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,
Departments of
*
Pediatrics and
Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611;
Children’s Memorial Hospital, Chicago, IL 60614; and
Department of Microbiology and Immunology, Loyola University Stritch School of Medicine, Maywood, IL 60153
The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked advertisement in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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genes isolated from a primary, unamplified KD
vascular cDNA library. Five sets of clonally related sequences were
identified, comprising 34% (15 of 44) of the isolated sequences.
Furthermore, point mutations consistent with somatic mutation were
detected in the related sequences. Next, using formalin-fixed coronary
arteries from two additional fatal KD cases, we sequenced VDJ junctions
of genes isolated by RT-PCR, and a restricted pattern of CDR3 usage
was observed in both. We conclude that the vascular IgA response in
acute KD is oligoclonal. The identification of an oligoclonal IgA
response in KD strongly suggests that the immune response to this
important childhood illness is Ag-driven. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20% of
untreated patients develop coronary artery aneurysms (1).
The majority of KD patients who are treated within the first 10 days of
illness with i.v. gammaglobulin and aspirin show a rapid resolution of
fever and inflammatory signs, and this treatment reduces the prevalence
of coronary artery abnormalities to 5% (2). The
mechanism of action of i.v. gammaglobulin in KD is unclear. The
symptoms of KD resolve over several weeks, even in untreated patients,
and recurrences are unusual, distinguishing the illness from autoimmune
vasculitic disorders. The sequelae of undiagnosed KD during childhood
appears to account for cases of acute myocardial infarction or sudden
death in young adults (3). Because the etiology of KD is
unknown, no diagnostic test is available, specific therapy cannot be
developed, and prevention is not feasible.
Clinical and epidemiologic features of KD strongly suggest an
infectious etiology (1). These include the young age group
affected, the clinical features of the illness, the occurrence of
epidemics with periodicity, and the geographic wave-like spread of
illness during epidemics (1). To date, traditional methods
to identify a microbial agent have failed to clarify the etiology of
KD. Recently, there has been interest in a superantigen etiology of KD
based upon possible selective expansion of V
2 and V8 T cell
receptor families in peripheral blood in acute KD (4, 5);
however, other investigators have been unable to confirm this finding
(6, 7, 8, 9, 10). Toxic shock syndrome toxin-1 or streptococcal
superantigens were proposed to be related etiologically to KD based
upon a single study (11) that has not been confirmed by
others (7, 8, 12, 13, 14).
We have reported the novel finding that IgA plasma cells infiltrate the
vascular wall during acute KD (15). In this study, we
examined the clonality of the IgA response in KD vascular tissue by
sequencing the CDR3 regions of genes isolated from vascular tissues
from three fatal acute KD patients. We report that the IgA produced in
acute KD vascular tissue is oligoclonal, consistent with an Ag-driven
immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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genes from a KD vascular cDNA library
KD vascular cDNA library.
Vascular tissue (750 mg) from the aorto-iliac junction was used to
isolate total RNA by the guanidium thiocyanate-acid phenol method
(16), and an aliquot of the total RNA was used to isolate
poly A+ RNA using Dynabeads oligo dT (Dynal, Oslo, Norway).
The tissue was obtained from patient A, a 10-year-old Caucasian male
with acute KD who died from a ruptured coronary artery aneurysm on day
13 of illness (15). An oligo dT-primed, directional cDNA
library in the phage expression vector 
ZAP was synthesized using a
cDNA synthesis kit (ZAP-cDNA synthesis kit; Stratagene, La Jolla, CA)
as previously reported (15). The primary, unamplified cDNA
library was used to isolate clones for sequencing of the VDJ
junctions.
Isolation of clones.
clones were identified from plates of the primary library by
hybridization with an probe. Initial filter lifts were hybridized
with a VH-C probe generated by RT-PCR of control spleen
RNA as previously described (15), in which products of six
separate PCR reactions using primers from the six leader sequences of
the human heavy chain variable regions with a primer from the constant
region of (17, 18) were mixed (Table I
). Only five of 11 clones initially
identified using this probe contained variable region. Because use of
this probe did not obviate the isolation of clones containing the
constant region of only, we incubated additional filter lifts of
primary library with an constant region probe derived from a
plasmid preparation of a cDNA library clone. PCR amplification of
isolated plaques was then performed using a primer in the pBluescript
vector (SK; Table I) and a primer in the 5' constant region of
(C; Table I), and clones that yielded a product large enough to
contain variable region (>300 bp) were sequenced. Both probes were
labeled with [32P]dCTP using a random priming method
(Random Primers DNA Labeling System; Life Technologies, Gaithersburg,
MD). Positive clones were plaque purified using standard methods
(19). The majority (two- thirds) of the phage clones were
excised using ExAssist helper phage (Stratagene) to yield pBluescript
phagemid for sequencing; the remaining one-third were PCR amplified and
directly sequenced as described below.
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was
used to sequence the VDJ junctions (primer CS; Table I
).
Isolation of additional full-length VH4-group 1 clones.
Additional full-length clones containing the CDR3 sequence of the
VH4-group 1 clones were isolated from the amplified vascular cDNA
library, to determine whether other related clones demonstrating
somatic mutation were present in the library. The oligonucleotide AR1
corresponding to this CDR3 sequence (Table I) was synthesized and
labeled with [
-32P]dATP (Amersham) using T4
polynucleotide kinase (Life Technologies, Gaithersburg, MD) by standard
methods (19). One microliter of amplified cDNA library
(titer, 1 x 1010/ml) was subjected to PCR
amplification using a VH4 leader primer and a primer in the
constant region of (Table I). Amplified product was cloned into the
pGem-T vector (Promega, Madison, WI), and individual clones were
subjected to colony hybridization with the oligonucleotide AR1 probe
(Table I) using standard methods (19). Hybridizing clones
were sequenced as above to confirm that they contained the
VH4-group 1 CDR3 sequence. The variable region of the
clones was assigned to a germline sequence by comparing the sequence
data with published human germline sequences. These clones are not
included in the list of clones identified as they were not isolated
from the primary library.
Analysis of genes from formalin-fixed, paraffin-embedded
coronary artery blocks
Tissue blocks.
Coronary artery tissue blocks were obtained from patients B
(11-month-old Caucasian male) and C (4-mo-old Caucasian female), two
fatal KD cases in which death occurred within 2 mo of onset of the
illness. Patient B was not treated with i.v. gammaglobulin because the
diagnosis of KD was not made prior to death. Patient C received i.v.
gammaglobulin therapy. Ten 8-µm sections from each block were
processed for RNA isolation. As a control source for the amplification
of a polyclonal population of genes, ten 8-µm sections of
paraffin-embedded, formalin-fixed spleen tissue from patient A were
also obtained for RNA processing.
RNA isolation. Ten 8-µm sections were processed for RNA isolation by first deparaffinizing with xylene, and washing the tissue with 95% ethanol. The tissue sections were resuspended in digestion buffer (1% SDS, 0.1 M Tris-HC1, pH 7.3, 25 µM EDTA, and 1 mg/ml proteinase K) and incubated at 50°C for 12 h with agitation. After phenol-chloroform extraction and ethanol precipitation, the nucleic acid pellet was resuspended in solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 0.1 M mercaptoethanol) and incubated at room temperature for 24 h. After phenol-chloroform extraction and ethanol precipitation, the pellet was treated with 20 U of DNase I and incubated at 37°C for 30 min, followed by repeat extraction, precipitation, and resuspension of the pellet in water for use in RT-PCR.
RT-PCR.
One-tenth volume of the isolated RNA was incubated with 120 pm random
hexamers (Amersham Pharmacia, Piscataway, NJ) for 10 min at 70°C, and
first-strand buffer (Life Technologies), 500 µM dNTP, 10 mM DTT, 27 U
RNAguard (Pharmacia), and 200 U Superscript II (Life Technologies) were
added. The reaction was incubated at room temperature for 10 min,
followed by 1 h at 42°C. PCR was performed in a standard
reaction (19) using a primer in the constant region of
and a primer in the third framework region of VH3 or
VH4 (Table I) for 40 cycles of denaturation at 94°C for 1
min, primer annealing at 58°C for 1 min, and extension at 72°C for
2 min. A semi-nested PCR amplification followed, using the third
framework primer and three J region primers (Table I) for 40 additional
cycles using the same cycling parameters. Pfu Turbo (Stratagene) was
used to generate PCR products instead of Taq polymerase, to minimize
copying errors. After performing runoff reactions as described below,
PCR products were cloned into PCR Script (Stratagene) according to the
manufacturer’s instructions, and clones were sequenced as above.
Runoff reactions.
To obtain a rapid overall assessment of the distribution of CDR3 sizes
in the population of genes amplified from the tissues, we adapted a
method previously reported for determining the CDR3 sizes in a
population of T cell receptor genes (20). This method uses
a fluorescent dye-labeled primer for a single cycle of primer annealing
and extension of the PCR products previously generated as above.
Products of the runoff reaction were analyzed on a DNA sequencer (PE
Biosystems 377XL, ABI Prism, Genescan 3.0, run mode 36C-2400,
well-to-read distance 36 cm). The runoff products migrate by size,
which depends on the length of the CDR3 region(s) amplified. Size
standards were made by performing runoff reactions on PCR products of
genes of known CDR3 size using a primer labeled with a different
fluorescent dye. Size standards were run in the same well with the
sample to be assayed. The primers used were designed at the start of
the third framework region (Table I). Primers for samples were labeled
with 6-FAM dye, and primers for size standards were labeled with
HEX dye.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene sequences from vascular
tissues from three fatal KD cases. First, we isolated sequences
from a primary, unamplified cDNA library derived from nonfixed KD
vascular tissue (15). Because additional unfixed KD
vascular tissues were not available, we isolated sequences by
RT-PCR from formalin-fixed, paraffin-embedded vascular tissues from two
additional fatal KD cases.
Analysis of gene sequences from the primary, unamplified KD
vascular cDNA library
The primary library titer was 2.5 x 106 plaques,
with >90% recombinants and 0.1% -actin clones. The library has at
least 0.1% immunoglobulin clones detected by immunoscreening with
125I-labeled anti-human Ig (Amersham) as previously
reported (15). The dimensions of the tissue piece were
approximately 5 x 3 x 30 mm (450 mm3). The mean
number of IgA plasma cells in a 10 mm x 3 mm x 0.005 mm
(0.15 mm3) piece of this tissue was 300, as determined by
calculating the average of IgA-positive cells in eight separate
FITC-stained sections of the tissue. Thus, a 0.005-mm tissue section
contained 2000 IgA plasma cells/mm3. However, because
the maximal diameter of a large plasma cell is 0.025 mm, the
same plasma cell might appear in up to five successive 0.005-mm
sections. Therefore, the number of IgA plasma cells in the tissue
more closely approximated 400 per mm3 (2000 ÷ 5). Thus, in
the original 450-mm3 piece of tissue, there would be
180,000 IgA plasma cells (400 cells/mm3 x 450
mm3).
We isolated 88 clones from the primary, unamplified library by
hybridization with an probe as described in Materials and
Methods. Forty-four of these 88 clones included the CDR3 region
and were sequenced. Ten clones were sequenced twice, using excised
phagemid and PCR, to ensure that Taq polymerase error did not introduce
random mutations into the products; identical CDR3 sequence was
obtained by both methods in all cases.
Distribution of VH genes in the KD vascular cDNA library by
family.
To determine whether expansion or deletion of a heavy chain variable
region (VH) family occurred in the IgA genes in the KD
tissue, isolated clones were grouped by VH family by
examination of the CDR1 and 2 regions and the framework regions and
comparing them to published VH family sequences
(21). Of the 44 clones, 42 were members of the
VH1, VH3, VH4, VH5,
VH6, and VH7 families as shown in Table II. The distribution of VH
family members in the genes in the KD cDNA library was similar to
that reported in the literature for adult peripheral blood B cells
(18, 22). More VH7 clones were present in the
KD vascular cDNA library than would be expected by comparison to
published data for adult controls (10 vs 2%, p = 0.05
by Fisher’s exact test); however, VH7 has been reported to
be frequently used in the infant cord blood repertoire
(23), and data for its use in childhood peripheral blood B
cells are lacking. The remaining two clones did not contain
sufficient framework 3 region to allow assignment to a family. Overall,
the distribution of VH families in KD vascular tissue
appears to be similar to that found in normal peripheral blood B
cells.
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genes in the vascular cDNA library were
polyclonal or oligoclonal, we sequenced the CDR3 regions. Sequence
identity between B cells in this region indicates clonal relatedness
(24, 25); expansion of B cells with the same VDJ
rearrangements and with evidence of somatic mutation are characteristic
features of an immune response to Ag (24, 25).
Sequence analysis of the 44 clones revealed two groups of clonally
related VH4 sequences (Fig. 1). The VH4-group 1 sequences
contain five members (clones C5v, E2, 5-1, 7-1a, and A2) and the
VH4-group 2 sequences contain two members (clones C2
and 4–5). As can be seen in Fig. 1A, the
VH4-group 1 sequences show evidence of somatic mutation
compared to germline sequence VH4.18, and clone A2 shows
evidence of somatic mutation compared to clones E2, 5-1, 7-1a, and C5v.
Amino acid sequences for this group are seen in Fig. 1B.
Similar evidence of somatic mutation is seen in the
VH4-group 2 sequences (Fig. 1C). Clones C2 and
4–5 show somatic mutation compared to germline DP-65 and to each
other. Fig. 1D shows amino acid sequences for this second
group. The identification of multiple clones with identical VDJ
rearrangements and evidence of somatic mutation strongly suggests an
oligoclonal, Ag-driven immune response in acute KD.
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). The
VH3-group 1 has three members (clones D3, 11-2, and 11-5),
the VH3-group 2 has two members (clones 2-2 and 13-1), and
the VH7-group 1 contains three members (clones 10-1, 7-1b,
and 10-4). The members of each group were identical to each other in
the regions sequenced. Therefore, these clones likely represent
sequences isolated from plasma cells that are highly represented in the
vascular tissue and were derived from the same original B cell that
underwent clonal expansion.
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C) in CDR3 that confers an amino
acid substitution (GA) (Fig. 1, A and B).
Clone A2 extends only to the start of FR3 and has two additional
nucleotide differences in FR3 compared to the other clones. Sequencing
of the VH4-group 1 was performed on excised phagemid rather
than on PCR-amplified product, ruling out PCR as a cause of these
mutations. Therefore, the sequence differences in these clones likely
represent point mutations introduced during somatic mutation in B cells
undergoing clonal expansion.
In a separate experiment, we performed PCR amplification of the
amplified cDNA library using a VH4 leader primer and a
primer in the constant region of , cloned the PCR products, and
hybridized clones with the oligonucleotide probe AR1 derived from the
VH4-group 1 CDR3 sequence. We obtained three full-length
clones with the expected CDR3 sequence, designated clones 17PCR, 28PCR,
and 39PCR (GenBank accession numbers AF247737–AF247739), all of which
had VH4 gene sequence more closely related to the germline
VH4.18 than the clones isolated from the primary library.
For example, clone 28PCR demonstrated 95% homology to germline
VH4.18, whereas the VH4-group 1 sequence E2
demonstrates 85% homology to germline VH4.18. The CDR3
sequence of the clones identified by PCR was similar to that of the
VH4-group 1 sequences, except for the last two amino acids
(HY in VH4-group 1, QH in the PCR clones). This provides
further support for the assignment of the VH4-group 1
sequences to the germline VH4.18, as the PCR clones had the
same VDJ rearrangement but higher homology to germline
VH4.18 than the VH4-group 1 set of clones. This
indicates the presence of additional clones of the same B cell lineage
in the cDNA library and provides further support for clonal expansion
with selection by Ag.
The VH4-group 1 sequences have undergone significant somatic mutation; the R (replacement)/S (silent) ratio in these sequences when compared to germline is 12:1 in the CDR regions compared to 13:7 (1.9) in the FR regions. This compares to the calculated R/S ratios for the VH4.18 germline gene of 4.5 for the CDR regions and 2.6 for the FR regions (27); observed number of CDR R mutations is significantly higher than expected, p < 0.001 using formula in Ref. 27 . These data collectively indicate that the VH4-group 1 sequences have undergone significant somatic mutation and have been clonally expanded, consistent with an Ag-driven immune response.
Other VDJ Rearrangements among isolated genes.
CDR3 sequence data for additional genes isolated from the vascular
cDNA library are given in Fig. 3. They
consist of three VH1 genes, 12 VH3 genes, seven
VH4 genes, two VH5 genes, two VH6
genes, and one VH7 gene.
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clones (five VH4-group 1, two
VH4-group 2, three VH3-group 1, two
VH3-group 2, and three VH7-group 1) in the
primary cDNA library. None of the clonally related CDR3 regions had
been entered previously into the GenBank/EMBL database. All 44
sequences are now entered into the GenBank/EMBL database under
accession numbers AF064878–AF064921.
Additional unfixed tissues were not available from other fatal KD cases
to allow for construction of other KD vascular libraries. Therefore,
methodology was developed to isolate and analyze gene sequences
from formalin-fixed, paraffin-embedded coronary artery tissues from
other fatal KD cases.
Analysis of formalin-fixed, paraffin-embedded KD coronary artery tissue blocks
We chose to amplify VH3 and VH4 gene
sequences because these families are most represented in the peripheral
blood B cell repertoire and consistently gave polyclonal CDR3 size
distribution patterns when RT-PCR and runoff reactions were performed
on genes from peripheral blood B cell RNA from controls (data not
shown). Moreover, RT-PCR amplification of RNA isolated from
paraffin-embedded, formalin-fixed spleen tissue from KD patient A
yielded polyclonal VH3 and VH4 CDR3 size
profiles (Fig. 4) as expected. Sequencing
of the spleen VH3 and VH4 PCR products
revealed a diverse repertoire of CDR3 genes (Fig. 5). These experiments demonstrate that
our procedure will detect a polyclonal distribution of sequences if
such sequences are present in the sample.
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400 IgA plasma cells in a 5-µm tissue section or, at a minimum,
1200 IgA plasma cells in the ten 8-µm tissue sections.
RT-PCR amplification of the coronary artery block from patient B did
not yield any visible VH3 product after reverse
transcription and nested PCR, but a reproducible VH4
product was obtained. The runoff reaction products of this
VH4 product are seen in Fig. 6; one single prominent band
corresponding to a CDR3 size of 33 nucleotides is seen. Nine clones
obtained from this PCR product were sequenced in the CDR3 region and
were identical (Fig. 7).
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and VH4 products;
runoff reaction products are seen in Fig. 8. Eleven VH3 clones and 12
VH4 clones were sequenced; each set of sequences within the
amplified VH family was identical in the CDR3 region
(Fig. 7). The CDR3 sequences from patients B and C are entered into the
GenBank database under accession numbers AF237658–AF237660. Thus, in
patients B and C, the usage of predominant VH3 and
VH4 sequences provides further evidence of an
oligoclonal response in the KD vascular wall.
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PCR products from
paraffin-embedded, formalin-fixed spleen from patient A was identical
to CDR3 sequence E1, which was isolated from the primary, unamplified
vascular cDNA library from KD patient A (Figs. 3 and 5). Isolation of
clones from the vascular library and from the formalin-fixed,
paraffin-embedded tissues were performed by different methods as
described above, at different times, and in different laboratories. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have demonstrated in this study that clonally related genes are
present in a primary, unamplified vascular cDNA library from patient A,
a child who died of fatal acute KD. Use of the primary, unamplified
cDNA library avoided potential amplification bias that might occur with
amplification of the primary cDNA library or copying errors that might
occur with use of PCR methods to generate the population of genes
to be studied. Strikingly, five clonally related sets of sequences
account for 34% of the sequences characterized from the primary,
unamplified vascular cDNA library. The most frequently isolated
sequence, the VH4-group 1, shows evidence of extensive
somatic mutation, with a R/S ratio of 12 in the CDRs and 1.9 in the FR
regions. It uses a JH1 gene, which has been reported to be
used by only 1% of human peripheral blood B cells (22, 26). The presence of these clones, which have undergone
somatic mutation and clonal expansion, supports an Ag-driven B cell
immune response in the vascular wall in acute KD.
In addition, we have shown an oligoclonal pattern of VH3
and VH4 gene usage in paraffin-embedded, formalin-fixed
coronary arteries from two additional fatal KD patients in whom one
CDR3 sequence predominated in the VH3 or
VH4 genes in the coronary artery tissues. Thus, evidence
of an oligoclonal IgA response was detected in vascular tissue of all
three KD patients. Using the same method, a polyclonal profile of
VH3 and VH4 gene usage in
paraffin-embedded, formalin-fixed KD spleen was demonstrated. However,
it is possible that the clonality of genes in KD spleen is somewhat
more restricted than are those in a spleen from a healthy patient; this
may be the case in spleen from any patient with an overwhelming
infection. To support this point, one identical CDR3 sequence was
obtained from 44 sequences isolated from a primary, unamplified
vascular cDNA library from KD patient A and from 20 VH4
sequences cloned from a paraffin-embedded, formalin-fixed spleen sample
from the same patient. The fact that an identical CDR3 sequence was
identified in a small number of sequences examined from vascular
tissue and spleen from the same patient implies that the KD spleen is
producing some of the same oligoclonal IgA Ab that are being made in
the vascular wall.
The representation of different VH family members in our vascular cDNA library is similar to that reported for human peripheral blood B cells (18, 22). We did not observe a significant expansion of a VH family without clonality nor depletion of a VH family, findings that might have suggested stimulation by a superantigen (32). Rather, we found evidence of clonally related sequences in the VH3, VH4, and VH7 families, consistent with an oligoclonal Ab response directed at various antigenic epitopes of an etiologic agent.
Although the presence of a set of two clonally related sequences from
our 44 clones isolated from the unamplified vascular cDNA library might
simply be due to isolation of the sequence from the same plasma cell by
chance, we believe this to be unlikely. First, the cDNA library was
prepared from total RNA originally isolated from tissue that contained
180,000 IgA plasma cells, as described in Materials and
Methods. Second, sequence data clearly indicate that the
VH4-group 2 set consists of two different members, as shown
in Fig. 1 C and D. These clones were sequenced as
excised pBluescript phagemids; we performed no PCR amplification with
its potential to introduce nucleotide replacement by Taq polymerase
error. Clone C2 is full length and clone 4-5 includes most of FR3. The
clones differ in sequence by four nucleotides in FR3 and one in CDR3,
indicating that they were produced by two different plasma cells (Fig. 1C). We cannot rule out that VH3-group 2 clones
2-2 and 13-1 are from the same plasma cell; clone 13-1 extends only to
the end of CDR2 and has the same nucleotide sequence as clone 2-2
throughout FR3.
We cannot exclude that a superantigen-driven T cell response occurs
concurrently with an Ag-driven B cell response in acute KD, but we
believe this to be unlikely. Data indicate that clonal expansion of
CD8+ T cells occurs in acute KD (6, 9). As
previously suggested (6), an apparent polyclonal increase
of V2 and V8 T cells in acute KD, suggesting activation by a
superantigen (4), may have actually resulted from failure
to separate CD4+ and CD8+ T cells, with the
presence of CD4+ T cells obscuring clonal expansion of
CD8+ cells. The data presented in this paper support an
Ag-driven immune response in KD. We hypothesize that the KD infectious
agent enters the host through the respiratory route and is recognized
and processed in the bronchus-associated lymphoid tissues.
Epidemiologic data in KD are consistent with a respiratory portal of
entry of the etiologic agent (1). B cells then switch to
IgA and enter the general circulation. The pathogen may enter the
bloodstream, resulting in the multisystem involvement characteristic of
KD (1), and also enter vascular tissues. IgA B cells may
enter the vascular walls with other inflammatory cells and there
differentiate into IgA plasma cells, probably under the influence of
locally produced cytokines. Our findings strongly support the
hypothesis of an Ag-driven immune response in KD, because we found both
clonally related sequences and evidence of somatic mutation within
the related clones in the vascular cDNA library. These findings are
characteristic of an Ag-driven immune response in a germinal center
(25). By probing the KD vascular cDNA library with an
oligonucleotide probe corresponding to the CDR3 sequence of the
VH4-group 1 set of genes, we identified additional
sequences of the same B cell lineage in the library, findings again
typical of an Ag-driven immune response in a germinal center. Because
germinal centers are not present in KD vascular tissue
(15), this suggests that IgA cells are undergoing somatic
mutation in the bronchus-associated lymphoid tissue or other lymphoid
tissue in response to Ag prior to migrating to the vascular tissue.
Our finding of an oligoclonal IgA response in the vascular wall in acute KD supports an Ag-driven immune response. The production of oligoclonal Abs in human tissues that are directed at the disease-causing pathogen is a feature of many infectious processes (33, 34). Our data support the hypothesis of a conventional Ag in the pathogenesis of KD.
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Acknowledgments |
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Footnotes |
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2 Address correspondence to Dr. Anne H. Rowley, Pediatrics W140, Ward 12-140, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611.
3 Abbreviation used in this paper: KD, Kawasaki Disease.
Received for publication March 27, 2000. Accepted for publication October 16, 2000.
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 S. Stagi, G. Simonini, L. Ricci, M. de Martino, and F. Falcini Coeliac disease in patients with Kawasaki disease. Is there a link? Rheumatology, July 1, 2006; 45(7): 847 - 850. [Abstract] [Full Text] [PDF]
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T. Kobayashi, Y. Inoue, K. Takeuchi, Y. Okada, K. Tamura, T. Tomomasa, T. Kobayashi, and A. Morikawa Prediction of Intravenous Immunoglobulin Unresponsiveness in Patients With Kawasaki Disease Circulation, June 6, 2006; 113(22): 2606 - 2612. [Abstract] [Full Text] [PDF]
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 A. H. Rowley, S. T. Shulman, F. L. Garcia, J. A. Guzman-Cottrill, M. Miura, H. L. Lee, and S. C. Baker Cloning the Arterial IgA Antibody Response during Acute Kawasaki Disease J. Immunol., December 15, 2005; 175(12): 8386 - 8391. [Abstract] [Full Text] [PDF]
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S. M. Benseler, B. W. McCrindle, E. D. Silverman, P. N. Tyrrell, J. Wong, and R. S.M. Yeung Infections and Kawasaki Disease: Implications for Coronary Artery Outcome Pediatrics, December 1, 2005; 116(6): e760 - e766. [Abstract] [Full Text] [PDF]
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J. W. Newburger, M. Takahashi, M. A. Gerber, M. H. Gewitz, L. Y. Tani, J. C. Burns, S. T. Shulman, A. F. Bolger, P. Ferrieri, R. S. Baltimore, et al. Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Statement for Health Professionals From the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, American Heart Association Pediatrics, December 1, 2004; 114(6): 1708 - 1733. [Abstract] [Full Text] [PDF]
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J. W. Newburger, M. Takahashi, M. A. Gerber, M. H. Gewitz, L. Y. Tani, J. C. Burns, S. T. Shulman, A. F. Bolger, P. Ferrieri, R. S. Baltimore, et al. Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Statement for Health Professionals From the Committee on Rheumatic Fever, Endocarditis and Kawasaki Disease, Council on Cardiovascular Disease in the Young, American Heart Association Circulation, October 26, 2004; 110(17): 2747 - 2771. [Abstract] [Full Text] [PDF]
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 F Falcini Vascular and connective tissue diseases in the paediatric world Lupus, February 1, 2004; 13(2): 77 - 84. [Abstract] [PDF]
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H. C. Meissner and D. Y. M. Leung Kawasaki Syndrome: Where Are the Answers? Pediatrics, September 1, 2003; 112(3): 672 - 676. [Full Text] [PDF]
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