Indolent lymphoma: the pathologist's viewpoint

Indolent lymphomas have recently been the object of numerous studies, which have focused on new aspects relevant both for the better comprehension of their histogenesis and the identification of new therapeutic strategies. As marginal-zone lymphoma (MZL) represents the category of indolent lymphomas that has obtained more benefit from such an approach, the authors focused on the most recent achievements and not yet solved controversies in this area. In spite of their postulated common derivation, the three categories of MZL of the WHO Classification appear dissimilar. In fact, they show significant molecular differences among them as well as a certain heterogeneity within each group. By no means, there is a cogent need of more refined tools to revise these neoplasms and to produce a more rational grouping. The recent identification of the IRTA gene family corresponding to IG-like receptors differentially expressed in B-cells might contribute to their better understandin

Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development

Machine learning approaches for predicting cardiovascular deconditioning induced by prolonged microgravity exposure

LAUREA MAGISTRALEIl decondizionamento cardiovascolare (CV) è una delle principali conseguenze dei voli spaziali e consiste in cambiamenti nella funzione e nella struttura del cuore e dei vasi sanguigni in risposta all’esposizione prolungata alla microgravità a cui il corpo umano è sottoposto. Ciò si traduce, con una marcata variabilità interindividuale, in ridotta capacità di esercizio e intolleranza ortostatica (OI), quantificate rispettivamente tramite massima potenza aerobica (VO2max [ml/kg/min]) e durata dell’Head-Up Tilt (HUT) test [min], che possono influenzare la capacità degli astronauti di svolgere compiti legati alla missione o costituire un problema quando vengono riesposti a un campo di gravità non terrestre. Poiché è stata dimostrata una relazione tra il decondizionamento CV indotto dalla microgravità e variazioni dell’attività e della variabilità elettrica cardiaca, in questa tesi è stato ipotizzato che informazioni ottenibili da una registrazione Holter ECG di 24 ore, facilmente ottenibile durante i voli spaziali, potrebbero essere predittive degli indicatori chiave del decondizionamento CV, permettendone un frequente monitoraggio. Utilizzando dati relativi a 87 soggetti partecipanti in sei campagne di riposo a letto Head-Down Tilt di diversa durata (cioè il principale analogo terrestre di microgravità), l'ipotesi di ricerca è stata testata sviluppando modelli di Machine Learning basati su variabili predittive calcolate da serie battito-battito e serie temporali estratte dal tracciato ECG di 24 ore, con l’obiettivo di predire il livello di decondizionamento CV in termini di VO2max e di durata dell’HUT test, misurati il giorno successivo all’acquisizione ECG. Variabili di breve e lungo termine del dominio del tempo, della frequenza, e non-lineari sono state calcolate da ogni tracciato ECG e impiegate in due strategie predittive: una basata su una singola registrazione ECG, senza quindi necessità di eseguire nessun test di esercizio massimale o di stimolazione ortostatica, e una specificamente progettata per applicazioni aerospaziali in cui sono stati considerati anche i risultati pre-volo (cioè la condizione base dell'astronauta). I risultati dei diversi modelli predittivi proposti hanno dimostrato la praticabilità dell'approccio predittivo ipotizzato, con una capacità di distinzione dei soggetti in diverse fasce di rischio di OI (circa 80% di accuratezza), ottenute tramite classificazione a posteriori delle predizioni, e una stima dei valori di VO2max (RMSE < 5 ml/kg/min) confrontabili con risultati di studi precedenti e potenzialmente utilizzabili per valutare la fattibilità delle missioni per i singoli astronauti e correggere le contromisure applicate durante il volo.Cardiovascular (CV) deconditioning is a major consequence of spaceflight and consists in changes in function and structure of heart and blood vessels in response to prolonged microgravity exposure to which the human body is subjected. This translates, with marked inter-individual variability, in reduced exercise capacity and orthostatic intolerance (OI), quantified by maximum aerobic power (VO2max [ml/kg/min]) and Head-Up Tilt (HUT) test duration [min], which may affect astronauts' ability to perform mission-related tasks or constitute a concern when re-exposed to a non-terrestrial gravity field. The driving hypothesis of this thesis was that, since a relationship was demonstrated between microgravity-induced CV deconditioning and changes in cardiac electrical activity and variability, information obtainable from a 24-hour Holter ECG recording, easily acquirable during spaceflight, might be predictive of key indicators of CV deconditioning, allowing for its frequent monitoring. Based on data from 87 subjects participating in six campaigns of Head-Down Tilt bed rest with different duration (i.e., the main ground-based microgravity analogue), the research hypothesis was tested by developing Machine Learning models based on predictive features calculated from beat-to-beat and time series extracted from the 24-hour ECG trace, aimed at predicting the level of CV deconditioning in terms of VO2max and HUT test duration, measured the day following the ECG acquisition. Short- and long-term time-domain, frequency-domain, and non-linear features were calculated from each ECG and used in two predictive strategies: one based on a single ECG recording, thus without the need to perform any maximal exercise or HUT tests, and one specifically designed for aerospace applications in which the pre-spaceflight results (i.e., baseline condition of the astronaut) were also considered. Results of the different proposed predictive models demonstrated the feasibility of the hypothesized prediction approach, with an ability to distinguish subjects into different risk intervals of OI (around 80% accuracy), obtained by a-posteriori classification of predictions, and an estimation of VO2max values (RMSE < 5 ml/kg/min) comparable with results of previous studies and that could potentially be used to assess mission feasibility for individual astronauts and to correct in-flight countermeasures

Modelling Electrostatic Interactions and Solvation in Chromatin: from the single nucleosome towards the chromatin fibre

Chromatin is a complex of proteins and DNA found in the nuclei of eukaryotic cells. It reinforces the DNA and its topology tunes DNA transcription and gene expression. It is formed by nucleosomes, structures composed of an octameric protein core and approximately 147 base pairs of DNA. Chromatin is an extremely complex system, the behaviour of which is ruled by both mechanical and electrostatic factors that are depend on its structure, and biomolecular interactions occurring in the cell nucleus. In this thesis, I analyse chromatin compaction from an electrostatic perspective and focus on the role of electrostatics and solvation as determinants of the topology of chromatin. I examine the effect of the histone tails and propose a methodology to connect electrostatic calculations to the structural and functional features of protein-DNA systems. This methodology can also be combined with coarse-grained representations. I study the electrostatic forces acting on the phosphate atoms of the DNA backbone. I investigate the electrostatic origins of effects such as different stages in DNA unwrapping, nucleosome destabilisation upon histone tail truncation, and the role of specific arginines and lysines undergoing Post - Translational Modifications. I find that the positioning of the histone tails can oppose the attractive pull of the histone core, locally deform the DNA, and tune DNA unwrapping. I conduct an analysis of the porosity of nucleosomes and related to the importance of solvation phenomena. I complement and support my computational findings on nucleosome electrostatic interactions experimental Zeta Potential and Dynamic Light Scattering measurements on single nucleosomes under varying ionic concentrations, providing information on the surface charge and the size of nucleosomes. I present a comprehensive study of the electrostatic interactions between nucleosome pairs sampling different translations and rotations. My analysis aims to provide a cohesive description of nucleosome electrostatic interactions in the chromatin fibre, combining information on the energetics of different relative positions of nucleosomes, especially in very tight packing situations. In addition to numerical estimates of electrostatic interaction energy of nucleosomes at different relative distances and orientations, obtained within the Poisson-Boltzmann framework, I present their approximation by analytical asymptotic expressions, where nucleosomes are approximated as monopoles and dipoles centred in dielectric spheres immersed in an electrolytic solution. I am able to identify a non-linearity region around the nucleosomes, and to exploit the fact that that in points outside that region the electrostatic potential can be described by the linearised Poisson-Boltzmann Equation

Variations in “rescuability” of immunoglobulin molecules from different forms of human lymphoma: implications for anti-idiotype vaccine development

Idiotypic (Id) vaccination has shown promising results in patients with follicular lymphoma (FL). However, it still remains unclear whether the same approach might be suitable for the treatment of other B-cell malignancies. For this reason, we recently performed an interim analysis of patients proposed to receive this treatment at our center. The feasibility of employing idiotype vaccines was evaluated for five different B-cell malignancies in their first relapse, both in terms of induction and fusion, as well as overall treatment. Our data suggest that, unlike follicular lymphoma (87%), this approach is not feasible to treat other B-cell malignancies (0–20%) such as mantle cell, small lymphocytic, diffuse large cell and Burkitt’s lymphoma (P < 0.01). The main difficulties encountered were technical problems related to the survival of idiotype-producing hybridomas (83%) and the early loss of idiotype production by growing hybridomas (17%). However, it remains possible that an idiotype vaccine might still be produced through molecular means for most, if not all cases of relapsing B-cell malignancies

Indolent lymphoma: the pathologist's viewpoint

Abstract Indolent lymphomas have recently been the object of numerous studies, which have focused on new aspects relevant both for the better comprehension of their histogenesis and the identification of new therapeutic strategies. As marginal-zone lymphoma (MZL) represents the category of indolent lymphomas that has obtained more benefit from such an approach, the authors focused on the most recent achievements and not yet solved controversies in this area. In spite of their postulated common derivation, the three categories of MZL of the WHO Classification appear dissimilar. In fact, they show significant molecular differences among them as well as a certain heterogeneity within each group. By no means, there is a cogent need of more refined tools to revise these neoplasms and to produce a more rational grouping. The recent identification of the IRTA gene family corresponding to IG-like receptors differentially expressed in B-cells might contribute to their better understanding

A synthetic biology approach for consistent production of plant-made recombinant polyclonal antibodies against snake venom toxins

Antivenoms developed from the plasma of hyperimmunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost-effective antivenom production based on plant-made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin-binding genetic information captured from peripheral blood lymphocytes of hyperimmunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant-made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice.Fil: Julve Parreño, Jose Manuel. Universidad Politécnica de Valencia; EspañaFil: Huet, Estefanía. Universidad Politécnica de Valencia; EspañaFil: Fernández del Carmen, Asun. Universidad Politécnica de Valencia; EspañaFil: Segura, Alvaro. Universidad de Costa Rica; Costa RicaFil: Venturi, Micol. Universidad Politécnica de Valencia; EspañaFil: Gandía, Antoni. Universidad Politécnica de Valencia; EspañaFil: Pan, Wei-Song. Universidad Politécnica de Valencia; EspañaFil: Albaladejo, Irene. Universidad Politécnica de Valencia; EspañaFil: Forment, Javier. Universidad Politécnica de Valencia; EspañaFil: Pla, Davinia. Instituto de Biomedicina de Valencia; EspañaFil: Wigdorovitz, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; ArgentinaFil: Calvete, Juan J.. Instituto de Biomedicina de Valencia; EspañaFil: Gutiérrez, Carlos. Universidad de Las Palmas de Gran Canaria; EspañaFil: Gutiérrez, José María. Universidad de Costa Rica; Costa RicaFil: Granell, Antonio. Universidad Politécnica de Valencia; EspañaFil: Orzáez, Diego. Universidad Politécnica de Valencia; Españ

Clinical implications of antigen transfer mechanisms from malignant to dendritic cells: Exploiting cross-priming

Expansion and activation of cytolytic T lymphocytes bearing high-affinity T-cell receptors specific for tumor antigens is a major goal of active cancer immunotherapy. Physiologically, T cells receive promitotic and activating signals from endogenous professional antigen-presenting cells (APC) rather than directly from malignant cells. This phenomenon fits with the broader concept of cross-presentation that earlier was demonstrated for minor histocompatibility and viral antigens. Many mechanisms have been found to be capable of transferring antigenic material from malignant cells to APC so that it can be processed and subsequently presented by MHC class I molecules expressed on APC. Dendritic cells (DC) are believed to be the most relevant APC mediating cross-presentation because they can take up antigens from apoptotic, necrotic, and even intact tumor cells. There exist specific molecular mechanisms that ensure this transfer of antigenic material: 1) opsonization of apoptotic bodies; 2) receptors for released heat shock proteins carrying peptides processed intracellularly; 3) Fc receptors that uptake immunocomplexes and immunoglobulins; and 4) pinocytosis. DC have the peculiar capability of reentering the exogenously captured material into the MHC class I pathway. Exploitation of these pieces of knowledge is achieved by providing DC with complex mixtures of tumor antigens ex vivo and by agents and procedures that promote infiltration of malignant tissue by DC. The final outcome of DC cross-presentation could be T-cell activation (cross-priming) but also, and importantly, T-cell tolerance contingent upon the activation/maturation status of DC. Artificial enhancement of tumor antigen cross-presentation and control of the immune-promoting status of the antigen-presenting DC will have important therapeutic implications in the near future

Potentiation of therapeutic immune responses against malignancies with monoclonal antibodies

Immunotherapeutic monoclonal antibodies (mAbs) can be defined as those that exert their functions by tampering with immune system cell molecules, causing an enhancement of antitumor immune responses. Some of these antibodies are agonistic ligands for surface receptors involved in the activation of lymphocytes and/or antigen-presenting cells, whereas others are antagonists of mechanisms that normally limit the intensity of immune reactions. Several mAbs of this category have been described to display in vivo antitumor activity in mouse models. Only anti–CTLA-4 (CD152) mAb has entered clinical trials, but the preclinical effects described for anti- CD40, anti-CD137 (4-1BB), anti-CD102 (intercellular adhesion molecule-2), and regulatory T cell-depleting mAbs should lead to their prompt clinical development. Their use in combination with immunizations against tumor antigens has been reported to be endowed with synergistic properties. This new group of antitumor agents holds promise for at least additive effects with conventional therapies of cancer and deserves intensive translational research