Molecular heterogeneity, detected by two electrophoretic micro procedures, of IgG in human cerebrospinal fluid (CSF).

Semi-automated electrophoretic procedures in the PhastSystem (Amersham Pharmacia Biotech) with micro polyacrylamide gels (PAGs) and SDS-PAG gradients were modified to analyze IgG in human cerebrospinal fluid (CSF) and matched serum samples with respect to the molecular IgG structure L-H-H-L. Isoelectric focusing (IEF) with specific immunofixation detected discrete IgG bands in CSF standing out against a polyclonal and monoclonal background pattern in CSF and serum; they were denoted oligoclonal bands (IgG OBs) (OB assay positive) indicating IgG synthesis in the central nervous system (CNS) of patients with subacute and chronic processes of inflammatory CNS disorders; assay was negative with identical (mirror) bands in CSF and serum for other CNS processes. IgG OBs were specified as lambda (kappa) IgG subfractions, precipitated with the anti-light (L) chains lambda (kappa) and anti-heavy (H) chain fragments (Fd, Fc, C(H)2) as well as with anti-F(ab')(2), and as duplex IgGs with kappa and lambda OBs at the same pI. With SDS-PAG gradient electrophoresis and specific immunofixation more than six IgG fractions were detected and classed according to apparent molecular weights of a S-sulfonated human IgG standard; they were characterized with the monospecific antibodies against the L and H chain fragments as 25, 50, 75, 100, 125 and 150 kD fractions containing combinations of L and H chains as well as mixtures of both L and H chain fragments of varying dimensions. Generally, this molecular IgG heterogeneity could not be connected with the IgG OB heterogeneity revealed by IEF; but single OBs in the strongly alkaline pH region of PAG may correspond to H fragments with basic pI. Nevertheless, evidence for the existence of both free L chains and the free H chain were revealed as specific OBs with IEF and with the anti-L and anti-H antibodies in the 25 kD and 50 kD fractions, respectively, of CSF samples of six patients with diverse CNS diseases. Further experiments are needed to elicit the origin of the molecular IgG heterogeneity during the immune response of subacute and chronic inflammatory processes in human CNS.

Quantification of beta-trace protein and detection of transferrin isoforms in mixtures of cerebrospinal fluid and blood serum as models of rhinorrhea and otorrhea diagnosis.

Different mixtures from a serum pool and a cerebrospinal fluid (CSF) pool were used as models to study CSF contamination in secretions by determining two CSF specific proteins: beta-trace protein (beta-TP) and the asialo-transferrin (a-Tf) band which was detected by isoelectric focusing (IEF) with Tf specific immunofixation. Beta-TP and total Tf were measured immunonephelometrically. Secretion/serum ratios of beta-TP content > 2.0 indicated CSF contaminations with > or = 5% (v/v) CSF; this was confirmed by detecting the a-Tf band by IEF. Reliable a-Tf bands were only revealed with secretion/serum rations of Tf contents < 0.1, indicating an interference of major sialo-Tf fractions with the a-TF band detection in the sample. For CSF detection in rhinorrhea and otorrhea, complementary use of beta-TP assay and a-Tf assay is recommended. Preanalytically, dilution or concentration of the sample as well as denaturation of Tf and beta-TP should be prevented by optimizing sample collection.

Flow cytometry of cerebrospinal fluid (CSF) lymphocytes: alterations of blood/CSF ratios of lymphocyte subsets in inflammation disorders of human central nervous system (CNS).

Flow cytometry was adapted to measure lymphocytes in human cerebrospinal fluid (CSF). The method was sufficiently precise, reproducible and accurate despite low cell counts. In lumbar CSF of controls with 500 to 3500 (10(3)/l) leukocytes, lymphocyte counts correlated with those in corresponding venous blood: blood/CSF ratios of approximately 2000 : 1 were found for total T cells (CD3+) and CD3+ HLA-DR-, CD3+4+, CD3+8+ subsets, ratios were increased for the lymphocyte subsets CD3+ HLA-DR+ < or = CD3+16+56+ < CD16+56+3- < CD8+3- << CD19+; CD8+4+ ratio was half of CD3+ ratio. Data indicate selective barriers (blood-brain and blood-CSF barriers) to blood lymphocyte subsets which favor the transfer of T subsets. Correlation of the subset ratios to the CD3+ ratio indicates distinct barrier properties which changed differently with acute and subacute inflammations and neuroimmunological diseases of central nervous system (CNS) in lumbar or ventricular CSF, but not with simple protein barrier disturbance. HLA DR+ T ratios were higher than HLA DR- T ratios only with controls and some neuroimmunological diseases. Lymphocyte barrier characteristics were related to protein leakage situated at the same barriers, indicating for the lymphocyte subsets selective transfer routes in control subjects and non-selective routes in patients with CNS inflammation where altered ratios revealed a mixture of both routes.

[Early summer meningoencephalitis. Extension of the endemic area to mid-Hessia]. / Frühsommermeningoenzephalitis (FSME). Ausbreitung des Endemiegebietes nach Mittelhessen.

Tick-borne encephalitis (TBE) is the most severe arbovirus disease transmitted by ticks. The mortality of the central European form is 0.7-2%. Active immunisation is recommended for endemic regions. We report on 4 patients with TBE acquired in Middle-Hessen between 1994 and 1997 (2 in 1997). After repeated CSF and serum testing the TBE-specific antibodies were found in all 4 cases. In one case there was also evidence for a prior infection with borrelia burgdorferi. The results of the initial CSF-analysis were atypical in 2 cases (high cell count of 136 cells/mm3, total protein up to 1.5 g/l). The endemic region for TBE has expanded in northern direction into Middle-Hessen, a region in which Lyme borreliosis is also endemic. Thus, true double infections are possible. This and the initially frequently atypical CSF-findings make the differential diagnosis difficult. Therefore, repetitive CSF and blood examinations are recommended.

Validation by isoelectric focusing of the anion-exchange isotransferrin fractionation step involved in determination of carbohydrate-deficient transferrin by the CDTect assay.

Serum concentration of carbohydrate-deficient transferrin (CDT) is used for laboratory diagnosis of chronic alcohol abuse. Using isoelectric focusing for validation of the initial isotransferrin fractionation step involved in the determination of CDT by the CDTect assay, we found a complete in vitro iron saturation of transferrin and sufficient stability of the transferrin iron load during column passage; effective separation of non-CDT-isotransferrins and CDT-isotransferrins at the microcolumns; partial coelution of trisialo-Fe2-transferrin, which did not significantly affect CDT measurement; partial retention of CDT-isotransferrins, especially disialo-Fe2-transferrin, which may cause falsely negative results for CDT at the upper reference limits; good precision of the isotransferrin fractionation step; and no significant effects of low concentrations of serum protein and transferrin. We strongly urge standardization of CDT analysis and suggest isoelectric focusing for validation of CDT analysis methods and verification of odd results.

Increased serum concentration of carbohydrate-deficient transferrin in patients with combined pancreas and kidney transplantation.

Serum concentration of carbohydrate-deficient transferrin (cCDT) is used for laboratory diagnosis and follow-up of chronic alcohol abuse. In analyzing by CDTect-RIA (Pharmacia) sera from outpatients with combined pancreas and kidney transplantation and no excessive alcohol consumption, we found above-normal values for cCDT and CDT/transferrin ratios (CDT/Tf) in more than half of the samples. Isoelectric focusing of these samples showed distinct bands of carbohydrate-deficient isotransferrins, supporting the abnormal findings from the CDTect assay. In contrast, diabetics and outpatients who had received only kidney transplants showed normal values for cCDT, CDT/Tf, and isotransferrin patterns. Increased serum Tf, sialidase-producing microorganisms, and immunosuppressive medication were eliminated as causes of these abnormal cCDT and CDT/Tf results. Successful pancreas transplantation leads to hyperinsulinemia and normoglycemia, in contrast to hypoinsulinemia and hyperglycemia in the patients who receive kidney transplants alone. These factors may have pathogenic importance for CDT increase, yielding results falsely interpreted as positive with respect to alcohol abuse in patients with combined pancreas and kidney transplantation.

Effect of separation conditions on automated isoelectric focusing of carbohydrate-deficient transferrin and other human isotransferrins using the PhastSystem.

To investigate the effect of automated isoelectric focusing conditions in the PhastSystem, e.g., the point of sample application, prerun and separation times, and minimized gels on isotransferrin band pattern, human sera were analyzed with native transferrin iron load, after iron saturation or iron depletion in vitro. Varying the focusing conditions we found (i) Point of sample application (anode, middle of the gel, cathode) strongly affected transferrin iron loss. It was greatest at the anode and least at the cathode. (ii) Without prerun, distinct transferrin iron loss also occurred. A short prerun time prevented iron loss, but increasing it did not improve transferrin iron load stability as stated by others. (iii) An inappropriately long separation time inevitably yielded iron loss. In conclusion, inappropriate isoelectric focusing conditions strongly affect iron load stability of isotransferrins (obviously via low pH within the gel), resulting in transferrin iron release and cofocusing of isotransferrins with different sialic acid or iron contents. For determination of carbohydrate-deficient transferrin, such conditions resulted in overestimation of the marker of chronic alcohol abuse. Our findings may be of guiding importance for isoelectric focusing of protein-ligand complexes. We recommend the procedure described for development of isoelectric focusing of protein-ligand complexes.

Apolipoprotein AI, AII, and AIV isoforms in plasma determined by automated isoelectric focusing with PhastSystem and immunofixation.

Distinct genetic variants of apolipoprotein (apo) AI have been shown to influence concentrations of high-density lipoprotein (HDL) cholesterol. The genetic polymorphism of apo AIV may modulate HDL-cholesterol, plasma triglycerides, and lipoprotein(a) concentrations. There is evidence for an antagonizing role of apo AII in reverse cholesterol transport. Since genetic polymorphisms and variants of these apolipoproteins are detectable by isoelectric focusing (IEF), we developed a rapid and easy automated method for IEF analysis of apos AI, AII, and AIV on self-made or commercially available gels, using the PhastSystem. Diluted plasma or serum samples (1 microL) are applied automatically onto the gel and IEF is carried out for 35-45 min. Afterwards, the apo A bands are precipitated by specific polyclonal antibodies and visualized by automated silver staining. This rapid procedure is suitable as a routine or screening method for IEF analysis of these major HDL apolipoproteins.

Laboratory diagnosis of radicular and pseudoradicular syndromes in cerebrospinal fluid (CSF): reliability of methods in consideration of pathogenetic aspects. Report on section "CSF diagnosis" of 4th Klagenfurter Neurology Workshop Conference on radicular and pseudoradicular syndromes, Klagenfurt, Austria, August 28-29, 1992.

Laboratory tests may be used to confirm the clinical differentiation of pseudoradicular syndromes and radicular syndromes. In the presence of pseudoradicular syndromes, CSF and blood samples yield no positive results with either non-specific or specific methods. Radicular syndromes give rise to positive findings; using non-specific methods they can be subdivided into inflammatory and non-inflammatory forms, with and without blood-nerve barrier impairment. Non-specific quantities of CSF routine diagnosis are total protein, albumin, leukocyte counts and differential cell count, L-lactate, intrathecal -IgG, -IgA, -IgM and immunoglobulin-class oligoclonal bands. Oligoclonal bands enable the highly sensitive differentiation of non-inflammatory from subacute-chronically inflammatory forms of radicular syndromes. Most of the specific quantities are the subject of current research, e.g. bacterial antigens, D-lactate, cultivation tests, polymerase chain reaction tests and pathogen-specific oligoclonal bands. Pathomechanisms affecting the permeability of the blood-nerve barrier to increasing concentrations of protein and to leukocyte subsets possibly explain the CSF findings in radicular and pseudoradicular syndromes.

Rapid determination of apolipoprotein E phenotypes from whole plasma by automated isoelectric focusing using PhastSystem and immunofixation.

The influence of the genetic apolipoprotein (apo) E isoforms on human plasma lipoproteins is well established. There is, however, still a need for a phenotyping procedure applicable in laboratories not specialized in lipid research. To this end, we developed a rapid, automated electrophoresis method for apoE phenotyping. Either self-made or commercially available precasted gels can be used. Fresh or frozen samples corresponding to 0.1 microliter of plasma are applied automatically after lipid extraction in a urea-containing buffer onto the gel and isoelectric focusing is carried out for 45 min. Thereafter, apoE bands are precipitated by specific polyclonal antibodies and visualized by automated silver staining. The method is reliable, easily and quickly performed, and not restricted to specialized laboratories.

Transport and production of cerebrospinal fluid (CSF) change in aging humans under normal and diseased conditions.

In three control groups of lumbar (L), cisternal (C), and ventricular (V) cerebrospinal fluid (CSF) with prealbumin contents of ventricular origin (CSF-V-prealbumin) within the reference range, indications for an increased transport of V-CSF into spinal space are presented in the elderly as well as an elevated production of V-CSF. Indications for a diminished V-CSF production, respectively, an increased transport of CSF-V-prealbumin into the spinal space were found in elderly patients suffering from diverse CNS diseases. As these alterations proved to be not disease-specific, the age-related increase of CSF transport was discussed with respect to brain atrophy and enlarged CSF spaces in the elderly central nervous system (CNS). CSF serum prealbumin content, an indicator of blood-brain-barrier function, increased with age similarly to albumin in the "normal" elderly, respectively with diverse CNS diseases. Age- and disease-related decreases of albumin and prealbumin contents in blood serum may lead to wrong interpretations.

Age-related alterations of the blood-brain-barrier (bbb) permeability to protein molecules of different size.

Transfer processes of differently sized protein molecules across the blood-brain-barrier (bbb) were studied in "normal" , respectively, diseased elderly humans. Lumbar CSF/serum ratio of albumin contents increased significantly with age in a control group, but it was diminished in patients suffering from inflammatory or non-inflammatory diseases. Ratio of alpha 2-macroglobulin contents increased with age, especially in the diseased elderly. Concentration ratios of lysozyme, orosomucoid, lactoferrin, IgG, IgA and IgM decreased with age in a control group and especially decreased in a patient group. The data are discussed with respect to two different transfer processes which appeared to be altered in age and/or disease processes.

On the detection of a blood-joint barrier for radiosulfate and [3H]glucosamine in single joints of aging rats.

A blood-joint barrier effect has been detected for two precursors of glycosaminoglycan biosynthesis (35SO4 and [3H]glucosamine) in single joints of adult Wistar rats by calculating the ratio between their blood plasma levels in the equilibrium range and their incorporation rates into joint tissues of knee joints (tibia and femur parts, patella) or femur and humerus heads. The barrier effect appears to be joint specific in the adult rat, because it was not found in joints of one month old rats and in tendon tissue. In 17 months old rats the barrier effect increased indicating a diminished supply of the joints with both precursors. Incorporation rates into the joint tissues, however, were higher than into the isolated glycosaminoglycans of aged rats indicating the barrier effect to be not the only cause for a diminished glycosaminoglycan biosynthesis in joint cartilage of aging rats.

Classical and modern methods of cerebrospinal fluid analysis. Report on the First All-German Symposium of the Society for Laboratory Medicine (FRG) and the Study Group for CSF Analysis and Clinical Neurochemistry of the Society for Psychiatry and Neurology (formerly GDR) in Marburg a. d. Lahn, October 5-6, 1990.

The aim of the symposium was to prepare an inventory of cerebrospinal fluid (CSF) analysis used in Germany, and to evaluate them in comparison with modern methods. From the large field of CSF analysis, four main topics were selected, all related to the practical application of the methods. The following conclusions were drawn: Classical techniques of cytodiagnosis are clinically important. Therefore, manual and mechanized techniques must be further improved with respect to counting, collection, and differentiating of CSF cells. As cytokines and complement factors are early mediators of diverse processes in CNS, highly sensitive techniques must be developed for their routine analysis e.g. in CNS inflammation. Recent efforts to detect specifically viral and bacterial agents (e.g. by polymerase chain reaction, Particle Counting Immuno-Assay, Enzyme Immuno-Assay) or antibodies (e.g. affinity-mediated immunoblot, specific antibody index) in CSF must be continued in order to develop definite and practicable assays for daily routine. For the detection of intrathecally produced antibodies, qualitative procedures appear to be more reliable then quantitative ones, provided that the former are highly sensitive and specific.

In-vivo biosynthesis of acid glycosaminoglycans with radiosulfate and [3H]glucosamine in single joints from rats of different age.

Administration intraperitoneally of 35SO4 plus D-[6-3H]glucosamine of similarly specific radioactivity to rats of different ages showed uptake rates and incorporation to be higher with 35SO4 in articular cartilage. Comparing the joints studied: knee joints (femur and tibia parts), humerus, and femur heads, cartilage cells appeared to be most metabolically active (age-dependently) in femur heads. All cells, in addition to glycoproteins, synthesized in vivo isomeric chondroitin sulfates and hyaluronate at different rates.

Flattening and/or expanding of pH gradients in isoelectric focusing gels exemplified with PhastSystem.

A simple method of flattening and/or expanding of pH gradients in isoelectric focusing is described for any pH interval desired: to modify pH gradients near one electrode a paper strip soaked with carrier ampholytes is applied onto the gel close to the opposite electrode. In order to flatten central parts of pH intervals paper strips are applied onto the gel at both electrodes. Conditions and criteria (e.g. amount and pH intervals of carrier ampholytes, width and localization of the paper strip, separation period) for optimization are presented with PhastSystem using ready-made gels with three different pH intervals and pI marker proteins (Pharmacia). Examples utilizing erythrocyte lysates are presented.

Changes with age in leucocyte counts, protein and IgG content of lumbar cerebrospinal fluid (CSF) of patients with inflammatory, ischemic, and hemorrhagic diseases or tumors of the central nervous system (CNS).

Parameters of blood-brain barrier (BBB) function and of humoral and cellular immune response were determined in lumbar CSF and blood serum samples from 717 patients and statistically compared to "healthy" controls (n = 190). Leucocyte counts (mainly mononuclear cells), total protein and IgG levels in CSF were significantly higher, and prealbumin fraction mostly lower in patients, besides other alterations of protein electrophoresis. Serum fractions (median) of protein electrophoresis were within the reference range. When parameters were correlated with age, a significant increase of total protein of 1.62 mg/dl and of IgG of 0.13 mg/dl per 10 years of age was found in lumbar CSF of controls. Elevated leucocyte counts did not correlate with age of patients, but they increased significantly with age, with respect to controls, to a higher extent in patients with polyneuritis, ischemia and injury; they decreased in patients with brain tumors and polyneuropathy. When compared to controls, elevated total protein levels significantly increased with age only with tumors and injury patients. In multiple sclerosis, IgG increased and beta-globulin fraction decreased significantly in CSF with age. The findings point to age-dependent alterations of BBB and immune response only in some patients groups which were different from those of controls.

Inhibitory and stimulating effects of various types and series of prostaglandins on in vitro biosynthesis of proteochondroitin-4,6-sulfate and protein and anaerobic glycolysis in calf rib cartilage.

The effect of various types of prostaglandins (PGs) have been studied in a semi-in-vitro system with cartilage slices of calf ribs. 0.1 mmol/1 PG B1, D2, E1, E2, F1 alpha, F2 alpha inhibit biosynthesis of Ch-4,6-S protein to a higher extent than 10 mumol/l; 1 and 2 series PG E and F (but not B) inhibit similarly, PG A2 inhibits twice as much. With biosynthesis of total protein 2-series PG A, B, E, F act more inhibitorily than 1-series PG. 10 mumol/l PG A2, E1, E2 produce cAMP-like effects, e.g. acceleration of biosynthesis of Ch-4,6-S protein and total protein as well as of anaerobic glycolysis; PG F2 alpha stimulates the first two anabolic processes, PG B1 only the second one. PG A1 stimulates Ch-4,6-S protein biosynthesis and anaerobic glycolysis, a cGMP-like effect. 20 mumol/l diBu-cAMP or cAMP (together with 0.1 mmol/l theophylline) produce stimulating and inhibitory effects on these three anabolic processes; both compounds produce additively positive or additively negative effects in connection with the inhibitory PG effects on these three anabolic processes.