Showing papers by "University of Milano-Bicocca published in 2000"

Dendritic Cells Discriminate between Yeasts and Hyphae of the Fungus Candida albicans Implications for Initiation of T Helper Cell Immunity in Vitro and in Vivo

Abstract: The fungus Candida albicans behaves as a commensal as well as a true pathogen of areas highly enriched in dendritic cells, such as skin and mucosal surfaces. The ability of the fungus to reversibly switch between unicellular yeast to filamentous forms is thought to be important for virulence. However, whether it is the yeast or the hyphal form that is responsible for pathogenicity is still a matter of debate. Here we show the interaction, and consequences, of different forms of C . albicans with dendritic cells. Immature myeloid dendritic cells rapidly and efficiently phagocytosed both yeasts and hyphae of the fungus. Phagocytosis occurred through different phagocytic morphologies and receptors, resulting in phagosome formation. However, hyphae escaped the phagosome and were found lying free in the cytoplasm of the cells. In vitro, ingestion of yeasts activated dendritic cells for interleukin (IL)-12 production and priming of T helper type 1 (Th1) cells, whereas ingestion of hyphae inhibited IL-12 and Th1 priming, and induced IL-4 production. In vivo, generation of antifungal protective immunity was induced upon injection of dendritic cells ex vivo pulsed with Candida yeasts but not hyphae. The immunization capacity of yeast-pulsed dendritic cells was lost in the absence of IL-12, whereas that of hypha-pulsed dendritic cells was gained in the absence of IL-4. These results indicate that dendritic cells fulfill the requirement of a cell uniquely capable of sensing the two forms of C . albicans in terms of type of immune responses elicited. By the discriminative production of IL-12 and IL-4 in response to the nonvirulent and virulent forms of the fungus, dendritic cells appear to meet the challenge of Th priming and education in C . albicans saprophytism and infections.

The nicotinic receptor β2 subunit is mutant in nocturnal frontal lobe epilepsy

Abstract: Clustered attacks of epileptic episodes originating from the frontal lobe during sleep are the main symptoms of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE, MIM 600513). Despite the clinical homogeneity, three forms of ADNFLE have been associated with chromosomes 20 (ENFL1; ref. 1), 15 (ENFL2; ref. 2) and 1 (ENFL3; ref. 3). Mutations of the gene encoding the neuronal nicotinic acetylcholine receptor α4 subunit (CHRNA4 ) have been found in ADNFLE-ENFL1 families4,5,6, but these mutations account for only a small proportion of ADNFLE cases7. The newly identified locus associated with ENFL3 harbours several candidate genes, including CHRNB2 (ref. 8), whose gene product, the β2 nicotinic acetylcholine receptor (nAChR) subunit, co-assembles with the α4 nAChR subunit to form the active receptor9.

Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis.

Abstract: Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I–restricted re-presentation of gp96-associated peptides and CTL activation; non–receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

Adrenergic and Reflex Abnormalities in Obesity-Related Hypertension

Abstract: Previous studies have shown that essential hypertension and obesity are both characterized by sympathetic activation coupled with a baroreflex impairment. The present study was aimed at determining the effects of the concomitant presence of the 2 above-mentioned conditions on sympathetic activity as well as on baroreflex cardiovascular control. In 14 normotensive lean subjects (aged 33. 5+/-2.2 years, body mass index 22.8+/-0.7 kg/m(2) [mean+/-SEM]), 16 normotensive obese subjects (body mass index 37.2+/-1.3 kg/m(2)), 13 lean hypertensive subjects (body mass index 24.0+/-0.8 kg/m(2)), and 16 obese hypertensive subjects (body mass index 37.5+/-1.3 kg/m(2)), all age-matched, we measured beat-to-beat arterial blood pressure (by Finapres device), heart rate (HR, by ECG), and postganglionic muscle sympathetic nerve activity (MSNA, by microneurography) at rest and during baroreceptor stimulation and deactivation induced by stepwise intravenous infusions of phenylephrine and nitroprusside, respectively. Blood pressure values were higher in lean hypertensive and obese hypertensive subjects than in normotensive lean and obese subjects. MSNA was significantly (P:<0.01) greater in obese normotensive subjects (49.1+/-3.0 bursts per 100 heart beats) and in lean hypertensive subjects (44.5+/-3.3 bursts per 100 heart beats) than in lean normotensive control subjects (32.2+/-2.5 bursts per 100 heart beats); a further increase was detectable in individuals with the concomitant presence of obesity and hypertension (62.1+/-3. 4 bursts per 100 heart beats). Furthermore, whereas in lean hypertensive subjects, only baroreflex control of HR was impaired, in obese normotensive subjects, both HR and MSNA baroreflex changes were attenuated, with a further attenuation being observed in obese hypertensive patients. Thus, the association between obesity and hypertension triggers a sympathetic activation and an impairment in baroreflex cardiovascular control that are greater in magnitude than those found in either of the above-mentioned abnormal conditions alone.

Integrin LFA-1 interacts with the transcriptional co-activator JAB1 to modulate AP-1 activity.

Abstract: Integrin adhesion receptors transduce signals that control complex cell functions which require the regulation of gene expression, such as proliferation, differentiation and survival. Their intracellular domain has no catalytic function, indicating that interaction with other transducing molecules is crucial for integrin-mediated signalling. Here we have identified a protein that interacts with the cytoplasmic domain of the beta2 subunit of the alphaL/beta2 integrin LFA-1. This protein is JAB1 (Jun activation domain-binding protein 1), a coactivator of the c-Jun transcription factor. We found that JAB1 is present both in the nucleus and in the cytoplasm of cells and that a fraction of JAB1 colocalizes with LFA-1 at the cell membrane. LFA-1 engagement is followed by an increase of the nuclear pool of JAB1, paralleled by enhanced binding of c-Jun-containing AP-1 complexes to their DNA consensus site and increased transactivation of an AP-1-dependent promoter. We suggest that signalling through the LFA-1 integrin may affect c-Jun-driven transcription by regulating JAB1 nuclear localization. This represents a new pathway for integrin-dependent modulation of gene expression.

FAS Engagement Induces the Maturation of Dendritic Cells (Dcs), the Release of Interleukin (Il)-1β, and the Production of Interferon γ in the Absence of IL-12 during Dc–T Cell Cognate Interaction: A New Role for FAS Ligand in Inflammatory Responses

Abstract: Ligation of the Fas (CD95) receptor leads to an apoptotic death signal in T cells, B cells, and macrophages. However, human CD34+–derived dendritic cells (DCs) and mouse DCs, regardless of their maturation state, are not susceptible to Fas-induced cell death. This resistance correlates with the constitutive expression of the Fas-associated death domain–like IL-1β–converting enzyme (FLICE)-inhibitory protein (FLIP) ligand. We demonstrate a new role of Fas in DC physiology. Engagement of Fas on immature DCs by Fas ligand (FasL) or by anti-Fas antibodies induces the phenotypical and functional maturation of primary DCs. Fas-activated DCs upregulate the expression of the major histocompatibility complex class II, B7, and DC–lysosome-associated membrane protein (DC-LAMP) molecules and secrete proinflammatory cytokines, in particular interleukin (IL)-1β and tumor necrosis factor α. Mature DCs, if exposed to FasL, produce even higher amounts of IL-1β. Importantly, it is possible to reduce the production of IL-1β and interferon (IFN)-γ during DC–T cell interaction by blocking the coupling of Fas–FasL with a Fas competitor. Finally, during cognate DC–T cell recognition, IL-12 (p70) could not be detected at early or late time points, indicating that Fas-induced, IFN-γ secretion is independent of IL-12.

Differential effects of corticosteroids during different stages of dendritic cell maturation.

Abstract: Dendritic cell (DC) maturation is a complex process involving many cell functions. We have studied how the exposure of DC to corticosteroids at different stages of DC maturation affects priming and the expansion of different subsets of CD4(+) T cells. Growth factor- dependent DC lines and fresh bone marrow-derived DC were used. When exposed to inflammatory stimuli, immature DC previously treated with dexamethasone were unable to undergo full maturation and were unable to prime Th1 cells efficiently. There was specific and significant reduction in the number of IFN-gamma-producing effector cell (shown by intracellular cytokine staining) and also in the amount of IFN-gamma produced. Interestingly, the number of IL-4-producing T cells and the amount of IL-4 synthesis was not significantly altered. Furthermore, multiple restimulation of T cells with these DC gave rise to a subpopulation of T regulatory cells (Tr1) which were negative for IFN-gamma and IL-4 but were IL-10 positive. In contrast, when DC were activated with lipopolysaccharide prior to dexamethasone treatment, the suppressive effect of glucocorticoids was not significant. Thus, the stage of DC maturation influences the inhibitory effect of corticosteroids. By arresting DC maturation, corticosteroids strongly reduce cell-mediated Th1 responses and allow the selective expansion of Tr1 cells.

Immature Dendritic Cells Acquire Cd8+Cytotoxic T Lymphocyte Priming Capacity upon Activation by T Helper Cell–Independent or–Dependent Stimuli

Abstract: The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo. Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4(+) T helper (Th) cells. Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro. Similarly, priming of CTL immunity in vivo in CD4-depleted mice was only observed if these mice were immunized with activated D1 cells. This study provides formal evidence that activation of DCs, induced by Th-independent as well as Th-dependent stimuli, is essential for efficient induction of CTL responses.

Push–Pull Organic Chromophores for Frequency‐Upconverted Lasing

Abstract: Properly designed organic molecules where a p-conjugated bridge is end-capped by an electron-donor and electron-withdrawing group (push‐pull chromophores) can show, under specific conditions, frequency-upconverted lasing emission. Materials based on these dyes are therefore able to convert the emission of a cheap and easily available infrared (IR) laser (e.g., 800 nm radiation) into more useful visible (vis) laser emission via a two-photon absorption induced fluorescence phenomenon. The design of the molecular structure, the substituent effect, and modulation of the electronic and geometric parameters can be used in order to optimize and tune frequency emission and conversion efficiency. Examples of some of the best dyes reported to-date and organic design strategies employed are discussed in this article.

HERG potassium channels are more frequently expressed in human endometrial cancer as compared to non-cancerous endometrium.

Abstract: HERG K+channels, besides contributing to regulate cardiac and neuronal excitability, are preferentially expressed in tumour cell lines of different histogenesis, where their role in the development and maintenance of the neoplastic phenotype is under study. We show here that both herg gene and HERG protein are expressed with high frequency in primary human endometrial cancers, as compared to normal and hyperplastic endometrium. RT-PCR and immunohistochemistry, using specific anti-HERG antibodies developed in our laboratory, were applied to tissue specimens obtained from 18 endometrial cancers and 11 non-cancerous endometrial tissues. herg RNA and HERG protein are expressed in 67% and 82%, respectively, of cancerous, while in only 18% of non-cancerous tissues. In particular, no expression was found in endometrial hyperplasia. Moreover, electrophysiological experiments confirmed the presence of functioning HERG channels on the plasma membrane of tumour cells. On the whole, these data are the first demonstration of the presence of HERG channels in primary human neoplasias, and could candidate HERG as a potential tool capable of marking cancerous versus hyperplastic endometrial growth. © 2000 Cancer Research Campaign http://www.bjcancer.com

Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs

Abstract: Biodistribution of nonviral cationic vector/DNA complexes was studied after systemic or intratracheal administration to the lungs and correlated with transgene expression. Intravenous injection in C57Bl/6 mice gave maximal and significant luciferase expression in the lungs with the cationic polymer PEI 22K/DNA complexes at the highest ratios of positive/negative charges versus DNA alone. While DOTAP/DNA complexes with high charge ratio determined lower but still significant luciferase activity versus uncomplexed DNA, GL-67A and PEI 25K mediated negligible luciferase expression. Labelled PEI 22K and DOTAP complexes were evenly distributed in the alveolar region, where GFP expression was revealed, while PEI 25K and GL-67A complexes were not detected, suggesting a different interaction of these complexes with the plasma membrane of endothelial cells. Following an intratracheal injection, the highest and significant levels of transfection were obtained with slightly positive PEI complexes as compared with DNA alone, whereas cationic lipid-based vectors, DOTAP and GL-67A, gave not significant luciferase activity. Both types of polyplexes gave similar levels of lung luciferase expression by targeting different airway cell populations. PEI 25K complexes determined high levels of GFP in the bronchial cells, confirming confocal data on fluorescent complexes internalization. PEI 22K complexes gave mainly high GFP signal in the distal tract of the bronchial tree, where tagged complexes were recovered. Fluorescent lipid complexes were found in aggregates in the lumen of bronchi totally (DOTAP) or partially (GL-67A) co-localizing with surfactant protein A. Results indicated that cationic polymers could overcome the surfactant barrier which inhibited airway cell transfection mediated by cationic lipids.

Aloe-Emodin quinone pretreatment reduces acute liver injury induced by carbon tetrachloride.

Abstract: Aloe contains several active compounds including aloin, a C-glycoside that can be hydrolyzed in the gut to form aloe-emodin anthrone which, in turn, is auto-oxidized to the quinone aloe-emodin. On the basis of the claimed hepatoprotective activity of some antraquinones, we studied aloe-emodin in a rat model of carbon tetrachloride (CCl 4 ) intoxication, since this xenobiotic induces acute liver damage by lipid peroxidation subsequent to free radical production. Twelve rats were treated with CCl 4 (3 mg/kg) intraperitoneally and six were protected with two intraperitoneally injections of aloe-emodin (50 mg/kg: CCl 4 +aloe-emodin); six other rats were only aloe-emodin injected (aloe-emodin) and six were untreated (control). Histological examination of the livers showed less marked lesions in the CCl 4 +aloe-emodin rats than in those treated with CCl 4 alone, and this was confirmed by the serum levels of L-aspartate-2-oxoglutate-aminotransferase (394±38.6 UI/l in CCl 4 , 280±24.47 UI/l in CCl 4 +aloe-emodin rats; P<0.05). We also quantified changes in hepatic albumin and tumour necrosis factor-a mRNAs. Albumin mRNA expression was significantly lower only in the liver of CCl 4 rats (P<0.05 versus control) and was only slightly reduced in the CCl 4 +aloe-emodin rats. In contrast tumour necrosis factor-a mRNA was significantly higher (P<0.05) in the CCl 4 than the control rats and almost equal in the CCl 4 +aloe-emodin, aloe-emodin and control groups. In conclusion, aloe-emodin appears to have some protective effect not only against hepatocyte death but also on the inflammatory response subsequent to lipid peroxidation.

Glucose- and arginine-induced insulin secretion by human pancreatic beta-cells: the role of HERG K(+) channels in firing and release.

Abstract: The human ether-a-go-go-related genes (herg) are expressed in tissues other than heart and brain where the HERG K(+) channels are known to regulate the repolarization of the heart action potential and the neuronal spike-frequency accommodation. We provide evidence that herg1 transcripts are present in human pancreatic islets that were used to study both insulin secretion and electrical activity with radioimmunoassay and single cell perforated patch-clamp techniques, respectively. Glucose- and arginine-induced islets insulin secretion data suggested a net increase of release under perfusion with antiarrhythmic drugs known to selectively block HERG channels. Indeed we could routinely isolate a K(+) current that was recognized as biophysically and pharmacologically similar to the HERG current. An analysis of the glucose- and arginine-induced electrical activity (several applications during 30 min) in terms of firing frequency and putative insulin release was done in control and in the presence of selective blockers of HERG channels: the firing frequency and the release increased by 32% and 77%, respectively. It is concluded that HERG channels have a crucial role in regulating insulin secretion and firing of human beta-cells. This raises the possibility that some genetically characterized hyperinsulinemic diseases of unknown origin might involve mutations in the HERG channels.

A Light Workflow Management System Using SimpleProcess Models

Abstract: Workflow management systems are considered a hot technology. Nevertheless, up to now they have not had the diffusion other packages such as productivity tools, E-mail systems and groupware platforms have. We believe that this fact is due to the many limitations of current workflow technology (weak support for changes; complex exception handling mechanisms; limited openness to and integrability with other system componentss …) and that radically new workflow management systems should be designed and developed in order to offer adequate products to the market. In this paper, we outline the main innovative features of the workflow management component of the Milano system making it highly flexible and adaptable. Particular attention is paid to its modelling framework, which is based on a class of net systems well supported by efficient algorithms, and to the services it offers to both workflow designers and actors. The most relevant aspects of the MILANO workflow management system are also illustrated through a realistic example.

Improving Flexibility of Workflow Management Systems

Abstract: In order to support both the redesign of a Business Process and its continuous improvement, the technology supporting it must be as flexible as possible. Since workflow management systems are the main technology for supporting Business Processes, they and, in particular, their modeling framework must satisfy a long list of apparently conflicting requirements: the models must be both cognitive artifacts and executable programs; they must be simple and yet able to support exceptions; they must support both static and dynamic changes. In this chapter, after briefly discussing the above requirements, we present the formal aspects of the modeling framework of the MILANO workflow management system. Its flexibility is based on a net-theoretical modeling framework which lets simple process models deliver a large class of services to its users.

Introduction to a theory of value coherent with the no-arbitrage principle

Abstract: This paper defines the value of a general claim based on agent's preferences and coherent with the No Arbitrage Principle. This Value is a non trivial extension of the certainty equivalent since it takes into consideration the possibility of partially hedging the risk carried by the claim. When the market is complete this Value is the unique no arbitrage price. When the risk may not even be partially covered, this Value is the certainty equivalent. Between these two cases just some of the risk may be hedged and the no arbitrage principle requires the price to lie in the “arbitrage interval”. The Value we propose is exactly designed to satisfy this condition.

Observation of the Alpha Particle ``Knock-On'' Neutron Emission from Magnetically Confined DT Fusion Plasmas

Abstract: Suprathermal fuel ions from alpha-particle knock-on collisions in fusion DT plasmas are predicted to cause a weak feature in the neutron spectrum of d+t-->alpha+n. The knock-on feature has been searched for in the neutron emission of high ( >1 MW) fusion-power plasmas produced at JET and was found using a magnetic proton recoil type neutron spectrometer of high performance. Measurement and predictions agree both in absolute amplitude and in plasma-parameter dependence, supporting the interpretation and model. Moreover, the results provide input to projecting alpha-particle diagnostics for future self-heated fusion plasmas.

Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture

Abstract: Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X-100 and fractionated by sucrose-gradient centrifugation to prepare a detergent-insoluble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1- 3 H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cross-linked to GM3, the cross-linking complexes being highly concentrated in the detergent-insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time-dependent, transient process so that CAV1 cross-linking to GM3 was hardly detectable after a 24-h chase followed the pulse time. After a 24-h chase, only the high molecular mass proteins cross-linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized. Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold-conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti-GM3 and anti-CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals. Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains.

Glutamate uptake is decreased in platelets from Alzheimer's disease patients.

Abstract: Because excitotoxicity may be involved in neurodegeneration in Alzheimer's disease, we investigated possible modifications of platelet glutamate uptake in AD patients. High-affinity glutamate uptake was studied in platelets from 35 Alzheimer's disease patients, 10 multi-infarct dementia patients, and 35 age-matched normal controls; it was decreased by 40% in platelets from Alzheimer's disease patients compared with controls and with multi-infarct dementia patients. Platelet glutamate uptake could be used as peripheral marker of glutamatergic involvement and as adjunctive diagnostic tool in Alzheimer's disease patients.

Molecular events of bacterial-induced maturation of dendritic cells.

Abstract: In order to protect the body from infectious microorganisms, mammals have developed powerful lines of defense, consisting in innate and adaptive immune responses. The innate response is phylogenetically more ancient and, for a long time, it has been considered to be broadly directed to microorganisms. However, the discovery of a new class of receptors, involved in recognition of patterns characteristic of groups of microorganisms (the toll-like receptor family) has re-evaluated the role of the innate immune system as a discriminating system. Indeed, there is increasing evidence that the induction of different types of effector adaptive responses are directed by the innate immune system after recognition of particular groups of pathogens. The central role of Dendritic cells (DC) in the induction of adaptive immune responses towards infectious agents has been extensively described, but, recently, a new role of DC as a link between the non-antigen- and the antigen-specific responses has been proposed. DC have, indeed, the capacity to recruit and activate cells of the innate immune system upon inflammation. Thus, understanding the interaction of bacteria with DC, and the early molecular events resulting from this interaction may shed some light on the mechanisms of initiation of the immune response to infectious agents and on aspects of invasiveness, pathogenicity, and the persistence of certain bacteria.

Risk Assessment for Honeybees from Pesticide-Exposed Pollen

Abstract: A method for assessing the risk for honeybees from pesticide exposure via pollen is proposed. Four pesticides, selected as markers, were monitored in pollen samples collected in two sampling areas, one located in an intensive agricultural area and the other far from direct pesticide impact. Analytical results were consistent with use patterns of the chemicals and their physico-chemical and persistence properties. For a preliminary estimate of bee exposure via pollen, both by ingestion and by contact, an exposure index was developed, based on physico-chemical properties, persistence and application rates. On the basis of the exposure estimates and acute toxicological data (ingestion and contact LD50), Toxicity Exposure Ratios (TERs) were calculated as indicators of the risk for honeybees due to this particular exposure route. TER values were compared to Hazard Quotient (HQ), calculated as the ratio between application rate and the LC50 value, according to European guidelines, showing a satisfactory agreement. The advantage of the above described procedures is that the environmental fate of the chemicals, and not only application rates, are taken into account. This approach may represent a preliminary tool for a comparative screening of the risk for pollinator insects due to this particular exposure route.