Multiple sclerosis de novo CNS IgG synthesis. Effect of CNS irradiation

Megavoltage CNS irradiation was given to 20 patients with clinically definite multiple sclerosis (MS) to determine if de novo CNS IgG synthesis could be eradicated. In all five patients given 1,200 rads, a transient reduction in the de novo CNS IgG synthesis rate was noted. In ten patients given 1,800 rads, the following occurred: a reduction in synthesis rate in three patients, a reduction followed by enhancement in two, only enhancement in four, and no change in one. In all five additional patients, a therapy of adrenocorticotropic hormone (ACTH) followed by prednisone in combination with 1,800 rads produced greater and more persistent decreases in CNS IgG synthesis, but did not block the enhancement effect. Only two of 19 patients who had abnormal CNS IgG synthesis rates had reductions to normal; no patients showed changes in the number or pattern CSF IgG oligoclones. Hence, no treatment eradicated de novo CNS IgG synthesis. A persistent decrease in CSF leukocytes occurred in all 20 patients due to the reduction of small lymphocytes (not dose related). The blood-brain-barrier to albumin concentration was transiently damaged in 11 of 15 patients given irradiation, but when patients were premedicated with ACTH/prednisone therapy, no damage was found. None of the patients demonstrated neurological improvement, change in the activity of their disease, or persistent adverse effects.     

Free light chains in the CSF in multiple sclerosis

Summary The presence of free light chains (FLC) was investigated in 32 patients with clinically definite or laboratory supported definite multiple sclerosis (MS), 2 patients with neurosyphilis and 10 normal controls. The detection of FLC in unconcentrated cerebrospinal fluid (CSF) was performed by means of agarose isoelectric focusing, followed by transfer of proteins to nitrocellulose membranes, double immunofixation, avidin-biotin amplification and peroxidase staining. Bands due to FLC were clearly demonstrated in the CSF of 28 MS patients; 3 of them showed only kappa FLC, 10 only lambda FLC, while 15 had both kappa and lambda FLC. The CSF of 4 MS patients was FLC negative. In both cases of neurosyphilis FLC bands were observed. FLC were never found in normal CSF. Among the indexes of intrathecal immunological activity (IgG oligoclonal bands, FLC, IgG index, intra-blood-brain barrier IgG synthesis rate, pleocytosis) the FLC proved to be the second most frequent abnormality in MS CSF, the presence of IgG oligoclonal bands being the first. In one MS case an FLC band was found, while all the other indexes of intrathecal IgG production were negative. A high correlation was found between an elevated number of FLC and pleocytosis. The presence of FLC in MS CSF seems to indicate a recent immunological stimulation leading to increased synthesis of FLC within the CNS.     

Comparison between agarose gel electrophoresis and isoelectric focusing of CSF for demonstration of oligoclonal immunoglobulin bands in neurological disorders

Isoelectric focusing and agarose gel electrophoresis of CSF and serum revealed similar frequencies of oligoclonal bands in multiple sclerosis (100% with both methods), infectious CNS disorders (38 and 23%) and other neurological diseases (8 and 10%). In selected cases with unsure CSF oligoclonal bands on agarose gel electrophoresis, isoelectric focusing displayed definite oligoclonal bands. In contrast to agarose gel electrophoresis, isoelectric focusing revealed evidence for oligoclonal bands in serum as well as in CSF in 41% of the multiple sclerosis patients, indicating diffusion from CSF to serum. In 4 cases with gammaglobulin bands appearing in both CSF and serum on agarose gel electrophoresis, isoelectric focusing revealed normal CSF and serum protein patterns at pH above 6.4 where most IgG is migrating.     

Immunoglobulin class and light chain type of oligoclonal bands in CSF in multiple sclerosis determined by agarose gel electrophoresis and immunofixation.

Agarose gel electrophoresis and immunofixation of CSF and serum from 39 patients with multiple sclerosis (MS) revealed oligoclonal IgG in the CSF in all cases and oligoclonal IgA and IgM in 1 patient each. IgG kappa bands only were found in 10 patients, while no patient had IgG lambda bands alone. IgG kappa bands predominated in 20 patients and IgG lambda bands in 5, while 4 patients had the same number of IgG kappa and IgG lambda bands. Twenty-seven patients also displayed IgG bands with kappa and lambda present simultaneously. Bands of free lambda chains were found in 7 patients, while free kappa chain bands were not seen. One or 2 faint IgG bands in 4 patients constituted the only serum abnormality. In 4 additional MS patients selected on the basis of normal findings on agarose gel electrophoresis of the CSF, immunofixation did not reveal oligoclonal Ig, while isoelectric focusing showed bands in 1. Immunofixation is recommended for proving the presence of oligoclonal Ig in CSF and for characterizing oligoclonal Ig into classes and types of light chains.     

Oligoclonal IgG in tears

We examined tears from patients with MS and systemic or eye diseases, and normal controls. Tears were isoelectrofocused on agarose gel, silver-stained, and immunofixated for IgG. In the cathodal portion of the gel (pH greater than or equal to 8.3), oligoclonal bands were detected in 14 of 21 (67%) MS tear samples. In most cases, these bands were not present in serum. Only 1 of 26 non-MS subjects showed two faint IgG tear bands, which were strongly present in serum. It appears that oligoclonal IgG bands can be detected in tears as well as in CSF of MS patients.     

The use and abuse of the cerebrospinal fluid IgG profile in the adult: A practical evaluation

Cerebrospinal fluid (CSF) samples from 150 patients with multiple sclerosis (MS) and 190 patients with other neurological disorders were examined with a battery of tests (the IgG profile) to detect quantitative and qualitative abnormalities in CSF IgG. The CSF IgG profile consisted of an IgG/albumin (IgG/alb) ratio, an IgG index, and examination by agarose gel electrophoresis (AGE) and isoelectric focusing (IF) to detect oligoclonal bands. The tests were compared for diagnostic accuracy, including false-positive results. The IgG/alb ratio was less reliable in confirming the diagnosis of MS, but no significant difference in accuracy was found among the other three methods. IF tended to identify more possible and probable MS cases than did AGE but gave a higher rate of false-positives. The IgG index and IgG synthesis rates showed no significant difference in their ability to identify MS patients. Steroid administration decreased the incidence of abnormal IgG/alb ratios and IgG indices, but not abnormal oligoclonal bands. Central nervous system (CNS) infections or immunological diseases involving the CNS produced a 28 to 40% incidence of abnormalities in the CSF. Neither the patient's age, sex, duration of illness, activity of disease, nor longitudinal course correlated with the CSF findings. A few (1%) control neurological patients had all components in the CSF IgG profile abnormal. For most routine clinical purposes the IgG index and AGE are sufficient for confirmation of diagnosis, and the IgG index was the best single test in our series.     

Clinical and CSF findings in multiple sclerosis patients with or without IgG oligoclonal bands at isoelectric focusing examination of CSF and serum proteins

The IgG oligoclonal bands in CSF can be found in high percentage of MS patients but the existence of a limited number of cases with CSF normal IgG profile is known as well. In the present study 63 out of 70 patients with definite MS and 24 out of 35 with probable MS had oligoclonal bands in the CSF at the isoelectric focusing examination. The 18 patients with normal CSF IgG pattern did not show any statistically significant difference as concerns the age at onset and the duration of the disease, the functional disability, the course of the disease and the quantitative CSF parameters (IgG index, IgG synthesis and serum/CSF albumin quotient). The oligoclonal pattern does not seem of value to discriminate different groups of MS patients but it remains essentially of great diagnostic importance in this disease.     

Comparison of agar gel electrophoresis and isoelectric focusing in multiple sclerosis and subacute sclerosing panencephalitis

Isoelectric focusing (IEF) and agar gel electrophoresis (AGE) were used to examine cerebrospinal fluid (CSF) and sera from patients with multiple sclerosis (MS) and subacute sclerosing panencephalitis (SSPE). All 15 SSPE samples and 29 of the 33 MS CSF samples showed oligoclonal IgG bands by AGE and IEF. Serum bands were more frequent in SSPE than in MS and were more commonly detected by IEF than by AGE. In MS CSF the number of bands on IEF correlated with: (1) disease duration, (2) CSF IgG, (3) CSF IgG/albumin ratio, and (4) central nervous system IgG synthesis. Serial studies revealed increases in IEF band number in 4 of 10 MS and 3 of 5 SSPE CSF specimens; fluctuations in band intensity were also noted. Densitometric scans of CSF IEF gels showed high, sharply angled IgG band peaks in SSPE; the CSF band peaks in MS were flatter and had higher background IgG. The SSPE pattern could be made to resemble the MS pattern through addition of normal polyclonal IgG to SSPE CSF. These findings suggest that in addition to oligoclonal IgG, polyclonal IgG is synthesized locally within the central nervous system in MS.     

Identification of IgG subclasses'' oligoclonal bands in multiple sclerosis CSF

IgG subclasses' oligoclonal bands in unconcentrated CSF from MS patients were detected by isoelectric focusing in agarose gel with subsequent immunoblotting using mouse monoclonal antibodies to human IgG subclasses and double-antibody avidin-biotin-alkaline phosphatase system. All MS CSF showed presence of oligoclonal bands specific to the IgG1 subclass; in addition, several of these samples also had oligoclonal bands specific to IgG3, IgG2, or IgG4, in order of decreasing frequency. Since the CSF of a greater number of MS patients showed oligoclonal bands specific to the IgG1 and IgG3 subclasses, the findings are consistent with those reported in patients with chronic viral infections and autoimmune diseases.     

Detection of oligoclonal IgG bands in unconcentrated CSF in multiple sclerosis and other neurological diseases by isoelectric focusing on ultrathin-layer polyacrylamide gel immunofixation and silver staining

Isoelectric focusing of proteins (IEF) in ultrathin-layer polyacrylamide gel (0.4 mm, PAG), followed by direct immunofixation with monospecific antiserum and silver staining, is a highly specific, sensitive and simple method for the demonstration of oligoclonal IgG in unconcentrated cerebrospinal fluid (CSF) samples (5-10 microliters). For the present method, the optimal concentrations of IgG in CSF samples are about 0.025-0.030 g/l, corresponding to the applied amount of 125-150 mg. In our testing of this method, oligoclonal IgG bands in CSF specimens were clearly demonstrated in 52 (96%) of 54 patients with clinically established definite diagnosis of multiple sclerosis (MS), in 4 (40%) of 10 patients with infectious diseases of the CNS, and in 9 patients (25%) of 38 with other neurological diseases. Abnormal patterns were also demonstrated in the serum of patients with MS (43%). Intrathecally synthesized IgG was mathematically calculated in 43 (80%) out of 54 patients with MS. This method appears to be a useful alternative for the demonstration of oligoclonal IgG bands in the unconcentrated CSF, especially when questionable or negative results arise by routine electrophoretic technique for oligoclonal bands detection.     

IgG synthesis rate in evaluation of multiple sclerosis in a community hospital

We obtained CSF and serum from 21 patients with definite MS in a community hospital. Ninety percent of patients had an elevated IgG synthesis rate, comparable with the sensitivity of oligoclonal bands (0.95) or the IgG index (0.90) and better than the IgG% (0.47) in definite MS. We found that no area hospital routinely employs the IgG synthesis rate. The low-sensitivity IgG% and the dimensionless IgG index can be abandoned. The linear IgG synthesis rate and oligoclonal band scanning should be the CSF tests of choice.     

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.     

Oligoclonal free kappa and lambda bands in the cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases. An immunoaffinity-mediated capillary blot study.

We describe an affinity-mediated capillary blotting technique for the detection of free kappa or lambda light chains in native cerebrospinal fluid (CSF) after isoelectric focusing in agarose gel. Interferences by light chains bound to immunoglobulins were carefully excluded. An absolute amount of 20-50 ng of free kappa or lambda Bence-Jones proteins were detectable by this method, under the form of several discrete bands with isoelectric points between 5 and 8.5. No free light chains were observed in CSF and sera from patients without neurological disorders (n = 26). Such bands were present in most CSF samples in the case of central nervous system (CNS) infections, except in aseptic meningitis. In a group of 48 multiple sclerosis (MS) patients, 44 (92%) displayed oligoclonal free kappa bands restricted to the CSF; oligoclonal IgG bands were observed in 40 cases, and oligoclonal free lambda bands in 33. In this group, the presence of CSF free light chain bands was highly correlated with their absolute levels (p less than 0.001). In other neurological diseases (n = 44), oligoclonal free kappa and free lambda bands were detected much more rarely, in seven (16%) and four (9%) cases respectively. Surprisingly, the CSF from three unrelated patients with Huntington's disease (out of five tested) contained both oligoclonal IgG and free kappa bands.     

Short communication CNS tumours: oligoclonal immunoglobulin D in cerebrospinal fluid and serum

Oligoclonal immunoglobulin D bands were detected by isoelectric focusing in 7 out of 25 unconcentrated cerebrospinal fluid (CSF) samples obtained from patients with tumours of the central nervous system (CNS). The tumours were confirmed by clinical and histological findings. Two patients with CNS malignancy had intrathecal synthesis of oligoclonal bands both IgD and IgG. Four patients with a variety of CNS tumours had a systemic IgD immune response but no oligoclonal IgG bands. One patient with the most malignant tumour histology had a systemic IgD response as well as local synthesis of IgG. The study reveals several new aspects regarding CNS tumours: they are immunologically active and are capable of invoking oligoclonal immunoglobulin production both within the CNS and systemically. Multiple immunoglobulin activation can be found in malignant CNS tumours, and systemic IgD production may occur independently from IgG synthesis and may represent an immune response to a neoantigen produced in the CNS compartment.     

The influence of high-dose prednisone medication on autoantibody specific activity and on circulation immune complex level in cerebrospinal fluid of multiple sclerosis patients

In agreement with the close correlation between intrathecal IgG production and anti-MBP (myelin basic protein) and anti-MAG (myelin-associated glycoprotein) antibody activity in the CSF of active MS cases, and parallel to the reduction of intrathecal IgG synthesis resulting from corticosteroids medication, we have found a significant reduction of anti-MBP and anti-MAG antibody activity expressed per 0.5 micrograms of CSF IgG in the same group of 40 MS patients subjected to high-dose prednisone therapy. Every patient received 3980 mg of prednisone over 54 days. In native CSF of 30% (21/70) of active MS cases, circulating immune complexes (CIC) were detected by C1q binding solid-phase RIA. There was no correlation between CIC level in the CSF or MS patients and 1. IgG index which was used as an indicator of intrathecal IgG synthesis, or 2. CSF anti-MBP specific antibody activity, or 3. CSF anti-MAG specific antibody activity. High-dose prednisone therapy resulted in a highly significant reduction of the CSF CIC level. CIC were also found in the CSF of patients affected with various chronic diseases of the CNS.     

Oligoclonal antibodies in the diagnosis of multiple sclerosis and other diseases of the nervous system

The detection of CSF oligoclonal IgG is one of the most useful laboratory tests to aid in the diagnosis of multiple sclerosis. Oligoclonal IgG is synthesized within the CNS and is usually detected in the CSF by agar and agarose gel electrophoresis or by isoelectric focusing. Oligoclonal IgG is present in about 90-95% in patients with clinically definite MS. There is a predominance of kappa light chains and IgG1 and IgG3 subclasses in oligoclonal IgG in MS. The specificity of oligoclonal IgG in this disease is still unknown, as antibodies, for instance against measles virus or myelin basic protein, represent only a minimal part of intrathecally synthesized IgG.     

An isoelectric focusing overlay study of the humoral immune response in Theiler's virus demyelinating disease

Oligoclonal immunoglobulin G (IgG) bands are a frequent feature of inflammatory diseases of the central nervous system (CNS). In multiple sclerosis (MS), cerebrospinal fluid (CSF) oligoclonal IgG bands are a potential clue to the pathogenesis of the disease; however, their particular antigenic target is unknown. We sought to characterize the IgG response in an experimental CNS persistent demyelinating infection by isoelectric focusing (IEF) studies of serum and CSF from mice infected with Theiler's murine encephalomyelitis virus (TMEV). Following IEF, we used a new technique in order to identify TMEV-specific antibodies; focused immunoglobulins were blotted onto nitrocellulose paper which was then overlaid with radiolabeled virus. Autoradiograms showed that most of the TMEV antibody was locally synthesized within the CNS since CSF, but not serum, TMEV antibody had an anodal distribution. CSF IEF TMEV antibody spectrotypes were very similar, presumably because the CSFs were collected from the same inbred mouse strain. CSF TMEV antibody displayed less restricted heterogeneity than the very restricted cathodal CSF oligoclonal IgG bands seen in MS. The new IEF immunoblotting antigen overlay technique will be a powerful detection system to probe for the antigenic target against which MS CSF IgG may be directed.     

The significance of abnormal immune responses in patients with multiple sclerosis

Early diagnosis of multiple sclerosis (MS) may be assisted by tests for the abnormal immune responses of the central nervous system (CNS) including oligoclonal IgG bands in the cerebrospinal fluid (CSF), increased CNS IgG synthesis, increased CNS antibody synthesis against multiple viruses and increased numbers of enlarged lymphoid cells in the CSF. Alterations in immunological responses are important in the pathogenesis of MS. Further studies are needed, however, to identify the antigen(s) and/or antibodies responsible for oligoclonal IgG in the CSF of MS patients. Also, the cause(s) for the other immunological abnormalities with diagnostic importance need to be identified. The increased synthesis of antibodies against multiple unrelated viruses suggests generalized alteration in the immune regulatory system. The etiology of MS might be multifactorial involving abnormal immunological responses, possibly precipitated by infectious agents acquired during childhood by genetically susceptible individuals. The immunological responses including alterations in myelin basic protein concentration, antimyelin antibody and immune complex activities in CSF, and in vitro stimulation, suppression and migration inhibition of blood lymphocytes appear to correlate with stage of MS and severity of CNS damage. Some of the tests may become useful in estimating the prognosis of the disease. Longitudinal studies are needed to clarify the sensitivity of the diagnostic and prognostic immunological tests and etiological significance of these abnormalities in MS.     

Oligoclonal IgG and free chains in multiple sclerosis demonstrated by thin-layer polyacrylamide gel isoelectric focusing and immunofixation

A modified technique of isoelectric focusing on thin-layer polyacrylamide gel followed by immunofixation with monospecific antisera was used to identify individual cerebrospinal fluid (CSF) and serum proteins and to define the oligoclonal reaction observed in multiple sclerosis (MS). "Normal" IgG gave about 20 to 30 bands at pH 3.5 to 9.5, IgA about 10 bands at pH 3.5 to 6.4, beta-trace protein a smear at pH 3.5 to 8.5, and gamma-trace protein 1 or 2 bands at pH 8.0, 9.5 or both. Up to 11 oligoclonal IgG bands migrating between pH 6.5 and 9.5 were found in CSF from 26 of 27 consecutive patients with MS and also in 20 of the corresponding sera, although at lower numbers and concentrations. In 26 patients, 1 or more of the bands corresponding to normal polyclonal IgG were stronger in CSF than in serum. These data support the hypothesis that two colonies of lymphocytes are activated intrathecally, one of them synthesizing oligoclonal and the other polyclonal IgG. Up to 11 mostly faint bands of free light chains, predominantly of lambda type and migrating between pH 3.5 and 9.5, were found in 8 of 9 CSF specimens from patients with MS.     

Measles virus antibodies in serum and cerebrospinal fluid in patients with multiple sclerosis and other neurological disorders,with special reference to measles antibody synthesis within the central nervous system

Measles virus hemagglutination-inhibiting (HI) and gel precipitating (GP) antibodies were determined in sera and cerebrospinal fluids (CSF) from 65 patients with multiple sclerosis (MS) and 65 patients with other neurological diseases. The serological results were correlated to content of immunoglobulin-G (IgG) and electrophoretic patterns of sera and CSF.Measles GP antibodies, identified as directed against measles virus ribonucleoprotein antigens, were detected in sera and in CSF from a significantly higher proportion of MS than of non-MS patients. No significant difference between the 2 groups of patients was found for measles HI antibodies.Reduced serum/CSF HI and/or GP antibody ratios were found in about one half of the MS patients and in 2 patients with chronic myelopathy. All patients with reduced antibody ratios had evidence of IgG synthesis within the central nervous system (CNS), as inferred from oligoclonal IgG patterns of the CSF. Reduced ratios of measles GP antibodies were 3 times as common as reduced ratios of HI antibodies. Immuno-electrophoretic assays indicated that the CSF GP antibodies were electrophoretically restricted in a number of MS patients.The results indicate that measles virus may be an active immunogen within the CNS in many MS patients and in some patients with chronic myelopathy, giving rise to an oligoclonal IgG antibody response.