Cerebrospinal fluid protein concentrations in children: age-related values in patients without disorders of the central nervous system.

BACKGROUND: The published reference values for cerebrospinal fluid (CSF) total protein concentrations in children suffer from two major drawbacks: (a) the age-related range often is too broad when applied to the steeply falling concentrations in early infancy; and (b) no values have been published for widely used dry chemistry methods. METHODS: We conducted a 2-year retrospective survey of CSF results obtained in a children's hospital with a dry chemistry-based method set up on the Vitros 700 analyzer. RESULTS: The data related to ambulatory children up to 16 years of age and term neonates with no clinical or biological signs of brain disease (n = 1074). Seven age groups with significantly different CSF protein values were identified, and their age-related percentiles (5th, 50th, and 95th) were determined. On the basis of the upper 95th percentile, from age 0 to 6 months the CSF protein concentrations fell rapidly from 1.08 to 0.40 g/L. A plateau (0.32 g/L) was reached from age 6 months to 10 years, followed by a slight increase (0.41 g/L) in the 10-16 years age range. CONCLUSIONS: These results imply that CSF total protein concentrations in the pediatric setting, particularly in infants, must always be interpreted with regard to narrow age-related reference values to avoid false-positive results.

Trophic response of the intestinal mucosa to inflammation in rats injected with turpentine: a study using pair-fed controls.

The effects of restricted food intake and acute inflammation on the small bowel were studied, Wistar rats (250 g) were given subcutaneous injections of turpentine (TR) and compared to two control groups, at 18, 42 and 66 h. One was fed ad libitum (C), the other was pair fed (PF) with TR. The TR and PF rats showed hypoplasia of the jejunal mucosa with decreased protein and DNA contents at 42 h and 66 h. The hypoplasia resulted in a reduced villus height that was significantly different from the controls at 66 h (C: 468 +/- 17, TR[66] : 376 +/- 20, PF[66] : 258 +/- 2.9 microm, P<0.001). This decrease in villus height was significantly greater in the PF rats than in the TR rats at 66 h. The crypt height/villus height (C/V) ratio in the PF rats was greater than in the TR group at all times. However, the protein and DNA contents in the TR group were significantly higher than in the PF group at 42 h and 66 h (TR/PF[42] : 29.5 +/- 1.9 vs 20.5 +/- 2.0, P< 0.001, [66]: 25.8 +/- 2.0 vs 16.6 +/- 1.3 mg/10 cm, P,< 0.001). Disaccharidase activities (sucrase and glucoamylase) per 10 cm jejunum at 66 h were significantly lower in the PF group than in the control and TR groups (sucrase mU/10cm[66] C : 3090 +/- 144, TR 2683 +/- 479, PF 1969 +/- 144, P,< 0.001; glucoamylase mU/10 cm[66] 237 +/- 25, TR 169 +/- 40, PF 123 +/- 5, P< 0.01). The N-aminopeptidase patterns in the TR and PF groups were similar. These data suggest that dietary restriction during acute inflammation is the main factor causing hypoplasia of the jejunal mucosa. However, acute inflammation has a trophic effect on the morphological and function of the mucosa. This effect is probably due to inflammatory mediators, whose synthesis is stimulated by turpentine.

False increase in C-reactive protein attributable to heterophilic antibodies in two renal transplant patients treated with rabbit antilymphocyte globulin.

Increased serum C-reactive protein (sCRP) is a sensitive marker of renal graft rejection. We describe the cases of two children with uncomplicated renal transplantation who had false-positive sCRP values on analyzers using rabbit anti-CRP but values within the reference range with anti-CRP from other animal species. Cross-reaction with heterophilic antibodies was suggested by clinical and biological signs of serum sickness and daily treatment with rabbit antilymphocyte globulin (ALG). The interference depended on the serum concentration of the cross-reactant and was removed by subtotal IgG adsorption to Protein A or Protein G or by immunoadsorption using rabbit ALG or total IgG in non-immune rabbit serum. Anti-rabbit IgG and IgM antibodies were detected in both patients. These are the first reported cases of cross-reaction with heterophilic antibodies in a turbidimetric CRP assay.

Effects of alimentary whole proteins versus their small peptide hydrolysates on liver and skeletal muscle during the acute inflammation phase in the rat.

Acute inflammation induces changes in liver proteins with an increase in synthesis of positive acute-phase proteins such as alpha1-acid glycoprotein (alpha1-AGP) and a decrease in synthesis of negative acute-phase proteins such as albumin. This is associated with muscle wasting, mediated by increased proteolysis and impaired protein synthesis. As protein metabolism can be altered in other situations (malnutrition, growth) by the form of the dietary nitrogen, we studied the effects of the molecular form of nitrogen on liver and skeletal muscle adaptation, looking at gene expression for two acute-phase proteins (albumin and alpha1-AGP) and a number of muscle proteins (alpha1-actin, ubiquitin and C9 proteasome subunit). Two groups of 24 Wistar rats (250 g) were injected S/C with 0.125 ml turpentine/rat and were fed one of two liquid diets. These diets had caloric, nitrogen, carbohydrate and lipid content but differed in the molecular form of the nitrogen source (whole protein [WP] versus peptide hydrolysate [PH]). Liver and muscle adaptation were studied at 18, 42 or 66 h after turpentine injection. Weight, deoxyribonucleic acid and protein content of the liver were significantly higher with the WP diet than with the PH diet at 42 h and 66 h. There was more alpha1-AGP messenger ribonucleic acid (mRNA) at 18 h and less albumin mRNA at 42 h. Thus, the PH diet causes a more rapid increase in alpha1-AGP mRNA content and a smaller decrease in albumin mRNA content after turpentine injection than the WP diet. However, the changes in plasma acute-phase proteins (albumin and alpha1-AGP) were similar with the two diets. In skeletal muscle, there was no change in mRNA levels for the C9 proteasome subunit at any time point with both diets compared to the controls. However, there were greater ubiquitin mRNA levels at 18|h and less alpha-actin mRNA levels at 18 h, 42 h and 66 h following turpentine injection in the two dietary groups than in the controls. These results suggest that the molecular form of nitrogen ingested regulates hepatic gene transcription or mRNA stability of acute-phase proteins, during the early period of inflammation, but did not affect the expression of muscle proteins, which was altered by turpentine injection. Post-transcriptional control of acute-phase protein genes may contribute to the maintenance of similar plasma levels.

Recovery processes after repeated supramaximal exercise at the altitude of 4,350 m.

We tested the hypothesis that prolonged exposure to high altitude would impair the restoration of muscle power during repeated sprints. Seven subjects performed two 20-s Wingate tests (WT1 and WT2) separated by 5 min of recovery, at sea level (N) and after 5-6 days at 4,350 m (H). Mean power output (MPO) and O2 deficit were measured during WT. O2 uptake (VO2) and ventilation (VE) were measured continuously. Blood velocity in the femoral artery (FBV) was recorded by Doppler ultrasound during recovery. Arterialized blood pH and concentrations of bicarbonate ([HCO3-]), venous plasma lactate ([La-]), norepinephrine ([NE]), and epinephrine ([Epi]) were measured before and after WT1 and WT2. MPO decreased between WT1 and WT2 by 6.9% in N (P < 0.05) and by 10.7% in H (P < 0.01). H did not further decrease MPO. O2 deficit decreased between WT1 and WT2 in H only (P < 0.01). Peak VO2 after WT was reduced by 30-40% in H (P < 0.01), but excess postexercise O2 consumption was not significantly lowered in H. During recovery in H compared with N, VE, exercise-induced acidosis, and [NE] were higher, [Epi] tented to be higher, [La-] was not altered, and [HCO3-] and FBV were lower. The similar [La-] accumulation was associated with a higher exercise-induced acidosis and a larger increase in [NE] in H. We concluded from this study that prolonged exposure to high altitude did not significantly impair the restoration of muscle power during repeated sprints, despite a limitation of aerobic processes during early recovery.

Separation and characterization of the main methylated nucleobases from nuclear, cytoplasmic and poly (A)+ RNA by high-performance liquid chromatography and mass spectrometry.

We were able to detect nine methylated nucleobases (3-methyluracil, 1-, 2-, 3- and 7-methylguanine, 1-, 2-, 3- and 6-methyladenine) in RNA from rat and calf liver, baker's yeast, Torula and Euglena cells by using reversed-phase high-performance liquid chromatography and thermospray mass spectrometry. Total cellular, nuclear, cytoplasmic and poly (A)+ RNA from rat liver showed marked methylation, mainly of 1- and 3- methylguanine, and 3- and 2-methyladenine. These bases were especially abundant in nuclear RNA and, to a lesser extent, in poly (A)+ RNA. In contrast, 7-methylguanine and 6-methyladenine were poorly represented in poly (A)+ RNA.

Effect of purified IgGs from HIV-1-infected and non infected individuals on immune activation.

The purification and analysis of IgGs from sera of HIV-1-infected and non infected individuals are reported. The effect of antibodies purified from sera of infected individuals on antigen-induced T cell proliferation was investigated in relation to their possible involvement in an autoimmune reaction in AIDS, in view of the previously unravelled striking peptide similarities between HIV-1 gp120 and the immunoregulatory CD4 and Fas molecules. However, our data do not allow definite conclusions to be drawn. The necessity of purifying antibodies against specific peptides to show their direct effect on T-cell activation is further stressed.

Changes in the glycoforms of rat alpha-1-acid glycoprotein during experimental polyarthritis.

We analyzed the carbohydrate moiety of purified alpha-1-acid glycoprotein (AGP) from Lewis adult male rats that were healthy (AGPh) or had experimental polyarthritis (AGPi). Sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after N-glycanase treatment showed that AGPi had a slightly lower molecular mass (43 kDa vs. 45 kDa for AGPh) due to a lesser carbohydrate content. Carbohydrate analysis of purified AGP showed a slight decrease in the sialyl and galactosyl molar ratio in polyarthritis. However, the same difference in AGPh and AGPi (i.e. 0.6 residue) between the sialyl and galactosyl molar ratio indicated more than one sialyl residue per complex-type branch. Affinity for concanavalin A (ConA) of the whole glycoprotein and released oligosaccharides showed a progression during polyarthritis towards more reactive glycoforms or more ConA-bound oligosaccharides. Anion-exchange HPLC of the ConA-fractionated oligosaccharides corroborated the decreased sialylation in polyarthritis. Taken together, these results suggest a fall in branched and sialylated oligosaccharides during experimental polyarthritis. These structural changes might be related to an increase in Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase activity described elsewhere in inflammatory states.

Oligosaccharide specificity of normal human hepatocyte alpha 1-3 fucosyltransferase.

A purified alpha 1-3 fucosyltransferase (alpha 1-3 FT) was recovered in the Golgi fraction of isolated hepatocytes from normal human liver tissue. The efficiency of purification was controlled by measurement of fucose transfer to asialotransferrin (for alpha 1-3 FT), to phenyl-beta-D-galactose (for alpha 1-2 FT) and to 2' fucosyl lactose (for alpha 1-3/4 FT). The initial hepatocyte isolation step got rid of 97% and 94% of alpha 1-2 and alpha 1-3/4 total liver FT, respectively. After Golgi enrichment (26-fold purification and a yield of 7.6%), alpha 1-3 FT extract expressed a specific activity of 2 pM/min per mg protein. When incubated in optimized conditions with type 1, 2 or 6 oligosaccharide acceptors (10 mM), hepatocellular alpha 1-3 FT efficiently transferred fucose to N-acetyllactosamine and its 3' sialylated derivative, but poorly to lactose. When incubated with neutral or sialylated biantennary N-glycans, the enzyme expressed the highest affinity (Km = 0.38 mM) for the 3'bisialylated derivative. This suggests that hepatocellular alpha 1-3 FT is involved in the synthesis of sialosyl Le(x) determinants on cirrhotic alpha 1AGP.

Determination of cortisol and associated glucocorticoids in serum and urine by an automated liquid chromatographic assay.

We describe a method for the determination of urinary free cortisol and glucocorticoids in plasma, used in the diagnosis of adrenal disorders, based on automated reverse-phase high-performance liquid chromatography (HPLC). The within-day and day-to-day CVs were less than 5.5 and 8.0%, respectively. The calibration curves for cortisol and 11-deoxycortisol were linear up to 2000 nmol/L. Cortisol concentrations as low as 3.5 nmol/L in 1 mL of plasma or urine can be measured. Correlation of HPLC results for 40 plasma specimens with those by radioimmunoassay showed r = 0.965. This method is sensitive and free from the interference habitually encountered in immunoassays, and can thus be proposed for research and as a potential reference method.

Striking similarities between HIV-1 Env protein and the apoptosis mediating cell surface antigen Fas. Role in the pathogenesis of AIDS.

We have designed a computer strategy in order to detect systematically peptidic sites with the potential of interfering with the immune regulatory processes. Applying this software to HIV-1 proteins has led us to unravel a few peptidic sites which could either act directly or be the targets of an auto-immune reaction during HIV-1 infection. We previously reported that the SLWDQ pentapeptide identity with a critical site of CD4 could trigger in HIV-1 infected individuals both an humoral and a cellular autoimmune reaction. In this study, we focused on surprising similitudes unravelled by our software Automat, between HIV-1/2 and another immunoregulatory molecule, the Fas protein which is also called the apoptosis-mediating cell-surface antigen.

Analysis of carbohydrate-mediated heterogeneity and characterization of N-linked oligosaccharides of glycoproteins by high performance capillary electrophoresis.

Capillary zone electrophoresis (CZE) has been investigated as an alternative method to analyze the carbohydrate moieties of glycoproteins. Carbohydrate-mediated microheterogeneity of the recombinant plasminogen activator (rt-PA) was examined. The glycoprotein was resolved in multiple electrophoretic species using CZE but the separation was complicated by adsorption of the molecules to the wall of the capillary. The influence of several parameters, such as pH, molarity of the buffer and addition of a cationic additive, on the separation of glycopeptides was investigated. High resolution and reproducible separations of rt-PA glycopeptides carrying hybrid and complex type chains were obtained using either a 100 mM phosphate buffer, pH 6.6, or a 100 mM Tricine buffer, pH 8.2, containing 1.25 mM of putrescine. N-Oligosaccharides from fetuin, t-PA and alpha 1-acid glycoprotein were separated within 20 min on the basis of both their sialic acid content and their structure. The use of an oligosaccharide fingerprinting technique, such as the present one, could have many applications in biotechnology to assess, for example, the consistency of production of a glycoprotein or for analytical glycoprotein chemistry.

Evaluation of the hepatotoxicological effects of a drug in an in vivo/in vitro model.

Both in vivo and in vitro models have certain disadvantages for the study of the chronic hepatotoxicity of drugs. The aim of this work was to evaluate a new approach based on an in vivo/in vitro model. After chronic in vivo treatment of rats with Vincamine and Vindeburnol (an eburnamenine derivative which exhibits hepatotoxic properties in man) liver cells were isolated, and functional and metabolic disorders (metabolic utilization of fructose and protein biosynthesis) were studied to determine injury. The results showed no modification of blood parameters, but a direct relationship between the dose of Vindeburnol administered in vivo and the metabolic disorders observed in vitro, evidencing the high sensitivity and reliability of this model.

Alterations of the glycan moiety of human alpha 1-acid glycoprotein in late-term pregnancy.

The carbohydrate moiety of purified alpha 1-acid glycoprotein (AGP) from healthy male adults (AGPn) and late-term pregnant women (AGPp) was analysed. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate before and after N-glycanase treatment showed that AGPp had a slightly higher molecular mass due to an enriched carbohydrate moiety. BIO-GEL P-4 and Concanavalin A (Con A)-Sepharose chromatography of the oligosaccharides released by hydrazinolysis and fractionated by high-voltage electrophoresis indicated a progression towards Con A-unbound oligosaccharides and towards larger glycans in pregnancy. Carbohydrate analysis of purified AGPp and AGPn and of the most increased oligosaccharide fraction (F4A) evidenced a decrease in the fucosyl molar ratio and a slight increase in the galactosyl, N-acetyl-glucosaminyl and N-acetyl neuraminyl ratios. These results suggest that AGP contains more highly branched oligosaccharides and/or additional N-acetyllactosamine-type oligosaccharides in pregnancy.

Semi-automated fluorometric assay for urinary total and B N-acetyl-beta-D-glucosaminidase: analytical investigation.

Total N-acetyl-beta-D-glucosaminidase (NAG) and NAG-B isoenzyme determination are determined in order to investigate renal tubular function. Here, we propose a semi-automated adaptation of Sandman's manual method for the Monarch centrifugal analyzer. Fluorescence of the released 4-methylumbelliferone is automatically read and results are directly expressed in nanokatal/l. Mean (1 SD) values expressed in nanokatal/millimole creatinine in urine from healthy female (n = 30) and male (n = 30) subjects were 8.2 (4.0) and 7.8 (2.9) for total NAG and 1.9 (1.4) and 1.5 (0.7) for the NAG-B isoenzyme activity. The assay of six patients with various renal disorders shows definite increase in total NAG and NAG-B isoenzyme activities.

An enzyme-linked immunoassay for the measurement of rat alpha 1-acid glycoprotein synthesized by cultured hepatocytes.

We have developed a sandwich ELISA to quantify rat alpha 1-acid glycoprotein (AGP). The assay correlated well with RID and the minimum detectable concentration was 1 microgram/l. The assay permits high sensitivity determinations of the rate of synthesis of AGP in vitro. The maximum mean rates observed were 1500 and 1800 ng/24 h/10(6) cells for hepatocytes cultured alone and co-cultured hepatocytes respectively and 39 ng/h/10(6) cells for isolated hepatocytes.

Microheterogeneity of the carbohydrate moiety of human alpha 1-acid glycoprotein in two benign liver diseases: alcoholic cirrhosis and acute hepatitis.

To determine whether alterations of the carbohydrate moiety of human alpha 1-acid glycoprotein constitute a marker of hepatic damage we studied purified alpha 1-acid glycoprotein from healthy individuals and two groups of patients with benign liver diseases: alcoholic cirrhosis and acute viral hepatitis. The results indicate: (1) increased concanavalin A-non reactive forms in cirrhosis and hepatitis, (2) a markedly increased proportion of fucosyl residues in all cirrhotic and some hepatitis patients. Although hyperfucosylation is generally considered to be a tumor marker, the observation here in the two benign liver diseases indicates that an increased fucosyl content should be considered as a more general expression of pathological glycoconjugate metabolism.

One-step purification of rat plasma alpha 1-acid glycoprotein by antibody affinity chromatography: application to normal and inflamed rat sera.

Most purification procedures used previously to isolate alpha 1-acid glycoprotein (AGP) from plasma can lead to some alterations in its carbohydrate moiety. An immunoaffinity chromatographic method is proposed for purifying in one step rat plasma AGP without any detectable modification of its glycan moiety. Crossed immunoaffinoelectrophoresis with concanavalin A before and after purification showed identical patterns, suggesting no glycan selection during the purification. In the same way no desialylation occurred during the purification step. This immunoaffinity chromatographic procedure provided evidence of a decreased level of fucosyl residues in turpentine oil rat plasma AGP compared with normal rat plasma AGP.