Multivalent Fcγ-receptor engagement by a hexameric Fc-fusion protein triggers Fcγ-receptor internalisation and modulation of Fcγ-receptor functions.

Engagement of Fcγ-receptors triggers a range of downstream signalling events resulting in a diverse array of immune functions. As a result, blockade of Fc-mediated function is an important strategy for the control of several autoimmune and inflammatory conditions. We have generated a hexameric-Fc fusion protein (hexameric-Fc) and tested the consequences of multi-valent Fcγ-receptor engagement in in vitro and in vivo systems. In vitro engagement of hexameric-Fc with FcγRs showed complex binding interactions that altered with receptor density and triggered the internalisation and degradation of Fcγ-receptors. This caused a disruption of Fc-binding and phagocytosis. In vivo, in a mouse ITP model we observed a short half-life of hexameric-Fc but were nevertheless able to observe inhibition of platelet phagocytosis several days after hexameric-Fc dosing. In cynomolgus monkeys, we again observed a short half-life, but were able to demonstrate effective FcγR blockade. These findings demonstrate the ability of multi-valent Fc-based therapeutics to interfere with FcγR function and a potential mechanism through which they could have a sustained effect; the internalisation and degradation of FcγRs.

Sclerostin antibody treatment enhances bone strength but does not prevent growth retardation in young mice treated with dexamethasone.

OBJECTIVE: Exposure to supraphysiologic levels of glucocorticoid drugs is known to have detrimental effects on bone formation and linear growth. Patients with sclerosteosis lack the bone regulatory protein sclerostin, have excessive bone formation, and are typically above average in height. This study was undertaken to characterize the effects of a monoclonal antibody to sclerostin (Scl-AbI) in mice exposed to dexamethasone (DEX). METHODS: Young mice were concomitantly treated with DEX (or vehicle control) and Scl-AbI antibody (or isotype-matched control antibody [Ctrl-Ab]) in 2 independent studies. Linear growth, the volume and strength of the bones, and the levels of bone turnover markers were analyzed. RESULTS: In DEX-treated mice, Scl-AbI had no significant effect on linear growth when compared to control treatment (Ctrl-Ab). However, in mice treated with DEX and Scl-ABI, a significant increase in trabecular bone at the femoral metaphysis (bone volume/total volume +117% versus Ctrl-Ab-treated mice) and in the width and volume of the cortical bone at the femoral diaphysis (+24% and +20%, respectively, versus Ctrl-Ab-treated mice) was noted. Scl-AbI treatment also improved mechanical strength (as assessed by 4-point bending studies) at the femoral diaphysis in DEX-treated mice (maximum load +60% and ultimate strength +47% in Scl-AbI-treated mice versus Ctrl-Ab-treated mice). Elevated osteocalcin levels were not detected in DEX-treated mice that received Scl-AbI, although levels of type 5b tartrate-resistant acid phosphatase were significantly lower than those observed in mice receiving DEX and Ctrl-Ab. CONCLUSION: Scl-AbI treatment does not prevent the detrimental effects of DEX on linear growth, but the antibody does increase both cortical and trabecular bone and improves bone mechanical properties in DEX-treated mice.

A rapid and cost-effective method for analysis of three common genetic risk factors for thrombosis.

A simple, rapid and cost-effective method for the analysis of three of the most widely screened genetic risk factors for thrombosis has been established. The protocol developed uses blood spots stored on filter paper (Guthrie spots) as well as DNA extracted from anticoagulated blood. The use of Guthrie spots taken at birth enables the retrospective study of patients who develop thrombotic complications without necessitating resampling. Following isolation of DNA, conventional fluorescence-labelled polymerase chain reaction (PCR) is performed using a thermostable DNA polymerase. Denatured, single-stranded PCR products are analysed on a semi-automated capillary-based genetic analyser, the data being stored electronically. This sensitive protocol obviates the need for endonuclease digestion and the associated gel running and documentation, and leads to a reduction in the recurrent costs of laboratory consumables.

Effects of hyaluronan on models of immediate and delayed hypersensitivity in the rat.

Others have previously shown that superoxide dismutase conjugated with hyaluronan (HA) retains enzymic activity but is non-immunogenic. Whether HA could be widely used to prevent sensitisation to protein/polypeptide therapeutics is not known. In this study we investigated the effects of HA on bovine serum albumin (BSA) and methylated BSA pleural reactions in sensitised rats (active Arthus and delayed hypersensitivity reactions respectively) and on a reverse passive Arthus reaction in which rats received an intravenous injection of rabbit immunoglobulin and intrapleural challenge with goat anti rabbit immunoglobulin. HA suppressed the active Arthus and delayed hypersensitivity models when administered at the time of sensitisation but only the delayed hypersensitivity model at the time of intrapleural challenge. HA did not modulate the reverse passive Arthus reaction. The results show no evidence that simple mixing of HA with antigens masks antigenic determinants. However, HA appeared to have suppressive effects on both antibody and cell-mediated immune reactions. Therefore it may not be necessary to conjugate protein/polypeptide therapeutics to HA in order to prevent immune sensitisation.

The effects of cyclooxygenase 2 inhibitors on cartilage erosion and bone loss in a model of Mycobacterium tuberculosis-induced monoarticular arthritis in the rat.

Selective cyclooxygenase 2 (COX 2) inhibitors NS-398 and nimesulide were investigated for their effects on patellar cartilage and bone content in a model of Mycobacterium tuberculosis (M.tb)-induced monoarticular arthritis in the rat. The protective/destructive properties of these nonsteroidal antiinflammatory drugs (NSAIDs) were compared with piroxicam, known to accelerate cartilage breakdown and reduce bone erosion in this model in comparison to untreated arthritic controls. Male CFHB Wistar rats were injected intraarticularly with heat killed M.tb into the left stifle joint, resulting in loss of patellar cartilage glycosaminoglycans (GAG), bone erosion and inflammation. The right stifle joint received saline. Animals were dosed daily, p.o., with NS-398 (1, 10 mg/kg), nimesulide (0.5, 5 mg/kg) or piroxicam (10 mg/kg). Four days after M.tb injection, patellar GAG content, bone weight and joint swelling were measured in drug-treated animals and untreated arthritic controls. Changes in the left joint were compared to the right. The expression and distribution of COX 2 protein was determined by immunocytochemistry in synovial tissue from arthritic controls over the time course. Focal accumulations of inflammatory cells were positively immunolabelled for COX 2 in the synovium from the left stifle joint of untreated arthritic animals, 6 h after injection of M.tb. Labeling of inflammatory cell infiltrates increased and was widespread in the synovium at 24 h. By day 4 fibroblasts were positively labelled for COX 2 in addition to polymorphonuclear and mononuclear leukocytes. Piroxicam and nimesulide at the higher dose significantly exacerbated M.tb-induced cartilage GAG loss while NS-398 was without effect. Both COX 2 inhibitors did not alter M.tb-induced patellar bone loss. In contrast, piroxicam significantly reduced bone loss. All COX inhibitors significantly reduced joint swelling. In conclusion, the selective inhibition of COX 2 may result in the amelioration of synovitis with a lowered risk of NSAID-induced cartilage damage in rheumatic disease.

Effects of diacerhein on granuloma induced cartilage breakdown in the mouse.

OBJECTIVE: Diacerhein, an anti-osteoarthritic agent, was tested for its ability to suppress synthesis of proinflammatory cytokines in a model of granuloma-induced cartilage breakdown. DESIGN: 50 TO mice received a subcutaneous implant of cotton-wrapped rat femoral head cartilage for a period of 2 weeks. Animals (N = 10/group) were dosed daily with either 6 mg/kg p.o. diclofenac or diacetylrhein at 5, 15 or 50 mg/kg p.o. in 0.1.ml 1% gum tragacanth which served as a control. Implanted cartilages were assayed for glycosaminoglycan (GAG) and hydroxyproline content. The surrounding granulomas were assayed for interleukin-1 alpha (IL-1 alpha), tumour necrosis factor-alpha (TNF-alpha) and IL-6. Statistical analysis was by Mann-Whitney U test. RESULTS: Diclofenac had no significant effect on GAG or hydroxyproline content of implanted cartilage or on granuloma cytokine concentrations. Diacerhein protected implanted cartilages against hydroxyproline loss, implanted control cartilages contained 220 micrograms hydroxyproline compared with diacerhein at 5, 15 and 50 mg/kg which produced a 21, 16 and 59% decrease in hydroxyproline loss compared with non-implanted controls (P < 0.05, 0.05 and 0.001) respectively. Diacerhein also protected against GAG loss at 5 mg/kg and 50 mg/kg, control cartilages contained 134 micrograms GAG compared with diacerhein at 5 mg/kg and 50 mg/kg which produced a 24 and 38% decrease in GAG loss respectively (P < 0.05 for both). Diacerhein significantly reduced granuloma interleukin-1 alpha content at 5 mg/kg (control level of 2.4 micrograms/ml reduced by 58%; P < 0.05), reduced TNF-alpha at 5 mg/kg and 15 mg/kg (reduced by 61%: P < 0.01 and 49%: P < 0.05 respectively; control level of 469 pg/ml) and reduced IL-6 at 15 mg/kg and 50 mg/kg (control level of 537 pg/ml reduced by 60 and 51%, respectively; P < 0.01 for both). CONCLUSIONS: The mechanism of the chondroprotective effects of diacerhein is not understood but may be explained by a reduction in the concentrations of proinflammatory cytokines.

Effects of deviant child behavior on parental alcohol consumption. Stress-induced drinking in parents of ADHD children.

Distress and ad lib alcohol consumption after interactions with child confederates were investigated in parents of children with externalizing disorders--attention-deficit hyperactivity disorder (ADHD), conduct disorder (CD), or oppositional defiant disorder (ODD). Sixty subjects interacted with boys trained to act like either normal children or children with ADHD/CD/ODD. Interactions with deviant confederates resulted in feelings of inadequacy and produced negative affect but had no effect on alcohol consumption. Post hoc analyses showed that parents with a family history of alcohol problems (FH+) showed increased drinking after interaction with a deviant confederate, compared with FH+ parents who interacted with the normal confederate. FH- parents showed the opposite pattern of results.

Effects of deviant child behavior on parental distress and alcohol consumption in laboratory interactions.

Levels of adult distress and ad lib alcohol consumption following interactions with child confederates were investigated in parents of children with no diagnosable psychiatric disorders. Sixty parents (20 married couples and 20 single mothers) interacted with boys trained to enact behaviors characteristic of either normal children or "deviant" children with externalizing behavior disorders--attention-deficit hyperactivity disorder (ADHD), conduct disorder (CD), and oppositional defiant disorder (ODD). Relative to the normal child role, interactions with deviant confederates were rated as significantly more unpleasant, resulted in feelings of role inadequacy, and produced significantly more anxiety, depression, and hostility. After the interactions, parents were given the opportunity to drink as much of their preferred alcoholic beverage as they desired while anticipating a second interaction with the same child. The participants consumed more alcohol following exposure to deviant as opposed to normal confederates.

Teacher ratings of DSM-III-R symptoms for the disruptive behavior disorders.

Ratings were collected on a rating scale comprised of the DSM-III-R diagnostic criteria for disruptive behavior disorders. Teacher ratings were obtained for 931 boys in regular classrooms in grades K through 8 from around North America. Means and standard deviations for attention-deficit hyperactivity disorder (ADHD), oppositional-defiant disorder (ODD), and conduct disorder (CD) scales are reported by age. Frequencies of DSM-III-R symptoms are reported by age, and suggested diagnostic cutoffs are discussed. A factor analysis revealed three factors: one reflecting ODD and several CD symptoms, one on which ADHD symptoms of inattention loaded, and one comprised of ADHD impulsivity/overactivity symptoms. Conditional probability analyses revealed that several hallmark symptoms of ADHD had very poor predictive power, whereas combinations of symptoms from the two ADHD factors had good predictive power. Combinations of ODD symptoms also had very high predictive power. The limited utility of teacher ratings in assessing symptoms of conduct disorder in this age range is discussed.

Relative efficacy of long-acting stimulants on children with attention deficit-hyperactivity disorder: a comparison of standard methylphenidate, sustained-release methylphenidate, sustained-release dextroamphetamine, and pemoline.

Twenty-two children with attention deficit-hyperactivity disorder underwent a double-blind, placebo-controlled, crossover evaluation of the efficacy of standard methylphenidate twice a day and comparable doses every morning of a sustained-release preparation of methylphenidate (SR-20 Ritalin), a sustained-release form of dextroamphetamine (Dexedrine Spansule), and pemoline. The children were participating in a summer treatment program in which they engaged in recreational and classroom activities. Dependent measures include evaluations of social behavior during group recreational activities, classroom performance, and performance on a continuous performance task. Results revealed generally equivalent and beneficial effects of all four medications. Dexedrine Spansule and pemoline tended to produce the most consistent effects and were recommended for 10 of the 15 children who were responders to medication. The continuous performance task results showed that all four medications had an effect within 2 hours of ingestion, and the effects lasted for 9 hours. The implications of these results for the use of long-acting stimulant medication in children with attention deficit-hyperactivity disorder are discussed.

Influence of hormones on the nicotinamide nucleotide coenzymes of adipose tissue.

The concentrations of the oxidized and reduced forms of the nicotinamide nucleotides were measured in the epididymal fat pads of normal, alloxan-diabetic and hypophysectomized rats. In both alloxan-diabetic rats and hypophysectomized rats the weight of the adipose tissue fell, as did the total content of NADH and NADPH; in addition, NAD(+) was decreased in the alloxan-diabetic group. Of these changes the most marked was in NADPH and this was the only significant difference when the results were expressed as nicotinamide nucleotides/mg. of tissue protein. The concentration of NADPH in the hypophysectomized rats was not altered by treatment with growth hormone but was restored to normal by treatment with thyroxine. These results are discussed in relation to the known effect of these hormonal conditions on lipid synthesis in adipose tissue.

Effect of alloxan-diabetes on multiple forms of hexokinase in adipose tissue and lung.

Comparison has been made of the effect of alloxan-diabetes on the multiple forms of hexokinase (EC 2.7.1.1) in adipose tissue and lung. Types I and II hexokinase were distinguished in adipose tissue by their different stabilities to heat treatment, which made it possible to determine the activity of each form spectrophotometrically; additional confirmatory evidence was obtained from starch-gel electrophoresis. Type II hexokinase was markedly depressed in adipose tissue from alloxan-diabetic rats. Lung contained types I, II and III hexokinase, type I predominating. There was no significant change in the pattern of these multiple forms of hexokinase in lung from alloxan-diabetic rats. These results are discussed in relation to current ideas that the insulin-sensitivity of a tissue may be correlated with the content of type II hexokinase.