Blood-brain-barrier and intrathecal immunoglobulin changes in tuberculous meningitis and demyelinating disorders: a preliminary report from a South Indian hospital

Sixty-three paired serum and CSF samples (15 controls and 48 patients) were studied for CSF albumin quotient, IgG index and synthesis rate. Control values for albumin quotient and IgG index were less than 10.0 and less than 0.6 respectively. IgG synthesis rate/day was calculated according to the Tourtellotte formula and a value of greater than 3.0 mg/day is considered high. Our findings in patients with central demyelinations, subacute sclerosing panencephalitis, Guillain Barré Syndrome and cerebrovascular accidents are comparable to earlier studies. Fifteen tuberculous meningitis patients were studied and could be separated into 2 groups, 9 with elevated albumin quotients and 6 with normal albumin quotients. Three patients with elevated albumin quotients and 4 with normal albumin quotients showed increased intrathecal IgG synthesis.     

Oligoclonal bands in cerebrospinal fluid: a comparative study of isoelectric focusing, agarose gel electrophoresis and IgG index

OBJECTIVE: To compare the sensitivity and specificity of isoelectric focusing (IEF) with immunofixation, agarose gel electrophoresis (AGE) and the IgG index in detecting intrathecally synthesized IgG in multiple sclerosis (MS) and in other nervous system disorders. MATERIALS AND METHODS: Cerebrospinal fluid (CSF) and serum from 147 patients with various nervous system diseases, 20 of whom had MS, were compared with IEF, AGE and the IgG index. RESULTS: CSF-restricted oligoclonal bands (OCB) were found in 20 of 20 patients with MS using IEF and in 9 of 20 using AGE. OCB were found in 12 patients with other nervous system disorders (OND) using IEF and 4 using AGE. The mean IgG index was 0.50 in OND and 0.96 in MS (P< 0.0001). Of 20 MS patients, 9 had an IgG index above the defined cut-off value of 0.72. CONCLUSIONS: IEF is about twice as sensitive as AGE in detecting OCB in MS. IEF is also far superior to the IgG index in determining intrathecal IgG synthesis.     

Intrathecal immunoglobulin synthesis. Comparison of various formulae with the 'gold standard' of isoelectric focusing

The recent introduction of a further new formula for calculating intrathecal Ig synthesis led us to compare the results of calculations obtained when using it and certain other older formulae with those of isoelectric focusing (IEF), since only the latter proves the intrathecal synthesis of oligoclonal proteins. On comparison, it was found that the results produced by the usual formulae were, without exception, inferior to those of isoelectric focusing. By using Receiver Operating Characteristic (ROC) curves, it is shown that calculations with the Reiber formula agree best with IEF. Virtually the same results are obtained with the "ln IgG index + 1" and "IgG index".     

On the diagnostic value of different CSF investigations in multiple sclerosis

Summary An assessment was made of the relative values of different method of calculating intrathecal IgG synthesis in multiple sclerosis. In 60 patients with definite, probable or possible multiple sclerosis the IgG/protein and IgG/albumin ratios, the IgG index, the Tourtellotte formula and the graphic evaluation according to Reiber were applied. All the patients had oligoclonal IgG in the CSF. Reiber's method was the most sensitive, yielding 66% of abnormal findings. Only the cell count showed significant differences between active and inactive stages. There was a correlation between the cell count and the daily rate of intrathecal IgG synthesis and between the latter and the number of oligoclonal bands in CSF, but none of these parameters correlated with duration of disease or the number of previous bouts.This work was supported by the Hermann and Lilly Schilling Foundation in the Stifterverband für die Deutsche Wissenschaft, Essen, Federal Republic of Germany     

Viral antibody activity of oligodonal and polyclonal immunoglobulins synthesized within the central nervous system in multiple sclerosis

Thin-layer polyacrylamide gel isoelectric focusing (PAG IEF), a very high capacity method for separating immunoglobulins (Ig), was performed on cerebrospinal fluid (CSF) and serum. It was followed by antigen immunofixation with measles, mumps, herpes simplex (HSV), and rubella virus antigens and anti-human Ig autoradiography in order to demonstrate viral antibodies in separated Ig zones. Two of 11 control patients and 21 of 25 patients with multiple sclerosis (MS) displayed one or more zones of viral antibodies in the CSF without any counterpart, or with distinctly fainter zones, in the serum. Such reaction patterns were taken to indicate the possibility of intrathecal antibody synthesis. Antibody synthesis to measles was found in one to five zones in 76% of the patients with MS; antibody zones were found to HSV in 36% of the patients, to mumps in 12%, and to rubella in 12%. In 36% of the patients, two or three different antibody specificities (of which one was always measles) were found simultaneously in individual autoradiogram zones. For all viral antibodies detected in the CSF autoradiograms, their counterparts in oligoclonal or polyclonal IgG zones (or both) were demonstrable by PAG IEF of the corresponding CSF. The majority of patients with MS also had one or more oligoclonal CSF IgG zones without known antibody specificity. Antigen immunofixation and autoradiography are mainly qualitative. It is not known whether the viral antibodies present in oligoclonal or polyclonal IgG zones in MS CSF reflect a polyclonal B cell activation, a disease-specific immune reaction, or both.     

血脑屏障完整性及鞘内免疫球蛋白合成在中枢神经系统疾病中的意义

目的探讨中枢神经系统疾病血脑屏障(BBB)的完整性和鞘内免疫球蛋白合成的意义。方法将研究对象分为8组(A组为10例正常对照组,B组为10例化脓性脑膜炎患者,C组为10例结核性脑膜炎患者,D组为20例病毒性脑(膜)炎患者,E组为8例脱髓鞘疾病患者,F组为10例格林巴利综合症患者,G组为10例蛛网膜下腔出血和脑出血患者,H组为14例缺血性脑血管病患者),采用速率散射比浊方法测定血清和CSF的白蛋白(Alb)和免疫球蛋白(IgG、IgMI、gA)含量,利用公式计算QAlbI、gG指数、IgG合成率,并用Protis软件进行数据和图形处理分析。结果 (1)各组QAlb异常率均较正常对照组增高,其中B、C、E、G组QAlb明显高于正常对照组(P0.01);(2)B、C、E、G组IgG指数和IgG合成率较正常对照组明显增高(P0.05或0.01),其中IgG指数以E组最高,IgG合成率以B、E组最高;(3)经Protis软件分析82例患者,正常35例,单纯BBB功能障碍20例,仅有鞘内合成8例,BBB功能障碍伴有鞘内合成19例。结论通过对CSF中蛋白质组分定量的分析,并利用Protis软件进行数据和图形处理,可以明确中枢神经系统疾病血脑屏障完整性和鞘内免疫球蛋白的合成,以及对某些中枢神经系统疾病的诊断和鉴别诊断具有重要的临床意义。     

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.     

Muscle antibodies in the cerebrospinal fluid from patients with myasthenia gravis

IgG antibodies to nicotinic acetylcholine receptor (AChR) and to a muscle antigen extracted by citric acid, were quantified in serum and cerebrospinal fluid (CSF) from 28 patients with myasthenia gravis, and the serum:CSF ratios compared with those of total IgG. Agarose-electrophoresis and calculations of the IgG index and Tourtellotte's formula were performed. No evidence of intrathecal antibody synthesis was demonstrated. Compared to the total IgG concentrations in serum and CSF, the CSF concentrations of IgG AChR antibodies were lower than expected.     

Progressive myoclonus epilepsy is not accompanied by humoral immune response within the central nervous system

CSF and serum from five patients with progressive myoclonus epilepsy taken on two occasions with about one month's interval were examined for intrathecal humoral immune response. The CSF IgG and IgA index values were normal, and no oligoclonal bands were detectable by agarose electrophoresis or polyacrylamide isoelectric focusing (IEF). Immunofixation of IEF separated IgG with four different viruses as possible antigens, and autoradiography did not reveal any intrathecal antibody production. In contradiction to previous reports, no evidence was obtained for a local humoral immune response in progressive myoclonus epilepsy.     

Intrathecal synthesis of IgM in neurological diseases: a comparison between detection of oligoclonal bands and quantitative estimation

Immunoglobulin (Ig) M concentration in cerebrospinal fluid (CSF) was measured in 30 reference subjects to determine reference ranges which were up to 0.41 mg/l for CSF IgM, 0.06 for IgM index, and 9 x 10(-4) mg/l for the Reiber empirical formula. Intrathecal IgM synthesis was then studied in 159 patients with different neurological diseases both by detecting CSF oligoclonal IgM bands and by determining the IgM index and Reiber's intrathecal IgM production values. At the same time, the state of blood-CSF barrier was evaluated in all patients by CSF polyacrylamide gel electrophoresis and by CSF/serum albumin ratio. Oligoclonal IgM bands were present mainly in patients with intrathecal humoral immune response, e.g. multiple sclerosis, other inflammatory nervous diseases, and infections of the central nervous system. Quantitative IgM ratios were, however, elevated in conditions not associated with local humoral immune response. Furthermore, quantitative ratios were elevated in patients with damaged blood-CSF barriers. Oligoclonal IgM bands are thus considered to be the most useful tool in detecting intrathecal synthesis of IgM.     

脑脊液寡克隆区带、24小时鞘内IgG合成率及IgG指数在脑脊膜血管型神经梅毒诊断中的应用研究

目的 探讨脑脊液寡克隆区带、24 h鞘内IgG合成率及IgG指数在脑脊膜血管型神经梅毒诊断中的应 用价值。 方法 回顾性分析2014年6月-2019年6月于首都医科大学附属北京地坛医院神经内科连续住院的 脑脊膜血管型神经梅毒患者的临床资料,对脑脊液甲苯胺红不加热血清试验（toluidine red unheated serum test,TRUST）、寡克隆区带、24 h鞘内IgG合成率及IgG指数诊断脑脊膜血管型神经梅毒的阳性率 和假阴性率进行分析。 结果 研究共纳入确诊的脑脊膜血管型神经梅毒患者102例。脑脊液寡克隆区带、24 h鞘内IgG合 成率及IgG指数检查的阳性率分别为93.1%、93.1%和91.2%,均高于TRUST检查的阳性率（71.6%,均 P＜0.001）。脑脊液寡克隆区带、24 h鞘内IgG合成率及IgG指数检查的假阴性率分别为6.9%、6.9%及 8.8%,均低于TRUST检查（28.4%,均P＜0.001）。 结论 脑脊液寡克隆区带、24 h鞘内IgG合成率及IgG指数均可用于脑脊膜血管型神经梅毒的诊断, 且上述指标的敏感性和假阴性率均优于脑脊液TRUST检测。     

Peripheral neuropathy in IgD myeloma. Cerebrospinal fluid paraprotein analysis in three cases

High resolution polyacrylamide gel isoelectric focusing (IEF), followed by direct immunofixation with anti-delta chains monospecific antibodies, were used to detect and identify IgD paraprotein in the cerebrospinal fluid (CSF) of 3 patients affected by IgD myeloma. Two of these patients presented a paraproteinemic neuropathy. Blood-brain barrier damage was investigated by means of CSF/serum albumin ratio. IgG index and CSF and serum IgD/albumin ratio were also evaluated. An intrathecal origin of the IgD paraprotein was excluded. The correlation between the presence of the paraprotein in the CSF and the possible neurological involvement was also examined.     

Oligoclonal immunoglobulin bands in cerebrospinal fluid of patients with Alzheimer''s disease and vascular dementia

We examined serum and cerebrospinal fluid (CSF) of 16 patients with Alzheimer's disease (AD), 28 patients with vascular dementia (VD), their age-matched controls and multiple sclerosis (MS) patients in order to evaluate the humoral immune response within the central nervous system both quantitatively and qualitatively. Intra-blood-brain barrier (BBB) protein synthesis was calculated by CSF IgG index. The presence of oligoclonal banding (OCB) was investigated with agarose isoelectric focusing (IEF) followed by immunoblotting with antihuman IgG. No patient with AD and only 4 patients with VD had slightly elevated IgG indexes, and no statistically significant differences in the indexes were found between the two groups. No bands were found in the CSF of AD patients but 3 VD patients had OCB in both serum and CSF. One VD patient had bands in serum but no bands in CSF. No kappa or lambda free light chains were found in those demented patients with demonstrable bands in the CSF and serum. No OCB were found in control sera and CSF. For comparison, the majority of patients with MS had OCB in CSF. Thus, no consistent increase of intrathecal protein synthesis was found in patients with AD and VD. Methodological differences explain at least part of the conflicting results published earlier.     

Analysis of the cerebrospinal fluid by isoelectrofocusing on agarose in multiple sclerosis

Intrathecal synthesis of immunoglobulins can be proved by means of two methods: quantitatively by immunoglobulins titration in CSF, the results expressed with several ratios; qualitatively by demonstration of oligoclonal distribution of gammaglobulins. IEF is the most sensitive of the qualitative methods. From a technical point of view Agarose Isoelectrofocusing seems to be a better method than polyacrylamide isoelectrofocusing and permits, when the interpretation is difficult, immunofixation into the gel. The authors report the results of a comparative study between the evaluation of the IgG Index and agarose isoelectrofocusing of 281 CSF divided into 113 CSF from patients with Multiple Sclerosis (MS) and 168 CSF from patients with other neurological diseases (OND). Sensitivity of IEF was higher than IgG index to prove intrathecal IgG synthesis: in the group of patients with MS, 91 p. 100 of CSF were abnormal instead of 72 p. 100 of IgG Index. In the group of patients with OND, abnormalities in IEF were low (5 p. 100) but the number of inflammatory diseases was poor. These results were similar with the findings of many authors using the same methods. In our opinion, IEF is the best technique which a specialized laboratory can use in routine to prove an immunoglobulin intrathecal synthesis.     

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.     

Antibodies against oligodendrocytes in serum and CSF in multiple sclerosis and other neurological diseases: 125I-protein A studies

Antibodies against oligodendrocytes were determined in pairs of unconcentrated CSF serum from 12 patients with multiple sclerosis (MS) and 25 control patients including 10 with aseptic meningoencephalitis (AM), using a 125I-protein A microassay. Antibody levels in serum and in CSF did not differ between MS and controls. Calculating the antibody index equal to (CSF/serum antibodies against oligodendrocytes):(CSF/serum albumin) in analogy to the CSF IgG index, thereby compensating for influence of serum antibody concentration as well as altered blood-brain barrier, no evidence was obtained for intrathecal antibody production in the patients with MS. Those with AM had higher antibody index values, probably reflecting intrathecal synthesis. Antibodies against oligodendrocytes seem to be a regular component of CSF and serum in neurological diseases; intrathecal antibody production is less frequent in MS than in AM.     

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.     

Sensitized immunofixation: a new technique for analyzing the oligoclonal pattern of CSF immunoglobulins

Intrathecal immunoglobulin synthesis is observed in more than 90% of all cases of multiple sclerosis, producing a specific CSF IgG oligoclonal electrophoretic pattern. The consensual method used as reference is isoelectric focusing (IEF). We developed a new CSF Ig analysis method by immunofixation (IF). The method includes an immunoenzymatic detection step performed directly on the gel allowing the use of unconcentrated CSF and avoiding the blotting step. The reliability of this method was established by the analysis of 210 CSF/serum pairs including defined, probable and possible MS, other inflammatory CNS diseases and controls (noninflammatory CNS diseases and peripheral nervous system diseases). Intrathecal IgG synthesis was detected in 95.5% of defined MS cases. The specificity for CNS inflammatory diseases including MS diagnosis, evaluated by comparison with controls, was 98.8%. This new method is quicker and visual interpretation is easier than with IEF. It is a semi-automated method that should be considered for standardization of CSF IgG analysis.     

Cerebrospinal fluid free kappa light chains versus IgG findings in neurological disorders: qualitative and quantitative measurements

In this study free kappa light chains in cerebrospinal fluid (CSF) were determined both by an affinity mediated capillary blotting technique after isoelectric focusing (IEF) in agarose gel and by a quantitative enzyme linked immunosorbent assay (ELISA). The free kappa results were compared with the IgG findings in 4 neurological patient groups with a distinct CSF IgG pattern: (1) CSF without oligoclonal IgG bands, (2) CSF with serum derived IgG bands, (3) CSF restricted IgG bands and (4) CSF restricted and serum derived IgG bands. Oligoclonal free kappa bands are nearly absent in CSF of groups 1 + 2, and present in 88% of group 3 and 84% of group 4 patients. We could also establish free kappa indices from specimens in the 4 groups in analogy to IgG indices. Group 1 had a median free kappa index of 1.1, group 2: 1.0 and groups 3 + 4: 10.0. The correspondence between immunoblot and index findings for free kappa is better than for IgG. Free kappa index is more sensitive but somewhat less specific than IgG index for establishing intrathecal immune production.     

Intrathecal synthesis of virus-specific oligoclonal antibodies in patients with enterovirus infection of the central nervous system

Summary Cerebrospinal fluid (CSF) and serum samples from six patients with enterovirus infections were investigated by isoelectric focusing (IEF) and affinity-mediated immunoblot (AMI) for the clonal distribution of entervirus-specific antibodies. In two patients with either acute meningitis or encephalitis and in one patient with a relapse of multiple sclerosis, oligoclonal IgG bands specific for enteroviruses were found predominantly in the CSF, revealing intrathecal synthesis of these antibodies. In three other patients with neurological symptoms probably unrelated to a current enterovirus infection, IEF and AMI disclosed nearly identical patterns of coxsackievirus-B-specific oligoclonal bands in the CSF and serum, indicating diffusion of these antibodies from the serum into the CSF. Although the number of patients in this study is small, the results suggest that intrathecally synthesized enterovirus-specific antibodies may be used as a means of identifying an enterovirus infection of the CNS.