Tumor-derived microvesicles modulate antigen cross-processing via reactive oxygen species-mediated alkalinization of phagosomal compartment in dendritic cells

Dendritic cells (DCs) are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs) enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS) accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies

Tumor derived Microvesicles enhance cross-processing ability of clinical grade Dendritic Cells

Tumor cells release extracellular microvesicles (MVs) in the microenvironment to deliver biological signals to neighbouring cells as well as to cells in distant tissues. Tumor-derived MVs appear to play contradictory role promoting both immunosuppression and tumor growth and both evoking tumor specific immune response. Recent evidences indicate that tumor-derived MVs can positively impact Dendritic Cells (DCs) immunogenicity by reprogramming DC antigen processing machinery and intracellular signaling pathways, thus promoting anti-tumor response. DCs are considered pivot cells of the immune system due to their exclusive ability to coordinate the innate and acquired immune responses, cross-present exogenous antigens and prime naïve T cells. DCs are required for the induction and maintenance of long-lasting anti-tumor immunity and their exploitation has been extensively investigated for the design of anti-tumor vaccines. However, the clinical grade culture conditions that are required to generate DCs for therapeutic use can strongly affect their functions. Here, we investigated the immunomodulatory impact of MVs carrying the MUC1 tumor glycoantigen (MVsMUC1) as immunogen formulation on clinical grade DCs grown in X-VIVO 15 (X-DCs). Results indicated that X-DCs displayed reduced performance of the antigen processing machinery in term of diminished phagocytosis and acidification of the phagosomal compartment suggesting an altered immunogenicity of clinical grade DCs. Pulsing DCs with MVsMUC1 restored phagosomal alkalinization, triggering ROS increase. This was not observed when a soluble MUC1 protein was employed (rMUC1). Concurrently, MVsMUC1 internalization by X-DCs allowed MUC1 cross-processing. Most importantly, MVsMUC1 pulsed DCs activated IFNγ response mediated by MUC1 specific CD8+ T cells. These results strongly support the employment of tumor-derived MVs as immunogen platforms for the implementation of DC-based vaccine

Alkalinization during re-oxygenation prevents functional damage by hyperglycaemic hypoxia

HYPERGLYCAEMIA impairs recovery from transient cerebral ischaemia: the importance of tissue acidification for this phenomenon has not been clarified in detail. We investigated this issue in a less complex in vitro preparation of isolated rat dorsal spinal roots exposed for 30 min to hyperglycaemic hypoxia. Peak height of compound action potentials recovered minimally in 5 mM bicarbonate. However, recovery was greatly improved by addition of the weak base trimethylamine during re-oxygenation. Addition of the weak acid propionate had no such effect. Cytoplasmic alkalinization improved recovery in a brief time window only: application of trimethylamine after 15 min of re-oxygenation was without beneficial effect. These data emphasize the importance of cytoplasmic acidification for neurophysiological recovery from hyper-glycaemic hypoxia during the initial period of re-oxygenation

H+ és HCO3- transzporterek szabályozása humán pancreas ductus sejtekben = Regulation of H+ and HCO3-Transporters in Human Pancreatic Duct Cells

A pancreas nedvben található HCO3- ionok kiválasztásáért a pancreas ductus sejtek felelősek. Jelen pályázatban célul tűztük ki, hogy molekuláris biológiai, transzdukciós és mikrofluorescens technikák felhasználásával megértsük a HCO3- szekréció szabályozását humán pancreas ductus (CFPAC-1) sejtekben. Eredményeink a következőkben foglalhatóak össze: (1) Sikerül Szegeden beállítottunk egy humán polarizált pancreas ductus sejt modellrendszert, melyet egy korábbi Wellcome Trust ösztöndíj keretén belül a Newcastle-i Egyetemen fejlesztettünk ki (2003-5). (2) Meghatároztuk a ducts sejtek sav és bázis transzportereinek molekuláris identitását. (3) A cisztás fibrózis transzmembrán konduktancia regulátor Cl- csatorna (CFTR) az SLC26 és SLC4 anion transzporterek és a Na+/H+ cserélők (NHEk) működésére is hatással van. (4) Bebizonyítottuk, hogy az apikális anion csere az SLC26A6 transzporter által mediált. (5) A bazolaterális anion csere az SLC4A2 transzporter által mediált, az apikális Na+/H+ csere független a NHE2 és NHE3 transzporterektől. (6) Stimulált szekréció során nem változik a SLC4A2 és az apikális NHE aktivitás. (7) A kis dózisú kenodezoxikolsav a CFTR Cl- transzportjától függetlenül stimulálja az apikális Cl-/HCO3- cserét. Vizsgálataink eredményei nagymértékben hozzájárulnak a HCO3- szekréció szabályozásának megértéséhez a beteg (cisztás fibrózis) és vad-típusú CFTR-ral transzduktált pancreas ductalis epitheliumban. | Pancreatic duct cells secrete the HCO3- ions found in pancreatic juice. The overall aim of the project was to understand how HCO3- secretion is regulated in human pancreatic duct (CFPAC-1) cells using molecular biology, transduction and microfluorescence techniques. Our results can be summarized as follows: (1) We set up a human pancreatic duct cell model system in Szeged that was originally developed at the University of Newcastle during my Wellcome Trust Fellowship (2003-5). (2) We determined the molecular identities of acid and base transporters in duct cells. (3) The cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) regulates the activities of SLC26 and SLC4 (previously AE) families of anion transporters and Na+/H+ exchangers (NHEs). (4) We?ve demonstrated that apical anion exchange in the duct cell is mediated by SCL26A6. (5) Basolateral anion exchange is mediated by SLC4A2 (previously AE2), apical Na+/H+ exchange is independent of NHE2 and NHE3. (6) The activities of SLC4A2 and apical NHE transporters are not influenced by stimulated secretion. (7) Small doses of chenodeoxycholate stimulate apical Cl-/HCO3- activity independently of CFTR?s transport of Cl-. Taken together, this project lead to an improved understanding of HCO3- secretory mechanisms in the diseased (cystic fibrosis) and corrected (with wild-type CFTR) pancreatic ductal epithelium

Urban Evolution: The Role of Water

The structure, function, and services of urban ecosystems evolve over time scales from seconds to centuries as Earth’s population grows, infrastructure ages, and sociopolitical values alter them. In order to systematically study changes over time, the concept of “urban evolution” was proposed. It allows urban planning, management, and restoration to move beyond reactive management to predictive management based on past observations of consistent patterns. Here, we define and review a glossary of core concepts for studying urban evolution, which includes the mechanisms of urban selective pressure and urban adaptation. Urban selective pressure is an environmental or societal driver contributing to urban adaptation. Urban adaptation is thesequential process by which an urban structure, function, or services becomes more fitted to its changing environment or human choices. The role of water is vital to driving urban evolution as demonstrated by historical changes in drainage, sewage flows, hydrologic pulses, and long-term chemistry. In the current paper, we show how hydrologic traits evolve across successive generations of urban ecosystems via shifts in selective pressures and adaptations over time. We explore multiple empirical examples including evolving: (1) urban drainage from stream burial to stormwater management; (2) sewage flows and water quality in response to wastewater treatment; (3) amplification of hydrologic pulses due to the interaction between urbanization and climate variability; and (4) salinization and alkalinization of fresh water due to human inputs and accelerated weathering. Finally, we propose a new conceptual model for the evolution of urban waters from the Industrial Revolution to the present day based on empirical trends and historical information. Ultimately, we propose that water itself is a critical driver of urban evolution that forces urban adaptation, which transforms the structure, function, and services of urban landscapes, waterways, and civilizations over time

Modulation of a sustained calcium current by intracellular pH in horizontal cells of fish retina.

A sustained high voltage-activated (HVA), nifedipine- and cadmium-sensitive calcium current and a sustained calcium action potential (AP) were recorded from horizontal cells isolated from catfish retina. pH indicator dyes showed that superfusion with NH4Cl alkalinized these cells and that washout of NH4Cl or superfusion with Na-acetate acidified them. HVA current was slightly enhanced during superfusion of NH4Cl but was suppressed upon NH4Cl washout or application of Na-acetate. When 25 mM HEPES was added to the patch pipette to increase intracellular pH buffering, the effects of NH4Cl and Na-acetate on HVA current were reduced. These results indicated that intracellular acidification reduces HVA calcium current and alkalinization increases it. Sustained APs, recorded with high resistance, small diameter microelectrodes, were blocked by cobalt and cadmium and their magnitude varied with extracellular calcium concentration. These results provide confirmatory evidence that the HVA current is a major component of the AP and indicate that the AP can be used as a measure of how the HVA current can be modified in intact, undialyzed cells. The duration of APs was increased by superfusion with NH4Cl and reduced by washout of NH4Cl or superfusion with Na-acetate. The Na-acetate and NH4Cl washout-dependent shortening of the APs was observed in the presence of intracellular BAPTA, a calcium chelator, IBMX, a phosphodiesterase inhibitor, and in Na-free or TEA-enriched saline. These findings provide supportive evidence that intracellular acidification may directly suppress the HVA calcium current in intact cells. Intracellular pH changes would thereby be expected to modulate not only the resting membrane potential of these cells in darkness, but calcium-dependent release of neurotransmitter from these cells as well. Furthermore, this acidification-dependent suppression of calcium current could serve a protective role by reducing calcium entry during retinal ischemia, which is usually thought to be accompanied by intracellular acidosis

Anti-Trypanosoma cruzi action of a new benzofuran derivative based on amiodarone structure

Chagas disease is a neglected tropical affection caused by the protozoan parasite Trypanosoma cruzi. There is no current effective treatment since the only two available drugs have a limited efficacy and produce side effects. Thus, investigation efforts have been directed to the identification of new drug leads. In this context, Ca2+ regulating mechanisms have been postulated as targets for antiparasitic compounds, since they present paramount differences when compared to host cells. Amiodarone is an antiarrhythmic with demonstrated trypanocidal activity acting through the disruption of the parasite intracellular Ca2+ homeostasis. We now report the effect of a benzofuran derivative based on the structure of amiodarone on T. cruzi. This derivative was able to inhibit the growth of epimastigotes in culture and of amastigotes inside infected cells, the clinically relevant phase. We also show that this compound, similarly to amiodarone, disrupts Ca2+ homeostasis in T. cruzi epimastigotes, via two organelles involved in the intracellular Ca2+ regulation and the bioenergetics of the parasite. We demonstrate that the benzofuran derivative was able to totally collapse the membrane potential of the unique giant mitochondrion of the parasite and simultaneously produced the alkalinization of the acidocalcisomes. Both effects are evidenced by a large increase in the intracellular Ca2+ concentration of T. cruzi.Fil: Pinto Martinez, Andrea. instituto de Estudios Avanzados; VenezuelaFil: Hernández Rodríguez, Vanessa. instituto de Estudios Avanzados; VenezuelaFil: Rodríguez Durán, Jessica Jenireth. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. instituto de Estudios Avanzados; VenezuelaFil: Hejchman, Elżbieta. Medical University of Warsaw; PoloniaFil: Benaim, Gustavo. Universidad Central de Venezuela; Venezuel

Use of water from small alluvial aquifers for irrigation in semi-arid regions.

Water from small alluvial aquifers constitutes an attractive and low-cost option for irrigation and rural development in Northeastern Brazil. Based on piezometric measurements, geochemical analyses and electrical conductivity estimates, the present case study identified the main processes determining the hydrosaline dynamics of an alluvial aquifer in a small watershed inserted in the crystalline bedrock of a semi-arid region in Ceará and evaluated the availability of water for irrigation. Accumulation of salts in soil are related to evaporative flux from the aquifer and is increased by irrigation from the groundwater of the alluvial aquifer. The water in these aquifers may be used for irrigation, but represents a risk of soil salinization and alkalinization. Integrated management of surface and underground water resources in the Forquilha watershed may help control irrigation water quality (salinity and residual alkalinity), thereby rationalizing the use of local reservoirs and minimizing losses from evaporation. It has to take into account the complex dynamic of salts and water between the reservoirs, release of water into the river, floods and irrigations

Activation of electroneutral K flux in Amphiuma red blood cells by N-ethylmaleimide. Distinction between K/H exchange and KCl cotransport.

Exposure of Amphiuma red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) resulted in net K loss. In order to determine whether net K loss was conductive or was by electroneutral K/H exchange or KCl cotransport, studies were performed evaluating K flux in terms of the thermodynamic forces to which K flux by the above pathways should couple. The direction and magnitude of the NEM-induced net K flux did not correspond with the direction and magnitude of the forces relevant to K conductance or electroneutral KCl cotransport. Both the magnitude and direction of the NEM-activated K flux responded to the driving force for K/H exchange. We therefore conclude that NEM-induced K loss, like that by osmotically swollen Amphiuma red blood cells, is by an electroneutral K/H exchanger. In addition to the above studies, we evaluated the kinetic behavior of the volume- and NEM-induced K/H exchange flux pathways in media where Cl was replaced by SCN, NO3, para-aminohippurate (PAH), or gluconate. The anion replacement studies did not permit a distinction between K/H exchange and KCl cotransport, since, depending upon the anion used as a Cl replacement, partial inhibition or stimulation of volume-activated K/H exchange fluxes was observed. In contrast, all anions used were stimulatory to the NEM-induced K loss. Since, on the basis of force-flow analysis, both volume-and NEM-induced K loss are K/H exchange, it was necessary to reevaluate assumptions (i.e., anions serve as substrates and therefore probe the translocation step) associated with the use of anion replacement as a means of flux route identification. When viewed together with the force-flow studies, the Cl replacement studies suggest that anion effects upon K/H exchange are indirect. The different anions appear to alter mechanisms that couple NEM exposure and cell swelling to the activation of K/H exchange, as opposed to exerting direct effects upon K and H translocation