Novel insights in the pathogenesis of congenital immunodeficiencies”

The immune system is a complex integrated network of chemical and cellular mediators that developed during evolution to defend the body from any form of chemical, traumatic or infective insult to their integrity. A proper immune response relies on the innate immunity, that is responsible for a first line of defense against aggression and the aspecific recognition of a limited repertoire of antigens, and, later, on the adaptative immunity which includes chemical and cellular mediators responsible for a more powerful and specific defensive response from any form of antigen. Alterations of any part of the immune response results in failure of host defense and, in particular, of immunodeficiency, autoimmunity and cancer predisposition. Recent evidence highlights that the skin participates in a host defenses either acting as a primary boundary for germs, as the principal site of environment–host interactions, or directly in the developmental process of the immune system. As a matter of fact, skin and skin annexa abnormalities, such as skin dryness, brittleness of hair, nail abnormalities and abnormal dentition, can be not infrequently associated with distinct forms of immunodeficiency and may be a warning sign of immunodeficiency, since both epidermal and thymic epithelium have ectodermal origin. Severe combined immunodeficiency diseases (SCIDs) represent a heterogeneous group of rare genetic syndromes responsible for severe dysfunctions of the immune system, which share similar clinical manifestations. SCID is the most severe form of inherited primary immunodeficiency (PID) and its prevalence is approximately 1:100,000 live births, with a higher prevalence in males (1). SCIDs are difficult to recognize clinically because so many different infectious scenarios can occur. Without a functional cellular and humoral immune system SCID patients are susceptible to recurrent infections such as severe bacterial, viral, or fungal infections early in life and often present with interstitial lung disease, chronic diarrhea, and failure to thrive. In 5 addition, some patients develop skin rashes, usually caused by maternal T cells transplacental engraftment during fetal life or by a wide autoreaction due to the activation of autologous T cells against skin components (2, 3). Patients affected with particular forms of PID show an increased susceptibility to cancer. In particular, a high cancer susceptibility has been reported for a rare form of PID called Ataxia Telangiectasia (A-T) whose clinical hallmark is represented by the cerebellar neurodegeneration with the loss of Purkinje cells. Recently, in a few clinical trial sit has been documented that a shortterm treatment with glucocorticoids (GCs) is able to partially rescue either the A-T neurological phenotype and lymphocytes proliferation, even though the mechanism of action has not yet been defined (4-7). Conventionally, SCIDs have been so far classified, on the basis of the involvement of different cell lines in the pathogenesis of the disease and of the subsequent different clinical immunological phenotypes related to a specific genetic defect. T cell–deficient but normal B cell (T−B+) SCID and both T cell– and B cell–deficient (T−B−) SCID, in the presence or absence of NK cells (8). This classification helps in directing molecular studies toward a certain genetic alteration, since it is representative of the stage where the blockage occurs during the differentiation process. More recently, advances in next generation DNA sequencing allowed new gene identification through whole exome or whole genome sequencing (WES, WGS) of several forms of PIDs of unknown causes making the genetic identification of immunodeficiency syndromes more efficient (9). Only in the last two years, using this technology 34 new gene defects have been identified. Most of these immunodeficiencies are rare, even though some of them occur more frequently than what previously reported, as documented by several groups (10). Based on the principle of massively parallel sequencing, NGS technology provides an advanced tool to 6 dramatically increase the speed at which DNA can be sequenced at a lower cost as compared to the traditional Sanger sequencing approach. In this context my PhD program has been focused on the study of some immunological disorders, in order to identify new scenarios in pathogenesis, diagnosis and therapeutic approaches. This thesis reports the results obtained during my PhD course in “Clinical and Experimental Medicine” (XXXI Cycle, years 2015-2018). During these years my research has been focused on the study of the following lines of research: • positive effect of oral betamethasone administration on the in vitro lymphocytes functionality in patients affected with Ataxia-Telangiectasia, and the identification of the molecular checkpoint responsible for the partial functional rescue in lymphocytes of the patients affected with this disease. • characterization of a novel immunodeficiency whose hallmarks are represented by high IgM levels, impaired B-cell homeostasis and cancer susceptibility, • autoimmune manifestations and the pathogenetic mechanism underlying autoimmunity in a specific PID

Childhood vaccinations: A pilot study on knowledge, attitudes and vaccine hesitancy in pregnant women

Background. The objective of this pilot study was to test a questionnaire aimed at assessing knowledge of and attitudes towards vaccination, as well as intention to vaccinate, among pregnant women. Methods. The questionnaire was self-administered by 49 pregnant women attending antenatal classes at three Family Centers in Rome. Results. Poor knowledge of vaccinations, inadequate attention from healthcare professionals, recurrent consultation of unreliable sources of information, and misconceptions about the side effects of vaccines, all contribute to vaccine hesitancy. Where appropriate, questionnaire sections were shown to be internally consistent. Conclusion. The questionnaire proved reliable and is suitable for further studies

Novel iodoacetamido benzoheterocyclic derivatives with potent antileukemic activity are inhibitors of STAT5 phosphorylation

Signal Transducer and Activator of Transcription 5 (STAT5) protein, a component of the STAT family of signaling proteins, is considered to be an attractive therapeutic target because of its involvement in the progression of acute myeloid leukemia. In an effort to discover potent molecules able to inhibit the phosphorylation-activation of STAT5, twenty-two compounds were synthesized and evaluated on the basis of our knowledge of the activity of 2-(3′,4′,5′-trimethoxybenzoyl)-3-iodoacetamido-6-methoxy benzo[b]furan derivative 1 as a potent STAT5 inhibitor. Most of these molecules, structurally related to compound 1, were characterized by the presence of a common 3′,4′,5′-trimethoxybenzoyl moiety at the 2-position of different benzoheterocycles such as benzo[b]furan, benzo[b]thiophene, indole and N-methylindole. Effects on biological activity of the iodoacetamido group and of different moieties (methyl and methoxy) at the C-3 to C-7 positions were examined. In the series of benzo[b]furan derivatives, moving the iodoacetylamino group from the C-4 to the C-5 or C-6 positions did not significantly affect antiproliferative activity. Compounds 4, 15, 20 and 23 blocked STAT5 signals and induced apoptosis of K562 BCR-ABL positive cells. For compound 23, the trimethoxybenzoyl moiety at the 2-position of the benzo[b]furan core was not essential for potent inhibition of STAT5 activation

Abnormal cell-clearance and accumulation of autophagic vesicles in lymphocytes from patients affected with Ataxia-Teleangiectasia

Ataxia-Teleangiectasia (A-T) is a neurodegenerative disorder due to mutations in ATM gene. ATM in the nucleus ensures DNA repair, while its role in the cytosol is still poorly clarified. Abnormal autophagy has been documented in other neurodegenerative disorders, thus we evaluated whether alteration in this process may be involved in the pathogenesis of A-T by analyzing the autophagic vesicles and the genes implicated in the different stages of autophagy. Through transmission electron microscopy (TEM) and immunofluorescence analysis we observed an accumulation of APs associated with a LC3 puncta pattern, and a reduced number of ALs. We also documented an increased expression of genes involved in AP and lysosome biogenesis and function, and a decrease of Vps18 expression, involved in their vesicular trafficking and fusion. mTORC1-controlled proteins were hyperphosphorylated in A-T, in keeping with an increased mTOR inhibitory influence of autophagy. Betamethasone is able to promote the degradation of SQSTM1, a biomarker of autophagy. Collectively, our results indicate that in cells from A-T patients, the APs maturation is active, while the fusion between APs and lysosomes is inappropriate, thus implying abnormalities in the cell-clearance process. We also documented a positive effect of Betamethasone on molecules implicated in autophagosome degradation

Epigenetics: an Opportunity to Shape Innate and Adaptive Immune Responses

Epigenetics connects genetic and environmental factors: it includes DNA methylation, histone post-translational modifications and the regulation of chromatin accessibility by non-coding RNAs, all of which control constitutive or inducible gene transcription. This plays a key role in harnessing the transcriptional programs of both innate and adaptive immune cells due to its plasticity and environmental-driven nature, piloting myeloid and lymphoid cell fate decision with no change in their genomic sequence. In particular, epigenetic marks at the site of lineage specific transcription factors and maintenance of cell type-specific epigenetic modifications, referred to as "epigenetic memory", dictate cell differentiation, cytokine production and functional capacity following repeated antigenic exposure in memory T cells. Moreover, metabolic and epigenetic reprogramming occurring during a primary innate immune response leads to enhanced responses to secondary challenges, a phenomenon known as "trained immunity". Here we discuss how stable and dynamic epigenetic states control immune cell identity and plasticity in physiological and pathological conditions. Dissecting the regulatory circuits of cell fate determination and maintenance is of paramount importance for understanding the delicate balance between immune cell activation and tolerance, in healthy conditions and in autoimmune diseases. This article is protected by copyright. All rights reserved

Unbalanced Immune System: Immunodeficiencies and Autoimmunity

Increased risk of developing autoimmune manifestations has been identified in different primary immunodeficiencies (PIDs). In such conditions, autoimmunity and immune deficiency represent intertwined phenomena that reflect inadequate immune function. Autoimmunity in PIDs may be caused by different mechanisms, including defects of tolerance to self-antigens and persistent stimulation as a result of the inability to eradicate antigens. This general immune dysregulation leads to compensatory and exaggerated chronic inflammatory responses that lead to tissue damage and autoimmunity. Each PID may be characterized by distinct, peculiar autoimmune manifestations. Moreover, different pathogenetic mechanisms may underlie autoimmunity in PID. In this review, the main autoimmune manifestations observed in different PID, including humoral immunodeficiencies, combined immunodeficiencies, and syndromes with immunodeficiencies, are summarized. When possible, the pathogenetic mechanism underlying autoimmunity in a specific PID has been explained

CD4+ T Cell Defects in a Mulibrey Patient With Specific TRIM37 Mutations

Mulibrey (muscle-liver-brain-eye) syndrome (MUL) is an autosomal recessive disorder caused by mutations in the TRIpartite motif (TRIM)37 gene, encoding for TRIM37 a member of the TRIM E3 ubiquitin ligase protein family. MUL patients are characterized by growth retardation, dysmorphic features, and a wide range of abnormalities affecting different organs. However, T-cell abnormalities have not been observed in MUL subjects, to date. Here we described the immunological features of a MUL child carrying recently identified TRIM37 mutations, a 17q22 deletion of maternal origin combined with a TRIM37 variant of paternal origin. Here we found quantitative and functional defects in CD4+ T cells fromthisMUL case. Low levels of TRIM37 protein were specifically detected in CD4+ T cells ofMUL patient and associated with their altered proliferation and cytokine production. Of note, both CD4+ and CD8+ T lymphocytes of MUL child displayed an effector memory phenotype compared with healthy children. This clinical case research highlighted the possible role of TRIM37 in the control of immune cell number and function, especially in CD4+ T cells. Finally, this study may contribute to the novel mechanistic studies aim of identifying, in depth, the role of the TRIM37 protein in the immune system

In Ataxia-Telangiectasia, Oral Betamethasone Administration Ameliorates Lymphocytes Functionality through Modulation of the IL-7/IL-7Rα Axis Paralleling the Neurological Behavior: A Comparative Report of Two Cases

Ataxia-Telangiectasia (A-T) is characterized by cerebellar neurodegeneration and immunodeficiency. Recent studies suggest that very low glucocorticoids (GCs) doses may help improve A-T neurological phenotype in some patients. Interestingly, in GCs studies an unexpected improvement of lymphocytes proliferation in some A-T patients has been observed. GCs are able to upregulate IL-7 Rα expression and rescue it from the recycling. In this study, we compared several immunological functions, including PBMC proliferative responses, cell activation events and IL-7/IL-7 Rα axis functionality, with the neurological behavior during an in-vivo GCs treatment between the most Responder patient to GC and the Non-Responder at all. During in-vivo GC treatment, we observed an increase of lymphocyte proliferation upon stimulation with PHA or IL-7 only in the Responder. This finding paralleled the increase in the surface expression of IL-7 R and up-regulation of the CD69 T-cell activation marker. Internalization and recycling of IL-7 R occurred properly only in the Responder. Microarray analysis revealed a remarkable difference in the DE-genes levels among Responder and Non-Responder, mostly concerning miRNAs and Multiple Complex families. Our findings suggest that the improvement of lymphocyte functionality, which correlates to the neurological behavior, is mediated through an effect of GCs on the IL-7/IL-7 Rα axis