Loss of the Oxygen Sensor PHD3 Enhances the Innate Immune Response to Abdominal Sepsis

Hypoxia and HIFs (HIF-1α and HIF-2α) modulate innate immune responses in the setting of systemic inflammatory responses and sepsis. The HIF prolyl hydroxylase enzymes PHD1, PHD2 and PHD3 regulate the mammalian adaptive response to hypoxia; however, their significance in the innate immune response has not been elucidated. We demonstrate in this study that deficiency of PHD3 (PHD3(-/-)) specifically shortens the survival of mice subjected to various models of abdominal sepsis because of an overwhelming innate immune response, leading to premature organ dysfunction. By contrast, this phenotype was absent in mice deficient for PHD1 (PHD1(-/-)) or PHD2 (PHD2(+/-)). In vivo, plasma levels of proinflammatory cytokines were enhanced, and recruitment of macrophages to internal organs was increased in septic PHD3-deficient mice. Reciprocal bone marrow transplantation in sublethally irradiated mice revealed that enhanced susceptibility of PHD3-deficient mice to sepsis-related lethality was specifically caused by loss of PHD3 in myeloid cells. Several in vitro assays revealed enhanced cytokine production, migration, phagocytic capacity, and proinflammatory activation of PHD3-deficient macrophages. Increased proinflammatory activity of PHD3-deficient macrophages occurred concomitantly with enhanced HIF-1α protein stabilization and increased NF-κB activity, and interference with the expression of HIF-1α or the canonical NF-κB pathway blunted their proinflammatory phenotype. It is concluded that impairment of PHD3 enzyme function aggravates the clinical course of abdominal sepsis via HIF-1α- and NF-κB-mediated enhancement of the innate immune response.     

Effect of the PARP-1 Inhibitor PJ 34 on Excitotoxic Damage Evoked by Kainate on Rat Spinal Cord Organotypic Slices

Excitotoxicity triggered by over-activation of glutamate receptors is thought to be an early mechanism of extensive neuronal death with consequent loss of function following lesion of spinal networks. One important process responsible for excitotoxic death is ‘parthanatos’ caused by hyperactivation of poly(ADP-ribose) polymerase (PARP) enzyme 1. Using rat organotypic spinal slices as in vitro models, the present study enquired if 2-(dimethylamino)-N-(5,6-dihydro-6-oxophenanthridin-2yl)acetamide (PJ 34), a pharmacological inhibitor of PARP-1, could counteract the excitotoxic damage evoked by transient application (1 h) of kainate, a potent analogue of glutamate. Kainate induced dose-dependent (1 μM threshold) neuronal loss (without damage to astrocytes) detected 24 h later via a PARP-1 dependent process that had peaked at 4 h after washout kainate. All spinal regions (ventral, central and dorsal) were affected, even though the largest damage was found in the dorsal area. Whereas PJ 34 did not protect against a large concentration (100 μM) of kainate, it significantly inhibited neuronal losses evoked by 10 μM kainate as long as it was co-applied with this glutamate agonist. When the application of PJ 34 was delayed to the washout time, neuroprotection was weak and regionally restricted. These data suggest that kainate-induced parthanatos developed early and was prevented by PJ 34 only when it was co-applied together with excitotoxic stimulus. Our results highlight the difficulty to arrest parthanatos as a mechanism of spinal neuron death in view of its low threshold of activation by kainate, its widespread distribution, and relatively fast development.     

Induction of intracellular ceramide by interleukin-1β in oligodendrocytes

The sphingomyelin pathway has been implicated in mediating the effect of several extracellular agents leading to important biochemical and cellular changes. The aim of this investigation is to study interleukin-1β (IL-1β) signaling in oligodendrocytes. For this purpose, the CG4 oligodendrocyte cells were differentiated and incubated with IL-1β. This treatment induced a time- and dose-dependent increase of the endocellular ceramide. To mimic the effect of the elevation of endogenous ceramide, the CG4 cells were treated with the ceramide analogue C2-ceramide. Cell survival, measured with the MTT assay, showed that, by increasing the concentration of ceramide, up to 40% of CG4 cells were dying within 6 h, similar data were obtained with the primary differentiated oligodendrocytes. Condensation of chromatin, nuclear fragmentation, and formation of apoptotic bodies indicated that apoptosis was the cause of death. Surprisingly, long-term exposure (72 h) to increasing concentrations of IL-1β, which increases intracellular ceramide, did not induce oligodendroglial cell death. These results show that an increase of intracellular ceramide is not sufficient to induce apoptosis in oligodendrocytes and that IL-1β signaling through the ceramide pathway in these cells can mediate functions other than programmed cell death. J. Cell Biochem. 66:532–541, 1997. © 1997 Wiley-Liss, Inc.     

An HGF-MSP chimera disassociates the trophic properties of scatter factors from their pro-invasive activity

Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP) have an intrinsic dual nature: they are trophic cytokines preventing apoptosis on one side and scatter factors promoting invasion on the other. For therapeutic use, their anti-apoptotic activity must be separated from their pro-invasive activity. To this end, we engineered chimeric factors containing selected functional domains of HGF and/or MSP in different combinations, and tested their biological activity. Here we present a chimeric cytokine derived from the alpha-chains of HGF and MSP, named Metron factor 1 for its ability to concomitantly activate the HGF receptor (Met) and the MSP receptor (Ron). We provide evidence that Metron factor 1 prevents apoptosis and stimulates cell proliferation at nanomolar concentrations, but is devoid of any pro-invasive activity. In an in vivo murine model of drug-induced nephrotoxicity, intravenous injection of recombinant Metron factor 1 prevented renal damage and preserved tubular integrity.     

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness‐induced CCN1 activates β‐catenin nuclear translocation and signaling and that this contributes to upregulate N‐cadherin levels on the surface of the endothelium, in vitro. This facilitates N‐cadherin‐dependent cancer cell–endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness‐induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.     

Aerobiology of Urticaceae pollen in Trieste (NE Italy)

The aerobiological behaviour of Urticaceae in Trieste and the correlations with the meteorological parameters were examined. Airborne pollen was collected from 1990 to 1999 using a Hirst type spore trap (Burkard) and the data interpretation was performed according to the standard method adopted by the Italian Aeroallergen Network. The main pollen season of Urticaceae in Trieste goes from mid-April to mid-September. The highest values occur in May and June. Although different seasonal patterns are found every year, the main peak occurs on average at the beginning of May, followed by other decreasing peaks until September. Thecumulative counts vary greatly over the years, with a mean value of 18.315 p/m³. The maximum annual total pollen grains was registered in 1996 and the minimum in 1991. Spearman's correlation was used to establish the relationship between the daily pollen counts and the daily meteorological data both considering their original quantitative values and transformed values according to their day by day changes. Daily pollen concentrations present usually positive correlation with temperature, negative with rainfall and wind speed and no correlation with humidity. Better results were obtained with transformed values.     

Towards genome-scale structure prediction for transmembrane proteins

In this paper we briefly review some of the recent progress made by ourselves and others in developing methods for predicting the structures of transmembrane proteins from amino acid sequence. Transmembrane proteins are an important class of proteins involved in many diverse biological functions, many of which have great impact in terms of disease mechanism and drug discovery. Despite their biological importance, it has proven very difficult to solve the structures of these proteins by experimental techniques, and so there is a great deal of pressure to develop effective methods for predicting their structure. The methods we discuss range from methods for transmembrane topology prediction to new methods for low resolution folding simulations in a knowledge-based force field. This potential is designed to reproduce the properties of the lipid bilayer. Our eventual aim is to apply these methods in tandem so that useful three-dimensional models can be built for a large fraction of the transmembrane protein domains in whole proteomes.