Autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus.

Abstract

Type 1 diabetes mellitus (DM) results from a cell-mediated autoimmune attack against pancreatic beta cells. Previous animal and clinical studies suggest that moderate immunosuppression in newly diagnosed type 1 DM can prevent further loss of insulin production and can reduce insulin needs.
To determine the safety and metabolic effects of high-dose immunosuppression followed by autologous nonmyeloablative hematopoietic stem cell transplantation (AHST) in newly diagnosed type 1 DM.
A prospective phase 1/2 study of 15 patients with type 1 DM (aged 14-31 years) diagnosed within the previous 6 weeks by clinical findings and hyperglycemia and confirmed with positive antibodies against glutamic acid decarboxylase. Enrollment was November 2003-July 2006 with observation until February 2007 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirão Preto, Ribeirão Preto, Brazil. Patients with previous diabetic ketoacidosis were excluded after the first patient with diabetic ketoacidosis failed to benefit from AHST. Hematopoietic stem cells were mobilized with cyclophosphamide (2.0 g/m2) and granulocyte colony-stimulating factor (10 microg/kg per day) and then collected from peripheral blood by leukapheresis and cryopreserved. The cells were injected intravenously after conditioning with cyclophosphamide (200 mg/kg) and rabbit antithymocyte globulin (4.5 mg/kg).
Morbidity and mortality from transplantation and temporal changes in exogenous insulin requirements (daily dose and duration of usage). Secondary end points: serum levels of hemoglobin A1c, C-peptide levels during the mixed-meal tolerance test, and anti-glutamic acid decarboxylase antibody titers measured before and at different times following AHST.
During a 7- to 36-month follow-up (mean 18.8), 14 patients became insulin-free (1 for 35 months, 4 for at least 21 months, 7 for at least 6 months; and 2 with late response were insulin-free for 1 and 5 months, respectively). Among those, 1 patient resumed insulin use 1 year after AHST. At 6 months after AHST, mean total area under the C-peptide response curve was significantly greater than the pretreatment values, and at 12 and 24 months it did not change. Anti-glutamic acid decarboxylase antibody levels decreased after 6 months and stabilized at 12 and 24 months. Serum levels of hemoglobin A(1c) were maintained at less than 7% in 13 of 14 patients. The only acute severe adverse effect was culture-negative bilateral pneumonia in 1 patient and late endocrine dysfunction (hypothyroidism or hypogonadism) in 2 others. There was no mortality.
High-dose immunosuppression and AHST were performed with acceptable toxicity in a small number of patients with newly diagnosed type 1 DM. With AHST, beta cell function was increased in all but 1 patient and induced prolonged insulin independence in the majority of the patients.

Full text

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PRELIMINARY
COMMUNICATION
Autologous Nonmyeloablative
Hematopoietic Stem Cell Transplantation
in Newly Diagnosed Type 1 Diabetes Mellitus
Ju ´lio C. Voltarelli, MD, PhD
Carlos E. B. Couri, MD, PhD
Ana B. P. L. Stracieri, MD, PhD
Maria C. Oliveira, MD, MSc
Daniela A. Moraes, MD
Fabiano Pieroni, MD, PhD
Marina Coutinho, MD, MSc
Kelen C. R. Malmegrim, PhD
Maria C. Foss-Freitas, MD, PhD
Belinda P. Simo ˜es, MD, PhD
Milton C. Foss, MD, PhD
Elizabeth Squiers, MD
Richard K. Burt, MD
T
of autodestruction is subclinical until
the amount of beta-cell mass is insuf-
ficient to maintain glucose homeosta-
sis. Thus, at the time of clinical diag-
nosis,approximately60%to80%ofthe
beta-cell mass has been destroyed.2
Type 1 DM comprises only 5% to
10% of all diabetic etiologies but is as-
sociated with a high frequency of vas-
cular complications and compromises
quality and expectancy of life.3,4Pa-
tients with type 1 DM depend on ex-
ogenousinsulinadministrationforsur-
vival and for control of long-term
complications. The best-established
treatmentistightcontrolofbloodglu-
cose achieved by frequent daily injec-
tions or continuous subcutaneous in-
YPE1 DIABETES MELLITUS(DM)
resultsfromacell-mediatedau-
toimmune attack against pan-
creatic beta cells.1The course
For editorial comment see p 1599.
Author Affiliations: Department of Clinical Medicine,
SchoolofMedicineofRibeira ˜oPreto,UniversityofSa ˜o
Paulo, Ribeira ˜o Preto, Brazil (Drs Voltarelli, Couri, Stra-
cieri, Oliveira, Moraes, Pieroni, Coutinho, Malmegrim,
Foss-Freitas,Simo ˜es,andFoss);Y’sTherapeuticInc,Bur-
lingame, Calif (Dr Squiers); and Division of Immuno-
therapy,NorthwesternUniversity,Chicago,Ill(DrBurt).
CorrespondingAuthor:JulioC.Voltarelli,MD,PhD,Re-
gionalBloodCenter(Hemocentro),CampusUSP,14051-
140 Ribeira ˜o Preto, Brazil (jcvoltar@fmrp.usp.br).
Context Type 1 diabetes mellitus (DM) results from a cell-mediated autoimmune
attack against pancreatic beta cells. Previous animal and clinical studies suggest that
moderate immunosuppression in newly diagnosed type 1 DM can prevent further loss
of insulin production and can reduce insulin needs.
Objective To determine the safety and metabolic effects of high-dose immunosup-
pression followed by autologous nonmyeloablative hematopoietic stem cell transplan-
tation (AHST) in newly diagnosed type 1 DM.
Design, Setting, and Participants A prospective phase 1/2 study of 15 patients
with type 1 DM (aged 14-31 years) diagnosed within the previous 6 weeks by clinical
findingsandhyperglycemiaandconfirmedwithpositiveantibodiesagainstglutamicacid
decarboxylase. Enrollment was November 2003-July 2006 with observation until Feb-
ruary2007attheBoneMarrowTransplantationUnitoftheSchoolofMedicineofRibeira ˜o
Preto, Ribeira ˜o Preto, Brazil. Patients with previous diabetic ketoacidosis were excluded
after the first patient with diabetic ketoacidosis failed to benefit from AHST. Hematopoi-
eticstemcellsweremobilizedwithcyclophosphamide(2.0g/m2)andgranulocytecolony-
stimulating factor (10 µg/kg per day) and then collected from peripheral blood by leu-
kapheresis and cryopreserved. The cells were injected intravenously after conditioning
with cyclophosphamide (200 mg/kg) and rabbit antithymocyte globulin (4.5 mg/kg).
Main Outcome Measures Morbidity and mortality from transplantation and tem-
poral changes in exogenous insulin requirements (daily dose and duration of usage).
Secondary end points: serum levels of hemoglobin A1c, C-peptide levels during the
mixed-meal tolerance test, and anti–glutamic acid decarboxylase antibody titers mea-
sured before and at different times following AHST.
Results During a 7- to 36-month follow-up (mean 18.8), 14 patients became insulin-
free (1 for 35 months, 4 for at least 21 months, 7 for at least 6 months; and 2 with late
response were insulin-free for 1 and 5 months, respectively). Among those, 1 patient re-
sumedinsulinuse1yearafterAHST.At6monthsafterAHST,meantotalareaunderthe
C-peptide response curve was significantly greater than the pretreatment values, and at
12 and 24 months it did not change. Anti–glutamic acid decarboxylase antibody levels
decreased after 6 months and stabilized at 12 and 24 months. Serum levels of hemoglo-
bin A1cwere maintained at less than 7% in 13 of 14 patients. The only acute severe ad-
verseeffectwasculture-negativebilateralpneumoniain1patientandlateendocrinedys-
function (hypothyroidism or hypogonadism) in 2 others. There was no mortality.
Conclusions High-dose immunosuppression and AHST were performed with ac-
ceptable toxicity in a small number of patients with newly diagnosed type 1 DM. With
AHST, beta cell function was increased in all but 1 patient and induced prolonged in-
sulin independence in the majority of the patients.
Trial Registration clinicaltrials.gov Identifier: NCT00315133
JAMA. 2007;297:1568-1576
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fusion of insulin, ie, intensive insulin
therapy.Thistreatmentreducestherisk
ofretinopathy,nephropathy,andneu-
ropathy by 35% to 90% when com-
pared with conventional therapy with
1 to 2 injections per day.5
Subgroup analysis of the Diabetes
ControlandComplicationsTrialshowed
that patients with a larger beta cell re-
serve demonstrable by serum C-
peptide levels presented a slower de-
clineoftheselevelsduringthestudyand
experienced fewer microvascular com-
plicationsthanpatientswithloworun-
detectable C-peptide concentrations.
Therefore, beta cell preservation is an-
other important target in the manage-
ment of type 1 DM and in the preven-
tion of its related complications.6
Many clinical trials have evaluated
theroleofimmunointerventioninpre-
ventingresidualbetacelllossbyblock-
ing the autoimmune response with
prednisone,7azathioprine,8,9predni-
sone plus azathioprine,10cyclospor-
ine,11antibodies against CD3,12,13heat
shockprotein,14andrabbitantithymo-
cyte globulin.15These therapies were
shown to induce a slower decline or
some improvement in C-peptide lev-
els when compared with placebo
groups.However,almostallpatientsre-
quired exogenous insulin use.
Since 1996, organ-threatening sys-
temiclupuserythematosus16andother
autoimmunediseases17havebeensuc-
cessfullytreatedwithhigh-doseimmu-
nosuppression followed by autolo-
gousnonmyeloablativehematopoietic
stem cell transplantation (AHST). Or-
ganfunctionwassalvagedandinmany
casesimprovedfollowingAHST.Inani-
mal models, allogeneic bone marrow
transplantation prevents both insuli-
tis and the development of type 1 DM
in susceptible strains of mice.18
On the basis of these observations,
we initiated a phase 1/2 study in No-
vember2003analyzingthesafety,meta-
boliceffects,andabilityofAHSTtopre-
servebetacellfunctioninpatientswith
newly diagnosed type 1 DM. Here we
reportthefirstprospectivetrial,toour
knowledge,ofstemcelltherapyinhu-
manDM.Wedescribe15patientswith
type1DM,submittedtoAHST,andob-
servedfrom7to36months(mean18.8
months) after treatment.
METHODS
Patients
Inclusion criteria were patients of both
sexes, aged 12 to 35 years, with a diag-
nosis of type 1 DM during the previous
6 weeks confirmed by measurement of
serumlevelsofanti–glutamicaciddecar-
boxylase (anti-GAD) antibodies. From
September2003toFebruary2007,more
than 100 patients were offered screen-
ingforenrollment(mostbye-mailortele-
phoneinterviews).Ofthosepatients,52
fulfilled the inclusion criteria and were
personallyinterviewed,15patientsopted
to participate, and all 15 were subse-
quently enrolled between November
2003 and July 2006 and observed until
February 2007 at the Bone Marrow
Transplantation Unit of the School of
Medicine of Ribeira ˜o Preto, Ribeira ˜o
Preto, Brazil.
The main reasons for not fitting the
inclusion criteria were the duration of
type 1 DM longer than 6 weeks or
previous episodes of diabetic ketoaci-
dosis. Concerns about the probable
adverse effects related to the immuno-
suppression were the main cause of
refusing study participation. The first
patientenrolledwasdiagnosedwithdia-
betic ketoacidosis and received hydro-
cortisone (200 mg) and methylpred-
nisolone (125 mg) to prevent rabbit
antithymocyte globulin reactions. This
patient’s continued insulin depen-
dence after AHST (see Results section)
resultedinmodificationoftheprotocol
to exclude patients with diabetic keto-
acidosis-onset diabetes and to remove
glucocorticoids from the immunosup-
pression regimen. Other exclusion cri-
teria were positive serology for human
immunodeficiency virus, hepatitis B or
C,andunderlyinghematologic,nephro-
logic,cardiac,psychiatricorhepaticdis-
ease. Serum levels of ?-human chori-
onic gonadotropin were determined in
all women to exclude pregnancy.
Participants were initially treated by
theirownphysiciansuntiladmissionto
thepresentstudy.Race/ethnicitywasself-
reportedandwasassessedbecauseofthe
diversity of the Brazilian population
along with its prevalence of black/
whitebiracialiy.HLAclassIItypingwas
performed at low/medium resolution
using reverse sequence-specific oligo-
nucleotideprobes(RSSOP-OneLambda,
Canoga Park, Calif), and at high reso-
lution using sequence–specific primers
(SSP, One Lambda). The study proto-
colwasapprovedbytheresearchethics
committeesofboththeUniversityHos-
pitaloftheSchoolofMedicineofRibeira ˜o
Preto and the Brazilian Ministry of
Health.Aninformedconsentaccording
totheDeclarationofHelsinkiwassigned
by patients or their parents.
Study Design
Key end points of the study were mor-
bidity and mortality from transplanta-
tion and temporal changes in exog-
enous insulin requirements (daily dose
and duration of usage). Secondary end
pointswereserumlevelsofhemoglobin
A1c, C-peptide levels during the mixed-
meal tolerance test, and anti-GAD anti-
body titers measured before and at dif-
ferent times following transplantation.
BloodsamplesforhemoglobinA1cde-
termination were collected after an
8-hour fast at pretreatment and every
3monthsthereafter.Bloodsamplesfor
the determination of C-peptide, an in-
direct measure of endogenous insulin
secretion,werecollectedinthefasting
state and every 30 minutes during a
2-hourmixed-mealtolerancetest.The
morning and evening doses of insulin
were withheld the day before the test
at pretreatment, 6 months, 1 year and
then yearly following AHST. Serum
anti-GADantibodiesweretitratedatthe
same intervals.
All patients were encouraged to
self-monitor blood glucose at least
twice daily (before and 2 hours after
different meals and/or at 3 AM)
between mobilization and the condi-
tioning phase and then indefinitely
after discharge from the hospital. Dur-
ing hospitalization, blood glucose
monitoring was performed before
meals and at bedtime. Insulin titration
was based on fasting before meals and
STEM CELL TRANSPLANTATION IN TYPE 1 DIABETES
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2 hours after meals with target blood
glucose levels of less than 120 mg/dL
(6.7 mmol/L) and less than 140
mg/dL (7.7 mmol/L), respectively.
The dose of insulin was reduced by
1-2 IU/mL if patients presented clini-
cal findings of hypoglycemia and/or
blood glucose levels less than 4.9
mmol/L (90 mg/dL).
Standard recommendations for life-
style modification (performing physi-
cal activities and a low-sugar diet) af-
ter AHST were made to all patients
irrespective of exogenous insulin use.
Intensiveinsulintherapywasthetreat-
ment of choice for all patients who
neededexogenousinsulin.Allchanges
in insulin doses were ordered by one
of the endocrinologists of the team
(C.E.B.C.).
Stem Cell Mobilization Regimen
Peripheral hematopoietic stem cells
were mobilized with cyclophospha-
mide and granulocyte colony-
stimulating factor (Leucin, Labora-
tory Bergamo, Sa ˜o Paulo, SP, Brazil).
Cyclophosphamide (2 g/m2) was in-
fused in 2 doses 12 hours apart in 250
mLofsalinesolutionover1hour.Uro-
protection was achieved with intrave-
nous saline infusion at 250 mL/h, ini-
tiated4hoursbeforecyclophosphamide
infusion and continued for 16 hours.
Mesna(sodium2-mercaptoethanesul-
fonate), 4 g/m2, was infused over 24
hours to bind toxic cyclophospha-
midemetabolitesinthebladder.Granu-
locyte colony-stimulating factor (10
µg/kg per day) was injected subcuta-
neouslystarting1dayaftercyclophos-
phamide infusion and continuing un-
til leukapheresis was completed.
Leukapheresis using a continuous-
flow blood cell separator was initiated
when the rebounding CD34?cells
reached10cells/µL.Apheresiswascon-
tinued daily until the number of har-
vestedprogenitorcellsreachedamini-
mumof3.0?106CD34?cells/kgbody
weight. Unmanipulated peripheral
bloodstemcellswerefrozenin10%di-
methyl sulfoxide in a rate-controlled
freezer and stored in the vapor phase
of liquid nitrogen.
Conditioning (Immune Ablative)
Regimen
Conditioningwasachievedwithcyclo-
phosphamideandantithymocyteglobu-
lin. Cyclophosphamide was given in-
travenously in divided doses of 50
mg/kg per day over 1 hour on days 5,
4, 3, and 2 before stem cell infusion.
Rabbit antithymocyte globulin (thy-
moglobulin, IMTIX Sangstat, Lyon,
France) was administered at a dose of
0.5mg/kgperdayonday5before,and
at a dose of 1 mg/kg per day on days 4,
3,2,and1beforestemcellinfusion.Ex-
ceptforthefirstpatient,prophylaxisof
antithymocyte globulin reactions was
donewithdexchlorpheniramine(6mg
by mouth) avoiding the use of gluco-
corticoids. Stem cell infusion was per-
formed on day 0 and granulocyte
colony-stimulatingfactor(5µg/kgper
day) was administered subcutane-
ously from day 5 after stem cell infu-
sionuntilneutrophilcountwasgreater
than 1000/µL.
Supportive Care
Patientswereisolatedinroomsequipped
with high-efficiency particulate air fil-
ters. After hospital admittance for con-
ditioning,antimicrobialprophylaxiswas
started with ciprofloxacin (500 mg ev-
ery 12 hours intravenously), acyclovir
(250mg/m2every8hoursbymouthun-
til day 35), amphotericin B (0.2 mg/kg
perdayintravenouslyand10mg/daero-
solized).Ciprofloxacinwasreplacedby
cefepime (2 g every 12 hours intrave-
nously)duringfebrileepisodes.Afteren-
graftment, antifungal prophylaxis was
changed to fluconazole (400 mg/d by
mouthuntilday60)andsulfamethoxa-
zole/trimethoprim(800/160mgevery12
hours by mouth 2 times per week) or
dapsone(100mg3timesperweek)was
given through day 60 for prevention of
Pneumocystis jiroveci pneumonia.
Weekly monitoring of cytomegalovi-
rus antigenemia in circulating neutro-
phils was performed until day 60.
Duringpretreatmentevaluation,se-
men samples were collected and fro-
zen in liquid nitrogen.Leuprolide ac-
etatedepot(3.75mgbyintramuscular
injection) was given to female pa-
tientstopreventmenstrualbleedingand
toprotectovarianfunction.Allwomen
opted to use oral contraceptive meth-
ods after AHST.
Laboratory Assessment
of Diabetic Status
Serum C-peptide levels were mea-
suredbyradioimmunoassayusingcom-
mercialkits(DiagnosticSystemsLabo-
ratories Inc, Webster, Tex). The lower
limit of detection was 0.1 ng/mL and
undetectedvalueswerereportedas0.1
ng/mL. Serum levels of anti-GAD an-
tibodiesweremeasuredbyradioimmu-
noassay using commercial kits (RSR
Limited, Cardiff, UK) and the results
wereconsideredpositiveifgreaterthan
1 U/mL. Hemoglobin A1cwas mea-
sured by low-pressure liquid chroma-
tography.
Statistical Analysis
Multiplecomparisonsoftotalareaun-
derthecurveofserumC-peptidemea-
sured during the mixed-meal toler-
ance test (during fasting and at 30, 60,
90,and120minutes)weremadeusing
amodelofmultipleregressionofmixed
effects for periods 0, 6, 12, and 24
monthsposttransplantation.Thesame
model was used to test anti-GAD ti-
ters. To present the mean variation of
hemoglobin A1clevels with time, a
model of linear regression of random
effects was constructed using the fol-
lowingequation:y=?0??1?log(time)
??2? [log(time)]2,inwhicheachpa-
rameter represents a random effect in
each patient. These models are char-
acterized to present residuals that are
normallydistributed.Dataanalysiswas
completed using PROC MIXED, SAS
statistical software, version 8 (SAS In-
stitute Inc, Cary, NC).
RESULTS
Fifteen patients aged 14 to 31 years
(mean 19.2 years) were enrolled in the
studybetweenNovember2003andJuly
2006. Individual demographic charac-
teristicsandfollow-upvariablesarelisted
in TABLE 1 and TABLE 2. Mean body
mass index (calculated as weight in ki-
lograms divided by height in meters
STEM CELL TRANSPLANTATION IN TYPE 1 DIABETES
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squared) at diagnosis was 19.8 (range,
16.6-23.4)andmeanplasmaglucosewas
391mg/dL(21.7mmol/L)(range,130-
612mg/dL[7.2-33.9mmol/L]).Allpa-
tientspresentedsymptomsofhypergly-
cemia(polyuria,polydipsia,andweight
loss) at diagnosis. Six patients pre-
sentedbothHLAhaplotypescharacter-
istic of high risk for type 1 DM, 7 pa-
tients presented 1 of those haplotypes
and 2 patients presented 0.
Time from diagnosis to mobilization
rangedfrom25to56days(mean,38.4)
and mean duration of hospital stay for
transplantation (from conditioning to
discharge) was 19.2 days (range, 15-
24). Mean number of infused CD34?
cells was 11.0?106/kg (range, 5.8-
23.1?106/kg). Neutrophil engraft-
ment(?500/µL)occurredbetweendays
8and10aftertransplantation(mean9.1
days)andplateletengraftment(?20000/
µL)wasdetectedbetweenday0andday
15 after transplantation (mean 11.4
days).
Most patients had febrile neutrope-
nia, nausea, vomiting, alopecia, and
Table 1. Pretreatment and Follow-up Variables of Patients With Type 1 Diabetic Mellitus Undergoing Autologous Nonmyeloablative
Hematopoietic Stem Cell Transplantation (Patient Demographics, HLA Type, Blood Glucose, Hemoglobin A1c, Weight Loss, Hyperglycemia
Symptoms, Body Mass Index)
Hemoglobin
A1cat
Pre-
transplantation,
%
1/M 24Biracial§ DRB1*03,
*04/DQB1
*0201,*0302
2/M 27 BlackDRB1*03,
*04/DQB1
*0201,*0302
3/M21 Biracial§ DRB1*03,
*04/DQB1
*0201,*0302
4/M 15 WhiteDRB1*01,
*07/DQB1
*0201,*0501
5/M16White DRB1*04,*10/
DQB1*0302,
*0501
6/M14 WhiteDRB1*01,*03/
DQB1*0201,
*0501
7/F20 WhiteDRB1*04,*12/
DQB1*0302,
*0301
8/M16 Biracial§ DRB1*03,*04/
DQB1*0201,
*0302
9/F18White DRB1*03,*13/
DQB1*0201,
*0602
10/F17 WhiteDRB1*01/
DQB1*0501
11/M16Biracial§ DRB1*03,*04/
DQB1*0201,
*0302
12/F14 Biracial§ DRB1*01,*04/
DQB1*0302,
*0501
13/M24 WhiteDRB1*03/
DQB1*0201
14/M31WhiteDRB1*04,*04/
DQB1*0302,
*0402
15/M16WhiteDRB1*01,*03/
DQB1*0201,
*0501
Mean
(SD) (5.1) (137.8) (1.38)
SI conversion factor: to convert glucose to mmol/L, multiply by 0.0555.
†Months since mobilization regimen.
‡Calculated as weight in kilograms divided by height in meters squared.
§Denotes patients who self-identified as having both black and white racial parentage.
Patient
No./Sex
Age,
yRace
HLA
Type
Blood
Glucose at
Diagnosis,
mg/dL
477
Weight
Loss at
Diagnosis,
%
14.0
Duration of
Symptoms of
Hyperglycemia,
d
35
Follow-
up,
mo†
12
Body Mass Index‡
Pre-
treatment
22.6
Pre-
mobilization
21.8
Pre-
conditioning
22.5
Last
Visit
21.97.6
5897.5 2.82 36 22.924.123.4 21.8
381 9.33.05 3219.0 19.619.619.9
3218.09.0 103223.0 22.422.322.3
404 7.714.5212517.5 19.019.0 18.6
5047.315.07 2223.4 23.323.4 21.5
391 10.011.0 20 2116.8 20.720.9 19.3
3145.416.0 50 18 17.618.9 17.918.0
330 6.7 7.014 1819.1 20.419.1 19.2
612 8.95.030 17 20.120.719.6 21.9
1305.45.05 1517.8 17.817.617.8
5818.1 7.07 1019.8 19.720.321.6
2698.1 9.1 149 18.418.9 18.218.3
2737.8 7.1148 22.121.521.5 22.1
29110.112.730716.6 17.617.817.2
19.2391.1 7.86 9.2
(4.4)
17.6
(13.5)
18.8
(9.2)
19.8
(2.4)
20.4
(1.9)
20.2
(2.0)
20.1
(1.8)
STEM CELL TRANSPLANTATION IN TYPE 1 DIABETES
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other common transplantation-related
complications due to the drugs used in
the mobilization and conditioning
(TABLE 3). Bilateral pneumonia of un-
identifiedetiologythatrequiredsupple-
mentary oxygen therapy and re-
spondedcompletelytobroad-spectrum
antibioticsoccurredinpatient2andwas
the only severe acute complication of
AHST.Duringlong-termfollow-up,pa-
tient 3 developed autoimmune hypo-
thyroidismandtransientrenaldysfunc-
tion associated with rhabdomyolysis, a
complication that was treated success-
fully with levothyroxine. Measure-
ments of gonadal function (follicle-
stimulating hormone and lutenizing
hormone in both sexes, testosterone in
men, and estradiol in women) were in
the normal range in 14 of 15 patients.
Patient 2 fathered a child 2 years after
transplantation(bynaturalmeans)and
patient10presentedmildhypergonado-
tropichypogonadismat12monthsfol-
lowing transplantation. There was no
mortality.
Thefirstpatientenrolledinthestudy
presented few minor complications of
transplantation(Table3).However,this
patient’s insulin requirements in-
creasedprogressivelyandat12months
following transplantation when he
abandoned follow-up, he was using a
dose 250% higher than his initial re-
quirement(1.7IU/kgperday).Hishe-
moglobin A1clevels were 7.6%, 8.2%,
8.9%,9.7%,and11.1%at0,3,6,9,and
12 months following transplantation,
respectively, and his C-peptide levels
werelowatstudyentry(basallevel,0.4
ng/mL;peakstimulatedlevel,notavail-
able) and did not increase after 1 year
(basal,0.3ng/mL;peakstimulatedlevel,
0.4ng/mL)(Table1andTable2).Anti-
GADantibodylevelswere36.0,9.9,and
7.7 U/mL at 0, 6, and 12 months fol-
lowing transplantation, respectively.
Since the protocol was changed after
treating this patient, his data were not
includedinthestatisticalanalysis.Thus,
hemoglobin A1c(FIGURE 1) and re-
sultsofC-peptidelevels(FIGURE2)re-
fersto14patientsfulfillingthesamese-
lection criteria and receiving the same
conditioning regimen.
Beforethemobilizationregimen,all
patients required exogenous insulin
(mean,0.38IU/kgperday,range,0.13-
0.58). By February 2007, 13 patients
were free from exogenous insulin for
1to35months(mean,16.2)(Table2).
Patient 7 used a fraction of the initial
insulindosefor20monthsanddiscon-
tinuedinsulinuseinJanuary2007.Pa-
tient 10 discontinued insulin tran-
siently during transplantation (from 2
days before to 7 days after), then re-
sumedinsulinuse(0.34IU/kgperday)
and after progressive reduction in its
dosediscontinuedinsulinagain1year
aftertransplantation.Patient11wasfree
from insulin from 3 days before trans-
Table 2. Pretreatment and Follow-up Variables of Type 1 Diabetic Patients Undergoing Autologous Nonmyeloablative Hematopoietic Stem
Cell Transplantation (Anti–Glutamic Acid Decarboxylase, C-Peptide, Insulin Dose, Insulin-Discontinuation Time, Insulin-Free Time)
Patient
No.
1
Anti–Glutamic Acid
Decarboxylase, U/mL*†
C-Peptide Fasting/
Peak Stimulated, ng/mL†
Insulin Dose,
IU/kg per Day
Time of
Insulin
Discontinu-
ation, d†
Not
discontinued
?6
?34
?2
Time
Free
From
Insulin,
mo
0
Diag-
nosis
36.0
6 mo
9.9
12 mo
7.7
24 mo36 mo
Pre-
treatment
0.4/NA
6 mo
0.4/0.4
12 mo
0.3/0.4
24 mo36 mo
Pre-
treat-
ment
0.48
Pre-
mobili-
zation
0.51
Pre-
condi-
tioning
0.79
2
3
4
5
6
7
8
9
10§
11?
12
13
14
15
Mean
(SD)
Abbreviation: NA, not available.
SI conversion factor: to convert C-peptide to nmol/L, multiply by 0.331.
*Statistical analysis of mean anti–glutamic acid decarboxylase values: P = .02 between pretreatment and 6 mo; P = .13 between 6 and 12 mo; P = .46 between 12 and 24 mo.
†Times related to stem cell infusion.
‡Empty cells denote follow-up times not yet reached by the respective patient.
§This patient had transient insulin discontinuation from 2 days prior until 7 days following stem cell infusion and insulin was discontinued again after 1 year (see Results).
?This patient was free from exogenous insulin from 3 days prior until 360 days following stem cell infusion and then resumed insulin use at the dose of 0.43 IU/kg per day (see Results).
¶Excluding patient 7.
49.0
1.1
22.0
51.0
17.0
4.0
48.0
102.0
44.0
11.0
11.0
24.0
37.0
21.1
31.8
(25.5)
19.0
0.0
20.0
51.0
4.6
14.0
9.5
31.0
19.0
4.4
10.0
21
29
20.0
0.0
20.0
24.0
1.6
9.0
6.4
30.0
13.0
6.5
17.0
0.0
17.0
41
23
‡
0.3/0.6
0.3/1.0
1.0/2.8
0.6/3.1
0.8/1.8
0.09/0.09
0.3/0.4
0.09/0.4
0.4/1.3
0.9/0.4
0.09/0.3
0.5/3.1
0.1/1.6
0.5/0.8
0.4/1.3
(0.3/1.0)
0.3/0.7
0.9/1.6
1.3/2.4
2.7/12.3
2.1/8.0
0.3/2.6
0.7/2.0
0.6/2.5
0.3/1.7
0.3/1.7
2.0/7.9
0.5/2.9
3.0/9.2
0.5/1.2
1.6/6.2
1.8/8.2
0.9/5.0
0.6/1.5
1.6/2.5
1.3/4.8
1.1/6.8
0.3/3.5
0.8/0.7
0.5/1.1
1.7/1.8
3.2/6.9
2.4/8.4
2.0/4.60.29
0.39
0.36
0.52
0.26
0.48
0.35
0.42
0.61
0.10
0.22
0.28
0.32
0.66
0.38
(0.14)
0.34
0.27
0.23
0.38
0.42
0.44
0.55
0.35
0.29
0.13
0.45
0.58
0.37
0.44
0.30
(0.12)
0.20
0.21
0.18
0.27
0.42
0.17
0.34
0.29
0.25
0.20
0.58
0.28
0.05
0.67
0.32
(0.20)
35
30
31
24
21
1
17
17
5
12
9
8
7
6
14.8
(11.2)
−1
−6
?610
0
−1
−2
−3
0
−2
−3
−1
17.3
(13.2)
12.5
(9.6)
18.7
(16.8)
1.1/4.0
(0.9/3.7)
1.0/3.7
(0.5/2.6)
1.9/4.5
(1.1/3.7)
1.7
(10)¶
STEM CELL TRANSPLANTATION IN TYPE 1 DIABETES
1572
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Page 6

plantationuntil360daysafter,whenin-
sulinusewasresumed(0.43IU/kgper
day)afteranupperrespiratorytractvi-
ral infection. The time course of indi-
vidualinsulindosesindifferentphases
is presented in Table 2.
All14patientstreatedaccordingtothe
same protocol (patients 2-15) com-
plied with blood glucose self-monitor-
ing and scheduled medical appoint-
ments. The time course of hemoglobin
A1cconcentrations of those patients is
presented in Figure 1. There was a sta-
tisticallysignificantreductionofhemo-
globinA1clevelsaftertransplantation.At
entry into the study, 11 of 14 patients
presented values above 7% and within
3 months after AHST, hemoglobin A1c
values were below this level and were
maintainedduringfollow-up(exceptfor
the relapsing patient 11).
The time course of fasting and peak
stimulated C-peptide levels and of the
area under the curve response curve
during mixed-meal tolerance test are
shown in Table 2 and Figure 2. Com-
pared with pretreatment levels, peak
stimulated C-peptide levels following
transplantationincreasedin11of13pa-
tients studied at 6 months, in 8 of 10
patients studied at 12 months, in 4 of
4patientsstudiedat24months,andin
1 patient studied at 36 months. Mean
peak stimulated C-peptide levels were
1.3 ng/mL at pretreatment and follow-
ing transplantation 4.0 ng/mL at 6
months, 3.7 ng/mL at 12 months, and
4.5 ng/mL at 24 months. The increase
at 24 months following transplanta-
tion was statistically significant com-
pared with all other time points
(Table 2). Mean area under the curve
ofC-peptidelevelsbeforetransplanta-
tion (92.0 ng/mL per 2 hours) showed
a statistically significant increase at 6
months following transplantation
(332.7ng/mLper2hours),whichwas
not different from 12 months (289.2
Table 3. Transplantation Complications and Gonadal Function Tests*
Patient
No.
Mobilization
Complications
Nausea,
vomiting,
pyoderma
Dysuria
Minor
Conditioning
Complications†
Anorexia, fever,
catheter
infection
Major
Conditioning
Complications
None
Late
Complications
None
Last Visit,
mo
12
Follicle-
Stimulating
Hormone,
mIU/mL‡
6.9
Luteinizing
Hormone,
mIU/mL§
9.8
Estradiol,
pg/mL
Testos-
terone,
ng/dL
315
1
2 Bilateral
pneumonia
(from day −2
to day ?14)
None
None36 8.02.5 495
3None Diarrhea, fever,
sinusitis,
skin rash
Fever, catheter
infection,
herpes
simplex, right
cephalic vein
thrombosis
Anorexia, fever,
urticaria
Anorexia, fever,
skin rash,
hypokalemia,
mucositis
Diarrhea, skin rash,
fluid overload
Diarrhea, skin rash
Anorexia, diarrhea,
fever
Skin rash
Rhabdomyolysis,
hypothyroidism
(day ? 360)
Leucopenia
32 4.84.2 379
4 Nausea,
vomiting
None 3213.3 8.0475
5NoneNone None25 9.02.8401
6 None
None None227.45.3 364
7NoneNoneNone2110.311.035
8
9
None
None
None
None
None
None
18
18
3.2
12.0
7.8
2.5
335
43
10NoneNoneHypogonadism
(day ?360)
None
None
None
None
17 31.4 14.8 38
11
12
13
14
Fever
None
Fever
Sialorrhea
Anorexia, fever
Epistaxis
Diarrhea, skin rash
Fever, skin rash,
fluid overload
Fever, skin rash
None
None
None
None
15
10
9
8
7.6
1.7
6.0
9.2
3.4
5.8
3.2
2.4
372
?20
289
676
15Nausea,
vomiting,
anorexia
NoneNone7 8.61.8 292
SI conversion factor: to convert estradiol to pmol/L, multiply by 3.671 (normal range, ?20). To convert testosterone to nmol/L, multiply by 0.0347 (normal range, ?250).
*Gonadal function tests were determined at the last visit. Estradiol levels were measured only in women in the follicular phase and testosterone levels were measured in men.
†All patients except 4, 5, 7, and 8 presented with nausea; vomiting presented in all except 4 and 6; all presented with alopecia.
‡Normal range: 0.9 to 15.
§Normal range: 1.3 to 13.0.
STEM CELL TRANSPLANTATION IN TYPE 1 DIABETES
©2007 American Medical Association. All rights reserved.
(Reprinted) JAMA, April 11, 2007—Vol 297, No. 14
1573
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Page 7

ng/mL per 2 hours) and 24 months
(270.3 ng/mL per 2 hours) (Figure 2).
Mean values of anti-GAD antibod-
ies at diagnosis and at 6, 12, and 24
monthsaftertreatmentwere31.8U/mL,
17.3U/mL,12.5U/mL,and18.7U/mL,
respectively (Table 2). Statistical dif-
ferences were observed between pre-
and and post-6-month titers but not
among posttreatment times. Anti-
GAD titers showed as negative in only
1 patient (patient 3) at 6 months post-
treatment, and continued to show as
negative at the 2-year-follow-up.
COMMENT
Many clinical trials have analyzed the
effect of various immunointervention
regimens in blocking autoimmune re-
sponse and preserving beta-cell func-
tion. Short chronic use (?12 months)
ofprednisone,7azathioprine,8,9azathio-
prineplusprednisone,10andcyclospor-
ine11in randomized controlled trials
producedvariabledegreesofimprove-
ment in C-peptide levels at the end of
follow-up compared with pretreat-
ment values. However, these effects
werenotmaintainedafterimmunosup-
pression was discontinued.7-11
Recentstudiesusingshort-termtreat-
ment with anti-CD3 monoclonal anti-
bodies or heat-shock protein showed
long-lasting improvements on C-
peptidelevels(upto18months),how-
ever with only partial improvement in
insulinusage.12-14Controlgroupsinthe
recent studies of immunointervention
(treatedwithintensiveinsulintherapy)
experiencedprogressivedeclinesofC-
peptide levels after study entry or af-
ter transient increase in its levels and
aparallelincreaseininsulinneeds.12-15
In our study, the increase of C-
peptide levels and reduction of hemo-
globin A1cwere maintained 2 years af-
ter insulin discontinuation, excluding
the acute effect of insulin therapy on
C-peptide concentrations and meta-
bolic control. The natural history of
type 1 DM was more altered in our
study than in other immunosuppres-
sion interventions because, different
from those studies, 14 of 15 or 93% of
our patients experienced variable per-
Figure 1. Hemoglobin A1cLevels and Periods Free From Exogenous Insulin Requirement
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 2
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 3
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 4
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 5
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 6
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 7
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 8
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 9
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 10
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 11
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 12
11
10
7
6
5
4
8
9
Hemoglobin A1c, %
Patient 13
11
10
7
6
5
4
8
9
036 9 12 15 18 21 24 27 30 33 36
After Transplantation, mo
Hemoglobin A1c, %
Patient 14
11
10
7
6
5
4
8
9
036 9 12 15 18 21 24 27 30 33 36
After Transplantation, mo
Hemoglobin A1c, %
Patient 15
Study
Entry
Study
Entry
Hemoglobin A1c
Free From Exogenous Insulin
Data from patient 1 were not included. Mean hemoglobin A1cvalues were adjusted with a model of linear re-
gressionofrandomeffectsbasedonthefollowingequation:y=7.8185−2.4237?log(time)?0.5512?[log(time)]2.
Differences between pretransplantation and all posttransplantation levels were statistically significant (P?.05).
Horizontal dotted lines indicate hemoglobin A1ctreatment goal ? 7%. Gray tint indicates end of follow-up.
STEM CELL TRANSPLANTATION IN TYPE 1 DIABETES
1574
JAMA, April 11, 2007—Vol 297, No. 14 (Reprinted)
©2007 American Medical Association. All rights reserved.
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Page 8

iodsofinsulinindependenceandmost
ofthemmaintainedthisstatusthrough-
out the follow-up.
Beta cell function in newly diag-
nosed type 1 DM is a measurable out-
come that predicts long-term clinical
status. Thus, preservation of beta-cell
mass can be expected to provide long-
termbenefits.6,19Thefirstpatientfailed
to show a clinical benefit probably be-
cause of a very low beta-cell reserve at
study entry, predicted by previous ke-
toacidosis that was further jeopar-
dized by the beta-cell apoptotic effect
of glucocorticoids used during condi-
tioning.20Mostofthesubsequent14pa-
tients treated without glucocorticoids
in the conditioning regimen demon-
strated increased beta-cell function
measured by C-peptide levels and be-
came insulin-independent for 1 to 35
months. Two patients (identified as 7
and 10) who initially remained on in-
sulin use shortly after transplantation
developedinsulinindependence20and
12 months after AHST, respectively,
probably secondary to progressive el-
evationsinC-peptidelevelsovertime.
Thereversewasseeninpatient11,who
presented a decline in C-peptide lev-
elsafter1yearandresumedinsulinuse
after that time. With the exception of
patient1,irrespectiveofinsulinuseall
others achieved and maintained peak
stimulatedC-peptidelevelsgreaterthan
0.60 ng/mL, which is known to be
associated with reduced prevalence of
diabeticcomplications.21Areaunderthe
curvelevelsofC-peptideincreasedsig-
nificantly after transplantation and
remained high up to 24 months there-
after.
All patients experienced common
transplantation-related complications
of high-dose immunosuppression and
only 1 patient presented a major
infectious complication. The low fre-
quency of severe acute complications
after AHST is expected in a group of
young patients with early-onset type 1
DM in contrast to other advanced
autoimmune diseases.16,17On the
other hand, 2 patients presented late
endocrine dysfunctions that could be
caused by autoimmune dysregulation
associated with the transplant proce-
dure22or by autoimmune polyendo-
crine syndrome frequently associated
with type 1 DM.23We cannot exclude
the occurrence of long-term compli-
cations related to high-dose cyclo-
phosphamide use.
The exact mechanism of action of
AHST in autoimmune disorders is not
fullyunderstood.Whetherthemecha-
nism is active or passive tolerance, ie,
T-regulatorycellsuppressionorclonal
deletion,isunknown.Inmultiplescle-
rosis, evidence supporting post-AHST
immune resetting includes an in-
creaseinthymus-derivednaiveTcells,
decreased central-memory T cells, in-
creased output of recent thymic emi-
grants,andrecoveryofadiversebutdis-
tinctT-cellreceptorrepertoirefollowing
AHST.24Detailedstudiesofimmunere-
constitutionareunderwayinthesepa-
tients to better understand the mecha-
nisms of action of AHST in new-onset
diabetes.Preliminarydatasuggestare-
setting of the immune system toward
atolerantphenotypebeyond1yearaf-
tertransplantation,asobservedinmul-
tiplesclerosis(K.C.R.M.andJ.C.V.,un-
published data, 2006). In the patients
of this study, persistence of anti-GAD
antibodies,evenatlowtiters,showsthat
theconditioningregimenwasnotfully
ablative for autoreactive B-cell clones
andconfirmsthatthemagnitudeofthe
humoral response is not predictive of
beta cell reserve or clinical response.19
Improvement of beta-cell function
after intensive immunosuppression
could be explained by regeneration of
beta cells from surviving beta cells or
from pancreatic or bone marrow stem
cells.25,26However, pancreatic stem
cells have not been clearly demon-
strated, and significant in vivo genera-
tion of islet cells from hematopoietic
stem cells was not observed in animal
models of type 1 DM18or in patients
with long-term type 1 DM treated
with allogeneic hematopoietic stem
cell transplantation for concomitant
blood disorders.27
Thisis,toourknowledge,thefirstre-
port of high-dose immunosuppression
followed by autologous nonmyeloabla-
tive hematopoietic stem cell transplan-
tation for human type 1 DM. Very en-
couraging results were obtained in a
small number of patients with early-
onsetdisease.Ninety-threepercentofpa-
tients achieved different periods of in-
sulin independence and treatment-
related toxicity was low, with no
mortality. Further follow-up is neces-
sary to confirm the duration of insulin
independenceandthemechanismsofac-
tion of the procedure. In addition, ran-
domizedcontrolledtrialsandfurtherbio-
logical studies are necessary to confirm
theroleofthistreatmentinchangingthe
natural history of type 1 DM and to
evaluatethecontributionofhematopoi-
etic stem cells to this change.
AuthorContributions:DrVoltarellihadfullaccesstoall
of the data in the study and takes responsibility for the
integrityofthedataandtheaccuracyofthedataanalysis.
Studyconceptanddesign:Voltarelli,Malmegrim,Foss,
Squiers, Burt.
Acquisitionofdata:Voltarelli,Couri,Stracieri,Oliveira,
Moraes, Pieroni, Coutinho, Malmegrim, Foss-Freitas,
Simo ˜es, Foss, Squiers.
Analysis and interpretation of data: Voltarelli, Couri,
Stracieri, Malmegrim, Foss-Freitas, Simo ˜es, Foss,
Squiers, Burt.
Drafting of the manuscript: Voltarelli, Couri, Stracieri,
Malmegrim, Simo ˜es, Squiers.
Critical revision of the manuscript for important in-
tellectual content: Voltarelli, Couri, Oliveira, Moraes,
Pieroni, Coutinho, Malmegrim, Foss-Freitas, Simo ˜es,
Foss, Squiers, Burt.
Figure 2. Time Course of Total Area Under
the Curve of C-Peptide Levels During
Mixed-Meal Tolerance Test
1000
300
600
500
400
700
800
900
200
100
0
C-Peptide AUC, ng/mL per 2 h
After Transplantation, mo
Pretreatment6 2412
Datafrompatient1werenotincluded.Statisticalanaly-
sis was performed using a model of multiple regres-
sion of mixed effects. P?.001 between pretreatment
and 6 months; P=.85 between 6 and 12 months;
P=.18 between 12 and 24 months following trans-
plantation. SI conversion factor: to convert C-
peptide to nmol/L, multiply by 0.331.
STEM CELL TRANSPLANTATION IN TYPE 1 DIABETES
©2007 American Medical Association. All rights reserved.
(Reprinted) JAMA, April 11, 2007—Vol 297, No. 14
1575
on April 18, 2007 www.jama.comDownloaded from

Page 9

Statistical analysis: Couri, Malmegrim, Squiers.
Obtainedfunding:Voltarelli,Malmegrim,Squiers,Burt.
Administrative, technical, or material support:
Voltarelli, Stracieri, Malmegrim, Foss, Squiers.
Studysupervision:Voltarelli,Malmegrim,Foss,Squiers.
Financial Disclosures: None reported.
Funding/Support: Research supported by the Brazil-
ian Ministry of Health, FAEPA-HCRP, FUNDHERP,
FAPESP, CNPq, FINEP, Genzyme Corporation, and
Johnson & Johnson-LifeScan–Brazil.
Role of the Sponsor: The funding organizations did
notparticipateinthedesignandconductofthestudy;
in the collection, management, analysis, and inter-
pretation of the data; or in the preparation, review,
or approval of the manuscript.
Acknowledgment: We are grateful to Edson Mar-
tinez, PhD, and Davi Aragon, MSc, Center for Quan-
titativeMethodsoftheSchoolofMedicineofRibeira ˜o
Preto, University of Sa ˜o Paulo (CEMEQ-FMRP-USP)
for statistical advice; to Lewis Joel Greene, PhD, and
ElettraGreene,BA,forEnglishreview;andtothemul-
tiprofessional team of the Bone Marrow Transplan-
tationUnitandtheRegionalBloodCenteroftheHos-
pital das Clı ´nicas of Ribeira ˜o Preto, University of Sa ˜o
Paulo, Brazil. Individuals named in this acknowledg-
mentreceivednocompensationfromafundingspon-
sor for their contribution to this article.
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