Similarity of autoimmune diseases based on the profile of immune complex antigens.

Since immune complexes (IC) are a direct product of immune response through the binding between antigen and antibody, the profile of antigen-associated ICs may depend on each autoimmune disease. In this report, we examined the similarity of four neurological autoimmune diseases, Alzheimer's disease and healthy donors, and seven connective tissue diseases based on the profiling of IC-associated antigens which were previously or recently identified by immune complexome analysis of cerebrospinal fluid (CSF) or serum samples. The similarity between each pair of two diseases was assessed by correlation coefficients as distance matrix with the use of detection frequency (i.e., the percentage of patients who were positive for a certain antigen in each disease) of each IC-associated antigen in a certain disease. Among 15 pairs of five diseases and healthy control examined by the analysis of CSF samples, only 1 pair of neuropsychiatric systemic lupus erythematosus and multiple sclerosis corresponds to the higher correlation value (r = 0.73) than 0.7. On the other hand, among seven connective tissue diseases examined by the analysis of serum samples, 12 of 21 pairs show high correlation value (r > 0.70). Our finding suggested that the profile of IC-associated antigens identified by immune complexome analysis of CSF samples can be useful for evaluating the similarity of neurological autoimmune diseases; however, not by that of serum samples.

Novel anti-suprabasin antibodies may contribute to the pathogenesis of neuropsychiatric systemic lupus erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is often difficult to diagnose and distinguish from other diseases, because no NPSLE-specific antibodies have been identified. We developed a novel proteomic strategy for identifying and profiling antigens in immune complexes in the cerebrospinal fluid (CSF), and applied this strategy to 26 NPSLE patients. As controls, we also included 25 SLE patients without neuropsychiatric manifestations (SLE), 15 with relapsing remitting multiple sclerosis (MS) and 10 with normal pressure hydrocephalus (NPH). We identified immune complexes of suprabasin (SBSN) in the CSF of the NPSLE group. The titer of anti-SBSN antibodies was significantly higher in the CSF of the NPSLE group compared to those of the SLE, MS and NPH groups. Microarray data showed that the senescence and autophagy pathways were significantly changed in astrocytes exposed to anti-SBSN antibodies. Our findings indicate that SBSN could be a novel autoantibody for the evaluation of suspected NPSLE.

Selective and Sensitive Mass Spectrometric Identification of Immune Complex Antigens in Cerebrospinal Fluid.

Comprehensive identification of immune complex antigens (IC-antigens) in cerebrospinal fluid (CSF) is useful to provide insights into pathophysiology and could form the basis for novel diagnostic and treatment strategies for central nervous system autoimmune diseases and other neurological disorders. Immune complexome analysis is the method for comprehensively identifying IC-antigens in biological fluids (such as serum and CSF). Here, we describe IC-antigens detection method; specifically, ICs in CSF are captured and are subjected to papain-digestion elution and tryptic digestion, and are analyzed by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS).

Antigen-antibody dissociation in Alzheimer disease: a novel approach to diagnosis.

With the ever-increasing population of aged individuals at risk of developing Alzheimer's disease (AD), there is an urgent need for a sensitive, specific, non-invasive, and diagnostic standard. The majority of efforts have focused on auto-antibodies against amyloid-beta (Abeta) protein, both as a potential treatment, and a reliable biomarker of AD pathology. Naturally occurring antibodies against Abeta are found in the CSF and plasma of patients with AD as well as healthy control subjects. To date, differences between diseased and control subjects have been highly variable. However, some of the antibody will be in preformed antigen-antibody complexes and the extent and nature of such complexes may provide a potential explanation for the variable results reported in human studies. Thus, measuring total amounts of antigen or antibody following unmasking is critical. Here, using a technique for dissociating antibody-antigen complexes, we found significant differences in serum antibodies to Abeta between AD and aged-matched control subjects. While the current study demonstrates the relevance of measuring total antibody, bound and unbound, against Abeta in AD, this technique may be applicable to diseases such as acquired immune deficiency syndrome and hepatitis B where determination of antigen and antibody levels are important for disease diagnosis and assessing disease progression.

Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model.

We have previously shown that chronic treatment with the monoclonal antibody m266, which is specific for amyloid beta-peptide (Abeta), increases plasma concentrations of Abeta and reduces Abeta burden in the PDAPP transgenic mouse model of Alzheimer's disease (AD). We now report that administration of m266 to PDAPP mice can rapidly reverse memory deficits in both an object recognition task and a holeboard learning and memory task, but without altering brain Abeta burden. We also found that an Abeta/antibody complex was present in both the plasma and the cerebrospinal fluid of m266-treated mice. Our data indicate that passive immunization with this anti-Abeta monoclonal antibody can very rapidly reverse memory impairment in certain learning and memory tasks in the PDAPP mouse model of AD, owing perhaps to enhanced peripheral clearance and (or) sequestration of a soluble brain Abeta species.

Immune complexes in serum and in cerebrospinal fluid in African trypanosomiasis. Correlation with polyclonal B cell activation and with intracerebral immunoglobulin synthesis.

The possible occurrence of immune complexes (IC) in serum and in cerebrospinal fluid (CSF) has been studied in 36 patients with African trypanosomiasis (Trypanosoma brucei gambiense). In serum, very high levels of IC were detectable by the (125)I-C1q-binding and by the conglutinin-binding assays with positive results in 94 and 87%, respectively, of untreated patients. Circulating IC were found in both early and late stages of the disease, without significant quantitative differences; their size was 15-25S. There was a significant negative correlation between C3 values and C1qBA. Our studies suggest that circulating IC occurring during trypanosomiasis may be the expression of a polyclonal B cell activation. Indeed, there was a significant correlation (P < 0.001) between the levels of circulating IC and either the levels of IgM (mean value 12.5+/-7.2 mg/ml) or with the levels of rheumatoid factor-like antiimmunoglobulin antibodies that were detected by solid phase radioimmunoassay in 74% of the patients.IC were detected in 31 of 35 CSF samples, with a marked elevation in patients with definite involvement of the central nervous system as compared with earlier stages of sleeping sickness. The occurrence of IC in CSF was not related to an impairment of the blood-brain barrier as shown by analysis of CSF/serum albumin ratios. The level of IC in CSF did not correlate with the serum level and, therefore, circulating IC do not appear to cross efficiently an unimpaired blood-brain barrier. The analysis of IgG, IgM, and albumin concentrations in serum and CSF demonstrates a marked intracerebral immunoglobulin synthesis in patients with manifestations of meningoencephalitis. There was a correlation between CSF-C1q binding assay and this local IgG synthesis. These data are consistent with a local formation of IC in CSF in patients with active meningoencephalitis. The results obtained in eight patients followed during therapy suggest that the presence of IC in CSF may be an indicator of a continuing central nervous system disease and that the quantitation of CSF-IC may be useful for monitoring patient care.

Detection of contactin-2 in cerebrospinal fluid (CSF) of patients with Alzheimer's disease using Fluorescence Correlation Spectroscopy (FCS).

OBJECTIVES: Alzheimer's disease (AD) is the most common cause of dementia in the world. As many AD biomarkers occur at rather low abundances in CSF or blood, techniques of very high sensitivity and accuracy are important as diagnostic tools in the clinic. Here, we aimed to provide proof of concept of the use of a single molecule detection technique, Fluorescence Correlation Spectroscopy (FCS) for detection of novel candidate biomarkers for AD. DESIGN AND METHODS: FCS detects the diffusion times of the antigen-antibody complexes in highly diluted sample solutions, thus eliminating the need of large sample volumes and allows estimating the concentration of the target antigen. We developed a FCS set-up for contactin-2, a neuronal cell adhesion molecule and a ligand of beta-secretase 1 (BACE1) and amyloid precursor protein (APP), the latter proteins being important players in AD. With this method, we investigated whether contactin-2 concentrations are changed after delayed storage and in patients with Alzheimer's disease. RESULTS: The FCS set-up for measuring contactin-2 in CSF had a lower limit of quantification (LLOQ) of 0.2ng/ml and intra- and inter-assay coefficients of variation (CVs) of 12.2% and 14.6% respectively. Contactin-2 levels were stable up to one week storage of CSF (n=3) at RT and 4°C. Further, contactin-2 levels were similar in probable AD patients (n=34, p=0.27) compared to patients with subjective cognitive decline (SCD) (n=11). CONCLUSIONS: FCS is a sensitive tool, which can be used for detecting biomarkers in the clinical setting using very low sample volumes (10µl) and can measure proteins in their native conformations in the body fluid.

Presence of rabies specific immune complexes in cerebro-spinal fluid can help in ante-mortem diagnosis of human paralytic rabies.

BACKGROUND: Human rabies presents in two clinical forms, viz. furious or encephalitic and paralytic. Clinical diagnosis of paralytic form is difficult and requires laboratory confirmation. Presently available diagnostic techniques are not very sensitive for ante-mortem confirmation of rabies. OBJECTIVE: In the present study, we investigated whether presence of rabies specific immune complexes in cerebro-spinal fluid (CSF) of paralytic rabies patients could help in ante-mortem diagnosis of rabies. STUDY DESIGN: A capture ELISA based on monoclonal antibodies to rabies nucleoprotein (N) and glycoprotein (G) was developed to detect immune complexes to rabies N and G proteins. We studied CSF samples collected ante-mortem from 30 suspected paralytic rabies patients in whom diagnosis was later confirmed by autopsy. We included 30 CSF samples from people undergoing spinal anesthesia as negative controls and 30 CSF samples from other viral encephalitis as disease controls. RESULTS: Twenty-three out of 30 CSF samples (76.6%) showed presence of immune complexes to both rabies N and G proteins. None of the negative controls and CSFs from other confirmed viral infections were positive. Thus, the results were 100% specific and the sensitivity of this test was 76.6%. CONCLUSIONS: Detection of immune complexes to rabies antigens may be used as one of the techniques for rapid ante-mortem diagnosis of human rabies.

Immune complexes in cerebrospinal fluid and serum of neurological patients. Possible intrathecal formation in bacterial meningitis and herpetic encephalitis.

We assayed immune complexes (IC) by Particle Counting ImmunoAssay in the serum and cerebrospinal fluid (CSF) of patients with various neurological disorders. In pyogenic meningitis, the levels of IC sharply increased 4-8 days after onset with a fall before the 10th day of the disease. In herpetic encephalitis the IC and antibody levels started to increase about 12 days after onset. The IC persisted at high values for 3-4 weeks, whereas the high antibody titers persisted for several months during the follow-up. In these 2 groups of patients IC were probably locally produced as indicated by the lack of correlation with the IC levels in the serum. We did not detect any significant increase of IC in the serum and CSF of patients with multiple sclerosis (N = 48) or with acute idiopathic polyradiculoneuritis (N = 11). Using another technique based on the determination of IgG and C4 in polyethylene glycol precipitates we also failed to detect any significant increase of IC in multiple sclerosis sera.

Characterization of immune complexes in multiple sclerosis by an antigen-specific immune complex radioimmunoassay.

Serum and cerebrospinal fluid samples from multiple sclerosis (MS) patients containing various levels of Clq-reactive immune complexes (IC) and samples from age- and sex-matched controls were tested by an antigen-specific IC radioimmunoassay which detects IC containing myelin membrane-related antigens. Positive reactivity in the assay was significantly associated with IC-containing MS sera (P less than 0.005) but such an association was not observed with MS cerebrospinal fluid. Analysis of longitudinal specimens revealed that the levels of the antigen-specific serum IC fluctuated with time. Significant correlation between serum levels of Clq-reactive IC and serum levels of IC containing myelin membrane-related antigens was observed (r = 0.62; P less than 0.001). Sucrose gradient centrifugation of sera from 1 patient showed that the IC had a peak density of 1.075 g/ml, indicating the presence of lipid material. The results suggest that serum IC of MS patients frequently contain myelin membrane-related antigens and that these antigens may be lipids or lipid-associated.

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.

Detection of antibodies against myelin basic protein and increased levels of HIV-IgG antibodies and HIV antigen after solubilization of immune complexes in sera and CSF of HIV infected patients.

Fifteen HIV seropositive patients were studied. It was possible to enhance detection of HIV antigen and HIV and myelin basic protein (MBP) antibodies after dissociation of immune complexes by acid hydrolysis. HIV p24 antigen was then detected in four patients, three of whom were previously antigen negative. In 14 patients the treatment resulted in increased anti-HIV IgG subclass levels. Anti-MBP IgG was detected in 12 patients. Intrathecal synthesis of anti-MBP IgG subclasses was found in eight patients, five of whom had symptoms from the central nervous system.

Detection of mycobacterial antigen and antibodies in the cerebrospinal fluid of patients with tuberculous meningitis.

An immunodiagnostic test for the detection of a soluble nonprotein mycobacterial antigen by reverse passive haemagglutination with IgM murine monoclonal antibody was developed. The test was used to analyse the cerebrospinal fluid of 89 patients with tuberculous meningitis (TBM) from India and 127 control subjects from India and the UK. The antigen was demonstrable in 88% of culture-positive and 73% of culture-negative TBM patients. However, it was also detected in 21% of Indian patients with pyogenic meningitis, and in 8% of Indian and 1% of UK control subjects. Antibodies binding to a soluble mycobacterial extract were detected at low titre in 68% of all subjects with TBM and in 37% of Indian cases of pyogenic meningitis. Because patients with TBM had raised levels of the antigen and of antibodies to the antigen, the possible role of immune complexes in the pathogenesis of the disease is briefly discussed.

Circulating immune complexes in patients with multiple sclerosis. A longitudinal study of serum and CSF by C1q and platelet binding tests.

Two hundred and twenty-eight paired serum and CSF samples collected from 31 patients with MS during a 2-3-year follow-up were analyzed for the presence of immune complexes (IC) by C1q RIA and PIPA (platelet [125I]protein A) techniques. One hundred and forty-four sera from 11 healthy individuals were analyzed as controls. In almost all patients (29/31) IC were detectable during some period of the disease, as tested by either of the techniques. The results obtained by C1q RIA and PIPA correlated positively with each other in MS when mean serum values of each patient were compared. The mean CSF IC levels detected by C1q RIA appeared to correlate to the mean IgG indexes, an indicator of the intrathecal rate of IgG synthesis. The amount of IC in serum and CSF fluctuated independently in MS. The results of the PIPA test for MS serum IC correlated significantly to the duration of the disease. The PIPA test results also showed that patients in stable or chronic phases of MS displayed IC in serum and CSF more often than patients with a relapsing/remitting course of disease but there was no clear correlation between fluctuations in IC levels in individual patients measured by C1q RIA or PIPA techniques and the disease course. Because of the lack of a clear correlation between the presence, quantity and fluctuation of IC and the clinical picture we suggest that those IC detected in the present study are probably not a precipitating factor in the pathogenesis of multiple sclerosis.

Idiotype anti-idiotype complexes in cerebrospinal fluids of multiple sclerosis patients.

The involvement of idiotype-anti-idiotype complexes in multiple sclerosis was approached by ultracentrifuge studies. Specimens of the immunoglobulin G fraction obtained from the cerebrospinal fluids of multiple sclerosis patients which contained distinct oligoclonal bands, were subjected to analytical ultracentrifugation. None of these samples revealed the presence of any detectable amount of oligomeric immunoglobulin G, namely dimer or trimer. By comparison with control mixtures containing known amounts of dimeric immunoglobulin, it was evaluated that the cerebrospinal fluid samples contain less than 5% dimer. These findings indicate that the spinal fluids of multiple sclerosis patients do not contain idiotype-anti-idiotype complexes to an extent that would account for the oligoclonal immunoglobulin bands as representing complementary idiotypes and anti-idiotypes.

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals.

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

Antigens, antibodies and immune complexes in cerebrospinal fluid of patients with cerebral gnathostomiasis.

Methods for the detection of antigens, antibodies and immune complexes in the cerebrospinal fluid (CSF) of patients with neurological manifestations suggestive of cerebral gnathostomiasis were developed, in the hope that they may be useful in confirming the diagnosis of Gnathostoma spinigerum infection. Gnathostoma antigens were determined by a sandwich enzyme linked immunosorbent assay (ELISA) using antibodies from rabbits immunized with the excretory/secretory (ES) antigens obtained from the in vitro supernatant fluid in which the third-stage G. spinigerum larvae were maintained. With a biotin streptavidin procedure, the presence of G. spinigerum antigens as low as 2 ng in one ml of CSF could be detected. An indirect ELISA was used for the quantitation of IgG antibodies in the paired serum and CSF of these patients. A complement consumption method was used for the detection of immune complexes in the concentrated CSF specimens. Of the 11 patients with clinical signs and symptoms suggestive of having G. spinigerum infection involving the central nervous system, only one patient had antigens detected in the CSF and in this one patient no antibody could be demonstrated. One other patient had immune complexes in her CSF. All remaining patients had IgG antibodies demonstrable in the CSF specimens. These data suggest that the detection of IgG antibodies in CSF is more reliable than the other two methods in confirming the diagnosis of cerebral gnathostomiasis.

Circulating immune complexes in cerebrospinal fluid of patients with tuberculous meningitis.

Circulating immune complexes (ICs) were isolated from cerebrospinal fluids (CSFs) of patients with tuberculous meningitis (TBM), non-tuberculous neurological diseases by a polyethylene glycol (PEG) precipitation method. Mycobacterium tuberculosis antigen 5 was detected in CICs of 30% patients with TBM, by sandwich ELISA. CIC level decreases during antituberculosis chemotherapy and therefore its detection can provide a method to monitor the therapeutic schedule in patients with TBM.

[A case of Sjögren's syndrome with mononeuritis associated with high levels of circulating immune complexes].

A 32-year-old woman with Sjögren's syndrome accompanied by mononeuritis of right lower extremity was described. She admitted Shinshu University Hospital with the chief complaints of Raynaud's phenomenon and generalized lymphadenopathy. One year before admission Raynaud's phenomenon of right hand was first noticed. One month later she complained of a gritty sensation in the eyes and severe dryness of the mouth. On admission, she had systemic lymphadenopathy. Neurological examination revealed decreased light touch and pinprick sensations and decreased deep tendon reflexes in right lower extremity. Lip biopsy revealed marked mononuclear cell infiltrates around the salivary glands. Laboratory data included IgG 3,007 mg/dl, circulating immune complexes 1,200 micrograms AHGeq/ml (Raji-cell ELISA), anti-nuclear antibody 2,560X (speckled pattern) and anti-SSA antibody 256X. The levels of total protein, IgG and immune complexes were increased in the CSF of this patient. Sural nerve biopsy revealed a decreased number of large myelinated fibers and perivascular lymphocyte infiltration of vasa nervorum. Direct immunofluorescent staining showed deposition of IgG and C3 in the endoneural vessel walls. The increased level of immune complexes were decreased in the sera of the patient, accompanied by remission. These results suggest that immune complexes may be involved in the pathogenesis of a mononeuritis associated with Sjögren's syndrome.

[Immune complexes IN blood and liquor in patients with spinal cord injuries]. / Immunnye kompleksy krovi i likvora u bol'nykh s travmoi spinnogo mozga.

Serum levels of circulating immune complexes (CIC) and those in the liquor were studied in patients with spinal trauma using enzyme immunoassay. CIC and clinical--laboratory findings characterizing the activity of the inflammation were consistent. Assessment of CIC factor in spinal trauma seems clinically valuable as it is involved in inflammation and tissue damage suggesting a prognosis of CIC-related pathology.