New approaches in management and treatment of primary immunodeficiencies and other genetic diseases.

During my PhD program in "Human Reproduction, Development and Growth" (XXVII Cycle, years 2011-2014), I contributed to the development of new approaches for the treatment of PIDs and other genetic diseases by advanced therapies, such as gene and cellular therapy. Moreover, I participated in the clinical and biological characterization of patients with immunodeficiencies and in the implementation of new approaches for the clinical management of children affected by hematological and immunological disorders undergoing bone marrow transplantation. My other lines of research have been focused on the use of new drugs in pediatric population, characterization of severe and unusual conditions and description of the State of the Art of known patologies in childhood

Relationship between severe obesity and gut inflammation in children: what's next?

Background: Preliminary evidence suggests an association between obesity and gut inflammation. Aims: To evaluate the frequency of glucose abnormalities and their correlation with systemic and intestinal inflammation in severely obese children. Patients and Methods: Thirty-four children (25 males; median age 10.8 ± 3.4 yrs) with severe obesity (BMI >95%) were screened for diabetes with oral glucose tolerance test (OGTT), systemic inflammation with C-reactive protein (CRP) and gut inflammation with rectal nitric oxide (NO) and faecal calprotectin. Results: BMI ranged from 23 to 44 kg/m2, and BMI z-score between 2.08 e 4.93 (median 2.69 ± 0.53). Glucose abnormalities were documented in 71% of patients: type 2 diabetes in 29%, impaired fasting glucose (IFG) in 58%, and impaired glucose tolerance (IGT) in 37.5%. Thirty-one patients (91%) were hyperinsulinemic. CRP was increased in 73.5% with a correlation between BMI z-score and CRP (p 0.03). Faecal calprotectin was increased in 47% patients (mean 77 ± 68), and in 50% of children with abnormal glucose metabolism (mean 76 ± 68 ìg/g), with a correlation with increasing BMI z-score. NO was pathological in 88%, and in 87.5% of glucose impairment (mean 6.8 ± 5 ìM). Conclusions: In this study, the prevalence of glucose abnormalities in obese children is higher than in other series; furthermore, a correlation is present between markers of systemic and intestinal inflammation and glucose abnormalities

NADPH Oxidase Deficiency: A Multisystem Approach

The immune system is a complex system able to recognize a wide variety of host agents, through different biological processes. For example, controlled changes in the redox state are able to start different pathways in immune cells and are involved in the killing of microbes. The generation and release of ROS in the form of an “oxidative burst” represent the pivotal mechanism by which phagocytic cells are able to destroy pathogens. On the other hand, impaired oxidative balance is also implicated in the pathogenesis of inflammatory complications, which may affect the function of many body systems. NADPH oxidase (NOX) plays a pivotal role in the production of ROS, and the defect of its different subunits leads to the development of chronic granulomatous disease (CGD). The defect of the different NOX subunits in CGD affects different organs. In this context, this review will be focused on the description of the effect of NOX2 deficiency in different body systems. Moreover, we will also focus our attention on the novel insight in the pathogenesis of immunodeficiency and inflammation-related manifestations and on the protective role of NOX2 deficiency against the development of atherosclerosis

Risk of Seizures in Children Receiving Busulphan-Containing Regimens for Stem Cell Transplantation

Busulphan (BU) is associated with neurotoxicity and risk of seizures. Hence, seizure prophylaxis is routinely utilized during BU administration for stem cell transplantation (SCT). We collected data on the incidence of seizures among children undergoing SCT in Italy. Fourteen pediatric transplantation centers agreed to report unselected data on children receiving BU as part of the conditioning regimen for SCT between 2005 and 2012. Data on 954 pediatric transplantation procedures were collected; of them, 66% of the patients received BU orally, and the remaining 34%, i.v. All the patients received prophylaxis of seizures, according to local protocols, consisting of different schedules and drugs. A total of 13 patients (1.3%) developed seizures; of them, 3 had a history of epilepsy (or other seizure-related pre-existing condition); 3 had documented brain lesions potentially causing seizures per se; 1 had febrile seizures, 1 severe hypo-osmolality. In the remaining 5 patients, seizures were considered not explained and, thus, potentially related to BU administration. The incidence of seizures in children receiving BU-containing regimen was very low (1.3%); furthermore, most of them had at least 1deither pre-existing or concurrentdassociated risk factor for seizures. 2014 American Society for Blood and Marrow Transplantation

ALPS-Like Phenotype Caused by ADA2 Deficiency Rescued by Allogeneic Hematopoietic Stem Cell Transplantation

Adenosine deaminase 2 (ADA2) deficiency is an auto-inflammatory disease due to mutations in cat eye syndrome chromosome region candidate 1 (CECR1) gene, currently named ADA2. The disease has a wide clinical spectrum encompassing early-onset vasculopathy (targeting skin, gut and central nervous system), recurrent fever, immunodeficiency and bone marrow dysfunction. Different therapeutic options have been proposed in literature, but only steroids and anti-cytokine monoclonal antibodies (such as tumor necrosis factor inhibitor) proved to be effective. If a suitable donor is available, hematopoietic stem cell transplantation (HSCT) could be curative. Here we describe a case of ADA2 deficiency in a 4-year-old Caucasian girl. The patient was initially classified as autoimmune neutropenia and then she evolved toward an autoimmune lymphoproliferative syndrome (ALPS)-like phenotype. The diagnosis of ALPS became uncertain due to atypical clinical features and normal FAS-induced apoptosis test. She was treated with G-CSF first and subsequently with immunosuppressive drugs without improvement. Only HSCT from a 9/10 HLA-matched unrelated donor, following myeloablative conditioning, completely solved the clinical signs related to ADA2 deficiency. Early diagnosis in cases presenting with hematological manifestations, rather than classical vasculopathy, allows the patients to promptly undergo HSCT and avoid more severe evolution. Finally, in similar cases highly suspicious for genetic disease, it is desirable to obtain molecular diagnosis before performing HSCT, since it can influence the transplant procedure. However, if HSCT has to be performed without delay for clinical indication, related donors should be excluded to avoid the risk of relapse or partial benefit due to a hereditary genetic defect

Hemophagocytic inflammatory syndrome in ADA-SCID: report of two cases and literature review

Hemophagocytic inflammatory syndrome (HIS) is a rare form of secondary hemophagocytic lymphohistiocytosis caused by an impaired equilibrium between natural killer and cytotoxic T-cell activity, evolving in hypercytokinemia and multiorgan failure. In the context of inborn errors of immunity, HIS occurrence has been reported in severe combined immunodeficiency (SCID) patients, including two cases of adenosine deaminase deficient-SCID (ADA-SCID). Here we describe two additional pediatric cases of ADA-SCID patients who developed HIS. In the first case, HIS was triggered by infectious complications while the patient was on enzyme replacement therapy; the patient was treated with high-dose corticosteroids and intravenous immunoglobulins with HIS remission. However, the patient required HLA-identical sibling donor hematopoietic stem cell transplantation (HSCT) for a definitive cure of ADA-SCID, without HIS relapse up to 13 years after HSCT. The second patient presented HIS 2 years after hematopoietic stem cell gene therapy (GT), secondarily to Varicella-Zoster vaccination and despite CD4+ and CD8+ lymphocytes’ reconstitution in line with other ADA SCID patients treated with GT. The child responded to trilinear immunosuppressive therapy (corticosteroids, Cyclosporine A, Anakinra). We observed the persistence of gene-corrected cells up to 5 years post-GT, without HIS relapse. These new cases of children with HIS, together with those reported in the literature, support the hypothesis that a major dysregulation in the immune system can occur in ADA-SCID patients. Our cases show that early identification of the disease is imperative and that a variable degree of immunosuppression could be an effective treatment while allogeneic HSCT is required only in cases of refractoriness. A deeper knowledge of immunologic patterns contributing to HIS pathogenesis in ADA-SCID patients is desirable, to identify new targeted treatments and ensure patients’ long-term recovery

ALPS-Like Phenotype Caused by ADA2 Deficiency Rescued by Allogeneic Hematopoietic Stem Cell Transplantation

Adenosine deaminase 2 (ADA2) deficiency is an auto-inflammatory disease due to mutations in cat eye syndrome chromosome region candidate 1 (CECR1) gene, currently named ADA2. The disease has a wide clinical spectrum encompassing early-onset vasculopathy (targeting skin, gut and central nervous system), recurrent fever, immunodeficiency and bone marrow dysfunction. Different therapeutic options have been proposed in literature, but only steroids and anti-cytokine monoclonal antibodies (such as tumor necrosis factor inhibitor) proved to be effective. If a suitable donor is available, hematopoietic stem cell transplantation (HSCT) could be curative. Here we describe a case of ADA2 deficiency in a 4-year-old Caucasian girl. The patient was initially classified as autoimmune neutropenia and then she evolved toward an autoimmune lymphoproliferative syndrome (ALPS)-like phenotype. The diagnosis of ALPS became uncertain due to atypical clinical features and normal FAS-induced apoptosis test. She was treated with G-CSF first and subsequently with immunosuppressive drugs without improvement. Only HSCT from a 9/10 HLA-matched unrelated donor, following myeloablative conditioning, completely solved the clinical signs related to ADA2 deficiency. Early diagnosis in cases presenting with hematological manifestations, rather than classical vasculopathy, allows the patients to promptly undergo HSCT and avoid more severe evolution. Finally, in similar cases highly suspicious for genetic disease, it is desirable to obtain molecular diagnosis before performing HSCT, since it can influence the transplant procedure. However, if HSCT has to be performed without delay for clinical indication, related donors should be excluded to avoid the risk of relapse or partial benefit due to a hereditary genetic defect

Hematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.</p

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies

Background: Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders.Objective: To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes.Methods: Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: T cell defects, Humoral defects and Other PIDs. The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology.Results: The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% (n = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage.Conclusions: NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene