MS and clinically isolated syndromes: shared specificity but diverging clonal patterns of virus-specific IgG antibodies produced in vivo and by CSF B cells in vitro.

BACKGROUND: Intrathecal synthesis of oligoclonal IgG antibodies against measles virus (MeV), varicella zoster virus (VZV) and herpes simplex virus type-1 (HSV-1) is a characteristic feature multiple sclerosis (MS). METHODS: We have used isoelectric focusing-immunoblot to define the clonal patterns of IgG and of IgG antibodies to MeV, VZV and HSV-1 in supernatants of in vitro cultures of peripheral blood lymphocytes (PBL) and cerebrospinal fluid (CSF) cells and in sera and CSF from three patients with MS and three patients with clinically isolated syndromes (CIS) suspective of demyelinating disease. RESULTS: In vitro synthesis of IgG by PBL was not detected in any patient. In contrast, in vitro synthesis by CSF cells of oligoclonal IgG and oligoclonal IgG antibodies to one or two of the three viruses tested was observed in all six patients. The clonal patterns of the in vitro synthesized IgG and virus specific IgG differed to varying extent from those synthesized intrathecally in vivo. However, in each patient, the in vitro and in vivo intrathecally produced antibodies displayed specificity for the same viruses. The addition of B cell activating factor (BAFF) had no effect on the amounts or clonal patterns of either total IgG or virus-specific IgG produced by CSF cells in vitro. CONCLUSION: Virus specific B cells capable of spontaneous IgG synthesis are clonally expanded in the CSF of patients with MS. The B-cell repertoire in CSF samples is only partially representative of the intrathecal B-cell repertoire.

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.

Cerebral vasculopathy associated with primary varicella infection.

A previously healthy 5-year-old boy developed cerebral vasculopathy, presenting as two episodes of acute hemiparesis 3 and 9 months, respectively, after a primary varicella infection (chickenpox). This association has not been reported before, to our knowledge, although cerebral vasculopathy is a well-known complication of herpes zoster ophthalmicus. The diagnosis was based on the presence of oligoclonal varicella-specific IgG in the cerebrospinal fluid and angiographic findings. Clinical and angiographic follow-up, and serial thymidine kinase activity levels in the cerebrospinal fluid suggested a self-limiting course of the virus-induced vasculopathy. Varicella zoster virus seems to be another potential causative agent to be considered in acute childhood hemiplegia.

Imprint immunofixation of electrofocused immunoglobulins.

An imprint immunofixation (IIF) technique for the characterization of electrofocused immunoglobulins (Ig) is described. Electrofocused proteins are blotted (imprinted) from the separating polyacrylamide gel to agarose gels by gel-to-gel overlays. The protein imprints are chemically fixed in the agarose gels with a solution of 46% methanol, 8% acetic acid and 46% water. The imprinted Ig are then identified radioimmunologically, using an indirect system with monoclonal mouse anti-human Ig antibodies in the first layer and 125I-labelled rabbit anti-mouse Ig in the second, followed by autoradiography. The method is sensitive and permits characterization of Ig in unconcentrated cerebrospinal fluid. By sequential imprinting, each separated specimen can be characterized for up to 10 separate antigenic determinants without loss of sensitivity.

Immunofluorescence staining of agarose-embedded cells. A new technique developed for immunological characterization of markers on a small number of cells.

A simple new method for the immunofluorescence staining of small numbers of cells is described: cell suspensions are mixed with low-temperature-gelling agarose at 37 degrees C and 2 microliter samples of agarose containing cells are dispensed onto multitest microslides precoated with agarose. The cells are subsequently stained by immunofluorescence techniques. Alternatively, the cell slides can be stored in liquid nitrogen until immunofluorescence staining is carried out. Since cells are entrapped within the agarose matrix, cell loss is prevented during staining and washing procedures. The method permits staining of as few as 250 cells for each marker, thus enabling simultaneous characterization of several separate cell markers in cerebrospinal fluid or other body compartments from which comparatively few cells are obtainable.

Quantitation of diphtheria and tetanus antibodies by reversed rocket immunoelectrophoresis.

A reversed rocket immunoelectrophoresis technique, applying serum samples to antigen-containing gel, is described for quantitation of human antibodies to diphtheria and tetanus. Lowering of the pH of the buffer from 8.6 to 5.2 increased the height of the rockets, but also resulted in more non-specific precipitation. This smudging could be removed by dextran sulphate precipitation of the sera, a necessity when quantitating sera with small concentrations of anti-bodies. The merits of this method are discussed in relation to other in vitro as well as in vivo methods. The method was found to be reproducible, rapid, cheap and sensitive, and to require small samples of serum.

HLA-DQA1 and HLA-DQB1 genes may jointly determine susceptibility to develop multiple sclerosis.

Serologic DR typing and genomic DRB1, DQA1, DQB1, DPA1, and DPB1 typing using sequence-specific oligonucleotides were performed in 69 multiple sclerosis (MS) patients and 181 healthy controls on in vitro amplified DNA. The frequencies of DR2 as well as the DR2-associated DQA1*0102 and DQB1*0602 alleles were increased whereas DR7 was decreased among MS patients. The distribution of DR4 subtypes as well as DP alleles were similar in patients and healthy controls. All but one of 23 DR4-positive MS patients carried the DQB1*0302 allele, whereas five of five DR7-positive MS patients carried the DQB1*0303 allele. Of the MS patients, 99% compared to 79% of the controls carried DQA1 alleles encoding glutamine at residue 34, while 97% of the MS patients compared to 72% of the controls carried DQB1 alleles encoding DQ beta chains sharing long polymorphic stretches. A combination of such DQA1 and DQB1 alleles was carried by 96% of the MS patients and 60% of the controls, suggesting an association between MS and a combination of particular DQA1 alleles and DQB1 alleles. The corresponding DQ alpha beta heterodimers may have in common an ability to bind a particular peptide.

Patients with multiple sclerosis carry DQB1 genes which encode shared polymorphic amino acid sequences.

Of 61 Norwegian multiple sclerosis patients tested, 59, i.e., 97%, were positive for at least one of the HLA specificities DR2, DR4, or DRw6. Typing with sequence-specific oligonucleotide probes revealed that the same 59 patients carried DR2-, DR4-, or DRw6-associated HLA-DQB1 genes which encode shared polymorphic amino acid sequences in the membrane-distal part of their HLA-DQ beta chains. This shared DQ beta polymorphism may explain previously reported DR associations and could thus be the primary HLA association in MS.

Herpes simplex virus encephalitis: intrathecal synthesis of oligoclonal virus-specific IgG, IgA and IgM antibodies.

Paired specimens of serum and CSF from seven patients with acute herpes simplex virus encephalitis were examined during the acute illness or the convalescent stage or during both stages. Imprint immunofixation analyses of viral antibodies separated by agarose electrophoresis and by electrofocusing disclosed intrathecal production of herpes simplex virus IgG antibodies in all seven patients, and of IgA and IgM antibodies in six and three of six patients, respectively. Intrathecal production of herpes simplex virus-specific IgG and IgA was observed in two patients from whom samples were collected after 1 year, while intrathecal production of virus-specific IgM was not demonstrated later than 5 weeks after onset. The intrathecally synthesized IgG and IgM, and to a lesser extent IgA antibodies displayed oligoclonal characteristics. Oligoclonal bands of IgG were observed in the CSF of all patients. Evidence is presented to show that the bulk of the oligoclonal CSF IgG represents herpes simplex virus-specific antibodies. Intrathecally synthesized populations of herpes simplex virus antibodies cross-reacting with varicella-zoster virus were identified in three of the patients.

Long-term persistence of intrathecal virus-specific antibody responses after herpes simplex virus encephalitis.

Paired sera and cerebrospinal fluids (CSF) from nine surviving patients were collected 4.5 to 8 years after acute herpes simplex (HS) virus encephalitis. Oligoclonal bands of IgG were detected in the CSF of all, and seven patients had an elevated CSF IgG index. Antibodies to HS, varicella-zoster (VZ), measles, and cytomegalo viruses were analysed by enzyme-linked immunosorbent assay (ELISA) and by imprint immunofixation (IIF) of specimens separated by electrophoresis and by thin-layer electrofocusing. Intrathecal synthesis of HS and VZ IgG antibodies was demonstrated in all and of measles IgG antibodies in one patient by both methods. Intrathecal synthesis of HS IgA antibodies was demonstrated by ELISA in three and by IIF in seven patients; the latter method also disclosed intrathecal synthesis of VZ IgA antibodies in two. No patient had intrathecal synthesis of viral IgM antibodies. The intrathecally synthesized antibodies demonstrated by IIF displayed oligoclonal characteristics. The IIF analyses as well as virus absorption tests indicated that the intrathecally synthesized VZ IgG and IgA antibodies could be explained as HS antibodies cross-reacting with VZV. The results indicate that a long-term persistence of intrathecal antibody responses to HS virus is a common feature after acute HS encephalitis. The intrathecal production of measles IgG antibodies in one case may reflect a similar persistence of non-specific immune responses induced during the acute 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.

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 patinets 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. Immunoelectrophoretic 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.

Intrathecal synthesis of virus-specific oligoclonal IgG, IgA and IgM antibodies in a case of varicella-zoster meningoencephalitis.

Varicella-zoster (VZ) virus meningoencephalitis was diagnosed in a 72-year-old man without other clinical signs of VZ infection, on the basis of intrathecal virus-specific IgG, IgA and IgM antibody responses demonstrated by imprint immunofixation (IIF) and by serological analyses of serum and CSF. The intrathecally produced antibodies displayed oligoclonal characteristics. The intrathecal production of VZ-IgG and -IgA antibodies persisted throughout the observation period of 20 months, while that of VZ-IgM antibodies was not detectable later than 3 months after onset. Part of the intrathecally produced VZ-IgG and -IgA antibody populations, but no IgM antibodies, were shown to cross-react with herpes simplex virus. Oligoclonal IgG bands were demonstrated in the CSF throughout the observation period. The bulk of the IgG bands was shown to represent VZ-specific antibodies.

Intrathecal complement activation in neurological diseases evaluated by analysis of the terminal complement complex.

The terminal complement complex (TCC) was determined in plasma and cerebrospinal fluid (CSF) from 208 neurological patients. Elevated CSF TCC levels were observed in higher frequencies in patients with infectious diseases (80%), radiculoneuritis (62%), multiple sclerosis (30%), and miscellaneous autoimmune diseases (27%) than in patients with miscellaneous non-inflammatory diseases (2-13%). The plasma level of TCC was significantly increased only in the infectious group. No positive correlation was observed between the plasma and the CSF TCC concentration in the whole patient population nor in subgroups divided according to blood-brain barrier function. Furthermore, the CSF TCC concentration did not correlate with the serum/CSF albumin ratio or with CSF total protein concentration when this was below 1.0 g/l. It is concluded that an elevated TCC concentration in CSF reflects intrathecal complement activation and that quantification of TCC in CSF may be a valuable supplement in the examination of neurological diseases.

The intrathecal immune response in the early stage of multiple sclerosis.

Sequential pairs of cerebrospinal fluid (CSF) and serum samples from 10 patients followed for 2.5-12 years after onset of unilateral optic neuritis (ON) were studied. Eight patients developed definite multiple sclerosis (MS) during the observation period. All patients had normal CSF protein patterns on agar or agarose gel electrophoresis at onset. Six patients developed oligoclonal immunoglobulin (Ig) bands in the CSF during the observation period. Imprint immunofixation of electrofocused specimens disclosed intrathecal synthesis of oligoclonal IgG antibodies to 1 or more of 6 viruses (measles, herpes simplex type 1, varicella-zoster, cytomegalo, mumps, rota) during the observation period in 8 patients. Changes in patterns of intrathecally synthesized viral antibodies, characterized by the appearance of "new" antibody populations and the waxing or waning of others were observed in 6 patients. The results suggest that the early stage of MS in some patients is associated with transient as well as permanent recruitment of B cell clones producing viral antibodies of different specificities.

HLA antigens in multiple sclerosis.

The frequency of the MLC activating HLA-Dw2 determinant was found significantly increased to 68% among 37 patients with long-standing multiple sclerosis, compared to 30.2% among healthy controls. A similar (or stronger) degree of association was found with an "Ia-like" B cell specificity, SOW, while the association with HLA-B7 was less apparent. No correlation between HLA-Dw2 and signs of local synthesis of oligoclonal IgG or measles or rubella virus antibodies in the CNS was observed.

Sex and age at diagnosis are correlated with the HLA-DR2, DQ6 haplotype in multiple sclerosis.

The HLA-DR2, DQ6 (i.e., HLA-DRB1*1501, DQA1*0102, DQB1*0602) haplotype contributes to the risk of developing multiple sclerosis (MS) in Caucasoids of Northern European heritage. A correlation between the clinical expression of MS and the presence of HLA-DR2, DQ6 has, however, not convincingly been shown. In this study conventional bivariate analysis and logistic regression analysis were used to study the relationship between HLA-DR2, DQ6 and four disease variables in a cohort of 286 Norwegian MS patients from the Oslo area. Logistic regression analysis showed that HLA-DR2, DQ6 was significantly more frequent among female than male patients (P=0. 0251), and was negatively correlated with age at diagnosis regardless of sex (P=0.0254). No significant correlation was observed between HLA-DR2, DQ6 and type of disease (relapsing-remitting versus primary chronic progressive MS) or presence/absence of oligoclonal bands in the cerebrospinal fluid.

Human immunodeficiency virus infection of the brain. II. Detection of intrathecally synthesized antibodies by enzyme linked immunosorbent assay and imprint immunofixation.

Sera and CSF from 29 patients in early and late stages of HIV infection were analysed for intrathecal antibody production. Elevated CSF-IgG indices indicating intrathecal IgG synthesis were demonstrated in 9 patients while 4 of 18 patients tested had oligoclonal IgG bands in the CSF. Analysis of HIV-specific antibodies by enzyme-linked immunosorbent assay (whole antigen and site-directed ELISA) and calculation of "antibody indices" (CSF/serum antibody quotient divided by CSF/serum albumin quotient) indicated intrathecal HIV antibody synthesis in 19 patients. Analysis of serum and CSF antibodies by an imprint immunofixation (IIF) method showed intrathecal synthesis of predominantly polyclonal HIV-IgG antibodies in 11 of 13 patients examined. IIF analysis of antibodies to six other infectious agents showed no intrathecal antibody production except in one patient who had minor fractions of intrathecally synthesized IgG antibodies to varicella zoster virus. The present results demonstrate that an intrathecal HIV-specific antibody response may be present in both early and late stages of HIV infection, and indicates that HIV may reach the brain at an early stage of infection.