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).

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.

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.

Identification and characterization of immune complexes in the cerebrospinal fluid of patients with spinal cord schistosomiasis.

The pathogenesis of neuroschistosomiasis is largely unknown. Available evidence suggests that it depends on the presence of parasite eggs in the nervous tissue and on the host's immune response. We investigated the presence of immune complexes (ICs) in the cerebrospinal fluid (CSF) of four patients with spinal cord schistosomiasis (SCS), and performed their characterization. ICs containing soluble egg antigen of Schistosoma mansoni (SEA) were found in the CSF of all the SCS patients. To our knowledge, this is the first evidence of ICs containing schistosomal antigens in the CSF of patients with SCS. Further studies are necessary to confirm our findings and investigate the possible roles of ICs in the pathogenesis of this disease.

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.

Immune complexes of auto-antibodies against A beta 1-42 peptides patrol cerebrospinal fluid of non-Alzheimer's patients.

The diagnostic potential of large A beta-peptide binding particles (LAPs) in the cerebrospinal fluid (CSF) of Alzheimer's dementia (AD) patients and non-AD controls (nAD) was evaluated. LAPs were detected by confocal spectroscopy in both groups with high inter-individual variation in number. Molecular imaging by confocal microscopy revealed that LAPs are heterogeneous superaggregates that could be subdivided morphologically into four main types (LAP 1-4). LAP-4 type, resembling a 'large chain of pearls', was detected in 42.1% of all nAD controls but it was virtually absent in AD patients. LAP-4 type could be selectively removed by protein A beads, a clear indication that it contained immunoglobulins in addition to beta-amyloid peptides (A beta 1-42). We observed a close correlation between LAPs and immunoglobulin G (IgG) concentration in CSF in controls but not in AD patients. Double labeling of LAPs with anti-A beta and anti-IgG antibodies confirmed that LAP-4 type consisted of A beta and IgG aggregates. Our results assign a central role to the immune system in regulating A beta1-42 homeostasis by clustering this peptide in immunocomplexes.

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.

Antithyroid antibodies in the CSF: their role in the pathogenesis of Hashimoto's encephalopathy.

Antithyroid antibodies and circulating immune complexes (CIC) were found in the CSF of six patients with Hashimoto's encephalopathy (HE) but not in the CSF of 21 controls. The synthesis of autoantibodies and CIC was intrathecal and their titers were independent of the patients' clinical status or therapy. Their presence in the CSF of patients with acute or subacute encephalopathy may be useful in diagnosing HE.

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.

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.

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.

[Cerebrospinal fluid sorption in the system of complex treatment of chronic cerebral ischemia]. / Likvorosorbtsiia v sisteme kompleksnogo lecheniia khronicheskoi tserebral'noi ishemii.

The cerebrospinal fluid was investigated in 16 patients with chronic cerebral ischemia. Reactions of the local immune system of the liquor was shown to change by the autoimmune type. Medical efficiency of cerebrospinal fluid sorption was proved and it can be considered a method of detoxication aimed at breaking the pathogenetic chain: formation of abundance of the autoantibodies--increased amount of the circulating immunocomplexes--damage of the cell membranes--discharge of deep antigens--appearance of a new generation of autoantibodies. Using cerebrospinal fluid sorption as a test for the detection of latent functional reserves of the neurons not changed irreversibly in the zone of reduced perfusion of the cerebral tissue is thought to be a perspective method.

Significance of mycobacterial immune complexes (IgG) in the diagnosis of tuberculous meningitis.

SETTING: Tuberculous meningitis (TBM) has high mortality, especially in children. Early accurate diagnosis and adequate treatment would reduce this mortality. Diagnosis of TBM remains an enigma because of low cerebrospinal fluid (CSF) culture positivity for Mycobacterium tuberculosis and weak clinical correlation with conventional immunoassays. OBJECTIVE: To evaluate significance of mycobacterial immune complexes (IgG) and anti-mycobacterial antibodies in the diagnosis of TBM. METHOD: CSF from TBM patients and various types of other neurological (both infectious and non-infectious) and non-neurological cases was studied for the presence of IgG and anti-mycobacterial antibodies using antigen capture (by anti-BCG) and multilayered ELISA (using M. tuberculosis soluble extract), respectively. RESULTS: IgG in CSF could be detected in 33 of 55 (60%) and anti-mycobacterial antibodies in 30 of 55 (55%) TBM cases. Presence of IgG, anti-mycobacterial antibodies or both could be detected in 45 of 55 (82%) of the TBM cases. Excepting three of the pyogenic meningitis CSF, none of the infectious (49), non-infectious neurological cases (30) and non-neurological controls (32) showed the presence of IgG or anti-mycobacterial antibodies. CONCLUSION: Detection of IgG along with anti-mycobacterial antibodies aids in diagnosis of a large proportion of TBM cases.

Diagnosis of tuberculous meningitis: a comparative analysis of 3 immunoassays, an immune complex assay and the polymerase chain reaction.

OBJECTIVE: To compare 3 immunoassays, an immune complex assay, and an application of the polymerase chain reaction (PCR) for the diagnosis of tuberculous meningitis (TBM). MATERIAL: Cerebrospinal fluid (CSF) from 33 patients with TBM and from 34 control patients with infectious and non-infectious CNS diseases was analysed. RESULTS: The antibody immunoassays were either nonspecific or insensitive. However, detection of mycobacterial IgG immune complexes correlated strongly with infection, as they were detected in the CSF from 64% of the patients with TBM compared to only 3 (9%) of the control samples. PCR analysis, using Mycobacterium tuberculosis-specific oligonucleotide primers, also strongly correlated with infection, as DNA was amplified from 54% of the samples from patients with TBM, but from only 2 (6%) of the control samples. Both 'false positive' samples were also positive in the immune complex assay and came from 2 patients with otogenic brain abscesses. It is conceivable that these patients suffered from otogenic tuberculosis with secondary non-mycobacterial meningitis. When combining the immune complex assay with DNA-detection by PCR, 100% of the culture positive and 74% of culture negative samples were found to be positive, while maintaining a high specificity. CONCLUSION: Parallel analysis to detect mycobacterial immune complexes and M. tuberculosis-specific DNA by PCR from the CSF of patients may offer a sensitive and specific tool for the diagnosis of TBM.

[The immunological reactions and enzymatic activity of the cerebrospinal fluid in intracranial complications following neurosurgical operations]. / Immunologicheskie reaktsii i énzimaticheskaia aktivnost' tserebrospinal'noi zhidkosti pri vnutricherepnykh oslozhnenii posle neirokhirurgicheskikh operatsii.

The specific features of immunological responses and enzymatic activity of cerebrospinal fluid in the development of intracranial infectious complications were outlined in 16 neurosurgical patients after surgical interventions. On days 1-3-5 days after surgery, the immunobiochemical spectrum of cerebrospinal fluid showed substantial changes associated with the impaired permeability of the blood-brain barrier and the stress-induced transition of biochemical functional systems on the minimum functioning during anaerobic energy supply. Increasing lipid peroxidation processes reflect the result of catabolic reactions and energy deficiency, followed by intensive cytolysis of leukocytes and nerve cells. With this, antibody synthesis and great rises in the formation of circulating immune complexes is the essence of sanogenetic mechanisms basically aimed at eliminating the released and bound antigen in the given time interval.

[The intrathecal synthesis of G-class immunoglobulins in neurological cancer patients]. / Sostoianie intratekal'nogo sinteza immunoglobulinov klassa G u neiroonkologicheskikh bol'nykh.

The authors show that brain tumors onset and relevant recurrences are closely related to enhanced synthesis of IgG in the liquor and of circulating immune complexes as well as to blocking factors registered in the liquor. The above facts illustrate autoimmune reactions involved in pathogenesis and recurrent growth of brain tumors. Surgical removal of the latter inhibits the intensity of IgG intrathecal synthesis.

Detection of immune complexes in the CSF of Japanese encephalitis patients: correlation of findings with outcome.

This paper reports the direct evidence for the presence of Japanese encephalitis virus (JEV)-specific immune complexes in the CSF of 31/185 confirmed patients for the first time. A monoclonal antibody-based capture ELISA was used for the detection of immune complexes. Amongst the 31 cases positive for immune complexes, 14 were positive for JEV IgM antibodies and/or neutralizing antibodies in the CSF, 6 were positive for viral antigen in the CSF and 11 for both antibody and antigen. Correlation of findings to final outcome revealed that the presence of immune complexes in CSF was significantly associated with death (p = 0.01).

Dermal, serological and CSF changes in amyotrophic lateral sclerosis with and without intrathecal interferon beta treatment.

In 12 patients with amyotrophic lateral sclerosis (ALS) participating in a therapeutic trial with intrathecally applied human fibroblast interferon-beta (IFN-beta) and in 9 untreated ALS patients, we found significantly elevated circulating serum IgG immune complexes (CIC), quantitative immunoglobulin changes, and creatine kinase (CK) elevation; CK reached significantly more often pathological levels in non-bulbar disease. Dermal ultrastructural changes were equally present in all treated as well as untreated ALS patients. Some time ago IL-6 was quantitatively cleaned out of the Fiblaferon-preparation. Erythrocyte sedimentation rate (ESR) rose during intrathecal IFN therapy in 9/10 ALS patients. In 4/4 adequately monitored motoneuron patients, this elevation coincided with a decrease of serum CK, while ESR and CK did not correlate in 60 non-ALS non-IFN neurological controls. Collagen ultrastructure, CSF total protein or barrier function, immune complexes, immunoglobulin quantitation and serum CK may contribute to differentiated diagnosis and should be included in future study protocols.