Modelling Down Syndrome leukaemia using transchromosomic ES cell lines.

PhDAMKL (acute megakaryoblastic leukaemia) accounts for at least 50% of all cases of acute myeloid leukaemia (AML) associated with Down Syndrome (DS). Every tenth neonate with DS develops Transient Myeloproliferative Disorder (TMD), a self-regressing neoplasia with features that closely resemble AMKL. Despite the vast majority of TMD cases self-regressing within a few weeks, approximately 30% of DS infants with TMD develop by the age of 2-4 years a more aggressive, full-blown AMKL. Both DS-TMD and DS-AMKL are associated with trisomy of human chromosome 21 (HSA21) and with acquired mutations of GATA-1 (a transcription factor essential for erythroid/megakaryocytic lineage specification) leading to the exclusive production of a short form of the protein known as GATA-1s. Additional molecular events involved in the progression from TMD to AMKL remain largely unknown. The aim of this project was to shed new light on the critical events involved in the pathogenesis of DS-TMD and AMKL utilizing an innovative in vitro model that mimics Down syndrome, a murine embryonic stem cell line carrying an extra copy of human chromosome 21 (HSA21). Using this transchromosomic ES cell system, I explored the effect of trisomy 21 (t21) on the generation of megakaryocytes in vitro, and showed that trisomic megakaryocyte precursors display increased levels of GATA-1 compared to euploid controls and exhibit the tendency of forming macroscopic colonies without overt GATA1 mutations. Furthermore, I genetically manipulated the transchromosomic ES cell system by retrovirally 4 overexpressing GATA1s and demonstrated that trisomy 21 is required for GATA-1s to exert its full hyperproliferative potential. The influence of the supernumerary HSA21 on the ontogenesis of haematopoietic stem cells (HSCs) from mesodermal precursors was also studied in the transchromosomic system. In this thesis, I present evidence that mesodermal colonies derived from transchromosomic ES cells give rise to an increased number of immature haematopoietic progenitors compared to euploid controls. I demonstrate that at least two independent genes on HSA21 contribute to this effect, and that trisomy of RUNX-1 (a master regulator of primitive haematopoiesis encoded on chromosome 21) is required for an increased haematopoietic commitment from the mesodemal precursors. This thesis shows that t21 influences haematopoiesis (in general) and the megakaryocytic lineage (in particular) at several levels and that it is responsible for an overall increase in levels of immature cells that are targets for acquisition of further leukaemogenic mutations. Finally, in this study I clarify the role of JAK3, a gene whose mutations have been reported in AMKL, in the progression from TMD to AMKL

Benefits of glucocorticoids in non-ambulant boys/men with Duchenne muscular dystrophy: A multicentric longitudinal study using the Performance of Upper Limb test

The aim of this study was to establish the possible effect of glucocorticoid treatment on upper limb function in a cohort of 91 non-ambulant DMD boys and adults of age between 11 and 26 years. All 91 were assessed using the Performance of Upper Limb test. Forty-eight were still on glucocorticoid after loss of ambulation, 25 stopped steroids at the time they lost ambulation and 18 were GC naive or had steroids while ambulant for less than a year. At baseline the total scores ranged between 0 and 74 (mean 41.20). The mean total scores were 47.92 in the glucocorticoid group, 36 in those who stopped at loss of ambulation and 30.5 in the naive group (p <0.001). The 12-month changes ranged between -20 and 4 (mean -4.4). The mean changes were -3.79 in the glucocorticoid group, -5.52 in those who stopped at loss of ambulation and -4.44 in the naive group. This was more obvious in the patients between 12 and 18 years and at shoulder and elbow levels. Our findings suggest that continuing glucocorticoids throughout teenage years and adulthood after loss of ambulation appears to have a beneficial effect on upper limb function. (C) 2015 The Authors. Published by Elsevier B.V

The potential role of advanced glycation end products in food allergy pathogenesis

prevalence has dramatically increased in the last two decades. Among dietary factors, it has been hypothesized that advanced glycation endproducts(AGEs), present at high level in junk food, could be involved in FA pathogenesis. AGEs are a heterogeneous group of compounds deriving from sugars(sweets and beverages), autoclaved/processed foods, microwaved foods, more roasted/barbecued meat. To evaluate the AGEs levels in FA children compared with healthy controls and subjects with respiratory allergy. Methods: We evaluated paediatric patients with challenge-proven FA, children with respiratory allergy(RA) and age and sex-matched healthy controls. Subcutaneous AGEs levels were evaluated through the AGE reader. Food-frequency questionnaires were evaluated in all study subjects. In vitro studies were performed on human enterocytes(Caco-2 cells) stimulated with 200 mg/ml of BSA-AGE for 24and48 hours to evaluate effects on gut barrier function: mucin2(mucus production), transpithelial electrical resistance(TEER), ZO-1, occludin expression(intestinal permeability). The direct effects elicited on peripheral blood mononuclear cells (PBMCs) after the treatment with 200 mg/ml of BSA-AGE for 48hours, 4and 7days of treatment were also evaluated. RESULTS: 115 subjects were evaluated and subdivided into 3 groups: group 1 patients with FA (n=31); group 2 patients with RA (n=18), group 3 healthy controls (n=66). The consumption of food containing AGEs was higher in subjects with FA compared to RA children and healthy controls (p<0.05). FA and RA children presented significant higher subcutaneous AGEs levels compared to healthy controls (p<0.05). Linear regression analysis confirmed a significant positive correlation between subcutaneous levels of AGEs and consumption of food containing AGEs. Human enterocytes exposed to BSA-AGE treatment showed a reduction of TEER, of Muc2 and tight junction proteins (Occludin and ZO-1). Moreover, the treatment with BSA-AGE on human PBMCs stimulates pro-inflammatory cytokines TNF-α and Th2 cytokines(IL-5 and IL-13)production , but it was unable to modulate IL-10 production. Finally, after7days of treatment with BSAAGE, we found a low percentage of proliferating CD4+T. CONCLUSIONS: Current hypotheses and models of FA do not adequately explain the dramatic increase observed in the last years

Malaria in an asylum seeker paediatric liver transplant recipient: diagnostic challenges for migrant population

Transplanted patients are particularly exposed to a major risk of infectious diseases due to prolonged immunosuppressive treatment. Over the last decade, the growing migration flows and the transplant tourism have led to increasing infections caused by geographically restricted organisms. Malaria is an unusual event in organ transplant recipients than can be acquired primarily or reactivation following immunosuppression, by transfusion of blood products or through the transplanted organ. We report a rare case of Plasmodium falciparum infection in a liver transplanted two years-old African boy who presented to one Italian Asylum Seeker Center on May 2019. We outlined hereby diagnostic challenges, possible aetiologies of post-transplantation malaria and finally we summarized potential drug interactions between immunosuppressive agents and antimalarials. This report aims to increase the attention to newly arrived migrants, carefully evaluating patients coming from tropical areas and taking into consideration also rare tropical infections not endemic in final destination countries

Timed rise from floor as a predictor of disease progression in Duchenne muscular dystrophy: An observational study

The role of timed items, and more specifically, of the time to rise from the floor, has been reported as an early prognostic factor for disease progression and loss of ambulation. The aim of our study was to investigate the possible effect of the time to rise from the floor test on the changes observed on the 6MWT over 12 months in a cohort of ambulant Duchenne boys.A total of 487 12-month data points were collected from 215 ambulant Duchenne boys. The age ranged between 5.0 and 20.0 years (mean 8.48 ±2.48 DS).The results of the time to rise from the floor at baseline ranged from 1.2 to 29.4 seconds in the boys who could perform the test. 49 patients were unable to perform the test at baseline and 87 at 12 month The 6MWT values ranged from 82 to 567 meters at baseline. 3 patients lost the ability to perform the 6mwt at 12 months. The correlation between time to rise from the floor and 6MWT at baseline was high (r = 0.6, p<0.01).Both time to rise from the floor and baseline 6MWT were relevant for predicting 6MWT changes in the group above the age of 7 years, with no interaction between the two measures, as the impact of time to rise from the floor on 6MWT change was similar in the patients below and above 350 m. Our results suggest that, time to rise from the floor can be considered an additional important prognostic factor of 12 month changes on the 6MWT and, more generally, of disease progression

The blockade of the transient receptor potential vanilloid type 1 and fatty acid amide hydrolase decreases symptoms and central sequelae in the medial prefrontal cortex of neuropathic rats

<p>Abstract</p> <p>Background</p> <p>Neuropathic pain is a chronic disease resulting from dysfunction within the "pain matrix". The basolateral amygdala (BLA) can modulate cortical functions and interactions between this structure and the medial prefrontal cortex (mPFC) are important for integrating emotionally salient information. In this study, we have investigated the involvement of the transient receptor potential vanilloid type 1 (TRPV1) and the catabolic enzyme fatty acid amide hydrolase (FAAH) in the morphofunctional changes occurring in the pre-limbic/infra-limbic (PL/IL) cortex in neuropathic rats.</p> <p>Results</p> <p>The effect of <it>N</it>-arachidonoyl-serotonin (AA-5-HT), a hybrid FAAH inhibitor and TPRV1 channel antagonist, was tested on nociceptive behaviour associated with neuropathic pain as well as on some phenotypic changes occurring on PL/IL cortex pyramidal neurons. Those neurons were identified as belonging to the BLA-mPFC pathway by electrical stimulation of the BLA followed by hind-paw pressoceptive stimulus application. Changes in their spontaneous and evoked activity were studied in sham or spared nerve injury (SNI) rats before or after repeated treatment with AA-5-HT. Consistently with the SNI-induced changes in PL/IL cortex neurons which underwent profound phenotypic reorganization, suggesting a profound imbalance between excitatory and inhibitory responses in the mPFC neurons, we found an increase in extracellular glutamate levels, as well as the up-regulation of FAAH and TRPV1 in the PL/IL cortex of SNI rats. Daily treatment with AA-5-HT restored cortical neuronal activity, normalizing the electrophysiological changes associated with the peripheral injury of the sciatic nerve. Finally, a single acute intra-PL/IL cortex microinjection of AA-5-HT transiently decreased allodynia more effectively than URB597 or I-RTX, a selective FAAH inhibitor or a TRPV1 blocker, respectively.</p> <p>Conclusion</p> <p>These data suggest a possible involvement of endovanilloids in the cortical plastic changes associated with peripheral nerve injury and indicate that therapies able to normalize endovanilloid transmission may prove useful in ameliorating the symptoms and central sequelae associated with neuropathic pain.</p

Reliability of the Performance of Upper Limb assessment in Duchenne muscular dystrophy

Abstract The Performance of Upper Limb was specifically designed to assess upper limb function in Duchenne muscular dystrophy. The aim of this study was to assess (1) a cohort of typically developing children from the age of 3 years onwards in order to identify the age when the activities assessed in the individual items are consistently achieved, and (2) a cohort of 322 Duchenne children and young adults to establish the range of findings at different ages. We collected normative data for the scale validation on 277 typically developing subjects from 3 to 25 years old. A full score was consistently achieved by the age of 5 years. In the Duchenne cohort there was early involvement of the proximal muscles and a proximal to distal progressive involvement. The scale was capable of measuring small distal movements, related to activities of daily living, even in the oldest and weakest patients. Our data suggest that the assessment can be reliably used in both ambulant and non ambulant Duchenne patients in a multicentric setting and could therefore be considered as an outcome measure for future trials

Migrants rescued on the Mediterranean Sea route: nutritional, psychological status and infectious disease control

Introduction: North Africa has become a key migratory hub where a large number of migrants attempt the journey by sea from the Libyan coastline to the south of Europe. In this humanitarian disaster scenario, the Mediterranean route has been one of the most used by illegal boats. Methodology: In this report, the state of physical and psychological health of a cluster of Eritrean migrants, escaped from Libya and rescued in the Mediterranean Sea after a shipwreck, was described by epidemiological, clinical and laboratory investigations. Results: Data suggest that despite the majority of the migrants being apparently in good health upon a syndromic surveillance approach, most of them suffered a decline in psychological status as well as severe malnutrition. The emergence of infectious diseases, related to poor living conditions during the journey, is not a rare event. Conclusion: The present report highlights the risks of failures of the syndromic medical approach in the setting of the extremely challenging migration route and underlines migrant frailties consequent to a prolonged journey and long period of detention. These stressors, which can degrade the initial health condition of traveling migrants, can lead to a premature "exhausted migrant effect" that should be carefully investigated in order to avoid the early emergence of diseases related to frailty

Loss of Function of TET2 Cooperates with Constitutively Active KIT in Murine and Human Models of Mastocytosis

Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM

High resolution and contrast 7 tesla MR brain imaging of the neonate

IntroductionUltra-high field MR imaging offers marked gains in signal-to-noise ratio, spatial resolution, and contrast which translate to improved pathological and anatomical sensitivity. These benefits are particularly relevant for the neonatal brain which is rapidly developing and sensitive to injury. However, experience of imaging neonates at 7T has been limited due to regulatory, safety, and practical considerations. We aimed to establish a program for safely acquiring high resolution and contrast brain images from neonates on a 7T system.MethodsImages were acquired from 35 neonates on 44 occasions (median age 39 + 6 postmenstrual weeks, range 33 + 4 to 52 + 6; median body weight 2.93 kg, range 1.57 to 5.3 kg) over a median time of 49 mins 30 s. Peripheral body temperature and physiological measures were recorded throughout scanning. Acquired sequences included T2 weighted (TSE), Actual Flip angle Imaging (AFI), functional MRI (BOLD EPI), susceptibility weighted imaging (SWI), and MR spectroscopy (STEAM).ResultsThere was no significant difference between temperature before and after scanning (p = 0.76) and image quality assessment compared favorably to state-of-the-art 3T acquisitions. Anatomical imaging demonstrated excellent sensitivity to structures which are typically hard to visualize at lower field strengths including the hippocampus, cerebellum, and vasculature. Images were also acquired with contrast mechanisms which are enhanced at ultra-high field including susceptibility weighted imaging, functional MRI, and MR spectroscopy.DiscussionWe demonstrate safety and feasibility of imaging vulnerable neonates at ultra-high field and highlight the untapped potential for providing important new insights into brain development and pathological processes during this critical phase of early life