Abstract
Article abstract-We investigated the possible mechanisms of paralysis and recovery in a patient with the acute motor axonal neuropathy (AMAN) pattern of the Guillain-Barre syndrome. The AMAN pattern of GBS is characterized clinically by acute paralysis without sensory involvement and electrodiagnostically by low compound motor action potential amplitudes, suggesting axonal damage, without evidence of demyelination. Many AMAN patients have serologic or culture evidence of recent Campylobacter jejuni infection. Pathologically, the most severe cases are characterized by wallerian-like degeneration of motor axons affecting the ventral roots as well as peripheral nerves, but some fatal cases have only minor changes in the roots and peripheral nerves, and some paralyzed patients with the characteristic electrodiagnostic findings of AMAN recover rapidly. The mechanism of paralysis and recovery in such cases has been uncertain. A 64-year-old woman with culture-proven Campylobacter upsaliensis diarrhea developed typical features of AMAN. She improved quickly following plasmapheresis. Her serum contained IgG anti-GM1 antibodies. The lipopolysaccharide of the organism bound peanut agglutinin. This binding was blocked by cholera toxin, suggesting that the organism contained the Gal(beta 1-3)GalNAc epitope of GM1 in its lipopolysaccharide. Motor-point biopsy showed denervated neuromuscular junctions and reduced fiber numbers in intramuscular nerves. In contrast, the sural nerve biopsy was normal and skin biopsy showed normal dermal and epidermal innervation. In AMAN the paralysis may reflect degeneration of motor nerve terminals and intramuscular axons. In addition, the anti-GM1 antibodies, which can bind at nodes of Ranvier, might produce failure of conduction. These processes are potentially reversible and likely to underlie the capacity for rapid recovery that characterizes some cases of AMAN.
NEUROLOGY 1997;48: 717-724
The term "Guillain-Barre syndrome" (GBS) has been used synonymously with acute inflammatory demyelinating polyneuropathy (AIDP). [1-3] However, it is increasingly likely that in some cases the immune attack is directed toward the axon rather than the myelin sheath. [4-9] Some of these "axonal" cases of GBS have nearly pure motor fiber involvement, a pattern that we have termed "acute motor axonal neuropathy" (AMAN). [10] This pattern, frequent in northern China, [10,11] is characterized by acute ascending paralysis, areflexia, and albumino-cytologic dissociation in cerebrospinal fluid (CSF) without sensory involvement. [10] Electrophysiologic studies show a reduction in amplitude or absence of compound muscle action potentials (CMAPs) with normal conduction velocities, distal latencies, F waves, and sensory nerve action potential (SNAP) amplitudes and velocities. [10]
Recent studies of AMAN have identified complement activation products and immunoglobulins on the nodal axolemma and in the periaxonal space of myelinated motor fibers, and macrophages can be seen invading nodes of Ranvier of motor fibers. [6-9] There is little internodal demyelination. In severe cases, many fibers undergo wallerian-like degeneration, which may begin as proximally as the ventral root exit zone. In contrast to these findings, in AIDP complement activation products have been found on the outer surface of Schwann cells, and macrophages phagocytose myelin at early stages of the disease. [12]
A high proportion of AMAN cases follow infection with Campylobacter jejuni. [10,11,13-19] The lipopolysaccharides of some of the organisms isolated from affected patients have been shown to contain Gal(beta 1-3)GalNAc epitopes in their glycoconjugates. [15,16,20] Anti-GM1 antibodies, which bind to this epitope, are found in the sera of many AMAN patients [11,17,21] as well as in the sera of some patients with other patterns of GBS. [15,16,22,23] These findings have led to the proposal that these cases represent "molecular mimicry," in which epitopes of the lipopolysaccharide (LPS) elicit antibodies that bind to axons and especially to the nodal axolemma, so that the nerve fiber is the inadvertent target of an immune response directed against the enteric organism.
An important but unexplained aspect of the AMAN syndrome is that some patients recover rapidly. [11] Most of the Chinese AMAN patients recover as rapidly as Chinese AIDP patients, [24] an observation that is incompatible with the extensive wallerian-like degeneration seen in the ventral roots from the most severe autopsy cases. [6,10] We have previously speculated that the explanation could lie either in motor fiber degeneration restricted to the axon terminals or in a physiologic failure of conduction. [6,7,11] This latter possibility has received support from recent data on the effects of anti-GM1 antibodies [9,25-29] and of Guillain-Barre sera [30] on the conduction of action potentials and on the function of axonal ion channels. [25]
In this report, we describe a patient from the United States whose case of GBS was preceded by gastrointestinal infection by Campylobacter upsaliensis. Her disease fulfilled the diagnostic criteria for AMAN. She had rapid recovery from her paralysis. The pathologic data suggest that motor nerve terminal degeneration with regeneration may underlie her rapidly reversible weakness.
Case report.
A 64-year-old American woman presented with diarrhea, with leg weakness developing on the seventh day. The diarrhea resolved with loperamide. She denied any paresthesias, meningismus, bladder or bowel problems, or foreign travel. On day 2 after onset of weakness, she was discharged home without diagnosis, using a walker. She had increased difficulty with her gait and began to experience weakness in her arms. She was admitted to Johns Hopkins Hospital on day 7 after onset of weakness with a presumptive diagnosis of Guillain-Barre syndrome. Physical examination on admission showed 5-/5 proximal and 4/5 distal arm strength and 3/5 proximal leg strength. Ankle flexion was 4-5/ and was easily overcome manually. She was unable to stand or walk (Hughes grade 4). Her reflexes were 2+ in the upper extremities, 1+ at the knee, and absent at the ankles. Her sensory examination was normal for pain, temperature, vibration, touch, and proprioception. Her CSF had 2 white cells/mm3, a protein content of 57 mg/dL, and a glucose content of 63 mg/dL.
After admission, her patellar reflexes disappeared and her biceps reflexes were reduced. She was treated with five courses of plasmapheresis, beginning on day nine. Her patellar reflexes returned after the second treatment (day 11), and she was able to stand with support after the fourth treatment (on day 15 of her neurologic illness). She was discharged to a rehabilitation facility on day 21 of her illness. At that time, she had 4/5 power in plantar flexion at the ankle.
Methods.
Bacteriology.
Stool cultures were performed by using charcoal containing selective media (Campy CSM, BBL, Cockeysville, MD) at 42 degrees C in a microaerobic environment. The isolate was identified by conventional biochemical testing, [31] ribotyping, [32,33] and flagellin gene typing. [34,35] Isotype-specific antibodies against Campylobacter were performed by using an ELISA assay as previously described. [36]
Characterization of the LPS of the isolate.
LPS was extracted with use of the hot phenol-water method described by Westphal and Jann. [37] The patient's serum, obtained 8 days after the onset of neurologic illness, was studied for the presence of anti-LPS antibodies. The LPS was run on 12% SDS-PAGE. The gels were then either fixed and silver stained [38] for LPS or transferred to PVDF membranes for immunoblotting. Nonspecific binding was blocked with 2% gelatin and 0.05% Tween in PBS at room temperature for an hour. To determine the presence of anti-LPS antibody in patient's sera and presence of GM1 and asialo GM1 ganglioside epitopes on the LPS, the blots were incubated with patient or control sera at a dilution of 1:1,000 to 1:5,000 at 4 degrees C overnight or horse-radish peroxide (HRP) conjugated cholera toxin (CT) or peanut agglutinin (PNA) at dilutions of 1:10,000 (40 ng/ml) and 1:5,000 (200 ng/ml), respectively. HRP-labeled specific secondary antibodies against different human immunoglobulin classes and IgG subclasses were used at a dilution of 1:2,000 at room temperature for an hour. An Escherichia coli LPS was used as control. The immunoblots were developed with the use of an enhanced chemiluminescent detection system.
Pathology.
A motor-point biopsy from the gastrocnemius muscle was performed on day 16 of illness. The muscle strip was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight and then transferred to buffer. The muscle was then cut into segments of approximately 1 cm. Cholinesterase staining was performed as previously described. [39] The segments with positive cholinesterase staining were identified and embedded in Epon. Onemicrometer semi-thin sections were stained with toluidine blue. Areas within intramuscular nerves and neuromuscular junctions were identified. Thin sections of end-platerich regions were obtained as previously described [39] and observed under a Hitachi electron microscope. Three-millimeter punch biopsy specimens of the skin on the lateral aspect of the heel and on the lateral calf were processed and stained for axons with antibodies against protein gene product 9.5 (PGP 9.5), a panaxonal stain, as previously described. [40]
Results.
Electrodiagnostic studies.
An electrophysiologic study 1 week after the onset of her weakness showed normal SNAP amplitudes and latencies. The motor nerve conduction studies showed an absent right peroneal nerve CMAP amplitude and markedly decreased left peroneal CMAP amplitude with normal conduction velocity and distal latency. The median and ulnar nerves also showed normal conduction velocity and distal latency with decreased CMAP amplitudes (Table 1). Needle electromyography (EMG) of multiple upper and lower extremities and paraspinal muscles showed fibrillations and positive sharp waves in the distal lower limb muscles bilaterally. The EMG recordings of muscles above the knees and in the right upper extremity were normal. Her second study 2 weeks after the onset of her weakness showed a return of right peroneal motor response, although it remained severely reduced in amplitude. Upper limb motor conduction studies continued to show reduced CMAP amplitudes with normal distal latency and nerve conduction studies.
Table 1. Nerve conduction study at 1 week and 2 weeks*
Pathology.
The intramuscular nerves from the gastrocnemius motor-point biopsy showed severe fiber loss and ongoing wallerian-like degeneration of most myelinated nerve fibers (Figure 1A, and Figure 1B). The unmyelinated nerve fibers and some small myelinated fibers in the intramuscular nerves remained intact (Figure 1C). The cholines-terase stain identified numerous neuromuscular junctions. Immunostaining with the antibody against PGP 9.5, a panaxonal stain, [40] revealed that almost all motor nerve terminals lacked innervation (Figure 2). Electron microscopy confirmed that the motor nerve terminals were absent from the neuromuscular junctions, and that the Schwann cells of the motor nerve terminals (telodendroglia) occupied the neuromuscular junctions (Figure 3).
Figure 1. Motor-point biopsy results showing intramuscular nerve. (A and B) Most of the myelinated fibers are lost. (C) The unmyelinated fibers and one small myelinated fiber remain intact.
Figure 2. (A and B) Combined PGP 9.5 and cholinesterase staining of muscle showing denervated neuromuscular junctions (blue). Some PGP-reactive debris remains (arrows), but the end plates are denervated. In contrast, (C) illustrates normal innervated neuromuscular junctions from mouse muscle.
Figure 3. Motor nerve terminals are absent from the neuromuscular junction, and the Schwann cells of the motor nerve terminals (telodendroglia) remain.
The sural nerve biopsy was entirely normal at the light and electron microscopic levels (Figure 4A), except for rare fibers undergoing wallerian-like degeneration (about one per fascicle, <1% in teased fibers). The myelinated fiber density was 9,700/mm2 (normal, 7,500 to 12,500). The nodes of Ranvier were normal ultrastructurally.
Figure 4. (A) Sural nerve biopsy results show normal myelinated and unmyelinated fibers. (B) Skin biopsy results show normal epidermal and dermal sensory nerve fibers. EPI = epidermis; DERM = dermis; P = papillae. Arrows indicate epidermal sensory nerve fibers.
The skin biopsies showed normal epidermal and dermal sensory nerve fibers (Figure 4B), and the fiber densities within the epidermis were 20.4 epidermal nerve fibers/mm epidermis (normal for a 60- to 69-year-old woman, 16.3 +/- 4) [40] (J. McArthur, A. Stocks, unpublished).
Bacteriology.
By the time of admission, her diarrhea had resolved, but her stool culture was positive for Campylobacter. The isolate, identified as C upsaliensis by using a combination of phenotypic and genotypic characteristics, [31-33] was found by ribosomal typing to have a pattern similar to that of eight other strains of C upsaliensis [32,33] (data not shown). Four of these other strains have been characterized by DNA:DNA hybridization. [41] Common bands shared by this patient's strain and by other C upsaliensis strains include the band at ca. 7 kb and the two bands at ca. 24 and 28 kb. The organism was not typable by the Penner O system. In particular, it was negative with the O:19 antiserum. [36] By flagellin gene typing, the organism gave a pattern distinct from C jejuni, but that was identical to three other human isolates of C upsaliensis strains [34,35] (data not shown).
Antibodies of the IgG, IgA, and IgM isotypes to C jejuni O1, O2, O3 surface antigens, as well as to the surface antigen and the LPS from the patient's C upsaliensis isolate, were detected in the patient's serum with an ELISA assay. [42]
Characterization of the Campylobacter LPS.
This strain of C upsaliensis showed a broad band of low molecular weight LPS (also called lipooligosaccharide) of approximately 10 to 14 kd by silver staining. A short series of high molecular weight LPS bands were visualized only by immunoblotting with the patient's serum but not by silver staining, a finding that has been reported in previous studies of C jejuni. [43] The immunoblotting results showed that the patient's serum had anti-LPS antibodies of IgA, IgM, and IgG classes. The IgG anti-LPS antibodies were present in highest titers. IgG2 was the major IgG subclass with anti-LPS binding, whereas IgG1 had only weak binding to LPS. The sera from two healthy controls did not react with the C upsaliensis LPS.
Glycoconjugate epitopes on LPS and antiglycoconjugate antibodies.
The immunoblots showed binding of PNA to a band of Campylobacter LPS (see Figure 5). Cholera toxin and the patient's serum blocked this binding. These results suggest the presence of Gal(beta 1-3)GalNAc (asialo-GM1-like) epitopes in the LPS of this particular Campylobacter strain. The E coli LPS bound neither PNA nor CT. The patient's sera were strongly positive for IgG anti-GM1 antibody (1:2,350) and had reactivity above background for IgG anti-GD1b (1:290) and IgG anti-GA1 antibody (1:235). IgM anti-GM1, GD1a, GD1b, GA1, and GA1b were not detected, nor was IgG anti-GD1a or GA1b.
Figure 5. Immunoblot of C upsaliensis LPS. Lane 1, PNA binds to a band. PNA binding is blocked by pre-incubation with unconjugated cholera toxin, 10 micro g/ml (lane 2) and patient's serum (lane 3).
Discussion.
The axonal forms of Guillain-Barre syndrome were initially identified in patients with a severe paralysis and slow recovery. [4,8,44] That course is consistent with the extensive wallerian-like degeneration of peripheral nerves documented in autopsied cases of the acute motor-sensory axonal form (AMSAN), [4,6,8] and with the need for axonal regeneration for recovery. However, in studies carried out in northern China, the median recovery time for individuals with the axonal form of GBS restricted to motor involvement (AMAN) was the same as that for AIDP patients. [24] In addition, some autopsied AMAN cases have only mild degeneration of motor fibers at the level of the spinal roots and peripheral nerves, yet they had experienced complete paralysis. [6,7]
Several possible structural explanations for the failure of conduction with neither demyelination nor axonal degeneration can be inferred from these previous observations on AMAN. Pathologic studies have demonstrated lengthening of the nodes of Ranvier of motor fibers, [7] recruitment of macrophages to overlie the nodes, [6,7] distortion of the paranodal myelin in some fibers, [7] opening of the periaxonal space of the internode to endoneurial fluid (presumably signifying loss of the insulating role of myelin), [9] and entry of macrophages into the periaxonal space. [6,7] Each of these structural changes might block conduction and could contribute to reversible failure of conduction. Two other quickly reversible mechanisms have been suggested: physiologic failure of conduction along motor axons due to binding of antibody to the nodal axolemma, [25] and wallerian-like degeneration restricted to the motor nerve terminals. [6,7] The present patient provides direct evidence supporting this last mechanism and indirect evidence consistent with the former hypothesis.
Motor nerve terminal degeneration.
This patient had extensive denervation in her paralyzed gastrocnemius, as indicated by the fibrillations on EMG, by the loss of motor nerve terminals in the neuromuscular junctions, and by extensive wallerian-like degeneration of myelinated axons within the intramuscular nerves. The unmyelinated as well as some small myelinated fibers in the intramuscular nerves were normal. As in previous AMAN cases, a cutaneous sensory nerve, the sural, was almost normal, with only rare degenerating fibers. Two lines of evidence suggest that the degeneration of the motor fibers was restricted to their most distal regions. First, denervation potentials were detected relatively early. If the denervation was secondary to a lesion at the level of the spinal root, denervation potential should have been delayed for up to 2 weeks. [45] Second, the short time to recovery of power in these muscles could not be explained by axonal regeneration over a long distance. Growing axons in humans regenerate at a rate of <1 mm/day; thus, early reinnervation would suggest that, at most, only a few centimeters are involved. An alternative explanation-reinnervation by terminal sprouting of surviving intact motor nerve fibers-is unlikely because of the extensive denervation present on the biopsy: virtually no motor axons remained.
In an individual case, terminal demyelination cannot be distinguished from terminal degeneration electrodiagnostically if the CMAPs cannot be recorded, as emphasized by recent reports. [46-48] Low CMAP amplitude can be due to distal demyelinating lesions causing conduction block. [46] However, if this were the case one would expect to see an increase in distal latency, reflecting the slowing of conduction through the demyelinated lesions. The findings in the present case document the loss of intramuscular motor fibers. The target of immune attack in AMAN appears to be the axolemma instead of the myelin. [6-9]
Serum-mediated failure of conduction.
Paralysis in AMAN could in theory also involve physiologic block of nerve transmission. The return of our patient's reflexes within hours of her plasmapheresis suggests the possibility of a rapidly reversible nerve transmission failure. A possible mechanism underlying this blockage is suggested by the observation that rabbit anti-GM1 antibodies can affect both sodium and potassium channels in voltage-clamped single fibers. [25] These effects on ion channels are consistent with the fact that GM1-reactive epitopes are highly enriched in the nodes of myelinated fibers [49-52] where the sodium channels are concentrated. In addition, our previous studies of AMAN autopsies have detected immunoglobulin and complement activation products bound to nodes of Ranvier, even in fibers with no other obvious pathology. [9] The present patient had anti-GM1 antibodies in her sera. Thus, an attractive reconstruction is that these antibodies both blocked conduction and resulted in complement-mediated fiber degeneration in other fibers.
The absence of a blood-nerve barrier in the motor nerve terminals may make this region particularly vulnerable to antibody-mediated disorders, such as AMAN appears to be. In contrast, at the ventral root level an antibody-mediated attack would be hindered by the blood-nerve barrier. A predilection for motor nerve terminal involvement may explain the high proportion of AMAN patients who recover rapidly; perhaps only the most severe cases go on to have the wallerian-like degeneration of ventral root fibers found at autopsy in some of the fatal cases. [6,10,52]
Bacteriology.
This is the first documented case of GBS following gastrointestinal infection with C upsaliensis, an organism recognized for several years to be a cause of diarrhea, [53] as well as of bacteremia in the pediatric population. [41,54] In some studies, C upsaliensis was more frequently isolated from patients with diarrheal illness than were other commonly recognized agents such as Shigella and Yersinia enterocolitica. [53] Whether C upsaliensis is frequently associated with GBS is unknown because most laboratories do not use procedures adequate for isolating this species from routine stool cultures. [31] It is noteworthy that the isolate was not typable by the Penner O system, providing further evidence [18,19] that axonal patterns of GBS are not exclusively associated with a restricted set of Penner types such as O:19.
The silver staining shows that C upsaliensis has both low and high molecular weight LPS structures. The low molecular weight LPS consists of lipooligosaccharide, whereas high molecular weight LPS consists of lipooligosaccharide as well as O-side chains. The presence of all three immunoglobulin classes of anti-LPS antibodies suggests an acute humoral immune response directed against C upsaliensis LPS antigens. This immune response is most likely initiated at the intestinal mucosal surface, as indicated by the presence of IgA anti-LPS antibodies. The fact that IgG anti-LPS antibodies had the highest titers in index case serum is consistent with the seroconversion studies [55] that have shown a fall in IgA and a rise in IgG anti-LPS antibodies by 2 weeks postinfection.
AMAN and anti-GM1 antibodies.
Several reports have previously noted the association between IgG anti-GM1 antibodies and acute motor axonal neuropathies. [15-18,21,22] In addition, the C jejuni strains associated with axonal patterns of GBS have been shown to contain GM1-like epitopes in their lipopoly-saccharides. The LPS from our C upsaliensis demonstrate binding to PNA, which is also known to bind to asialo-GM1-like epitopes. Interestingly, cholera toxin interfered with PNA binding but did not itself bind to the LPS. This can be due to the insensitivity of our assay on this LPS, or because the GM1 epitope on the LPS may be masked. The presence of high IgG anti-GM1 and anti-GA1 antibody in our patient suggests that these antibodies are directed against the Gal(beta 1-3)GalNAc on asialo-GM1 epitope of the LPS. Alternatively, the LPS may be sialated in vivo, thereby changing the asialo-GM1 to a GM1-like epitope. The present data provide further evidence supporting the link between GM1 reactivity and AMAN. [16,56]
Proposed pathogenesis of AMAN.
The present patient illustrates the possible pathophysiologic mechanisms involved in AMAN. She was infected with an organism that carried on its LPS the Gal(beta 1-3)GalNAc epitope. The host response caused the formation of crossreacting antibodies against GM1 or GM1-like structures. As shown in previously reported cases, antibody is deposited at the nodes of Ranvier, where these gangliosides are particularly enriched. Whether or not Gal(beta 1-3)GalNAc proves to be the pathophysiologically relevant epitope, these observations suggest that sensitization to LPS can stimulate anti-axonal antibodies. Antibody binding may be sufficient to impair conduction at the nodes of Ranvier, and it can be followed by complement activation and macrophage infiltration. Despite this immune attack against axons, most motor nerve fibers in the spinal roots and nerves remain structurally intact. The distal-most motor nerves where the blood-nerve barrier is deficient may be most vulnerable, rapidly losing CMAP amplitude and F waves. Recovery may be due to a reversal of physiologic failure of conduction or to regeneration of degenerated distal motor nerves. In severe cases, widespread secondary wallerian-like degeneration occurs, portending a slow or incomplete recovery.
Addendum: There is an apparent discrepancy in our LPS immunoblotting results, in which cholera toxin did not bind to LPS (cholera toxin-HRP 40 ng/ml) but was able to block PNA binding (unconjugated cholera toxin 10 micro g/ml). This is explained by our recent experiment that showed cholera toxin binding to C. upsaliensis LPS in an ELISA performed with cholera toxin concentration of 1-10 micro g/ml. This indicates that higher concentrations than that used for immunoblots are required to demonstrate cholera toxin binding to this LPS. However, immunoblotting with a concentration similar to that used in ELISA (1 micro g/ml) yielded uninterpretable results due to high background levels.
Acknowledgments
The authors wish to thank Peter Hauer and Huong Ung for technical assistance and Mabel Ann Nicholson from the Centers for Disease Control and Prevention for providing the Campylobacter upsaliensis strains and serotyping.
- Copyright 1997 by Advanstar Communications Inc.
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