Lab-on-a-chip and integrated strategies in tumor immunotheraphy

Background 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

Identification of new HIV-1 Gag-specific cytotoxic T lymphocyte responses in BALB/c mice

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How Can We Help You?: An Instagram-Based Online Self-Help for Eating Disorders

In recent years, there has been a noticeable increase in online self-help treatments and peer-support programs for eating disorders. The possibility of easily accessing them anytime makes these programs an important support tool and an influencing source for increasing motivation to change. The aim of this work is to describe the #How can we help you? project, its initial feedback received from users, and its future directions. Researchers and clinicians developed an Instagram profile (Dicci Come Aiutarti) based on psychoeducation, aimed at orienting those suffering from a self-reported eating disorder towards clinical care, providing information about eating concerns and related constructs, and increasing motivation for treatment and illness awareness. The contents shared are based on narratives about people who had recovered from an eating disorder, importance and ability to change, and nutrition management. We have provided an overview of the needs of the Instagram profile users, a description of the main interactions recorded since the profile was opened, and examples of the unmet needs shared by users in direct messages. Future directions of the project concern the definition and formalization of the type of support provided by developing a psychoeducational and integrated program and also, the formulation of a research protocol able to assess the usability, effectiveness, and satisfaction of the Instagram profile. © 2023 by the authors

High-pressure balloon dilatation in children: our results in 30 patients with POM and the implications of the cystoscopic evaluation

Primary Obstructive Megaureter (POM) is a common cause of hydronephrosis in children with spontaneous resolution in most cases. High-Pressure Balloon Dilatation (HPBD) has been proposed as a minimally invasive procedure for POM correction in selected patients. The aim of the paper is to review our experience with HPBD in patients with POM. We performed a retrospective study in a single Centre collecting data on patients' demographics, diagnostic modalities, surgical details, results and follow-up. In particular, the endoscopic aspect of the orifice permitted the identification of 3 patterns: adynamic ureteral segment, stenotic ureteric ring and pseudoureterocelic orifice. We performed HPBD in 30 patients over 6 years. We had 23 patients with adynamic distal ureteral segment (type 1), 4 with stenotic ring (type 2) and 3 with ureterocelic orifice (type 3). In 3 patients (10%) the guidewire did not easily pass into the ureter requiring ureteral stenting or papillotomy. Post-operative course was uneventful. Five patients (3 pseudoureterocelic) required open surgery during follow-up. HPBD for the treatment of POM is a safe and feasible procedure and it can be a definitive treatment of POM. Complications are mainly due to double J stent and none of our patients had symptoms related to vescico-ureteral reflux. The aspect of the orifice, identified during cystoscopy, seems to correlate with the efficacy of the dilatation: type 1 and 2 are associated with good and excellent results respectively; type 3 do not permit dilatation in almost all cases requiring papillotomy. HPBD can be performed in selected patients of all paediatric ages as first therapeutic line. The presence of a pseudoureterocelic orifice or long stenosis might interfere with the ureteral stenting and seems associated with worse outcomes

Serum IgG antibodies from pregnant women reacting to mimotopes of simian virus 40 large T antigen, the viral oncoprotein

Simian virus 40 (SV40) large T antigen (LT) coding sequences were revealed in different human samples, whereas SV40 antibodies (Ab) were detected in human sera of cancer patients and healthy individuals, although with a lower prevalence. Previous studies carried out by the neutralization assay gave a SV40 seroprevalence, in the general population, up to 8%, although higher rates, 12%, were detected in kidney transplant children, in a group of HIV-positive patients, and in healthy females. In this study, serum samples from pregnant women, together with those from non-pregnant women, were analyzed to check the prevalence of IgG Ab reacting to SV40 LT antigens. Serum samples were collected from pregnant and non-pregnant women, with the same mean age. Women were in the range of 15-48 years old. Samples were assayed by an indirect ELISA employing specific SV40 LT mimotopes as antigens, whereas functional analysis was performed by neutralization of the viral infectivity in cell cultures. As a control, sera were analyzed for Ab against BK polyomavirus (BKPyV), which is a human polyomavirus homologous to SV40. Statistical analyses employed chi-square with Yates' correction, and Student's t tests. Indirect ELISAs indicated that pregnant women tested SV40 LT-positive with a prevalence of 17% (23/134), whereas non-pregnant women had a prevalence of 20% (36/180) (P > 0.05). Ab against BKPyV were detected with a prevalence of 80% in pregnant women and with a prevalence of 78% in non-pregnant women. These data indicate that SV40 infects at a low prevalence pregnant women. We may speculate that SV40, or a close human polyomavirus still undetected, could be transmitted from mother to fetus

Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine

Intellectual Property, Open Science and Research Biobanks

In biomedical research and translational medicine, the ancient war between exclusivity (private control over information) and access to information is proposing again on a new battlefield: research biobanks. The latter are becoming increasingly important (one of the ten ideas changing the world, according to Time magazine) since they allow to collect, store and distribute in a secure and professional way a critical mass of human biological samples for research purposes. Tissues and related data are fundamental for the development of the biomedical research and the emerging field of translational medicine: they represent the “raw material” for every kind of biomedical study. For this reason, it is crucial to understand the boundaries of Intellectual Property (IP) in this prickly context. In fact, both data sharing and collaborative research have become an imperative in contemporary open science, whose development depends inextricably on: the opportunities to access and use data, the possibility of sharing practices between communities, the cross-checking of information and results and, chiefly, interactions with experts in different fields of knowledge. Data sharing allows both to spread the costs of analytical results that researchers cannot achieve working individually and, if properly managed, to avoid the duplication of research. These advantages are crucial: access to a common pool of pre-competitive data and the possibility to endorse follow-on research projects are fundamental for the progress of biomedicine. This is why the "open movement" is also spreading in the biobank's field. After an overview of the complex interactions among the different stakeholders involved in the process of information and data production, as well as of the main obstacles to the promotion of data sharing (i.e., the appropriability of biological samples and information, the privacy of participants, the lack of interoperability), we will firstly clarify some blurring in language, in particular concerning concepts often mixed up, such as “open source” and “open access”. The aim is to understand whether and to what extent we can apply these concepts to the biomedical field. Afterwards, adopting a comparative perspective, we will analyze the main features of the open models – in particular, the Open Research Data model – which have been proposed in literature for the promotion of data sharing in the field of research biobanks. After such an analysis, we will suggest some recommendations in order to rebalance the clash between exclusivity - the paradigm characterizing the evolution of intellectual property over the last three centuries - and the actual needs for access to knowledge. We argue that the key factor in this balance may come from the right interaction between IP, social norms and contracts. In particular, we need to combine the incentives and the reward mechanisms characterizing scientific communities with data sharing imperative

Preclinical emergence of vandetanib as a potent antitumour agent in mesothelioma: molecular mechanisms underlying its synergistic interaction with pemetrexed and carboplatin

BACKGROUND: Although pemetrexed, a potent thymidylate synthase (TS) inhibitor, enhances the cytoytoxic effect of platinum compounds against malignant pleural mesothelioma (MPM), novel combinations with effective targeted therapies are warranted. To this end, the current study evaluates new targeted agents and their pharmacological interaction with carboplatin-pemetrexed in human MPM cell lines. METHODS: We treated H2052, H2452, H28 and MSTO-211H cells with carboplatin, pemetrexed and targeted compounds (gefitinib, erlotinib, sorafenib, vandetanib, enzastaurin and ZM447439) and evaluated the modulation of pivotal pathways in drug activity and cancer cell proliferation. RESULTS: Vandetanib emerged as the compound with the most potent cytotoxic activity, which interacted synergistically with carboplatin and pemetrexed. Drug combinations blocked Akt phosphorylation and increased apoptosis. Vandetanib significantly downregulated epidermal growth factor receptor (EGFR)/Erk/Akt phosphorylation as well as E2F-1 mRNA and TS mRNA/protein levels. Moreover, pemetrexed decreased Akt phosphorylation and expression of DNA repair genes. Finally, most MPM samples displayed detectable levels of EGFR and TS, the variability of which could be used for patients' stratification in future trials with vandetanib-pemetrexed-carboplatin combination. CONCLUSION: Vandetanib markedly enhances pemetrexed-carboplatin activity against human MPM cells. Induction of apoptosis, modulation of EGFR/Akt/Erk phosphorylation and expression of key determinants for pemetrexed and carboplatin activity contribute to this synergistic interaction, and, together with the expression of these determinants in MPM samples, warrant further clinical investigation