175 research outputs found
Lab-on-a-chip and integrated strategies in tumor immunotheraphy
No full textBackground
While conventional chemotherapy and radiation therapy have improved the survival of many cancer patients, there are still major disadvantages associated with these treatments such as high toxicity and drug-resistance. The possibility to manipulate the immune system to recognize and kill tumor cells is very attractive despite numerous obstacles remaining to be overcome. In particular, the ability of the immune system to destroy disseminated metastases in a specific way makes immunotherapy an attractive alternative to conventional therapies. Nevertheless, other unconventional technologies emerged in recent years seem to be very promising; in particular the analysis and monitoring of single cell-to-cell interactions and the capability to individually control single cells by Lab-on-a-chip devices have become of great interest in different areas of life sciences. These new technologies, in combination with progresses reached in anti-tumor vaccines, could be useful to improve immune T cell responses against tumor antigen for a more efficient immunotherapy.
Aims
This thesis focuses on two tumor immunotherapy issues: 1) design, realization and validation of innovative Lab-on-a-chip devices for immune system study, that allows single tumor cell and effector cell interaction, detection and isolation; 2) identification of molecular mechanisms that prevent EBV-associated tumors (e.g. Burkitt’s lymphoma) recognition by T cells and study of their potential correction by specific treatments. The main goal of this study remains indeed the evaluation of an integrated strategy for immunotherapy development enhancing for malignancies treatment.
Methods
Biocompatibility test, generation of memory CTL cultures, 51Cr release assay, IFN-Elispot, proteasomes purification, western blot assay, enzymatic assay, immunofluorescence, RT-PCR.
Main Results
As concerns the first part of the thesis, a main achievement was the design of Lab-on-a-chip platform that combined microfluidics and electronics together, consisting in a matrix of
up to thousand microwells where living cells can be deposited. Subsequently, different materials have been evaluated to identify the most biocompatible ones for biosensor manufacturing. Once developed Lab-on-a-chip prototype, it has been tested from a functional point of view. In particular, it has been demonstrated that the biosensor is able to isolate and trap single cells inside microwells by dielectrophoresis, that recovered cells are still alive and that their biological functions and gene expression remain unaltered. Furthermore, tumor cell lysis by immune effector cells could be successfully monitored inside device microwells, showing that biosensor could be used for cell to cell interaction studies.
Regarding the second aim of this thesis, it has been identified a new epitope-specific T cell response against EBV nuclear antigen 1 (EBNA1). It has also been demonstrated that CTLs specific for another EBNA1-derived epitope (referred as HPV) are detectable in the majority of HLA-B35 individuals, and recognize EBV-transformed B lymphocytes (LCL) but not Burkitt’s lymphoma (BL). Afterwards LCL and BL have been compared for their antigen processing machinery, demonstrating that one of the major differences was at the proteasome level; indeed, proteasomes from BL cells have displayed a far lower chymotryptic and tryptic-like activities. Interestingly, it has also been shown that treatment with proteasome inhibitors partially restored the capacity of BL cells to present the HPV epitope.
Conclusions
The results achieved in single cell manipulation and cell to cell analysis interaction by Lab-on-a-chip technology, and the findings reached to improve BL immune recognition, represent an implementation of innovative tools that could allow important progresses in cancer diagnosis and immunotherapy
“I fear the secrets of time”. Excerpts from Sogar Papageien überleben uns
No full textPresented here, in the Italian translation by Elisa Destro, are several excerpts from the novel Sogar Papageien überleben uns (2010) by German-Russian author Olga Martynova.Si presentano qui, nella traduzione italiana di Elisa Destro, alcuni estratti dal romanzo Sogar Papageien überleben uns (2010) dell\u27autrice russo-tedesca Olga Martynova
Novel oleanolic vinyl boronates: synthesis and antitumor activity
No full textA series of novel oleanane-type pentacyclic triterpenoids bearing a boronate ester moiety at C3 have been synthesized by palladium-catalyzed cross-coupling of bis(pinacolato)diboron with vinyl triflates, in the presence of base, and these compounds were fully characterized by 1D and 2D NMR techniques. Evaluation of their antiproliferative effects on a panel of hematological-based and solid tumor cell lines identified three active oleanolic vinyl boronates that inhibited the growth of leukemia (Jurkat, K562), Burkitt's lymphoma (Jijoye), cervix (Hela), colon (SW480), and ovary (SKOV-3) cancer cells without concomitant inhibition of non-tumoral human fibroblasts. Their mechanisms of action were investigated on the leukemia Jurkat cell line. The results show that the incorporation of boron in the oleanolic acid core combined with the presence of amide bonds afforded compounds with desirable biological effects such as apoptosis induction and inhibition of proteasomal activity on tumor cells, which makes them potential templates for further development in the anticancer drug setting
Experimental and theoretical charge density distribution of the colossal magnetoresistive transition metal sulfide FeCr2S4
No full textThe total charge density distribution rho(r) of the colossal magnetoresistive transition metal sulfide FeCr2S4 was evaluated through a multipole formalism from a set of structure factors obtained both experimentally, by means of single crystal high-quality x-ray diffraction data collected at T=23 K, and theoretically, with an extended-basis unrestricted Hartree-Fock periodic calculation on the experimental geometry. A full topological analysis, followed by the calculation of local energy
density values and net atomic charges, was performed using the quantum theory of atoms in molecules. The experimental and theoretical results were compared. Good agreement was found for the topological properties of the system, as well as for the atomic net charges and the nature of the chemical bonds. An analysis of the electron density rho(r), its Laplacian L(r), and the total energy density H(r) at the bond critical points was employed to classify all the interactions that resulted as predominantly closed shell (ionic) in nature. The topological indicators of the bonded interactions for Fe are distinct from those for Cr. The Fe–S bond distances were found to be 0.145 Å shorter than the ideal values computed on the basis of Shannon’s crystal radii, much shorter than the Cr–S distances with respect to their ideal Shannon lengths. Concomitantly, rho(r) and H(r) at the bond critical points are greater for Fe–S interactions, indicating that the local concentration of charge density in the internuclear region is larger for the tetrahedrally coordinated iron than for the octahedrally coordinated chromium. The isosurface in the real space for L(r)=0 was plotted for both iron and chromium, pointing out the local zones of valence shell charge concentration and relating them to the partial d-orbital occupancy of the two transition metal atoms
Legami intergenerazionali e identificazioni inconsce nel romanzo L'ottava vita (per Brilka) di Nino Haratischwili
No full textThe article aims to investigate the representation of the transgenerational transmission of traumatic experiences in the novel Das achte Leben (für Brilka) (2014) by the Georgian-born author Nino Haratischwili, by drawing on psychoanalytic and epigenetic studies on the subject, as well as cultural studies on memory and post-memory. The article highlights psychic processes such as phantasmatic projections and telescopic identifications of the unconscious, which denote a disturbance in genealogy – ascribable to unprocessed grief and trauma – and transmitted from one generation to another on an unconscious level as a void. At the same time, the essay also examines the therapeutic journey that needs to be undertaken by the descendants of the family at the centre of the work in order to re-engage with their past history. If, on the one hand, the narrative is full of gaps and circular structures (a symptom of an unspeakable trauma that cannot be expressed and always returns to itself), on the other hand, the blank pages at the end, as well as the circular structure of the frame, have a positive meaning – that of (post-)memory, which attempts to return to its origins in order to create a new, meaningful narrative and, with it, a new life
Progress in the understanding of drug-receptor interactions. Part 2, experimental and theoretical electrostatic moments and interaction energies of an angiotensin II receptor antagonist (C30H30N6O3S)
No full textA combined experimental and theoretical charge density study of an angiotensin II receptor antagonist (1) is presented focusing on electrostatic properties such as atomic charges, molecular electric moments up to the fourth rank and energies of the intermolecular interactions, to gain an insight into the physical nature of the drug-receptor interaction. Electrostatic properties were derived from both the experimental electron density (multipole refinement of X-ray data collected at T=17 K) and the ab initio wavefunction (single molecule and fully periodic calculations at the DFT level). The relevance of SO and SN intramolecular interactions on the activity of 1 is highlighted by using both the crystal and gas-phase geometries and their electrostatic nature is documented by means of QTAIM atomic charges. The derived electrostatic properties are consistent with a nearly spherical electron density distribution, characterised by an intermingling of electropositive and -negative zones rather than by a unique electrophilic region opposed to a nucleophilic area. This makes the first molecular moment scarcely significant and ill-determined, whereas the second moment is large, significant and highly reliable. A comparison between experimental and theoretical components of the third electric moment shows a few discrepancies, whereas the agreement for the fourth electric moment is excellent. The most favourable intermolecular bond is show to be an NHN hydrogen bond with an energy of about 50 kJ mol-1. Key pharmacophoric features responsible for attractive electrostatic interactions include CHX hydrogen bonds. It is shown that methyl and methylene groups, known to be essential for the biological activity of the drug, provide a significant energetic contribution to the total binding energy. Dispersive interactions are important at the thiophene and at both the phenyl fragments. The experimental estimates of the electrostatic contribution to the intermolecular interaction energies of six molecular pairs, obtained by a new model proposed by Spackman, predict the correct relative electrostatic energies with no exceptions
α,β-Unsaturated <i>N</i>-Acylpyrrole Peptidyl Derivatives: New Proteasome Inhibitors
No full textBecause of the encouraging results obtained using vinyl ester derivatives, we synthesized and tested a novel series of peptide-based proteasome inhibitors bearing a new pharmacophore unit at the C-terminal. N-Acylpyrrole moiety is a potential substrate for Michael addition by catalytic threonine. Several analogues have demonstrated a selective inhibition of the multicatalytic complex β1 subunits, the capacity to permeate cellular membrane, and good pharmacokinetics properties
Characterization of an human leucocyte antigen A2-restricted Epstein-Barr virus nuclear antigen-1-derived cytotoxic T-lymphocyte epitope
No full textSummary The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is regularly expressed in all proliferating virus-infected cells and is therefore an interesting target for immunotherapy. Alleles of the human leucocyte antigen (HLA) -A2 family are dominantly expressed in Caucasians so we sought to identify EBNA1-specific cytotoxic T-lymphocyte (CTL) responses restricted through this allele. We report on the characterization of the LQTHIFAEV (LQT) epitope. LQT-specific memory CTL responses were reactivated in three of 14 healthy EBV seropositive donors (21%) whereas responses to HLA-A2-restricted epitopes, two derived from LMP2 and one from EBNA3A, were detected in 93%, 71% and 42% of the donors, respectively. The LQT-specific CTL clones did not lyse EBV-carrying lymphoblastoid cell lines and Burkitt's lymphoma cell lines nor EBNA1-transfected Burkitt's lymphoma cells but specifically released interferon-γ upon stimulation with HLA-matched EBNA1-expressing cells and this response was enhanced by deletion of the Gly-Ala repeat domain that inhibits proteasomal degradation. The poor presentation of the endogenously expressed LQT epitope was not affected by inhibition of peptidases that trim antigenic peptides in the cytosol but full presentation was achieved in cells expressing a trojan antigen construct that releases the epitope directly into the endoplasmic reticulum. Hence, inefficient proteasomal processing appears to be mainly responsible for the poor presentation of this epitope. © 2009 Blackwell Publishing Ltd
N-Terminal-prolonged vinyl ester-based peptides as selective proteasome b1 subunit inhibitors
No full textThe synthesis and biological properties of vinyl ester peptide-based molecules bearing linear N-terminal
amino acids are reported. Compounds were tested in vitro for their capacity to inhibit the chymotryptic-,
tryptic-like, and post-acidic activities of the proteasome. Some analogues showed selective inhibition of
post-acidic (PGPH) activity, which is attributed to the b1 subunit. Interestingly, active compounds demonstrated
higher inhibitory activity toward ‘standard’ proteasomes than toward immunoproteasomes.
The inhibitory potency was found to be related to the amino acidic sequence and to the length of the
N-terminal residues. The new inhibitors demonstrated resistance to plasmatic proteases and a good
capacity to permeate the cell membrane
Identification of new HIV-1 Gag-specific cytotoxic T lymphocyte responses in BALB/c mice
No full textAbstract Background As HIV-specific cytotoxic T cells play a key role during acute and chronic HIV-1 infection in humans, the ability of potential anti-HIV vaccines to elicit strong, broad T cell responses is likely to be crucial. The HIV-1 Gag antigen is widely considered a relevant antigen for the development of an anti-HIV vaccine since it is one of the most conserved viral proteins and is also known to induce T cell responses. In the majority of studies reporting Gag-specific cellular immune responses induced by Gag-based vaccines, only a small number of Gag T cell epitopes were tested in preclinical mouse models, thus giving an incomplete picture of the numerous possible cellular immune responses against this antigen. As is, this partial knowledge of epitope-specific T cell responses directed to Gag will unavoidably result in a limited preclinical evaluation of Gag-based vaccines. Results In this study we identified new Gag CD8+ T cell epitopes in BALB/c mice vaccinated with the HIV-1 Gag antigen alone or in combination with the HIV-1 Tat protein, which was recently shown to broaden T cell responses directed to Gag. Specifically, we found that CTL responses to Gag may be directed to nine different CTL epitopes, and four of these were mapped as minimal CTL epitopes. Conclusion These newly identified CTL epitopes should be considered in the preclinical evaluation of T cell responses induced by Gag-based vaccines in mice.</p
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