STEM CELLS AND DEVELOPMENT 17:423–439 (2008)
© Mary Ann Liebert, Inc.
Original Research Report
Peripherally Administered Human Umbilical Cord Blood Cells
Reduce Parenchymal and Vascular ?-Amyloid Deposits
in Alzheimer Mice
WILLIAM V. NIKOLIC,1,6HUAYAN HOU,1,6TERRENCE TOWN,2YUYAN ZHU,3
BRIAN GIUNTA,1CYNDY D. SANBERG,3JIN ZENG,1DEYAN LUO,1JARED EHRHART,1
TAKASHI MORI,1,4PAUL R. SANBERG,5and JUN TAN1,5
Modulation of immune/inflammatory responses by diverse strategies including amyloid-? (A?) im-
munization, nonsteroidal anti-inflammatory drugs, and manipulation of microglial activation states
has been shown to reduce Alzheimer’s disease (AD)-like pathology and cognitive deficits in AD trans-
genic mouse models. Human umbilical cord blood cells (HUCBCs) have unique immunomodulatory
potential. We wished to test whether these cells might alter AD-like pathology after infusion into
the PSAPP mouse model of AD. Here, we report a marked reduction in A? levels/?-amyloid plaques
and associated astrocytosis following multiple low-dose infusions of HUCBCs. HUCBC infusions
also reduced cerebral vascular A? deposits in the Tg2576 AD mouse model. Interestingly, these ef-
fects were associated with suppression of the CD40–CD40L interaction, as evidenced by decreased
circulating and brain soluble CD40L (sCD40L), elevated systemic immunoglobulin M (IgM) levels,
attenuated CD40L-induced inflammatory responses, and reduced surface expression of CD40 on
microglia. Importantly, deficiency in CD40 abolishes the effect of HUCBCs on elevated plasma A?
levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased
phagocytosis of A?. Furthermore, sera from HUCBC-infused PSAPP mice significantly increased
microglial phagocytosis of the A?1-42peptide while inhibiting interferon-?-induced microglial CD40
expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of re-
combinant CD40L protein. These data suggest that HUCBC infusion mitigates AD-like pathology
by disrupting CD40L activity.
1Rashid Laboratory for Developmental Neurobiology, Silver Child Development Center, Department of Psychiatry & Behav-
ioral Medicine, University of South Florida, Tampa, FL 33613.
2Maxine Dunitz Neurosurgical Institute and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles,
CA 90048, and Department of Immunology, Yale University School of Medicine, New Haven, CT 06520.
3Saneron CCEL Therapeutics, Inc, Tampa, FL 33612.
4Institute of Medical Science, Department of Pathology, Saitama Medical Center/University, Kawagoe, Saitama 350-8550,
5Center for Excellence in Aging and Brain Repair, Department of Neurosurgery, University of South Florida, Tampa, FL 33613
6William V. Nikolic and Huayan Hou contributed equally to this project.
ized by deposition of amyloid-? peptide (A?) in the brain
parenchyma. A? plaques are potent activators of both mi-
croglia and astrocytes, central nervous system (CNS)-res-
ident immunocompetent cells that respond to cerebral
amyloidosis by chronic low-level proinflammatory acti-
vation . Although it was once thought that activation
of microglia and astrocytes in the AD brain was an
epiphenomenon and not a pathoetiological contributor to
AD, more recent studies have suggested that the A?-me-
diated inflammatory cascade is an etiological perpetrator
in AD. For example, therapeutic strategies aimed at ma-
nipulating this inflammatory cascade, including A? im-
munization [2–4], nonsteroidal anti-inflammatory drugs
(NSAIDs) [5–9], and modulation of microglial activation
[10–14], are able to reduce AD-like pathology and im-
prove behavioral impairment in Alzheimer’s transgenic
mouse models and, in some cases, reduce AD pathology
Previously, we showed that the CD40–CD40 ligand
(CD40L) interaction plays a critical role in A?-induced
proinflammatory microglial activation . Moreover,
we have demonstrated that disruption of this signaling
pathway reduces cerebral A? deposits in the Tg2576
mouse model of AD and improves cognitive deficits in
PSAPP AD mice [12–15]. The implication of CD40–
CD40L interaction in AD-associated brain inflammatory
process is supported from studies demonstrating in-
creased expression of CD40 and CD40L in and around
?-amyloid plaques in AD brain [16,17]. Recently,
Desideri and colleagues  reported that circulating sol-
uble CD40L (sCD40L) levels are significantly increased
in AD patients versus healthy elderly controls, further
supporting a role for this receptor/ligand dyad in the
pathogenesis of AD.
Human umbilical cord blood cells (HUCBCs) have
been shown to oppose the proinflammatory T helper cell
type 1 (Th1) response, as demonstrated in an animal
model of stroke where HUCBC infusion promoted a
strong anti-inflammatory T helper 2 (Th2) response .
Importantly, this effect was associated with reduced in-
farct volume and rescue of behavioral deficit .
HUCBC infusion has also shown therapeutic benefits in
other neuroinflammatory conditions, including multiple
sclerosis, amyotrophic lateral sclerosis, age-related mac-
ular degeneration, and Parkinson’s disease [20–22]. In
AD preclinical models, administration of these cells to
the PSAPP mice was associated with extension of life-
span, although high doses were administered in this par-
On the basis of these lines of evidence, we investigated
LZHEIMER DISEASE (AD) is the most common pro-
gressive dementia and is pathologically character-
whether multiple low-dose administrations of HUCBCs
to AD transgenic mouse models could reduce AD-like
pathology through suppression of deleterious inflamma-
tory responses involving the CD40 pathway. To address
this possibility, we infused both double-transgenic
PSAPP mice and Tg2576 mice with HUCBCs and then
examined cerebral parenchymal and vascular A? levels/
?-amyloid deposits, astrocytosis, microgliosis, and CD40
MATERIALS AND METHODS
Animals and administration of HUCBCs
HUCBCs (95–98% mononuclear cells) were provided by
Saneron CCEL Therapeutics, Inc. (Tampa, FL). Transgenic
PSAPP (APPswe, PSEN1dE9) and Tg2576 mice were obtained
from the Jackson Laboratory (Bar Harbor, ME) [24,25] and
Taconic, Inc. (Germantown, NY) , respectively, and were
treated intravenously (i.v.) with HUCBCs (100,000 cells/
mouse) or phosphate-buffered saline (PBS) biweekly for the
first 2 months and monthly for the remaining 4 months (n ?
10/group, 5 males and 5 females). Mice were treated starting
at 7 months of age (after appreciable A? deposits), and blood
was collected by submandibular bleeding at 0, 2, 4, and 6
months to monitor plasma cytokines, sCD40L and A? levels
throughout the study. We analyzed brains of these mice for A?
deposits and gliosis at 13 months of age (when these mice man-
ifest well-established AD-like pathology, including A? deposits
and gliosis). We treated PSAPP mice deficient in CD40 and
controls at 8 weeks of age (preliminary studies showed that we
can clearly detect plasma A? levels at this age) with HUCBCs.
Blood samples were collected by submandibular bleeding at the
second month after the treatment. Animals were housed and
maintained in the College of Medicine Animal Facility at the
University of South Florida (USF), and all experiments were
performed in compliance with protocols approved by USF In-
stitutional Animal Care and Use Committee.
Mice were anesthetized with isofluorane and perfused tran-
scardially with ice-cold physiological saline containing heparin
(10 U/ml). Brains were rapidly isolated and quartered using a
mouse brain slicer (Muromachi Kikai, Tokyo, Japan). The first
and second anterior quarters were homogenized for western blot
analysis, and the third and fourth posterior quarters were used
for microtome or cryostat sectioning . Brains were then
fixed in 4% paraformaldehyde in PBS at 4°C overnight and rou-
tinely processed in paraffin at a core facility at the Department
of Pathology (USF College of Medicine). Five coronal sections
from each brain (5 ?m thickness) were cut with a 150-?m in-
terval [for cingulate cortex (CC) bregma ?0.10 mm to ?0.82
mm; for entorhinal cortex (EC) and hippocampus (H), bregma
?2.92 mm to ?3.64 mm]. Sections were routinely deparaf-
finized and hydrated in a graded series of ethanol before pre-
blocking for 30 min at ambient temperature with serum-free
NIKOLIC ET AL.
protein block (Dako Cytomation, Carpinteria, CA). A? im-
munohistochemical staining was performed using anti-human
amyloid-? antibody (4G8) in conjunction with the VectaStain
Elite ABC kit (Vector Laboratories, Burlingame, CA) coupled
with diaminobenzidine substrate. Congo Red staining was done
according to standard practice using 10% (wt/vol) filtered
Congo Red dye cleared with alkaline alcohol. These sections
were rinsed 3? for 5 min each in 70% ethanol, hydrated for 5
min in PBS, and mounted in Vectashield fluorescence mount-
ing medium (Vector Laboratories). ?-Amyloid plaques positive
for 4G8 or Congo Red were detected under bright field using
an Olympus BX-51 microscope. A? burden was determined by
quantitative image analysis. Briefly, images of five 5-?m sec-
tions (150 ?m apart) through each anatomic region of interest
(hippocampus and neocortex) were captured and a threshold op-
tical density was obtained that discriminated staining from
background. Manual editing of each field was used to eliminate
artifacts. Data are reported as percentage of immunolabeled area
captured (positive pixels divided by total pixels captured).
Quantitative image analysis was performed by a single exam-
iner (T.M.) blinded to sample identities.
Double immunofluorescence for A? and CD40 was per-
formed using rat anti-mouse CD40 (1:1,000; Pharmingen, Los
Angeles, CA) and rabbit anti-pan A? (1:100; Biosource Inter-
national, Inc.) with overnight incubation followed by incuba-
tion at ambient temperature with goat anti-rat immunoglobulin
G (IgG) fluorescein isothiocyanate (FITC, 1:50; PharMingen)
and donkey anti-rabbit Alexa Fluor555(1:500; Invitrogen, Carls-
bad, CA) for 45 min. Double immunofluorescence for A? and
activated astrocytes was performed using a biotinylated human
amyloid-? monoclonal antibody (4G8; 1:100, Signet Labora-
tories, Dedham, MA) and glial fibrillary acidic protein (GFAP)
polyclonal antibody (1:500, DAKO). Normal rabbit, normal
mouse serum (isotype control), or PBS (0.1 M, pH 7.4) were
used instead of primary antibody or avidin-biotin-peroxidase
complex (ABC) reagent as negative controls. Quantitative im-
age analysis was done based on a previous method  with
minor modifications. Images were acquired as digitized tagged-
image format files to retain maximum resolution using an
Olympus BX-60 microscope with an attached digital camera
system (DP-70, Olympus, Tokyo, Japan), and digital images
were routed into a Windows PC for quantitative analyses using
SimplePCI software (Compix, Inc. Imaging Systems, Cranberry
Township, PA). The cingulate cortex region was captured from
the image of the cortex adjacent to the sagittal fissure, and the
entorhinal cortex region was captured from the image of the
cortex ventral to the entorhinal fissure. In images from cingu-
late and entorhinal regions, the cortical edge was not included
so that the full anatomic region of interest could be captured.
The hippocampal region was captured from between a portion
of the CA1 subfield of the pyramidal cell layer and the la-
cunosum molecular layer. The anatomical locations and bound-
aries of the regions analyzed were based on those previously
defined . Images of five 5-?m sections through each ana-
tomic region of interest were captured, and a threshold optical
density was obtained that discriminated staining from back-
ground. Each anatomic region of interest was manually edited
to eliminate artifacts. For “burden” analyses, data are repre-
sented as percentage of immunolabeled area captured (positive
pixels) relative to the full area captured (total pixels).
Flow cytometric and western blot analyses
of CD40 expression
For flow cytometric analysis of microglial CD40 expres-
sion, primary cultured microglial cells were plated in six-well
tissue culture plates at 5 ? 105cells/well and incubated with
interferon-? (IFN-?, 100 ng/ml) in the presence or absence of
serum derived from HUCBC- or PBS-infused individual
PSAPP mice. Twelve hours after incubation, microglial cells
were washed with flow buffer, consisting of PBS containing
0.1% (wt/vol) sodium azide, and 2% (vol/vol) fetal calf serum
(FCS), and resuspended in 250 ?l of ice-cold flow buffer for
fluorescence-activated cell sorting (FACS) analysis, according
to methods described previously . Briefly, cells were prein-
cubated with anti-mouse CD16/CD32 monoclonal antibody
(clone 2.4G2, PharMingen) for 10 min at 4°C to block non-
specific binding to Fc receptors. Cells were then centrifuged
at 5,000 ? g, washed three times with flow buffer, and then
incubated in flow buffer with hamster anti-mouse CD40-FITC
or isotype control antibody-FITC (1:100 dilution; PharMin-
gen). After 30 min of incubation at room temperature, cells
were washed twice with flow buffer, resuspended in 250 ?l of
flow buffer and analyzed by a FACScan™ instrument (Becton
Dickinson, Franklin Lakes, NJ). A minimum of 10,000 cells
were accepted for FACS analysis. Cells were gated based on
morphological characteristics using CellQuest™ software
(Beckton Dickinson) such that apoptotic and necrotic cells
were not accepted for FACS analysis. Percentages of positive
(CD40-expressing) cells were calculated as follows: for each
treatment, the mean fluorescence value for the isotype-matched
control antibody was subtracted from the mean fluorescence
value for the CD40-specific antibody.
For western immunoblotting analysis of brain CD40 expres-
sion, mouse brain homogenates were prepared from HUCBC-
and PBS-infused PSAPP mice, as previously described .
An aliquot corresponding to 100 ?g of total protein of each
sample was separated by sodium dodecyl sulfate polyacryl-
amide gel electrophoresis (SDS-PAGE) and transferred elec-
trophoretically to immunoblotting polyvinylidene difluoride
(PVDF) membranes. Nonspecific antibody binding was blocked
with 5% nonfat dry milk for 1 h at room temperature in Tris-
buffered saline (TBS; 20 mM Tris and 500 mM NaCl, pH 7.5).
Subsequently, membranes were first hybridized with rabbit anti-
CD40 antibody (1:1,500 dilution; StressGen, Victoria, Canada)
for 2 h and then washed three times in TBS. Immunoblotting
was by an anti-rabbit horseradish peroxidase (HRP)-conjugated
IgG secondary antibody as a tracer. The luminol reagent was
used to develop the blots. To demonstrate equal loading, the
same membranes were then stripped with ?-mercaptoethanol
stripping solution (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and
100 mM ?-mercaptoethanol) and reprobed with mouse mono-
clonal antibody to actin. Densitometric analysis was done as
previously described  using a FluorS Multiimager with
Quantity One™ software (BioRad, Hercules, CA).
HUCBCs REDUCE AD-LIKE PATHOLOGY BY SUPPRESSING CD40L ACTIVITY
NIKOLIC ET AL.
A? and cytokine enyzme-linked immunosorbent assays
Mouse brains were isolated under sterile conditions on ice
and placed in ice-cold lysis buffer as previously described .
Brains were then sonicated on ice for approximately 3 min, al-
lowed to stand for 15 min at 4°C, and centrifuged at 15,000
rpm for 15 min. This fraction represented the detergent-soluble
fraction. Detergent-insoluble A?1-40,42 species were further
subjected to acid extraction of brain homogenates in 5 M guani-
dine buffer , followed by a 1:5 dilution in lysis buffer.
A?1-40,42was detected in brain homogenates prepared with lysis
buffer or in plasma samples at a 1:10 or 1:5 dilution, respec-
tively, in dilution buffer consisting of PBS ? 1% bovine serum
albumin (BSA) ? phenylmethylsulfonyl fluoride (PMSF).
A?1-40,42 was quantified in these samples using our own
A?1-40,42enzyme-linked immunosorbent assay (ELISA) kits
 and further evaluated with commercially available
A?1-40,42ELISA kits (IBL-America, Minneapolis, MN) in ac-
cordance with the manufacturer’s instructions, except that stan-
dards included 0.5 M guanidine buffer in some cases to facili-
tate A? aggregation. A?1-40,42were represented as pg/ml of
plasma and pg/mg of total protein (mean ? SD).
Cell suspensions of splenocytes from individual mice were
prepared as previously described  and passed in 0.5-ml ali-
quots into 24-well plates at 3 ? 106/ml. These cells were treated
for 48 h with concanavalin A (ConA, 5 ?g/ml). Supernatants
were then collected and assayed by interleukin-10 (IL-10), tu-
mor necrosis factor-? (TNF-?), and IL-12(p70) cytokine
ELISA kits in strict accordance with the manufacturer’s in-
structions (R&D Systems, Minneapolis, MN). The Bio-Rad
protein assay was performed to measure total cellular protein
from each of the cell groups under consideration just prior to
quantification of cytokine release by ELISA, and cytokine se-
cretion was expressed as pg/mg total cellular protein (mean ?
SD). To verify whether stimulation of splenocytes produced any
between-groups differences on cell death that might account for
altered cytokine profiles, lactate dehyrogenase (LDH) release
assay was carried out as described , and LDH was not de-
tected in any of the wells studied. ELISAs for IgM and IgG
antibodies were carried out as previously described . Opti-
cal densities were determined by a microplate reader at 450 nm.
The ratio of IgM to IgG was calculated using optical density
values, and then the average ratio for each group was deter-
mined (mean ? SD). Brain tissue-derived (from the detergent-
soluble brain homogenate fraction) and serum-derived (plasma)
samples were analyzed for sCD40L (Bener MedSystems,
Burlingame, CA), IL-4, IL-10, IL-2, IFN-?, TNF-?, IL-1?, IL-
12 (p70), and transforming growth factor-? (TGF-?) cytokines
by Bioplex assays (Bio-Rad Laboratories) according to the man-
Microglial phagocytosis assay
Primary cultures of murine microglia were established as pre-
viously described [11,33]. For fluorometric analysis of FITC-
A?1–42, primary murine microglia were seeded at 1 ? 105
cells/well (n ? 6 for each condition) in 24-well tissue-culture
plates containing 0.5 ml of complete RPMI-1640 medium. These
cells were treated for 60 min with “aged” A?1-42conjugated
with FITC (Biosource International) . In the presence of
FITC-A?1-42, microglia were then co-treated with serum (1/200,
1/400, 1/800 dilution) derived from HUCBC- or PBS-infused
individual PSAPP mice in the presence or absence of CD40L
protein (2 ?g/ml). Microglia were rinsed three times in A?-free
complete medium, and media were exchanged with fresh A?-
free complete medium for 10 min both to allow for removal of
nonincorporated A? and to promote concentration of the A?
into phagosomes. Extracellular and cell associated FITC-A?
were quantified using an MSF reader (SpectraMax®, Molecular
Devices, Sunnyvale, CA) with an emission wavelength of 538
nm and an excitation wavelength of 485 nm. A standard curve
from 0 nM to 500 nM of FITC-A? was run for each plate. To-
tal cellular proteins were quantified using the Bio-Rad protein
assay. The mean fluorescence values for each sample were de-
termined by fluorometic analysis. Relative fold change values
were calculated as the mean fluorescence value for each exper-
imental sample over control. In this manner, both extracellular
and cell associated FITC-A? were quantified. To determine the
extent to which cell death might have influenced phagocytic ac-
tivity in the various treatment groups, we performed LDH re-
lease assay, and no significant cell death was detected over the
3 h timeframe in any of the treatment groups (p ? 0.05).
Primary culture peripheral macrophages were collected from
3-month-old wild-type mice by infusing their peritoneal cavity
with ice-cold PBS following a 4-day intraperitoneal (i.p.) in-
jection with 1 ml of 3% (wt/vol) brewer’s thyoglycollate re-
suspended in PBS. Cells were pooled following the isolation to
decrease variance. Then they were plated in culture medium
(RPMI-1640; 10% FBS and antibiotics) to give 1.5 ? 106
cells/well in six-well plates. Cells were incubated overnight at
37°C under 5% CO2in a humidified incubator, and nonadher-
ent cells were removed by washing twice with PBS at 37°C.
Following the removal of nonadherent cells, the remaining cells
were tested for A? phagocytosis as described above with the
addition of 1:200, 1:400, and 1:800 dilution of sera derived from
HUCBC-treated mice, sera derived from PBS-treated mice, as
well as supernatants from cultured HUCBCs.
Data are presented as mean ? SD. All statistics were calcu-
lated using one-way analysis of variance (ANOVA) for multi-
ple comparisons. A p value of ?0.05 was considered signifi-
cant. The statistical package for the social sciences release
10.0.5 (SPSS Inc., Chicago, Illinois) was used for all data anal-
Cerebral parenchymal and vascular ?-amyloid
plaques are reduced in AD transgenic mice
peripherally infused with HUCBCs
Previous work in a mouse model of stroke has shown
that HUCBC infusion results in significant reduction in
infarct volume as well as rescue of behavioral deficits as-
sociated with decreased proinflammatory cytokine pro-
duction . We sought to determine whether HUCBC
HUCBCs REDUCE AD-LIKE PATHOLOGY BY SUPPRESSING CD40L ACTIVITY
(95–98% mononuclear cells) infusion could impact A?-
associated pathology in PSAPP double-transgenic mice.
These animals were injected i.v. with HUCBCs (100,000
cells/mouse) beginning at 7 months of age (when ?-amy-
loid deposits have already accumulated). At 13 months
of age, mice were sacrificed and evaluated for changes
in AD-like pathology. We chose to administer multiple
low doses of HUCBCs because, in our pilot studies, we
observed that this strategy was superior compared to a
single high dose of HUCBCs on reducing cerebral amy-
loidosis in Tg2576 mice (data not shown). HUCBC in-
fusion in PSAPP mice resulted in marked reduction of
cerebral ?-amyloid pathology as assayed by A? antibody
(4G8) immunohistochemistry (Fig. 1A) and Congo Red
histochemistry (Fig. 1C). Quantitative image analysis re-
vealed statistically significant differences for each brain
region examined (p ? 0.001) between PSAPP mice in-
fused with HUCBCs (PSAPP/HUCBC) and PSAPP mice
peripherally infused with PBS (PSAPP/PBS) for both A?
antibody (Fig. 1B) and Congo Red staining (Fig. 1D).
Furthermore, ELISA analysis of brain extracts showed
that levels of both detergent-soluble and -insoluble
A?1-40,42peptides were reduced in PSAPP mice infused
with HUCBCs (by 62% and 70%, respectively; Fig. 1E).
A t-test for independent samples revealed significant be-
tween-groups differences for each group examined (p ?
Given that peripheral administration of HUCBCs re-
duces cerebral parenchymal A? deposits and brain A?
levels in PSAPP mice, we wished to investigate the im-
pact of HUCBC infusion on cerebral amyloid angiopa-
thy (CAA), which is characterized by A? deposits in the
cerebral vasculature and is known to occur in 83% of AD
patients . For this analysis, we used the Tg2576
mouse model of AD, which is known to manifest copi-
ous A? deposits in cerebral vessels at 15–20 months of
age [35–39]. We peripherally infused these mice with
HUCBC or controls (PBS vehicle treatment or no treat-
ment) (n ? 10, 5 males and 5 females per group) at 12
months of age using the identical procedure above. Six
months thereafter, these mice were sacrificed for analy-
ses of cerebral parenchymal or vascular ?-amyloid de-
posits by Congo Red histochemistry. As shown in Fig.
1F, Tg2576 mice receiving HUCBC treatment demon-
strated reduction of both cerebral parenchymal and vas-
cular Congo Red deposits compared with controls. Quan-
titative image analysis revealed statistically significant
differences between Tg2576/HUCBC and Tg2576/PBS
or nontreated control groups when examining total
(78%), vascular (86%), or parenchymal (74%) Congo
Red staining (p ? 0.001; Fig. 1G). No significant differ-
ence was revealed between Tg2576/PBS and nontreated
Tg2576 control mice (p ? 0.05). In addition, we also an-
alyzed cerebral A? levels/?-amyloid deposits by A?
ELISA and A? antibody immunohistochemistry, and we
obtained statistically significant results similar to those
observed in HUCBC-infused PSAPP mice (p ? 0.001;
data not shown).
Reduced CD40?microglia and GFAP?
astrocytes in PSAPP mice peripherally
infused with HUCBCs
Previously, it has been suggested that brain inflam-
mation resulting from activated microglia and astrocytes
contributes to ?-amyloid plaque formation, and we have
previously shown that ligation of microglial CD40 en-
ables activation in response to A? peptides [11,42,43].
To investigate whether HUCBCs could inhibit brain in-
flammation, we examined co-localization of ?-amyloid
deposits with CD40?microglia (an in vivo microgliosis
marker ) or reactive GFAP?astrocytes by immuno-
histochemistry and western blot analyses in PSAPP mice.
As shown in Fig. 2A, CD40?microglial cells were re-
duced in the PSAPP/HUCBC-infused group. Quantita-
tive image analysis revealed statistically significant re-
ductions when comparing PSAPP/HUCBC-infused and
PSAPP/PBS-infused groups for both A? and CD40 stain-
ing in hippocampal dentate gyrus and CA1 regions
(**p ? 0.001) (Fig. 2B). Western blot analysis of CD40
expression showed a statistically significant decrease in
brain homogenates from HUCBC-infused PSAPP mice
(p ? 0.001) (Fig. 2C). Furthermore, immunohistochem-
istry/histochemistry and immunofluorescence analyses
showed reductions in ?-amyloid-associated astrocytosis
in PSAPP/HUCBC mice versus PSAPP/PBS-treated
mice (Fig. 2D), and morphometry revealed reductions for
neocortex and hippocampus by 84% and 86%, respec-
tively in PSAPP/HUCBC mice (p ? 0.001) (Fig. 2E).
Increased plasma A? levels correlate with
decreased CD40–CD40L interaction in
HUCBC-infused PSAPP mice
Previously, we have shown that administration of neu-
tralizing CD40L antibody to PSAPP mice results in in-
creased levels of plasma A? concomitant with reduced
cerebral A?/?-amyloid pathology, suggesting that de-
pletion of CD40L promotes brain-to-blood clearance of
A? . It is well-known that the CD40–CD40L inter-
action promotes proinflammatory Th1 and opposes anti-
inflammatory Th2 immune responses [44,45]. In addi-
tion, HUCBC treatment has been shown to be an
immunoregulator in an animal model of stroke [19,46].
We investigated whether reduction of cerebral A? lev-
els/?-amyloid deposits in HUCBC-infused PSAPP mice
might result from increased brain-to-blood clearance of
A?, and be associated with suppression of the proin-
flammatory CD40–CD40L interaction. We probed indi-
vidual blood samples from PSAPP mice infused with
NIKOLIC ET AL.
HUCBCs or PBS for A?1-40,42and sCD40L. ELISA re-
vealed increased plasma A?1-40,42 levels in PSAPP/
HUCBC mice that correlated inversely with decreased
levels of plasma sCD40L in these animals (Figs. 3A–C).
One-way ANOVA followed by post hoc comparison re-
vealed significant differences between PSAPP/HUCBC-
infused and PSAPP/PBS-infused mice for plasma
A?1-40,42levels and plasma sCD40L levels at each time
point indicated (Figs. 3A–C) (**p ? 0.001). It is well es-
tablished that CD40–CD40L interaction on B cells is re-
quired for IgM to IgG antibody class switching. There-
fore, we went on to evaluate the functional consequence
of HUCBC-mediated suppression of the CD40–CD40L
interaction on IgM and IgG titers in mouse blood samples
paraffin-embedded coronal brain sections from the cingulate cortex (CC), hippocampus (H), and entorhinal cortex (EC) were
stained with monoclonal human A? antibody 4G8 (A) or Congo Red (C). Percentages (plaque area/total area) of A? antibody-
immunoreactive deposits (B) or of Congo Red-stained deposits (D) were calculated by quantitative image analysis (mean ? SD;
n ? 10, 5 females and 5 males per group). (E) A? ELISA analysis was carried out for both levels of detergent-soluble A?1-40,42
(top panel) or 5 M guanidine-extracted A?1-40,42(bottom panel). Data are represented as mean ? SD of A?1-40,42(pg/mg pro-
tein). Mouse paraffin-embedded coronal brain sections from hippocampal regions of Tg2576 mice were stained with Congo Red
(F). Positions of the hippocampal subfields CA1, CA3, and dentate gyrus (DG) are indicated (upper left panel). Arrows indicate
A? deposit-affected vessels. (G) Percentages (% labeled area) of Congo Red-stained plaques/vessels were quantified by image
analysis (mean ? SD; n ? 10, 5 females and 5 males), and percentage reduction is indicated.
Cerebral A?/?-amyloid pathology is reduced in PSAPP and Tg2576 mice peripherally infused with HUCBCs. Mouse
HUCBCs REDUCE AD-LIKE PATHOLOGY BY SUPPRESSING CD40L ACTIVITY
NIKOLIC ET AL.
obtained at the time of sacrifice. ELISA data revealed a
significantly increased ratio of IgM to IgG in PSAPP/
HUCBC mice when compared to control (Fig. 3D, *p ?
0.05), suggesting that the CD40 signaling pathway is func-
tionally suppressed in HUCBC-infused PSAPP mice. It
has been recently reported that CD40 deficiency in APP
transgenic mice confers a decrease in A?/?-amyloid loads
. Although to a lesser extent than PSAPP/CD40?/?
mice, we also found that PSAPP/CD40?/?mice do clearly
manifest ?-amyloid deposits (data not shown), allowing
us to test whether administration of HUCBC to PSAPP/
CD40?/?mice resulted in further amelioration of amy-
loidosis in these animals. Thus, we treated PSAPP/
CD40?/?at 8 weeks of age with HUCBCs or PBS (con-
trol) and assayed circulating A? levels, which correlate
with cerebral amyloid levels in transgenic AD mice .
Results indicate no further benefit of HUCBC in PSAPP/
CD40?/?mice on enhanced A?plasma levels (Figs. 3E,F;
p ? 0.05), a presumed indicator of A? brain-to-blood ef-
flux. These data suggest that HUCBCs mediate beneficial
effects on reduction of amyloidosis via reducing CD40
pathway bioactivity, and are consistent with our previous
studies showing that genetic or pharmacologic ablation of
CD40–CD40L interaction mitigates AD-like pathology in
transgenic mice [11,15].
We hypothesized that, if HUCBCs mediated reduced
amyloidosis by reducing CD40 pathway activity, this
should be associated with a shift from pro- to anti-in-
flammatory cytokines in HUCBC-infused PSAPP mice.
Consistent with this hypothesis, we found that plasma
levels of the anti-inflammatory cytokines IL-4 and IL-10
were increased in HUCBC-infused PSAPP mice (Fig.
4A, **p ? 0.001). Furthermore, primary splenocytes
from HUCBC-infused PSAPP mice showed reduced pro-
inflammatory TNF-? and IL-12 (p70) and increased anti-
inflammatory IL-10 secretion (Fig. 4B, **p ? 0.001).
We also analyzed brain cytokine levels by ELISA, and
results showed statistically significant increases in anti-
inflammatory TGF-?1 and IL-10 levels in PSAPP/
HUCBC-infused mouse brain homogenates (Fig. 4C;
**p ? 0.001). Consistent with our data showing reduc-
tion in circulating sCD40L after HUCBC treatment, we
also measured sCD40L in brain homogenates and found
a significant decrease in PSAPP/HUCBC mice compared
to control (Fig. 4D, **p ? 0.001).
HUCBCs inhibit microglial CD40 expression and
enhance in vitro phagocytosis of A? peptides
We and others have previously shown that microglial
CD40 expression is important for CNS inflammatory re-
sponses [11,12,15–17,42,43], and IFN-? is a strong in-
ducer of microglial CD40 expression [48–50]. To inves-
tigate whether a soluble factor secreted following
HUCBC infusion could modulate microglial expression
of CD40, we treated primary microglial cells with serum
from HUCBC- or PBS-infused individual PSAPP mice
in the presence of IFN-? (100 ng/ml) for 8 h. We then
examined CD40 expression by FACS analysis. As shown
in Fig. 5A, sera derived from HUCBC-infused PSAPP
mice significantly inhibited IFN-?-induced microglial
CD40 expression compared to controls (p ? 0.001).
However, this effect was not directly mediated by
HUCBCs or human adult mononuclear cells (HAMNCs),
but was rather due to a soluble circulating factor pro-
duced by HUCBC-infused PSAPP mice (Fig. 5A).
We and others have shown that stimulation of mi-
croglial CD40 results in impaired A? phagocytic activ-
ity  and promotion of microgial neurotoxic inflam-
matory responses . Thus, we wished to examine
whether HUCBCs could enhance microglial phagocyto-
sis of the A? peptide. We prepared primary cultures of
adult microglia from HUCBC- and PBS-infused PSAPP
mice according to previously described methods , and
then subjected these cells to A? phagocytosis assay us-
ing native or A? antibody-opsonized fluorescent-tagged
A?1-42(FITC-A?1-42) according to our previously de-
scribed methods . As shown in Fig. 5B, when mea-
suring fluorescein isothiocyanate (FITC)-tagged A?1-42
in cell supernatants or lysates, one-way ANOVA fol-
lowed by post hoc comparison showed a significant in-
crease in A? uptake by microglia derived from HUCBC-
versus PBS-infused PSAPP mice (**p ? 0.001). Inter-
estingly, the presence of A? IgG (2.5 ?g/ml)  sig-
nificantly enhanced A? uptake by PSAPP/HUCBC- ver-
sus PSAPP/PBS-derived microglial cells (##p ? 0.001).
Given that sera from HUCBC-infused PSAPP mice
suppressed IFN-?-induced microglial CD40 expression,
we wished to test if the sera could increase microglial
A? phagocytosis. We incubated primary cultures of
neonatal microglia with serum from individual PSAPP/
HUCBC- versus PSAPP/PBS mice at 1:200, 1:400, and
1:800 dilutions in the presence of FITC-A?1-42. We found
that sera at the 1:200 dilution markedly enhanced mi-
croglial phagocytosis of A?1-42peptide, which was op-
posed by the presence of recombinant mouse CD40L pro-
tein at 2 ?g/ml (Fig. 5C).
In addition, we wished to test if sera-derived from
HUCBC-treated mice could increase peripheral macro-
phage phagocytic activity. We incubated both sera de-
rived from HUCBCs and PBS-treated animals at 1:200,
1:400, and 1:800 dilutions with primary macrophage
cells from wild-type mice in six-well tissue-culture
plates in the presence of 300 nM FITC-A?1-42as de-
scribed above. We found that sera at the 1:200 dilution
significantly enhanced macrophage phagocytosis of
A?1-42peptide (Fig. 5D) (**p ? 0.01 with n ? 4 for
each treatment group presented). However these effects
were not observed in cultured HUCBC media (data not
HUCBCs REDUCE AD-LIKE PATHOLOGY BY SUPPRESSING CD40L ACTIVITY
rescence was performed on mouse brain coronal paraffin sections prepared from PSAPP mice infused with HUCBC or PBS. The
red signal indicates A??(top panels); green indicates CD40?(middle panels), and merged images (bottom panels) reveal co-lo-
calization of CD40 and A?. DAPI (blue) was used as a nuclear counterstain. (B) Immunofluorescence intensity for A? and CD40
was determined. (C) Western blot analysis shows reduced CD40 expression in brain homogenates from PSAPP/HUCBC versus
PSAPP/PBS mice as indicated (actin was used as an internal reference control). Densitometry analysis shows the ratio of CD40
to actin as indicated below the figure. (D) Immunohistochemistry analysis shows GFAP staining (top panel), and immunofluo-
rescence (bottom panel) reveals co-localization of GFAP (red signal) and A? (green signal). (E) Morphometric analysis results
(mean GFAP/?-amyloid double positive plaques per mouse ? SD) are shown for the neocortex and the hippocampus of
PSAPP/HUCBC versus PSAPP/PBS mice. Percent reduction of plaques double positive for GFAP and A? in PSAPP/HUCBC
mice is indicated.
?-Amyloid-associated microgliosis and astrocytosis are reduced in HUCBC infused-PSAPP mice. (A) Immunofluo-
NIKOLIC ET AL.
On the basis of genetic, biochemical, and post mortem
evidence, A? peptides are key etiological contributors to
AD pathogenesis . In addition to parenchymal A?
deposits, deposition of A? in the cerebral vasculature
(known as CAA) is a pathological feature of AD, and oc-
curs with 83% frequency in AD patients [34,54–56]. A?
has been shown to mediate proinflammatory and neu-
rodegenerative changes, and oligomeric forms of the pep-
tide are neurotoxic . It is well documented that brain
inflammatory mechanisms mediated by reactive glia are
activated in response to A? plaques [1,33,58,59]. Ex-
pression profiles of two such proinflammatory molecules,
CD40 and CD40L, are markedly increased in and around
A? plaques in AD patients and in mouse models of the
disease [16,17], and genetic or pharmacologic blockade
of the CD40–CD40L interaction reduces AD-like pathol-
ogy in transgenic AD mice , suggesting an etiologic
role of this receptor/ligand dyad in the disease [12,14].
In a recent clinical report, it was found that circulating
sCD40L levels are significantly increased in AD patients
, suggesting that peripheral as well as brain dysreg-
ulation of the CD40 pathway occurs in AD. We have pre-
viously shown that CD40 ligation promotes proinflam-
matory activation of microglia and reduces microglial
phagocytosis of A? peptide in vitro [11,33], supporting
a mechanistic explanation for reduced AD-like pathology
after blocking the CD40–CD40L interaction .
HUCBCs have been shown to down-regulate the proin-
flammatory Th1 response in an animal model of stroke
, and have also shown to be of therapeutic benefit in
other neuroinflammatory/neurodegenerative conditions
[20–22]. On the basis of this evidence, we sought to
examine their putative therapeutic value in mitigating AD-
like pathology in transgenic mice. After HUCBC infusion,
treated mice exhibited diminished cerebral A?/?-amyloid
pathology and down-regulation of proinflammatory re-
sponses in the brain and in the periphery. Based on the
conspicuous role of the CD40–CD40L interaction in me-
diating brain proinflammatory response and exacerbating
AD-like pathology, we investigated whether HUCBC-me-
diated reduction of AD-like pathology might be associated
with alteration in this receptor/ligand dyad. Our results
show decreased expression of microglial CD40 and re-
duction in both CNS and peripheral sCD40L concomitant
with HUCBC-induced diminished AD-like pathology,
raising the possibility that disruption of the CD40–CD40L
shown from blood (plasma) for A?1-40(A), A?1-42(B), sCD40L (C), and IgM/IgG (D). Data are presented as mean ? SD (n ?
10) for A?1-40, A?1-42, or sCD40L (pg/ml plasma). Arrows below the panels show the time for each peripheral infusion with
HUCBC or PBS. (D) Data are presented as a ratio of IgM to IgG in blood (plasma) from mice at the 6th month following the
treatment. A? ELISA analysis for A?1-40(E) and A?1-42(F) in blood (plasma) derived from PSAPP/CD40?/?or PSAPP/CD40?/?
mice at the 2nd month, following the third HUCBC infusion. Data in E and F are presented as mean ? SD (n ? 4, 2 males and
2 females) of A?1-40or A?1-42(pg/ml plasma).
HUCBC infusion results in CD40-dependent increased plasma A? levels in PSAPP mice. ELISA analysis results are
HUCBCs REDUCE AD-LIKE PATHOLOGY BY SUPPRESSING CD40L ACTIVITY
interaction may be responsible for mitigation of AD-like
pathology in this scenario. To address this hypothesis di-
rectly, we treated PSAPP mice homozygous deficient for
CD40 with HUCBC and assayed circulating A? levels as
a marker of brain-to-blood A? efflux, and results showed
no further benefit of HUCBC in these mice.
Here, we demonstrate that infusion of the HUCBC
mononuclear fraction into PSAPP and Tg2576 mice re-
sults in reduced levels of both soluble and insoluble brain
A?1-40,42concomitant with increased plasma A?1-40,42
levels. Past studies have suggested brain-to-blood clear-
ance mechanisms that selectively remove A? from the
brain, potentially reducing A? levels in normal as well
as AD patient brains [47,60–63]. Experiments in rat mod-
els demonstrating clearance of A?1-40peptide from the
brain via the blood–brain barrier (BBB) support this no-
tion [62–64]. Vascular endothelial cells, which are im-
portant BBB constituents, express CD40 [14,28,49,65],
and we now show that sCD40L is reduced in blood
plasma from HUCBC-treated PSAPP mice, raising the
possibility that interruption of CD40–CD40L interaction
at the level of cerebrovascular endothelial cells may pro-
mote brain-to-blood clearance of A?. Furthermore, re-
duced circulating sCD40L levels in HUCBC-treated
PSAPP mice raises the possibility that inflammatory cy-
tokines produced by the CD40–CD40L interaction on en-
dothelial cells are reduced. This idea is consistent with
our finding of a shift toward anti-inflammatory cytokines
in the CNS after HUCBC infusion. Interestingly, we also
demonstrate that CAA, which is present in 83% of AD
patients , is reduced by 68% after HUCBC treatment
in Tg2576 AD mice. This result shows that reduction in
parenchymal A? does not come at the cost of increased
vascular A? deposits, unlike a model in which TGF-?1
overexpression reduces parenchymal plaques but in-
creases vascular A? deposits [66,67].
In vitro HUCBC studies have shown that these cells
secrete soluble factors that have beneficial effects
[46,68]. For example, supernatants from cultured
HUCBCs promote survival of NT2 neural cells and pe-
ripheral blood mononuclear cells cultured under condi-
tions designed to induce cell stress and limit protein syn-
thesis . Additionally, HUCBCs have been shown to
produce a number of neurotrophic factors and cytokines
that modulate inflammatory responses, including nerve
growth factor, colony stimulating factor-1, thrombopoi-
etin, and IL-11 [19,69,70]. Previous reports have shown
that HUCBC entry into the brain is not required to pro-
mote neuroprotection , and that recovery following
brain injury is mediated through peripheral responses
shown for plasma-derived (A), splenocyte culture-derived (B), brain tissue-derived cytokines (C), and brain tissue-derived sCD40L
(D). Data are presented as mean ? SD (n ? 10) values of cytokines (pg/ml plasma or medium) (A and B) or fold increase of cy-
tokines over control (untreated) mice (C and D).
HUCBC infusion promotes anti-inflammatory/Th2 responses and decreases sCD40L in the CNS. ELISA results are
NIKOLIC ET AL.
sion and promote A? microglial/macrophage phago-
cytic activity. (A) FACS analysis for CD40 expression
in primary wild-type neonatal microglial cells treated
with cultured medium from HUCBCs or human adult
mononuclear cells (HAMNCs), or serum from individ-
ual PSAPP/HUCBC or PSAPP/PBS mice following
IFN-? challenge. Data are presented as percentage of
CD40 expressing cells (mean ? SD; n ? 5). (B–D) Mi-
croglial/macrophage phagocytosis assay results for ex-
tracellular and cell-associated FITC-A?1-42, which was
detected using a fluorometer. Data are represented as
the relative fold of mean ? SD fluorescence over con-
trol for each sample (n ? 4 for each condition pre-
sented). Primary microglial cells from adult PSAPP/
HUCBC or PSAPP/PBS mice (B), wild-type neonatal
microglia (C), and primary peripheral macrophages (D)
HUCBCs modulate microglial CD40 expres-
. We did not detect infiltration of HUCBCs into brain
parenchyma, either at 4 h after HUCBC administration
or at the time of mouse sacrifice (data not shown), mak-
ing it unlikely that these cells were directly involved in
ameliorating cerebral amyloidosis. Therefore, we hy-
pothesized that a soluble factor produced after HUCBC
infusion in the periphery was responsible for reduced
AD-like pathology and inflammatory response. To test
this, we: (1) measured cytokines in blood plasma, spleen,
and brains from HUCBC- or PBS-treated PSAPP mice,
(2) evaluated the impact of sera from these treated mice
on IFN-?-induced microglial CD40 expression, and (3)
assayed A? phagocytosis in vitro in neonatal microglia
treated with sera from HUCBC/PSAPP or PBS/PSAPP
mice and in adult microglial cultures derived from these
mice. Results generally show a shift from proinflamma-
tory Th1-type cytokines toward anti-inflammatory Th2
cytokines in tissues from HUCBC-treated PSAPP mice.
Furthermore, sera from HUCBC-treated mice are able to
reduce microglial CD40 expression and enhance A?
phagocytosis by these cells. Finally, adult microglia from
HUCBC-treated PSAPP mice have increased capacity to
When taken together, the above results suggest that, in
addition to promoting brain-to-blood A? efflux, HUCBC
infusion promotes production of a peripheral anti-in-
flammatory soluble factor that is likely able to cross the
BBB and affect microglial A? clearance. Previous re-
ports have show that soluble factors, including heat-shock
proteins and pro-inflammatory cytokines, are capable of
modulating A? phagocytosis by microglia [73,74], and
our previous work has shown that microglial CD40–
CD40L interaction retards A? phagocytosis/clearance
. Nonsaturable BBB transport mechanisms have been
described for a number of cytokines including TNF-?
(which is transported via TNF receptors) and IL-1, and
other soluble factors such as leukemia inhibitory factor,
chemoattractant-1, and epithelial growth factor .
Thus, it remains possible that soluble factors produced
by the host in response to HUCBC treatment gain access
to the brain via the BBB and encounter microglia. Ulti-
mately, we propose that infused HUCBCs exert their
effect on reducing cerebral amyloidosis by causing the
host to secrete a soluble factor that acts by reducing
sCD40L–CD40 interaction on microglia, which then pro-
motes microglial clearance of A?. This mechanism is
supported by our observations of: (1) reduced brain lev-
els of sCD40L in HUCBC-infused PSAPP mice, (2) re-
duced CD40 expression on microglia cultured in the pres-
ence of HUCBC-infused PSAPP mouse sera, (3)
increased A? phagocytosis/removal by microglia cul-
tured in the presence of HUCBC-infused PSAPP mouse
sera or cultured from adult PSAPP/HUCBC mice, and
(4) our previous observations that microglial CD40 liga-
tion shifts these cells away from a A? phagocytic phe-
notype and toward a proinflammatory response .
Future studies designed to identify this soluble factor
are warranted and may yield additional pharmacothera-
peutic target(s). Additionally, our observation of no fur-
ther therapeutic benefit of HUCBCs when administered
to PSAPP/CD40?/?mice establishes a CD40 pathway-
dependent mechanism for HUCBC therapeutic benefit on
reduction of cerebral amyloidosis. These results dovetail
with our previous studies showing that the CD40–CD40L
interaction mitigates AD-like pathology in transgenic
Recently, it was shown that peripheral macrophages
are able to infiltrate the brain and limit cerebral amyloi-
dosis in AD mice after irradiation, suggesting that
hematogenously derived macrophages are efficient at
phagocytosing and clearing A? deposits . However,
earlier studies have shown that brain-resident microglia
are also able to phagocytose/clear A? [77–79]. We did
not detect the presence of brain-infiltrating macrophages
in the current experimental paradigm. Specifically, we
stained for CD40 (a marker for both macrophages and
microglia), and noted the presence of process-bearing
cells that morphologically resembled microglia in and
around A? plaques (see Fig. 2A). Also, we did not ob-
serve vascular “cuffing” that would suggest the presence
of infiltrating macrophages that are frequently observed
in other CNS inflammatory conditions such as experi-
mental autoimmune encephalomyelitis . Further-
more, our results provide evidence that both primary cul-
ture microglia and macrophages posses the ability for
enhanced A? phagocytosis following in vitro stimulation
with sera derived from HUCBC-treated animals. This, too,
is consistent with peripheral immunomodulation of the
CD40–CD40L interaction by HUCBC treatment. Addi-
tionally, given the difficulties inherent to discriminating
macrophages from microglia, and the ability of periph-
eral macrophages to engraft into the CNS and take up a
microglial phenotype after brain injury , it remains
possible that peripheral macrophages may contribute to
reduced cerebral amyloidosis after HUCBC treatment.
In this report, we have shown that HUCBC infusion
ameliorates AD-like pathology, including reductions in:
(1) cerebral A? levels/?-amyloid pathology, (2) CAA,
and (3) brain inflammation including CD40?-activated
microglia and GFAP?-activated astrocytes. These effects
of HUCBCs were associated with increased brain-to-
blood efflux of A? and a shift from proinflammatory Th1
to anti-inflammatory Th2 cytokines both in the brain and
in the periphery, similar to what we observed after A?
immunization [31,82,83]. In addition, HUCBC infusion
of PSAPP mice reduces both CNS and circulating
sCD40L levels, and sera from these mice is able to pro-
mote microglial A? phagocytosis. When taken together,
our results provide the basis for a novel immunomodu-
latory strategy for AD using HUCBCs.
HUCBCs REDUCE AD-LIKE PATHOLOGY BY SUPPRESSING CD40L ACTIVITY
This work was supported by the Johnnie B. Byrd, Sr.
Alzheimer’s Center & Research Institute (J.T.), the Na-
tional Institutes of Health/National Institute on Aging
(NIH/NIA, R41AG031586, J.T.), the NIH/National In-
stitute of Neurological Disorders and Stroke (NINDS,
R01NS048335, J.T.); Cryo-Cell International, Inc. and
Saneron CCEL Therapeutic, Inc. T.T. is supported by an
Alzheimer Association grant and an NIH/NIA “Pathway
to Independence” award (K99 AG029726). We thank
Dr. Huntington Potter and Dr. Alison Willing for help-
P.R.S. is a cofounder and J.T. is a consultant for
Saneron CCEL Therapeutics, Inc. and are inventors on a
patent application submitted by USF. P.R.S. was not in-
volved in any data acquisition and analysis.
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HUCBCs REDUCE AD-LIKE PATHOLOGY BY SUPPRESSING CD40L ACTIVITY