Kawasaki disease (KD) is a multisystem vasculitis that primarily affects the coronary arteries of young children. A number of epidemiological and clinical observations suggest that KD is caused by an infectious agent, with suggestions ranging from Staphylococci, Streptococci, Mycoplasma or Chlamydia, [1] [2] [3] [4] to viruses such as adenovirus, parvovirus or Epstein-Barr virus. [5] [6] [7] However, no single causative pathogen has been consistently demonstrated 8 in the nasopharynx, oropharynx, skin, or faeces of patients with KD. 9, 10 Internal tissues of the airway or of the gastrointestinal (GI) tract may also be entry or colonization sites of the potential causative agents, but these have not been investigated in detail. Intense interest has recently centered on novel human coronavirus messenger RNA (mRNA) detected in the respiratory secretions of some children with KD; 11 however, other investigators have not been able to confirm this finding. 12, 13 We have hypothesized that the mucosa of the upper GI tract could be involved in KD because of the role of the GI tract as an immunological organ constantly exposed to microorganisms and other agents. We have previously observed increased numbers of CD4 + T cells and human leucocyte antigen (HLA)-DR + cells, and fewer CD8 + T cells, in the gut of patients with KD compared with controls. 14 Consequently, we carried out a microbiologic investigation of the small intestine and showed that the range of bacterial species adhering to the lumen of the jejunum of patients with KD was quite different from that of controls. 15 Notably, five strains of Streptococci and two strains of Staphylococci [both species are known to be common sources of superantigen (sAg)] were isolated only from KD patients. We have also investigated T-cellreceptor (TCR) Vb2 expression in the small intestinal I M M U N O L O G Y O R I G I N A L A R T I C L E mucosa of KD patients 14 and found that these cells were selectively increased in the mucosa of patients in the acute phase of KD compared with controls.
On the basis of these findings, we carried out microbiological and molecular biological studies focused on the biological activity of microorganisms detected on the alimentary tract surfaces of KD children in the acute phase of the disease. We focused, in particular, on heat shock protein (hsp) and superantigenic activity, in view of previous data indicating that they might have a role in KD. [17] [18] [19] Materials and methods
The study received ethical approval from Juntendo University Hospital in Tokyo. All families of the patients and control subjects had given their consent to participate in the study.
Nineteen patients with KD (14 boys and five girls, 5 months to 8 years of age) were enrolled in our study between February 2004 and June 2006; their diagnoses were made in accordance with the clinical criteria for KD (Table 1) . They had been hospitalized within 7 days of the onset of fever. All the patients except for one (patient no. 7) received intravenous c-globulin at a dose of 2 g/kg. Coronary artery involvement was demonstrated in patients 1, 2 and 17. Giant aneurisms were found in patient 2, who died of myocardial infarction. The other patients had no evidence of persistent cardiac abnormal lesions, as assessed by echocardiography.
Jejunal swab specimens and peripheral blood samples were taken for analysis from all KD subjects and, as controls, from 15 patients with food-sensitive enteropathies in remission. 20 We considered our control subjects to be a normal population with respect to the jejunal microflora for the reason stated in our previous studies. [14] [15] [16] Microbiologic study on the jejunal surfaces Swab specimens of the jejunal surfaces were acquired from a piece of jejunal mucosa taken using a sterile paediatric Crosby-type capsule. Details of the microbiologic examination are given elsewhere. 14-16 None of the subjects or controls were treated with antibiotics before the jejunal biopsies were taken.
Bacteria were cultured in brain-heart infusion medium at 37°for 24 hr. Bacterial culture supernatants (BCS) were collected by centrifugation and then filtered through a 0Á22-lm pore membrane to remove the bacteria.
Blood samples were taken from all subjects when KD was at the acute phase (before drug treatment) and 12-16 days after the onset of fever (i.e. during the convalescent phase). Blood specimens from children with food-sensitive enteropathies in the convalescent phase were taken simultaneously with their biopsies.
Peripheral blood mononuclear cell co-culture with BCS Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples using the standard procedure of density-gradient centrifugation, and 200 ll (containing 1 · 10 5 PBMCs) was transferred, in triplicate, to each well of a round-bottomed 96-well plate (Becton Dickinson, Franklin Lakes, NJ) and then incubated, for 72 hr at 37°in 5% carbon dioxide, in the presence or absence of BCS at a final concentration of 0Á25%.
For screening for the presence of hsps in BCS, BCSinduced proliferative activity of PBMCs, obtained from convalescent-phase KD patients, was compared with that of PBMC from controls because hsps could induce significant cell-proliferative activity; [17] [18] [19] however, this cell-proliferative activity appeared to be weaker than that induced by sAg or by other potentially immunodominant proteins. Significant cell-proliferative activity of BCS, containing hsp, to PBMC was defined by a stimulatory index (SI) of more than 3Á0. 18, 19 Flow cytometric analysis Superantigenic activity analysis in BCS has been described previously. 21, 22 We looked for expansion of Vb2 and Vb8 T cells because Vb2 T-cell proliferation in KD patients has been reported previously, 21, 22 and Vb8 T cells were known to be expanded by a clinical isolate of Streptococcus pyogenes toxin used as a positive control. Monoclonal antibodies (mAbs) were obtained from PharMingen (San Diego, CA). Data were analyzed using CELLQUEST Ò software (Becton Dickinson, San Jose, CA).
In order to detect bacterial hsps in BCS and human endogenous hsp molecules in the PBMC supernatant co-cultured with BCS, in comparison with controls, Western blot analysis was performed using plasma samples from the subjects as antibodies to those proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in 10% gels (ATTO Corporation, Tokyo, Japan), as described previously. 23 
Proteins in a BCS sample were separated by SDS-PAGE and electrotransferred to immobilon P, polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membrane was incubated at 37°for 40 min in 100-fold dilutions of plasma samples obtained from patients in the acute and convalescent phases of KD, or from controls, as 
Proteins were visualized by staining with Coomassie Ò Brilliant Blue. The target proteins were excised from the gels, trypsinized and analyzed using a matrix-assisted laser desorption time-of-flight mass spectrometer (Voyager DE Pro; Applied Biosystems, Tokyo, Japan). A list of the corrected mass peaks provided details for peptide mass fingerprinting (PMF). Proteins were identified in the National Center for Biotechnology Information (NCBI) database through PMF using the MS-FIT program in protein prospector (http://prospector.ucsf.edu).
To measure the concentrations of cytokines and human endogenous hsp60 in PBMCs co-cultured with BCS, enzyme-linked immunosorbent assays (ELISAs) were performed using mAbs according to the manufacturer's instructions. ELISA kits for interferon-c (IFN-c), tumour necrosis factor-a (TNF-a) and interleukin-10 (IL-10) were purchased from Sanquin Research (Amsterdam, the Netherlands) and those for human endogenous hsp60 were obtained from Stressgen (Ann Arbor, MI). The detection limits were 0Á5 IU/ml for IFN-c, 10 pg/ml for TNF-a, 4Á0 pg/ml for IL-10 and 3 ng/ml for hsp60. Absorbance was read at 450 nm against air using a microplate reader (Bio-Rad, Hercules, CA).
The paired Student's t-test was used to analyze statistical significance between subjects with KD and controls. Values of P < 0Á05 were considered significant.
To detect hsp in BCS, BCS-induced proliferative activity on PBMCs was performed. All bacteria isolated from all KD patients gave culture supernatants that induced significant proliferative activity (SI > 3Á0) in autologous PBMCs (Table 1) . We performed antibioticsensitivity testing on the strains producing BCS that caused significant proliferation of PBMCs, considering the possibility of treatment of these bacteria if they partici-pated in the onset of disease, and found that they were all resistant to commonly used antibiotics ( Table 2) . No strains of bacteria isolated from control children produced BCS that induced significant proliferative activity of autologous PBMCs.
To determine superantigenic activity in PBMCs stimulated with BCS, the expression of TCRs Vb2 and Vb8 was determined on cultured cells. Streptococcus sanguinis of KD patients 11 and 13, Streptococcus oralis of patients 6, 8 and 10, and Streptococcus mitis of patients 1, 2, 4, 14, 15, 17 and 18, and all staphylococcal strains of patients 3, 5, 9, 12, 16 and 19 produced an expansion of T-cell populations expressing Vb2 (Fig. 1) . None of the other strains, including Neisseria mucosa, induced selective Vb expansion in T-cell populations from children with KD or from controls.
The antibody responses to bacterial products that induced significant proliferative activity, as shown in Table 1 , in host plasma, were analyzed by Western blotting using plasma obtained from 19 patients during acute and convalescent stages of KD. Some plasma samples obtained from patients during the convalescent stage of KD, after treatment with immunoglobulin, showed vigorous antibody reactivity against 60 000-70 000 molecular weight (MW) proteins that were not seen in acute plasma; however, interestingly, this phenomenon was not observed in Ó 2009 Blackwell Publishing Ltd, Immunology, 128, 511-520 the BCS originating from superantigenic strains, in which a small quantity of 60 000-70 000 MW proteins was seen in plasma from patients in the acute phase of KD (Fig. 2a,b) . Furthermore, in control plasma samples this reactivity was not seen to any bacterial products originating from the jejunal mucosa of control or KD patients. Because of a lack of blood samples it was impossible to examine all the combinations and therefore representative reactivity patterns of several samples are demonstrated in Fig. 2c .
The 60 000-70 000 MW target proteins were identified in the NCBI database through PMF using the MS-Fit program. All were identified as hsp60 originated from each organism with high sequence coverage of more than 30% (Table 3) . Interestingly, those hsp60 molecules were derived from only Gram-negative strains of bacteria in KD patients 1, 2, 3, 4, 6, 7, 10, 12, 13, 14, 15, 17 and 18, whereas some Gram-positive cocci yielded no hsps or the others had already secreted them before the onset of KD. The most frequently detected protein was hsp60, hitherto reported to be derived from Neisseria spp. in convalescent plasma from 8 of 19 (42%) patients with KD.
It has been previously suggested that the increased expression of endogenous hsp may contribute to the abnormally activated immune system of KD patients via molecular mimicry between bacterial hsps and human hsps. [17] [18] [19] To demonstrate the production of endogenous hsp by PBMCs reacting to each strain of bacteria, the presence of human hsp60 in PBMC culture supernatant, co-cultured with BCS which induced significant proliferative activity, was investigated by ELISA. The production of endogenous hsp60 was clearly evident in the supernatants originating from Gram-negative microbes compared with those from Gram-negative microbes and controls (Fig. 3) .
We analyzed homology between hsp60 derived from bacteria such as Neisseria, Acinetobacter and human endogenous hsp60 according to the NCBI database, CLUSTALW W Multiple Sequence Alignments and the manufacturer's reference. Homology between the 66-86 amino acid sequence of bacterial hsp60, originating from both N. mucosa and Acinetobacter iwoffii, and the 90-110 amino acid sequence of human hsp60 (50%), presumably recognized by effector T cells in the disease, 19 was found, as shown in Table 4 , indicating cross-reactivity between Figure 3 . Evaluation of endogenous human heat shock protein 60 (hsp60)produced from peripheral blood mononuclear cells (PBMCs) co-cultured with bacterial culture supernatants (BCS). A significantly higher content of endogenous hsp60 [determined by enzyme-linked immunosorbent assay (ELISA)] was found in the supernatants originating from the Gram-negative strains than in those from the Grampositive strains and the controls, which contained only a small amount of human hsp60. those two molecules related to a possible pathogenic role of hsp-specific T cells in KD. Likewise, high homology was observed between the 222-230 amino acid sequence of bacterial hsp60 and the 246-254 amino acid sequence of human hsp60 (67%), which are demonstrated to have binding specificities to HLA-DR4. 24 Similarly, the 259-267 amino acid sequence of bacterial hsp60 and the 283-291 amino acid sequence of human hsp60 showed homology (56%), and are known to show high binding affinity to HLA-DR8.
The cytokine response of PBMCs to bacterial products showed that pro-inflammatory cytokines IFN-c and TNF-a were elicited in patients with KD, but not in the controls (Fig. 4) . As a recent report proposed that increased levels of IL-10 induced by self-hsp60 molecules could lead to anti-inflammatory process in children with juvenile idiopathic arthritis, we also investigated the concentration of IL-10 in supernatants. 25 Interestingly, microbial products, except for sAg, elicited a significantly higher IL-10 response in PBMC from KD, unlike those from controls.
It has been postulated that the primary cause of KD is an infectious agent that elicits cytokine secretion; these cytokines target vascular endothelial cells, producing cell-surface neoantigens, and antibodies produced against these antigens may then target the vascular endothelium, resulting in a cascade of events leading to vascular damage. Most of the searches for an aetiologic agent in KD have been carried out for a single infectious agent. However, a more plausible underlying principle is that there is cooperation between different mechanisms of different causative bacteria, and a final common pathway of immune activation is responsible for the clinical disorder and complications. 8, 26 Recently, the issue has been raised as to whether the provoking antigen in KD might be a conven- Interferon-c (IFN-c) and tumour necrosis factor-a (TNF-a) production was induced in a BCS obtained from a significant strain, but not in the control supernatants (P < 0Á01 versus controls; those P values were omitted in the figure for a tidy view). Microbial products except for superantigen (sAg) elicited a significantly higher interleukin-10 (IL-10) response in PBMCs from patients with Kawasaki disease, unlike those from the controls (P < 0Á01).
tional antigen or a sAg. [27] [28] [29] [30] Although the debate continues regarding the mechanism of initial immune activation, the most likely scenario is that sAg and conventional peptide antigens work together to direct a persistent immune response leading to coronary artery damage. 26 A unifying model proposed that a microbe with superantigenic activity initiates massive activation of the developing immune system. A subpopulation of sAgresponsive T cells is rescued from apoptosis as a result of interaction with costimulatory signals between antigenpresenting cells and a conventional peptide antigen. This peptide antigen may be derived from itself or an infectious mimic of itself. The immune response is perpetuated locally where the self-antigen is found on the coronary vessels.
In terms of the current study we have provided data consistent with two of these hypotheses, namely that bacteria unique to the upper bowel of patients with KD have sAg activity and that supernatants of these bacteria contain hsps, which induce T-cell division and production of pro-inflammatory cytokines. One may be concerned with the description of S. oralis, S. sanguinis and S. mitis as superantigenic strains because they are generally not known as superantigenic strains. Very few reports on the sAg activity of S. mitis have been published, 31, 32 and sAg activity for S. oralis and S. sanguinis has not yet been confirmed, except for some interesting findings suggesting the possibilities of toxic shock-like syndrome caused by viridans streptococci to which those strains belong and sAg-related toxigenic conversion between streptococcus species. 33, 34 However, these findings did not directly support the production of sAg by those strains. Further investigations in this respect are obviously required. Autologous PBMCs may be problematic because T cells bearing a certain TCR would be stimulated in an oligoclonal manner and would eventually become anergic. However, we demonstrated that PBMCs obtained from patients during the convalescent phase of KD had the capacity to respond to sAg, probably because some T-cell subsets bearing Tb2 were not anergized or the T cells had never been exposed to sAg in these young children. The reason why the IFN-c and TNF-a production induced by BCS containing sAg was not markedly higher than that induced by BCS without sAg may be explained likewise in that a handful of T cells which respond to sAg in the convalescent sample could produce moderately high amounts of those cytokines.
Previous theories have proposed that hsp60 signals mediates T-cell activation through Toll-like receptor 4 on antigen-presenting cells, including GI dendritic cells, 35 although there are also reports to the contrary. 36 Heat shock protein 60 molecules may also function as conventional peptide antigens. [17] [18] [19] It is an attractive concept that this cross-reactivity between specific epitopes of bacterial and human hsps could play a role in vascular dam-age characteristic of KD, as humoral and cellular reactivity against hsp65/63 has been described in more common vascular diseases such as atherosclerosis, 37 which may share a common pathogenic mechanism involving the immune response against hsp65/63; however, the etiologic agents that produce bacterial hsp, leading to the onset of KD, are unknown.
To identify the possible aetiologic pathogen(s) and to elucidate the pathogenic mechanisms, we focused on microbiota that can produce hsps and are present on the GI tract surfaces of children with acute KD on the basis of the findings obtained from our previous studies. [14] [15] [16] The agents demonstrated here are rich in variety, consistent with our hypothesis that many different agents may be related to the onset of KD. Based on our findings in this study, the most likely candidates for bacteria associated with the pathogenesis might be Gram-negative microbes, such as N. mucosa, in cooperation with plural Gram-positive cocci having superantigenic activity. Furthermore, bacterial hsp60-70 seemed to possess a certain homology, together with self-hsps. Recent studies suggest that an interaction between bacterial and self-hsp antibody responses to non-self epitopes might be promoted by the concomitant generation of anti-inflammatory cytokines, such as IL-10, induced by self-epitopes. 35 Accumulating evidence from epidemiological or immunopathological studies has suggested that hsp60 autoantibodies can cross-react with bacterial and self-hsp60 and may induce cytotoxic damage of stressed endothelial cells, resulting in coronary atherosclerosis. [38] [39] [40] [41] These theories seem applicable to the pathogenesis of coronary lesions of KD, in view of the high expression of hsp60 in endothelial cells, together with the serological detection of bacterial and self-hsp in KD patients, demonstrated by some investigators. [17] [18] [19] Vascular surface-expressed hsp60, transferred from the cytoplasm or mitochondria following induction by bacterial hsp60 stimulation, can be recognized by circulating anti-hsp autoantibodies or cytotoxic T lymphocytes targeting autoantigens.
The incidence rate of coronary lesions in patients with KD may depend upon how strongly causative agents can induce the initial immune activation which elicits autoreactive T cells and, importantly, how much self-hsp molecules they can evoke from the cytoplasm or mitochondria to the vascular surface. Interestingly Gram-negative microbes appeared to trigger more self-hsp than Gram-positive cocci. Furthermore, Gram-negative microbes, such as N. mucosa, which co-exist with Grampositive cocci, have been isolated in KD patients with vascular involvements.
The amino acid sequences of Neisseria hsp60 (221-231 and 255-269), which were found in this study, had high homology (60%, 50%, respectively) with self-hsp60 (246-256 and 280-294), which are core epitopes of a protein with a high capability of binding human class II molecules, respectively. 25 These sequences were demonstrated to have high affinity with HLA-DRB1*0802 and *0401, respectively, by DR-peptide binding assays. Interestingly, these DR alleles are frequently found specifically in the Japanese population (24Á5%, 40Á4%, respectively). 24 This may account for the high incidence of KD among Japanese people. Other explanations suggest Toll-like receptor 4 42 or hsp polymorphism, 43, 44 both of which can affect the production control of self-hsp as an autoantigen.
We included children with food-sensitive enteropathies as controls; however, it is not entirely clear whether they are appropriate controls. Such types of studies may be inevitably under limitations because it is nearly impossible to conduct them in age-matched healthy controls, for ethical reasons.
Immunoglobulin is helpful in the acute phase of the illness, but there is a failure rate of approximately 10% and an adverse effect rate close to 30%. Additionally immunoglobulin is expensive, at approximately $100 per gram. 45 Antibiotic treatments targeting the causative bacteria may yield a great deal of reliability and economical benefits for the treatment for KD if the more detailed pathogenic mechanisms of the disease can be defined by further studies.