Multiplex serum cytokine monitoring as a prognostic tool in rheumatoid arthritis.

OBJECTIVE: Early optimized therapy of rheumatoid arthritis (RA) results in improved outcomes. The initiation of optimized therapy is hindered by the difficulty of early diagnosis and the limitations of current disease activity and therapeutic response assessment tools. Identifying patients requiring early combination DMARD/biologic therapy is currently a significant clinical challenge given the lack of definitive prognostic criteria. Since cytokines are soluble intracellular signaling molecules that modulate disease pathology in RA, we tested the recent conjecture that en mass serum cyto-kine measurement and monitoring will provide a useful tool for effective therapeutic management in RA. METHODS: We assayed the levels of 16 serum cytokines in 18 RA patients treated prospectively with methotrexate and from 18 unaffected controls. Specific mechanistic aspects of inflammatory pathology in the periphery could be discerned on a patient-specific basis from patients' serum cytokine profiles, information that may aid in the design of anti-cytokine biologic therapy. A serum Cytokine Activity Index (CAI) was also created using multi-variant analysis methods. RESULTS: Distinct cytokines were significantly elevated in RA patients relative to controls, and three distinct clusters with correlations to disease activity were identified. The Cytokine Activity Index correlated well with the therapeutic res-ponse; responders and non-responders in this cohort were distinguishable as early as one month post initiation of methotrexate therapy, well before clinical assessments of response are commonly completed. CONCLUSION: Clinical assessment tools could be derived from this approach that may provide a means to continually track patients, allowing intervention strategies to be better evaluated on a patient-specific basis and to identify residual cytokine activity that could be used to guide combination therapy.

L-DOPA-quinone inactivates tryptophan hydroxylase and converts the enzyme to a redox-cycling quinoprotein.

Tryptophan hydroxylase, the initial and rate limiting enzyme in the biosynthesis of serotonin (5-HT), is inactivated by the quinone of L-DOPA. L-DOPA itself has no effect on enzyme activity. The inactivation of tryptophan hydroxylase could be prevented by glutathione (GSH), dithiothreitol, cysteine, and ascorbic acid but not by scavengers of hydrogen peroxide (catalase), hydroxyl radical (DMSO), or superoxide (superoxide dismutase). All cysteinyl residues within tryptophan hydroxylase are modified after treatment with L-DOPA-quinone as revealed by loss of DTNB-reactivity and formation of cysteinyl-DOPA residues. L-DOPA-quinone also converts tryptophan hydroxylase to a redox-cycling quinoprotein. These results suggest a possible mechanism of 5-HT neuronal damage in Parkinson's Disease by a redox-cycling quinoprotein.

Tyrosine hydroxylase is inactivated by catechol-quinones and converted to a redox-cycling quinoprotein: possible relevance to Parkinson's disease.

Quinone derivatives of DOPA, dopamine, and N-acetyldopamine inactivate tyrosine hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the catecholamine neurotransmitters. The parent catechols are inert in this capacity. The effects of the catecholquinones on tyrosine hydroxylase are prevented by antioxidants and reducing reagents but not by scavengers of hydrogen peroxide, hydroxyl radicals, or superoxide radicals. Quinone modification of tyrosine hydroxylase modifies enzyme sulfhydryl groups and results in the formation of cysteinyl-catechols within the enzyme. Catecholquinones convert tyrosine hydroxylase to a redox-cycling quinoprotein. Quinotyrosine hydroxylase causes the reduction of the transition metals iron and copper and may therefore contribute to Fenton-like reactions and oxidative stress in neurons. The discovery that a phenotypic marker for catecholamine neurons can be converted into a redox-active species is highly relevant for neurodegenerative conditions such as Parkinson's disease.

Inactivation of tryptophan hydroxylase by nitric oxide: enhancement by tetrahydrobiopterin.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by the nitric oxide generators sodium nitroprusside, diethylamine/nitric oxide complex, and S-nitroso-N-acetylpenicillamine. Physiological concentrations of tetrahydrobiopterin, the natural and endogenous cofactor for the hydroxylase, significantly enhance the inactivation of the enzyme caused by each of these nitric oxide generators. The substrate tryptophan does not have this effect. The chemically reduced (tetrahydro-) form of the pterin is required for the enhancement, because neither biopterin nor dihydrobiopterin is effective. The 6S-isomer of tetrahydrobiopterin, which has little cofactor efficacy for tryptophan hydroxylase, does not enhance enzyme inactivation as does the natural 6R-isomer. A number of synthetic, reduced pterins share with tetrahydrobiopterin the ability to enhance nitric oxide-induced inactivation of tryptophan hydroxylase. The tetrahydrobiopterin effect is not prevented by agents known to scavenge hydrogen peroxide, superoxide radicals, peroxynitrite anions, hydroxyl radicals, or singlet oxygen. On the other hand, cysteine partially protects the enzyme from both the nitric oxide-induced inactivation and the combined pterin/nitric oxide-induced inactivation. These results suggest that the tetrahydrobiopterin cofactor enhances the nitric oxide-induced inactivation of tryptophan hydroxylase via a mechanism that involves attack on free protein sulfhydryls. Potential in vivo correlates of a tetrahydrobiopterin participation in the inactivation of tryptophan hydroxylase can be drawn to the neurotoxic amphetamines.

Tryptophan hydroxylase: cloning and expression of the rat brain enzyme in mammalian cells.

A cDNA encoding full-length tryptophan hydroxylase was produced by reverse transcriptase-PCR from rat brain mRNA and expressed transiently in a human fibroblast cell line. Catalytic activity was low unless transfected cells were grown in the presence of FeSO4. Recombinant tryptophan hydroxylase was found almost exclusively within the soluble compartment of the cell and was dependent on tryptophan and tetrahydrobiopterin for activity. The catalytic activity of recombinant tryptophan hydroxylase was stimulated > 25-fold by Fe(II) and to a somewhat lesser extent by the polyanions heparin and phosphatidylserine. The enzyme was inhibited by desferrioxamine and dopamine, both of which complex iron. When extracts from transfected cells were subjected to sucrose gradient centrifugation and analytical gel filtration, the recombinant enzyme behaved the same as the native enzyme from brain. A monoclonal antibody against phenylalanine hydroxylase that cross-reacts with brain tryptophan hydroxylase was capable of immunoprecipitating the recombinant hydroxylase from solution. These data indicate that recombinant tryptophan hydroxylase expressed in mammalian cells is assembled into tetramers of approximately 220,000 daltons. Its catalytic and physical properties appear to be very similar to those of the native enzyme from brain.

Expression and deletion mutagenesis of tryptophan hydroxylase fusion proteins: delineation of the enzyme catalytic core.

cDNAs encoding the full-length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1-98) or catalytic (amino acids 99-444) domains of the enzyme, were cloned and expressed as glutathione S-transferase fusion proteins in E. coli. The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione-agarose. The full-length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathione S-transferase-tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity (Vmax) than the brain enzyme. Analysis of full-length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99-444 of the full-length enzyme, contains the sequence motifs needed for subunit assembly. Both wild-type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full-length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase.

Inactivation of brain tryptophan hydroxylase by nitric oxide.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by nitric oxide (NO) and by the NO generators sodium nitroprusside, diethylamine/NO, S-nitroso-N-acetylpenicillamine, and S-nitrosocysteine. The inactivation occurs in an oxygen-free environment and is enhanced by dithiothreitol and ascorbic acid. Protection against the effect of NO on tryptophan hydroxylase is afforded by oxyhemoglobin, reduced glutathione, and exogenous Fe(II). Catalase partially protects the enzyme from NO-induced inactivation, whereas both superoxide dismutase and uric acid are without effect. These findings indicate that tryptophan hydroxylase is a target for NO and suggest that critical iron-sulfur groups in this enzyme serve as the substrate for NO-induced nitrosylation of the protein, resulting in enzyme inactivation.

Synovial fluid lactic acid in septic and nonseptic arthritis.

We determined lactic acid levels by the lactic dehydrogenase method in synovial fluid of 41 patients with various rheumatic diseases, to test the concept that significantly elevated values were diagnostic of septic arthritis. Nine patients had septic arthritis, 15 rheumatoid arthritis (RA), and the remainder miscellaneous conditions. In another 9 patients with different rheumatic diseases, including 1 with septic arthritis, synovial fluid lactic acid was determined by both the lactic dehydrogenase and gas-liquid chromatography methods. There was a wide scatter of values among patients with septic and nonseptic inflammatory arthritis, and much overlap occurred. We could not differentiate septic arthritis from RA on the basis of synovial fluid lactic acid levels. Results were similar with both procedures for determining lactic acid levels.

Immunoprotein deposition in synovial tissue in Reiter's syndrome.

The aetiology of Reiter's syndrome (RS) is unknown. In order to evaluate the role of immunological mechanisms in this disease we performed synovial biopsies on 12 patients with RS looking for deposition of immunoglobulins and complement components in synovial tissue. By immunofluorescent techniques 11 synovia were found to have immunoprotein deposition. IgM deposition was found around vessels in 8 synovia and in the interstitial tissue in 4. C3 was present perivascularly in 11 cases; in 4 of those there was also staining in the interstitial tissue. No immunoproteins were found in infiltrating or synovial lining cells. The finding of immunoproteins in the synovium of the majority of patients with RS suggests that immunological mechanisms are involved in the pathogenesis of this disease.