Cerebrospinal fluid complement activation in neurological diseases.

Laser nephelometry (LN) is a rapid and very sensitive method for simultaneous determination of albumin, immunoglobulins, C3c and C4 in diluted serum and paired cerebrospinal fluid (CSF) samples. It is very useful in routine analyses. Determination of C3c and C4 covers classical as well as alternative pathways of complement activation. In CSF, they are mostly derived from and related to serum values. Under physiological conditions, the addition of intrathecal C4 synthesis is likely. The incidence of complement activation within CSF is also influenced by the method of choice (native molecules, activation products and complexes, inhibitors) and the mode of interpretation of results according to the functional state of the blood-brain barrier (BBB). Calculation of indexes and the modified Reiber's graph method are valid means of detection of complement activation within CSF. Complement activation within CSF was confirmed in 36% (111/302) of neurological patients examined; in 55% (48/87) of patients with inflammatory and demyelinating diseases, in 40% (37/94) of patients with CNS infections and complications, in 33% (4/12) of patients with motor neuron diseases, in 27% (11/40) of patients with spinal cord compression and sequelae, in 25% (8/32) of patients with neoplastic disease, and in 17% (6/37) of patients with cerebrovascular accidents.

Comparison of the values of basic fibroblast growth factor determined by an immunoassay in the sera of patients with traumatic brain injury and enhanced osteogenesis and the effects of the same sera on the fibroblast growth in vitro.

In patients with severe traumatic brain injury, the early healing of fractures is accompanied by hypertrophic callus formation or heterotopic ossifications, which might even result in ankylosis of the affected joints. Analysis of the sera of patients with traumatic brain injury revealed post-traumatic dynamic changes of basic fibroblast growth factor immunoreactivity, similar to those observed during fracture healing associated with enhanced osteogenesis. The aim of this study was to determine whether such changes in basic fibroblast growth factor concentrations could be related to the phenomenon of enhanced osteogenesis. Basic fibroblast growth factor immunoreactivity was determined (using an IEMA kit) in the sera of patients with traumatic brain injury and bone fractures (n = 8) and in the sera of patients with either traumatic brain injury alone (n = 10) or bone fractures alone (n = 7), and the effects of these sera on L929 fibroblast growth were analysed in vitro. The results did not prove a causative relationship between the changes of basic fibroblast growth factor immunoreactivity and in vitro growth promoting effects of the sera. However, it is apparent that, in addition to changes in the growth-promoting activity and basic fibroblast growth factor concentration of serum, other as yet unknown post-traumatic changes can cause enhanced osteogenesis.

Interleukin 4, IgG and oligoclonal IgG in aqueous humor of cataract patients.

We measured interleukin 4, total IgG, IgG-albumin relative concentration ratio, and oligoclonal IgG in aqueous humors of patients with uncomplicated senile cataracts and cataracts complicated by previous uveitis of unknown etiology. The values of all mentioned parameters are significantly elevated in aqueous humors of patients with complicated cataracts. The possible implications of our findings are briefly discussed.

Precipitation and characterization of an aluminosilicate from AlCl3-Na2SiO3-HCl in serum, of interest for Alzheimer disease.

A precipitation experiment was performed with human serum to model aluminosilicate formation in brains of patients with Alzheimer disease. Aluminum and (or) silicate ions were added to serum in a 1:2 molar ratio at pH 7.4. Precipitates formed immediately and were left for 24 h at 37 degrees C before filtration. Silicate and aluminosilicate formed precipitates with human serum proteins albumin, transferrin, and IgG. In untreated samples, the IgG/albumin ratio increased slightly compared with the ratio in dried serum. Diethylbarbiturate-washed precipitates had a significantly lower protein content than did untreated ones. The IgG/albumin ratio increased considerably in the sample containing aluminosilicate. We conclude that IgG is the sodium dodecyl sulfate-soluble human protein most firmly bound to the aluminosilicate matrix. From 27Al magic-angle-spinning nuclear magnetic resonance (MAS NMR), a pronounced peak was found at 52.79 ppm and a minor peak at 0.53 ppm, suggesting that 4-coordinated aluminum predominates and that 6-coordinated aluminum is present in a smaller proportion. The 29Si MAS NMR spectrum shows a poorly ordered structure. The aluminosilicate formed also contains the cations Na+ > K+ > Ca2+ > Mg2+ and anions Cl- > PO4(3-). Rather than looking for aluminum toxicity to explain the effects of Alzheimer disease, one should consider that by precipitating such a composite phase, the balance of cations, anions, and proteins in human serum is changing.

Diagnostic significance of methemoglobin determination in colorless cerebrospinal fluid.

The presence of various heme derivatives can be demonstrated spectrophotometrically in colorless cerebrospinal fluid (CSF). Because of the high sensitivity of the method, it may detect compounds that reflect a "traumatic tap" rather than a disease process. However, the presence of methemoglobin excludes the possibility of a hemorrhagic CSF being caused by traumatic lumbar puncture. Here we describe a highly sensitive spectrophotometric method involving measurement at the Soret band (400-420 nm) to detect methemoglobin (greater than or equal to 15%) in trace amounts of hemoglobin mixture (less than 0.3 mumol/L). We demonstrated methemoglobin in colorless CSF samples in 9% of 454 patients with cerebrovascular pathology and in 4% of 449 patients with other neurological diseases (n = 449). In a group of 21 patients with verified acute cerebral hematomas, methemoglobin was confirmed in 66% of colorless CSF samples after ultrafiltration. We conclude that routine spectrophotometric analysis of all CSF samples is very useful, allowing detection of xanthochromic compounds in patients with small cerebral and subdural hematomas as well as in those with minimal subarachnoid hemorrhages, hemorrhagic infarctions, or bleedings from aneurysms and neoplasms.

Gangliosides in the human brain development and aging.

In this study, brain gangliosides in prenatal and postnatal human life were analyzed. Immunohistochemically, the presence of "c"-pathway of gangliosides (GQ1c) in embryonic brain was only recorded at 5 weeks of gestation. Biochemical results indicated a twofold increase in human cortex ganglioside concentration between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except cerebellar cortex, which was characterized by increasing GT1b. In this developmental period, GD3 was found to be localized in the ventricular zone of the cortical wall. After birth, GD1b ganglioside in neuropil of granular cell layer corresponding to growing mossy fibers was expressed in cerebellar cortex. Between birth and 20/30 years of age, a cerebral neocortical difference of ganglioside composition was observed, characterized by lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In cerebellar cortex, GD1b and GT1b fractions decreased with aging.

Gangliosides of human cerebrospinal fluid in various neurologic diseases.

Simultaneous profile determination and quantification of human cerebrospinal fluid (CSF) gangliosides in various neurologic diseases (n = 71) was examined. Gangliosides were extracted with methanol/chloroform from clinically available amounts of CSF (4-5 ml), then separated and quantified by high-performance thin-layer chromatography (HPTLC) and direct densitometry. Based on chromatographic comparison with standards, the percentage of lipid-bound NeuAc positive fractions in 'normal' CSF samples were: GM1 (II3 NeuAc-GgOse4Cer) (3%); GD3 (II3 NeuAc2-Lac-Cer) (4%); GD1a (IV3 NeuAc, II3 NeuAc-GgOse4 Cer) (15%); X1 (3%); GD1b (II3(NeuAc)2-GgOse4 Cer) (16%); X2 (4%); GT1b (IV3 NeuAc, II3(NeuAc)2-GgOse4-Cer) (40%); and GQ1b (IV3(NeuAc)2, II3(NeuAc)2-GgOse4-Cer (15%). Similarity between CSF and CSF and human cerebellar cortex, particularly in proportion of "b" series gangliosides (GQ1b, GT1b, GD1b), could be observed. A higher proportion of GD1a ganglioside, with decreased GQ1b was found in infancy. The total ganglioside content (mean +/- 2 SD) varied between 645-894 micrograms/l. Significant alterations of the CSF ganglioside profile, with an increase in less polar gangliosides, GM3 and GD3, correlated with the blood-brain barrier dysfunction (CSF hemorrhages, compressive syndrome), or some malignant processes (metastatic brain melanoma). A statistically significant increase in the content of total CSF gangliosides was found in the following groups of patients as compared to controls: (1) ischemic cerebrovascular accident (CVI) with good outcome (P less than 0.02); (2) peripheral neuropathy and polyneuropathy (P less than 0.001) and (3) intravertebral discopathy (P less than 0.05). A significant decrease in the content of total CSF gangliosides was found in CVI group with lethal outcome (P less than 0.05).

Determination of gangliosides in human cerebrospinal fluid by high-performance thin-layer chromatography and direct densitometry.

A method for the separation and quantification of a complex ganglioside mixture from a clinically available amount (5 ml) of human cerebrospinal fluid (CSF) is described. After reduction of the CSF volume by ultrafiltration, gangliosides are extracted with methanol/chloroform, then separated and quantified by high performance thin layer chromatography (HPTLC) and direct densitometry. For purification of crude ganglioside extract, the method of choice was microdialysis against water. Recovery for the present method including all methodological steps was 78%. No delective loss of gangliosides was demonstrated. The CSF ganglioside pattern from 'normal' CSF samples resembles that of brain gangliosides, particularly cerebellum gangliosides. Based on chromatographic comparison with standards, the percentages of lipid-bound NeuAc-positive fractions were: GM1 = II3NeuAc-GgOse4Cer (3%), GD3 = II3NeuAc2-Lac-Cer (3%), GD1a = IV3NeuAc,II3NeuAc-GgOse4Cer (15%), X1 (3%), GD1b = II3(NeuAc)2-GgOse4Cer (16%), X2 (3%), GT1b = IV3NeuAc,II3NeuAc2-GgOse4-Cer (41%), and GQ1b = IV3NeuAc2-,II3NeuAc2-GgOse4-Cer (16%). The total ganglioside content varied between 616-944 micrograms/l. Within-run and between-run assay precision (relative standard deviation) for 'normal' pooled CSF ranged from 0.04 to 0.12 for the predominant CSF ganglioside fractions (GD1a, GD1b, GT1b, GQ1b), and from 0.13 to 0.23 for the less pronounced fractions (GM1, GD3).

Study of serum and cerebrospinal fluid enzymes in diagnosis and differential diagnosis of cerebrovascular diseases.

In the paper are presented the results of total enzyme activity investigation: GOT, GPT, LDH and CPK, and of the CPK isoenzymes in the cerebrospinal fluid of 148 examinees and in the serum of 67 examinees with an acute stroke, who were treated at the Intensive Care Unit of the Department of Neurology and Institute of Neuropathology, Clinical Medical center "Rebro". The aim was to determine the reliability of the applied methods in the diagnosis of cerebrovascular diseases, particularly in the differential diagnosis of cerebral hemorrhage, ischemia and subarachnoidal hemorrhage. The highest frequency of pathologic findings of the tested enzymes in the whole group of patients with CVA was obtained in the determination of the CPK total activity assessment, then followed the assessments of LDH activity, isoenzyme CPK profile, GOT and finally GPT activity. A larger number of pathologic findings of all mentioned enzymes and CPK isoenzymes was found in the group of patients with ICH. In the patients with ICH and ISI, who survived stroke a higher incidence of normal findings of the total enzymic activities was found, while in those who died from ICH a higher incidence of pathologic findings could not be established. The correlations between the total CPK activity in the serum and in the cerebrospinal fluid does not exist, as well as the correlation between the CPK isoenzyme profile in the serum and CSF.

Measurement of intraocular IgA and IgM synthesis and filtration through the blood-aqueous barrier in cataract patients.

The authors have tested two formulas by means of which the filtrated and the intraocularly (intrathecally) synthesized fraction of particular protein can be distinguished. The laser nephelometric method was applied to determine the serum (IgG, IgA, IgM, C3, albumin), aqueous humor (IgA, IgM, albumin) and CSF (IgG, IgM, C3, albumin) protein values in 42 subjects to test two models in physiological conditions. It was confirmed that the best results were achieved using the formula after Stambuk. Computer simulation performed to compare the formulas after Stambuk and Reiber & Felgenhauer showed a strong correlation (r greater than 0.98) between the calculated filtrated fractions of both models in a wide range of different barrier permeabilities. Laser nephelometry proved to be a useful method to determine low IgA and IgM values in aqueous humor.

Detection of IgG oligoclonal bands in unconcentrated CSF by isoelectric focusing in ultrathin polyacrylamide gel, direct antiserum immunofixation and silver nitrate staining.

Isoelectric focusing of proteins in ultrathin polyacrylamide gel (0.4 mm), followed by direct immunofixation with monospecific antisera and silver nitrate staining, is a highly specific, sensitive and simple method for the detection of oligoclonal IgG in unconcentrated CSF samples. The ultrathin polyacrylamide gels have several advantages, i.e. significantly smaller amounts of reagents are required, and thinner gel can be more efficiently cooled, resulting in higher resolution and shorter running, washing, staining and destaining times. Direct immunofixation in the gel, a time-saving and simple step, increases the sensitivity and specificity of the method. We reduced the samples to 5-10 microliters. For the present method, the optimal concentration of IgG was 0.025-0.030 g/l. It is possible to detect oligoclonal IgG bands at an IgG concentration corresponding to the applied amount of 80-100 ng. In our testing of this method, oligoclonal bands in CSF specimens were clearly demonstrated in 33 (97%) out of 34 patients with definite multiple sclerosis, in 16 (42%) out of 38 patients with infectious diseases of the central nervous system and in 11 (18%) out of 58 patients with other neurological disorders. The method appears to be a useful alternative for the demonstration of oligoclonal IgG bands in unconcentrated CSF samples, and can be recommended for use in the CSF laboratory routine.

Detection of oligoclonal IgG bands in unconcentrated CSF in multiple sclerosis and other neurological diseases by isoelectric focusing on ultrathin-layer polyacrylamide gel immunofixation and silver staining.

Isoelectric focusing of proteins (IEF) in ultrathin-layer polyacrylamide gel (0.4 mm, PAG), followed by direct immunofixation with monospecific antiserum and silver staining, is a highly specific, sensitive and simple method for the demonstration of oligoclonal IgG in unconcentrated cerebrospinal fluid (CSF) samples (5-10 microliters). For the present method, the optimal concentrations of IgG in CSF samples are about 0.025-0.030 g/l, corresponding to the applied amount of 125-150 mg. In our testing of this method, oligoclonal IgG bands in CSF specimens were clearly demonstrated in 52 (96%) of 54 patients with clinically established definite diagnosis of multiple sclerosis (MS), in 4 (40%) of 10 patients with infectious diseases of the CNS, and in 9 patients (25%) of 38 with other neurological diseases. Abnormal patterns were also demonstrated in the serum of patients with MS (43%). Intrathecally synthesized IgG was mathematically calculated in 43 (80%) out of 54 patients with MS. This method appears to be a useful alternative for the demonstration of oligoclonal IgG bands in the unconcentrated CSF, especially when questionable or negative results arise by routine electrophoretic technique for oligoclonal bands detection.