Abstract
Background: Clinical and serologic studies suggest that Guillain–Barré syndrome (GBS) and atypical GBS with preserved muscle stretch reflexes (MSRs) form a continuous spectrum as well as do Fisher syndrome (FS), FS/GBS overlap, Bickerstaff brainstem encephalitis (BBE), BBE/GBS overlap, acute ophthalmoparesis (AO), ataxic GBS, and acute oropharyngeal palsy.
Objective: To clarify the spectrum of neurologic disorders that occur subsequent to Campylobacter jejuni enteritis.
Methods: We recruited patients with various neurologic conditions and from whom C jejuni was isolated. Bacterial features were investigated.
Results: Diagnoses for the patients from whom C jejuni was isolated were GBS (n = 90), FS (n = 22), MSR-preserved GBS (n = 10), FS/GBS (n = 6), BBE (n = 1), BBE/GBS (n = 2), AO (n = 3), ataxic GBS (n = 1), and acute oropharyngeal palsy (n = 3). Isolates from MSR-preserved GBS were similar to those of GBS in serotype (HS:19), genotype (lipo-oligosaccharide [LOS] locus class A or B, cst-II genotype [Thr51]), and GM1 or GD1a epitope expression on LOS. FS/GBS overlap, BBE, BBE/GBS overlap, AO, ataxic GBS, and acute oropharyngeal palsy isolates were similar to those of FS in serotype (HS:2 or HS:4-complex), genotype (LOS locus class A or B, cst-II genotype [Asn51]), and GQ1b epitope expression on LOS.
Conclusions: The bacterial findings support the proposal that Guillain–Barré syndrome (GBS) and muscle stretch reflex–preserved GBS comprise a continuous spectrum as well as do Fisher syndrome (FS), FS/GBS overlap, Bickerstaff brainstem encephalitis (BBE), BBE/GBS overlap, acute ophthalmoparesis, ataxic GBS, and acute oropharyngeal palsy.
Campylobacter jejuni is isolated from many patients with Guillain–Barré syndrome (GBS) and Fisher syndrome (FS),1 but comprehensive studies of other neurologic disorders have yet to be made. To clarify the spectrum of neurologic disorders that may occur after C jejuni enteritis, we recruited patients who had various neurologic conditions and from which C jejuni was isolated, and analyzed their clinical features.
Patients with GBS subsequent to C jejuni enteritis often have anti-GM1 and anti-GD1a immunoglobulin G (IgG) antibodies,2,3 whereas those with FS have anti-GQ1b IgG antibodies, which cross-react with GT1a (anti-GQ1b/GT1a IgG antibodies).4 Penner serotyping showed that in Japan, GBS is associated with HS:19, and FS is associated with HS:2 and the HS:4-complex.1 Lipo-oligosaccharide (LOS) is a major cell-surface structure expressed by C jejuni, which is divided into several LOS locus classes based on organization of the LOS biosynthesis genes. GBS and FS are associated with classes A and B.5–7 C jejuni sialyltransferase, Cst-II, present in classes A and B, functions in the biosynthesis of ganglioside-like LOS, and variation in the nucleotide sequence of the cst-II gene affects its activity.8 Cst-II (Thr51) produces GM1-like and GD1a-like LOSs,8 and cst-II (Thr51) strains are associated with GBS.9 In contrast, Cst-II (Asn51) produces GT1a-like, GD1c-like, and GD3-like LOSs, in which each terminal trisaccharide is identical to that of GQ1b (figure E-1 on the Neurology Web site at www.neurology.org),8,10–12 and cst-II (Asn51) strains are associated with FS.9 To clarify the pathogenesis and identify the nosologic position of each neurologic disorder, we examined the bacterial features of isolates obtained from patients with various neurologic conditions.
Methods.
Patients and C jejuni strains.
From May 1996 to April 2005, we received approximately 9,900 requests from Japanese physicians to test serum antiganglioside antibodies from patients who had GBS (n = 3,358), FS (n = 1,114), chronic inflammatory demyelinating polyneuropathy (CIDP) (n = 1,070), acute disseminated encephalomyelitis (n = 34), encephalitis (n = 115), and encephalopathy (n = 22). On receipt of the serum samples, we requested the primary physicians to obtain stool cultures and send them to the Tokyo Metropolitan Institute of Public Health. When C jejuni was isolated at the hospitals, we asked that they be sent to the institute. Patients' clinical features were reviewed in an application form, as were their medical records both at admission and at discharge (obtained from each primary physician) to recruit C jejuni–isolate cases as well as to ascertain diagnoses. Information was obtained on age, sex, onset month, antecedent infectious symptoms, initial symptoms, neurologic signs during the illness, and CSF findings. When medical records did not contain adequate information, questionnaires were faxed to the physicians concerned.
Diagnoses of GBS and CIDP were based on established criteria.13,14 GBS-like patients who did not fulfill the criteria because of normal to brisk muscle stretch reflexes (MSRs) throughout the illness were considered to have “MSR-preserved GBS.” Patients seen at Dokkyo Medical University Hospital were classified as having acute motor axonal neuropathy (AMAN) or acute inflammatory demyelinating polyneuropathy if they fulfilled the published electrodiagnostic criteria.15 Diagnoses of FS, BBE, and acute ophthalmoparesis (AO) without ataxia were based on our published criteria.16 Patients with ophthalmoplegia and severe limb weakness (3 or less on the Medical Research Council scale) were considered to have overlapping FS and GBS (FS/GBS), and those with consciousness disturbance, in addition to ophthalmoplegia and severe limb weakness, were considered to have overlapping BBE and GBS (BBE/GBS). FS-like patients who did not fulfill the criteria because they had no external ophthalmoplegia throughout the illness were classified as having “ataxic GBS.”17 Pharyngeal–cervical–brachial weakness-like patients who did not fulfill the criteria because they had no cervical–brachial weakness throughout the illness were classified as having “acute oropharyngeal palsy.”18,19
Penner serotyping was performed as described previously.1 The C jejuni LOS locus class (A to F) was determined in a PCR performed with the specific primers for each locus.5 The cst-II gene genotype (Asn/Thr51) was determined by direct sequencing of the PCR fragment.9
ELISA and thin-layer chromatography with immunostaining.
Serum IgG and IgM antibodies to GM1, GD1a, GT1a, and GQ1b (figure E-1) were measured by ELISAs as described elsewhere.10 In brief, serum samples diluted 1:500 were placed in separate microtiter plate wells. The mean value for triplicate reference wells without antigen was subtracted from the mean value for triplicate wells of each sample, and the optical density (OD492) was assessed. An OD492 of less than 0.1 was judged to be negative. An OD492 of 0.1 to 0.5 was categorized as 1+; 0.5 to 1.0, 2+; 1.0 to 1.5, 3+; 1.5 to 2.0, 4+; 2.0 to 2.5, 5+; and 2.5 or more, 6+.
Crude LOS was prepared as described elsewhere.10 Whether ganglioside epitopes (GM1, GD1a, and GQ1b) were present on the C jejuni LOS was determined by thin-layer chromatography with immunostaining of the sera from patients with GBS (S6960 [anti-GM1], S5174 [anti-GD1a]) and FS (S7577 [anti-GQ1b/GT1a]). To determine the reactivity of patients' sera with each C jejuni LOS, thin-layer chromatography plates with serum diluted 1:100 were incubated, then incubated again with peroxidase-conjugated anti-human γ-chain–specific antibodies (Dako, Denmark; 1:250).
Statistical analysis.
Differences in group frequencies were compared by the χ2 or Fisher exact test (two-tailed) with SPSS 12.0J software (SPSS Inc., Chicago, IL). Differences in medians were examined by the Mann–Whitney U test. A p value of less than 0.05 was considered significant.
Analysis of strains GC033, GC057, and GC124.
Overnight growth (C jejuni isolates GC033, GC057, and GC124) on each agar plate was treated as described elsewhere.10 O-deacylated LOS samples were analyzed by capillary electrophoresis-electrospray ionization mass spectrometry. Isolation of genomic DNA from the isolates was done with a DNeasy Tissue kit (Qiagen Inc., CA). Genes involved in the biosynthesis of the LOS outer core were amplified using an Advantage 2 PCR enzyme system (BD Biosciences Clontech, Palo Alto, CA). The 7.2-kb region (from waaC to cst-II) was sequenced by means of custom-made primers used previously to sequence this locus in multiple C jejuni strains.8 DNA sequencing was performed with a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Quebec, Canada). Products were analyzed in an ABI 3100 Genetic Analyzer (Applied Biosystems).
Results.
Clinical and serologic features.
We found 141 C jejuni–isolate patients, two of whom were excluded because of inadequate information. Diagnoses for the other 139 patients were GBS (n = 90, 65%), FS (n = 22, 16%), MSR-preserved GBS (n = 10, 7%), FS/GBS (n = 6, 4%), BBE (n = 1, 0.7%), BBE/GBS (n = 2, 1.4%), AO (n = 3, 2.1%), ataxic GBS (n = 1, 0.7%), acute oropharyngeal palsy (n = 3, 2.1%), and CIDP (n = 1, 0.7%).
Because the clinical and serologic features of C jejuni–isolate patients with GBS or FS have already been reported,1 we focused on the other patients. Table 1 shows the clinical and serologic features of patients with MSR-preserved GBS, FS/GBS, BBE, BBE/GBS, AO, ataxic GBS, and acute oropharyngeal palsy. We have tentatively called last six “FS-related conditions.” The sex ratio, frequency of antecedent diarrhea, interval from onset of antecedent diarrhea to neurologic symptom onset, and frequency of CSF albuminocytologic dissociation, as well as seasonal distribution, did not differ significantly between GBS and MSR-preserved GBS or between FS and FS-related conditions, whereas the age distribution did differ significantly between FS and FS-related conditions. None of the patients with GBS and MSR-preserved GBS showed sensory involvement. Those factors did not differ between FS and FS/GBS or between FS/GBS and GBS. Based on electrodiagnostic criteria, of the patients seen at our hospital, two with MSR-preserved GBS and one with GBS were classified as having AMAN.
Table 1 Campylobacter jejuni–isolate patients of whose diagnoses were neither Guillain–Barré nor Fisher syndromes
There were no significant differences in the positive frequencies of anti-GM1 or anti-GD1a IgG antibodies between GBS (89%) and MSR-preserved GBS (90%) or in those of anti-GQ1b/GT1a IgG antibodies between FS (91%) and FS-related conditions (93%) (table 2). Anti-GM1 or anti-GD1a IgG antibodies were positive in FS (18%), FS/GBS (50%), and GBS (89%), and anti-GQ1b/GT1a IgG antibodies in FS (91%), FS/GBS (83%), and GBS (6%) (tables 1 and 2). None of the antiganglioside antibodies were detected in the CIDP patient.
Table 2 Clinical profiles of Campylobacter jejuni–isolate patients
Bacterial features.
Because the bacterial features of C jejuni isolated from GBS and FS have been reported,5 we focused on isolates from the other conditions. Eight strains obtained from the 10 C jejuni–isolate patients with MSR-preserved GBS and 11 from the 25 patients with FS-related disorders underwent bacterial analyses. Seven of the 8 isolates from the MSR-preserved GBS patients had bacterial features in common with those of GBS; serotypes of HS:19, LOS locus class A or B, a cst-II (Thr51) content, and GM1 or GD1a epitope on LOS (table 3).1,5,9,10 In contrast, 9 of the 11 isolates from FS-related condition patients had bacterial features in common with those of FS isolates; serotypes of HS:2 or the HS:4-complex, LOS locus class A or B, cst-II (Asn51) content, and GQ1b epitope expression on LOS. Serum IgGs from these patients reacted with each LOS, whereas serum IgG from the CIDP patient did not (figure 1).
Table 3 Characteristics of Campylobacter jejuni isolates
Figure 1. Serum reactivity with Campylobacter jejuni lipo-oligosaccharide isolates. A, resorcinol staining; B, immunostaining of patients' sera with C jejuni lipo-oligosaccharides. Lane 1, GC159 (Bickerstaff brainstem encephalitis); lane 2, GC057 (acute ophthalmoparesis); lane 3, GC216 (ataxic Guillain–Barré syndrome); lane 4, GC183 (acute oropharyngeal palsy); lane 5, GC111 (chronic inflammatory demyelinating polyneuropathy).
Mass spectrometric analysis of the O-deacylated LOS from C jejuni GC033 (FS-related), GC124 (BBE/GBS-related), and GC057 (AO-related) gave rise to spectra that were consistent with the presence of multiple mass species. The differences in observed masses within each strain were the result of variation in lipid A composition and, in the case of GC057, to the presence of one or two terminal sialic acids (tables E-1, E-2, and E-3).
GC033 and GC124 had the same mass species; therefore, we propose that both express a GD1c mimic (figure 2A). The presence of two sialic acids forming a chain was confirmed by tandem mass spectrometry and a search for the precursor ion at m/z 581 (data not shown). We speculate that two sialic acids are substituted on the terminal galactose because these strains have a Cj1135 allele (putative glucosyltransferase, GenBank accession numbers DQ536321 and DQ536322) that can transfer a glucose to heptose II, the heptose substituted with the inner galactose. The presence of glucose on heptose II prevents sialylation of the inner galactose, indicative that the two sialic acids are on the terminal galactose.20
Figure 2. Proposed lipo-oligosaccharide outer core structures based on capillary-electrophoresis electrospray ionization mass spectrometry analysis of O -deacylated lipo-oligosaccharide samples from Campylobacter jejuni isolates. (A) GC033 (Fisher syndrome) and GC124 (overlapping Bickerstaff brainstem encephalitis and Guillain–Barré syndrome); (B) GC057 (acute ophthalmoparesis). Gal = galactose; NeuAc = N -acetylneuraminic acid; GalNAc = N -acetylgalactosamine; Hep = l-glycero-d-manno-heptose; Glc = glucose; Kdo = 3-deoxy-d-manno-2-octulosonic acid; PEA = phosphoethanolamine.
Mass spectrometric analysis of the O-deacylated LOS sample from GC057 showed mass species with either one or two sialic acids (table E-2). The presence of two sialic acids forming a chain was confirmed by tandem mass spectrometry and a search for the precursor ion at m/z 581 (data not shown). We speculate that the mass species with one sialic acid corresponds to a GM1b mimic and species with two sialic acids to a GD1c mimic (figure 2B) and that only the terminal galactose is substituted by sialic acid because GC057 also has a Cj1135 allele that can transfer glucose to heptose II in its inner core (see above and GenBank accession number DQ438950). Tandem mass spectrometry data also was consistent with the absence of phosphoethanolamine or phosphate on heptose I (which is linked to the 3-deoxy-d-manno-2-octulosonic acid) of the GC057 LOS. Heptose I is substituted by a phosphate or a phosphoethanolamine in all C jejuni LOS structures so far reported.20 Consequently, the absence of these substitutions on the heptose I of the LOS of C jejuni GC057 is an important observation.
Discussion.
Serologic determinations may include false-positive cases, and C jejuni isolation is the gold standard for the diagnosis of bacterial infections. Over a 9-year period, we found 139 patients from whom C jejuni was isolated and whose medical histories were available for clinical analysis, although the latent period between preceding intestinal infection and the neuropathy onset often exceeds the excretion period of viable C jejuni cells in stools.1 GBS, FS, MSR-preserved GBS, FS/GBS, BBE, BBE/GBS, AO, ataxic GBS, acute oropharyngeal palsy, and CIDP were diagnosed. Encephalopathy after C jejuni enteritis has been reported,21 but no C jejuni isolates were found in 290 patients who had encephalopathy, encephalitis, or acute disseminated encephalomyelitis.
By chance, C jejuni enteritis may be concurrent with various diseases. When the bacterium is isolated from patients who have neurologic disorders, it is difficult to judge whether it had a causative role. Epidemiologic studies, however, have shown that C jejuni infection is related to GBS and FS,10,22 and experimental studies suggest that antiganglioside IgG antibodies induced by ganglioside-mimicking C jejuni LOS cause both GBS and FS.9,23 Our study has showed that each C jejuni LOS was recognized by the IgGs from patients with MSR-preserved GBS or FS-related conditions, indicative that as in GBS and FS, IgG antibodies induced by a ganglioside-like LOS are active in the development of those conditions. In contrast, LOS was not recognized by the IgG from a CIDP patient. Moreover, because only one C jejuni strain was isolated from more than 1,000 patients with CIDP, isolation might have occurred by chance. Our observations do not support the speculation that C jejuni infection induces the development of CIDP.24,25
Patients who experienced acute paralytic syndrome after gastrointestinal illness but had normal to brisk MSR have been reported, but the nosologic position of the syndrome is not clear.26 We earlier reported four C jejuni–isolate patients who had acute progressive motor weakness and preserved MSR.27 They had AMAN and anti-GM1 IgG antibodies, as did patients who had GBS subsequent to C jejuni enteritis.2 We therefore proposed that the diagnostic criteria for GBS be extended to require hyporeflexia or areflexia as a hallmark. This, our larger study, showed that the clinical, serologic, and bacteriologic features of GBS are similar to those of MSR-preserved GBS, supportive evidence that both conditions are part of a continuous spectrum. Predominantly the HS:19 and cst-II (Thr51) strains were isolated from the MSR-preserved GBS as well as GBS patients,5 and the GM1 or GD1a epitope was expressed in both of these C jejuni isolates. Moreover, anti-GM1 and anti-GD1a IgG antibodies were positive in both conditions. The hyperreflexia mechanism in AMAN is not known, but dysfunction of the inhibitory system via spinal interneurons may increase motor neuron excitability.28 Inflammation of the spinal anterior roots may lead to disruption of the blood–CNS barrier, allowing antiganglioside antibodies access to antigens near anterior horn cells, especially in intramedullary collateral branches to the inhibitory interneurons. These findings suggest that host factors such as antibody accessibility, rather than bacterial ones, determine MSR.
Because the clinical and serologic features of FS are similar to those of FS/GBS, BBE, BBE/GBS, AO, ataxic GBS, and acute oropharyngeal palsy, the latter conditions have been considered to be FS related. Moreover, in each condition, antecedent C jejuni infection has been suggested serologically. Several patients from whom C jejuni was isolated have been reported. Our study provides evidence that these are FS-related conditions from the bacterial as well as the patients' standpoint.
Because of areflexia and CSF albuminocytologic dissociation, FS is considered a GBS variant.29 This is strongly indicated by clinical observations that some patients who present with FS progress to GBS.30 Moreover, it is supported by our serologic observations that some FS/GBS patients carry IgG antibodies against GM1 and GD1a, as well as against GQ1b, which are reasonable findings. For example, a GT1a-like LOS is synthesized by Cst-II (Asn51) via GM1-like and GD1a-like LOSs, and an FS isolate (CF93-6) carries GM1-like and GD1a-like LOSs as well as a GT1a-like LOS.8,10 The results shown in tables 1 and 2 suggest that C jejuni strains bearing cst-II (Asn51) induce the synthesis of anti-GM1 or anti-GD1a IgG antibodies, as well as anti-GQ1b/GT1a IgG antibodies, and that FS/GBS develops in some patients, whereas the same strains may induce only anti-GQ1b/GT1a IgG antibodies and the development of FS in others. Host genetic factors may determine which autoantibodies and clinical presentation occur.
BBE is characterized by consciousness disturbance as well as ophthalmoplegia and ataxia. The nosologic relationship of BBE to FS has long been debated. Anti-GQ1b IgG antibodies are present in BBE as in FS.4,31 This was confirmed in patients with C jejuni–isolated BBE in our study. The immunologic profile common to FS and BBE supports a common pathogenesis. BBE etiology is speculated to be similar to that of GBS based on evidence of prodromal upper respiratory infection, areflexia, and CSF albuminocytologic dissociation.32 Some patients experience limb weakness, considered the result of overlapping AMAN.33 These clinical findings indicate that BBE and GBS are closely related, as are BBE and FS. Our study showed that the bacterial characteristics of a BBE isolate and BBE/GBS isolates were those of FS, not GBS isolates. The three BBE and BBE/GBS isolates belonged to the HS:2 or HS:4-complex and had the GQ1b epitope characteristic of FS isolates.5 This is evidence that BBE and FS comprise parts of a continuous spectrum. BBE can be positioned as FS associated, having the apparent CNS sign of consciousness disturbance. Rather than bacterial factors, host factors such as antibody accessibility may determine whether the clinical presentation is FS or BBE, as in GBS and MSR-preserved GBS. As in FS that overlaps GBS, host genetic factors may determine the autoantibodies produced and whether the clinical presentation is BBE or BBE/GBS.
Acute onset of external ophthalmoplegia is a cardinal FS feature.29 Four-fifths of FS cases studied started with diplopia, and the median period for the disappearance of ataxia was 1 month, and that of ophthalmoplegia was 3 months.30 This temporal profile suggests that AO without ataxia is a mild form of FS, which is supported by serologic observations that patients with AO as well as those with FS carry anti-GQ1b IgG antibodies.4,34 Our study confirmed that C jejuni–isolate AO patients had anti-GQ1b IgG antibodies. One AO isolate (GC057) belonged to HS:2 and had cst-II (Asn51) and a GQ1b epitope, characteristic of FS isolates.5 These AO bacterial features are further evidence that AO and FS are parts of a continuous spectrum. Host rather than bacterial factors, such as the amounts or affinities of autoantibodies produced, may determine whether the clinical presentation is AO or FS.
Ataxic GBS, originally described by Richter,17 is characterized by severe ataxia of the cerebellar type with no or minimal ophthalmoplegia. Clinical findings of hyporeflexia or areflexia, distal paresthesias, and CSF albuminocytologic dissociation indicate that the condition is a GBS variant. Some patients with ataxic GBS carry anti-GQ1b IgG antibodies.35 The fact that ataxic GBS and FS have an autoantibody in common suggests that they form a continuous spectrum. Patient 23 with ataxic GBS also had anti-GQ1b IgG antibodies, and the isolate (GC216) had the cst-II (Asn51) genotype and expressed a GQ1b epitope on the LOS. Bacterial findings also support the speculation that ataxic GBS and FS are parts of a continuous spectrum, and that host rather than bacterial factors determine whether the clinical presentation is ataxic GBS or FS. Interestingly, the ganglioside composition of the neuromuscular junctions differs among mouse strains.36 Some humans may not express GQ1b in their oculomotor nerves, although most do, as well as in their primary sensory neurons.4,37 Immunohistochemical investigations of a large number of autopsy case studies are needed to clarify this.
One-fourth of patients with FS studied had bulbar palsy,30 but acute oropharyngeal palsy is characterized by oropharyngeal weakness without ophthalmoplegia and limb weakness.18 The acute oropharyngeal palsy patients carried anti-GQ1b/GT1a IgG antibodies. C jejuni was isolated from Patient 24, who had anti-GQ1b/GT1a IgG antibodies. That isolate (GC183), which belonged to the HS:4-complex, had the cst-II (Asn51) genotype and a GQ1b epitope on the LOS. Another isolate (GC229) had the cst-II (Thr51) genotype and did not carry a GQ1b epitope, but Patient 25 had anti-GQ1b IgG antibodies and serum IgG bound to its LOS. Whether the GC229 isolate actually functions in the induction of anti-GQ1b antibodies is unknown. Immunochemical analyses have shown that patients' lower cranial nerves had both GQ1b and GT1a,38 but some humans may not express GQ1b in their oculomotor nerves (as discussed above). Host factors, such as antigen distribution, may determine whether the clinical presentation is AO, ataxic GBS, or acute oropharyngeal palsy.
Mass spectrometry showed that three C jejuni isolates from FS, BBE/GBS, and AO patients had GD1c-like LOS with terminal trisaccharides identical to those of GQ1b and GT1a (figures 2 and E-1). Another C jejuni strain (PG 836) isolated from a patient with FS also had a GD1c-like LOS.12 These findings are further evidence that BBE/GBS and AO are related to FS and that host rather than bacterial factors determine the clinical presentation. In conclusion, the bacterial characteristics of FS are similar to those of FS/GBS, BBE, BBE/GBS, AO, ataxic GBS, and acute oropharyngeal palsy—additional evidence that these are FS-related conditions. The bacterial genotype defines whether GBS, FS, or the related conditions will develop with a role for the patient in defining the specific clinical presentation.
Acknowledgment
The authors thank Ms. Maki Okazaki (Department of Neurology and Research Institute for Neuroimmunological Diseases, Dokkyo Medical University School of Medicine), Ms. Saiko Koike (Institute for Medical Science Dokkyo Medical University), Mr. Denis Brochu, Ms. Anna Cunningham, and Ms. Sonia Leclerc (Institute for Biologic Sciences, National Research Council of Canada) for technical assistance.
Footnotes
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Additional material related to this article can be found on the Neurology Web site. Go to www.neurology.org and scroll down the Table of Contents for the November 28 issue to find the title link for this article.
*These authors contributed equally to this work.
Supported in part by grants from the Uehara Memorial Foundation to M.K.; the Naito Foundation to M.K.; Dokkyo Medical University School of Medicine to M.K. (no. 2005-01-2); the Mizutani Foundation for Glycoscience to N.Y.; a grant for Scientific Research (B) (KAKENHI 16390254 to N.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Research Grants on Measures for Intractable Diseases (1724360 and 17243601 to N.Y.) from the Ministry of Health, Labor and Welfare of Japan; and a grant from the Human Frontier Science Program (RGP0038/2003-C to M.G. and N.Y.).
Disclosure: The authors report no conflicts of interest.
Received May 10, 2006. Accepted in final form August 9, 2006.
References
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