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Antibodies against Helicobacter pylori were detected in the cerebrospinal fluid obtained from patients with guillain-barr syndrome.

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Antibodies against
Helico bucter pylori
Were Detected in the
Cerebrospinal Fluid
Obtained from Patients with
Guillain-Barri: Syndrome
Susumu Chiba, MD, PhD,* Toshiro Sugiyama, MD, PhD,t
Hiroyuki Matsumoto, MD, PhD,* Kazunari Hisano, MD,$
Teruhito Awakawa, MD,$ Kazumi Hiura, MD,*
Masaki Saitoh, MD,* and Kohzoh Imai, MD, PhD$
We examined the antibodies against Helicobacter pylori
proteins in the cerebrospinal fluid (CSF) of 7 patients
with Guillain-Bard syndrome (GBS). Crude H. pylori
antigens, fractionated heat shock protein (HSP), and urease B (UB) from H. pylori antigens were separated by
SDS-PAGE. With Western blot analysis, four of seven
CSF samples had several IgG antibodies against H. pylori
proteins, including HSP and UB. No cross reactivity
against Campylobacter jejuni was observed. These antibodies may be involved in the immune responses of patients with GBS.
Chiba S, Sugiyama T , Matsumoto H, Hisano K,
Awakawa T , Hiura K, Saitoh M, Imai K.
Antibodies against Helicobacter pylori were
detected in the cerebrospinal fluid obtained from
patients with Guillain-Bard syndrome.
Ann Neurol 1998;44:686-688
Guillain-Bard syndrome (GBS) is an acute inflammatory demyelinating disorder of the peripheral nervous
system characterized by an acute infection which precedes the clinical disease by days to weeks. Recently,
Campylobacter jejuni was claimed to be a predominant
precipitating agent which may also trigger an immune
response to glycoconjugates of peripheral myelin in patients with GBS. Yuki and his colleagues'22described 5
individuals with a severe axonal form of GBS associated with C. jejztni enteritis and extremely high titers of
serum IgG anti-GM1 antibodies.
From the *Department of Neurology, $First Department of Internal
Medicine, School of Medicine, Sapporo Medical University, and
?Third Department of Internal Medicine, School of Medicine,
Hokkaido University, Sapporo, Japan.
Received Jun 24, 1997, and in revised form Apr 17, 1998. Accepted
for publication Apr 17, 1978.
Address correspondence to Dr Chiba, Department of Neurology,
School of Medicine, Sapporo Medical University, Minami 1 Jo,
Nishi 16 chome, Chuo-ku, Sapporo 060, Japan.
O n the other hand, infection with Helicobacter pylori
leads to diverse clinical and pathological outcomes such
as gastroduodenal ulcer or chronic gastritis in human^.^
Recent reports indicated a possible correlation between
H. pylori infection and increased risk of coronary heart
disease and stroke.* In this study, we detected a set of
unique antibodies against H. pylori in the cerebrospinal
fluid (CSF) of 4 of 7 patients with GBS using Western
blot analysis.
Materials and Methods
CSF samples were obtained from 7 patients (4 men, 3
women; mean age, 42 years) with GBS in the acute phase and
5 patients (1 man, 4 women) with definite multiple sclerosis
(MS) as disease controls. The diagnosis of GBS was based on
the clinical criteria defined by Asbury and C ~ r n b l a t h .An
electrophysiological examination disclosed three axonal and
four demyelinating forms. H. pylori strains isolated from patients who had been histologically proven to have gastritis
were used as antigens. The microorganisms were inoculated
from fresh blood agar plates into 200 ml of Brucelh broth
supplemented with 5% fetal calf serum. Cultures were incubated for 48 hours with shaking at 37°C in an anaerobic jar
containing a Campy Pouch (Becton Dickinson, Cockeysville,
MD). Crude antigens were extracted from H. pylori by adding 0.2 M of glycine hydrochloride (pH 2.2) for 20 minutes
at room temperature. Heat shock protein (HSP) and urease
B (UB) from the crude H. pylori antigens were fractionated
by continuous elution electrophoresis using a Model 49 1
Prep-cell (Bio-Rad, Tokyo, Japan). The fractionated antigens
were identified by monoclonal anti-UB antibody (Institute of
Immunology, Tokyo, Japan) and polyclonal anti-HSP antibody.' One hundred micrograms of the crude antigen and 1
p g each of fractionated HSP and UB were separated by
SDS-PAGE using 12% acrylamide gel. Western blot nitrocellulose was applied in a blotting chamber (Bio-Rad) according to the standard protocol. After blotting, the membrane was incubated overnight in phosphate-buffered saline
(PBS) solution containing 3% bovine serum albumin. The
membrane was cut into small strips, and each strip was incubated with a 20-fold dilution of CSF in 0.05% Tween 20
in PBS solution overnight. T o compare the antibody profile
between CSF and serum, we also used a 200-fold dilution of
serum from 3 patients who had been proven to have antibodies against H. pylori antigens in CSF. After three washes
with 0.05% Tween 20 in PBS solution, the strips were overlayered for 4 hours with rabbit anti-human IgG conjugated
with horseradish peroxidase at a 1:lOOO dilution (0.5%
Tween 20 in PBS solution). After washing (3 times with and
1 time without 0.05% Tween 20 in PBS), developing solution containing 4-chloro- 1-naphthol and H,O, was added.
To confirm the specificity of the CSF antibodies, a crude
Escbericbia coli antigen was used in every sample and a crude
C. jejttni antigen was also used in three samples. We examined not only five samples of CSF in which antibodies were
absorbed by mixing with the crude H. pylori antigen for 1
hour at 37°C but also five CSF samples of patients with MS
as a disease control.
Copyright 0 1998 by the American Neurological Association
Figure 1 shows the profile of the Western immunoblot
analysis. Western blot analysis using the crude H. pylori
antigen disclosed that four (3 axonal forms and 1 demyelinating form) of seven CSF samples obtained from
patients with GBS had several IgG antibodies against
H. pylori antigens, whose molecular weight ranged
from between 60 and 70 kd to near 90 kd. Comparing
the antibody profile between CSF and serum, serum
from every case had antibodies recognizing numerous
proteins whose molecular weights ranged between 25
and 120 kd (Fig 2). After CSF samples had been absorbed with the crude H. pylori antigens, all of these
antibodies disappeared. As regards the subfractions of
H. pylori antigen, we were able to identify specific
bands that were compatible with antibodies against
HSP (66 kd) and UB (62-65 kd). We could not confirm the 90-kd protein, which was thought to be vacuolating cytotoxin.
No reactions were found by Western blot analysis
either between the CSF samples of GBS patients and
the crude E. coli or C. jejuni antigens, respectively, (see
Fig 2) or between the CSF samples of MS patients and
crude H. pylori antigens. These results indicate that a
set of unique antibodies against H. pylori antigens, including HSP and UB, exists in the CSF of patients
with GBS in the acute phase.
H. pylori is a curved or spiral gram-negative microaerophilic organism which was first isolated from a human
gastric biopsy specimen in 1983.' Histological gastritis
and H. pylori infection commonly occur in the stomach of apparently healthy persons and increase in prevalence with advancing age.' The pathophysiological
Fig 1. Reactiviiy of cerebrospinalJuid antibodies against
crude Helicobacter pylori antigens, fiactionated heat shock
protein, and urease B in patients with Guillain-Barrk
97 KDa66 KO*-
45 KO.-
Fig 2. The different projles of antibodies between cerebrospinal jluid and serum and no redctions either between the CSF
of patients with Guillain-Bard syndrome and the crude
Escherichia coli or Campylobacter jejuni antigens, respectively.
role of H. pylori infection in gastroduodenal diseases,
including gastric malignancy, has been discussed. In
addition, recent reports suggest a possible expanded
role of H. pylori infection in the development of coronary heart disease, stroke, and slun diseases.*,'
Regarding the pathophysiology of GBS, current evidence supports the belief that immunological mechanisms are of importance in the development of GBS
with special reference to elevated serum antibody titers
against various types of gangliosides and glycolipids.
Recent studies have suggested that C. jejuni may be the
most common preceding pathogen in close conjunction with anti-GM1 and GD1b antibodies. Antibodies
against the other gangliosides such as GM2, G D l a ,
GTlb, G Q 1b, sialylparagloboside and G M I b have also
been reported as etiological factors of GBS."
H. pylori has been classified into the genus Campylobacter. From the immunological aspect, Kostrzynska
et all* demonstrated that H. pylori proteins were antigenically cross reactive with C. jejuni and with the
flagellins of a wide range of H. pylori-like organisms
but not with Escherichia, Vibrio, Aeromonas, or Salmonella species by Western blot analysis. We could not
find cross-reactive antibodies against crude C. jejuni
antigens in CSF samples having H. pylori antibodies,
Etiologically, detected anti-HSP antibody may play a
role in the pathophysiology of GBS. This 66-kd HSP
is thought to be a member of the HSP 60 family.
KaufmannI2 proposed a model in which y6 T cells reactive with highly conserved HSP are involved in autoimmune reactions. Furthermore, considerable attention has been focused on HSP that has an ability to
stimulate a strong immune response even in neurological disorders such as MS.13
Brief Communication: Chiba et al: CSF Antibodies against H. pylori in GBS 687
Our study is the first in which anti-H. pylori antibodies were detected in the CSF of patients with GBS
of a predominantly axonal form. We were also able to
find specific antibodies in CSF against fractionated
HSP from H. pylori independent of serum antibodies.
Nevertheless, the pathophysiological role of these antibodies in the development of GBS remains unclear.
1. Yuki N, Yoshino H , Sato S, Miyatake T. Acute axonal polyneuropathy associated with anti-GM1 antibodies following
Campylobacter enteritis. Neurology 1990;40: 1900-1902
2. Yuki N, Sato S, lnuzuka T, et al. Axonal degeneration in the
Guillain-Barre syndrome and anti-GM1 ganglioside antibodies.
Muscle Nerve 1992;15:116 (Letter)
3. Parsonnet J, Friedman DG, Vandersteen DP, et al. Helicobacter
pylori infection and the risk of gastric carcinoma. N Engl J Med
4. Whincup PH, Mendall MA, Perry IJ, et al. Prospective relations between Helicobacter pylori infection, coronary heart disease, and stroke in middle aged men. Heart 1996;75:568-572
5. Asbury AK, Cornblath DR. Assessment of current diagnostic
criteria for Guillain-Bad syndrome. Ann Neurol 1990;
6. Yokota K, Hirai Y, Haque M, et al. Heat shock protein produced by Helicobacter pylori. Microbiol lmmunol 1994;38:403405
7. Warren JR, Marshall BJ. Unidentified curved bacilli on gastric
epithelium in active chronic gastritis. Lancet 1983;1:1273-1275
8. Dooley CP, Cohen H , Fitzgibbons PL, et al. Prevalence of Helicobacter pylori infection and histological gastritis in asymptomatic persons. N Engl J Med 1989;321:1562-1566
9. Rebora A, Drago F, Parodi A. May Helirobacter pylori be important for dermatologists? Dermatology 1995;191:6-8
10. Kusunoki S, Iwamori M, Chiba A, et al. G M l b is a new member of antigen for serum antibody in Guillain-Barre syndrome.
Neurology 1996;47:237-242
11. Kostrzynska M , Berts JD, Austin JW, Trust TJ. Identification,
characterization, and special localization of two flagellin species
in Helicobacter pylori flagella. J Bacteriol 1991;173:937-946
12. Kaufmann SHE. Heat shock protein and the immune response.
Immunol Today 1990;11:129-136
13. Georgopoulos C, McFarland H. Heat shock proteins in multiple sclerosis and orher autoimmune diseases. lmmunol Today
Ganaxolone, a Selective,
High-Minity Steroid
Modulator of the
y -Aminobutyric Acid-A
Receptor, Exacerbates
Seizures in Animal Models
of Absence
0. Carter Snead 111, MD
Ganaxolone (3a-hydroxy-3fi-methyl-5a-pregnan-20-one)
is a novel neurosteroid which has anticonvulsant properties in a number of seizure models as well as the ability
to enhance function of the y-aminobutyric acid-A
(GABAA) receptor complex via a neurosteroid binding
site. The object of these experiments was to ascertain the
efficacy of ganaxolone against absence seizures. Ganaxolone was assessed in the low-dose pentylenetetrazol
(PTZ) and the y-hydroxybutyric acid (GHB) model of
absence seizures in rats. Ganaxolone pretreatment resulted in a significant prolongation of absence seizure in
both the PTZ and GHB models. Further, ganaxolone in
doses above 20 mg/kg alone produced bilaterally synchronous spike wave discharges (SWDs) associated with
behavioral arrest. These data suggest that augmentation
of GABAA receptor complex function by neurosteroids
has the potential to result in or exacerbate absence seizures.
Snead OC 111. Ganaxolone, a selective, highaffinity steroid modulator of the y-aminobutyric
acid-A receptor, exacerbates seizures in
animal models of absence.
Ann Neurol 1998;44:688-691
Neuroactive steroids are endogenous metabolites and
synthetic analogues of certain steroid hormones that
rapidly alter the excitability of neurons by direct actions on ligand-gated ion channels.' Specifically, the
endogenous 3a-hydroxy ring A-reduced metabolites of
progesterone, allopregnanolone (3o1,5a-P) and pregnanolone (3o1,5p-P) produce a powerful enhancement
of GABA, receptor-mediated responses in vitro2 by in-
From the Department of Pediatrics, Faculty of Medicine, University
of Toronto, Division of Neurology, Hospital for Sick Children, Toronto, Ontario, Canada.
Received Jan 27, 1998, and in revised form Apr 14. Accepted for
publication Apr 21, 1998.
Address correspondence to D r Snead, 555 University Avenue, Toronto, Ontario, M5G 1x8, Canada.
Copyright 0 1998 by the American Neurological Association
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