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Acute cytomegalovirus infection and IgM anti-GM2 antibody
Journal of Neurological Sciences 154 (1998) 14–17
Acute cytomegalovirus infection and IgM anti-GM2 antibody a, a,b Nobuhiro Yuki *, Yumi Tagawa a
Department of Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo, Japan b Third Department of Internal Medicine, Kagawa Medical School, Kagawa, Japan Received 21 February 1997; accepted 5 June 1997
Abstract Guillain-Barre´ syndrome (GBS) sometimes is preceded by cytomegalovirus (CMV) infection. Irie et al. (J. Neuroimmunol. 1996;68:19–26) reported that three patients with GBS subsequent to CMV infection had IgM and IgG anti-GM2 antibodies. In our larger study, the IgMs from the CMV-associated GBS patients showed significantly higher anti-GM2 antibody titers than anti-GM3, anti-GD1a, anti-GD1b, anti-GD2, anti-GD3, anti-GT1b, anti-GQ1b, and anti-SGPG antibody titers. None of the anti-glycosphingolipid antibody titers differed significantly from the others in the IgGs from the CMV-associated GBS patients. However, IgM anti-GM2 antibody frequently was present in GBS patients who were not preceded by CMV infection and non-GBS patients with acute CMV infection. Our results did not support the conclusion of Irie et al. that anti-GM2 antibodies were closely associated with acute CMV infection in GBS, but acute CMV infection, with and without GBS, was associated with IgM anti-GM2 antibody. 1998 Elsevier Science B.V. Keywords: Guillain-Barre´ syndrome; Cytomegalovirus; Anti-ganglioside antibody; GM2
1. Introduction
2. Materials and methods
Guillain-Barre´ syndrome (GBS) develops 1–3 weeks after various infections, e.g. those of Campylobacter jejuni and human cytomegalovirus (CMV). GBS subsequent to C. jejuni enteritis significantly is associated with anti-GM1 antibody (Rees et al., 1995; Visser et al., 1995), although no patients with C. jejuni enteritis have anti-GM1 antibodies (Yuki et al., 1990; Koblar et al., 1991). In contrast, Irie et al. (1996) detected IgM and IgG anti-GM2 antibodies in three patients with GBS following CMV infection. They reported that none of 48 GBS patients who were not preceded by CMV infection had the anti-GM2 antibodies, and that only one of six non-GBS patients with acute CMV infection had the anti-GM2 antibodies. To examine whether IgM and IgG anti-GM2 antibodies are associated with GBS subsequent to CMV infection, we investigated serum antibodies to glycosphingolipids in CMV-associated GBS and controls.
2.1. CMV serology
*Corresponding author: Department of Neurology, Dokkyo University School of Medicine, Kitakobayashi 880, Mibu, Shimotsuga, Tochigi 321-02, Japan. Fax: 181 282 865884; e-mail: [email protected] 0022-510X / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0022-510X( 97 )00174-3
Serum samples were taken from 113 GBS patients and 66 patients with Fisher’s syndrome (FS) within 4 weeks of the onset of neurologic symptoms. Normal control (NC) serum samples were obtained from 67 healthy volunteers. An enzyme-linked immunosorbent assay (ELISA) on antihuman IgM antibody-coated plates (Cytomegalo IgM (II)EIA ‘SEIKEN’) was used to check for the presence of IgM anti-CMV antibody according to the manufacturer’s instructions (Denka Seiken, Tokyo, Japan). Positive IgM antibody to CMV is suggestive of a primary infection, but reactivation of latent CMV infection cannot be excluded.
2.2. Anti-glycosphingolipid antibodies 2.2.1. Glycosphingolipids GM1, GD1a, GD1b, and GT1b were gifts from Dr Yasuo Suzuki (Shizuoka University, Shizuoka, Japan). GM2 was purchased from Funakoshi (Tokyo, Japan).
N. Yuki, Y. Tagawa / Journal of Neurological Sciences 154 (1998) 14 – 17
15
GM3, GD3, and GQ1b were obtained from Dia-Iatron (Tokyo, Japan). GalNAc-GD1a was prepared from bovine brain as described elsewhere (Yuki et al., 1996b). GD2 was purchased from Sigma (St. Louis, MO, USA). Sulfated glucuronyl paragloboside (SGPG) was provided by Dr Toshio Ariga (Virginia Commonwealth University, Virginia, USA). Asialo-GM1 (GA1) and asialo-GM2 (GA2) were gifts from Dr Takao Taki (Tokyo Medical and Dental University, Tokyo, Japan).
were not followed by neurologic disorders, four CMVassociated FS patients, 12 patients with C. jejuni-isolated GBS who did not have IgM anti-CMV antibody, and 23 NCs without acute CMV infection. Comparisons of the titers of anti-GM2 antibody and the other anti-glycosphingolipid antibodies were made with the Wilcoxon signed-ranks test. Analyses of the differences between groups were done with Fisher’s exact test. Statistical calculations were made with Stat View 4.0 software.
2.2.2. ELISA As described elsewhere (Yuki, 1996a), sera from nine CMV-associated GBS patients were tested for antibodies to glycosphingolipids GM1, GM2, GM3, GD1a, GalNAcGD1a, GD1b, GD2, GD3, GT1b, GQ1b, and SGPG. For the IgM anti-GM2 antibody assay, serum samples were taken from 10 patients with acute CMV infection who
2.2.3. Thin-layer chromatography-immunostaining The bovine brain ganglioside mixture and authentic samples of GM2, GalNAc-GD1a, GD2, GA1, and GA2 were separated on thin-layer chromatography (TLC) plates. The solvent system was chloroform-methanol-12 mM MgCl 2 (5:4:1, by volume). The plates were overlaid with sera (1:500 dilution) from two patients with CMV-
Table 1 Clinical features of, and anti-glycosphingolipid antibody in, patients with cytomegalovirus-associated Guillain-Barre´ syndrome Patient no.
1
2
3
4
5
6
7
8
9
Age/sex Prodrome symptoms
28/F Fever, cough, sore throat Present Absent Present 2 Facial palsy
21/F Diarrhea
27/M Fever, chill
20/F Diarrhea
21/F Fever, cough
17/F Absent
Not recorded Present Present 4 Facial palsy Bulbar palsy Dysesthesia Hypalgesia
29/F Fever, chill, diarrhea Not recorded Absent Absent 2 Absent
64/M Fever, cough
Present Present Present 2 Facial palsy Bulbar palsy Dysesthesia Hypalgesia Hypesthesia Vibration sense Impairment Demyelinating
48/F Sore throat, chill Present Absent Present 4 Facial palsy Bulbar palsy Dysesthesia
Present Absent Present 4 Absent
Not recorded Absent Absent 4 Facial palsy Bulbar palsy Dysesthesia Hypesthesia
Demyelinating
Demyelinating
Hepatosplenomegaly Atypical lymphocyte Abnormal liver enzyme Maximal disability grade a Cranial nerve involvement Sensory disturbance
Dysesthesia Hyperalgesia Hypesthesia
Electrophysiologic diagnosis IgM antibody titer Anti-GM2 Anti-GalNAc-GD1a Anti-GM1 Anti-GM3 Anti-GD1a Anti-GD1b Anti-GD2 Anti-GD3 Anti-GT1b Anti-GQ1b Anti-SGPG IgG antibody titer Anti-GM2 Anti-GalNAc-GD1a Anti-GM1 Anti-GM3 Anti-GD1a Anti-GD1b Anti-GD2 Anti-GD3 Anti-GT1b Anti-GQ1b Anti-SGPG
Demyelinating
Present Present Present 4 Facial palsy Bulbar palsy Dysesthesia Hypalgesia Hypesthesia Vibration sense Impairment Demyelinating
64 000 2000 500 0 500 0 2000 0 0 0 1000
8000 1000 0 2000 2000 500 500 500 0 1000 1000
8000 1000 0 0 0 0 500 500 0 0 0
4000 1000 0 0 0 0 0 0 0 0 2000
4000 0 0 0 0 0 0 0 0 0 0
2000 1000 2000 8000 500 0 0 0 0 0 1000
0 2000 0 0 0 0 0 0 0 0 0
0 1000 0 0 0 0 0 0 0 0 0
a
Disability scale according to Winer et al. (1988).
Present Present Present 5 Facial palsy Bulbar palsy Dysesthesia Hypalgesia Vibration sense Impairment
Absent
Dysesthesia Vibration sense Impairment
Demyelinating
Normal
Demyelinating
Demyelinating
2000 1000 2000 500 2000 1000 500 1000 500 0 4000
1000 2000 1000 500 1000 0 2000 0 2000 500 0
500 2000 500 0 0 0 0 0 0 500 0
500 500 2000 0 0 0 0 0 0 0 0
500 500 500 500 0 0 1000 500 500 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 500 0
500 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
16
N. Yuki, Y. Tagawa / Journal of Neurological Sciences 154 (1998) 14 – 17
associated GBS (patients 1 and 2 in Table 1) and a patient with acute CMV infection who was not followed by neurologic disorders, kept at 48C overnight then washed. Peroxidase-conjugated anti-human m-chain-specific antibodies (Dako, Denmark; 1:1000 dilution) were added, after each plate was incubated at room temperature for 2 h then washed and developed with 4-chloro-1-naphtol.
3. Results
3.1. CMV-associated GBS Nine of the 113 GBS patients (8%) and four of the 66 FS patients (6%) had serum IgM anti-CMV antibody which was not present in any of the 67 NCs. None of the 12 GBS patients from whom C. jejuni had been isolated had IgM anti-CMV antibody. The clinical features of the GBS patients are given in Table 1. Patient 2 was pregnant. Patient 8 had hepatocellular carcinoma. The others had been healthy before developing GBS.
3.2. IgM anti-GM2 antibody in CMV-associated GBS The IgMs from the CMV-associated GBS patients showed significantly higher anti-GM2 antibody titers than anti-GM3, anti-GD1a, anti-GD1b, anti-GD2, anti-GD3, anti-GT1b, anti-GQ1b, and anti-SGPG antibody titers (Table 1; Wilcoxon signed-ranks test, P,0.05). The IgM anti-GM2 antibody titers were decreased during the convalescent phase (data not shown). TLC-immunostaining showed that the IgMs from patients 1 and 2 reacted strongly with GM2 and faintly with GalNAc-GD1a which carries the same terminal trisaccharide as GM2 (Fig. 2).
Fig. 2. Binding of serum IgMs from patients with cytomegalovirus (CMV)-associated Guillain-Barre´ syndrome (GBS) and controls. Lane 1, bovine brain ganglioside mixture (GM1, GD1a, GD1b, and GT1b) and authentic samples of GD2, asialo-GM1 (GA1), and asialo-GM2 (GA2). Lane 2, authentic samples of GM2 and GalNAc-GD1a. Panel A, thinlayer chromatography plate stained with the orcinol / sulfuric acid reagent for hexose. Panels B–E, immunostained chromatograms that had been overlaid with sera from two patients with CMV-associated GBS (panels B and C), a patient with acute CMV infection who was not followed by neurologic disorders (panel D), and a patient with GBS who was not preceded by CMV infection (panel E). The IgMs from the patients with CMV-related GBS reacted strongly with GM2 and faintly with GalNAcGD1a. The IgM from patient 1 reacted weakly with GA2. The IgM from the patient with acute CMV infection reacted strongly with GM2. The IgM from the patient with GBS who was not preceded by CMV infection, which was used as control with anti-ganglioside antibody of different specificity, reacted strongly with GA1 and weakly with GM1.
The IgM from patient 1 reacted weakly with GA2. None of the anti-glycosphingolipid antibody titers differed significantly from the others in the IgGs from the CMV-associated GBS patients (Table 1). Not only the CMV-associated GBS patients but also the acute CMV infection patients and the C. jejuni-isolated GBS patients more frequently had IgM anti-GM2 antibody (more than 500) than the NCs (Fisher’s exact test, P, 0.001; Fig. 1). The frequency of IgM anti-GM2 antibody did not differ significantly between CMV-associated GBS and acute CMV infection (Fisher’s exact test, P50.09). TLC-immunostaining showed that the IgM from the nonGBS patient with acute CMV infection reacted strongly with GM2 (Fig. 2).
4. Discussion
Fig. 1. Distribution of serum IgM anti-GM2 antibody titers. CMV, acute cytomegalovirus (CMV) infection without neurologic disorders (n510); CMV-GBS, CMV-associated Guillain-Barre´ syndrome (n59); CMV-FS, CMV-associated Fisher’s syndrome (n54); C. jejuni-GBS, C. jejuniisolated GBS (n512); NC, normal control (n523).
We found positive results for IgM anti-CMV antibody in 8% of the GBS patients and 6% of the FS patients. Visser et al. (1996) reported that their patients with CMV-associated GBS were young and often women. Our nine patients also were young (mean age, 30) and frequently women (78%). Their patients often showed involvement of cranial nerves. Seven of our patients had facial or bulbar palsy (78%), but none of the nine patients had ophthalmoplegia. Our nine patients with CMV-associated GBS had higher IgM anti-GM2 antibody titers as compared to other IgM anti-glycosphingolipid antibody titers. We, however,
N. Yuki, Y. Tagawa / Journal of Neurological Sciences 154 (1998) 14 – 17
showed that IgM anti-GM2 antibody frequently was present in GBS patients who were not preceded by CMV infection and non-GBS patients with acute CMV infection. TLC-immunostaining confirmed that high IgM anti-GM2 antibody activity was present in the non-GBS patient with acute CMV infection. These results did not support the conclusion of Irie et al. (1996) that IgM and IgG anti-GM2 antibodies were closely associated with CMV-related GBS, but acute CMV infection, with and without GBS, was associated with IgM anti-GM2 antibody. Whether IgM anti-GM2 antibody functions in the development of GBS is unknown.
Acknowledgments This research was supported in part by a Research Grant for Neuroimmunological Diseases from the Ministry of Health and Welfare of Japan.
References Irie, S., Saito, T., Nakamura, K., Kanazawa, N., Ogino, M., Nukazawa, T., Ito, H., Tamai, Y., Kowa, H., 1996. Association of anti-GM2 antibodies in Guillain-Barre´ syndrome with acute cytomegalovirus infection. J. Neuroimmunol. 68, 19–26.
17
Koblar, S.A., Gregson, N.A., Hughes, R.A.C., Doherty, P., Walsh, F.S., 1991. Campylobacter neuropathy. Neurology 41, 1327–1328. Rees, J.H., Gregson, N.A., Hughes, R.A.C., 1995. Anti-ganglioside GM1 antibodies in Guillain-Barre´ syndrome and their relationship to Campylobacter jejuni infection. Ann. Neurol. 38, 809–816. ´ F.G.A., Meulstee, J., Rothbarth, P.Ph., Visser, L.H., van der Meche, Jacobs, B.C., Schmitz, P.I.M., van Doon, P.A. and the Dutch GuillainBarre´ Study Group, 1996. Cytomegalovirus infection and GuillainBarre´ syndrome: the clinical, electrophysiologic, and prognostic features. Neurology 47, 668–673. ´ F.G.A., van Doon, P.A., Meulstee, J., Visser, L.H., van der Meche, Jacobs, B.C., Oomes, P.G., Kleyweg, R.P. and the Dutch GuillainBarre´ Study Group, 1995. Guillain-Barre´ syndrome without sensory loss (acute motor neuropathy): a subgroup with specific clinical, electrodiagnostic and laboratory features. Brain 118, 841–847. Winer, J.B., Hughes, R.A.C., Osmond, C., 1988. A prospective study of acute idiopathic neuropathy. I. Clinical features and their prognostic value. J. Neurol. Neurosurg. Psychiatry 51, 605–612. Yuki, N., Yoshino, H., Sato, S., Miyatake, T., 1990. Acute axonal polyneuropathy associated with anti-GM1 antibodies following Campylobacter enteritis [see comments]. Neurology 40, 1900–1902. Comment in: Neurology 41, 1327–1328. Comment in: Neurology 41, 1530–1531. Yuki, N., 1996a. Acute paresis of extraocular muscles associated with IgG anti-GQ1b antibody. Ann. Neurol. 36, 668–672. Yuki, N., Taki, T., Handa, S., 1996b. Antibody to GalNAc-GD1a and GalNAc-GM1b in Guillain-Barre´ syndrome subsequent to Campylobacter jejuni enteritis. J. Neuroimmunol. 71, 155–161.
Acute cytomegalovirus infection and IgM anti-GM2 antibody a, a,b Nobuhiro Yuki *, Yumi Tagawa a
Department of Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo, Japan b Third Department of Internal Medicine, Kagawa Medical School, Kagawa, Japan Received 21 February 1997; accepted 5 June 1997
Abstract Guillain-Barre´ syndrome (GBS) sometimes is preceded by cytomegalovirus (CMV) infection. Irie et al. (J. Neuroimmunol. 1996;68:19–26) reported that three patients with GBS subsequent to CMV infection had IgM and IgG anti-GM2 antibodies. In our larger study, the IgMs from the CMV-associated GBS patients showed significantly higher anti-GM2 antibody titers than anti-GM3, anti-GD1a, anti-GD1b, anti-GD2, anti-GD3, anti-GT1b, anti-GQ1b, and anti-SGPG antibody titers. None of the anti-glycosphingolipid antibody titers differed significantly from the others in the IgGs from the CMV-associated GBS patients. However, IgM anti-GM2 antibody frequently was present in GBS patients who were not preceded by CMV infection and non-GBS patients with acute CMV infection. Our results did not support the conclusion of Irie et al. that anti-GM2 antibodies were closely associated with acute CMV infection in GBS, but acute CMV infection, with and without GBS, was associated with IgM anti-GM2 antibody. 1998 Elsevier Science B.V. Keywords: Guillain-Barre´ syndrome; Cytomegalovirus; Anti-ganglioside antibody; GM2
1. Introduction
2. Materials and methods
Guillain-Barre´ syndrome (GBS) develops 1–3 weeks after various infections, e.g. those of Campylobacter jejuni and human cytomegalovirus (CMV). GBS subsequent to C. jejuni enteritis significantly is associated with anti-GM1 antibody (Rees et al., 1995; Visser et al., 1995), although no patients with C. jejuni enteritis have anti-GM1 antibodies (Yuki et al., 1990; Koblar et al., 1991). In contrast, Irie et al. (1996) detected IgM and IgG anti-GM2 antibodies in three patients with GBS following CMV infection. They reported that none of 48 GBS patients who were not preceded by CMV infection had the anti-GM2 antibodies, and that only one of six non-GBS patients with acute CMV infection had the anti-GM2 antibodies. To examine whether IgM and IgG anti-GM2 antibodies are associated with GBS subsequent to CMV infection, we investigated serum antibodies to glycosphingolipids in CMV-associated GBS and controls.
2.1. CMV serology
*Corresponding author: Department of Neurology, Dokkyo University School of Medicine, Kitakobayashi 880, Mibu, Shimotsuga, Tochigi 321-02, Japan. Fax: 181 282 865884; e-mail: [email protected] 0022-510X / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0022-510X( 97 )00174-3
Serum samples were taken from 113 GBS patients and 66 patients with Fisher’s syndrome (FS) within 4 weeks of the onset of neurologic symptoms. Normal control (NC) serum samples were obtained from 67 healthy volunteers. An enzyme-linked immunosorbent assay (ELISA) on antihuman IgM antibody-coated plates (Cytomegalo IgM (II)EIA ‘SEIKEN’) was used to check for the presence of IgM anti-CMV antibody according to the manufacturer’s instructions (Denka Seiken, Tokyo, Japan). Positive IgM antibody to CMV is suggestive of a primary infection, but reactivation of latent CMV infection cannot be excluded.
2.2. Anti-glycosphingolipid antibodies 2.2.1. Glycosphingolipids GM1, GD1a, GD1b, and GT1b were gifts from Dr Yasuo Suzuki (Shizuoka University, Shizuoka, Japan). GM2 was purchased from Funakoshi (Tokyo, Japan).
N. Yuki, Y. Tagawa / Journal of Neurological Sciences 154 (1998) 14 – 17
15
GM3, GD3, and GQ1b were obtained from Dia-Iatron (Tokyo, Japan). GalNAc-GD1a was prepared from bovine brain as described elsewhere (Yuki et al., 1996b). GD2 was purchased from Sigma (St. Louis, MO, USA). Sulfated glucuronyl paragloboside (SGPG) was provided by Dr Toshio Ariga (Virginia Commonwealth University, Virginia, USA). Asialo-GM1 (GA1) and asialo-GM2 (GA2) were gifts from Dr Takao Taki (Tokyo Medical and Dental University, Tokyo, Japan).
were not followed by neurologic disorders, four CMVassociated FS patients, 12 patients with C. jejuni-isolated GBS who did not have IgM anti-CMV antibody, and 23 NCs without acute CMV infection. Comparisons of the titers of anti-GM2 antibody and the other anti-glycosphingolipid antibodies were made with the Wilcoxon signed-ranks test. Analyses of the differences between groups were done with Fisher’s exact test. Statistical calculations were made with Stat View 4.0 software.
2.2.2. ELISA As described elsewhere (Yuki, 1996a), sera from nine CMV-associated GBS patients were tested for antibodies to glycosphingolipids GM1, GM2, GM3, GD1a, GalNAcGD1a, GD1b, GD2, GD3, GT1b, GQ1b, and SGPG. For the IgM anti-GM2 antibody assay, serum samples were taken from 10 patients with acute CMV infection who
2.2.3. Thin-layer chromatography-immunostaining The bovine brain ganglioside mixture and authentic samples of GM2, GalNAc-GD1a, GD2, GA1, and GA2 were separated on thin-layer chromatography (TLC) plates. The solvent system was chloroform-methanol-12 mM MgCl 2 (5:4:1, by volume). The plates were overlaid with sera (1:500 dilution) from two patients with CMV-
Table 1 Clinical features of, and anti-glycosphingolipid antibody in, patients with cytomegalovirus-associated Guillain-Barre´ syndrome Patient no.
1
2
3
4
5
6
7
8
9
Age/sex Prodrome symptoms
28/F Fever, cough, sore throat Present Absent Present 2 Facial palsy
21/F Diarrhea
27/M Fever, chill
20/F Diarrhea
21/F Fever, cough
17/F Absent
Not recorded Present Present 4 Facial palsy Bulbar palsy Dysesthesia Hypalgesia
29/F Fever, chill, diarrhea Not recorded Absent Absent 2 Absent
64/M Fever, cough
Present Present Present 2 Facial palsy Bulbar palsy Dysesthesia Hypalgesia Hypesthesia Vibration sense Impairment Demyelinating
48/F Sore throat, chill Present Absent Present 4 Facial palsy Bulbar palsy Dysesthesia
Present Absent Present 4 Absent
Not recorded Absent Absent 4 Facial palsy Bulbar palsy Dysesthesia Hypesthesia
Demyelinating
Demyelinating
Hepatosplenomegaly Atypical lymphocyte Abnormal liver enzyme Maximal disability grade a Cranial nerve involvement Sensory disturbance
Dysesthesia Hyperalgesia Hypesthesia
Electrophysiologic diagnosis IgM antibody titer Anti-GM2 Anti-GalNAc-GD1a Anti-GM1 Anti-GM3 Anti-GD1a Anti-GD1b Anti-GD2 Anti-GD3 Anti-GT1b Anti-GQ1b Anti-SGPG IgG antibody titer Anti-GM2 Anti-GalNAc-GD1a Anti-GM1 Anti-GM3 Anti-GD1a Anti-GD1b Anti-GD2 Anti-GD3 Anti-GT1b Anti-GQ1b Anti-SGPG
Demyelinating
Present Present Present 4 Facial palsy Bulbar palsy Dysesthesia Hypalgesia Hypesthesia Vibration sense Impairment Demyelinating
64 000 2000 500 0 500 0 2000 0 0 0 1000
8000 1000 0 2000 2000 500 500 500 0 1000 1000
8000 1000 0 0 0 0 500 500 0 0 0
4000 1000 0 0 0 0 0 0 0 0 2000
4000 0 0 0 0 0 0 0 0 0 0
2000 1000 2000 8000 500 0 0 0 0 0 1000
0 2000 0 0 0 0 0 0 0 0 0
0 1000 0 0 0 0 0 0 0 0 0
a
Disability scale according to Winer et al. (1988).
Present Present Present 5 Facial palsy Bulbar palsy Dysesthesia Hypalgesia Vibration sense Impairment
Absent
Dysesthesia Vibration sense Impairment
Demyelinating
Normal
Demyelinating
Demyelinating
2000 1000 2000 500 2000 1000 500 1000 500 0 4000
1000 2000 1000 500 1000 0 2000 0 2000 500 0
500 2000 500 0 0 0 0 0 0 500 0
500 500 2000 0 0 0 0 0 0 0 0
500 500 500 500 0 0 1000 500 500 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 500 0
500 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
16
N. Yuki, Y. Tagawa / Journal of Neurological Sciences 154 (1998) 14 – 17
associated GBS (patients 1 and 2 in Table 1) and a patient with acute CMV infection who was not followed by neurologic disorders, kept at 48C overnight then washed. Peroxidase-conjugated anti-human m-chain-specific antibodies (Dako, Denmark; 1:1000 dilution) were added, after each plate was incubated at room temperature for 2 h then washed and developed with 4-chloro-1-naphtol.
3. Results
3.1. CMV-associated GBS Nine of the 113 GBS patients (8%) and four of the 66 FS patients (6%) had serum IgM anti-CMV antibody which was not present in any of the 67 NCs. None of the 12 GBS patients from whom C. jejuni had been isolated had IgM anti-CMV antibody. The clinical features of the GBS patients are given in Table 1. Patient 2 was pregnant. Patient 8 had hepatocellular carcinoma. The others had been healthy before developing GBS.
3.2. IgM anti-GM2 antibody in CMV-associated GBS The IgMs from the CMV-associated GBS patients showed significantly higher anti-GM2 antibody titers than anti-GM3, anti-GD1a, anti-GD1b, anti-GD2, anti-GD3, anti-GT1b, anti-GQ1b, and anti-SGPG antibody titers (Table 1; Wilcoxon signed-ranks test, P,0.05). The IgM anti-GM2 antibody titers were decreased during the convalescent phase (data not shown). TLC-immunostaining showed that the IgMs from patients 1 and 2 reacted strongly with GM2 and faintly with GalNAc-GD1a which carries the same terminal trisaccharide as GM2 (Fig. 2).
Fig. 2. Binding of serum IgMs from patients with cytomegalovirus (CMV)-associated Guillain-Barre´ syndrome (GBS) and controls. Lane 1, bovine brain ganglioside mixture (GM1, GD1a, GD1b, and GT1b) and authentic samples of GD2, asialo-GM1 (GA1), and asialo-GM2 (GA2). Lane 2, authentic samples of GM2 and GalNAc-GD1a. Panel A, thinlayer chromatography plate stained with the orcinol / sulfuric acid reagent for hexose. Panels B–E, immunostained chromatograms that had been overlaid with sera from two patients with CMV-associated GBS (panels B and C), a patient with acute CMV infection who was not followed by neurologic disorders (panel D), and a patient with GBS who was not preceded by CMV infection (panel E). The IgMs from the patients with CMV-related GBS reacted strongly with GM2 and faintly with GalNAcGD1a. The IgM from patient 1 reacted weakly with GA2. The IgM from the patient with acute CMV infection reacted strongly with GM2. The IgM from the patient with GBS who was not preceded by CMV infection, which was used as control with anti-ganglioside antibody of different specificity, reacted strongly with GA1 and weakly with GM1.
The IgM from patient 1 reacted weakly with GA2. None of the anti-glycosphingolipid antibody titers differed significantly from the others in the IgGs from the CMV-associated GBS patients (Table 1). Not only the CMV-associated GBS patients but also the acute CMV infection patients and the C. jejuni-isolated GBS patients more frequently had IgM anti-GM2 antibody (more than 500) than the NCs (Fisher’s exact test, P, 0.001; Fig. 1). The frequency of IgM anti-GM2 antibody did not differ significantly between CMV-associated GBS and acute CMV infection (Fisher’s exact test, P50.09). TLC-immunostaining showed that the IgM from the nonGBS patient with acute CMV infection reacted strongly with GM2 (Fig. 2).
4. Discussion
Fig. 1. Distribution of serum IgM anti-GM2 antibody titers. CMV, acute cytomegalovirus (CMV) infection without neurologic disorders (n510); CMV-GBS, CMV-associated Guillain-Barre´ syndrome (n59); CMV-FS, CMV-associated Fisher’s syndrome (n54); C. jejuni-GBS, C. jejuniisolated GBS (n512); NC, normal control (n523).
We found positive results for IgM anti-CMV antibody in 8% of the GBS patients and 6% of the FS patients. Visser et al. (1996) reported that their patients with CMV-associated GBS were young and often women. Our nine patients also were young (mean age, 30) and frequently women (78%). Their patients often showed involvement of cranial nerves. Seven of our patients had facial or bulbar palsy (78%), but none of the nine patients had ophthalmoplegia. Our nine patients with CMV-associated GBS had higher IgM anti-GM2 antibody titers as compared to other IgM anti-glycosphingolipid antibody titers. We, however,
N. Yuki, Y. Tagawa / Journal of Neurological Sciences 154 (1998) 14 – 17
showed that IgM anti-GM2 antibody frequently was present in GBS patients who were not preceded by CMV infection and non-GBS patients with acute CMV infection. TLC-immunostaining confirmed that high IgM anti-GM2 antibody activity was present in the non-GBS patient with acute CMV infection. These results did not support the conclusion of Irie et al. (1996) that IgM and IgG anti-GM2 antibodies were closely associated with CMV-related GBS, but acute CMV infection, with and without GBS, was associated with IgM anti-GM2 antibody. Whether IgM anti-GM2 antibody functions in the development of GBS is unknown.
Acknowledgments This research was supported in part by a Research Grant for Neuroimmunological Diseases from the Ministry of Health and Welfare of Japan.
References Irie, S., Saito, T., Nakamura, K., Kanazawa, N., Ogino, M., Nukazawa, T., Ito, H., Tamai, Y., Kowa, H., 1996. Association of anti-GM2 antibodies in Guillain-Barre´ syndrome with acute cytomegalovirus infection. J. Neuroimmunol. 68, 19–26.
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