Acute Phase Proteins in Cerebrospinal Fluid from Dogs with Naturally-Occurring Spinal Cord Injury.

Spinal cord injury (SCI) affects thousands of people each year and there are no treatments that dramatically improve clinical outcome. Canine intervertebral disc herniation is a naturally-occurring SCI that has similarities to human injury and can be used as a translational model for evaluating therapeutic interventions. Here, we characterized cerebrospinal fluid (CSF) acute phase proteins (APPs) that have altered expression across a spectrum of neurological disorders, using this canine model system. The concentrations of C-reactive protein (CRP), haptoglobin (Hp), alpha-1-glycoprotein, and serum amyloid A were determined in the CSF of 42 acutely injured dogs, compared with 21 healthy control dogs. Concentrations of APPs also were examined with respect to initial injury severity and motor outcome 42 d post-injury. Hp concentration was significantly higher (p<0.0001) in the CSF of affected dogs, compared with healthy control dogs. Additionally, the concentrations of CRP and Hp were significantly (p=0.0001 and p=0.0079, respectively) and positively associated with CSF total protein concentration. The concentrations of CRP and Hp were significantly higher (p=0.0071 and p=0.0197, respectively) in dogs with severe injury, compared with those with mild-to-moderate SCI, but there was no significant correlation between assessed CSF APP concentrations and 42 d motor outcome. This study demonstrated that CSF APPs were dysregulated in dogs with naturally-occurring SCI and could be used as markers for SCI severity. As Hp was increased following severe SCI and is neuroprotective across a number of model systems, it may represent a viable therapeutic target.

Biomarkers and diagnosis; protein biomarkers in serum of pediatric patients with severe traumatic brain injury identified by ICAT-LC-MS/MS.

This report is a feasibility study on the utility of gel-free proteomics in identifying peripheral biomarkers of brain injury. The study was performed in six pediatric patients admitted to the intensive care unit for severe traumatic brain injury (TBI). Serum samples collected at admission (less than 8 h after injury) were used for determining the levels of S100beta by enzyme-linked immunosorbent assay (ELISA) and for proteomics analyses. Serum samples were depleted of high abundant albumin and immunoglobulin, and were compared to a pooled reference from several healthy individuals. After labeling and separation on an ionic column, six different serum fractions were analyzed using Isotope-Coded Affinity Tag (ICAT), followed by tandem mass spectrometry (MS/MS) protein sequencing and identification. Ninety-five unique, differentially expressed proteins were identified, including several with a likely brain origin. Several proteins with pattern similarity to S100beta identified by hierarchical clustering could be considered for evaluation in a larger patient sample as potential peripheral markers of TBI.

CSF and serum orosomucoid (alpha-1-acid glycoprotein) in patients with multiple sclerosis: a comparison among particular subgroups of MS patients.

INTRODUCTION: To compare cerebrospinal fluid (CSF) and serum orosomucoid (alpha-1-acid glycoprotein-AAG) concentrations in various subgroups of patients with multiple sclerosis (MS). MATERIALS AND METHODS: CSF and serum AAG concentrations, AAG quotient (i.e., CSF AAG/serum AAGx10(3)) and index were determined in a group of 59 patients with clinically definite or probable MS. Patients were subdivided according to the disease form, disease severity according to an expanded disability status scale (EDSS), its treatment, disease duration and sex. RESULTS: CSF AAG was increased in 52.5% of the patients and AAG quotient even in 64.4%. An increase in the CSF AAG concentration, as well as in AAG quotient and index, appear only after several years of disease duration, while no significant correlation with age has been found. This suggests that CSF AAG changes in MS represent a secondary, unspecific phenomenon and that this protein is not relevant for the aethiopathogenesis of the disease. Nevertheless, the finding of subnormal CSF AAG levels in some MS patients in remission (never observed in those in the attack) implies the possibility that CSF AAG may be used as a "state marker" in MS. Serum AAG levels were significantly lower in secondary progressive form and in severely disabled patients. This observation suggest that serum AAG values determination might have some prognostic significance. Further studies are, however, needed. Serum AAG should be investigated in parallel with other CSF and serum protein fractions in order to establish a pannel of examinations enabling multiple statistical analyses. This approach may lead to the finding of a "complex state marker" enabling thus to evaluate more precisely disease course in individual patients and to accept appropriate therapeutic measures.

[Usefulness of establishing chosen acute phase proteins concentrations in serum and cerebrospinal fluid for differential diagnosis and monitoring of purulent meningitis in adults. I]. / Przydatnosc oznaczania stezen wybranych bialek ostrej fazy w surowicy krwi i plynie mózgowo-rdzeniowym do diagnostyki róznicowej i monitorowania przebiegu ropnych zapalen opon mózgowo-rdzeniowych u doroslych. I. Stezenie bialek ostrej fazy w surowicy krwi.

40 adult patients were examined: 24 with purulent meningitis and 16 with lymphocytic meningitis. The control group consisted of 100 healthy people. In purulent meningitis patients in the 1st, 3rd, 5th, 7th, 14th and 28th day of the disease, concentrations of the following acute phase proteins were measured in serum: C-reactive protein, alpha 1-antitripsin, alpha 1-orosomucoid, alpha 2-ceruloplasmin, alpha 2-macroglobulin and alpha 2-haptoglobin. In lymphocytic meningitis patients concentrations of the above mentioned acute phase proteins were measured only in the 1st day of the disease. Usefulness of establishing alpha 2-haptoglobin, alpha 1-antitripsin, alpha 2-ceruloplasmin and particularly C-reactive protein concentrations for differential diagnosis of purulent and lymphatic meningitis was proved. Evaluation of C-reactive protein and alpha 1-antitripsin concentration kinetics proved to be fully useful for monitoring of seriousness of the course of purulent meningitis, and together with evaluation of the clinical condition of the patient it can constitute a valuable marker of effectiveness of the disease treatment.

[Usefulness of establishing chosen acute phase proteins concentrations in serum and cerebrospinal fluid for differential diagnosis and monitoring of purulent meningitis in adults. II]. / Przydatnosc oznaczania stezen wybranych bialek ostrej fazy w surowicy krwi i plynie mózgowo-rdzeniowym do diagnostyki róznicowej i monitorowania przebiegu ropnych zapalen opon mózgowo-rdzeniowych u doroslych. II. Stezenie bialek ostrej fazy w plynie mózgowo-rdzeniowym.

40 adult patients were examined: 24 with purulent meningitis and 16 with lymphocytic meningitis. In the course of purulent meningitis concentrations of the following acute phase proteins were measured in the cerebrospinal fluid: C-reactive protein, alpha 1-antitripsin, alpha 1-orosomucoid, alpha 2-ceruloplasmin, alpha 2-macroglobulin and alpha 2-haptoglobin in the 1st, 3rd, 5th, 7th, 14th and 28th day of the disease. In lymphocytic meningitis patients concentrations of the above mentioned acute phase proteins were measured only in the 1st day of the disease. Full usefulness of establishing concentrations of all the above mentioned acute phase proteins within the first five days of the purulent meningitis for differential diagnosis of purulent and lymphatic meningitis was proved. Evaluation of concentration kinetics of acute phase proteins in cerebrospinal fluid for monitoring of the course of purulent meningitis is of a limited value.

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.

Isolation, purification, and characterization of an acidic protein in the cerebrospinal fluid of central nervous system disease.

Proteins from 21 cerebrospinal fluid (CSF) samples (14 derived from neurological cases and 7 from normal individuals) and 15 serum samples (11 from neurological cases and 4 from normal individuals) were analyzed by two-dimensional electrophoresis. Protein mapping revealed a very acidic protein (Ac-P) at about pH 3.5 in the 71% of CSF samples from neurological cases. However, no serum sample contained Ac-P. Ac-P was isolated and purified, and determined to be a glycoprotein containing a large amount of carbohydrate, with molecular weight 42,000 and isoelectric point 2.7-3.3. The amino acid composition of Ac-P was consistent with alpha 1-acid glycoprotein (AGP), and Ac-P was responsive to a commercial anti-AGP antiserum in the radial immunodiffusion test. The known polymorphism of AGP suggests some differences in physicochemical properties such as molecular weight and isoelectric point between AGP in serum and in CSF. Quantitative analysis of Ac-P (AGP) and total protein levels in CSF showed a partial interdependence. Ac-P may be a useful marker for detecting a pathological conditions of the central nervous system.

Polyclonal antibody-based enzyme-linked immunosorbent assay of alpha 1-acid glycoprotein.

This is a noncompetitive enzyme-linked immunosorbent assay for measuring low concentrations (2 to 100 micrograms/L) of human alpha 1-acid glycoprotein (AGP; orosomucoid). The method is based on a simple "sandwich" technique involving polyclonal rabbit antisera against AGP. Mean within-run and total (between-run) CVs were 6.2% and 9.7%, respectively. Analytical recovery, tested in various biological fluids, averaged 101%. The technique has been successfully applied to diluted biological fluids such as bronchoalveolar lavage, cerebrospinal and amniotic fluids, and hepatocyte-culture supernates. Because of its analytical validity and the commercial availability of the reagents, this assay is suitable for large-scale determinations of AGP concentrations in those biological fluids in which its concentration is relatively low.

Comparison of concentration of orosomucoid in serum and cerebrospinal fluid in different neurological diseases.

We compared orosomucoid levels in cerebrospinal fluid (CSF) in patients with aseptic meningitis, multiple sclerosis, ischemic and hemorrhagic stroke, CNS-affecting leukemia/lymphoma, and CNS-tumor with the levels in a reference group not having neurologic diseases. Because of possible blood brain barrier damage, we corrected for orosomucoid derived from serum by using the orosomucoid index, i.e. (CSF/serum orosomucoid)/(CSF/serum albumin). Elevated CSF orosomucoid was found in several diseases. In no case, however, was there any evidence of intrathecal synthesis of the protein. We concluded that CSF orosomucoid determination, when used as the only, measure, is of limited clinical value.

[Diagnostic value of alpha 1-acid glycoprotein in the cerebrospinal fluid]. / Zur diagnostischen Wertigkeit des sauren-alpha 1-Glykoproteins im Liquor cerebrospinalis.

Samples from 1415 neurological patients were used to study the diagnostic value of acid alpha 1-glycoprotein in the lumbar cerebrospinal fluid. The value of analysis for this substance is considered to lie in the detection of non-specific acute-phase reactions of various causes in the CNS with special reference to barrier problems. The large number of pathologically elevated values associated with acute inflammations of the meninges and cerebral parenchyme is particularly striking. The results of the studies are analyzed on the basis of comprehensive control investigations (n = 559).

Correlation between protein data in normal lumbar CSF and morphological findings of choroid plexus epithelium: a biochemical corroboration of barrier transport via tight junction pores.

Specific "reference areas" were derived from relationships between the proteins prealbumin, albumin, alpha 1-acid glycoprotein, alpha 2-macroglobulin, total transferrin, IgG, IgA, IgM, and the corresponding total protein in normal lumbar CSF samples. The procedure for calculating the boundary lines of these reference areas was carried out on the basis of double standard deviations in subgroups with total protein differences of 50 ml/l within the whole range of 150-400 ml/l CSF. The resulting biochemical data, hydrodynamic radii of the individual proteins investigated, and van Deurs' and Koehler's morphological findings on the existence of pores in the barrier-forming tight junctions of the choroid plexus epithelium could be surprisingly well correlated with one another, although these morphological findings were obtained in choroid plexus of the rat brain. The correlation allowed the conclusion that proteins undergo ultrafiltration via a pattern of tight junction pores with various diameters. However, the molecular mechanism seems to include an additional facilitating component.