not symptomatic babies, favorablyalteredthenatural
historyof thedisease. Secondly, it maybebecauseHSCTitself maybe
less effective in the CNS of MPS II than in that of MPS I even at the
early stage of disease. Zheng et al. reported a reduction in the num-
ber of vacuolated neurons and migration of hematopoietic donor
cells to the brain of MPS I mice with retrovirally transduced bone
marrow [14], and Ellinwood et al. reported a signicant decrease
in the brain GAG levels of MPS I felines after BMT [13]. In addi-
tion, Bikenmeier et al. reported a dramatic reduction in storage
of accumulated substrate in perivascular and meningeal cells,
but not in neurons and scattered glial cells, in the brain of
MPS VII mice after BMT [46]. In our study, there were many dis-
K. Araya et al. / Molecular Genetics and Metabolism 98 (2009) 255263 261
tended cells with accumulated substrate and only a few IDS-po-
sitive microglia/monocytes in the CNS of our patient. The ab-
sence of IDS-immunoreactivity in oligodendrocytes or neurons
can be due to either the true absence of IDS in these cells (the
absence of effective transfer of enzyme) or the limitation of
the immunohistochemical method to detect IDS present at low
concentrations in these cells. Our ndings might explain the rea-
son why HSCT fails to arrest neurological progression in MPS II.
However, despite our results and those of the previous studies
described above, evidence for the reason for this issue has been
insufcient yet; because there have been no studies about the
brain pathology of any type of MPS patients, not model animals,
after HSCT. Therefore, the issue may need more neuropathologi-
cal studies on human MPS patients after HSCT.
Our study is unique and valuable in evaluating the efcacy of
HSCT by detecting the donor-derived cells; since there has been
only one report documenting the fate of donor-derived cells after
HCST in patients with inherited metabolic diseases. Schonberger
et al. reported that immunohistochemical staining for adrenoleu-
kodystrophy (ALD, MIM #300100) protein revealed no differ-
ences between the brain of a 15-year-old ALD patient at
76 days post-HSCT and that of the control [17]. Their results
are in sharp contrast with the study by Yamada et al., who doc-
umented that little ALD protein was detected in the brain of an
ALD model mouse at 6 months post-HSCT [47]. After evaluation
of the accumulated substrate and IDS enzyme activity, we con-
clude that only a few donor-derived cells had penetrated into
the CNS of our MPS II patient at 10 months post-HSCT and that
the number of donor-derived cells was insufcient for metabolic
improvement. However, our ndings showing the existence of
donor cells in the brain parenchyma at 10 months post-CBSCT
suggest the potential of HSCT for treatment of MPS II.
The issue of HSCT for LSD patients requires detailed discussion
regarding the indication, timing, and conditioning regimen. There-
fore, the accumulation of neuropathological evidence, such as our
study, is very valuable for establishment of evidence-based proto-
cols of HSCT for these patients.
Acknowledgments
We thank Dr. Kinuko Suzuki, Tokyo Metropolitan Institute of
Gerontology, for critical reading of the manuscript; Dr. Akemi
Tanaka, Department of Pediatrics, Osaka City University Gradu-
ate School of Medicine, for the gift of reagents and advice on
the IDS enzyme assay; and Dr. Kazuhiko Bessho and Dr. Hidet-
oshi Taniguchi, Department of Pediatrics, Osaka University
Graduate School of Medicine, for their advice and technical
supervision.
This research was supported by a grant from Research on Mea-
sures for Intractable Diseases, Japanese Ministry of Health, Welfare
and Labor (to N.S.); and Grants-in-Aid for Scientic Research C
from the Ministry of Education, Culture, Sports, Science, and Tech-
nology of Japan (No. 19591205 to I.M. and No. 18790715 to K.K.S.).
We obtained unxed-frozen cerebrum samples from an 11-
year-old untreated MPS II patient and a 12-year-old non-MPS II
control from the NICHD Brain and Tissue Bank for Developmental
Disorders at the University of Maryland, Baltimore, MD, USA. The
role of the NICHD Brain and Tissue Bank is to distribute tissue;
and, therefore, it cannot endorse the studies performed or the
interpretations of results.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ymgme.2009.05.006.
References
[1] C. Hunter, A rare disease in two brothers, Proc. R. Soc. Med. 10 (1917) 104116
(Sect Study Dis Child).
[2] R.D. Lillie, H.M. Fullmer, Polysaccharides; Mucins, in: Histopathologic Technic
and Practical Histochemistry, fourth ed., McGraw-Hill, New York, 1976, pp.
611678.
[3] K. Suzuki, K. Suzuki, Lysosomal diseases, in: S. Love, D.N. Louis, D.W. Ellison
(Eds.), Greenelds Neuropathology, vol. I, eighth ed., Hodder Arnold, London,
2008, pp. 515599.
[4] K. Nagashima, H. Endo, K. Sakakibara, Y. Konishi, K. Miyachi, J.J. Wey, Y. Suzuki,
J. Onisawa, Morphological and biochemical studies of a case of
mucopolysaccharidosis II (Hunters syndrome), Acta Pathol. Jpn. 26 (1976)
115132.
[5] M. Kurihara, K. Kumagai, K. Goto, M. Imai, S. Yagishita, Severe type Hunters
syndrome. Polysomnographic and neuropathological study, Neuropediatrics
23 (1992) 248256.
[6] C.W. Hale, Histochemical demonstration of acid polysaccharides in animal
tissues, Nature 157 (1946) 802.
[7] B.C. Portmann, R.J. Thompson, E.A. Roberts, A.C. Paterson, Genetic and
metabolic liver disease, in: A.D. Burt, B.C. Portmann, L.D. Ferrell (Eds.),
MacSweens Pathology of the Liver, fth ed., Churchill Livingstone,
Philadelphia, 2007, pp. 199326.
[8] E. Neufeld, J. Muenzer, The mucopolysaccharidoses, in: C.R. Scriver, A.L.
Beaudet, W.S. Sly, D. Valle, B. Childs, K.W. Kinzler, B. Vogelstein (Eds.), The
Metabolic & Molecular Bases of Inherited Disease, vol. III, eighth ed., McGraw-
Hill, New York, 2001, pp. 34213452.
[9] J.E. Wraith, M. Scarpa, M. Beck, O.A. Bodamer, L. De Meirleir, N. Guffon, A.
Meldgaard Lund, G. Malm, A.T. Van der Ploeg, J. Zeman, Mucopolysaccharidosis
type II (Hunter syndrome): a clinical review and recommendations for
treatment in the era of enzyme replacement therapy, Eur. J. Pediatr. 167
(2008) 267277.
[10] V.K. Prasad, J. Kurtzberg, Emerging trends in transplantation of inherited
metabolic diseases, Bone Marrow Transplant. 41 (2008) 99108.
[11] J.C. Fratantoni, C.W. Hall, E.F. Neufeld, Hurler and Hunter syndromes: mutual
correction of the defect in cultured broblasts, Science 162 (1968) 570572.
[12] S.U. Walkley, M.A. Thrall, K. Dobrenis, M. Huang, P.A. March, D.A. Siegel, S.
Wurzelmann, Bone marrow transplantation corrects the enzyme defect in
neurons of the central nervous system in a lysosomal storage disease, Proc.
Natl. Acad. Sci. USA 91 (1994) 29702974.
[13] N.M. Ellinwood, M.A. Colle, M.A. Weil, M.L. Casal, C.H. Vite, S. Wiemelt, C.W.
Hasson, T.M. OMalley, X. He, U. Prociuk, L. Verot, J.R. Melniczek, A. Lannon,
G.D. Aguirre, V.W. Knox, S.M. Evans, M.T. Vanier, E.H. Schuchman, S.U.
Walkley, M.E. Haskins, Bone marrow transplantation for feline
mucopolysaccharidosis I, Mol. Genet. Metab. 91 (2007) 239250.
[14] Y. Zheng, N. Rozengurt, S. Ryazantsev, D.B. Kohn, N. Satake, E.F. Neufeld,
Treatment of the mouse model of mucopolysaccharidosis I with retrovirally
transduced bone marrow, Mol. Genet. Metab. 79 (2003) 233244.
[15] J.R. Hobbs, K. Hugh-Jones, A.J. Barrett, N. Byrom, D. Chambers, K. Henry, D.C.
James, C.F. Lucas, T.R. Rogers, P.F. Benson, L.R. Tansley, A.D. Patrick, J.
Mossman, E.P. Young, Reversal of clinical features of Hurlers disease and
biochemical improvement after treatment by bone-marrow transplantation,
Lancet 2 (1981) 709712.
[16] A. Will, A. Cooper, C. Hatton, I.B. Sardharwalla, D.I. Evans, R.F. Stevens, Bone
marrow transplantation in the treatment of alpha-mannosidosis, Arch. Dis.
Child. 62 (1987) 10441049.
[17] S. Schonberger, P. Roerig, D.T. Schneider, G. Reifenberger, U. Gobel, J. Gartner,
Genotype and protein expression after bone marrow transplantation for
adrenoleukodystrophy, Arch. Neurol. 64 (2007) 651657.
[18] C.R. Cogle, A.T. Yachnis, E.D. Laywell, D.S. Zander, J.R. Wingard, D.A. Steindler,
E.W. Scott, Bone marrow transdifferentiation in brain after transplantation: a
retrospective study, Lancet 363 (2004) 14321437.
[19] S. Tokimasa, H. Ohta, S. Takizawa, S. Kusuki, Y. Hashii, N. Sakai, M. Taniike, K.
Ozono, J. Hara, Umbilical cord-blood transplantations from unrelated donors
in patients with inherited metabolic diseases: single-institute experience,
Pediatr. Transplant. 12 (2008) 672676.
[20] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with
the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265275.
[21] Y.V. Voznyi, J.L. Keulemans, O.P. van Diggelen, A uorimetric enzyme assay for
the diagnosis of MPS II (Hunter disease), J. Inherit. Metab. Dis. 24 (2001) 675
680.
[22] J. Hindman, E. Cotlier, Glycosidases in normal human leukocytes and
abnormalities in G M1-gangliosidosis, Clin. Chem. 18 (1972) 971975.
[23] Y. Nakamura, M. Carlson, K. Krapcho, R. White, Isolation and mapping of a
polymorphic DNA sequence (pMCT118) on chromosome 1p [D1S80], Nucleic
Acids Res. 16 (1988) 9364.
[24] K. Kasai, Y. Nakamura, R. White, Amplication of a variable number of tandem
repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and
its application to forensic science, J. Forensic Sci. 35 (1990) 11961200.
[25] S.M. Smith, Y. Zhang, M. Jenkinson, J. Chen, P.M. Matthews, A. Federico, N. De
Stefano, Accurate, robust, and automated longitudinal and cross-sectional
brain change analysis, Neuroimage 17 (2002) 479489.
[26] S.M. Smith, M. Jenkinson, M.W. Woolrich, C.F. Beckmann, T.E. Behrens, H.
Johansen-Berg, P.R. Bannister, M. De Luca, I. Drobnjak, D.E. Flitney, R.K. Niazy, J.
Saunders, J. Vickers, Y. Zhang, N. De Stefano, J.M. Brady, P.M. Matthews,
262 K. Araya et al. / Molecular Genetics and Metabolism 98 (2009) 255263
Advances in functional and structural MR image analysis and implementation
as FSL, Neuroimage 23 (Suppl. 1) (2004) S208S219.
[27] J.T. Chen, D.L. Collins, H.L. Atkins, M.S. Freedman, A. Galal, D.L. Arnold, Brain
atrophy after immunoablation and stem cell transplantation in multiple
sclerosis, Neurology 66 (2006) 19351937.
[28] T. Yoshizumi, G.E. Gondolesi, C.A. Bodian, H. Jeon, M.E. Schwartz, T.M. Fishbein,
C.M. Miller, S. Emre, A simple new formula to assess liver weight, Transplant.
Proc. 35 (2003) 14151420.
[29] L. Vedolin, I.V. Schwartz, M. Komlos, A. Schuch, A.C. Puga, L.L. Pinto, A.P. Pires,
R. Giugliani, Correlation of MR imaging and MR spectroscopy ndings with
cognitive impairment in mucopolysaccharidosis II. AJNR, Am. J. Neuroradiol.
28 (2007) 10291033.
[30] L.E. Maquat, Nonsense-mediated mRNA decay in mammals, J. Cell Sci. 118
(2005) 17731776.
[31] J.H. Chang, S.P. Lin, S.C. Lin, K.L. Tseng, C.L. Li, C.K. Chuang, G.J. Lee-Chen,
Expression studies of mutations underlying Taiwanese Hunter syndrome
(mucopolysaccharidosis type II), Hum. Genet. 116 (2005) 160166.
[32] K. Romisch, Endoplasmic reticulum-associated degradation, Annu. Rev. Cell
Dev. Biol. 21 (2005) 435456.
[33] N. Davoust, C. Vuaillat, G. Androdias, S. Nataf, From bone marrow to microglia:
barriers and avenues, Trends Immunol. 29 (2008) 227234.
[34] D.W. Kennedy, J.L. Abkowitz, Kinetics of central nervous system microglial and
macrophage engraftment: analysis using a transgenic bone marrow
transplantation model, Blood 90 (1997) 986993.
[35] B. Ajami, J.L. Bennett, C. Krieger, W. Tetzlaff, F.M. Rossi, Local self-renewal can
sustain CNS microglia maintenance and function throughout adult life, Nat.
Neurosci. 10 (2007) 15381543.
[36] E.J. McKinnis, S. Sulzbacher, J.C. Rutledge, J. Sanders, C.R. Scott, Bone marrow
transplantation in Hunter syndrome, J. Pediatr. 129 (1996) 145148.
[37] J.M. Resnick, W. Krivit, D.C. Snover, J.H. Kersey, N.K. Ramsay, B.R. Blazar, C.B.
Whitley, Pathology of the liver in mucopolysaccharidosis: light and electron
microscopic assessment before and after bone marrow transplantation, Bone
Marrow Transplant. 10 (1992) 273280.
[38] J.M. Resnick, C.B. Whitley, A.S. Leonard, W. Krivit, D.C. Snover, Light and
electron microscopic features of the liver in mucopolysaccharidosis, Hum.
Pathol. 25 (1994) 276286.
[39] T. Ochiai, K. Ito, H. Shichino, M. Chin, H. Mugishima, Ultrastructural ndings of
cutaneous nerves in patients with Hunters syndrome following hematopoietic
stem cell transplant, Med. Mol. Morphol. 38 (2005) 118122.
[40] E.G. Shapiro, L.A. Lockman, M. Balthazor, W. Krivit, Neuropsychological
outcomes of several storage diseases with and without bone marrow
transplantation, J. Inherit. Metab. Dis. 18 (1995) 413429.
[41] J.J. Malatack, D.M. Consolini, E. Bayever, The status of hematopoietic stem cell
transplantation in lysosomal storage disease, Pediatr. Neurol. 29 (2003) 391403.
[42] W. Krivit, Allogeneic stem cell transplantation for the treatment of lysosomal and
peroxisomal metabolic diseases, Springer Semin. Immunopathol. 26 (2004) 119132.
[43] P.J. Orchard, B.R. Blazar, J. Wagner, L. Charnas, W. Krivit, J. Tolar, Hematopoietic
cell therapy for metabolic disease, J. Pediatr. 151 (2007) 340346.
[44] N. Guffon, Y. Bertrand, I. Forest, A. Fouilhoux, R. Froissart, Bone marrow
transplantation in children with Hunter syndrome: outcome after 7 to 17
years, J. Pediatr. 154 (2009) 733737.
[45] C. Peters, E.G. Shapiro, J. Anderson, P.J. Henslee-Downey, M.R. Klemperer, M.J.
Cowan, E.F. Saunders, P.A. deAlarcon, C. Twist, J.B. Nachman, G.A. Hale, R.E.
Harris, M.K. Rozans, J. Kurtzberg, G.H. Grayson, T.E. Williams, C. Lenarsky, J.E.
Wagner, W. Krivit, Hurler syndrome. II. Outcome of HLA-genotypically
identical sibling and HLA-haploidentical related donor bone marrow
transplantation in fty-four children. The Storage Disease Collaborative
Study Group, Blood 91 (1998) 26012608.
[46] E.H. Birkenmeier, J.E. Barker, C.A. Vogler, J.W. Kyle, W.S. Sly, B. Gwynn, B. Levy,
C. Pegors, Increased life span and correction of metabolic defects in murine
mucopolysaccharidosis type VII after syngeneic bone marrow transplantation,
Blood 78 (1991) 30813092.
[47] T. Yamada, Y. Ohyagi, N. Shinnoh, H. Kikuchi, M. Osoegawa, H. Ochi, J. Kira, H.
Furuya, Therapeutic effects of normal cells on ABCD1 decient cells in vitro and
hematopoietic cell transplantation in the X-ALD mouse model, J. Neurol. Sci.
218 (2004) 9197.
K. Araya et al. / Molecular Genetics and Metabolism 98 (2009) 255263 263