Oligoclonal IgG bands in patients with HTLV-I associated myelopathy--detection by agarose isoelectric focusing, transfer to cellulose nitrate and immunoperoxidase staining

The patient with HTLV-I associated myelopathy (HAM) shows a quite uniform clinical picture characterized by slowly progressive spastic paraparesis, slight sensory disturbances and urinary frequency, and the pathogenetic relationship between spastic paraparesis and HTLV-I was established. Since then, the role of the virus in causing myelopathy has drawn increasing attention. However, we have little information about cerebrospinal fluid (CSF) abnormalities in patients with HAM. Analysis of CSF oligoclonal bands (OB) in 22 patients with HAM was reported. All of 22 patients had typical clinical signs and symptoms of HAM with high titers of anti-HTLV-I antibodies in the serum by particle agglutination method. And these antibodies against HTLV-I were confirmed by enzyme-linked immunosorbent assay and western blot. Detection or characterization of CSF OB was done by high resolution agarose gel (HARG) electrophoresis with silver staining and immunofixation method with immunostaining. Other method for detection OB was by agarose isoelectric focusing (IEF), transfer to cellulose nitrate and immunoperoxidase staining (Olsson, 1984). CSF OB was detected in 13 of 22 patients with HAM by the method of immunofixation, using HRAG. All of CSF OB reacted with peroxidase conjugated goat anti-human IgG serum. More than 3 oligoclonal bands were not detected in HRAG electrophoresis. However, CSF OB was detected in all of 22 patients by the method of Olsson (IEF, in agarose, double-antibody peroxidase labelling and avidin-biotin amplification). The majority of patients with HAM had at least 5 or more OB in the region between pH 6.8 and 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration in children of oligoclonal IgG bands in unconcentrated CSF using agarose isoelectric focusing and immunolabeling

Agarose isoelectric focusing, followed by protein transfer to cellulose nitrate membrane and double-antibody avidin-biotin peroxidase staining (avidin-biotin agarose isoelectric focusing), was used to demonstrate oligoclonal IgG bands in unconcentrated cerebrospinal fluid (CSF) and serum; 161 consecutive pediatric patients, ages 6 months to 16 years with a variety of mainly neurologic disorders, were studied. The procedure was standardized for agarose isoelectric focusing (AIF) using 5 microliter specimens containing 125 ng of IgG. Oligoclonal bands were found in the CSF of 12% of the patients; bands were found simultaneously in the CSF and serum of 10% of the patients, mostly those with nervous system infections, but also those with central nervous system tumors, seizures, or migraine. In about 50% of positive cases, oligoclonal bands constituted the only CSF abnormality, reflecting an abnormal humoral immune response within the CSF-central nervous system compartment. Avidin-biotin AIF can be recommended as an integrated part of routine CSF examinations in children.     

Identification of light chain type bands in CSF and serum oligoclonal IgG from patients with multiple sclerosis

The light (L) chain types (kappa and lambda) of oligoclonal IgG bands of matching CSF and serum from 10 MS patients were identified in immunofixation after isoelectric focusing in polyacrylamide gel. Each specimen showed 10-15 oligoclonal bands in pH region of 7.5-9.3. In 7 MS CSF and 5 sera a greater number of oligoclonal IgG bands were of kappa (kappa)-type whereas in 3 CSF and 2 sera the majority was of lambda (lambda)-type. In 3 sera a clearcut correlation of bands with either type of L chain was not observed due to diffuse staining background. Only a small number of oligoclonal IgG bands in 7 of 10 CSF and serum pairs had identical isoelectric points and the same type of L chain. The results show that the individual MS patient had oligoclonal IgG bands in serum, differ with respect to number, isoelectric point and L chain type from the oligoclonal IgG profile seen in the patient's CSF.     

Spastic paraparesis and sensory disturbance improved by prednisolone therapy]

We reported a 65-year-old man whose sister was suffering from HTLV-I-associated myelopathy (HAM) and who presented slowly progressive spastic paraparesis, sensory disturbance in the feet, tremors and cerebellar ataxia. He was also positive for serum anti-HTLV-I antibody. He first showed a head tremor at the age of 3 years. He developed a spastic and ataxic gait when aged 15 years, and it became difficult for him to walk at the age of 50 years. Examination at 65 years showed a spastic and ataxic gait and scanning speech. Hyper-reflexia and Bahinski's signs were observed. Sensation in the feet was decreased. The anti-HTLV-I antibody titer in the serum was 1:512 by the PA method, and Western blot analysis revealed bands of P19, P24, P28 and P32. Examination of the cerebrospinal fluid (CSF), including oligoclonal bands, gave normal results. The CSF was negative for anti-HTLV-I antibody. CT and MRI of the head showed cerebellar atrophy. His sister was 60 years old. She had developed a spastic gait at the age of 15 years. Sensory defects and bladder dysfunction developed when aged 35 years. Hyper-reflexia, Babinski's sign and foot clonus were observed. Sensation in the feet was decreased. The urinary residual volume was increased. Ataxia was not observed. The anti-HTLV-I antibody titer in the serum was 1:8,192 by the PA method, and Western blot analysis revealed bands of p24, p28 and p32. Examination of the CSF, including oligoclonal bands, gave only normal results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid-responsive myeloneuropathy in a man dually infected with HIV-1 and HTLV-I

Two human retroviruses, HIV-1 and HTLV-I, have been associated with myelopathies in addition to other neurologic disorders. We report an American dually infected with HIV-1 and HTLV-I who developed steroid-responsive myeloneuropathy. This 28-year-old bisexual man developed interstitial pneumonitis and a transient midthoracic sensory level followed by the evolution of a slowly progressive spastic paraparesis and sensorimotor neuropathy. Serologic studies demonstrated coinfection with both HIV-1 and HTLV-I. Peripheral blood absolute CD4 count was persistently within the normal range. Cranial MRI was normal and spinal MRI showed T3-T10 atrophy. Serial CSF analyses demonstrated marked intrathecal synthesis of anti-HTLV-I IgG, lymphocytic pleocytosis, elevated protein and immunoglobulin G, and oligoclonal bands. HIV-1 was isolated from CSF but not from peripheral nerve. Lymphoproliferative studies confirmed spontaneous proliferation in both blood and CSF. Soluble interleukin 2 receptor and soluble CD8 were greatly elevated in blood and CSF when compared with patients with HIV-related vacuolar myelopathy and seronegative patients with other causes of myelopathy. Nerve biopsy showed epi- and endoneurial CD8+ lymphocytic infiltration without vasculitis; muscle biopsy showed features of acute and chronic denervation. A 6-week course of prednisone produced sustained improvement in leg strength and walking times. We speculate that the myeloneuropathy was caused by HTLV-I in the setting of coinfection with HIV-1.     

Agarose isoelectric focusing, antiserum immunofixation and silver staining for detection of oligoclonal bands in unconcentrated cerebrospinal fluid

Unconcentrated cerebrospinal fluid (CSF) and corresponding serum from 30 patients with multiple sclerosis (MS), 30 with other neurological disease and 30 controls suffering from tension headache or psychoneurosis, were examined for oligoclonal IgG bands by initial separation employing agarose isoelectric focusing (AIF) followed by a modified procedure of immunofixation with monospecific antiserum and silver staining. This method is specific for demonstration of IgG and has a limit for detection of 0.4 microgram of IgG. Comparing the results with those obtained by AIF followed by capillary blotting to nitrocellulose membrane, double antibody peroxidase labeling and avidin-biotin amplification, both methods revealed similar frequencies of positive findings for oligoclonal IgG bands in the three patient groups. AIF followed by antiserum immunofixation and silver staining is a simple, sensitive and specific method for detection of oligoclonal IgG in unconcentrated CSF.     

HTLV-I-associated myelopathy endemic in Texas-born residents and isolation of virus from CSF cells

We report three Texas-born patients with spastic paraparesis and well-documented infection with HTLV-I. CSF examination showed moderate pleocytosis, protein elevation, and elevated IgG index. Oligoclonal bands were present in two patients. On MRI, one patient had frontal lobe lesions that were low intensity on T1- and high intensity on T2-weighted images. HTLV-I immunoblot studies of serum and CSF revealed reactivity to p19, p24, p53, gp46, or gp68 from all three patients. Titration studies of serum and CSF antibodies on ELISA and immunoblot assays indicated an intrathecal virus-specific response. HTLV-I-specific p19 antigen capture assay and polymerase chain reaction (PCR) demonstrated HTLV-I in lymphocyte cultures derived from each patient's peripheral blood mononuclear cells (PBMC) or CSF cells. Using HTLV-I- and HTLV-II-specific pol and gag primers, PCR studies of PBMC cells obtained directly from the patients demonstrated that the patients were infected with HTLV-I and not HTLV-II. These three cases are to our knowledge the only US cases in whom virus isolation from the CSF has been accomplished. Importantly, two patients may be the first US cases of myelopathy arising from endemic infection.     

Multiple sclerosis. Electrofocused "bands" of oligoclonal CSF IgG do not carry antibody activity against measles, varicella-zoster or rotaviruses

Electrofocused serum and cerebrospinal fluid (CSF) specimens from patients with multiple sclerosis (MS) were analysed for immunoglobulins (Ig) and for antibodies to measles, varicella-zoster and rotaviruses by an imprint immunofixation method. Patterns of intrathecally synthesized antibodies to the 3 viruses differed from patterns of oligoclonal IgG in the CSF. A variable proportion of virus antibody bands (average 19% for measles antibodies, 8% for varicella-zoster antibodies, 31% for rotavirus antibodies) displayed isoelectric points identical to bands of IgG, but absorption with measles, varicella-zoster and rotavirus antigens produced no change in the bands of IgG and no quantifiable decrease of the CSF IgG. The results confirm previous evidence that the intrathecally synthesized viral antibodies so far demonstrated in MS are not carried by the oligoclonal bands of CSF IgG and account for only a minor fraction of the CSF IgG.     

Oligoclonal IgG bands in cerebrospinal fluid. Principles for demonstration and interpretation based on findings in 1114 neurological patients

Unconcentrated cerebrospinal fluid (CSF) and serum samples from 1114 consecutive patients were examined for presence of oligoclonal IgG bands (OB) by agarose isoelectric focusing (AIF) followed by protein transfer to nitrocellulose membrane, immunolabeling, and avidinbiotin-peroxidase staining (avidin-biotin AIF). Oligoclonal bands were demonstrated in CSF from all 58 patients with multiple sclerosis (MS), eight of 29 with aseptic nervous system infections, and 9% of 1014 with other neurological disorders (OND) considered as noninflammatory at primary clinical evaluation. Comparative examination of all specimens in another laboratory by conventional AIF after concentration of CSF revealed lower frequencies of OB in all diagnostic groups. In addition to the high sensitivity of avidinbiotin AIF, which enables detection of OB by separation of 5 microL of unconcentrated CSF even when the CSF IgG level is around the lower normal range, the procedure also has optimal specificity since IgG exclusively is detected. Avidin-biotin AIF may be the method preferred for routine examination of CSF for OB. Demonstration of OB in CSF is valuable especially in MS, where, in contrast to diagnostic aids such as evoked potentials and neuro-imaging, it establishes inflammatory type of nervous system involvements. Oligoclonal IgG bands in CSF from patients with OND reflect intrathecal immune response and should lead to investigations of infectious etiology.     

HTLV-I-associated myelopathy: oligoclonal immunoglobulin G bands contain anti-HTLV-I p24 antibody

Human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM) and tropical spastic paraparesis belong to a new group of neurological diseases associated with retroviral infection. An HTLV-I-like virus has recently been implicated in multiple sclerosis as well. We studied paired cerebrospinal fluid and serum specimens from HAM and multiple sclerosis patients by isoelectric focusing and an isoelectric focusing HTLV-I p24 overlay technique to clarify the role of HTLV-I in these diseases. We detected oligoclonal bands by isoelectric focusing with silver-staining in cerebrospinal fluid, but not serum, from all 5 HAM and all 9 multiple sclerosis patients. An isoelectric focusing HTLV-I p24 overlay technique demonstrated anti-p24 antibody in HAM cerebrospinal fluid at a different pI distribution than that seen in paired serum, indicating local synthesis of anti-p24 antibody within the central nervous system. Oligoclonal bands in HAM cerebrospinal fluid corresponded in pI distribution to anti-p24 antibody activity, suggesting the presence of an ongoing HTLV-I infection in the central nervous system. Multiple sclerosis patients had no evidence of anti-HTLV-I activity by p24 radioimmunoprecipitation assay, Western immunoblots, or isoelectric focusing HTLV-I p24 overlay analysis. Our data support a role for HTLV-I as an etiological agent in HAM, but not in multiple sclerosis.     

Oligoclonal IgG in multiple sclerosis and subacute sclerosing panencephalitis brains

IgG was obtained from multiple sclerosis (MS) and subacute sclerosing panencephalitis (SSPE) brain materials by elution at neutral and acid pH, and by freezing and thawing. Serum, cerebrospinal fluid (CSF), and brain eluates were compared by isoelectric focussing (IEF) with sensitive peroxidase-anti-IgG staining.In 3 MS cases, different plaques or regions of the same brain had IgG patterns with some common and some different bands. Pooled white matter eluates contained the summation of bands seen in individual plaques or regions. Comparison of serum, CSF, and neutral and acid brain eluate IgG patterns showed many common bands but also unique bands.In two SSPE cases, eluates from different regions of the same brain showed virtually identical IgG patterns. Comparisons of serum, CSF, and neutral and acid eluates of the same brain also showed a common pattern of bands.Similarities in IgG patterns of sera, CSF, and regional brain eluates, from single cases of SSPE, suggest a common response to the same antigen in all regions or compartments. Differences in IgG patterns of sera, CSF, pooled brain eluates, and plaques or regional eluates from single cases of MS, suggest: either that (a) all MS oligoclonal IgG is ‘nonsense’ antibody in terms of disease pathogenesis, or (b) much of the MS oligoclonal IgG is ‘nonsense’ antibody, present as part of a specific oligoclonal immune reaction.     

Two cases of atypical HTLV-I associated myelopathy (HAM)

We report two cases of HTLV-I associated myelopathy (HAM) who showed high HTLV-I antibody titers with clinically atypical neurological symptoms compared with typical HAM originally reported by Osame et al. Case 1 is a 59 year-old-woman who showed Shy-Drager syndrome-like symptoms such as a slowly progressive gait disturbance, pyramidal and extra-pyramidal symptoms, an orthostatic hypotension and a sweating disturbance. The anti HTLV-I antibody titer was highly positive in both her serum and cerebrospinal fluid (CSF), and there was also a high level of oligoclonal immunoglobulin in her CSF. These symptoms improved slightly with steroid therapy. Therefore, it was suspected that this neurological condition was associated with HTLV-I, which means that HTLV-I can be associated not only with myelopathy but also with various other neurological symptoms. The second case is a 52-year-old woman who had a myelopathy with a slowly progressive course. She had suffered from a transient optic neuritis 5 years before admission that had improved completely with steroid therapy. She had highly positive anti HTLV-I antibody in both her serum and CSF, and also showed a high level of oligoclonal immunoglobulin in her CSF. With administration of steroids, the sensory disturbances and abnormal findings in the CSF improved slightly. Koprowski et al reported that in some MS patients they found positive anti HTLV-I antibody and furthermore proved the presence of CSF cells which hybridized with a HTLV-I probe. They suggested the presence of an unknown HTLV-related agent which may be a pathogenic factor in some subtypes of MS. The transient optic neuritis responding to steroid therapy and the following transverse myelopathy, as seen in case 2, are highly characteristic of MS. Thus, some clinical features of HAM may be very similar to MS.     

Immunoblot detection of oligoclonal anti-myelin basic protein IgG antibodies in cerebrospinal fluid in multiple sclerosis

Migration properties and occurrence of antibodies against myelin basic protein (MBP) in paired CSF and serum specimens from patients with multiple sclerosis (MS) were demonstrated after agarose isoelectric focusing, immunoblot transfer, and immunoperoxidase staining. Oligoclonal IgG antibody bands directed against MBP were found in the CSF of 9 of 28 patients with MS (32%), but not in the CSF of any of 34 patients with other neurologic diseases. No serum showed anti-MBP antibody bands. The CSF anti-MBP antibodies migrated to the anodal region of the IgG area in a different fashion from oligoclonal IgG and anti-measles IgG antibodies, which were detected in parallel. The anti-MBP bands were transient in three of seven patients whom we studied consecutively. Enzyme-linked immunosorbent assay (ELISA) of serum and CSF for detection of IgG reactivity against MBP showed absorbance values above 2 standard deviations of controls in 44% of the MS patients and in 21% of those with other neurologic diseases. Results of this assay correlated partly with those of the immunoblot assay. ELISA positive and immunoblot negative results might be due to a broad polyclonal anti-MBP antibody response.     

Qualitative evidence of anti-Yo-specific intrathecal antibody synthesis in patients with paraneoplastic cerebellar degeneration

We investigated the presence of anti-Yo-specific oligoclonal antibody bands in cerebrospinal fluid (CSF) and serum samples of 9 patients with anti-Yo syndrome and 11 controls. Isoelectric focusing combined with affinity blotting, revealed anti-Yo-specific intrathecal antibody synthesis in all patients with anti-Yo syndrome: Four patients had positive anti-Yo-specific oligoclonal IgG bands in CSF which were not demonstrable in their sera; five CSF/serum pairs showed additional, more intensive, oligoclonal bands in CSF compared to the corresponding serum. Interestingly, four patients with absence of oligoclonal bands of total IgG in CSF revealed positive anti-Yo-specific oligoclonal bands in the same sample. This speaks for a higher sensitivity of detection of oligoclonal bands using an affinity blot loaded with Yo-specific antigen compared to an affinity blot coated with anti-human IgG used for the detection of oligoclonal bands of total IgG. In conclusion, the presence of anti-Yo-specific oligoclonal IgG bands in CSF which were absent, or less strong, in patients sera provides qualitative evidence of anti-Yo-specific IgG synthesis by intrathecal B-cell clones. These results could be of interest in detection of intrathecal-specific IgG synthesis in nervous system infectious diseases provided that the target antigen is known.     

Immunoglobulin abnormalities in the cerebrospinal fluid during bacterial meningitis

11 patients with bacterial meningitis, examined during the course of the disease for immunoglobulin (Ig) abnormalities in the cerebrospinal fluid (CSF), all had an increased CSF IgM index equal to (CSF/serum IgM):(CSF/serum albumin), indicating intrathecal IgM production. Seven patients had a slightly increased CSF IgG index, and 7 a slightly increased IgA index. Six of the 11 patients had an increased IgM index in the presence of normal indices for IgG and IgA. Follow-up revealed the return of these values to normal. Four patients had identical oligoclonal IgG bands in the CSF and serum, probably representing a systemic immune response, but in only one case were oligoclonal bands suggestive of intrathecal IgG production found. No oligoclonal IgA response was demonstrable in the 4 patients examined. Antigen-immunofixation or antigen-absorption studies revealed evidence of a specific, intrathecal IgG antibody response in only 2 patients, while a search for IgG antibodies against aetiologically unrelated bacterial and viral antigens was negative. With the exception of IgM production, therefore, a humoral intrathecal immune response is less common in bacterial than in aseptic meningitis.     

GAD65 IgG autoantibodies in stiff person syndrome: clonality, avidity and persistence

Background and purpose:  Persistent intrathecal production of IgG autoantibodies against glutamic acid decarboxylase 65 (GAD65 IgG) and oligoclonal IgG of undetermined specificity has been reported in stiff person syndrome (SPS).
Methods:  To chart the avidity and clonal patterns of GAD65 IgG, we performed scatchard plot of binding characteristics and isoelectric focusing-immunoblot of cerebrospinal fluid (CSF) and serum from five SPS patients.
Results:  Oligoclonal GAD65 IgG bands, predominantly restricted to the IgG1 subclass, were detected in CSF and serum in all patients. The distribution of GAD65-specific IgG bands in serum and CSF revealed intrathecal synthesis of oligoclonal GAD65 IgG in all five patients, whilst radioimmunoassay demonstrated intrathecal synthesis of GAD65 IgG in four. The binding avidity of GAD65 IgG from CSF was more than 10 times higher than in serum in two of the patients but did not differ substantially in the remaining three. These differences were not related to symptom severity. The pattern of oligoclonal GAD65 IgG bands in CSF and serum in three patients examined remained unchanged for up to 7 years after symptom debut.
Conclusion:  This study confirms the persistent systemic and intrathecal production of GAD65-specific IgG in SPS, and further shows that this immune response is oligoclonal and mediated by a stable population of affinity maturated B cell clones.     

Pattern and concentration of IgG in cerebrospinal fluid in neurosarcoidosis

Reports have suggested that the pattern of CSF IgG differentiates neurosarcoidosis from multiple sclerosis. We examined CSF and serum of 7 patients with neurosarcoidosis to determine concentrations of IgG and albumin and the presence of oligoclonal bands. Our results showed that neurosarcoidosis may have associated abnormalities of IgG synthesis and oligoclonal bands present in CSF, but without a consistent pattern.     

Intrathecal production of oligoclonal IgM and IgG in CNS sarcoidosis

A longitudinal study of multiple paired CSF and serum specimens from a patient with CNS sarcoidosis revealed high CSF IgM and IgG indices as well as oligoclonal IgM and IgG bands in CSF reflecting intrathecal IgM and IgG production. The antibody specificity of intrathecally-produced IgM and IgG remained undefined despite analysis for antibodies against mycobacterium tuberculosis and Kveim suspension. Steroid treatment induced rapid and complete clinical remission, and also decrease of CSF IgM and IgG antibodies, while oligoclonal IgM and IgG persisted in CSF. Repeated determinations of these CSF variables together with cell count and CSF/serum albumin ratio as a variable of blood-brain barrier function, might be useful in assessing effect of therapy in CNS sarcoidosis.     

Antigen-specific oligoclonal IgG in AIDS-related cytomegalovirus and toxoplasma encephalitis

Objective — We retrospectively studied serum and cerebrospinal fluid (CSF) specimens from AIDS patients with either Cytomegalovirus (2 cases) or Toxoplasma gondii (5 cases) encephalitis. The samples, which had previously proved to be negative for total IgG oligoclonal bands (OCBs), were investigated for antigen-specific OCBs directed to the disease-related opportunistic agent. Material & methods — Paired serum and CSF samples from the given AIDS patients were considered. We undertook affinity immunoblotting of either virus- or protozoan-specific IgG onto antigen-coated nitrocellulose paper after protein separation by agarose isoelectric focusing (IEF). Results — Antigen-specific OCBs to the disease-related opportunistic agent were detected in serum and in CSF samples from all the patients. Conclusions — During overt AIDS, routine IEF methods may fail to detect OCBs, probably because nonspecific polyclonal hypergammaglobulinemia, which is typical of this disease, reduces their visibility. Our IEF/immunoblotting profiles are characterized by identical serum and CSF bands. The detection of antigen-specific OCBs may support the diagnosis of some opportunistic infections of the central nervous system in AIDS.     

Intra-blood-brain barrier IgG synthesis in cerebral cysticercosis

Patients with central nervous system cysticercosis show elevated binding of cerebrospinal fluid (CSF) IgG to homogenized cysticercus. To determine whether any of the CSF IgG was the result of de novo intra-blood-brain barrier (BBB) synthesis, CSF and serum samples from six patients were examined for elevated rate of synthesis and oligoclonal bands. Five of the six patients had increased intra-BBB IgG synthesis rate and four of these patients also had oligoclonal IgG bands present in the CSF that were absent in the serum. These results demonstrate intra-BBB IgG synthesis similar to that observed in other infectious and inflammatory diseases of the central nervous system.