Studio immunologico per l'individuazione di pazienti trapiantati per cirrosi HBV relata che potrebbero sospedere la terapia con immunoglobine specifiche (HBIG)

Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT) is associated with poor graft and patient survival. Treatment with HBV-specific immunoglobulins (HBIG) in combination with nucleos(t)ide analogs is effective in preventing HBV reinfection of the graft and improving OLT outcome. However, the combined immunoprophylaxis has several limitations, mainly the high cost and the lack of standard schedules about duration. So far, the identification of markers able to predict the reinfection risk is needed. Although the HBV-specific immune response is believed to play an essential role in disease outcome, HBV-specific cellular immunity in viral containment in OLT recipients is unclear. To test whether or not OLT recipients maintain robust HBV-specific cellular immunity, the cellular immune response against viral nucleocapsid and envelope-protein of HBV was assessed in 15 OLT recipients and 27 individuals with chronic and 24 subjects with self-limited HBV infection, respectively. The data demonstrate that OLT recipients mounted fewer but stronger clusters of differentiation (CD)8 T cell responses than subjects with self-limited HBV infection and showed a preferential targeting of the nucleocapsid antigen. This focused response pattern was similar to responses seen in chronically infected subjects with undetectable viremia, but significantly different from patients who presented with elevated HBV viremia and who mounted mainly immune responses against the envelope protein. In conclusion, virus-specific CD4 T cell–mediated responses were only detected in subjects with self-limited HBV infection. Thus, the profile of the cellular immunity against HBV was in immune suppressed patients similar to subjects with chronic HBV infection with suppressed HBV-DNA.

Algal wastewater treatment and biomass producing potential: nutrient removal efficiency and cell physiological responses

Microalgae are sun - light cell factories that convert carbon dioxide to biofuels, foods, feeds, and other bioproducts. The concept of microalgae cultivation as an integrated system in wastewater treatment has optimized the potential of the microalgae - based biofuel production. These microorganisms contains lipids, polysaccharides, proteins, pigments and other cell compounds, and their biomass can provide different kinds of biofuels such as biodiesel, biomethane and ethanol. The algal biomass application strongly depends on the cell composition and the production of biofuels appears to be economically convenient only in conjunction with wastewater treatment. The aim of this research thesis was to investigate a biological wastewater system on a laboratory scale growing a newly isolated freshwater microalgae, Desmodesmus communis, in effluents generated by a local wastewater reclamation facility in Cesena (Emilia Romagna, Italy) in batch and semi - continuous cultures. This work showed the potential utilization of this microorganism in an algae - based wastewater treatment; Desmodesmus communis had a great capacity to grow in the wastewater, competing with other microorganisms naturally present and adapting to various environmental conditions such as different irradiance levels and nutrient concentrations. The nutrient removal efficiency was characterized at different hydraulic retention times as well as the algal growth rate and biomass composition in terms of proteins, polysaccharides, total lipids and total fatty acids (TFAs) which are considered the substrate for biodiesel production. The biochemical analyses were coupled with the biomass elemental analysis which specified the amount of carbon and nitrogen in the algal biomass. Furthermore photosynthetic investigations were carried out to better correlate the environmental conditions with the physiology responses of the cells and consequently get more information to optimize the growth rate and the increase of TFAs and C/N ratio, cellular compounds and biomass parameter which are fundamental in the biomass energy recovery.

Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

Roles of Ecdysone signaling in cell survival and epithelium morphogenesis during Drosophila melanogaster development

In Drosophila the steroid hormone ecdysone regulates a wide range of developmental and physiological responses, including reproduction, embryogenesis, postembryonic development and metamorphosis. Drosophila provides an excellent system to address some fundamental questions linked to hormone actions. In fact, the apparent relative simplicity of its hormone signaling pathways taken together with well-established genetic and genomic tools developed to this purpose, defines this insect as an ideal model system for studying the molecular mechanisms through which steroid hormones act. During my PhD research program I’ve analyzed the role of ecdysone signaling to gain insight into the molecular mechanisms through which the hormone fulfills its pleiotropic functions in two different developmental stages: the oogenesis and the imaginal wing disc morphogenesis. To this purpose, I performed a reverse genetic analysis to silence the function of two different genes involved in ecdysone signaling pathway, EcR and ecd.

Analysis of the memory B cell response against glycoconjugate vaccines

The development of vaccines directed against polysaccharide capsules of S. pneumoniae, H. influenzae and N. meningitidis have been of great importance in preventing potentially fatal infections. Bacterial capsular polysaccharides are T-cell-independent antigens that induce specific antibody response characterized by IgM immunoglobulins, with a very low IgG class switched response and lack of capability of inducing a booster response. The inability of pure polysaccharides to induce sustained immune responses has required the development of vaccines containing polysaccharides conjugated to a carrier protein, with the aim to generate T cell help. It is clear that the immunogenicity of glycoconjugate vaccines can vary depending on different factors, e.g. chemical nature of the linked polysaccharide, carrier protein, age of the target population, adjuvant used. The present study analyzes the memory B cell (MBC) response to the polysaccharide and to the carrier protein following vaccination with a glycoconjugate vaccine for the prevention of Group B streptococcus (GBS) infection. Not much is known about the role of adjuvants in the development of immunological memory raised against GBS polysaccharides, as well as about the influence of having a pre-existing immunity against the carrier protein on the B cell response raised against the polysaccharide component of the vaccine. We demonstrate in the mouse model that adjuvants can increase the antibody and memory B cell response to the carrier protein and to the conjugated polysaccharide. We also demonstrate that a pre-existing immunity to the carrier protein favors the development of the antibody and memory B cell response to subsequent vaccinations with a glycoconjugate, even in absence of adjuvants. These data provide a useful insight for a better understanding of the mechanism of action of this class of vaccines and for designing the best vaccine that could result in a productive and long lasting memory response.

Transcriptional responses of the Helicobacter pylori cag pathogenicity island

The severity of Helicobacter pylori infections largely depends on the genetic diversity of the infecting strain, and particularly on the presence of the cag pathogenicity island (cag-PAI). This virulence locus encodes a type-IV secretion system able to translocate in the host cell at least the cag-encoded toxin CagA and peptidoglycan fragments, that together are responsible for the pathogenic phenotype in the host. Little is known about the bacterial regulators that underlie the coordinated expression of cag gene products, needed to assemble a functional secretion system apparatus. To fill this gap, a comprehensive analysis of the transcriptional regulation of the cag-PAI operons was undertaken. To pursue this goal, a robust tool for the analysis of gene expression in H. pylori was first implemented. A bioluminescent reporter system based on the P. luminescens luxCDABE operon was constructed and validated by comparisons with transcriptional analyses, then it was systematically used for the comprehensive study and mapping of the cag promoters. The identification of bona fide cag promoters had permitted to pinpoint the set of cag transcriptional units of the PAI. The responses of these cag transcriptional units to metabolic stress signals were analyzed in detail, and integrated with transcription studies in deletion mutants of important H. pylori virulence regulators and protein-DNA interaction analyses to map the binding sites of the regulators. Finally, a small regulatory RNA cncR1 encoded by the cag-PAI was identified, and the 5’- and 3’-ends of the molecule were mapped by primer extension analyses, northern blot and studies with lux reporter constructs. To identify regulatory effects exerted by cncR1 on the H. pylori gene expression, the cncR1 knock out strain was derived and compared to the parental wild type strain by a macroarray approach. Results suggest a negative effect exerted by cncR1 on the regulome of the alternative sigma54 factor.

Histone deacetylase inhibitors in adult patients with diffuse large b-cell lymphoma relapsed/refractory: a phase II study with Panobinostat

Backgrounds:Treatment of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) not eligible to high dose therapy represents an unmet medical need. Panobinostat showed encouraging therapeutic activity in studies conducted in lymphoma cell lines and in vivo in patients with advanced hematologic malignancies.Purpose:FIL-PanAL10 (NCT01523834) is a phase II, prospective multicenter trial of the Fondazione Italiana Linfomi (FIL) to evaluate safety and efficacy of single agent Panobinostat as salvage therapy for R/R DLBCL patients and to evaluate a possible relationships between response and any biological features. Patients and Methods:Patients with R/R DLBCL were included. The treatment plan included 6 induction courses with Panobinostat monotherapy followed by other 6 courses of consolidation. The primary objective was to evaluate Panobinostat activity in terms of overall response (OR); secondary objectives were: CR rate, time to response (TTR), progression-free survival (PFS), safety and feasibility of Panobinostat. We included evaluation of the impact of pharmacogenetics, immunohistochemical patterns and patient’s specific gene expression and mutations as potential predictors of response to Panobinostat as explorative objectives. To this aim a pre-enrollment new tissue biopsy was mandatory. ResultsThirty-five patients, 21 males (60%), were enrolled between June 2011 and March 2014. At the end of induction phase, 7 responses (20%) were observed, including 4 CR (11%), while 28 patients (80%) discontinued treatment due to progressive disease (PD) in 21 (60%) or adverse events in 7 (20%). Median TTR in 9 responders was 2.6 months (range 1.8-12). With a median follow up of 6 months (range 1-34), the estimated 12 months PFS and OS were 27% and 30.5%, respectively. Grade 3-4 thrombocytopenia and neutropenia were the most common toxicities (in 29 (83%) and 12 (34%) patients, respectively. Conclusions The results of this study indicate that Panobinostat might be remarkably active in some patients with R/R DLBCL, showing durable CR

In vitro characterisation and expansion of human regulatory T cells for their in vivo application in the induction of tolerance in haematopoietic stem cell and solid organ transplantation

Solid organ transplantation (SOT) is considered the treatment of choice for many end-stage organ diseases. Thus far, short term results are excellent, with patient survival rates greater than 90% one year post-surgery, but there are several problems with the long term acceptance and use of immunosuppressive drugs. Hematopoietic Stem Cells Transplantation (HSCT) concerns the infusion of haematopoietic stem cells to re-establish acquired and congenital disorders of the hematopoietic system. The main side effect is the Graft versus Host Disease (GvHD) where donor T cells can cause pathology involving the damage of host tissues. Patients undergoing acute or chronic GvHD receive immunosuppressive regimen that is responsible for several side effects. The use of immunosuppressive drugs in the setting of SOT and GvHD has markedly reduced the incidence of acute rejection and the tissue damage in GvHD however, the numerous adverse side effects observed boost the development of alternative strategies to improve the long-term outcome. To this effect, the use of CD4+CD25+FOXP3+ regulatory T cells (Treg) as a cellular therapy is an attractive approach for autoimmunity disease, GvHD and limiting immune responses to allograft after transplantation. Treg have a pivotal role in maintaining peripheral immunological tolerance, by preventing autoimmunity and chronic inflammation. Results of my thesis provide the characterization and cell processing of Tregs from healthy controls and patients in waiting list for liver transplantation, followed by the development of an efficient expansion-protocol and the investigation of the impact of the main immunosuppressive drugs on viability, proliferative capacity and function of expanded cells after expansion. The conclusion is that ex vivo expansion is necessary to infuse a high Treg dose and although many other factors in vivo can contribute to the success of Treg therapy, the infusion of Tregs during the administration of the highest dose of immunosuppressants should be carefully considered.