Suppression of tumour necrosis factor production from mononuclear cells by a novel synthetic compound, CLX-090717.

OBJECTIVES: To evaluate the clinical efficacy of a novel synthetic peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonist, CLX-090717, in several in vitro cell culture systems and murine CIA, an experimental model of RA. METHODS: Peripheral blood monocytes purified by elutriation, and rheumatoid synovial cells isolated from clinical tissue were cultured with CLX-090717 and TNF-alpha release was measured. Molecular mechanism of action was analysed by western blotting and electrophoretic mobility shift assay. Thioglycollate-elicited murine peritoneal macrophages were cultured with CLX-090717 and lipopolysaccharide (LPS)-induced TNF-alpha release was assayed. Therapeutic studies were done in mice with established arthritis by evaluating clinical parameters and histology. In addition, type II collagen response of lymphocytes from mice with CIA was examined. RESULTS: CLX-090717 significantly inhibited spontaneous TNF-alpha release by RA synovial membrane cells, as well as LPS-induced TNF-alpha release from human and murine monocytic cells. Inhibition of TNF-alpha in monocytes was mediated partially through a nuclear factor-kappaB (NF-kappaB)-dependent pathway, as judged by sustained levels of IkappaBalpha in cytosolic extracts and a reduced level of LPS-induced NF-kappaB activity in nuclear extracts. CLX-090717 reduced clinical signs of arthritis and damage to joint architecture when administered therapeutically to arthritic mice. Mechanisms of action in CIA involved the reduction in proliferation of arthritic lymphocytes to antigen in vitro as well as reduced TNF-alpha release. CONCLUSIONS: Our data suggest that the synthetic compound CLX-090717 has potential as a small molecular weight anti-inflammatory therapeutic for chronic inflammatory conditions.

Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo.

Pulmonary fibrosis is the common end stage of a number of pneumopathies. In this study, we examined the ability of the human cytokine, relaxin, to block extracellular matrix deposition by human lung fibroblasts in vitro, and to inhibit lung fibrosis in a bleomycin-induced murine model. In vitro, relaxin (1-100 ng/ml) inhibited the transforming growth factor-beta-mediated over-expression of interstitial collagen types I and III by human lung fibroblasts by up to 45% in a dose-dependent manner. Relaxin did not affect basal levels of collagen expression in the absence of TGF-beta-induced stimulation. Relaxin also blocked transforming growth factor-beta-induced upregulation of fibronectin by 80% at the highest relaxin dose tested (100 ng/ml). The expression of matrix metalloproteinase-1, or procollagenase, was stimulated in a biphasic, dose-dependent manner by relaxin. In vivo, relaxin, at a steady state circulating concentration of approximately 50 ng/ml, inhibited bleomycin-mediated alveolar thickening compared with the vehicle only control group (P < 0.05). Relaxin also restored bleomycin-induced collagen accumulation, as measured by lung hydroxyproline content, to normal levels (P < 0.05). In summary, relaxin induced a matrix degradative phenotype in human lung fibroblasts in vitro and inhibited bleomycin-induced fibrosis in a murine model in vivo. These data indicate that relaxin may be efficacious in the treatment of pathologies characterized by lung fibrosis.

Alzheimer's disease: beta-amyloid precursor protein mRNA expression in mononuclear blood cells.

beta-Amyloid precursor protein (beta APP) mRNA was examined in peripheral mononuclear blood cells (PMBCs) in Alzheimer's disease, Down's syndrome and control subjects. Total RNA from PMBCs was reverse transcribed and then amplified using the polymerase chain reaction (PCR). The 3 major beta APP transcripts were expressed in PMBCs from all subjects. These results suggest that PMBCs could be a circulating source for abnormal amyloid deposition in the brain and in peripheral tissues.

Abnormalities in premigratory granule cells in the weaver cerebellum defined by monoclonal antibody OZ42.

The immunoreactivity in OZ42, a neural cell specific antibody that recognizes premigratory cerebellar granule cells, was examined in early postnatal wild-type and weaver mouse cerebella. We find that the OZ42-positive staining in the external granular layer (EGL) is first seen at postnatal day 1 in the most posterior and ventral aspect of midline cerebellum in the wild-type and heterozygous weaver mouse. By postnatal day 4 strong immunoreactivity is observed in the EGL of all cerebellar lobules. This staining is localized to a band of immunoreactive cells present at the interface of the EGL and the molecular layer (ML). In the homozygous weaver cerebellum, OZ42-positive staining is not seen until postnatal day 3. In the postnatal day 4 weaver cerebellum, immunoreactivity is considerably ligther than in littermate control cerebella, and found throughout the width of the EGL (i.e., not localized to the EGL-ML interface). This study demonstrates that the expression of a specific marker of granule cell development is abnormal in the granule cell population of the homozygous weaver mouse, a population of cells known to be intrinsically affected by the action of this mutant gene. In the light of previous studies, which have shown that the weaver phenotype is identifiable as early as the day of birth, and that the OZ42-antigen may be involved with the development process of axonal growth, it is reasonable to suggest that the weaver mutation results in an abnormality in the ability of granule cells to produce and/or stabilize axons.

Evidence from studies employing radioactively labelled fatty acids that the stimulation of flux through the diacylglycerol pool is an early action of vasopressin on hepatocytes.

1. In isolated hepatocytes prelabelled with [14C]-arachidonic, -stearic, -linoleic, -oleic or -palmitic acids, vasopressin increased the amount of radioactivity present in diacylglycerols. The largest increase was observed in cells labelled with arachidonic or stearic acids. 2. In cells prelabelled with [14C]- or [3H]-arachidonic acid, the onset of the increase in radioactivity in diacylglycerols induced by vasopressin was slow, the increase was partly dependent on the presence of extracellular Ca2+, and was associated with an increase in radioactivity present in phosphatidic acid which was more rapid in onset. Vasopressin decreased the amount of [3H]arachidonyl-phosphatidylinositol 4,5-bisphosphate, but the magnitude of this decrease was less than 10% of the observed increase in radioactivity in [3H]arachidonyl-diacylglycerol. 3. The concentration of vasopressin which gave half-maximal increase in [14C]arachidonyl-diacylglycerol at low extracellular Ca2+ was 10-fold higher than that which gave half-maximal stimulation of 45Ca2+ efflux. Phenylephrine, but not glucagon, also increased the amount of [14C]arachidonyl-diacylglycerol. 4. It is concluded that an early action of vasopressin on the liver cell is to increase the flux of carbon from phospholipids, including the phosphoinositides, to diacylglycerols.

Transiently expressed, neural-specific molecule associated with premigratory granule cells in postnatal mouse cerebellum.

A rat monoclonal antibody (OZ42), raised against immature mouse granule cells, recognizes a region of the external granular layer of postnatally developing cerebellar cortex. This region, about three cells thick, is adjacent to the developing molecular layer and contains postmitotic, premigratory granule cells. The OZ42 reactivity commenced near postnatal day 3 (P3), the deep external granular layer was strongly reactive by P10 and this level was maintained while granule cells remained in the external granular layer (approximately P15). Isolated immature granule cells in cytospin preparations specifically reacted with OZ42. Reactivity was extranuclear and was substantially reduced when cells were prepared by trypsinization, suggesting that at least some of the antigen is associated with the outer surface of the plasma membrane. Other postnatal reactivity to OZ42 (P0 to P3) was found in a band of cells in the deep cortical layers overlying the corpus callosum through the entorhinal cortex, terminating adjacent to the hippocampus. Reactivity in some regions of the corpus callosum and anterior commissure was seen from P0 to P5. No reactivity of non-neural tissues was observed at any stage. In the embryo there was extensive staining of the CNS and PNS at E10 and E14, which was largely gone by E16. Weaver mutant mice examined for reactivity to OZ42 showed that the granule cell death and cerebellar disorganization in P10 homozygous mutants was associated with a substantial decrease in OZ42 reactivity in the external granular layer. At P14 and P20, OZ42 reactivity in the weaver external granular layer was restricted to single cells, rather than an entire layer of cells, further indicating that the OZ42 antigen is present on granule cells rather than the substratum. By Western analysis of non-reducing SDS-PAGE gels, OZ42 recognized a single band with the molecular weight between 120 and 145 kD in P10, but not adult cerebellum and BALB/c mice. An OZ42-specific band at 60-70 kD was also seen under reducing conditions and occasionally in non-reducing conditions. These bands were not recognized by antibodies against NCAM, L1 and AMOG. Immunoprecipitation and cross-blocking with antiserum to TAG-1 suggested that OZ42 recognized the same molecule in the mouse cerebellum that has been described in embryonic rat and mouse spinal cord. The developmentally regulated expression of the neural-specific molecule recognized by OZ42 in the postnatal cerebellum suggests it my be involved with the early stages of granule cell axon elongation.

A transient increase in diacylglycerols is associated with the action of vasopressin on hepatocytes.

Vasopressin induced a transient increase of 50% in the total concentration of diacylglycerols (determined by g.l.c.) in isolated hepatocytes. The increase was maximal at 0.25 min, and the concentration of diacylglycerols in cells treated with vasopressin had returned to the basal value by 4 min. No change in the concentration of diacylglycerols was observed after the treatment of cells with glucagon. The dependency of this effect on the concentration of vasopressin was similar to that of the effect of the hormone on 45Ca2+ efflux measured at 0.1 mM extracellular Ca2+. Vasopressin increased the proportion of arachidonic acid and stearic acid and decreased the proportion of oleic acid present in the diacylglycerols. In hepatocytes prelabelled with [14C]arachidonic acid, vasopressin increased the amount of [14C]diacylglycerol. The effects of vasopressin on the total concentration of diacylglycerols and [14C]diacylglycerol were mimicked by an exogenous phospholipid phosphodiesterase (phospholipase C) from Clostridium perfringens. The results are consistent with the conclusion that the transient increase in diacylglycerols induced by vasopressin is caused by the rapid hydrolysis of both the phosphoinositides and one or more other phospholipids.

Relaxin induces vascular endothelial growth factor expression and angiogenesis selectively at wound sites.

Relaxin is a reproductive hormone that has historically been characterized as being responsible for pubic ligament loosening and cervical ripening. Recently, relaxin has been associated with neovascularization of the endometrial lining of the uterus, potentially via specific induction of vascular endothelial growth factor. Previously conducted clinical studies using partially purified porcine relaxin have described relaxin's ability to stimulate the healing of ischemic wounds, suggesting that relaxin may also have angiogenic effects at sites of ischemic wound healing. In the present study, relaxin's angiogenic effects in the context of wound repair were tested in rodent models of angiogenesis and wound healing. Relaxin showed an ability to stimulate new blood vessel formation, particularly at ischemic wound sites, and to induce both vascular endothelial growth factor and basic fibroblast growth factor specifically in cells, presumably including macrophages, collected from wound sites. Resident macrophages collected from nonwound sites, such as the lung, did not show altered expression of these cytokines following relaxin administration. Because angiogenic wound cells are frequently macrophages, THP-1 cells, a cell line of monocyte lineage that binds relaxin specifically, were tested for and shown to induce vascular endothelial growth factor and basic fibroblast growth factor in response to relaxin. In conclusion, relaxin may be useful in the treatment of ischemic wounds by stimulating angiogenesis via the induction of vascular endothelial growth factor and basic fibroblast growth factor in wound macrophages.