Frequency and relevance of IgM intrathecal synthesis in multiple sclerosis

Using a new ELISA method we have measured the IgM concentration in the serum and the cerebrospinal fluid CSF from 110 neurological patients. Among there, 41 had multiple sclerosis (MS), 48 other inflammatory diseases (OID), including 30 AIDS, and 21 non-inflammatory neurological diseases (NID). A highly significant correlation was established between results with native IgM and the dithiothreitol reduced IgM. An intrathecal synthesis (ITS) of IgM was detected using the CSF IgM/CSF albumin ratio, the IgM index and a quantitative formula in 33 patients: nine MS, 23 OID (including 18 AIDS) and one NID. The frequency of IgM ITS was 22% in MS patients, 48% in the OID (60% in AIDS) and 5% in the NID groups. This ITS was not impaired by an increase in serum IgM concentration or by a blood–CSF barrier damage. These facts confirm that intrathecal immunity is not a “steady-state” related to the general immunity but a specific response restricted to the central nervous system. Conversely, CSF IgM increase and IgM ITS were closely related (p < 10^?6). In addition, IgM ITS and IgG ITS were found to be highly correlated in OID, especially in AIDS patients: such correlation was not observed in the MS group. No significant correlations were observed between IgM ITS and any of the clinical parameters in MS patients. These results suggest the probable specificity of IgM ITS in MS patients.     

Anti-herpes simplex type 1 activity in IgG subclasses produced systemically and intrathecally in patients with herpes encephalitis

Summary The role of the humoral immune response in herpes simplex encephalitis (HSE) is largely unknown. The finding that herpes simplex virus type 1 (HSV 1) induced IgG Fc receptor binds to all IgG subclasses except IgG 3 prompted an investigation of anti-HSV activity in IgG subclasses from serum and cerebrospinal fluid (CSF) in ten patients with proven or highly probable HSE by means of a monoclonal antibody IgG subclass-specific solid-phase radioimmunoassay (SPRIA). In contrast to serum, CSF contained no or low anti-HSV IgG titres during the first 2 weeks of disease in five of seven patients tested. The IgG titres rose thereafter for at least 4 weeks after the start of illness and remained high in both serum and CSF up to 393 days. The anti-HSV IgG subclass distribution in serum was IgG 1 (ten of ten), IgG 2 (two of ten), IgG 3 (six of ten), and IgG 4 (six of ten). Two patients had a simultaneous anti-HSV IgG 3 and IgG 4 response. With the exception of one patient lacking anti-HSV IgG 4 and two patients lacking anti-HSV IgG 2, the subclass distribution in CSF was the same as in serum. The anti-HSV subclass distribution in sera from ten seropositive patients without evidence of recent herpes infection did not differ from that of the HSE patients, except that five of ten patients had simultaneous anti-HSV IgG 3 and IgG 4 responses. Thus we could not correlate the anti-HSV subclass response in patients with HSE with the subclass preference of the HSV-induced Fc receptor.     

Features of intrathecal immunoglobulins in patients with multiple sclerosis

We have analyzed immunoglobulin (Ig) isotypes and IgG subclasses in cerebrospinal fluid (CSF) and serum of patients with multiple sclerosis (MS) and other neurological diseases to determine whether different Ig isotype patterns correlate with clinical or paraclinical findings and CSF B cell populations. Intrathecal IgG1 synthesis was elevated in MS patients. An increased intrathecal IgM production was found in patients with a higher cerebral MRI lesion burden, whereas other clinical and paraclinical parameters were not associated with a specific Ig isotype or subclass profile. Finally, intrathecal IgG production (IgG1 and IgG3) correlated with the presence of mature B cells and plasma blasts.     

The effect of large-dose prednisone therapy on IgG subclasses in multiple sclerosis

The effect of large-dose prednisone therapy (3960 mg over 56 days) on IgG subclasses in the cerebrospinal fluid and sera, as well as on their intrathecal synthesis, was studied in 15 patients with clinically definite multiple sclerosis. The concentration of IgG subclasses was measured using ELISA with monoclonal antibodies against human IgG subclasses, secondary biotinylated antibody and avidin-biotin-peroxidase complex. There was a decrease of IgG1, IgG3 and IgG4 in the CSF of MS patients after the treatment, but the differences did not reach statistical significance. The IgG index was decreased about 34% (p<0.01) after the therapy. This was mainly due to diminished synthesis of IgG1 and IgG3. The significance of IgG subclasses in the pathogenesis of MS is discussed.     

IgG1-IgG4 subclasses in the cerebrospinal fluid and blood serum and their synthesis in the central nervous system in multiple sclerosis]

IgG1, IgG2, IgG3 and IgG4 subclasses were detected in cerebrospinal fluid and sera from 32 patients with MS. The CSF levels of IgG1 and IgG3 subclasses in MS patients were significantly higher than those of controls. There were no statistical differences between the values of IgG subclasses in MS and control sera. The IgG1 and IgG3 indices were significantly higher in MS patients than in controls showing that among four IgG subclasses IgG1 ang IgG3 were synthesized in the central nervous system of patients with MS. The elevation of IgG1 and IgG3 indices in MS patients was found more frequently (in about 90% of patients) than the elevation of the general IgG index (in 72% of patients).     

The study of anti-oligodendrocyte antibody in sera, and CSF of patients with multiple sclerosis

We studied anti-oligodendrocyte antibody in sera and CSF from patients with multiple sclerosis (MS) and other neurological diseases (OND) by enzyme-linked immunosorbent assay (ELISA). Oligodendrocytes were isolated by percoll density gradient from brains of 4 week-old rat and cultured in poly-1-lysine-coated 96 well microwell plate. After overnight culture, oligodendrocytes were fixed in 0.5% glutaraldehyde and stored at -20 degrees C. ELISA was performed using peroxidase-conjugated goat anti-human IgG (Fab')2 as usual. Serum and CSF were examined at dilution of 1:400 and 1:2 respectively, and O.D. was read at 490 nM. In sera from patients with MS and OND, the titer of anti-oligodendrocyte antibody was significantly higher than those from normal controls. However, there was no significant difference between MS and OND. Significantly higher titer of anti-oligodendrocyte antibody was observed in CSF from patients with meningoencephalitis and polyradiculoneuropathy. However, comparing CSF anti-oligodendrocyte antibody per CSF IgG, there was no significant difference among each group. There was no significant correlation between the cytotoxicity index of sera and anti-oligodendrocyte antibody level. There might be additional cytotoxic factor other than anti-oligodendrocyte antibody. Our data support the idea anti-oligodendrocyte antibody is not specific to MS, and the role of anti-oligodendrocyte antibody in the pathogenesis of MS is secondary.     

IgG subclasses and their intrathecal synthesis in patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis may be an autoimmune disease. In this paper IgG subclasses levels in the CSF and sera and their intrathecal synthesis were studied. IgG subclasses levels were determined by ELISA method using monoclonal antibodies against human IgG subclasses, secondary biotinylated antibody and avidin-biotin-peroxidase complex. There was statistically significant elevation of IgG1 and IgG3 subclasses in the CSF of ALS patients. In sera of patients with ALS, IgG2 level was diminished, but there was no statistical difference in other IgG subclasses. IgG1 and IgG3 indices were elevated in patients with ALS, detecting synthesis of these subclasses in the CNS. General IgG index value did not differ from the control value. The results support the concept that autoimmune mechanisms may play a role in the pathogenesis of ALS.     

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.     

Absence of IgG1 response in the cerebrospinal fluid of relapsing neuromyelitis optica

The authors studied immunoglobulin (Ig) G subclasses in the CSF and sera of patients with relapsing neuromyelitis optica (RNMO) and typical multiple sclerosis (MS). Although the total IgG concentrations were elevated in the CSF of patients with RNMO and MS, IgG1% and IgG1 index were significantly elevated only in patients with MS. The absence of the CSF IgG1 responses in the patients with RNMO may suggest less Th1 immunity and may also explain the rarity of oligoclonal IgG bands in patients with this disease.     

Specific in vitro IgG subclass synthesis and lymphocyte proliferation responses in herpes virus encephalitis

Cerebrospinal fluid, peripheral blood lymphocytes (PBL) and sera from 5 patients with herpes simplex encephalitis (HSVE), 3 with varicellae zoster (VZV) meningoencephalitis and 5 with encephalitis of unknown origin (NUD) were analyzed. Lymphocytes from both blood and CSF were shown to synthesize anti-VZV IgG subclasses in VZV meningoencephalitis and anti-HSV IgG subclasses in HSVE. The subclass patterns of CSF and in vitro synthesized anti-viral IgG were similar, suggesting that a considerable portion of the antiviral IgG subclasses detected are synthesized in the CNS compartment. Antigen presentation in vitro seemed to produce a heterologous IgG4 and/or 3 response in 3 patients. Lymphocyte proliferation was detectable in response to HSV and VZV, respectively.     

Increased reactivity to HTLV-I in inflammatory nervous system diseases

The presence of IgG antibodies reacting with purified and disrupted human T-lymphotropic virus type I (HTLV-I) was examined by an indirect enzyme-linked immunosorbent assay (ELISA) in sera from 49 patients with multiple sclerosis (MS), 21 patients with aseptic meningoencephalitis (AM), 12 patients with Guillain-Barré syndrome (GB), and 30 patients with tension headache (TH). This was also assessed in the concentrated cerebrospinal fluid (CSF) of most of these patients, as well as in sera of 60 blood donors (BD). Standardized amounts of serum IgG and CSF IgG were used in ELISA. For sera, higher reactivity with HTLV-I was found in all four patient groups compared with the BD group, but no significant differences were observed among the four groups. There was higher reactivity with HTLV-I in the CSF of patients with MS, AM, and GB compared to findings in patients with TH. Ten serum (2 MS, 3 GB, 3 TH, 2 BD) and 3 CSF (1 MS, 1 GB, 1 TH) specimens considered positive by ELISA for HTLV-I were found negative on confirmatory Western blot analysis. We extended this study to analyze the in vitro production of anti-HTLV-I-IgG antibodies by the 24-hour cultivation of unstimulated lymphocytes from peripheral blood and CSF of 6 additional patients with MS directly in HTLV-I antigen-coated wells of microtiter plates. This was followed by determination of specific antibodies by ELISA in the same wells. No antibody production was measurable. Our data do not favor the hypothesis of an HTLV-I-related human retrovirus in the etiology of MS.     

IgG1,3 and 4 oligoclonal bands in multiple sclerosis and other neurological diseases

Cerebrospinal fluid (CSF)samples from 10 patients with Multiple Sclerosis (MS) and 7 with other neurological diseases (OND) were studied in order to detect oligoclonal restriction of IgG subclasses 1,3 and 4. Agarose isoelectric focusing (AGA-IEF) followed by Western capillary blotting and immunoperoxidase staining with specific monoclonal antibodies were used. All MS samples showed oligoclonal IgG1 and 6 of them also had IgG3 or IgG4 bands. In the OND group only patients with subacute sclerosing panencephalitis (SSPE) and Guillain-Barré disease (GBD) showed CSF oligoclonal patterns for IgG subclasses. Our results demonstrate that in MS CSF other IgG subclasses beside IgG1 may display an oligoclonal pattern. The finding of more than one subclass in the same band indicates a microheterogeneous composition of these oligoclonal bands.

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Sommario Allo scopo di rilevare un'eventuale restrizione oligoclonale per le sottoclassi IgG1,3 e 4 sono stati studiati campioni di liquido cerebrospinale (LCS) ottenuti da 10 pazienti con Sclerosi Multipla (SM) definita e da 7 pazienti con altre malattie neurologiche (AMN). La metodica utilizzata è stata l'isoelectric focusing in agarosio seguita da Western capillary blotting su nitrocellulosa e colorazione con immunoperossidasi mediante anticorpi monoclonali. Tutti i campioni di SM mostrarono IgG1 oligoclonali e sei di essi presentarono contemporaneamente bande di IgG3 o di IgG4. Tra le AMN solo i pazienti con Panencefalite Sclerosante Subacuta e sindrome di Guillain-Barré mostrarono bande oligoclonali formate da sottoclassi di IgG. I nostri dati dimostrano che nel LCS di SM altre sottoclassi di IgG, oltre alle IgG1, possono essere oligoclonali. La presenza di più di una sottoclasse nella stessa banda depone per una microeterogeneità di queste bande oligoclonali.

     

多发性硬化患者血清和脑脊液Epstein-Barr病毒抗体检测的意义

目的探讨多发性硬化(MS)患者血清和脑脊液(CSF)中EpsteinBarr(EB)病毒IgG抗体检测的意义。方法采用酶联免疫吸附法检测MS患者65例、其他神经科疾病(OND组)患者71例、非神经科疾病(NND组)患者42例的血清和CSF中EB病毒核抗原、壳抗原和早期抗原的IgG抗体,并进行分析比较。根据血清抗体检测结果的组合,分析各组中病毒初次感染、既往感染和病毒重新激活的情况。结果3组患者血清EB病毒核抗原、壳抗原IgG抗体阳性率均>90%,差异无显著性(均P>0.05)。MS组EB病毒早期抗原IgG抗体阳性率(46.2%)明显高于其他两组(18.3%,9.5%,均P<0.05)。MS组病毒感染重新激活的比率(46.2%)明显高于其他两组(18.3%,9.5%,均P<0.05)。3组CSF病毒抗体阳性率差异无显著性(均P>0.05)。结论MS患者活动性的EB病毒感染较多,EB病毒感染重新激活的比例很高。     

IgG allotypes and subclasses in Norwegian patients with multiple sclerosis

Multiple sclerosis (MS) is a multifactorial disease in which genetic and environmental factors apparently have a major influence on the susceptibility and course of the disease. In the present study we have investigated the genetic basis and subclass levels of IgG in MS. Hundred and thirty-six Norwegian patients with MS and 92 controls were genotyped for IgG allotypes of the GM and KM systems. IgG and IgG subclasses were quantified in sera from 115 MS patients and 20 controls. Neither GM nor KM allo-, haplo- or genotypes were significantly correlated with susceptibility, severity or course of the disease. The G1M (3) (3), G2M (23) (23) and G3M (5) (5) allotypes were significantly correlated with high serum levels of IgG3, whereas high IgG2 levels were correlated with G1M (3) (3) and G2M (23) (23) in both patients and controls. Serum levels of IgG subclasses were not significantly correlated with course or severity of the disease. The results indicate no major role for IgG allotypes or IgG subclass levels in the pathogenesis of MS.     

Antiviral IgM and IgG subclasses in varicella zoster associated neurological syndromes.

The varicella zoster virus (VZV) and herpes simplex virus (HSV) IgGl-4 subclasses were compared in serum and cerebrospinal fluid (CSF) of 22 patients with VZV-associated neurological symptoms, 12 patients with HSV-associated neurological symptoms and 14 controls. The clinical syndromes of the VZV-associated diseases comprised meningo-encephalitis, myelitis, myelopathies and polyneuropathies, mostly with a favourable outcome. A characteristic finding was an intrathecal synthesis of VZV IgG1 and HSV-3. Commonly also IgG2 and 4 were seen in CSF of VZV patients. Their intrathecally synthesised HSV IgG was restricted to IgG1. VZV IgG3 occurred in serum and/or CFS together with VZV IgM in 14 cases and may be a marker of recent VZV replication. In patients with HSV-associated neurological disease, a multi-IgG subclass HSV response and concomitant VZV antibodies restricted to IgG1 was found. Intrathecal synthesis of both HSV and VZV IgG occurred in 20 patients. Detection of two or more VZV or HSV specific IgG subclasses synthesised intrathecally identified the aetiological agent in 19 of these 20 cases.     

The glycopeptide CSF114(Glc) detects serum antibodies in multiple sclerosis

Synthetic glycopeptides have the potential to detect antibodies in multiple sclerosis (MS). In the present study, we analyzed the antibodies (IgM class, IgG class and IgG subclasses) to the synthetic glycopeptide CSF114(Glc) in the serum of 186 MS patients, 166 blood donors (BDs), 25 patients affected by meningitis/encephalitis, 41 affected by systemic lupus erythematosus (SLE) and 49 affected by rheumatoid arthritis (RA). The IgM antibody level to CSF114(Glc) was significantly increased in MS patients versus BDs (p<0.001) or versus other autoimmune diseases (SLE or RA, p<0.001). The IgG response was restricted to the subclass IgG2. IgM antibodies to CSF114(Glc) were found in 30% of relapsing/remitting MS patients and, at lower levels, in subjects affected by meningitis/encephalitis. The study of antibodies to CSF114(Glc) is a new, potential immunological marker of 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.     

Detection and isolation of immune complexes in multiple sclerosis cerebrospinal fluid

Immune complexes were studied in the cerebrospinal fluid (CSF) of 20 multiple sclerosis (MS) and 20 other neurological disease (OND) patients using polyethylene glycol precipitation; ten samples from each group were also examined using gel chromatography followed by ELISA. Polyethylene glycol detected predominantly IgG and IgM complexes in 13 of 20 MS samples and four of 20 OND samples. Intact MS complexes ranged in size from 230 to 340 kDa and contained 64 and 53 kDa antigens. Gel chromatography detected IgA complexes in eight of ten MS samples and one of ten OND samples; these complexes appeared to consist of polymeric IgA rather than true antigen. Chromatography detected IgG complexes in nine of ten MS and four of ten OND samples. Intact MS complexes ranged from 240 to 320 kDa and contained 200 and 150 kDa antigens. This study suggests that immune complexes are a very frequent finding in the CSF of MS patients and are in sufficient quantity to visualize on gel electrophoresis.     

Serum and cerebrospinal fluid antibodies against myelin basic protein and their IgG subclass distribution in multiple sclerosis.

IgG class antibodies reactive with myelin basic protein (MBP) were determined by enzyme-linked immunosorbent assay (ELISA) in serum and cerebrospinal fluid (CSF) of 37 patients with multiple sclerosis and a control group of 32 patients with tension headache or psychoneurosis. Using standardised amounts of IgG from CSF and serum in ELISA, significantly higher mean antibody levels were found in CSF as well as in serum from the patients with multiple sclerosis. Ten (27%) of the multiple sclerosis CSF samples and 15 (41%) of the multiple sclerosis sera revealed anti MBP antibody levels exceeding 2 SD of the control group. Seven patients (19%) showed exclusive or higher levels of anti MBP antibodies in CSF, suggesting synthesis within the central nervous system. Analysis by ELISA for IgG subclasses of anti MBP antibodies revealed that they were restricted to IgG 1 in four patients and IgG 3 in one.     

Specific IgG subclass reactivity in herpes simplex encephalitis

Summary Serum and cerebrospinal fluid (CSF) samples from 19 patients with a previous diagnosis of herpes simplex virus encephalitis (HSVE), from 14 patients with a previous diagnosis of non HSVE encephalitis and from 21 healthy subjects were examined to detect IgG subclasses 1–4 reactive with herpes simplex virus (HSV), cytomegalovirus (CMV) and varicella zoster virus (VZV). Antibodies to HSV were detected in CSF and serum from the 14 HSVE-patients with a reactivated HSV infection and from 3 of the 5 patients with a primary HSV infection. The predominant subclass pattern was an early HSV-specific IgG1 rise, followed by IgG3 and, more seldom, IgG4; HSV IgG2 was rarely seen. In HSVE patients, HSV IgG3 was absent in early samples and usually appeared 10–20 days after onset of disease. In 14 out of 16 seropositive healthy controls, on the other hand, HSV IgG3 was present in the CSF. Rising VZV IgG levels in serum and CSF were found in 11 HSVE patients. Eight of them showed signs of intrathecal VZV IgG1 synthesis. The VZV IgG reactivity was restricted to IgG1 in 7 of these whereas the HSV IgG subclass response also included IgG3 or 4. The appearance of several HSV IgG subclasses appeared to serve as a marker of HSV infection in spite of the serological VZV reaction, usually restricted to VZV IgG1. Intrathecal synthesis of the quantitatively minor HSV IgG3 and 4 subclasses was detected earlier than intrathecal synthesis of total HSV IgG, dominated by IgG1 in 4 patients with HSVE.