Rapid-onset clinical and mechanistic effects of anti-C5aR treatment in the mouse collagen-induced arthritis model.

Preclinical evidence supports targeting the C5a receptor (C5aR) in rheumatoid arthritis (RA). To support ongoing clinical development of an anti-C5aR monoclonal antibody, we have investigated for the first time the mechanism of action and the pharmacodynamics of a blocking anti-murine C5aR (anti-mC5aR) surrogate antibody in mouse collagen-induced arthritis (CIA). First, efficacy was demonstrated in a multiple-dose treatment study. Almost complete inhibition of clinical disease progression was obtained, including reduced bone and cartilage destruction in anti-mC5aR-treated mice. Then, the mechanism of action was examined by looking for early effects of anti-mC5aR treatment in single-dose treatment studies. We found that 48 h after single-dose treatment with anti-mC5aR, the neutrophil and macrophage infiltration into the paws was already reduced. In addition, several inflammatory markers, including tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-17A were reduced locally in the paws, indicating reduction of local inflammation. Furthermore, dose-setting experiments supported a beneficial clinical effect of dosing above the C5aR saturation level. In conclusion, these preclinical data demonstrated rapid onset effects of antibody blockade of C5aR. The data have translational value in supporting the Novo Nordisk clinical trials of an anti-C5aR antibody in rheumatoid arthritis patients, by identifying potential biomarkers of treatment effects as well as by providing information on pharmacodynamics and novel insights into the mechanism of action of monoclonal antibody blockade of C5aR.

Characterization of the interleukin-17 isoforms and receptors in lesional psoriatic skin.

BACKGROUND: Th17 cells are a lineage of proinflammatory T helper cells producing interleukin (IL)-17. The importance of Th17 cells in inflammation and autoimmunity has now been recognized. The IL-17 cytokine family consists of six isoforms (IL-17A-IL-17F) whereas five members of the IL-17 receptor (IL-17R) family have been identified (IL-17RA-IL-17RE). OBJECTIVES: To characterize the expression of the IL-17 isoforms and receptors in lesional and nonlesional psoriatic skin. Methods Keratome and punch biopsies taken from patients with psoriasis were examined by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction in order to measure the IL-17 isoforms and receptors. RESULTS: We demonstrated significantly increased mRNA expression of IL-17A, IL-17C and IL-17F in psoriatic skin. In contrast, the mRNA expression of IL-17B and IL-17D was significantly decreased in lesional compared with nonlesional skin, while IL-17E mRNA was undetectable. The increased mRNA expression of IL-17A, IL-17C and IL-17F was paralleled by an increased protein accumulation of these cytokines in psoriatic skin. Analysis of the IL-17R mRNA expression revealed significantly impaired mRNA expression of IL-17RB, IL-17RC, IL-17RD and IL-17RE in lesional psoriatic skin, whereas the mRNA expression of IL-17RA was similar in lesional and nonlesional psoriatic skin. CONCLUSIONS: This study characterizes the mRNA profile of the IL-17 isoforms and receptors in psoriatic skin lesions. Furthermore, we demonstrate for the first time augmented protein levels of IL-17A, IL-17C and IL-17F in psoriatic skin lesions, indicating a possible role for IL-17C in addition to IL-17A and IL-17F in the pathogenesis of psoriasis.

Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes.

An accelerated rate of glucose transport is among the most characteristic biochemical markers of cellular transformation. To study the molecular mechanism by which transporter activity is altered, cultured rodent fibroblasts transfected with activated myc, ras, or src oncogenes were used. In myc-transfected cells, the rate of 2-deoxy-D-glucose uptake was unchanged. However, in cells transfected with activated ras and src oncogenes, the rate of glucose uptake was markedly increased. The increased transport rate in ras- and src-transfected cells was paralleled by a marked increase in the amount of glucose transporter protein, as assessed by immunoblots, as well as by a markedly increased abundance of glucose transporter messenger RNA. Exposure of control cells to the tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) for 18 hours had a similar effect of increasing the rate of glucose transport and the abundance of transporter messenger RNA. For ras, src, and TPA, the predominant mechanism responsible for activation of the transport system is increased expression of the structural gene encoding the glucose transport protein.

Severely impaired adipsin expression in genetic and acquired obesity.

Adipsin, a serine protease homolog, is synthesized and secreted by adipose cells and is found in the bloodstream. The expression of adipsin messenger RNA (mRNA) and protein was analyzed in rodents during metabolic perturbations and in several experimental models of obesity. Adipsin mRNA abundance is increased in adipose tissue during fasting in normal rats and in diabetes due to streptozotocin-induced insulin deficiency. Adipsin mRNA abundance decreased during the continuous infusion of glucose, which induces a hyperglycemic, hyperinsulinemic state that is accompanied by an increased adipose mass; it is suppressed (greater than 100-fold) in two strains of genetically obese mice (db/db and ob/ob), compared to their congenic counterparts, and is also reduced when obesity is induced chemically by injection of monosodium glutamate into newborn mice. Circulating adipsin protein is decreased in these animal models of obesity, as determined by immunoblotting with antisera to adipsin. Little change in adipsin expression is observed in a model of obesity obtained by pure overfeeding of normal rats (cafeteria model). These data suggest a possible role for adipsin in the above-mentioned disordered metabolic states, and raise the possibility that adipsin expression may be used to distinguish obesities that arise from certain genetic or metabolic defects from those that result from pure overfeeding.

Effect of sucrose overfeeding on Na,K-ATPase-mediated 86Rb uptake in normal and ob/ob mice.

When normal mice have their usual chow diets supplemented by free access to a solution of 10% sucrose, their caloric intake increases by about 30%. We have used this model to explore the effects of sucrose overfeeding on Na,K-ATPase-mediated cation transport. After 5 days of sucrose supplementation, Na,K-ATPase mediated K uptake is increased by 88% in liver slices and 26% in intact soleus muscles of these animals. Ob/ob mice are hyperphagic on an ad libitum chow diet, and they display even greater increments in caloric intake than do thin controls when similarly allowed access to sucrose. Despite hyperphagia while on a chow diet, Na,K-ATPase-mediated K uptake by liver slices of ob/ob mice is not significantly different from that of their thin littermates. In addition, Na,K-ATPase-mediated K uptake into liver slices of ob/ob mice does not significantly increase while on sucrose supplements. These findings demonstrate the influence of dietary factors on Na,K-ATPase-mediated ion transport in liver and muscle of normal mice, and suggest that ob/ob mice may have an impairment in such dietary control. These observations suggest an important role for nutritional factors in the overall regulation of monovalent cation transport, and may also have relevance for our understanding of the cellular mechanisms of dietary thermogenesis.

Multiple factors influence insulin-like growth factor-I binding to human skin fibroblasts.

The binding of [125I]insulin-like growth factor-I ([125I]IGF-I) to human skin fibroblasts (HSF) is regulated by multiple factors. In monolayers of HSF, IGF-I binds to both the type I IGF receptor and IGF-binding proteins (BPs) associated with the cell surface. [125I]IGF-I binding to both of these proteins depends markedly on the sodium chloride concentration of the binding buffer. In monolayers of HSF, replacing 120 mM NaCl with isoosmotic concentrations of sucrose increases binding of [125I]IGF-I by 2- to 6-fold. Enhancement of [125I]IGF-I binding in the absence of sodium chloride is also seen in HSF in suspension, in human erythrocytes, in monolayers of HEP G2 cells and FRTL5 cells, and in membranes prepared from human placentae. Kinetic analysis of [125I]IGF-I binding to HSF monolayers reveals that association rates are increased and dissociation rates are decreased in the absence of sodium chloride. The binding of [125I]alpha IR-3, a monoclonal antibody to the human type I IGF receptor, to monolayers and suspensions of HSF also depends on the sodium ion concentration; it is 5- to 7-fold higher in the absence of sodium chloride. Binding of [125I]IGF-I to monolayers of HSF also depends on NaCl under conditions where alpha IR-3 saturates the type I IGF receptor but does not affect IGF-BPs. These findings demonstrate that sodium chloride has a marked effect on the interaction of IGF-I with the type I IGF receptor in the plasma membrane and with BPs associated with the surface of intact HSFs. Since an effect is also evident in membranes prepared from intact tissues (human placenta), occurs at 4 C, and occurs with cells devoid of BPs, a mechanism involving receptor or BP translocation seems unlikely, at least as the sole explanation for these findings. Sodium ions (and other ions) may induce a conformational change in the receptor and BPs and cause decreased availability of both the IGF-I-binding site and the alpha IR-3 epitope on the receptor and the IGF-binding site on the BP.

Reduced adipsin expression in murine obesity: effect of age and treatment with the sympathomimetic-thermogenic drug mixture ephedrine and caffeine.

Adipsin gene expression is greatly diminished in certain forms of genetic and acquired obesity. In the present study we evaluate the time course for the development of adipsin deficiency in obesity and its regulation by the sympathomimetic-thermogenic drug mixture ephedrine and caffeine. Previously, it was unknown whether adipsin deficiency occurred before or after the development of massive obesity. In the first series of experiments in which mice were treated with monosodium glutamate (MSG) for the first week of life, we demonstrate that adipsin deficiency occurs early in the development of MSG-induced obesity as evidenced by decreased circulating adipsin concentrations by 1 week of age and deficient adipsin mRNA levels in white adipose tissue (WAT) by 2 weeks. In db/db mice, diminished circulating adipsin was noted at 2 weeks of age. In both models, decreased adipsin gene expression precedes the development of marked obesity. Little is known about the factors which regulate adipsin gene expression in obesity. Common to the ob/ob, db/db and MSG models is diminished thermogenesis and sympathetic nervous system activity. In a second series of experiments we sought to determine whether adipsin deficiency in obesity could be corrected by treatment with ephedrine and caffeine (E+C), a sympathomimetic-thermogenic mixture previously shown to increase thermogenesis and reverse obesity in some models. In the present study, E+C treatment of MSG obese mice reversed obesity and markedly increased serum adipsin and adipsin mRNA levels in WAT and brown adipose tissue (BAT). In ob/ob mice, however, E+C treatment produced a negligible increase in adipsin mRNA levels in WAT and BAT as well as serum adipsin concentrations and this correlated with only a very small decrease in obesity. Thus, the ability of E+C to increase adipsin gene expression correlated with its ability to reverse obesity in these two models. Finally, the effect of E+C on adipsin gene expression may not be exerted directly on the fat cell since treatment of cultured 3T3-F442A adipocytes and isolated rat adipocytes in primary culture produced no effect on adipsin mRNA or secreted protein despite a lipolytic effect as measured by increased glycerol release. In summary, decreased adipsin gene expression occurs early in the development of MSG and db/db obesity and is markedly increased in the MSG model by the sympathomimetic-thermogenic drug mixture, E+C, which also reverses obesity. Elucidation of the factors responsible for these effects may enhance our understanding of fat cell gene regulation and obesity.

Insulin stimulation of aminoisobutyric acid transport in human skin fibroblasts is mediated through both insulin and type I insulin-like growth factor receptors.

A monoclonal antibody to the human type I insulin-like growth factor (IGF-I) receptor (alpha IR-3) was used to distinguish actions of insulin and IGF-I that are mediated through insulin as opposed to IGF-I receptors on human skin fibroblasts. Both insulin and IGF-I stimulate uptake of the nonmetabolized alanine analog alpha-aminoisobutyric acid (AIB) in these cells. alpha IR-3 inhibited AIB uptake stimulated by both of these hormones in a dose-dependent manner. However, the pattern of hormone action in the presence of alpha IR-3 differed for the two hormones. In the case of IGF-I, alpha IR-3 potently inhibited AIB uptake at low hormone concentrations, but this inhibition was overcome by high hormone concentrations, consistent with impairment of IGF-I action through the IGF-I receptor. In the case of insulin, the action of low concentrations (i.e. 10 ng/ml) was not inhibited, but that of higher insulin concentrations was, suggesting a dual receptor mechanism of cell stimulation by insulin. alpha IR-3 will be an important tool in further studies of the biology of the IGF-I receptor in normal and abnormal human cells.

Kennedy's disease: unusual molecular pathologic and clinical features.

Two patients with the Kennedy's disease (KD) mutation have been identified in the Newcastle Brain Tissue Bank. One of these patients had presenile dementia as a prominent clinical feature, previously undescribed in KD. The pathologic substrate underlying the cognitive changes in this patient included neuronal depletion and gliosis in the hippocampus and subcortical gliosis in the prefrontal region. Immunostaining for macrophage markers showed evidence for subtle corticospinal tract pathology in both cases. In contrast to the molecular pathologic features found in ALS, surviving motor neurons in the two KD cases showed no evidence of ubiquitinated inclusions or alterations in neurofilament phosphorylation.

Cytokine involvement in predicting clinical graft-versus-host disease in allogeneic bone marrow transplant recipients.

An in vitro skin explant model has been used to predict the severity of acute graft-versus-host disease (GVHD) in 34 HLA-identical bone marrow transplant recipients (correlation coefficient 0.6 p < 0.001). Supernatants from HLA-matched patient/donor mixed lymphocyte cultures (MLCs) were analysed for levels of tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). High levels of both cytokines correlated with the development of GVHD grades II or above (p < 0.05). The supernatants were also tested for induction of class II MHC antigen expression on third party skin and results correlated with clinical outcome in 17 of 22 cases (77%) (correlation coefficient 0.65, p < 0.001). The results suggest that measurement of both TNF alpha and IFN gamma in HLA-matched MLC supernatants is of predictive value and that the skin explant model is a useful model for studying the aetiology of GVHD in humans.

Novel insertion in the KSP region of the neurofilament heavy gene in amyotrophic lateral sclerosis (ALS).

The abnormal assembly and accumulation of neurofilaments (NF) in the perikarya and proximal axons of motor neurones is a characteristic of ALS. Deletions in the KSP repeat region of the NF-H gene have previously been reported in seven patients with sporadic ALS. Here we report the identification of a novel 84 bp insertion in the NF-H gene. This leads to an extra four KSP repeat elements in a highly conserved repetitive region of the gene. Although neurofilament mutations are only associated with a very small proportion of ALS cases, this insertion provides further support of a role for neurofilaments in the pathogenesis of ALS.

CNS tissue Cu/Zn superoxide dismutase (SOD1) mutations in motor neurone disease (MND).

DNA extracted from CNS tissue of 79 cases of motor neurone disease (MND) was screened by single strand conformation analysis (SSCA) and heteroduplex analysis (HA) for mutations in the Cu/Zn superoxide dismutase (SOD1) gene. The aims were to determine whether somatic mutations of SOD1 may underlie some cases of MND and to characterize the genetic abnormalities by sequencing, for subsequent correlation with the molecular pathological phenotype. In 3 cases a point mutation was found in exon 4: E100G in one familial case, and I113T in two cases (one familial, one sporadic). Two cases had previously undescribed mutations in the 3' untranslated region (3'UTR) of SOD1 and one case had a single base substitution in the intronic sequence upstream from exon 2. None of these patients had a positive family history. Non-CNS tissue was available for 3 out of the 6 cases in whom changes were found. In all 3 the same changes were consistently found in both CNS and non-CNS tissue, excluding the presence of somatic mutations in SOD1. We investigated many MND blood samples and normal controls for the presence of the 3'UTR deletions. We found the 4 bp deletion in 1/90 sporadic MND patients and 1/209 non-MND controls. If the 3'UTR deletions are pathogenic, they would have to operate via a loss of the function mechanism, and further work is necessary to define their significance.

Poly(ADP-ribose) polymerase is found in both the nucleus and cytoplasm of human CNS neurons.

There is evidence that inhibitors of poly(ADP-ribose) polymerase (PARP) may be therapeutically useful in neurodegenerative diseases. Using immunocytochemistry, we have investigated the distribution of PARP in the human CNS. Some neuronal groups showed cytoplasmic staining in addition to the expected staining of nuclei. Considerable variation between different neuronal groups was noted: motor neurons in the spinal cord showed greatest cytoplasmic staining, whereas staining was virtually absent in other neurons, notably in the hippocampus. These results indicate that PARP can be associated with sub-cellular components other than the nucleus, and may indicate additional roles for this enzyme.

Human health implications of environmental contaminants in Arctic Canada: a review.

This paper assesses the impact on human health of exposure to current levels of environmental contaminants in the Canadian Arctic, and identifies the data gaps that need to be filled by future human health research and monitoring. The concept of health in indigenous groups of the Arctic includes social, cultural, and spiritual dimensions. The harvesting, sharing and consumption of traditional foods are an integral component to good health among Aboriginal people influencing both physical health and social well-being. Traditional foods are also an economic necessity in many communities. Consequently, the contamination of country food raises problems which go far beyond the usual confines of public health and cannot be resolved by health advisories or food substitutions alone. The primary exposure pathway for the contaminants considered in this paper is through the traditional northern diet. For the Inuit, the OCs of primary concern at this time from the point of view of exposure are chlordane, toxaphene, and PCBs. Exposures are higher in the eastern than in the western region of the North. For Dene/Metis, exposure to OCs is in general below a level of concern. However, estimated intake of chlordane and toxaphene has been found to be elevated for certain groups and is a cause for concern if exposures are elevated on a regular basis. The developing foetus and breast-fed infant are likely to be more sensitive to the effects of OCs and metals than individual adults and are the age groups at greatest risk in the Arctic. Extensive sampling of human tissues in the Canadian north indicate that a significant proportion of Dene, Cree and Inuit had mean maternal hair mercury levels within the 5% risk-range proposed by the WHO for neonatal neurological damage. Based on current levels, lead does not appear to pose a health threat while cadmium is likely only a major risk factor for heavy smokers or consumers of large amounts of organ meats. Consumers of traditional foods are exposed to an approximately seven-fold higher radiation dose than non-consumers of traditional foods due predominantly to the bioaccumulation of natural radionuclides in the food chain. Risk determination for contaminants in country food involves a consideration of the type and amounts of food consumed and the sociocultural, nutritional, economic, and spiritual benefits associated with country foods. Risk management options that minimize the extent to which nutritional and sociocultural aspects of Aboriginal societies are compromised must always be considered.

A comparison of children's electric wheelchairs.

During the past few years, the Occupational Therapists at the Kitchener-Waterloo Rotary Children's Centre have explored and prescribed electric wheelchairs for handicapped children from 2-19 years of age. This paper presents a comparison of three children's electric wheelchairs, the Bec, Solo, and Everest and Jennings.

Identifying sources and estimating glandular output of salivary TIMP-1.

OBJECTIVE: Tissue inhibitor of metalloproteinases 1 (TIMP-1) has been identified as a potential biomarker in diseases such as cancer, cardiovascular diseases and diabetes. Since TIMP-1 resides in most tissues and bodily fluids, we evaluated the potential of using saliva to obtain reproducible TIMP-1 measurements in a non-invasive manner. MATERIAL AND METHODS: Samples of unstimulated and stimulated whole saliva and saliva collected from individual glands were analysed for TIMP-1 content. A TIMP-1 ELISA was validated for use in saliva testing and the most optimal sampling and handling procedures for reproducible measurements identified. Western blotting and MALDI-TOF mass spectrometry were used for confirmatory analyses. RESULTS: The TIMP-1 ELISA was found suitable for saliva measurements. All saliva secretions contained TIMP-1, but in different concentrations ranging from 2.81 ng/mL in submandibular/sublingual saliva to 173.88 ng/mL in parotid saliva. TIMP-1 concentrations were influenced to a varying degree by fluctuations in flow. We found the lowest output in submandibular/sublingual saliva stimulated with 0.5% citric acid (3.56 ng/min) and highest output in chewing-stimulated whole saliva (267.01 ng/min). CONCLUSION: This study shows that saliva contains authentic TIMP-1, the concentration of which was found to depend on gland type and salivary flow. Stimulated whole saliva is suggested as a reliable and easily accessible source for TIMP-1 determinations in bodily fluids.

Identification of alternatively spliced TIMP-1 mRNA in cancer cell lines and colon cancer tissue.

TIMP-1 is a promising new candidate as a prognostic marker in colorectal and breast cancer. We now describe the discovery of two alternatively spliced variants of TIMP-1 mRNA. The two variants lacking exon 2 (del-2) and 5 (del-5), respectively, were identified in human cancer cell lines by RT-PCR. The del-2 variant was, furthermore, detected in extracts from 12 colorectal cancer tissue samples. By western blotting additional bands of lower molecular mass than full-length TIMP-1 were identified in tumor tissue, but not in plasma samples obtained from cancer patients. The two splice variants of TIMP-1 may hold important clinical information, and either alone or in combination with measurement of full-length TIMP-1 they may improve the prognostic and/or predictive value of TIMP-1 analyses.