Miple1 and miple2 encode a family of MK/PTN homologues in Drosophila melanogaster

Midkine (MK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. To date, this family of proteins has been implicated in multiple processes, such as growth, survival, and migration of various cells, and has roles in neurogenesis and epithelial–mesenchymal interaction during organogenesis. In this report, we have characterized two members of the MK/PTN family of proteins in Drosophila, named Miple1 and Miple2, from Midkine and Pleiotrophin. Drosophila miple1 and miple2 encode secreted proteins which are expressed in spatially restricted, nonoverlapping patterns during embryogenesis. Expression of miple1 can be found at high levels in the central nervous system, while miple2 is strongly expressed in the developing midgut endoderm. The identification of homologues of the MK/PTN family in this genetically tractable model organism should allow an analysis of their function during complex developmental processes. C. Englund, A. Birve, and L. Falileeva contributed equally to this work.     

Stretch-grown axons retain the ability to transmit active electrical signals

Little is known about extensive nervous system growth after axons reach their targets. Indeed, postnatal animals continue to grow, suggesting that axons are stretched to accommodate the expanding body. We have previously shown that axons can sustain stretch-growth rates reaching 1 cm/day; however, it remained unknown whether the ability to transmit active signals was maintained. Here, stretch-growth did not alter sodium channel activation, inactivation, and recovery or potassium channel activation. In addition, neurons generated normal action potentials that propagated across stretch-grown axons. Surprisingly, Na and K channel density increased due to stretch-growth, which may represent a natural response to preserve the fidelity of neuronal signaling.     

RX871024 reduces NO production but does not protect against pancreatic beta-cell death induced by proinflammatory cytokines

The imidazoline compound RX871024 reduces IL-1beta-induced NO production thereby protecting against IL-1beta-induced beta-cell apoptosis. The aim of this study was to evaluate whether imidazolines RX871024 and efaroxan protect beta-cells against death in the presence of a combination of the cytokines IL-1beta, IFNgamma, and TNFalpha. To address this issue, experiments involving different methods for detection of cell death, different concentrations of the cytokines, and a variety of conditions of preparation and culturing of ob/ob mouse islets and beta-cells have been carried out. Thoroughly performed experiments have not been able to demonstrate a protective effect of RX871024 and efaroxan on beta-cell death induced by the combination of cytokines. However, the inhibitory effect of RX871024 on NO production in ob/ob mouse islets and beta-cells was still observed in the presence of all three cytokines and correlated with the decrease in p38 MAPK phosphorylation. Conversely, efaroxan did not affect cytokine-induced NO production. Our data indicate that a combination of pro-inflammatory cytokines IL-1beta, IFNgamma, and TNFalpha, conditions modelling those that take place in type 1 diabetes, induces pancreatic beta-cell death that does not directly correlate with NO production and cannot be counteracted with imidazoline compounds.     

G-CSF prevents the progression of atherosclerosis and neointimal formation in rabbits

Granulocyte colony-stimulating factor (G-CSF) prevents left ventricular remodeling after myocardial infarction, but its effect on atherosclerosis is unknown. We examined two kinds of rabbit atherosclerosis models. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHL-MI) rabbits were treated with G-CSF or saline for 7 days from 14 months old. The vascular injury models were created by inflating angioplasty balloon in the iliac artery of rabbits and were divided into G-CSF and saline group. G-CSF significantly reduced the stenosis score of coronary artery and lipid plaque area of thoracic aorta in WHHL-MI rabbits at 4 weeks after the treatment. In the vascular injury model, G-CSF significantly prevented an increase in neointima/media ratio at 4 weeks after the treatment. G-CSF accelerated the reendothelialization of denuded arteries, and the pretreatment with nitric oxide synthase inhibitor significantly inhibited it. These results suggest that G-CSF has a therapeutic potential for the progression of atherosclerosis.     

SHP-1 inhibits LPS-mediated TNF and iNOS production in murine macrophages

Several lines of evidence have suggested that protein tyrosine phosphatases, including CD45 and SHP-1, regulate macrophage activation. Macrophages from mice lacking SHP-1 (motheaten mice) are hyper-responsive to many stimuli, suggesting that SHP-1 may negatively regulate macrophage activation. Herein we report that the repressible/inducible over-expression of wild-type SHP-1 in a subclone of RAW 264.7 macrophages (RAW-TT10 cells) inhibited both TNF secretion and iNOS protein accumulation in response to stimulation with lipopolysaccharide (LPS) and recombinant murine interferon-gamma and led to diminished LPS-mediated tyrosine phosphorylation of vav1. In contrast, expression of a truncated SHP-1 construct previously shown to interfere with endogenous SHP-1 function modestly augmented LPS-mediated TNF and iNOS production and did not inhibit vav1 tyrosine phosphorylation. Taken together, these data provide the first direct evidence that SHP-1 inhibits macrophage activation by LPS and suggest that this effect may be mediated in part by dephosphorylation of vav1.     

Interleukin-4 improves the migration of human myogenic precursor cells in vitro and in vivo

Different molecules are available to recruit new neighboring myogenic cells to the site of regeneration. Formerly called B cell stimulatory factor-1, IL-4 can now be included in the list of motogenic factors. The present report demonstrates that human IL-4 is not required for fusion between mononucleated myoblasts but is required for myotube maturation. In identifying IL-4 as a pro-migratory agent for myogenic cells, these results provide a mechanism which partly explains IL-4 demonstrated activity during differentiation. Among the different mechanisms by which IL-4 might enhance myoblast migration processes, our results indicate that there are implications of some integrins and of three major components of the fibrinolytic system. Indeed, increases in the amount of active urokinase plasminogen activator and its receptor were observed following an IL-4 treatment, while the plasminogen activator inhibitor-1 decreased. Finally, IL-4 did not modify the amount of cell surface alpha5 integrin but increased the presence of beta3 and beta1 integrins. This integrin modulation might favor myogenic cell migration and its interaction with newly formed myotubes. Therefore, IL-4 co-injection with transplanted myoblasts might be an approach to enhance the migration of transplanted cells for the treatment of a damaged myocardium or of a Duchenne Muscular Dystrophy patient.     

Mechanism of action differences in the antitumor effects of transmembrane and secretory tumor necrosis factor-alpha in vitro and in vivo

The proinflammatory cytokine tumor necrosis factor-alpha (TNFα) exists naturally in two forms, a 26 kDa transmembrane form (TM-TNFα), and a 17 kDa secretory form (S-TNFα). The biological roles for each of these forms of TNFα in tumor killing have not been completely elucidated. Therefore, in this study, three different recombinant retroviral vectors, wild-type TNFα, solely secretable TNFα mutant, and uncleavable transmembrane TNFα mutant, were constructed by molecular techniques and stably transfected into a murine hepatic carcinoma cell line (H22). TNFα, either secreted in cell culture supernatants by secretable TNFα mutant- or wild-type TNFα-producing tumor cells, or as a treansmembrane form expressed on the cell surface of uncleavable TNFα mutant- or wild-type TNFα-synthesizing tumor cells, was demonstrated to be cytotoxic against the TNF sensitive L929 cell line. The H22 cells transfected with the three different forms of TNFα were shown to kill parental H22 cells in an in vitro cytotoxicity assay [effect/target (E/T) ratio-dependent manner], and their maximal killing rates were ~38–43% at E/T ratio of 5:1. The injection of total 2.5×10⁵ mixed cells containing transfected and parental H22 tumor cells at different ratios into syngeneic mice resulted in the inhibition of tumor growth with a maximal inhibition rates of ~57~72% at E/T ratio of 5:1. A transient weight loss was found in mice bearing solely secretable TNFα mutant producing tumors, whereas no obvious side effects were seen in mice bearing uncleavable TNFα mutant or wild-type TNFα expressing tumors. Finally, we demonstrate that tumors secreting S-TNFα promoted the subsequent infiltration of CD4⁺ T cells, and to a lesser extent CD8⁺ T cells, to the tumor site. The TM-TNFα expressing tumors up-regulated Fas (CD95) expression and inhibited the expression of tumor metastasis associated molecule CD44v3. These results suggest that S-TNFα and TM-TNFα kill cancer cells in vivo through different mechanisms of action. We conclude that the non-secreted form of TNFα may be an ideal candidate for cancer gene therapy due to its therapeutic potential and lowered side effect profile.     

Temperature-sensitive gating in a descending visual interneuron, DCMD

Activity in neural circuits can be modified through experience-dependent mechanisms. The effects of high temperature on a locust visual interneuron (the descending contralateral movement detector, DCMD) have previously been shown to be mitigated by prior exposure to sub-lethal, elevated temperatures (heat shock, HS). Activity in the DCMD is reduced at high temperature in naïve animals (control), whereas HS animals show a maintained spike count at all temperatures. We examined whether this finding was due to direct effects of temperature on visual processing, or whether other indirect feedback mechanisms were responsible for the observed effect in the DCMD. Activity in the DCMD was elicited using a computer-generated looming image, and the response was recorded extracellularly. The temperature of visual processing circuits contributes directly to HS-induced plasticity in the DCMD, as maintaining the brain at 25°C during a thoracic temperature ramp eliminated the high frequency activity associated with HS. Removing ascending input by severing the thoracic nerve cord reduced DCMD thermosensitivity, indicating that indirect feedback mechanisms are also involved in controlling the DCMD response to increased thoracic temperature. Understanding how thermosensitive feedback within the locust affects DCMD function provides insight into critical regulatory mechanisms underlying visually-guided behaviors.     

The ratio of P40 monomer to dimer is an important determinant of IL-12 bioactivity

IL-12 is a 75 kDa heterodimer (IL12p70) comprised of independently regulated disulfide-linked 40 kDa (p40) and 35 kDa (p35) subunits. The p40 subunit exists extracellularly as a monomer (IL12p40) or dimer (IL12(p40)2) and can antagonize the action of IL12p70. Given the disagreement in the literature over the physiologic roles for IL12p70, IL12p40, and IL12(p40)2, we asked whether the bioactivity of IL-12 depended only on the concentration of the IL12p70 subunit alone or whether the relative concentrations of IL12p70, IL12p40, and IL12(p40)2 and their competitive binding with the IL-12 receptor are essential for determining IL-12 bioactivity under simulated human physiologic conditions. A mathematical model for IL-12 bioactivity was created by incorporating the production of IL12p70, IL12p40, and IL12(p40)2 by mature human DC and the interaction of these species with the IL-12 receptor. Using this model, we explored the effects of IFN-gamma, IL-4, and PGE2 concentrations on the bioactivity of IL-12. The simulations suggest that the concentration of IL12p70 alone is not indicative of IL-12 bioactivity; rather, the bioactivity of IL-12 produced by mature DC depends on IL12p70, IL12p40, and IL12(p40)2 production and their competitive interaction with the IL-12 receptor. In addition to the typically measured quantities of total p40 (IL12p40 + IL12(p40)2) and IL12p70, the ratio of IL12p40 to IL12(p40)2 is an equally important, yet underreported, determinant of IL-12 bioactivity.