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Canine leukocyte adhesion deficiency colony forinvestigation of novel hematopoietic therapies
Kate E. Creevya, Thomas R. Bauer Jr.a, Laura M. Tuschonga, Lisa J. Embreea,Lyn Colendab, Kevin Coganb, Matthew F. Starostb,
Mark E. Haskinsc, Dennis D. Hicksteina,*
aExperimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Building 10, Rm 12C-116, 10 Center Drive, Bethesda, MD 20892, USAbOffice of Research Services, Veterinary Resource Program, National Institutes of Health, Bethesda, MD 20892, USA
cDepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Received 20 December 2002; received in revised form 10 March 2003; accepted 10 March 2003
Abstract
The genetic immunodeficiency disease canine leukocyte adhesion deficiency (CLAD) was originally described in juvenile
Irish Setters with severe, recurrent bacterial infections. CLAD was subsequently shown to result from a mutation in the
leukocyte integrin CD18 subunit which prevents leukocyte surface expression of the CD11/CD18 complex. We describe the
development of a mixed-breed CLAD colony with clinical features that closely parallel those described in Irish Setters. We
demonstrate that the early identification of CLAD heterozygotes and CLAD-affected dogs by a combination of flow cytometry
and DNA sequencing allows the CLAD-affected animals to receive life-saving antibiotic therapy. The distinct clinical phenotype
in CLAD, the ability to detect CD18 on the leukocyte surface by flow cytometry, and the history of the canine model in marrow
transplantation, enable CLAD to serve as an attractive large-animal model for the investigation of novel hematopoietic stem cell
and gene therapy strategies.
Published by Elsevier B.V.
Keywords: Canine; Adhesion; Leukocyte
1. Introduction
Irish Setter dogs with the genetic immunodeficiency
disease canine leukocyte adhesion deficiency (CLAD)
experience recurrent life-threatening bacterial infec-
tions due to marked defects in the ability of their
leukocytes to adhere to vessel walls and migrate to the
site of infection (Trowald-Wigh et al., 1992, 2000).
CLAD results from a mutation in the common CD18
subunit of the leukocyte integrin family of adhesion
molecules, which is required for surface expression of
the CD11/CD18 molecules (Fig. 1) (Kijas et al., 1999;
Springer et al., 1984).
CLAD was first described in the late 1970s in the Irish
Setter and was referred to as ‘‘canine granulocytopathy
Veterinary Immunology and Immunopathology 94 (2003) 11–22
Abbreviations: CLAD, canine leukocyte adhesion deficiency;
HOD, hypertrophic osteodystrophy; LAD, leukocyte adhesion
deficiency; DLA, dog leukocyte antigen; FACS, fluorescent
activated cell sorting; VNTR, variable number of tandem repeats* Corresponding author. Tel.: þ1-301-594-1718;
fax: þ1-301-402-5054.
E-mail address: [email protected] (D.D. Hickstein).
0165-2427/03/$ – see front matter. Published by Elsevier B.V.
doi:10.1016/S0165-2427(03)00057-6
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syndrome’’ (Renshaw et al., 1975). Subsequent stu-
dies established the autosomal recessive nature of
inheritance of the disorder, as well as the severity
of the clinical phenotype, with terminal infectious
episodes in most instances (Renshaw and Davis,
1979). In the late 1980s CLAD was demonstrated
in an Irish Setter-cross-bred dog to be due to a
deficiency in surface expression of the CD11/CD18
leukocyte adhesion molecules (Giger et al., 1987).
Subsequently, two additional reports described the
clinical phenotype of CLAD in Irish Setters in Sweden
(Trowald-Wigh et al., 1992, 2000). Although CLAD
was first characterized in Irish Setters, it has now been
described in Irish Red And White Setters as well
(Debenham et al., 2002; Foureman et al., 2002).
In CLAD-affected dogs, the initial clinical signs
manifest shortly after birth with the development of
omphalitis, followed by frequent, severe bacterial
infections, typically accompanied by fever (Tro-
wald-Wigh et al., 2000). Despite antibiotic therapy,
CLAD-affected dogs in the community usually die or
are euthanized within the first few months of life
(Trowald-Wigh et al., 2000).
CLAD results from a single point mutation in the
CD18 gene, which results in a missense mutation of a
highly conserved cysteine residue (C36S) (Kijas
et al., 1999). This structural defect in CD18 pre-
cludes surface expression of the CD11/CD18 com-
plex (Fig. 1).
The phenotype of CLAD closely parallels the clin-
ical phenotype of the human disease leukocyte adhe-
sion deficiency (LAD). Children with LAD display
life-threatening bacterial infections (Anderson et al.,
1985; Anderson and Springer, 1987). In LAD, hetero-
geneous molecular defects in the CD18 molecule are
responsible for the disease (Kishimoto et al., 1987).
Fig. 1. Schematic of normal and mutant CD18 molecules in heterodimer formation and surface expression. The normal CD18 forms a
heterodimer with the CD11 subunit and is inserted into the leukocyte cell membrane, as depicted in the top panel. In CLAD, the mutant canine
CD18 is unable to dimerize properly with the CD11 subunits, resulting in the failure of the CD11/CD18 complex to be expressed on the
leukocyte membrane surface depicted in the bottom panel.
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Hematopoietic stem cell transplantation provides the
only definitive therapy for LAD (Thomas et al., 1995).
CLAD represents an attractive model for the inves-
tigation of novel hematopoietic stem cell transplant
regimens as well as hematopoietic stem cell gene
therapy approaches. The dog has been used exten-
sively for the development of clinical marrow trans-
plant protocols for more than 30 years (Storb and
Deeg, 1986; Storb et al., 1967, 1997). More recently,
dogs have been used to test novel gene therapy
approaches (Kiem et al., 1999; Schuening et al.,
1989). In this report, we describe the derivation and
detailed characterization of an outbred CLAD colony,
and the advantages of this model for the testing of
novel hematopoietic stem cell therapies.
2. Materials and methods
2.1. Dogs
All dogs were housed in facilities approved by the
American Association for Accreditation of Laboratory
Animal Care on the NIH Bethesda campus, the NIH
Animal Center inPoolesville, MD,and the University of
Pennsylvania School of Veterinary Medicine. Dogs
were immunized against parvovirus, distemper virus,
canine infectious hepatitis virus, and rabies virus.
Research protocols were approved by the Institutional
Animal Care andUse Committee of the NationalCancer
Institute, National Institutes of Health, Bethesda, MD.
Research was conducted according to principles out-
lined in the Guide for Laboratory Animal Facilities and
Care, provided by the National Academy of Sciences.
2.2. Pedigree
CLAD dogs used in this study were derived from a
breeding colony established from two Irish Setter het-
erozygous males. These heterozygous sibling males
were mated to normal mongrel females to generate
mixed-breed heterozygous males and females. These
heterozygotes were subsequently outbred to additional
normal dogs as shown in Fig. 2 (Pedigree/Draw#,
Population Genetics Laboratory, Southwest Foundation
for Biomedical Research, San Antonio, TX). These F1
Fig. 2. Partial pedigree of the CLAD colony derived from two founder male heterozygous littermates (dogs 1 and 2). Circles represent
females, and squares represent males. Normal dogs are depicted as open figures, heterozygotes are denoted by half-filled figures, and CLAD-
affected homozygotes are shown by filled figures. CLAD-affected animals were produced in the F2 generation. The colony mean coefficient of
inbreeding is 0.025.
K.E. Creevy et al. / Veterinary Immunology and Immunopathology 94 (2003) 11–22 13
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breedings were carried out at the NIH Animal Center in
Poolesville, MD, and at the University of Pennsylvania
School of Veterinary Medicine.
2.3. Litter management
In F2 litters all pups were identified and typed for
CLAD status at 7–10 days of age. Subcutaneous
microchips (AVID Friendchip#, Norco, CA) were
placed in all pups at this time to provide definitive
identification within the litter and the colony. An
aliquot of peripheral blood was used for white blood
cell count, flow cytometric analysis, DNA sequence
confirmation, and histocompatibility testing. White
blood cell counts were obtained on a commercial
automated counter (Baker 9118 CP, Biochem Immu-
nosystems, Bologna, Italy).
2.4. Flow cytometry
Peripheral blood leukocytes were analyzed by flow
cytometry. For these studies, red blood cells were
lysed with ACK lysing buffer (BioSource Interna-
tional, Camarillo, CA), leukocytes were counted and
standardized to a concentration of 5 � 106 cells/ml in
phosphate buffered saline (BioSource International,
Camarillo, CA) þ1% bovine albumin (ICN Biome-
dicals Inc., Aurora, OH), and aliquots were then
stained with fluorescein isothiocyanate (FITC)-
labelled mouse anti-human CD18 monoclonal anti-
body (Dako Corporation, Carpinteria, CA). FITC-
labelled mouse anti-human isotype IgG1 antibody
was used as a negative control (Dako Corporation,
Carpinteria, CA). For each sample, 50,000 cells were
analyzed using a FACScan (Becton Dickson, Moun-
tain View, CA). Dead cells were gated out using
7-amino actinomycin D (Sigma–Aldrich, St. Louis,
MO) added to cells at 1 mg/ml. Commercial software
(FlowJo#, TreeStar Inc., San Carlos, CA) was used to
analyze the results. Neutrophils were gated, and ana-
lyzed on a histogram with an age-matched known
normal and known heterozygote, in both linear and log
fluorescence expression.
2.5. DNA sequencing for CLAD mutation
Genomic DNA was isolated from peripheral blood
leukocytesusingtheWizardGenomicDNAPurification
Kit (Promega Corp., Madison, WI). Using primers
specific for canine genomic DNA that flank the site of
the CLAD mutation, a 442 base pair PCR product was
amplified using AmpliTaq Gold DNA Polymerase
(Applied Biosystems, Foster City, CA). After purifi-
cation using Microcon Spin Columns (Amicon, Milli-
pore, Bedford, MA), the template was commercially
sequenced (ACGT Inc., Northbrook, IL).
2.6. Radiographic findings
Skeletal radiographs were obtained from affected
animals during periods of clinically symptomatic
lameness or limb pain (CGR model SPG512S, Balti-
more, MD) and were developed on commercial X-ray
film (Kodak, Rochester, NY).
2.7. Canine histocompatibility testing
Genomic DNA from canine peripheral blood leu-
kocytes was isolated using a Genomic DNA purifica-
tion kit (Promega Corp.) according to manufacturer’s
protocols. DNA was subjected to PCR amplification
in a reaction containing a Cy5-endlabelled (Synthe-
gen LLC, Houston, TX) reverse primer and Platinum
Taq DNA Polymerase (Invitrogen, Carlsbad, CA).
Each sample was amplified separately using primer
sets to two highly polymorphic microsatellite mar-
kers: C.2200, near the class I dog leukocyte antigen
(DLA) major histocompatibility complex loci, or to
C.2202, near the Class II DLA loci, to ensure com-
plete DLA representation (Wagner et al., 1996).
Samples were amplified in an Applied Biosystems
GeneAmp PCR System 9700 and analyzed using a
Visible Genetics MicroGene Clipper according to the
manufacturer’s directions.
2.8. Preparation of bone for histological analysis
Bone tissue from the distal ulna and radius was
taken from a 2-month-old CLAD female euthanized
as a result of intractable infections that did not respond
to antibiotic therapy. The bone samples were
immersed in neutral buffered 10% formalin for 24 h
then transferred to a formic acid solution for decalci-
fication. Bone samples were then rinsed liberally with
tap water, dehydrated through a series of graded alco-
hols to xylene, and embedded in paraffin. Samples
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were cut into 5 mm sections and stained with hema-
toxylin and eosin.
3. Results
3.1. Establishment of a colony, and phenotypic/
genotypic identification
Two Irish Setter male CLAD heterozygous litter-
mates, who had the identical heterozygous genetic
defect at the CD18 locus (C36S), served as the foun-
ders for the establishment of the CLAD colony. These
two founder males are designated 1 and 2 (Fig. 2). This
molecular defect is the only CD18 mutation reported
in CLAD (Foureman et al., 2002; Kijas et al., 1999). In
the litter from which these males were obtained, three
female heterozygotes were also identified, as well as
one affected male pup, who displayed typical clinical
signs of CLAD and was euthanized at 4 months of age.
The two founder males were bred to four unrelated,
genotypically normal mongrel females to produce the
F1 generation. Two of these matings are depicted
(Fig. 2). Heterozygous males and females from these
breedings were used for further outbred F1 and F2
crosses. For clarity, the generation within the pedigree
is defined by the dam. CLAD-affected animals are
shown in Fig. 2 as filled figures, CLAD heterozygotes
as half-filled figures, and normal dogs as open figures.
CLAD heterozygotes selected for subsequent breed-
ing had few or no siblings in the colony, or displayed a
unique genotype by variable number of tandem
repeats (VNTR) analysis of the dog DLA loci. The
Fig. 3. Clinical manifestations characteristic of the CLAD phenotype. (A) Size difference of a CLAD-affected pup (left) and a normal
littermate (right) at 4 weeks of age. (B) Omphalitis and umbilical abscess in a 2-week-old pup. (C) Umbilical abscess in a 3-week-old pup. (D)
Metaphyseal swelling of the left carpal joint typical of HOD in a CLAD-affected pup at 3 months of age.
K.E. Creevy et al. / Veterinary Immunology and Immunopathology 94 (2003) 11–22 15
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establishment of the pedigree also served to confirm
the autosomal recessive nature of the mutation (Fig. 2).
3.2. Clinical features of CLAD
Privately-owned CLAD pups typically presented to
a veterinarian as juveniles with severe, recurrent, life-
threatening bacterial infections (Renshaw et al., 1975;
Trowald-Wigh et al., 2000). The clinical phenotype of
the mixed-breed CLAD dogs in our colony closely
paralleled the phenotype described previously in cases
of CLAD among client-owned dogs. Characteristics of
the clinical phenotype of CLAD are shown (Fig. 3). A
failure to gain weight at a rate commensurate with
littermates represented an initial indication of CLAD
(Fig. 3, panel A, compare the CLAD pup on the left
with its normal littermate on the right). Second, all
newborn pups in our colony who were ultimately
diagnosed with CLAD displayed delayed umbilical
cord detachment and, in several CLAD pups, the
delayed umbilical cord detachment progressed to
omphalitis and umbilical abscessation (Fig. 3, panels
B and C) (Table 1). The umbilical abscesses required
both drainage of purulent exudate, and prolonged
treatment with parenteral antibiotics. In addition, all
CLAD dogs developed fevers within the first 8 weeks
of life (Table 1). Several CLAD pups in our colony
also developed hypertrophic osteodystrophy (HOD)
with marked physeal swelling and lameness (Fig. 3,
panel D). This syndrome of HOD is described further.
The first pup with CLAD in our colony developed a
series of life-threatening infections. Thereafter, we
treated all CLAD pups with prophylactic, broad-spec-
trum antibiotics upon confirmation of the diagnosis of
CLAD. These episodes of pyrexia responded initially
to the institution of antibiotic therapy. Although pro-
phylactic therapy might be expected to alter the
clinical course, CLAD pups in our colony continued
to experience frequent infections, which responded
more slowly to antibiotic therapy with each subse-
quent episode.
Leukocytosis has been previously described in
CLAD, and was strongly correlated with the clinical
phenotype of CLAD in our colony (Table 1). The
leukocytosis developed as early as 1 week of age in
the CLAD dogs and consisted almost exclusively of
neutrophils. This leukocytosis ranged from a moderate
increase of 37,300/ml to a marked increase of 338,000/ml
(normal range 4000–15,500/ml) (Table 1).
3.3. Hypertrophic osteodystrophy
Lameness secondary to hypertrophic osteodystro-
phy (HOD) developed in the majority of the CLAD
Table 1
Summary of clinical signs, radiographic findings, and concurrent peripheral leukocyte counts in seven CLAD-affected pups
Dog
number
Age Clinical signs Radiographic
findings
White blood
cell count (cells/ml)
57 10 days Omphalitis 57.5 � 103
6 weeks RF lameness, pain HOD RF 39.7 � 103
13 weeks RH lameness, pain, fever, anorexia HOD RH 37.3 � 103
15 weeks R/LH lameness, pain, fever, anorexia HOD of R/L stifles, tarsi, elbows, carpi 60.4 � 103
88 9 days Omphalitis 112.0 � 103
7 weeks Pustular dermatitis 53.6 � 103
8 weeks Fever, lethargy 47.4 � 103
90 9 days Umbilical abscess 338.0 � 103
10 weeks Fever, anorexia, lethargy 47.0 � 103
95 5 days Umbilical abscess 113.0 � 103
9 weeks Arthralgia, joint swelling, fever HOD of R/L stifles, tarsi 60.9 � 103
102 9 weeks Emesis, fever, lethargy 49.8 � 103
103 9 weeks Fever, anorexia, lethargy 70.9 � 103
11 weeks Fever, anorexia, lethargy 58.8 � 103
107 9 weeks RF lameness, fever, lethargy HOD of R/L carpi, elbows 37.0 � 103
109 9 weeks R carpal pain/swelling, fever, lethargy 46.5 � 103
Abbreviations: R, right; L, left; F, forelimb; H, hindlimb; HOD, hypertrophic osteodystrophy.
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dogs in our colony, and was treated supportively with
non-steroidal anti-inflammatory drugs (NSAID’s).
HOD is a clinical diagnosis manifesting as migratory
metaphyseal pain, swelling, tenderness, fever and
lameness (Abeles et al., 1999). Radiographic lesions
provide definitive diagnosis of the syndrome, and
include radiolucent metaphyseal intramedullary foci,
with a radiopaque band bordering the physis (‘‘dou-
ble physis sign’’), and flared metaphyseal regions,
with deposition of periosteal new bone in one or
more concentric layers (‘‘periosteal cuffing sign’’)
(Fig. 4, compare the normal radiograph in panel A
with the radiograph of the CLAD animal with HOD
in panel B). Secondary angular limb deformities are
common sequelae of HOD and were exhibited in
several of our CLAD-affected animals (Abeles et al.,
1999; Woodard, 1982). The majority of CLAD
dogs within our colony experienced recurrent epi-
sodes of HOD commencing at approximately 2 mon-
ths of age. In the bone sections from a CLAD dog
with HOD who was euthanized at 2 months of age
due to intractable infections, there was microscopic
evidence of focal bone necrosis, loss of trabecular
bone and irregularly arranged new bone formation
(Fig. 4, panel D). The distorted medullary trabecu-
lar bone architecture with HOD was particularly
Fig. 4. Radiographic and histologic evidence of hypertrophic osteodystrophy in CLAD. (A) Lateral radiograph of the normal distal radius and
ulna from a 4-month-old dog. (B) Lateral radiographs of the distal radius and ulna of a 4-month-old CLAD dog with HOD. There is marked
soft tissue swelling, and a flared metaphysis with increased radiopacity at the physeal margin. There is a radiolucent area of intramedullary
lysis within the distal ulna indicated by the arrow. (C) Normal metaphysis with normal bone trabeculae displaying organized, linear areas of
bone formation (H and E staining, 100�). (D) Bone histopathology from the CLAD dog with HOD demonstrating loss of bone trabeculae with
disorganized new bone formation (H and E staining, 100�).
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apparent when contrasted with the microscopic sec-
tion of normal medullary trabecular bone (Fig. 4,
panel C).
3.4. Flow cytometry for analysis of CD18
expression
To identify normal, CLAD heterozygous, and
CLAD-affected dogs, peripheral blood leukocytes
from 7–10-day-old pups were analyzed by flow
cytometry for CD18 expression. Although CD18 is
expressed on all leukocytes in association with the
leukocyte integrin CD11 subunits, the distinction
among the normal, heterozygote, and CLAD-
affected phenotypes on flow cytometry is best
observed by analysis of the neutrophils, because
the CD11/CD18 complex is one of the predominant
surface receptors of the neutrophil. The results of
analysis of peripheral blood neutrophils from a
representative litter are displayed (Fig. 5). While
CLAD-affected animals were readily apparent in
both linear and log fluorescence expression, histo-
gram analysis of linear fluorescence highlighted the
difference in CD18 expression between normal and
heterozygous animals. Normal neutrophils expressed
high levels of CD18, neutrophils from CLAD hetero-
zygotes demonstrated slightly lower levels of CD18
surface expression, and neutrophils from CLAD-
affected animals lacked surface expression of
CD18 (Fig. 5).
3.5. DNA sequence analysis of the CLAD
mutation
DNA sequencing at the CD18 genomic locus was
used to confirm the status of the CLAD alleles. The G
to C base pair substitution at codon 36 denotes the
CLAD allele (Fig. 6) (Kijas et al., 1999). CLAD-
affected animals are homozygous for the mutant allele
(Fig. 6, bottom panel).
Fig. 5. Flow cytometric analysis of neutrophil CD18 expression from a CLAD litter produced by the mating of two CLAD heterozygous dogs.
Peripheral blood leukocytes from 9-day-old puppies were stained with a FITC-labelled anti-CD18 monoclonal antibody and analyzed by linear
fluorescence.
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3.6. Dog histocompatibility testing
Histocompatibility testing of all dogs in the colony
was accomplished using two microsatellite (VNTR)
markers, C.2200 and C.2202, which are closely linked
to the dog leukocyte antigen (DLA) Classes I and II
loci, and which are highly polymorphic (Wagner et al.,
1996). F2 crosses between heterozygotes were gov-
erned, in part, by pursuit of a diploid VNTR marker
mismatch between mates. That is, given two equally
distantly-related heterozygous male potential mates
for a given heterozygous female, the male who dif-
fered more substantially at the two VNTR loci was
selected as the mate. Crosses between animals who
were haploidentical at the VNTR loci, or who differed
in fragment size at any VNTR allele by fewer than
2 bp, were avoided whenever possible. These efforts
were intended to maintain as outbred a colony as
possible, and to maximize tissue type diversity, in
an attempt to mirror the situation in humans. VNTR
typing of carrier animals was used to identify those
who would be most useful additions to the breeding
colony and was also used to screen the littermates of
affected animals for potential hematopoietic stem cell
donors. The VNTR type of the F2 litter whose flow
cytometric profile is displayed in Fig. 4 is listed
(Table 2).
4. Discussion
We describe the establishment and characteriza-
tion of a mixed-breed CLAD colony. Despite the
severity of the clinical phenotype in the homozygous
state, knowledge of the precise molecular defect in
the CD18 subunit in CLAD permitted early and
definitive identification of CLAD-affected dogs, as
well as CLAD heterozygotes, using flow cytometry
and DNA sequencing. This allowed appropriate anti-
biotic therapy to be instituted in CLAD-affected
dogs within the first few days of life. Histocompat-
ibility typing, which was performed at the time that
Fig. 6. DNA sequence analysis of a normal, a CLAD heterozygote,
and a CLAD-affected dog is shown. DNA was obtained from
peripheral blood leukocytes, and sequenced with big dye termina-
tion sequencing. The G to C transposition was present on both
alleles in the homozygous CLAD-affected dog.
Table 2
VNTR identification of 8 littermate pups, generated by the mating of two CLAD heterozygotes
Dog Allele 1 (bp) Allele 2 (bp) VNTR
MHC Class I
C.2200
MHC Class II
C.2202
MHC Class I
C.2200
MHC Class II
C.2202
94 524 439 A 536 413 M AM
95 524 439 A 528 426 C AC
96 538 462 H 536 413 M HM
97 538 462 H 536 413 M HM
98 524 439 A 536 413 M AM
100 538 462 H 528 426 C HC
101 524 439 A 528 426 C AC
102 538 462 H 536 413 M HM
Alphabetical abbreviations for VNTR type are arbitrary. In this litter the father was VNTR type CM, and the mother was VNTR type AH.
K.E. Creevy et al. / Veterinary Immunology and Immunopathology 94 (2003) 11–22 19
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CLAD-affected and heterozygous dogs were identi-
fied, facilitated the retention of appropriately mat-
ched littermates. Detailed histocompatibility typing
using VNTR markers linked to the DLA loci also
allowed dogs with diverse tissue types to be purpose-
bred in the colony.
There are several features of CLAD that enable it to
serve as model for the development of novel hema-
topoietic approaches to therapy. First, the close clin-
ical parallel of the clinical phenotype of CLAD to the
phenotype of LAD indicates that therapeutic
approaches that are successful in CLAD are likely
to be relevant to therapy in LAD. Second, affected
CLAD dogs are homozygous for the identical mole-
cular defect in the CD18 subunit, which results in a
distinct clinical entity in which different therapies can
be assessed. Third, there are distinct and quantitative
parameters in CLAD to test for correction of the
disease phenotype. Fourth, the canine model is well
established as an experimental system in which novel
hematopoietic approaches to therapy, such as hema-
topoietic stem cell transplantation and hematopoietic
stem cell gene therapy, can be evaluated.
The clinical phenotype in CLAD closely resembles
that of LAD (Anderson et al., 1985). Omphalitis is
frequently a presenting sign in both CLAD and LAD,
as is the clinical picture of severe gingivitis, lympha-
denopathy, poor wound healing, and episodes of pyr-
exia and anorexia (Anderson et al., 1985). Infections
in both humans with LAD and dogs with CLAD are of
sufficient severity to be lethal if not treated promptly
with antibiotics. Thus, therapeutic approaches that
reverse the disease phenotype in CLAD would be
expected to be efficacious in LAD.
The genotype–phenotype relationship in CLAD and
the molecular defect in CLAD have recently been
shown to be a TGT to TCT change in codon 36 that
results in a missense amino acid change from a highly
conserved cysteine residue to a serine residue in the
extracellular domain of CD18 (Kijas et al., 1999).
Since this mutation most likely arose in a single
founder dog, and has been propagated through the
breed, all CLAD dogs reported have been homozy-
gous for the identical mutation (Debenham et al.,
2002; Foureman et al., 2002; Kijas et al., 1999).
CLAD in dogs is inherited in the same autosomal
recessive manner as the disease in humans. The tight
genotype–phenotype relationship in CLAD results in a
uniform clinical picture in untreated animals, against
which therapeutic interventions can be assessed.
The third feature of CLAD that makes it attractive
as a candidate disease for hematopoietic stem cell
therapy is the presence of distinct markers of disease.
These disease markers in CLAD include: (1) the
distinct clinical phenotype of frequent, life-threaten-
ing infections; (2) the lack of CD18-positive leuko-
cytes in the peripheral blood; and (3) the presence of a
marked elevation in the total peripheral blood leuko-
cyte count as a proxy for disease. Regarding the first
point, reversal of the disease phenotype remains the
cornerstone of any therapy in CLAD. Since most
CLAD dogs die or are euthanized by 4 months of
age due to infection, this outcome represents a clear
end-point against which future therapies can be com-
pared. In the second instance, since CD18-positive
leukocytes are not present in the peripheral blood of
CLAD dogs, the presence of CD18-positive leuko-
cytes in a CLAD dog, either from an allogeneic
hematopoietic stem cell transplantation, or from an
infusion of autologous, CD18 gene-corrected cells,
could be correlated with reversal of the disease phe-
notype. The presence and number of CD18-positive
leukocytes in the peripheral blood is easily detected
and quantified by flow cytometry. The third marker of
disease in CLAD is the peripheral blood white blood
cell count. This value is easily measured. A persistent
peripheral blood leukocytosis, typically characterized
by a mature neutrophilia, is readily apparent from an
early age in CLAD and LAD.
Children with LAD and dogs with CLAD, as well as
mice in which the CD18 gene has been disrupted by
gene targeting, exhibit a marked peripheral blood
leukocytosis, as well as myeloid hyperplasia in the
bone marrow (Anderson et al., 1985; Mizgerd et al.,
1997; Trowald-Wigh et al., 2000). The leukocytosis
could result from the inability of leukocytes to margin-
ate and egress from the vasculature due to CD18
deficiency. Alternatively, the leukocytosis could be
reactive in nature due to the inability of the leukocytes
to respond to chronic bacterial infection. Although the
precise mechanism for this leukocytosis remains
unclear, it appears that even low numbers of CD18-
positive leukocytes may reverse the leukocytosis,
which suggests that similar low numbers of CD18-
positive leukocytes may reverse the disease phenotype
(Horwitz et al., 2001).
20 K.E. Creevy et al. / Veterinary Immunology and Immunopathology 94 (2003) 11–22
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The previous history of the canine model itself in
the field of marrow transplantation and hematopoietic
stem cell gene therapy recommends CLAD as a model
for testing new approaches to hematopoietic stem cell
therapy. Dogs have been used successfully for the
development of clinical marrow transplant protocols
for more than 30 years (Storb and Deeg, 1986; Storb
et al., 1967, 1999). The dog major histocompatibility
complex has been defined by molecular and serolo-
gical techniques, regimens for conditioning prior to
stem cell transplant have been developed, canine anti-
CD34 monoclonal antibodies directed against hema-
topoietic stem cells are available, as are a variety of
antibodies which identify canine leukocyte subsets
(McSweeney et al., 1998). More recently, gene trans-
fer techniques involving canine hematopoietic stem
cells have been reported (Kiem et al., 1999). Impor-
tantly, insights into issues of hematopoiesis and trans-
plantation derived from canine studies have been
successfully extrapolated to humans.
The CLAD model described in this report now
provides a disease-specific, large-animal model for
assessing new therapeutic approaches that should be
directly applicable to LAD and other human hemato-
poietic stem cell disorders.
Acknowledgements
Kris Eckard and the staff at the NIH Animal Facility
at Poolesville, and Patty O’Donnell and the veterinary
staff at the School of Veterinary Medicine at the
University of Pennsylvania. Dr Haskins’ research
was supported by NIH grants RR 02512.
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