The demonstration of locally synthesized herpes simplex IgG antibodies in CSF by a Sepharose 4B linked enzyme immunoassay.

A newly developed peroxidase-linked immunoassay is described which is sensitive enough to quantify herpes simplex antibodies in serum and cerebrospinal fluid diluted to an IgG level of 1 mg/dl. Thus, a comparison of photometric signals allows the direct detection of specific antibodies which have been secreted by activated tissue B lymphocytes into the CSF compartment during the humoral immune phase of herpes simplex encephalitis. The technique utilizes urea-Triton-dissolved virus antigens covalently bound to Sepharose 4B pearls. A highly specific sandwich antibody was purified by immune absorption column chromatography and labelled in its protected state. In the majority of cases the antibody level increased around the 10th day, to reach its maximum a few days after. In some cases however the serum levels gradually rose over a period of several weeks. The antibody levels in the CSF increase uniformly at the same time, irrespective of the general immune response and soared up to higher than serum levels within a few days. Local antibody production may persist for years so that late diagnosis of herpes encephalitis becomes possible with a single side by side test of serum and CSF from the patient.

Differentiation of the humoral immune response in herpes simplex encephalitis.

Six patients with herpes simplex encephalitis were investigated. Antibody activities against different viral antigens were determined in serum and cerebrospinal fluid with an enzyme-linked immunosorbent assay after adjusting both fluids to identical immunoglobulin G concentrations. In serum, the strength of the antiviral reaction remained low and no qualitative changes became detectable. In cerebrospinal fluid, at first locally synthesized antibodies were directed against the same antigens as in serum. Ten days later, the intrathecal reaction increased with additional antibodies against at least two antigens. Once established, this expanded heterogeneity remained stable during the course of the disease.

Locally synthesized antibodies in cerebrospinal fluid of patients with AIDS.

Twelve patients with AIDS were examined by enzyme-linked immunosorbent assay for antibody activity of IgG in serum and CSF. Two patients were only anti-HTLV III antibody carriers (stage I), two had lymphadenopathy syndrome (stage II) and eight had manifest AIDS (stage III). Eight of the 12 patients had 2- to 8-fold higher antibody titres in CSF than in serum, indicating that anti-HTLV III antibodies were produced in the nervous system. One of these patients with obviously locally synthesized anti-HTLV III antibodies in CSF belonged to stage I, two to stage II and five to stage III. Only four of these eight patients also showed locally synthesized IgG in CSF as measured by laser-nephelometry. In contrast, 61 controls with normal CSF (12), impaired blood-CSF barrier (12) multiple sclerosis (12) and various infections of the CNS other than HTLV III (25), the last two groups with locally synthesized IgG in CSF, all revealed negative results. It appears possible that locally synthesized anti-HTLV III antibodies in CSF are a sensitive and early indicator of an HTLV III infection of the nervous system.

The detection of activated cerebrospinal fluid B lymphocytes by peroxidase conjugated antibodies.

A method for the preparation of a potent and specific peroxidase-labelled antihuman IgG antibody is described. CSF cells from patients with various neurological diseases were stained. Only in cases of inflammatory diseases was IgG detectable. The stained cells were so-called lymphoid cells. As the demonstrable IgG can be regarded as a product of intracellular synthesis, it is justified to call these cells activated B lymphocytes. The relative portion of activated B cells tends to be high in the first half of the mononuclear phase and depends upon the type of disease. It is low in most cases of bacterial and benign viral meningitis whereas high B cell numbers were found in Bannwarth meningopolyneuritis, mumps meningitis and herpes simplex encephalitis. Albeit there exists a time lag between the maximal number of activated B lymphocytes and the demonstration of the secretory immunoglobulin fraction, quantitative correlation between these two inflammation parameters were demonstrated in cases of acute meningoencephalitis. These correlations were not seen in chronic inflammatory diseases.

Acute encephalitis with intrathecal synthesis of measles antibodies.

In two cases of acute encephalitis, there was an intrathecal antibody synthesis against measles virus, as determined with an enzyme-linked immunosorbent assay. The clinical findings were not consistent with a usual measles infection, subacute sclerosing panencephalitis or acute multiple sclerosis.

Intrathecal humoral immune reaction in zoster infections.

Intrathecal humoral immune reaction in 26 patients with a reactivation of varicella-zoster virus was analyzed. 11 suffered from ganglionitis, in 7 cases an additional affection of the lower motor neuron was demonstrable. In 8 patients, meningitis, myelitis or cerebral infarctions by zoster angiitis were diagnosed. Intrathecal immune reaction in ganglionitis was weak whereas an intense IgG synthesis became demonstrable in all meningomyelitis/cerebral infarction cases. As demonstrated by immunoblotting, in early stages of the disease immune reaction in serum and cerebrospinal fluid differed only quantitatively. In the further course, most intrathecally synthesized antibodies were directed against low molecular antigens whereas serum pattern did not change. In some patients, additional antibodies not detectable in serum were demonstrable. Though intensity markedly differed, no qualitative differences between the immune response in ganglionitis and more widespread zoster infections of the CNS were detectable.

Cerebrospinal fluid virus antibodies. A diagnostic indicator for multiple sclerosis?

Specific antibody activities (antibody per weight unit IgG) of serum and CSF against a broad variety of viruses were compared in multiple sclerosis and certain inflammatory diseases of the central nervous system, e.g. neurosyphilis, as well as herpes simplex and zoster encephalitis. No "unspecific" antiviral activities within the CSF compartment were found in the non-MS diseases. The most frequent antibodies locally produced were directed against measles, rubella and zoster antigens. A diagnostic test with these three viruses would give positive results in about 80% of patients with MS. This finding is not as frequent as the oligoclonal pattern of the CSF gamma-globulins but would have a considerably greater diagnostic significance.

Clinical experience with beta-trace protein as a marker for cerebrospinal fluid.

beta-Trace protein is an immunological marker for the detection of cerebrospinal fluid traces. The aim of the study was to evaluate the predictive value of the beta-trace protein test. We investigated 145 specimens for cerebrospinal fluid using beta-trace protein. All case notes were retrospectively reviewed and tabulated for indication and procedure with focus on outcome. The specimens were analyzed by immunoelectrophoresis. beta-Trace protein was detected in 62 specimens and was absent in 83. In correlation with the clinical course, the clinical findings, the results of high-resolution computed tomography of the paranasal sinuses or petrous bone, computed tomographic cisternography, magnetic resonance imaging, indium 111-pentetic acid radionuclide cisternography, intraoperative findings, and visualization using sodium-fluorescein, there were no false-positive results. In 6 cases, a false-negative result occurred. The overall accuracy was 95.68%. The beta-trace test has a specificity of near 100% and a sensitivity of 91.17%. Analysis of beta-trace protein is a valuable test and has some advantages in comparison with the beta2-transferrin assay.

Traces of perilymph detected in epipharyngeal fluid: perilymphatic fistula as a cause of sudden hearing loss diagnosed with beta-trace protein (prostaglandin D synthase) immunoelectrophoresis.

The incidence of perilymphatic fistula as cause of sudden hearing loss is not known. We present a case with sudden unilateral hearing loss associated with a positive beta-trace protein test of an epipharyngeal fluid sample. The patient presented with sudden sensorineural hearing loss on the right side. A stapedotomy had been performed nine months previously due to otosclerosis. Intravenous therapy for the treatment of sudden hearing loss was unsuccessful. At the time of sudden hearing loss, epipharyngeal fluid was collected using a Raucocel sinus pack. Investigation using rocket immunoelectrophoresis showed the presence of beta-trace protein. Upon repeating tympanoscopy there was no obvious labyrinthine fluid egress, but the oval window was sealed with fibrin sponge and fibrin glue. The patient's hearing improved over a period of five months.

Low molecular solutes and the blood cerebrospinal fluid barrier.

An attempt is made to evaluate those portions of low molecular hydrophilic solutes that obey apparent passive transfer principles at the blood cerebrospinal fluid (CSF) barrier under steady state conditions. Twenty-four electrolytes, metabolites, and amino acids are determined in serum and CSF obtained simultaneously. The correlation found between the serum/CSF concentration ratios and the molecular radii of most compounds is indicative of the prevalence of passive transfer processes in CSF formation, as has been already demonstrated for proteins. The level of some constituents in the primary filtrate may be secondarily increased by influx from the adjacent extracellular space, e.g. glutamine and creatinine, or decreased by specific uptake from the CSF compartment, e.g. glycine. The CSF levels of all compounds are dependent upon the actual state of the blood CSF barrier as evaluated by the serum/CSF concentration ratios of albumin and alpha 2-macroglobulin. The results do not support the assumption that CSF may be used to test the permeability state of the blood brain barrier propery.

Beta trace-protein as marker for cerebrospinal fluid fistula.

The detection of beta trace-protein with the help of an easy-to-perform Ouchterlony test is strongly indicative of liquorrhea. The protein has been purified from pooled cerebrospinal fluid and an antiserum has been produced in rabbits. An open CSF fistula was diagnosed in 24 cases and there was no false positive result.

Protein analysis of subdural hygroma fluid.

The pathogenesis of posttraumatic subdural hygroma still remains largely unknown. One of the suggested pathological mechanisms is the traumatic development of an arachnoid tear and the subsequent efflux of CSF into the subdural space. We performed a multifactorial analysis of the hygroma fluid obtained at operation in comparison to the simultaneously taken plasma and lumbar CSF. The results of the protein analysis support the CSF origin of the subdural hygroma fluid.

Intrathecal synthesis of virus antibodies: a diagnostic test for multiple sclerosis.

Intrathecally synthesized antibodies against measles and rubella were determined in 221 patients with multiple sclerosis (MS) and in 476 control cases. A local production was detectable in more than 80% of the MS cases but in none of the control group. These results show that the demonstration of intrathecally synthesized antibodies against neurotropic viruses not related with respective infections may serve as helpful test in the diagnosis of MS.

[Beta-trace protein in diagnosis of cerebrospinal fluid fistula]. / Beta-trace-Protein in der Diagnostik der Liquorfistel.

Beta-trace protein is a lipocalin that was recently identified as prostaglandin D synthase and represents a major constituent of human cerebrospinal fluid. Beta-trace protein, similar to beta 2-transferrin, has been used as an immunological marker for the detection of cerebrospinal fluid. Between 1982 and 1999, 130 specimens from 101 patients with suspected cerebrospinal fluid leaks of the anterior or lateral skull base have been investigated for beta-trace protein. The specimens were analyzed by one-dimensional immunoelectrophoresis using Laurrell's electroimmunoassay. In all, beta-trace protein could be detected in 57 specimens and was absent in 73. All case notes were studied retrospectively. In correlation with the clinical course and the findings of CT of the paranasal sinuses, high resolution CT of the petrous bones, CT cisternography, NMR, 111In-DTPA radionuclide cisternography, intraoperative findings, visualization of sodium-fluorescein stained cerebrospinal fluid using endoscopic blue light or testing for glucose, there was no false-positive result. A false-negative result occurred in two cases. These findings show that the beta-trace test has a sensitivity of 97.3% and a specificity of nearly 100%. Our findings show that analysis of beta-trace protein can be a valuable test for detecting CSF leakage and has certain advantages in comparison with the beta 2-transferrin assay.

A new nephelometric assay for beta-trace protein (prostaglandin D synthase) as an indicator of liquorrhoea.

OBJECTIVES: To determine the sensitivity and specificity of a nephelometric beta-trace protein assay for the diagnosis of liquorrhoea. METHODS: One hundred and forty clinical samples with suspected liquorrhoea were analysed by a newly developed nephelometric assay. An established electroimmunoassay served as a reference method. The sensitivity and specificity of the beta-trace nephelometric assay were calculated by a 2x2 contingency table for 10 different versions of a dichotomised nephelometric variable. In 52 patients (79 samples), the nephelometric findings were validated by referring to the clinical diagnosis based on the course of the disease, imaging techniques, and surgical inspection. RESULTS: Given a specificity of 100%, a beta-trace protein concentration of 6 mg/l or higher in a sample indicated liquorrhoea with a sensitivity of 92% compared with the reference method and of 93% compared with the clinical evaluation. The relation between the electroimmunoassay and the nephelometric assay was highly significant (p<0.001). CONCLUSIONS: The nephelometric beta-trace protein assay is a simple and rapid method for the detection of liquorrhoea with high sensitivity and specificity and may facilitate the diagnosis of fistulas leaking CSF.

Concentrations of hepatocyte growth factor in cerebrospinal fluid under normal and different pathological conditions.

Hepatocyte growth factor (HGF) and its specific receptor, MET, are expressed in the developing and adult mammalian brain. Recent studies have shown a neurotrophic activity of HGF in the nervous system. The present study focused on HGF concentrations in the cerebrospinal fluid (CSF) and serum in normal persons and in different central nervous system (CNS) diseases considering blood-CSF barrier (BCB) function. Concentrations of HGF were analyzed using an enzyme-linked immunosorbent assay (ELISA). HGF was present in normal human CSF (346+/-126 pg/ml) representing approximately half of the HGF serum concentrations. The CSF HGF levels were not significantly changed in chronic CNS disease and in aseptic meningitis (419+/-71 pg/ml), but significantly increased in patients with bacterial meningitis (6101+/- 5200 pg/ml). The HGF levels in CSF were not influenced by increased serum concentrations in patients with normal or mildly affected BCB function. The results show that HGF is present in normal CSF and does not appear to cross the CSF barrier significantly unless it is severely disrupted. So far, strong increases of HGF concentration in CSF are only present in acute bacterial meningitis.