Extended evaluation of the safety and efficacy of GAD treatment of children and adolescents with recent-onset type 1 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: The aim of this study was to investigate the safety and efficacy of alum formulated glutamic acid decarboxylase GAD(65) (GAD-alum) treatment of children and adolescents with type 1 diabetes after 4 years of follow-up. METHODS: Seventy children and adolescents aged 10-18 years with recent onset type 1 diabetes participated in a phase II, double-blind, randomised placebo-controlled clinical trial. Patients identified as possible participants attended one of eight clinics in Sweden to receive information about the study and for an eligibility check, including a medical history. Participants were randomised to one of the two treatment groups and received either a subcutaneous injection of 20 µg of GAD-alum or placebo at baseline and 1 month later. The study was blinded to participants and investigators until month 30. The study was unblinded at 15 months to the sponsor and statistician in order to evaluate the data. At follow-up after 30 months there was a significant preservation of residual insulin secretion, as measured by C-peptide, in the group receiving GAD-alum compared with placebo. This was particularly evident in patients with <6 months disease duration at baseline. There were no treatment-related serious adverse events. We have now followed these patients for 4 years. Overall, 59 patients, 29 who had been treated with GAD-alum and 30 who had received placebo, gave their informed consent. RESULTS: One patient in each treatment group experienced an episode of keto-acidosis between months 30 and 48. There were no treatment-related adverse events. The primary efficacy endpoint was the change in fasting C-peptide concentration from baseline to 15 months after the prime injection for all participants per protocol set. In the GAD-alum group fasting C-peptide was 0.332 ± 0.032 nmol/l at day 1 and 0.215 ± 0.031 nmol/l at month 15. The corresponding figures for the placebo group were 0.354 ± 0.039 and 0.184 ± 0.033 nmol/l, respectively. The decline in fasting C-peptide levels between day 1 and month 1, was smaller in the GAD-alum group than the placebo group. The difference between the treatment groups was not statistically significant. In those patients who were treated within 6 months of diabetes diagnosis, fasting C-peptide had decreased significantly less in the GAD-alum group than in the placebo-treated group after 4 years. CONCLUSION/INTERPRETATION: Four years after treatment with GAD-alum, children and adolescents with recent-onset type 1 diabetes continue to show no adverse events and possibly to show clinically relevant preservation of C-peptide. TRIAL REGISTRATION: ClinicalTrials.gov NCT00435981 FUNDING: The study was funded by The Swedish Research Council K2008-55X-20652-01-3, Barndiabetesfonden (The Swedish Child Diabetes Foundation), the Research Council of Southeast Sweden, and an unrestricted grant from Diamyd Medical AB.

Thromboxane generation by human peripheral blood polymorphonuclear leukocytes.

Human peripheral blood polymorphonuclear leukocytes were stimulated to generate thromboxane B2 in a time- and concentration-dependent fashion upon exposure to serum-treated zymosan particles. Conversion by stimulated PMN of [14C] arachidonic acid to [14C]thromboxane B2 was confirmed by thin-layer radiochromatography, radio-gas chromatography, and mass spectrometry. Generation of thromboxane B2 was independent of platelet contamination and could be inhibited by the cyclooxygenase inhibitor, indomethacin. Cells rendered incapable of ingesting particles by treatment with cytochalasin B generated comparable amounts of thromboxane B2. These results suggest that human peripheral blood polymorphonuclear leukocytes synthesize thromboxanes in response to surface stimulation independently of phagocytosis.

Microsomal prostaglandin E synthase-1 and 5-lipoxygenase: potential drug targets in cancer.

There is strong evidence for a role of prostaglandin (PG)E(2) in cancer cell proliferation and tumour development. In PGE(2) biosynthesis, cyclooxygenases (COX-1/2) convert arachidonic acid to PGH(2), which can be isomerized to PGE(2) by PGE synthases, including microsomal PGE synthase-1 (MPGES-1). Data describing genetic deletions of MPGES-1 are reviewed. The results suggest that MPGES-1 is an alternative therapeutic target for cancer cells and tumours that express this enzyme. Several compounds that target COX-2 or MPGES-1 also inhibit 5-lipoxygenase. This may be advantageous for treatment of some forms of cancer.

Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation.

Arachidonic acid plays a central role in a biological control system where such oxygenated derivatives as prostaglandins, thromboxanes, and leukotrienes are mediators. The leukotrienes are formed by transformation of arachidonic acid into an unstable epoxide intermediate, leukotriene A4, which can be converted enzymatically by hydration to leukotriene B4, and by addition of glutathione to leukotriene C4. This last compound is metabolized to leukotrienes D4 and E4 by successive elimination of a gamma-glutamyl residue and glycine. Slow-reacting substance of anaphylaxis consists of leukotrienes C4, D4, and E4. The cysteinyl-containing leukotrienes are potent bronchoconstrictors, increase vascular permeability in postcapillary venules, and stimulate mucus secretion. Leukotriene B4 causes adhesion and chemotactic movement of leukocytes and stimulates aggregation, enzyme release, and generation of superoxide in neutrophils. Leukotrienes C4, D4, and E4, which are released from the lung tissue of asthmatic subjects exposed to specific allergens, seem to play a pathophysiological role in immediate hypersensitivity reactions. These leukotrienes, as well as leukotriene B4, have pro-inflammatory effects.

Prostaglandins: enzymatic analysis.

By, means of al specific nicotinamide-adeninie dinucleotide-dependent prostaglanidin dehydrogenase from swine lung, an enizymatic method has been developed for the assay of prostaglandins. The method permits analysis with a lower limit of 10(-12) mole of prostaglandin.

Leukotrienes and lipoxins: structures, biosynthesis, and biological effects.

Arachidonic acid is released from membrane phospholipids upon cell stimulation (for example, by immune complexes and calcium ionophores) and converted to leukotrienes by a 5-lipoxygenase that also has leukotriene A4 synthetase activity. Leukotriene A4, an unstable epoxide, is hydrolyzed to leukotriene B4 or conjugated with glutathione to yield leukotriene C4 and its metabolites, leukotriene D4 and leukotriene E4. The leukotrienes participate in host defense reactions and pathophysiological conditions such as immediate hypersensitivity and inflammation. Recent studies also suggest a neuroendocrine role for leukotriene C4 in luteinizing hormone secretion. Lipoxins are formed by the action of 5- and 15-lipoxygenases on arachidonic acid. Lipoxin A causes contraction of guinea pig lung strips and dilation of the microvasculature. Both lipoxin A and B inhibit natural killer cell cytotoxicity. Thus, the multiple interaction of lipoxygenases generates compounds that can regulate specific cellular responses of importance in inflammation and immunity.

Stereochemistry in the formation of 9-hydroxy-10,12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid from linoleic acid by fatty acid cyclooxygenase.

9-Hydroxy-10,12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid are formed from linoleic acid upon incubation with the microsomal fraction of homogenates of the sheep vesicular gland (Hamberg, M. and Samuelsson, B. (1967) J. Biol. Chem. 242, 5344-5354. This communication is concerned with the stereochemical aspects of the conversion. The ratio between the 9- and 13-hydroxy isomers was 77:23. Steric analysis of the individual isomers showed that the hydroxyl group of both isomers had mainly the L configuration, i.e. 9L:9D, 79:21 and 13L:13D, 9- and 13-hydroxyoctadecadienoates which had largely lost the tritium label (6% and 7% retention of tritium relative to precursor, respectively) showing that the hydrogen which is removed from C-11 during the conversion has the L (pro-S) configuration.

Metabolism of 8,11,14-eicosatrienoic acid in human platelets.

The following labeled compounds were isolated and identified after incubation of 8,11,14-eicosatrien [1-14C] oic acid with human platelets: 12-L-hydroxy-8,10,14-eicosatrienoic acid, 8,11,12-trihydroxy-9,14-eicosadienoic acid, 8,9,12-trihydroxy-10,14-eicosadienoic acid, 12-L-hydroxy-8,10-heptadecadienoic acid, prostaglandin E1, prostaglandin D1, and 8-(1-hydroxy-3-oxopropyl)-9,12-dihydroxy-10-heptadecenoic acid (thromboxane B1).

Regulation of the human leukocyte 5-lipoxygenase: stimulation by micromolar Ca2+ levels and phosphatidylcholine vesicles.

Human leukocyte 5-lipoxygenase (EC 1.13.11.12) is unique among the human lipoxygenase not only in its requirement for free ionized calcium, but also in its regulation by a membrane-associated stimulatory factor, the 100,000 x g pellet. In the present study, phosphatidylcholine (PC) vesicles, in the absence of 100,000 x g pellet, exhibited a dose-dependent stimulatory activity on the 5-lipoxygenase, which was at least as effective as the 100,000 x g pellet. Furthermore, the enzyme was activated by isolated human neutrophil plasma membranes and to a lesser degree by endoplasmic reticulum. The chemoattractant peptide fMet-Leu-Phe (0.1 microM), GTP (10 microM), toxin from bacterium Bordetella pertussis (islet activating protein, 5 micrograms/ml) and their various combinations were unable to modulate the enzymatic activity of the 5-lipoxygenase. Stimulation of the 5-lipoxygenase by relatively low levels of free ionized calcium was observed both in the presence of the pellet and PC vesicles: maximal stimulation was seen at about 10 microM Ca2+. The human leukocyte leukotriene A4 synthase activity also exhibited a similar requirement for free calcium ions. The present study indicates that the membrane-associated stimulatory factor of the human leukocyte 5-lipoxygenase may be replaced by PC vesicles. Moreover, the 5-lipoxygenase and leukotriene A4 synthase activities require significantly lower Ca2+ levels for maximal activation than has been reported previously.

Isolation and identification of lipoxygenase products from the rat central nervous system.

Leukotrienes B4, C4, D4 and E4, together with five monohydroxyeicosatetraenoic acids, were isolated after incubation of chopped rat brain tissue with ionophore A23187. The monohydroxyeicosatetraenoic acids were 5-hydroxy-6,8,11,14-eicosatetraenoic acid, 9-hydroxy-5,7,11,14-eicosatetraenoic acid, 11-hydroxy-5,8,12,14-eicosatetraenoic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. Identification of the compounds was performed using reversed-phase high-performance liquid chromatography, ultraviolet spectroscopy and gas chromatography-mass spectrometry. Formation of the compounds was inhibited by micromolar concentrations of nordihydroguaiaretic acid. Indomethacin specifically inhibited the formation of 11-hydroxy-5,8,12,14-eicosatetraenoic acid, suggesting that this compound was produced as a by-product during cyclooxygenase-catalyzed prostaglandin synthesis.

Antagonism of the prostaglandin endoperoxide imhibition of hormone-stimulated adenylate cyclase by guanosine triphosphate and 5'-guanylyl-imidodiphosphate.

The prostaglandin endoperoxide prostaglandin H2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid) inhibits basal and hormone-stimulated adenylate cyclase in fat cell ghosts. This inhibition by prostaglandin H2 has been found to be antagonized by GTP and Gpp(NH)p. Dose response studies have shown GTP and Gpp(nh)p to be maximally effective at 3.3 muM, the lowest concentration tested. Although the system is exceedingly sensitive to modulation by GTP or Gpp(NH)p UTP, CTP, GMP, and cyclic GMP did not antagonize the antihormone activity of prostaglandin H2. Kinetic studies indicate that the GTP or Gpp(NH)p antagonism of prostaglandin H2 is observable on initial rates of cyclic AMP synthesis, and persists throughout the adenylate cyclase measurements. Preincubation of fat cell ghosts with GTP followed by washing and resuspension results in a prostaglandin H2-sensitive adenylate cyclase system. However, the same preincubation experiment with Gpp(NH)p produces an irreversible antagonism of the prostaglandin H2 inhibition of hormone-stimulated adenylate cyclase. It is suggested that prostaglandin H2 stabilizes the fat cell adenylate cyclase system in a state that is resistant to hormone stimulation, and GTP or Gpp(NH)p overcome this stabilization.

Characterization of cerebroside (monoglycosylceramide) from the sea anemone, Metridium senile. Identification of the major long-chain base as an unusual dienic base with a methyl branch at a double bond.

1. Cerebroside of the sea anemone, Metridium senile, has been isolated (0.6 mg/g dry tissue weight) and structurally characterized. 2. The structure was shown by mass spectrometry, NMR spectroscopy and degradative studies as beta-glucopyranosylceramide. The major fatty acids were 16 : 0 and 20 : 0 D-2-hydroxy fatty acids. The major base was a novel base, D-erythro-1,3-dihydroxy-2-amino-9-methyl-trans-4, trans-8-octadecadiene. 3. Some unusual fatty acids of marine origin are suggested to originate in this long-chain base by metabolic conversion. 4. The implication of the methyl branch position of the base on our current view of sphingolipid function in the plasma membrane is discussed.

Hemoprotein catalysis of leukotriene formation.

Incubation of various hemoproteins with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid resulted in formation of epimeric 5(S),12-dihydroxy-6,8,10,14 -eicosatetraenoic acids and epimeric 8,15(S)-dihydroxy-5,9,11,13 -eicosatetraenoic acids, respectively. These dihydroxy acids were earlier recognized as nonenzymatic hydrolysis products of 5(S),6-oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) and 14,15(S)-oxido-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene A4). These allylic epoxides could be isolated as such from the hemoprotein incubations, and most probably they are intermediates in formation of the dihydroxy acids.

Conformational analysis of lipoxin A, lipoxin B and their trans-isomers.

Lipoxin A and lipoxin B (LXA and LXB) are formed from the oxygenation of arachidonic acid by interactions between the 5- and 15-lipoxygenases of human leukocytes. Each compound displays highly stereospecific biological actions. Here, we present a computational description of the following compounds: lipoxin A, (5S,6R,15S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; 11-trans-lipoxin A, (5S,6R,15S)-trihydroxy-7,9,11,13-trans-eicosatetraenoic acid; lipoxin B, (5S,14R,15S)-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid; and 8-trans-lipoxin B, (5S,14R,15S)-trihydroxy-6,8,10,12-trans-eicosatetraenoic acid. The analyses considered van der Waals energy, electrostatic interactions, torsional potential, and alterations in electrostatic forces. Additional analyses were carried out with each of the four compounds forming complexes with one calcium ion. Each compound gave very different conformers. Both lipoxin A and lipoxin B can form globular conformations, while their all-trans isomers form rigid extended structures. When complexes with each of these compounds and one calcium ion were examined (i.e., (LXA)2Ca: (11-trans-LXA)2Ca), both LXA and LXB formed several flexible conformations including crumpled, wrapped or extended conformations. In this situation, LXA showed a higher probability than LXB to wrap around one Ca2+. In contrast, the two all-trans isomers always lead to extended conformations. Results from the present study illustrate that changes in the stereochemistry of LXA and LXB lead to unique conformations which may underlie the different biological actions of these compounds. Moreover, they indicate that the conformations of eicosanoids can change while in aqueous or hydrophobic environments (i.e., biomembranes).

Serum factors regulate 5-lipoxygenase activity in maturating HL60 cells.

5-Lipoxygenase activity in DMSO-differentiated HL60 cells is regulated by human serum. The serum effect depended on the differentiation state of the cells. For a stimulatory effect to occur, it was required that the cells had been treated with DMSO before addition of serum. After this regimen, the HL60 cells acquired the same high 5-LO activity as found for human neutrophils isolated from peripheral blood (about 9-times higher than for HL60 cells treated only with DMSO). On the other hand, when serum was added together with DMSO and present during the entire differentiation period (seven days), or withdrawn after the first four days, the 5-LO activity did not increase. 5-LO activity of HL60 cells covaried with the expression of the CD14 molecule, a marker for myeloid cell maturation which was recently identified as a receptor for the complex of LPS and LPS-binding protein. These serum effects on 5-LO activity were only observed for intact cells. The prominent increase in 5-LO activity induced by serum was not concomitant with similar changes in the expression of 5-LO or 5-LO-activating protein (FLAP), as judged from analyses of immunoreactive protein and mRNA. Also, the high 5-LO activity induced by serum was rather insensitive to the drug MK886 under our standard assay conditions, which included addition of exogenous arachidonic acid (40 microM). The results indicate that additional cellular components of importance for 5-LO activity in HL60 cells become operative after serum treatment, and that mere expression of 5-LO and FLAP is insufficient for high 5-LO activity in intact cells.

Main structures of the Forssman glycolipid hapten and a Leb-like glycolipid of dog small intestine, as revealed by mass spectrometry. Difference in ceramide structure related to tissue localization.

Two glycolipids of dog small intestine, one with Forssman activity and one with Leb-like activity, have been characterized by mass spectrometry of methylated, and methylated and reduced (LiAlH4) derivatives. The Forssman glycolipid was conclusively shown to be a pentaglycosylceramide with the carbohydrate sequence hexosamine-hexosamine-hexose-hexose-hexose-ceramide, and with sphingosine (dihydroxy base) as major long-chain base and normal fatty acids as the only fatty acids. The Leb-like glycolipid was a hexaglycosyl-ceramide with sequence fucose-hexose-[fucose-] hexosamine-hexose-hexose-ceramide and with phytosphingosine (trihydroxy base) as major long-chain base and only 2-hydroxy fatty acids as fatty acids. The difference of two hydroxy groups in the ceramide between the two glycolipids may be related to a different tissue localization. As shown by immunofluorescense study the Forssman activity was associated with the lamina propria and the Leb-like activity to the glandular epithelium of dog small intestine.

Recombinant mouse leukotriene A4 hydrolase: a zinc metalloenzyme with dual enzymatic activities.

Recombinant mouse leukotriene A4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of zinc/mol of enzyme. Apparent kinetic constants (Km and Vmax) for the conversion of leukotriene A4 to leukotriene B4 (at 0 degree C, pH 8) were 5 microM and 900 nmol/mg per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide. Km and Vmax for this reaction (at 37 degrees C, pH 8) were 680 microM and 365 nmol/mg per min, respectively. Apo-leukotriene A4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A4 resulted in a dose-dependent inactivation of both enzyme activities.

Identification of a novel 7-cis-11-trans-lipoxin A4 generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of lipoxins A4 and B4.

Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 microM) to human neutrophils led to the formation of both lipoxin A4 and lipoxin B4 as well as a novel 5,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A4 and lipoxin B4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA4 (7-cis-11-trans-LXA4). LXA4, 11-trans-LXA4, 7-cis-LXA4 and 7-cis-11-trans-LXA4 all evoked dose-dependent (0.1-10 microM) contractions of the guinea pig lung strip, whereas 6-cis-LXB4 and 6-cis-8-trans-LXB4 relaxed this preparation. LXA4 and 7-cis-LXA4 were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.

The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies.

Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A2 kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A2 kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Gal alpha 1-4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of a Gal alpha 1-3(Fuc alpha 1-2)Gal beta-HexNAc-Gal alpha 1-4Gal beta-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant blood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which might explain the low expression of type-4 chain blood group B heptaglycosylceramides in human blood group B kidneys.

p38 MAP kinase mediates stress-induced leukotriene synthesis in a human B-lymphocyte cell line.

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.