Journal of Translational Medicine
Open Access
Stem Cell Therapy for Autism
Abstract
Autism spectrum disorders (ASD) are a group of neurodevelopmental conditions
whose incidence is reaching epidemic proportions, afflicting approximately 1
in 166 children. Autistic disorder or autism is the most common form of ASD.
Although several neurophysiological alterations have been associated with
autism, immune abnormalities and neural hypoperfusion appear to be broadly
consistent. These appear to be causative since correlation of altered
inflammatory responses, and
hypoperfusion with symptology is reported. Mesenchymal stem cells (MSC) are
in late phases of clinical development for treatment of graft versus host
disease and Crohn's Disease, two conditions of immune dysregulation. Cord
blood CD34+ cells are known to be potent angiogenic stimulators, having
demonstrated positive effects in not only peripheral ischemia, but also in
models of cerebral ischemia. Additionally, anecdotal clinical cases have
reported responses in autistic children receiving cord blood CD34+ cells. We
propose the combined use of MSC and cord blood CD34+cells may be useful in
the treatment of autism.
Background
Autism spectrum disorders (ASD) are reaching epidemic proportions, believed
to affect approximately 1 in 166 children. Autism, Asperger's syndrome,
Rett's disorder, and childhood disintegrae disorder are all encompassed by
the term ASD. Autism is the most prevalent ASD, characterized by
abnormalities in social interaction, impaired verbal and nonverbal
communication, and repetitive, obsessive behavior. Autism may vary in
severity from mild to disabling and is believed to arise from genetic and
environmental factors. While symptomology of autism may be noted by
caregivers around 12–18 months [1], definitive diagnosis generally occurs
around 24–36 months, however in some cases diagnosis may be made into
adulthood. Determination of autism is performed using the DSM-IV-TR, or
other questionnaires and tests. Children with autism appear withdrawn,
self-occupied, and distant. Inflexibility in terms of learning from
experiences and modifying patterns to integrate into new environments is
characteristic of autism. Depending on degree of severity, some children
with autism may develop into independent adults with full time employment
and self sufficiency; however this is seldom the case. Current treatments
for autism can divided into behavioral, nutritional and medical approaches,
although no clear golden standard approach exists. Behavioral interventions
usually include activities designed to encourage social interaction,
communication, awareness of self, and increase attention. Nutritional
interventions aim to restrict allergy-associated dietary components, as well
as to supplement minerals or vitamins that may be lacking. Medical
interventions usually treat specific activities associated with autism. For
example, serotonin reuptake inhibitors (SSRI's) such as fluoxetine,
fluvoxamine, sertraline, and clomipramine, are used for treatment of anxiety
and depression. Some studies have shown that SSRI's also have the added
benefit of increasing social interaction and inhibiting repetitive behavior.
Typical antipsychotic drugs such as thioridazine, fluphenazine,
chlorpromazine, and haloperidol have been showed to decrease behavioral
abnormalities in autism. Atypical antipsychotics such as risperidone,
olanzapine and ziprasidone have also demonstrated beneficial effect at
ameliorating behavioral problems. Autism associated seizures are mainly
treated by administration of anticonvulsants such as carbamazepine,
lamotrigine, topiramate, and valproic acid. Attention
deficient/hyperacti
(Ritalin®). Currently, numerous clinical trials are being conducted with
interventions ranging from hyperbaric oxygen, to administration of zinc, to
drugs exhibiting anti-inflammatory properties. Unfortunately, no clear
understanding of autism's pathogenic mechanisms exists, and as a result
numerous strategies are being attempted with varying degrees of success. In
this paper we examine two pathologies associated with autism – hypoperfusion
to the brain and immune dysregulation – and propose a novel treatment: the
administration of CD34+ umbilical cord cells and Mesenchymal cells.
Hypoperfusion of brain in autism
Children with autism have been consistently shown to have impaired, or
subnormal CNS circulation, as well as resulting hypoxia. Defects include
basal hypoperfusion, and decreased perfusion in response to stimuli that
under normal circumstances up regulates perfusion. In numerous studies the
areas affected by hypoperfusion seem to correlate with regions of the brain
that are responsible for functionalities that are abnormal in autism. For
example, specific temporal lobe areas associated with face recognition,
social interaction, and language comprehension, have been demonstrated to be
hypoperfused in autistic but not control children. The question of cause
versus effect is important. If temporal lobe ischemia is not causative but
only a symptom of an underlying process, then targeting this pathology may
be non-productive from the therapeutic perspective. However this appears not
to be the case. It is evident that the degree of hypoperfusion and resulting
hypoxia correlates with the severity of autism symptoms. For example,
statistically significant inverse correlation has been demonstrated between
extent of hypoxia and IQ. Supporting a causative effect of hypoperfusion to
autism development, Bachavelier et al reviewed numerous experimental reports
of primate and other animal studies in which damage causing hypoperfusion of
temporal areas was associated with onset of autism-like disorders. It is
also known that after removal or damage of the amygdala, hippocampus, or
other temporal structures induces either permanent or transient
autistic-like characteristics such as unexpressive faces, little eye
contact, and motor stereotypies occurs. Clinically, temporal lobe damage by
viral and other means has been implicated in development of autism both in
adults, and children. Evidence suggests that hypoperfusion and resulting
hypoxia is intimately associated with autism, however the next important
question is whether reversion of this hypoxia can positively influence
autism. In autism the associated hypoxia is not predominantly apoptotic or
necrotic to temporal neurons but associated with altered function.
Hypoperfusion may contribute to defects not only by induction of hypoxia but
also allowing for abnormal metabolite or neurotransmitter accumulation.
This is one of the reasons why glutamate toxicity has been implicated in
autism and a clinical trial at reversing this using the inhibitor of
glutamate toxicity, Riluzole, is currently in progress. Conceptually the
augmentation of perfusion through stimulation of angiogenesis should allow
for metabolite clearance and restoration of functionality. Although not
well defined, cell death may also be occurring in various CNS components of
autistic children. If this were the case, it is possible that neural
regeneration can be stimulated through entry of neuronal progenitor cells
into cell cycle and subsequent differentiation. Ample evidence of neural
regeneration exists in areas ranging from stroke, to subarachinoidal
hemorrhage, to neural damage as a result of congenital errors of metabolism.
Theoretically, it is conceivable that reversing hypoxia may lead to
activation of self-repair mechanisms. Such neural proliferation is seen
after reperfusion in numerous animal models of cerebral ischemia. The
concept of increasing oxygen to the autistic brain through various means
such as hyperbaric medicine is currently being tested in 2 independent
clinical trials in the US. However, to our knowledge, the use of cell
therapy to stimulate angiogenesis has not been widely used for the treatment
of autism.
Immune deregulation in autism
The fundamental interplay between the nervous system and the immune system
cannot be understated. Philosophically, the characteristics of self/non self
recognition, specificity, and memory are only shared by the immune system
and the nervous system. Physically, every immune organ is innervated and
bi-directional communication between neural and immune system cells has been
established in numerous physiological systems. In autism, several
immunological abnormalities have been detected both in the peripheral and
the central nervous systems. Astroglial cells, or astrocytes, surround
various portions of the cerebral endothelium and play a critical role in
regulating perfusion, and blood brain barrier function. Astrocytes are
capable of ediating several immunological/
of various toll like receptors (TLR) on astrocytes endows the ability to
recognize not only bacterial and viral signals but also endogenous "danger"
signals such as heat shock proteins, fibrinogen degradation products, and
free DNA. Physiologically, astrocytes play an important protective role
against infection, generating inflammatory cytokines such as TNF-alpha,
IL-1beta, and IL-6. Through secretion of various chemokines such as CXCL10,
CCL2 and BAFF, astrocytes play an important role in shaping adaptive immune
responses in the CNS. Astrocytes have antigen presenting capabilities and
have been demonstrated to activate T and B cell responses against exogenous
and endogenous antigens. Although astrocytes play a critical role against
CNS infection, these cells also have potential to cause damage to the host
when functioning in an aberrant manner. For example, various neurological
diseases are associated with astrocyte overproduction of inflammatory
agents, which causes neural malfunction or death. In amyotrophic lateral
sclerosis (ALS), astrocyte secretion of a soluble neurotoxic substance has
been demonstrated to be involved in disease progression. Astrocyte
hyperactivation has been demonstrated in this disease by imaging, as well as
autopsy studies. In multiple sclerosis, astrocytes play a key role in
maintaining autoreactive responses and pathological plaque formation. In
stroke, activated astrocytes contribute to opening of the blood brain
barrier, as well as secrete various neurotoxic substances that contribute to
post infarct neural damage. Vargas et al compared brain autopsy samples
from 11 autistic children with 7 age-matched controls. They demonstrated an
active neuroinflammatory process in the cerebral cortex, white matter, and
notably in cerebellum of autistic patients both by immunohistochemistr
morphology. Importantly, astrocyte production of inflammatory cytokines was
observed, including production of cytokines known to affect various neuronal
functions such as TNF-alpha and MCP-1. CSF samples from living autism
patients but not controls also displayed upregulated inflammatory cytokines
as demonstrated by ELISA. The potent effects of inflammatory cytokines on
neurological function cannot be underestimated. For example, patients
receiving systemic IFN-gamma therapy for cancer, even though theoretically
the protein should not cross the blood brain barrier, report numerous
cognitive and neurological abnormalities. In fact, IFNgamma, one of the
products of activated astrocytes, has been detected at elevated levels in
the plasma of children with autism. Mechanistically, inflammatory mediators
mediate alteration of neurological function through a wide variety of
different pathways, either directly altering neuron activity or indirectly.
For example, the common neurotoxin used in models of Parkinson's Disease,
MPTP is believed to mediate its activity through activation of IFN-gamma
production, leading to direct killing of dopaminergic neurons in the
substantia nigra. This is evidenced by reduced MPTP neuronal toxicity in
IFN-gamma knockout mice or by addition of blocking antibodies to IFN-gamma.
In terms of indirect effects of IFN-gamma, it is known that this cytokine
activates the enzyme 2, 3-indolaminedeoxyge
small molecule neurotoxins such as the kynurenine metabolites 3OH-kynurenine
and quinolinic acid which have been implicated in dementias associated with
chronic inflammatory states. T cell and B cell abnormalities have been
reported systemically in autistic children. These have included systemic T
cell lymphopenia, weak proliferative responses to mitogens, and deranged
cytokine production. At face value, lymphopenia would suggest general immune
deficiency and as a result little inflammation, however, recent studies have
demonstrated that almost all autoimmune diseases are associated with a state
of generalized lymphopenia (reviewed by Marleau and Sarvetnick).
Autoimmune-like pathophysiology appears to be prevalent in autism and
several lines of reasoning suggest it may be causative. Firstly, numerous
types of autoantibodies have been detected in children with autism but not
in healthy or mentally challenged controls. These include antibodies to
myelin basic protein, brain extracts, Purkinje cells and gliadin extracted
peptides, neutrophic factors, and neuron-axon filament and glial fibrillary
acidic protein. Secondly, family members of autistic children have a higher
predisposition towards autoimmunity compared to control populations. Hinting
at genetic mechanisms are observations that specific HLA haplotypes seem to
associate with autism. Another genetic characteristic associated with autism
is a null allele for the complement component C4b. Both HLA haplotypes as
well as complement component gene polymorphisms have been strongly
associated with autoimmunity. It is known that autoimmune animals have
altered cognitive ability and several neurological abnormalities. Thirdly,
autism has been associated with a peculiar autoimmune-like syndrome that is
still relatively undefined. Mucosal lesions in the form of chronic
ileocolonic lymphoid nodular hyperplasia characterized by lymphocyte
infiltration, complement deposition, and cytokine production have been
described uniquely to children with autism but not healthy controls or
cerebral palsy patients. This inflammatory condition is associated not only
with lesions on the intestinal wall, but also in the upper GI tract.
Although several characteristics of this condition are shared with Crohn's
Disease, one unique aspect is eosinophilic infiltrate, which seems to be
associated with dietary habits of the patient. Systemic manifestation of the
immune deregulation/
elevated levels of inflammatory cytokines such as IFN-gamma, IL-12, and
TNF-alpha. Indication that a relevant inflammatory response is ongoing is
provided by observation that the macrophage product neopterin is observed
elevated in children with autism. Inhibited production of anti-inflammatory
cytokines such as IL-10 and TGF-beta has also been observed in children with
autism, thus suggesting not only augmentation of inflammatory processes but
also deficiency of natural feedback inhibitor mechanisms. The systemic
effects of a chronic inflammatory process in the periphery may result in
production of soluble factors such as quinilonic acid, which have neurotoxin
activity. Ability of cellular immune deregulation to affect neural function
can occur independent of cell trafficking, as was demonstrated in animal
studies in which T cell depletion was associated with cognitive loss of
function that was reversible through T cell repletion [79]. Localized
inflammation and pathological astrocyte activation has been directly
demonstrated to be associated with pathogenesis in autism. Clinical trials
of inflammatory drugs have demonstrated varying degrees of success. For
example, in an open labeled study of the anti-inflammatory PPARgamma agonist
pioglitozone in 25 children, 75% reported responses on the aberrant behavior
checklist. Other interventions aimed at reducing inflammation such as
intravenous immunoglobulin administration reported inconsistent results,
however a minor subset did respond significantly. Clinical trials are
currently using drugs off-label for treatment of autism through inhibiting
inflammation such as minocycline, n-acetylcysteine, or ascorbic acid and
zinc. Despite the desire to correct immune deregulation/
in autism, to date, no approach has been successful.
Treatment of hypoperfusion defect by umbilical cord blood CD34+ stem cell
administration
Therapeutic angiogenesis, the induction of new blood vessels from
preexisting arteries for overcoming ischemia, has been experimentally
demonstrated in peripheral artery disease, myocardial ischemia, and stroke.
Angiogenesis is induced through the formation of collateral vessels and has
been observed in hypoperfused tissues. This process is believed to be
coordinated by the oxygen sensing transcription factor hypoxia inducible
factor-1 (HIF-1). During conditions of low oxygen tension, various
components of the transcription factor dimerize and coordinately translocate
into the nucleus causing up regulation of numerous cytokines and proteins
associated with angiogenesis such as SDF-1, VEGF, FGF, and matrix
metalloproteases. The potency of tissue ischemia stimulating angiogenesis is
seen in patients after myocardial infarction in which bone marrow angiogenic
stem cells mobilize into systemic circulating in response to ischemia
induced chemotactic factors. The angiogenic response has also been
demonstrated to occur after cerebral ischemia in the form of stroke and is
believed to be fundamental in neurological recovery. For example, in models
of middle cerebral artery occlusion, endogenous angiogenesis occurs which is
also involved in triggering migration of neural stem cells into damaged area
that participate in neuroregeneration. The association between neural
angiogenesis and neurogenesis after brain damage is not only
temporally-linked but also connected by common mediators, for example, SDF-1
secreted in response to hypoxia also induces migration of neural
progenitors. Angiogenic factors such as VEGF and angiopoietin have been
implicated in post ischemia neurogenesis. While recovery after cerebral
ischemia occurs to some extent without intervention, this recovery is can be
limited. Methods to enhance angiogenesis and as a result neurogenesis are
numerous and have utilized approaches that up regulate endogenous production
of reparative factors, as well as administration of exogenous agents. For
example, administration of exogenous cytokines such as FGF-2,
erythropoietin, and G-CSF, has been performed clinically to accelerate
healing with varying degrees of success. A promising method of increasing
angiogenesis in situations of ischemia is administration of cells with
potential to produce angiogenic factors and the capacity to differentiate
into endothelial cells themselves. Accordingly, the use of CD34+ stem cells
has been previously proposed as an alternative to growth factor
administration. Therapeutic administration of bone marrow derived CD34+
cells has produced promising results in the treatment of end-stage
myocardial ischemia, as well as a type of advanced peripheral artery disease
called critical limb ischemia [99]. Additionally, autologous peripheral
blood CD34+ cells have also been used clinically with induction of
therapeutic angiogenesis. Of angiogenesis stimulating cell sources, cord
blood seems to possess CD34+ cells with highest activity in terms of
proliferation, cytokine production, as well as endothelial differentiation.
Cord blood has been used successfully for stimulation of angiogenesis in
various models of ischemia. In one report, the CD34+, CD11b+ fraction, which
is approximately less than half of the CD34+ fraction of cord blood, was
demonstrated to possess the ability to differentiate into endothelial cells.
In another report, VEGF-R3+, CD34+ cells demonstrated the ability to
differentiate into endothelial cells and were able to be expanded 40-fold
expansion. The concentration of this potential endothelial progenitor
fraction in cord blood CD34+ cells is approximately tenfold higher as
compared to bone marrow CD34+ cells (1.9% +/- 0.8% compared to 0.2% +/-
0.1%) [103]. Administration of cord blood CD34+ cells into immune
compromised mice that underwent middle cerebral artery ligation reduced
neurological deficits and induce neuroregeneration, in part through
secretion of angiogenic factors. Several studies have confirmed that
systemic administration of cord blood cells is sufficient to induce
neuroregeneration. Given the potency of cord blood CD34+ cells to induce
angiogenesis in areas of cerebral hypoperfusion, we propose that this cell
type may be particularly useful for the treatment of autism, in which
ischemia is milder than stroke induced ischemia, and as a result the level
of angiogenesis needed is theoretically lower. However at face value,
several considerations have to be dealt with. Firstly, cord blood contains a
relatively low number of CD34+ cells for clinical use. Secondly, very few
patients have access to autologous cord blood; therefore allogeneic cord
blood CD34+ cells are needed if this therapy is to be made available for
widespread use. There is a belief that allogeneic cord blood cells can not
be used without immune suppression to avoid host versus graft destruction of
the cells. Numerous laboratories are currently attempting to expand cord
blood CD34+ cells, achieving varying degrees of success. Expansion methods
typically involve administration of cytokines, and or feeder cell layers.
The authors have developed a CD34+ expansion protocol that yields up to
60-fold expansion with limited cell differentiation.
This expansion method involves numerous growth factors and conditioned
medium, however is performed under serum free conditions (manuscript in
preparation)
authors with expanded CD34+ cells under local ethical approval with varying
degrees of success. Since other groups are also generating CD34+ expansion
technologies, we do not anticipate number of CD34+ cells to be a problem.
Safety concerns regarding allogeneic CD34+ cells are divided into fears of
graft versus host reactions, as well as host versus graft. The authors of
the current paper have recently published a detailed rationale for why
administration of cord blood cells is feasible in absence of immune
suppression [111]. Essentially, GVHD occurs in the context of lymphopenia
caused by bone marrow ablation. Administration of cord blood has been
reported in over 500 patients without a single one suffering GVHD if no
immune suppression was used. Although host versus graft may conceptually
cause immune mediated clearing of cord blood cells, efficacy of cord blood
Cells in absence of immune suppression has also been reported. Accordingly,
we believe that systemic administration of expanded cord blood derived CD34+
cells may be a potent tool for generation of neoangiogenesis in the autistic
brain.
Immune modulation by Mesenchymal stem cells
The treatment of immune deregulation in autism is expected to not only cause
amelioration of intestinal and systemic symptomology, but also to profoundly
influence neurological function. Reports exist of temporary neurological
improvement by decreasing intestinal inflammation through either antibiotic
administration or dietary changes. Although, as previously discussed, some
anti-inflammatory treatments have yielded beneficial effects, no clinical
agent has been developed that can profoundly suppress inflammation at the
level of the fundamental immune abnormality. We believe Mesenchymal stem
cell administration may be used for this purpose. This cell type, in
allogeneic form, is currently in Phase III clinical studies for Crohn's
disease and Phase II results have demonstrated profound improvement.
Mesenchymal stem cells are classically defined as "formative pluripotential
blast cells found inter alia in bone marrow, blood, dermis and periosteum
that are capable of differentiating into any of the specific types of
Mesenchymal or connective tissues. These cells are routinely generated by
culture of bone marrow in various culture media and collection of the
adherent cell population. This expansion technique is sometimes used in
combination with selection procedures for markers described above to
generate a pure population of stem cells. An important characteristic of
Mesenchymal stem cells is their ability to constitutively secrete immune
inhibitory factors such as IL-10 and TGF-b while maintaining ability to
present antigens to T cells. This is believed to further allow inhibition of
immunity in an antigen specific manner, as well as to allow the use of such
cells in an allogeneic fashion without fear of immune-mediated rejection.
Antigenspecific immune suppression is believed to allow these cells to shut
off autoimmune processes. Further understanding of the immune inhibitory
effects of Mesenchymal stem cells comes from the fact that during T cell
activation, two general signals are required for the T cell in order to
mount a productive immune response, the first signal is recognition of
antigen, and the second is recognition of costimulatory or coinhibitory
signals. Mesenchymal cells present antigens to T cells but provide a
coinhibitory signal instead of a co-stimulatory signal, thus specifically
inhibiting T cells that recognize them, and other cells expressing similar
antigens. Supporting this concept, it was demonstrated in a murine model
that Mesenchymal stem cell transplantation leads to permanent donor-specific
immunotolerance in allogeneic hosts and results in long-term allogeneic skin
graft acceptance. Other studies have shown that Mesenchymal stem cells are
inherently immunosuppressive through production of PGE-2, interleukin-
expression of the tryptophan catabolizing enzyme indoleamine 2,
3,-dioxygenase as well as Galectin-1. These stem cells also have the
ability to non-specifically modulate the immune response through the
suppression of dendritic cell maturation and antigen presenting abilities.
Immune suppressive activity is not dependent on prolonged culture of
Mesenchymal stem cells since functional induction of allogeneic T cell
apoptosis was also demonstrated using freshly isolated, irradiated,
Mesenchymal stem cells. Others have also demonstrated that Mesenchymal stem
cells have the ability to preferentially induce expansion of antigen
specific T regulatory cells with the CD4+ CD25+ phenotype. Supporting the
potential clinical utility of such cells, it was previously demonstrated
that administration of Mesenchymal stem cells inhibits antigen specific T
cell responses in the murine model of multiple sclerosis, experimental
autoimmune encephalomyelitis, leading to prevention and/or regression of
pathology. Safety of infusing Mesenchymal stem cells was illustrated in
studies administering 1–2.2 × 106 cells/kg in order to enhance engraftment
of autologous bone marrow cell. No adverse events were associated with
infusion, although level of engraftment remained to be analyzed in
randomized trials. The ability of Mesenchymal stem cells on one hand to
suppress pathological immune responses but on the other hand to stimulate
hematopoiesis leads to the possibility that these cells may also be useful
for treatment of the defect in T cell numbers associated with autism.
Practical clinical entry
We propose a Phase I/II open labeled study investigating combination of cord
blood expanded CD34+ cells together with Mesenchymal stem cells for the
treatment of autism. Such a trial would utilize several classical
instruments of autism assessment such as the Aberrant Behavior Checklist and
the Vineland Adaptive Behavior Scale (VABS) for assessment of symptomatic
effect. Objective measurements of temporal lobe hypoperfusion, intestinal
lymphoid hypertrophy, immunological markers and markers of hypoxia will be
included. In order to initiate such an investigation, specific
inclusion/exclusion criteria will be developed taking into account a
population most likely to benefit from such an intervention. Criteria of
particular interest would include defined hypoxia areas, as well as frank
clinical manifestations of inflammatory intestinal disease. Markers of
inflammatory processes may be used as part of the inclusion criteria, for
example, elevation of C-reactive protein, or serum levels of TNF-alpha,
IL-1, or IL-6 in order to specifically identify patients in whom the
anti-inflammatory aspects of stem cell therapy would benefit. More stringent
criteria would include restricting the study to only patients in which T
cell abnormalities are present such as ex vivo hypersecretion of interferon
gamma upon anti-CD3/CD28 stimulation, as well as deficient production of
immune inhibitory cytokines such as IL-10 and TGF-beta. One of the authors
has utilized both CD34+ and Mesenchymal stem cells clinically for treatment
of various diseases. In some case reports, the combination of CD34+ and
Mesenchymal stem cells was noted to induce synergistic effects in
neurological diseases, although the number of patients are far too low to
draw any conclusions. We propose to conduct this study based on the
previous experiences of our group in this field, as well as numerous other
groups that have generated anecdotal evidence of stem cell therapy for
autism but have not published in conventional journals. We believe that
through development of a potent clinical study with appropriate endpoints,
much will be learned about the pathophysiology of autism regardless of trial
outcome.
Click below to review the entire document including references; from the
Journal of Translational Medicine published June of 2007 titled "Stem Cell
Therapy for Autism"
http://www.translat
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StemCells subscribers may also be interested in these sites:
Children's Neurobiological Solutions
http://www.CNSfoundation.org/
Cord Blood Registry
http://www.CordBlood.com/at.cgi?a=150123
The CNS Healing Group
http://groups.yahoo.com/group/CNS_Healing
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