Tryptophan-immobilized column adsorbs immunoglobulin G anti-GQ1b antibody from Fisher's syndrome
A new approach to treatment
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Abstract
Sera from patients with Fisher's syndrome in the acute phase contain immunoglobulin (Ig)G anti-GQ1b ganglioside antibody. Removal of the autoantibody should lead to earlier recovery with less residual neurologic involvement. A tryptophan- or phenylalanin-immobilized polyvinyl alcohol gel column (IM-TR 350 or IM-PH 350) semiselectively adsorbs such autoantibodies as rheumatoid factor, anti-DNA antibody, or anti-acetylcholine receptor antibody. A batchwise adsorption test showed that an IM-TR gel adsorbed a larger amount of the IgG anti-GQ1b antibody than did an IM-PH column. Several patients with Fisher's syndrome therefore were given immunoadsorbent therapy using the IM-TR column without adverse reactions. An ex vivo plasma perfusion study done with the IM-TR column confirmed that it effectively adsorbs the IgG anti-GQ1b antibody. Results of adsorption tests done with various amino acid-immobilized gels suggest that both the hydrophobic force of the side chain and the anionic charge of the carboxylic acid in tryptophan are important in the adsorption of the autoantibody by the IM-TR gel. Immunoadsorption using the IM-TR column, which does not need replacement fluids, offers an alternative type of plasmapheresis for Fisher's syndrome.
NEUROLOGY 1996;46: 1644-1651
Fisher's syndrome (FS) is characterized by the acute onset of ophthalmoplegia, ataxia, and areflexia. It is considered a variant of Guillain-Barre syndrome (GBS) because one-third of FS patients eventually experience profound weakness, sometimes accompanied by respiratory failure, that requires mechanical ventilation. [1] Although the prognosis for FS generally is good, recovery occurring after a mean of 10 weeks, residual symptoms such as ataxia, ophthalmoplegia, and facial weakness are present in about 10% of the patients. [2] In most reports in the literature, no specific treatment has been given for FS. [2] For GBS, plasma exchange is the first treatment for which short-term and long-term benefits have been demonstrated. [3-5] A favorable response to plasma exchange also has been reported in some cases of FS, [6,7] but no controlled trials have been conducted. A major disadvantage of using standard plasma exchange is the unselected removal of all the plasma proteins, making replacement with allogeneic human proteins a necessity. The inherent and potentially serious risks of protein replacement are anaphylactic reactions and the transfer of viral diseases such as hepatitis and acquired immunodeficiency syndrome. A more selective method therefore is needed to remove pathogenic autoantibodies without the necessity of replacing plasma proteins.
Recent studies have shown that sera from patients in the acute phase of FS contain the immunoglobulin (Ig)G anti-GQ1b ganglioside antibody. [8-12] Campylobacter jejuni, an antecedent agent for FS development, has the GQ1b epitope. [13,14] GQ1b is enriched in human oculomotor, trochlear, and abducens nerves. [11] Sera containing anti-GQ1b antibody trigger the failure of acetylcholine release from motor nerve terminals in a mouse phrenic-nerve/diaphragm preparation. [15] Sera with anti-GQ1b antibody have toxicity against rat dorsal root ganglion neurons. [16] These facts suggest that the IgG anti-GQ1b antibody induced by microorganism infection functions in the development of FS. Removal of the autoantibody should lead to the earlier recovery of FS patients with less residual neurologic involvement.
A tryptophan- or phenylalanin-immobilized polyvinyl alcohol (PVA) gel column (IM-TR 350 or IM-PH 350, Asahi Medical, Tokyo, Japan) effectively adsorbs such autoantibodies as rheumatoid factor, anti-DNA antibody, and anti-acetylcholine receptor antibody. [17-20] Because the immunoadsorbents do not remove albumin, this adsorption therapy does not require substitution fluids. Some reports hint that treatment in which affinity columns are used may elicit beneficial response in GBS and FS. [21-24] I therefore examined in vitro whether the IM-TR and IM-PH gels adsorb anti-ganglioside antibodies in plasma from patients with various neurologic disorders. On the basis of the results, several patients with FS underwent immunoadsorbent therapy that used the IM-TR 350 column. I tested whether the column effectively adsorbed the IgG anti-GQ1b antibody ex vivo. I also investigated the mechanism of the semiselective adsorption of the autoantibody by the IM-TR gel.
Methods.
Batchwise adsorption study 1.
Plasma.
Plasma samples that had high anti-ganglioside antibody titers were selected. Blood samples were taken in double-filtration plasmapheresis from patients with GBS (n = 11), FS (n = 8), Bickerstaffs brainstem encephalitis (BBE) (n = 2), acute ophthalmoparesis without ataxia (n = 2), acute [25] or chronic sensory ataxic neuropathy (n = 3), multifocal motor neuropathy (n = 1), and amyotrophic lateral sclerosis-like disorder after ganglioside therapy [26] (n = 1).
Adsorbents.
The tryptophan-immobilized PVA (IM-TR) gel, phenylalanine-immobilized PVA (IM-PH) gel, and nonmodified PVA gels were provided by Asahi Medical (Tokyo, Japan). The amino bases of tryptophan and phenylalanine are linked covalently to a beaded PVA resin Figure 1. [17,18]
Figure 1. Adsorbents conjugated with various amino acids. (1) IM-TR, (2) IM-PH, (3) tryptophan-polyvinyl alcohol (PVA), (4) tryptamine-PVA, (5) histidine-PVA, (6) threonine-PVA, (7) serine-PVA.
ELISA.
ELISA was done as described elsewhere with minor modifications. [27] Briefly, 10 pmol of each ganglioside (GM2, GM1, GD1a, GD1b, GT1b, or GQ1b) was placed in individual wells of microtiter plates. Each plasma sample was diluted serially commencing at 1:100, and then the plates were incubated at 4 degrees C overnight. Peroxidase-conjugated anti-human gamma- or mu-chain-specific antibodies (Tago, Burlingame, CA; 1:5,000 dilutions) were added, and the plates were kept at room temperature for 2 hours. Each sample was tested in triplicate. Anti-ganglioside antibody titer (1:X) was the highest plasma dilution at which the value was 0.1 or greater.
Batchwise adsorption.
Plasma samples (n = 28) were mixed with each gel (ratio of gel to plasma, 1:6) for 1 hour at 37 degrees C. The anti-ganglioside antibody titers of the plasma before and after adsorption were determined by ELISA. The concentrations of the immunoglobulins (IgG, IgA, IgM), complements (C3, C4), and albumin in the plasma were measured by the standard analytical methods.
Immunoadsorption therapy.
Patients.
Eleven patients with FS and one patient with GBS underwent adsorption therapy with IM-TR 350, IM-PH 350, or both Table 1, Table 2. All had given their informed consents to receive this treatment. None of the patients with FS had limb weakness. The GBS patient (patient 8) had ophthalmoplegia and ataxia.
Table 1. Immunoadsorption therapy. Continued in (Table 2)
Table 2. Continued from (Table 1).
Ex vivo plasma perfusion through the adsorbent column.
During adsorption therapy, cell components were separated through membrane-type plasma separators. The plasma separator membrane Plasmaflo OP-05 (Asahi Medical) consists of polyethylene. Plasmacure PS-06 (Kuraray, Osaka, Japan) and Sulflux FS-05 (Kaneka, Osaka, Japan) consist of polysulfone. Plasma was passed through the IM-TR 350 or IM-PH 350 column and returned to the blood line. Each column held 350 mL of an IM-TR or IM-PH gel. Plasma samples taken longitudinally from the inlet and outlet of the affinity column were tested for IgG anti-GQ1b antibody titers. The concentrations of immunoglobulins, complements, albumin, and fibrinogen also were measured.
Batchwise adsorption study 2.
The amino bases of tryptamine, histidine, threonine, and serine were covalently linked to a beaded PVA resin Figure 1. A PVA resin was prepared to which the carboxylic acid of tryptophan was linked [tryptophan-PVA, Figure 1 (3)]. These gels were gifts from Asahi Medical. Six volumes of plasma from each FS patient (n = 6) was mixed with one volume of gel at 37 degrees C for 1 hour. The IgG anti-GQ1b antibody titers in the plasma were tested before and after adsorption.
Results.
Batchwise adsorption study 1.
The IM-TR gel adsorbed greater amounts of IgG anti-ganglioside antibodies [anti-GM1 (n = 9), anti-GD1a (n = 6), anti-GD1b (n = 8), and anti-GQ1b (n = 14)] than did the IM-PH and nonmodified PVA gels (Wilcoxon signed-ranks test, anti-GM1, anti-GD1a, anti-GD1b, p < 0.05; anti-GQ1b, p < 0.01) Figure 2A. In contrast, adsorption ability of IgM antiganglioside antibodies by the IM-TR was not different from that by the IM-PH and PVA gels Figure 2B. Although the IM-TR gel adsorbed larger amounts of IgG, IgA, IgM, C3, and C4 than did the IM-PH and PVA gels (n = 6, Wilcoxon signed-ranks test, p < 0.05), its adsorption of albumin did not differ (data not shown).
Figure 2. Batchwise adsorption using the nonmodified PVA gel, IM-TR gel, and IM-PH gel. (A) IgG anti-ganglioside antibodies. (B) IgM anti-ganglioside antibodies. The ability to adsorb anti-ganglioside antibody is expressed as [(preadsorption antibody titer)/(postadsorption antibody titer)].
Immunoadsorption Therapy.
IgG anti-GQ1b antibody titers at the outlet of the IM-TR column were less than those at the column inlet (n = 9, Wilcoxon signed-ranks test, p < 0.01). The autoantibody titers were reduced significantly after each session (n = 10, Wilcoxon signed-ranks test, p < 0.01). The ex vivo plasma perfusion study revealed that the adsorption of IgG anti-GQ1b antibody by the IM-TR column (n = 9) was superior to that by IM-PH (n = 7) (Wilcoxon signed-ranks test, p < 0.05) Figure 3A. In each session, the removal of IgG anti-GQ1b antibody by the IM-TR column (n = 10) was superior to that by IM-PH (n = 8) (Wilcoxon signed-ranks test, p < 0.01) Figure 3B. The IM-TR column retained larger amounts of IgG and C4 than did the IM-PH column (Wilcoxon signed-ranks test, p < 0.05) Figure 3C. Both columns did not adsorb albumin Figure 3C. Longitudinal studies of representative patients (patients 2 and 3) are shown in Figure 4.
Figure 3. (A) Ex vivo plasma perfusion through IM-TR and IM-PH columns. The ability to adsorb the autoantibody is expressed as [(IgG anti-GQ1b antibody titer at the inlet of the column at the start)/(titer at the outlet of the column)]. Bar, SE. *p < 0.05. (B) Removal of IgG anti-GQ1b antibody by the IM-TR and IM-PH columns. The ability to remove the autoantibody is expressed as [(the IgG anti-GQ1b antibody titer taken at the inlet of the column at the start)/(titer at the inlet at the end)]. Bar, SE. **p < 0.01. (C) Removal of IgG, IgA, IgM, C3, C4, and fibrinogen. The reduction rates were calculated as follows: (preadsorption concentration minus the postadsorption concentration)/(preadsorption concentration). Bar, SE. *p < 0.05.
Figure 4. Longitudinal ex vivo plasma perfusion using IM-TR. The ability to adsorb the IgG anti-GQ1b antibody is expressed as [(the IgG anti-GQ1b antibody titer at the inlet of the column)/(the titer at the outlet of the column)]. The reduction rates of IgG, IgA, IgM, and albumin were calculated as (preperfusion concentration minus the postperfusion concentration)/(preperfusion concentration).
No plasma proteins were required as substitution fluids. Patient 8 complained of nausea during each session. No adverse reactions occurred in the other patients during and after 47 sessions using the IM-TR 350 column and 17 sessions using the IM-PH 350; no hypotension, hypertension, bradycardia, dizziness, chills, fever, skin rash, hematoma, nausea, vomiting, or tetany occurred.
Hemolysis was seen in the plasma separator with two sessions in patient 6 when the FS-05 separator was used and in one session with patient 9 when PS-06 was used. The cause of the hemolysis was not clear as the transmembrane pressure of the separator was not increased. Restricted flow occurred in patient 10 in each session with the IM-TR 350 column. No other technical problems, such as membrane rupture or vascular access difficulties, occurred.
Batchwise adsorption test 2.
The IM-TR gel adsorbed larger amounts of IgG anti-GQ1b antibody than did the nonmodified PVA, the IM-PH, tryptophan-PVA, tryptamine-PVA, histidine-PVA, threonine-PVA, and serine-PVA gels (n = 6, Wilcoxon signed-ranks test, p < 0.05) Figure 5.
Figure 5. Batchwise adsorption of the IgG anti-GQ1b antibody by various amino acid-immobilized gels. The ability to adsorb IgG anti-GQ1b antibody is expressed as [(preadsorption antibody titer)/(postadsorption antibody titer)]. Bar, SE.
Discussion.
Plasmapheresis given patients with GBS and FS includes plasma exchange, double-filtration plasmapheresis, and immunoadsorption. In Japan, about half of the patients who received plasmapheresis were treated with double-filtration plasmapheresis, one-fourth with plasma exchange, and the rest immunoadsorption. [28] When neurologists in Japan choose adsorption therapy, they usually use an IM-PH column, [22,24] but there is no theoretical basis for the use of the IM-PH column rather than the IM-TR column. The neurologist who treated patient 1 Table 1 using the IM-PH column sent me samples of the patient's plasma to test whether IM-PH removed the IgG anti-GQ1b antibody. I found that IM-PH column therapy did not reduce the IgG anti-GQ1b antibody titers in the patient's plasma (reported by Yamawaki et al at the 5th meeting of the European Neurological Society, 1995). This clinical observation led me to study whether the IM-PH gel treatment removes anti-ganglioside antibodies.
Both the IM-PH and IM-TR gels adsorb rheumatoid factor and anti-DNA antibodies. [17,18] But, whereas the IM-TR gel adsorbs anti-acetylcholine receptor antibody, the IM-PH does not. [19,20] I therefore studied in vitro which gel adsorbs anti-ganglioside antibodies. Goto et al. [29] recently reported that both gels highly adsorbed the anti-GM1 or anti-GD1b antibody in the plasma from two patients with GBS and a patient with chronic inflammatory demyelinating polyneuropathy. Their study showed that the IM-PH and IM-TR gels adsorbed both the IgG and IgM anti-ganglioside antibodies. My larger study, however, showed that the IM-PH gel adsorbs IgG anti-ganglioside antibodies less effectively than the IM-TR gel Figure 2A and that both gels are less effective in removing IgM anti-ganglioside antibodies Figure 2B. Whereas the immunoglobulin class of the anti-GQ1b antibody in FS patients usually is IgG, [8-12] that of the anti-ganglioside in GBS patients is IgG, IgM, or both. [30,31] I therefore assumed that the IM-PH column should not be used for GBS and FS but that IM-TR therapy could be indicative for FS.
FS and GBS are considered to be closely related because some patients who presented with FS progressed to GBS. [1] The value of plasma exchange in GBS is well established [3-5] but that of intravenous immunoglobulin therapy has yet to be shown. [32] Steroid treatment is ineffective in GBS. [33,34] I therefore recommended plasma exchange as the treatment of first choice for FS and for GBS. Most neurologists did not, however, try to treat the patients using plasma exchange mainly because this therapy requires large amounts of replacement fluids. I then advise the use of adsorption therapy using the IM-TR column on the basis of the results of my adsorption test Figure 2A. Moreover, because of the results of the ex vivo study done on patients 2 and 3 Table 1, Figure 4, I subsequently urge neurologists to choose immunoadsorption, which does not require plasma proteins. Nine of 11 patients agreed to undergo one or two sessions using the IM-PH column. This ex vivo study confirmed that the IM-TR column had a greater degree of IgG anti-GQ1b antibody adsorption than did the IM-PH column Figure 3, A and B. Although the result does not negate the effectiveness of IM-PH therapy, it showed no theoretical basis for the use of the IM-PH column. As previously reported, [21-24] immunoadsorption therapy was tolerated without severe clinical complications in the 12 patients treated.
The subclasses of the IgG anti-GQ1b antibody are restricted to IgG1 and IgG3. [12,27] The IM-TR column adsorbed not only IgG1 and IgG3 but IgG2 and IgG4 as well. [21] This shows that semiselective adsorption of the autoantibody by the IM-TR gel does not depend on the IgG subclass. I then postulated that the IM-TR gel combines with the IgG anti-GQ1b antibody by physicochemical interaction. Hydrophobic forces of the side chains of amino acids are 3,400 cal/mol in tryptophan, 2,500 in phenylalanine, 500 in histidine, 400 in threonine, and -300 in serine. [35] The IM-TR gel adsorb the autoantibody more effectively than the IM-PH, histidine-PVA, threonine-PVA, and serine-PVA gels Figure 5. The IM-TR gel also had a greater degree of the autoantibody adsorption than did the tryptophan-PVA and tryptamine-PVA gels Figure 1 and Figure 5. These results suggest that both the hydrophobic force of the side chain and the anionic charge of the carboxylic acid in tryptophan are important in the adsorption of the autoantibody by the IM-TR gel. Hydrophobic residues and cationic charges may be abundant on the autoantibody surface. The actual mechanism of the affinity between the IM-TR gel and the IgG anti-GQ1b antibody has yet to be clarified.
The IgG anti-GQ1b antibody is associated with BBE, [36] acute ophthalmoparesis without ataxia, [11] and GBS with ophthalmoplegia, [9,11] as well as with FS. These diseases that show external ophthalmoplegia should be designated the "IgG anti-GQ1b antibody syndrome.'' The autoantibodies from patients with BBE, acute ophthalmoparesis, and GBS with ophthalmoplegia were adsorbed by the IM-TR gel as well as the autoantibody of FS Figure 2A. I propose that adsorption therapy that uses an IM-TR column should be considered an alternative form of plasmapheresis for the IgG anti-GQ1b antibody syndrome. Double-blind, randomized, prospective, multicenter trials are required to show whether this treatment prevents deterioration such as respiratory insufficiency and leads to earlier recovery with less neurologic deficits.
Acknowledgments
I thank Drs T. Yamawaki, H. Ichikawa, K. Deguchi, K. Obara, T. Orimo, T. Koide, Y. Inatomi, and K. Chida for providing the clinical information on the patients.
- Copyright 1996 by Advanstar Communications Inc.
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