The circadian <Emphasis Type="Italic">Clock</Emphasis> mutant mouse: impaired masking response to light

Synchronization of an internal clock (entrainment) and a direct response to light (masking) are complementary ways of restricting activity of an animal to day or night. The protein CLOCK has an important role in the oscillatory mechanism of mammalian pacemakers. Our data show that it is also involved in masking responses. Mice with the Clock/Clock mutation reduced their wheel running less than wildtypes when given 1-h light pulses of light (2–1,600 lx) in the night. With dimmer lights (<2 lx), there were no significant differences between mutant and wildtype mice. Impaired masking responses to light in Clock/Clock mice were confirmed in tests with ultradian light–dark cycles (3.5:3.5 h and 1:1 h). Tests with pulses of light longer than 1 h revealed that, although the mutants responded more slowly to light, they sustained the suppression of activity over the course of the 3-h tests better than wildtypes.     

Glomerular proliferation during early stages of diabetic nephropathy is associated with local increase of sphingosine-1-phosphate levels

In this study, the effects of short-term diabetes (4 days) on rat renal glomerular cells proliferation and the potential involvement of sphingolipids in this process were investigated. Immunohistochemical analysis showed that streptozotocin (STZ)-induced diabetes promoted increased intra-glomerular hyperplasia, particularly marked for mesangial cells. This was associated with a concomitant increase in neutral ceramidase and sphingosine-kinase activities and the accumulation of the pro-proliferative sphingolipid sphingosine-1-phosphate, in glomeruli isolated from kidney cortex of STZ-treated rats. These results suggest a possible involvement of sphingolipid metabolites in the glomerular proliferative response during the early stages of diabetic nephropathy.     

Bimodal effect of advanced glycation end products on mesangial cell proliferation is mediated by neutral ceramidase regulation and endogenous sphingolipids

Advanced glycation end-products (AGE) are generated by chronic hyperglycaemia and may cause diabetic microvascular complications such as diabetic nephropathy. Many factors influence the development of diabetic nephropathy; however, dysregulation of mesangial cell (MC) proliferation appears to play an early and crucial role. In this study, we investigated the effects of AGE on rat MC proliferation and the involvement of sphingolipids in the AGE response. Results show a bimodal effect of AGE on MC proliferation. Thus, low AGE concentrations (<1 microm) induced a significant increase (+26%) of MC proliferation, whereas higher concentrations (10 microm) markedly reduced it (-24%). In parallel, AGE exerted biphasic effects on neutral ceramidase expression and activity. Low AGE concentrations increased neutral ceramidase activity and expression, whereas high AGE concentrations showed opposite effects. Surprisingly, neutral ceramidase modulation did not result in changes of ceramide levels. However, the AGE (10 microm)-inhibitory effect on MC proliferation was associated with accumulation of sphingosine and was specifically prevented by blocking glucosylceramide synthesis, suggesting that the high AGE concentration effects are mediated by sphingosine and/or glycolipids. On the other hand, treatment of cells with low AGE concentrations led to an increase of sphingosine kinase activity and sphingosine-1-phosphate production that drove the increase of MC proliferation. Interestingly, in glomeruli isolated from streptozotocin-diabetic rats, a time-dependent modulation of ceramidase activity was observed as compared with controls. These results suggest that AGE regulate MC growth by modulating neutral ceramidase and endogenous sphingolipids.     

Isolation of progenitor cells from cord blood using adhesion matrices

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (PCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in αMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue® assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45⁺, CD105⁺, CD14⁺, CD49a⁺, CD49f⁺, CD44⁺ and CD34⁻. The highest incidence of PCs was observed on fibronectin and polystyrene. Passages were CD45⁻, CD14⁻, CD34⁻ and weakly CD105⁺. Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing Vit D₃ and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.     

Hyperglycemia and glucosamine-induced mesangial cell cycle arrest and hypertrophy: Common or independent mechanisms?

The Hexosamine Pathway (HP) is one hypothesis proposed to explain glucose toxicity and the alterations observed during the course of diabetic microvascular complication development. Glucosamine is a precursor of UDP-N-Acetylglucosamine (UDP-GlcNAc), the main product of the HP that has often been used to mimic its activation. The transfer of a UDP-GlcNAc residue onto proteins (O-GlcNAc modification) represents the final step of the HP and is considered as a major mechanism by which this pathway exerts its signalling effects. While it is well accepted that the HP promotes extracellular matrix accumulation in the context of diabetic nephropathy, its involvement in the perturbations of cell cycle progression and hypertrophy of renal cells has been poorly investigated. Nevertheless, in a growing number of studies, the HP and O-GlcNAc modification are emerging as important regulators of cell cycle progression. This review will focus on the role of glucosamine and O-GlcNAc modification in cell cycle regulation in the context of diabetic nephropathy. Special emphasis will be given into the role of the HP as a potential mediator of the effects of high glucose on the perturbations of renal cell growth.     

A human IgE bispecific antibody shows potent cytotoxic capacity mediated by monocytes

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper–mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity–mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.     

A randomized controlled trial to assess the safety and efficacy of silymarin on symptoms,signs and biomarkers of acute hepatitis

PurposeMilk thistle or its purified extract, silymarin (Silybum marianum), is widely used in treating acute or chronic hepatitis. Although silymarin is hepatoprotective in animal experiments and some human hepatotoxic exposures, its efficacy in ameliorating the symptoms of acute clinical hepatitis remains inconclusive. In this study, our purpose was to determine whether silymarin improves symptoms, signs and laboratory test results in patients with acute clinical hepatitis, regardless of etiology.MethodsThis is a randomized, placebo-controlled trial in which participants, treating physicians and data management staff were blinded to treatment group. The study was conducted at two fever hospitals in Tanta and Banha, Egypt where patients with symptoms compatible with acute clinical hepatitis and serum alanine aminotransferase (ALT) levels >2.5 times the upper limit of normal were enrolled. The intervention consisted of three times daily ingestion of either a standard recommended dose of 140 mg of silymarin (Legalon^®, MADAUS GmbH, Cologne, Germany), or a vitamin placebo for four weeks with an additional four-week follow-up. The primary outcomes were symptoms and signs of acute hepatitis and results of liver function tests on days 2, 4 and 7 and weeks 2, 4, and 8. Side-effects and adverse events were ascertained by self-report.ResultsFrom July 2003 through October 2005, 105 eligible patients were enrolled after providing informed consent. No adverse events were noted and both silymarin and placebo were well tolerated. Patients randomized to the silymarin group had quicker resolution of symptoms related to biliary retention: dark urine (p=0.013), jaundice (p=0.02) and scleral icterus (p=0.043). There was a reduction in indirect bilirubin among those assigned to silymarin (p=0.012), but other variables including direct bilirubin, ALT and aspartate aminotransferase (AST) were not significantly reduced.ConclusionsPatients receiving silymarin had earlier improvement in subjective and clinical markers of biliary excretion. Despite a modest sample size and multiple etiologies for acute clinical hepatitis, our results suggest that standard recommended doses of silymarin are safe and may be potentially effective in improving symptoms of acute clinical hepatitis despite lack of a detectable effect on biomarkers of the underlying hepatocellular inflammatory process.     

Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions

Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria (Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure-function relationship of LLO and (2) its role in intra- and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed.     

A highly conserved amino-terminal region of sonic hedgehog is required for the formation of its freely diffusible multimeric form

Although members of the Hedgehog (Hh) family were initially described as morphogens, many of these early conclusions were based on experiments that used non-physiologically relevant forms of Hh. Native Hh is modified by cholesterol (HhNp) and palmitate. These hydrophobic modifications are responsible for the ability of Hh to associate with cellular membranes, a property that initially appeared inconsistent with its ability to act far from its site of synthesis. Although it is now clear that Hh family members are capable of acting directly in long-range signaling, the form of Hh capable of this activity remains controversial. We have previously provided evidence for a freely diffusible multimeric form of Sonic Hedgehog (Shh) termed s-ShhNp, which is capable of accumulating in a gradient fashion through a morphogenic field. Here, we provide further evidence that s-ShhNp is the physiologically relevant form of Shh. We show that the biological activity of freely diffusible ShhNp resides in its multimeric form and that this multimeric form is exceedingly stable, even to high concentrations of salt and detergent. Furthermore, we now validate the Shh-Shh interactions previously observed in the crystal structure of human Shh, showing that a highly conserved amino-terminal domain of Shh is important for the formation of s-ShhNp. We also conclusively show that palmitoylation is required for s-ShhNp formation. Thus, our results identify both protein-protein and protein-lipid interactions that are required for s-ShhNp formation, and provide the first structural analyses supporting the existence of Shh multimers.