Identifizierung autoreaktiver T-Zellen bei Patienten mit Pemphigus Vulgaris mittels MHC-II-Dextramere

Pemphigus vulgaris (PV) gehört zur Gruppe der bullösen Autoimmunerkrankungen. Autoreaktive Dsg3 spezifische T-Zellen spielen eine zentrale Rolle in der PV Pathogenese durch die Anpassung der gegen Dsg3 gerichteten Antikörper-Produktion. Vor allem wird die Erhaltung der immunologischen Selbst-Toleranz von regulatorischen T-Zellen kontrolliert, deren Reduktion der Anzahl oder Funktionalität zu einer autoreaktiven Immunantwort führt. Der Fokus vorliegender Arbeit ist die Anwendung der Dextramer-Dsg3-Peptide-Färbung zur Detektion lebendig peripherer CD45 + CD19 - CD14 - CD3 - CD4 + Dsg3-Peptide-spezifischer T-Zellen. Nach der Stimulation mit rekombinantem Dsg3(aa1-566) von aus PV-Patienten und Probanden erworbenen PBMC wurde die Reaktivität der letztgenannten T-Zellen-Subpopulation gegen Dextramer-Dsg3-Peptide-Mix (Dsg3(aa190-204), Dsg3(aa206-220), Dsg3(aa254-268), Dsg3(aa378-392)) und gegen einzelne Dextramer-Dsg3-Peptide mittels durchflusszytometrischen Messung untersucht. Insgesamt gesehen ist das Ausmaß der Dextramer-Dsg3-Peptide-Reaktivität bei PV-Patienten höher als bei gesunden Probanden, man kann jedoch nur einen signifikanten Unterschied (*p=0,0263) zwischen diesen zwei Gruppen beobachten, wenn die Dextramer-Pool Färbung angewandt wird. Infolge der Stratifizierung nach Therapie ist die Reaktivität gegen Dextramer-Dsg3-Peptide-pool bei PV Patienten ohne Therapie nicht signifikant höher als bei Patienten unter Therapie. Der Vergleich zwischen aktiven PV Patienten und in Remission PV Patienten zeigt ebenfalls keinen signifikanten Unterschied. Interessanterweise zeigen PV Patienten eine höhere Reaktivität gegen„Class_II-associated invariant chain peptide“ (CLIP) als gesunde Kontrollen. Einerseits stellt die Dextramer-Färbung eine hervorragende Methode zur Detektion antigenspezifischer T-Zellen dar, andererseits ermöglicht die Dextramer-Technologie, spezifische HLA-restringierte Epitope zu untersuchen. Infolgedessen kann es zu einem vollständigeren Verständnis der Rolle von autoreaktiven CD4+ T-Zellen hinsichtlich der Pathogenese der Autoimmunantwort kommen und zusätzlich verfolgt werden, wie das Epitop-Spreading unterschiedliche Krankheitsstadien charakterisiert. Darüber hinaus ermöglicht die mittels Dextramer Antigen-Spezifizität-Bestimmung den Erhalt wichtiger Informationen hinsichtlich der T-Zellen Reaktivität, die für die Entwicklung der Immuntherapie mit regulatorischen T-Zellen verwendet werden könnte, wie z.B. Chimäre Antigenrezeptoren (CAR) Therapie

Increased expression and activity of p75NTR are crucial events in azacitidine-induced cell death in prostate cancer

The high affinity nerve growth factor (NGF) NGF receptor, p75NTR, is a member of the tumor necrosis factor (TNF) receptor superfamily that shares a conserved intracellular death domain capable of inducing apoptosis and suppressing growth in prostate epithelial cells. Expression of this receptor is lost as prostate cancer progresses and is minimal in established prostate cancer cell lines. We aimed to verify the role of p75NTR in the azacitidine-mediated antitumor effects on 22Rv1 and PC3 androgen-independent prostate cancer cells. In the present study, we reported that the antiproliferative and pro-apoptotic effects of 5-azacytidine (azacitidine) were more marked in the presence of physiological concentrations of NGF and were reduced when a blocking p75NTR antibody or the selective p75NTR inhibitor, Ro 08-2750, were used. Azacitidine increased the expression of p75NTR without interfering with the expression of the low affinity NGF receptor TrkA and induced caspase 9-dependent caspase 3 activity. Taken together, our results suggest that the NGF network could be a candidate for future pharmacological manipulation in aggressive prostate cancer

Tetrachloroethene recovery and hazard reduction of spent powders from dry cleaning process

Dry cleaning facilities using perchloroethylene produce a solid waste consisting of spent filtering powders with a high content of residual perchloroethylene, together with dyes and non-volatile residues. Untreated spent powders, classified as hazardous waste, cannot be disposed in landfill and incineration represents the only viable alternative. In this study, together with a full characterisation of the waste, the removal and recovery of the residual perchloroethylene by means of different heat treatments was investigated. In particular, tests of distillation and stripping with air and steam were carried out, evaluating the effectiveness of the treatments by quantifying the residual perchloroethylene in the samples treated. The results obtained show that the spent filtering powders contained about 25% wt. of perchloroethylene and that the maximum perchloroethylene recovery was obtained by steam stripping; approximately 98% after only 50 minutes. However, this treatment accounted for the production of a liquid mixture containing perchloroethylene and of a solid waste that required a further washing with boiling water to decrease the residual organic content below the eligibility criteria for landfill disposal

Biological denitrification of high-nitrate wastewaters: A comparison between three electron donors

Wastewaters discharged by several industrial activities, such as synthetic fibers, mineral processing, fertilizers, metal finishing, and ammunitions and explosives industries, have an high-salinity content and are characterized by a very high concentration of nitrates (more than 3 g/L). The treatment of these wastewaters generally deals in an anoxic biological process performed in activated sludge reactors (ASR). Due to the practical absence of an organic component, the treatment involves the addition of an external source of carbon, as electron donor for denitrification reactions. In addition, explosives industries wastewaters are characterized by low pH (2-3), since nitrates are generally discharged as nitric acid, and this induces a further difficulty in reactor operation, due to the extreme sensitivity of denitrifying biomass to pH conditions. In this paper the results of an experimentation performed in a laboratory scale anoxic ASR treating a highnitrate wastewater, simulating the explosives and ammunitions industries wastewaters, are presented and discussed. Three different carbon sources (methanol, acetic acid and sucrose) were compared, and the conditions to achieve the maximum removal of nitrogen were assessed. The ratio C:N to be maintained in the reactor to optimize cell growth and denitrification rate was also investigated. Copyright © 2013, AIDIC Servizi S.r.l

Production and characterization of adsorbent materials from sewage sludge by pyrolysis

The conventional sludge disposal options include landfill, application to farmland and forestry and incineration. However, since in the last decade sewage sludge and industrial sludge are being generated in increasing amounts due to the rapid urbanization and industrialization, a growing interest has been devoted in developing cost effective and renewable disposal alternatives. Among them, the manufacture of adsorbents to remove metals from water and wastewater appears to be promising, also considering the high cost of commercial carbons. In this paper copper, zinc and cadmium removal from wastewater using adsorbents produced from pyrolysis of sewage sludge is investigated in comparison with commercial adsorbents. The kinetic of the pyrolytic process was studied, and the adsorbents produced under different pyrolysis conditions were characterized. The adsorption capacity of the pyrolyzed material were estimated in batch tests performed in an activated sludge reactor. Results show that the adsorbent materials obtained by sewage sludge pyrolysis increased organic matter removal in activated sludge systems, and limited the inhibition effects of heavy metals. In addition, a chemical activation of the sludge before the pyrolysis resulted in an increase of the adsorption capacity of the obtained adsorbent. Copyright © 2013, AIDIC Servizi S.r.l

Dual PI3K/mTOR inhibitor, XL765 (SAR245409), shows superior effects to sole PI3K [XL147 (SAR245408)] or mTOR [rapamycin] inhibition in prostate cancer cell models

Deregulation of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway contributes to prostate cancer development and progression. Here, we compared the in vitro effects of the dual PI3K/mTOR inhibitor (XL765) with those observed with the sole PI3K (XL147) or mTOR (rapamycin) inhibition in 2 non-tumor prostate epithelial cell lines, 8 prostate cancer cell lines, and 11 prostate cancer cell derivatives. We demonstrated that the XL765 treatment showed superior and proliferative effects of XL147 or rapamycin. The XL765 effects were associated to increasing the chromosome region maintenance 1 (CRM1)-mediated nuclear localization of glycogen synthase kinase 3 beta (GSK3β) and Foxo-1a with higher induction of apoptosis when compared to those observed in XL147 and rapamycin treatments. IC50 values were calculated in phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-positive and PTEN-negative cell lines as well as after PTEN transfection or PTEN downmodulation by siRNA strategy revealing that the presence of this protein was associated with reduced sensitivity to PI3K and mTOR inhibitors. The comparison of IC50 values was also calculated for androgen-dependent and -independent cell lines as well as after androgen receptor (AR) transfection or the AR downmodulation by siRNA strategy revealing that androgen independence was associated with enhanced responsiveness. Our results provide a rationale to use the dual PI3K/Akt/mTOR inhibitors in hormone-insensitive prostate cancer models due to the overactivity of PI3K/Akt/mTOR in this disease condition

Exolectrogenic activity of a green microalgae, Chlorella vulg.

Bio-photovoltaic cells (BPVs) are a new photo-bio-electrochemical technology for harnessing solar energy using the photosynthetic activity of autotrophic organisms. This is a new technology for the production of sustainable and "clean" energy. Currently power outputs from BPVs are generally low and suffer from scarce efficiencies. However, a better understanding of the electrochemical interactions between the autotrophic microorganisms and conductive materials will be likely to lead to increased power yields. In the current study, the green microalgae Chlorella vulgaris was investigated for exoelectrogenic activity. To assess the exoelectrogenic activity of C. vulgaris a particular bio-photovoltaic cell was designed and built. The most important element is represented by the electrode configuration, based on inexpensive materials, with the anode immersed in the cultural broth and the cathode exposed to the atmosphere. This configuration represents a very interesting simplification for the cell design, furthermore allowing a simple illumination of the algal culture via a light source positioned above the cell, perpendicular to the electrode surface. This new kind of bioelectrochemical system does not need organic substrate and mediators, and the net production of CO2 is zero. This device was then characterized by measuring the electrical performance of the BPV. A power density of 14 μW/m2 was recorded, revealing interesting potentialities for green unicellular algae fuelled BPVs

XPS spectra analysis of Ti2+, Ti3+ions and dye photodegradation evaluation of titania-silica mixed oxide nanoparticles

TiO2-SiO2 mixed oxides have been prepared by the sol–gel technique from tetrabutyl orthotitanate and tetraethyl orthosilicate. The prepared materials were characterized by x-ray diffraction, scanning electron microscopy, energy dispersive x-ray spectroscopy, nitrogen physisorption, Fourier-transform infrared spectroscopy (FT-IR) and x-ray photoelectron spectroscopy (XPS). The results indicate that the TiO2-SiO2 mixed oxides have a large surface area and a nanoscale size. FT-IR spectra show that Ti atoms are bonded to silica by oxygen bridging atoms in Ti-O-Si bonds. The titanium valence states in TiO2-SiO2 mixed oxides were investigated by XPS, and their spectra report the presence of Ti2+ and Ti3+ cations for high silica concentration, suggesting the formation of oxygen vacancies. The photocatalytic activity of the prepared materials has been evaluated for the photodegradation of methylene blue (MB). The mixed oxides were activated by means of a UV light source, and the concentration of MB was monitored by UV–Vis spectroscopy. The synthesized TiO2-SiO2 shows significantly higher MB removal efficiency in comparison with that of the commercial TiO2 Degussa, P25

Angiotensin-converting-enzyme inhibition counteracts angiotensin II-mediated endothelial cell dysfunction by modulating the p38/SirT1 axis

Objective: Oxidative stress has been linked to endothelial dysfunction and angiotensin II stimulates the reactive oxygen species production contributing to several cardiovascular diseases. We have studied the chain of events induced by angiotensin-converting-enzyme (ACE) activation in vascular umbilical vein endothelial cells (HUVECs) by using an ACE inhibitor such as zofenoprilat. Methods: We used specific assay to measure the superoxide anion production, tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, and western blot for protein analysis in the study. Results: Zofenoprilat counteracts the superoxide anion production and cell apoptosis induced by angiotensin I treatment by blocking the extrinsic caspase cascade, NF-kB and p38 activation. p38 inhibitor SB203580 reverted the angiotensin II oxidant effects while the p38 constitutively activation, by MKK6 transfection, abrogated the zofenoprilat effects. Characterizing the zofenoprilat downstream effector we found that zofenoprilat reverted the SirT-1 downregulation induced by angiotensin II. p38 activation by angiotensin II was strictly correlated with SirT1 protein downregulation; SB203580 significantly prevented SirT1 downregulation induced by angiotensin II while the p38 constitutive activation abolished SIRT1 protein basal levels. p38 directly bound SirT1 sequestering it in the cytoplasm. SirT1 inhibition by sirtinol annulled zofenoprilat action while SirT1 overexpression reverted the cytotoxic effects of angiotensin II. Finally, zofenoprilat negatively controlled angiotensin I receptor protein expression through SirT1. Conclusion: The p38-SirT1 axis is found markedly relevant in modulating the cardiovascular benefit deriving from ACE-inhibitors and might represent a novel target for innovative drugs in cardiovascular prevention. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins

Inhibition of B-cell proliferation and antibody production by mesenchymal stromal cells is mediated by T cells

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs), endowed with immunosuppressive and anti-inflammatory properties, represent a promising tool in immunoregulatory and regenerative cell therapy. Clarifying the interactions between MSCs and B-lymphocytes may be crucial for designing innovative MSC-based strategies in conditions in which B cells play a role, including systemic lupus erythematosus (SLE) and rejection of kidney transplantation. In this study, we show that, both in healthy subjects and in patients, in vitro B-cell proliferation, plasma-cell differentiation, and antibody production are inhibited by BM-derived MSCs when peripheral blood lymphocytes are stimulated with CpG, but not when sorted B cells are cultured with MSCs+CpG. Inhibition is restored in CpG+MSC cocultures when sorted T cells are added to sorted B cells, suggesting that this effect is mediated by T cells, with both CD4(+) and CD8(+) cells playing a role. Moreover, cell–cell contact between MSCs and T cells, but not between MSCs and B cells, is necessary to inhibit B-cell proliferation. Thus, the presence of functional T cells, as well as cell–cell contact between MSCs and T cells, are crucial for B-cell inhibition. This information can be relevant for implementing MSC-based therapeutic immune modulation in patients in whom T-cell function is impaired