IL-10 Cytokine as Potential Biomarkers in Women with Fibromyalgia Who Underwent to a Short Balneotherapy Treatment

Abstract Fibromyalgia (FM) is a nonarticular rheumatic syndrome that leads to diffuse myalgia, sleep disturbances and morning stiffness. Balneotherapy has been shown an effective strategy to improve the health conditions of patients; however, the treatment follow-up is based on patient report due to the lack of biomarkers. Thus, this study evaluated the application of cytokines and phosphoglycerate mutase I (PGAM-I) to monitoring FM patient underwent to balneotherapy treatment. Eleven healthy and eleven women with FM were submitted to daily sessions of balneotherapy during 10 days. Clinical and quality of life parameters were assessed through a FIQ questionnaire. Blood levels of TNF-(, interleukins (IL-1, IL-2 and IL-10) and PGAM-I expression in patients' saliva were also evaluated. Patients with FM showed significant improvements in their clinical status after treatment. Also, FM patients has IL-10 levels lower than healthy women and the balneotherapy increased the expression of this cytokine in both groups, concomitantly to pain relief. Although inflammatory cytokines (IL-1, IL-2 and TNF-() were more expressed in FM patients than healthy patients their levels did not reduce after treatment. A slight increase of PGAM-I expression was observed. In conclusion, IL-10 levels could be a useful biomarker to balneotherapy follow-up of FM patients. However, these findings must be analyzed in a larger number of patients in order to validate IL-10 as an effective biomarker.

Characterization of a new phosphatase from Plasmodium.

Plasmodium falciparum malaria is the most important parasitic disease worldwide, responsible for an estimated 1 million deaths annually. Two P. falciparum genes code for putative phosphoglycerate mutases (PGMases), a widespread protein group characterized by the involvement of histidine residues in their catalytic mechanism. PGMases are responsible for the interconversion between 2 and 3-phosphoglycerate, an intermediate step in the glycolysis pathway. We have determined the crystal structures of one of the P. falciparum's PGMases (PfPGM2) and a functionally distinct phosphoglycerate mutase from Cryptosporidium parvum, a related apicomplexan parasite. We performed sequence and structural comparisons between the two structures, another P. falciparum enzyme (PfPGM1) and several other PGM family members from other organisms. The comparisons revealed a distinct conformation of the catalytically active residues not seen in previously determined phosphoglycerate mutase structures. Furthermore, characterization of their enzymatic activities revealed contrasting behaviors between the PfPGM2 and the classical cofactor-dependent PGMase from C. parvum, clearly establishing PfPGM2 as a phosphatase with a residual level of mutase activity. Further support for this function attribution was provided by our structural comparison with previously characterized PGM family members. Genetic characterization of PGM2 in the rodent parasite Plasmodium berghei indicated that the protein might be essential to blood stage asexual growth, and a GFP tagged allele is expressed in both blood and zygote ookinete development and located in the cytoplasm. The P. falciparum PGM2 is either an enzyme implicated in the phosphate metabolism of the parasite or a regulator of its life cycle.

Serum proteomic-based analysis for the identification of a potential serological marker for autoimmune hepatitis.

In the present study, by using a serologic proteomic analysis, we identified phosphoglycerate mutase isozyme B (PGAM-B) as a putative target of autoantibodies in autoimmune hepatitis (AIH). To evaluate whether the identified autoantigen is crucial for AIH, we cloned PGAM-B cDNA and expressed the recombinant protein in Escherichia coli. The soluble PGAM-B was purified by affinity chromatography and used as a coating antigen to determine the frequency of the PGAM-B-autoantibodies (PGAM-B-Abs) in patients with AIH and primary biliary cirrhosis (PBC) as well as chronic hepatitis B (CHB), chronic hepatitis C (CHC), and healthy donors by ELISA. Our study showed that the autoantibody to PGAM-B was predominantly present in AIH patients and 70.04% (50/71) of the tested AIH sera reacted to PGAM-B. The frequency of autoantibodies to PGAM-B is much higher in patients with AIH than in patients with PBC, CHB, CHC, and normal control. The data were further confirmed by using 1-DE Western blot analysis. Our study presents the first description of this protein as a candidate of diagnostic marker for AIH.

Effects of thyroid hormone and hypoxia on 2,3-bisphosphoglycerate, bisphosphoglycerate synthase and phosphoglycerate mutase in rabbit erythroblasts and reticulocytes in vivo.

OBJECTIVES: The effects of triiodothyronine (T(3)) and hypoxia on 2,3-bisphosphoglycerate (2,3-BPG) studied in vitro are unclear. To clarify these effects we selected a more physiologic approach: the in vivo study in rabbits. We also present the changes produced by T(3) and hypoxia on phosphoglycerate mutase (PGAM), which requires 2,3-BPG as a cofactor, and 2,3-BPG synthase (BPGS), the enzyme responsible for 2,3-BPG synthesis in erythroblasts and reticulocytes. METHODS: Hyperthyroidism was induced by daily T(3) injection (250 microg/kg), hypoxia by a mixture of 90% nitrogen and 10% oxygen and hypothyroidism by propylthiouracil (PTU) added to drinking water. RESULTS: Both T(3) administration and hypoxic conditions increased 2,3-BPG levels and BPGS mRNA levels and activity in erythroblasts but not in reticulocytes. Unlike BPGS, both PGAM mRNA levels and activity were increased in erythroblasts and reticulocytes under hyperthyrodism and hypoxia. The antihormone PTU produced opposite effects to T(3). CONCLUSION: The results presented here suggest that both hyperthyroidism and hypoxia modulate in vivo red cell 2,3-BPG content by changes in the expression of BPGS. Similarly, the changes in PGAM activity are also explained by changes in its expression.

A new stroke marker as detected by serum phosphoglycerate mutase B-type isozyme.

The diagnostic significance of phosphoglycerate mutase (PGM) B-type isozyme activity capable of being inducible under hypoxia at the gene level as a serum marker for cerebral stroke was investigated. The normal level (mean +/- 2 SD) in human serum was determined to be 38 +/- 18 units/L. Within 2 h after the onset of cerebral stroke (n = 65), B-type isozyme activity was elevated to 68 +/- 36 units/L, and retained to be higher level until 1-3 days. Serum B-type isozyme activities of 52 survival cases and 13 dead cases, being judged at the period of 1-2 months after the onset, were retrospectively compared; B-type isozyme activity that had been measured within 24 h after the onset was significantly higher (81 +/- 42 units/L) for the dead cases than for survival cases (57 +/- 27 units/L) with P < 0.05. These results suggest that serum PGM B-type isozyme has the potential as a novel marker for diagnosis of cerebral stroke and its severity.

Effect of oxalate and malonate on red cell metabolism.

The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

31P NMR spin-transfer in the phosphoglyceromutase reaction.

The rate of exchange of phosphoryl groups between 2- and 3-phosphoglycerate catalysed by (a) high concentrations (approximately equal to 5.0 mg protein ml-1) of rabbit muscle phosphoglyceromutase and (b) lysed human erythrocytes was measured using saturation and inversion transfer techniques with 31P-NMR spectroscopy. This is the first reported application of these techniques to a study of this particular enzymic reaction either in vitro or in situ in a cell cytosol. Selective irradiation of resonances was achieved by the DANTE pulse sequence which had not previously been used for spin-transfer studies. New equilibrium exchange theory was developed for the simplest model of a two-reactant enzyme-catalysed reaction and this was used to calculate turnover rates for the enzymes. There was a close similarity between the turnover rates calculated from the spin-transfer data obtained from the systems in vitro and in situ and those obtained by conventional enzymic assays, at low enzyme concentrations. This suggested an absence of any homogeneous enzyme-enzyme interactions which modify the kinetics at high protein concentrations either in lysates or in the system in vitro.

Plasma phosphoglycerate mutase as a marker of muscular dystrophy.

An elevation of phosphoglycerate mutase (PMG) has been detected in the blood plasma of the genetically dystrophic chicken and in Duchenne muscular dystrophy (DMD) patients. In the dystrophic chicken, plasma PGM in the pectoral muscle was simultaneously depressed to less than one-half that of the normal chicken. In a group of 9 DMD patients, plasma PGM activity was found to be significantly raised above the normal range. A survey of a small group of plasma specimens from human fetuses at risk for muscular dystrophy also suggested that PGM merits investigation as a potential adjunct to other diagnostic indices.

Evidence for the presence of a hybrid of phosphoglyceromutase/bisphosphoglyceromutase in the red cells: partial characterization of the hybrid.

Purified phosphoglyceromutase was hybridized in vitro with pure biphosphoglyceromutase . The hybrid showed an electrophoretic mobility identical to that of the intermediate band of red cell phosphoglyceromutase activity in the hemolysate patterns. Electrophoretic tests showed that the partially purified hybrid displayed both bisphosphoglyceromutase and phosphoglyceromutase activities and that the sample contained also a small portion of non-hybrid bisphosphoglyceromutase . By constrast to a non-hybrid mixture of the two purified mutases the hybrid exhibited heat instability of bisphosphoglyceromutase activity and neutralization of phosphoglyceromutase activity by anti- bisphosphoglyceromutase antibody.

Changes in the activity and isozyme patterns of glycolytic enzymes during stimulation of normal human lymphocytes with phytohaemagglutinin.

A study has been made of the effects of phytohaemagglutinin on the gene expression of the glycolytic enzymes in cultured human lymphocytes. All the enzymes were found to show an average increase in activity of between 160% and 360% in stimulated cells, but the increases were greater for the enzymes comprising the second half of the pathway. The enzyme activities in stimulated cells, cultured for 72 h, were similar to the activities measured in long-term lymphoid lines. Starch-gel electrophoresis was used to examine the isozyme patterns of the enzymes before and after exposure of the lymphocytes to PHA. Six of the enzymes showed isozyme patterns unchanged by stimulation. Four of the enzymes, aldolase, triosephosphate isomerase, enolase and lactate dehydrogenase, showed different isozyme patterns in stimulated cells from those seen in uncultured or unstimulated cells. The electrophoretic results showed a good correlation in isozyme pattern between uncultured lymphocytes and cultured unstimulated lymphocytes, and between PHA-stimulated lymphocytes and long-term lymphoid lines.

Active site phosphohistidine peptides from red cell bisphosphoglycerate synthase and yeast phosphoglycerate mutase.

Bisphosphoglycerate synthase (glycerate-1,3-P2 yields glycerate-2,3-P2) and phosphoglycerate mutase (glycerate-3-P formed from glycerate-2-P) are both phosphorylated by substrates at a histidine residue forming covalent intermediates which have been shown to function in the phosphoryl transfer reactions catalyzed by these enzymes (Rose, Z. B., and Dube, S. (1976) J. Biol. Chem. 251, 4817--4822). We have phosphorylated bisphosphoglycerate synthase from horse red blood cells with [U-32P]glycerate-2,3-P2, digested with trypsin, and purified the phosphopeptide. The amino acid sequence of the phosphohistidine peptide has been determined to be: His-Gly-Gln-Gly-Ala-Trp-Asn-Lys. In like manner, a phosphohistidyl peptide has now been purified from yeast phosphoglycerate mutase, for which the amino acid sequence is known (Winn, S. I., Watson, H. C., Fothergill, L. A., and Harkins, R. N. (1977) Biochem. Soc. Trans. 5, 657-659). The amino acid composition of the phosphopeptide indicates that histidine-8 was phosphorylated. The sequence of this peptide is closely homologous with the active site peptide from bisphosphoglycerate synthase. In yeast phosphoglycerate mutase, the denatured phosphoenzyme hydrolyzes with a single rate constant of 2.02 X 10(-4) s-1 at pH 3, 45 degrees C. The relevance of these observations to the enzymatic mechanism is discussed.

Metabolism of red blood cells in chronic renal failure. I. Glycolytic enzyme levels.

This paper starts a series on red blood cell (RBC) metabolism in patients with chronic renal failure (CRF). The glycolytic enzyme levels and in vitro half-lives of these patients' RBCs were determined. A number of enzymes (hexokinase, glucose-6-phosphate isomerase, fructose-6-phosphate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase) showed higher activities than in normal control RBCs. Other enzyme activities were normal. These results were discussed and several possible mechanisms considered. We favour the point of view of a shortened life span of the RBCs in CRF, making the most unstable enzymes of the glycolytic sequence appear increase as compared with normal controls.