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Axonal neuropathy with monoclonal IgG kappa that binds to a neurofilament protein.

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Axonal Neuropathy with Monoclonal
IgG Kappa That Binds to a Neurofilament
Raffaello Nemni, M D , Maria L. Feltri, MD, Raffaella Fazio, MD, Angelo Quattrini, M D ,
Isabella Lorenzetti, BD, Massimo Corbo, M D , and Nicholas Canal, DM
We report a 74-year-old woman with a slowly progressive sensory motor axonal neuropathy and a monoclonal IgG-K
that bound to a 68-kd axonal protein identified as the low molecular weight neurofilament protein. T h e sera of control
subjects and disease controls did not bind to neurofilament protein.
Nemni R, Feltri ML, Fazio R, Quattrini A, Lorenzetti I, Corbo M, Canal N. Axonal neuropathy with
monoclonal IgG kappa that binds to a neurofilament protein. Ann Neurol 1990;28:361-364
In paraproteinernic neuropathies, the monoclonal protein has been noted to bind to rnyelin-associated glycoprotein (MAG) [11, other glycoproteins [2], two
glycolipids {3, 41, and chondroitin sulfate C [5]. We
describe a patient with a slowly progressive sensory
motor axonal neuropathy associated with an IgG kappa
(IgG-K) monoclonal gammopathy. Absorption with
whole peripheral nerve homogenate removed the
spike from the patient’s serum. By immunoblot, the
bound to an axonal protein of
monoclonal I ~ G - K
68-kd molecular weight identified as the low molecular
weight neurofilament protein (NF-L).
Case Report
A 74-year-old woman was referred with a diagnosis of sensory-motor polyneuropathy that was slowly progressive for 1
year with symmetrical weakness of the lower limbs and
paresthesias. At the time of the admission, she required a
cane to walk. Neurological examination revealed reduced
muscle strength in the legs and absent deep tendon reflexes.
Sensation to pinprick, light touch, and temperature was normal. Sensation to vibration was diminished to the knees.
Heel-to-shin coordination was impaired.
The following laboratory data were normal: blood count;
tests for liver, kidney, and thyroid function; electrolytes;
muscle enzymes; cryoglobulins; vitamin BI2;antinuclear antibodies; latex fixation; and urinalysis. Chest roentgenogram
and x-ray studies of the long bones and complete myelogram
were also normal. The protein content of the cerebrospinal
fluid was normal, and there were no cells.
Serum protein electrophoresis revealed a small homogeneous “spike,” which was identified in immunoelectro-
From the Department of Neurology, University of Milan, Istimto
Scientific0 S. Raffaele, Milan, Italy.
Received Nov 30, 1989, and in revised form Feb 23 and Apr 3,
1990. Accepted for publication Apr 4, 1990.
phoresis as an IgG-K immunoglobulin. Serum IgG was 2,680
mgidl, IgM was 180 mg/dl, and IgA was 290 mg/dl. Free K
light chains were present in the urine.
Electrophysiological investigations revealed denervation
patterns in tested muscles, and relatively preserved conduction velocities in all four limbs. Motor (MAP) and sensory
action potential (SAP) amplitudes were decreased; peroneal
nerve MAP was 4 mV (normal > 5 mV), ulna nerve MAP
was 7 mV (normal > 10 mV), median nerve SAP was 12 pV
(normal > 18 kV), and sural nerve SAP was 7 pV (normal >
9 pV). Distal motor latencies (DL) were slightly increased;
ulnar nerve DL was 3.8 msec (normal [+SD) 2.8 + 0.3
msec), and peroneal nerve DL was 6 msec (normal 4.3 + 0.5
Histopathological study of a sural nerve biopsy at the ankle showed no inflammatory cells or deposits of pathological
material. Semithin plastic sections showed a mild degree of
endoneurial fibrosis and large myelinated fiber loss. Many
residual large myelinated fibers had abnormal changes in the
axoplasm, atrophic axons, or both. Many clusters of small
myelinated fibers, a sign of regeneration after axonal degeneration, were present in the nerve (Fig 1).
The endoneurial area was normal. The total number of
myelinated fibers was 5,234 (normal 5,200-9,500), and their
density was 6,543imm (normal 5,400-8,600). Smalldiameter myelinated fibers were increased in percentage,
with 97.8% of all fibers measuring less than 8 km (normal
66.4-77.2%). Numerous clusters (total number 281, normal
< 38) were responsible for this increase. Electron microscopy confirmed primary axonal damage with early disintegration of neurofilaments (Fig 2). Immunocytochemical studies
using peroxidase-conjugated anti-human affinity-purified
antibodies (Southern Biotechnology Associates, Inc) diluted
1:2,000 showed a small amount of IgG-K in the axons.
Address correspondence to Dr Nemni, Department of Neurology,
S . Raffaele Hospital, Via Olgettina, 60-20132 Milan, Italy.
Copyright 0 1990 by the American Neurological Association
Fig I . Transverse section of sural nerve. Large myelinated fbers
are diminished in number. There are several clusters of myelinatedjibers (arrows), indicating regeneration of nerve fibersfollowing axonal injury. (Semithin section of epoxy-embeaedtiJ,w e , toluidine bhe; x 200.)
Fig 3. immunohihochemistry ofperlusedfrozen section of normal
rat sciatic nerve incubated with the patient’s serum. The .rerum
was diluted 1 :1.280 and stained by goat anti-human igG
peroxihse conjugated. ( 1 s - u selectively immunostained axons;
X 600.)
Fig 2. Sural nerve biopsy specimen. A myelinated fiber with
early dissolution of nez/rofkzments. The mimtubules appear relatively preserved. Bar = 1 pm.
Fig 4. immunohastochemistry of perlased frozen Jection of normal
rat cerebellum incubated with the patient’s serum. The serum
was diluted I : I .280and Jtained by goat anti-human igG
peroxidase conjugated. UgG-uselectiveb immunostained Purkinje cell bodies; X 600.1
Rats were perfused with freshly depolymerized 4%
paraformaldehyde in 0.1 M sodium acetate buffer p H 6.0,
and the dissected pieces of sciatic nerve and cerebellum were
quickly frozen by immersion in liquid nitrogen. Sections
were cut on a cryostatic microtome, incubated with the patient’s serum, and stained by the peroxidase-antiperoxidase
method [6]. I&-K
selectively immunostained axons in the
sciatic nerve (Fig 3) and Purkinje cell bodies but no other
neurons in the cerebellar cortex (Fig 4)up to 1: 1,280 dilu-
tion. Such immunocytochemical reactivity is typical of antibodies to neurofilaments.
Preparation of Antigens
Human sciatic nerves were obtained at autopsies 12 hours
after death. Lyophilized whole peripheral nerve homogenate
was used for absorption studies. Axonal proteins were isolated as previously described [7] for Western immunoblot
362 Annals of Neurology Vol 28 No 3 September 1990
Pig 5 . Binding of
1gG-K to low molrcular %eight neurofilament
(NF-LJ at serum diIution of 1 : N O . (Lane A) molecular weight
standzrd; (lane B) NF-L pwparation; (lane C ) immunostained
with serum IgG; (lane D) with K light chain>;(lane E) with A
light chains. (limes A and B. ammido black I OB staining; Lanes
C , D, and E, alkaline phosphatase-conjugated assay.)
Absorption Studies
Aliquots (0.1 mi) of the patient’s serum were absorbed with
3 mg lyophilized whole peripheral nerve homogenate overnight at 4°C. Sera from 9 normal volunteers, 6 patients with
diabetic neuropathy, and 9 patients with polyneuropathy and
IgG monoclonal gammopathy were examined. The sera were
analyzed by serum protein electrophoresis before and after
absorption. All the experiments were repeated three times.
Incubation of the sera with whole peripheral nerve
homogenate completely removed the monoclonal IgG-K
band but did not remove the M-protein from the other 9
patients with monoclonal gammopathy of unknown significance. Control serum electrophoretic patterns (9 normal volunteers and 6 diabetic patients) were not modified by
Axonal proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, using a 10% polyacrylamide separating gel, and then transferred onto nitrocellulose sheets. Nitrocellulose was stained with amido black
10B, incubated with the patient’s serum at a dilution of
1: 500, and counterstained with goat anti-human alkaline
phosphatase-conjugated anti-human IgG, or anti-human K
or lambda (A) light chains at a dilution of 1: 3,000.
bound to one protein band. It had a mobility of 68
kd (Fig 5 ) and was identified as NF-L. No reaction was seen
with sera from the other 9 patients with monoclonal gammopathy of unknown significance, the 9 normal subjects, or
reacting with NF-L was
the 6 diabetic patients. The I&-K
detected by goat anti-human K light chains but not by goat
anti-human A light chains, indicating that it was a monoclonal
The results demonstrate that the patient’s I ~ G - K
monoclonal antibody binds to NF-L. There is evidence
that some monoclonal antibodies are involved in the
pathogenesis of the neuropathy associated with monoclonal gammopathy of unknown significance. In patients with monoclonal IgM that binds to MAG, deposits of IgM are found on myelin sheaths of the
residual fibers and demyelination correlates with antimyelin activity [8]. Demyelination of feline sciatic
nerve was induced by intraneural injection of serum
from patients with neuropathy and anti-MAG IgM 19).
Features of the human neuropathy have been reproduced in mice by intraperitoneal injection of purified
IgG M-protein [lo].
Autoantibodies to neurofilaments have been detected in sera from patients with degenerative neurological diseases [11) and also in sera from normal
subjects 1123. In these cases, the autoantibodies to
neurofilament are polyclonal, mainly IgG, and directed
at multiple epitopes resembling antibodies generated
in a secondary immune response to a foreign antigen.
They also cross-react with the other intermediate filaments 1131. Therefore, low titer polyclonal antibodies
to neurofilaments seem to be common constituents of
the human antibody repertoire. In most cases, they are
directed at the 200-kd high molecular weight neurofilament protein, and fewer persons had low titer antibodies to the middle and low molecular weight neurofilament proteins [ 141. Our patient differs greatly from
this situation, since she has a high titer of monoclonal
autoantibodies to NF-L.
Human monoclonal IgM with autoantibody activity
against intermediate filaments has been reported 1151.
This activity was detected in the course of a study of
the properties of monoclonal IgM molecules produced
by patients with Waldenstrom macroglobulinemia with
peripheral neuropathy. In a recent study, high titers of
anti-MAG antibodies were always associated with neuropathy, while low titers of anti-MAG antibodies were
also detected in control patients [16]. It is possible that
antibodies that are commonly present at low titer may
play a pathogenic role at higher concentrations, as has
been found for cold Wlutinin antibodies 1171.
We believe that in our patient the monoclonal IgGK may play a pathogenic role in causing the progressive
axonal polyneuropathy.
1. Latov N, Braun PE, Gross RB, et al. Plasma cell dyscrasia and
peripheral neuropathy: identification of the myelin antigens that
react with the human paraproteins. Proc Natl Acad Sci USA
2. Frail DE, Edwards AM, Braun PE. Molecular characteristics of
the epitope in myelin associated glycoprotein that is recognized
by a monoclonal IgM in human neuropathy patients. Mol Immunol 1984;21:72 1-725
3. Ilyas AA, Quarles RH, Maclntosh TD, et al. IgM in a human
neuropathy related to paraproteinemia binds to a carbohydrate
determinant in the myelin-associated glycoprotein and to a ganglioside. Proc Natl Acad Sci USA 1984;81:1225-1229
4. Freddo L, Ariga T, Saito M, et al. The neuropathy of plasma cell
Nemni et al: Neuropathy and Monoclonal Gammopathy
dyscrasia: binding of IgM-M-proteins to peripheral nerve glycolipids. Neurology 1985;35:1420-1424
5. Sherman WH, Latov N , Hays AP, et al. Monoclonal IgMK
antibody precipitating with chondroitin sulfate C from patients
with axonal polyneuropathy and epidermolysis. Neurology
6. Sternberger LA. The unlabeled antibody peroxidase antiperoxidase (PAP) method. In: Sternberger LA, ed. Immunocytochemistry. New York: John Wiley, 1979:104-169
7. R u n g MS, Schlaepfer WW, Williams RC. Isolation and characterization of neurofilaments from mammalian brain. Biochemistry 1981;20:170-175
8. Takatsu M, Hays AP, Latov N, et al. Imrnunufluorescence study
of patients with neuropathy and IgM M-proteins. Ann Neurol
9. Hays AP, Latov N , Takatsu M, Sherman WH. Experimental
demyelination of nerve induced by serum of patients with neuropathy and an anti-MAG IgM-protein. Neurology 1987;37:
10. Besinger VA, Toyka KV, Anzil AP, et al. Myeloma neuropathy: passive transfer from man to mouse. Science 1981;
11. Toh BH, Gibbs CJ Jr, Gajdusek DC, et al. The 200 and 150
kDa neurofilament proteins react with IgG autoantibodies from
patients with Kuru, Creutzfeldt-Jakob disease, and other
neurologic diseases. Proc Natl Acad Sci USA 1985;82:3485-
12. Stefansson K, Marton IS,Dieperink ME, et al. Circulatory
autoantibodies to the 200,000-Dalton protein of neurofilaments
in the serum of healthy individuals. Science 1985;228:11171119
13. Luca FC, Bloom GS, Vallee RB. A monoclonal antibody that
cross-reacts with phosphorylated epitopes on two microtubuleassociated proteins and two iieurofilament polypeptides. Proc
Natl Acad Sci USA 1986;83:1006-1010
14. Braxton DB, Williams M, Kamali D , et al. Specificity of human
anti-neurofilament autoantibodies. J Neuroimmunol 1989;21:
15. Dellagi K, Brouet JC, Perreau J, Paulin D. Human monoclonal
IgM with autoantibody activity against intermediate filaments.
Proc Natl Acad Sci USA 1982;79:446-450
16. Nobile Orazio E, Francomano E, Daverio R, et al. Anti-myelinassociated glycoprotein IgM antibody titers in neuropathy associated with macroglobulinemia. Ann Neurol 1989;26:543-550
17. Pmzansky W, Katz A. Cold agglutinins-antibodies with biological diversity. Clin Immunol Rev 1984;3:131-168
Below is the correct version of Table 4 from Brust
JCM, Dickinson PCT, Hughes JEO, Holtzman R".
T h e diagnosis and treatment of cerebral mycotic aneurysms. Ann Neurol 1990;27:238-246.
Table 4. Aneuvysm Behavior in Relation to Antibiotic
Behavior During Therapy
Bled before treatment, early excision
Bled before treatment, then enlarged during
Bled during treatment, then continued to
Bled during treatment after enlarging
Bled during treatment, then early excision
Bled following treatment, then early excision
Never bled, early excision
Never bled, but enlarged or unchanged
during or after treatment
Never bled, became smaller or disappeared
during or after treatment
No. of
364 Annals of Neurology Vol 28 No 3 September 1990
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kappa, axonal, monoclonal, neuropathy, protein, igg, bindi, neurofilament
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