Adenoviral vectors infect B lymphocytes in vivo.

B cells are the antibody-producing arm of the adaptive immune system and play a critical role in controlling pathogens. Several groups have now demonstrated the feasibility of using engineered B cells as a therapy, including infectious disease control and gene therapy of serum deficiencies. These studies have largely utilized ex vivo modification of the cells. Direct in vivo engineering would be of utility to the field, particularly in infectious disease control where the infrastructure needs of ex vivo cell modification would make a broad vaccination campaign highly challenging. In this study we demonstrate that engineered adenoviral vectors are capable of efficiently transducing murine and human primary B cells both ex vivo and in vivo. We found that unmodified human adenovirus C5 was capable of infecting B cells in vivo, likely due to interactions between the virus penton base protein and integrins. We further describe vector modification with B cell-specific gene promoters and successfully restrict transgene expression to B cells, resulting in a strong reduction in gene expression from the liver, the main site of human adenovirus C5 infection in vivo.

Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing.

Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.

Gene therapy for primary immunodeficiency.

Gene therapy is now being trialled as a therapeutic option for an expanding number of conditions, based primarily on the successful treatment over the past two decades of patients with specific primary immunodeficiencies (PIDs) including severe combined immunodeficiency and Wiskott-Aldrich syndrome and metabolic conditions such as leukodystrophy. The field has evolved from the use of gammaretroviral vectors to more sophisticated lentiviral platforms that offer an improved biosafety profile alongside greater efficiency for hematopoietic stem cells gene transfer. Here we review more recent developments including licensing of gene therapies, use of gene corrected autologous T cells as an alternative strategy for some PIDs and the potential of targeted gene correction using various gene editing platforms. Given the promising results of recent clinical trials, it is likely that autologous gene therapies will become standard of care for a number of devastating diseases in the coming decade.

Older Adults' Risk Perception during the COVID-19 Pandemic in Lombardy Region of Italy: A Cross-sectional Survey.

During COVID-19 pandemic, older adults are the segment of the population at higher health risk. Given the important role the risk perception has in influencing both the behaviors and psychological well-being, it appears useful exploring this factor in this segment of the population. Despite different studies already described the factors influencing the risk perception, few focused on older adults. For this reason, we investigated risk perception in 514 people over 60 years during the lockdown. We administered a structured interview collecting socio-demographic information, sources of information used, actions undertaken to avoid contagion, and risk perception. Risk perception related to COVID-19 was significantly lower than the perceived risk associated with other threats, and it was correlated to the number of sources of information used but not to the actions undertaken. Furthermore, we found higher risk perception in who knew infected persons, and a negative correlation between the risk perception and age, with the over 75 perceiving a lower risk of getting infected compared to the younger participants. Our results should be taken as informative for future studies. Indeed, further studies on the older adults and the risk perception during emergencies are needed to better orient both communication and supporting strategies.

NEIL1 is a candidate gene associated with common variable immunodeficiency in a patient with a chromosome 15q24 deletion.

We report the first patient with an interstitial deletion of chromosome 15q24.1-q24.3 associated with common variable immunodeficiency (CVID). The 18-year old female patient's clinical and immunological phenotype was compared with 8 additional previously published patients with chr15q24 deletions. A CGH analysis estimated the deletion to be 3.767Mb in size (chr15: 74,410,916-78,178,418) and the result was confirmed using qRT-PCR. We defined an immune-related commonly deleted region (ICDR) within the chromosomal band 15q24.2, deleted in all four patients with different forms of antibody deficiencies. Mutations in the 14 genes within this ICDR were not identified in the remaining allele in our patient by WES and gene expression analyses showed haploinsufficiency of all the genes. Among these genes, we consider Nei Like DNA Glycosylase 1 (NEIL1) as a likely candidate gene due to its crucial role in B-cell activation and terminal differentiation.

Comparison of imported Plasmodium ovale curtisi and P. ovale wallikeri infections among patients in Spain, 2005-2011.

Sequencing data from Plasmodium ovale genotypes co-circulating in multiple countries support the hypothesis that P. ovale curtisi and P. ovale wallikeri are 2 separate species. We conducted a multicenter, retrospective, comparative study in Spain of 21 patients who had imported P. ovale curtisi infections and 14 who had imported P. ovale wallikeri infections confirmed by PCR and gene sequencing during June 2005-December 2011. The only significant finding was more severe thrombocytopenia among patients with P. ovale wallikeri infection than among those with P. ovale curtisi infection (p = 0.031). However, we also found nonsignificant trends showing that patients with P. ovale wallikeri infection had shorter time from arrival in Spain to onset of symptoms, lower level of albumin, higher median maximum core temperature, and more markers of hemolysis than did those with P. ovale curtisi infection. Larger, prospective studies are needed to confirm these findings.

Human skin-derived keratinocytes and fibroblasts co-cultured on 3D poly ε-caprolactone scaffold support in vitro HSC differentiation into T-lineage committed cells.

In humans, the thymus is the primary lymphoid organ able to support the development of T cells through its three-dimensional (3D) organization of the thymic stromal cells. Since a remarkable number of similarities are shared between the thymic epithelial cells (TECs) and skin-derived keratinocytes and fibroblasts, in this study we used human keratinocytes seeded with fibroblasts on the 3D poly ε-caprolactone scaffold to evaluate their ability to replace TECs in supporting T-cell differentiation from human haematopoietic stem cells (HSCs). We observed that in the multicellular biocomposite, early thymocytes expressing CD7(+)CD1a(+), peculiar markers of an initial T-cell commitment, were de novo generated. Molecular studies of genes selectively expressed during T-cell development revealed that TAL1 was down-regulated and Spi-B was up-regulated in the cell suspension, consistently with a T-cell lineage commitment. Moreover, PTCRA and RAG2 expression was detected, indicative of a recombinant activity, required for the generation of a T-cell receptor repertoire. Our results indicate that in the multicellular biocomposite, containing skin-derived elements in the absence of thymic stroma, HSCs do start differentiating toward a T-cell lineage commitment. In conclusion, the construct described in this study exerts some properties of a lymphoid organoid, suitable for future clinical applications in cell-based therapies.

Role of the common γ chain in cell cycle progression of human malignant cell lines.

The γ-chain (γc) is a transducing element shared between several cytokine receptors whose alteration causes X-linked severe combined immunodeficiency. Recently, a direct involvement of γc in self-sufficient growth in a concentration-dependent manner was described, implying a direct relationship between the amount of the molecule and its role in cell cycle progression. In this study, we evaluate whether γc expression could interfere in cell cycle progression also in malignant hematopoietic cells. Here, we first report that in the absence of γc expression, lymphoblastoid B-cell lines (BCLs) die at a higher extent than control cells. This phenomenon is caspase-3 independent and is associated to a decreased expression of the antiapoptotic Bcl-2 family members. By contrast, increased expression of γc protein directly correlates with spontaneous cell growth in several malignant hematopoietic cell lines. We, also, find that the knockdown of γc protein through short interfering RNA is able to decrease the cell proliferation rate in these malignancies. Furthermore, an increased expression of all D-type cyclins is found in proliferating neoplastic cells. In addition, a direct correlation between the amount of γc and cyclins A2 and B1 expression is found. Hence, our data demonstrate that the amount of the γc is able to influence the transcription of genes involved in cell cycle progression, thus being directly involved in the regulatory control of cell proliferation of malignant hematopoietic cells.

De novo 13q12.3-q14.11 deletion involving BRCA2 gene in a patient with developmental delay, elevated IgM levels, transient ataxia, and cerebellar hypoplasia, mimicking an A-T like phenotype.

We report on a child with a de novo deletion of approximately 12 Mb detected through array comparative genomic hybridization (CGH). The deletion involved chromosome bands 13q12.3-13q14.11 and determined the loss of ≥50 genes. A second deletion on chromosome 12p11.3p11.22 of 43-167 kb, including about 12 genes, was unlikely of clinical relevance because inherited from the asymptomatic father. The child had developmental delay, dysmorphisms, and many features reminiscent of ataxia-telangiectasia (A-T), as cerebellar ataxia, oculocutaneus telangiectasia, and recurrent upper airway infections. Atraumatic fractures of the metatarsus were noted. Moreover, this is a rare case of 13q deletion syndrome associated with peripheral blood white cells radiosensitivity to bleomycin, reminiscent of what previously reported on X-ray hypersensitivity of fibroblasts from patients with alterations of this chromosome. The immunological evaluation revealed increased IgM serum levels and a low proliferative response to mitogens, PHA, and CD3 cross-linking (CD3 XL). After 12 years of age only a mild dysmetria persisted, while the proliferative response to mitogens became normal by 9 years of age.

From murine to human nude/SCID: the thymus, T-cell development and the missing link.

Primary immunodeficiencies (PIDs) are disorders of the immune system, which lead to increased susceptibility to infections. T-cell defects, which may affect T-cell development/function, are approximately 11% of reported PIDs. The pathogenic mechanisms are related to molecular alterations not only of genes selectively expressed in hematopoietic cells but also of the stromal component of the thymus that represents the primary lymphoid organ for T-cell differentiation. With this regard, the prototype of athymic disorders due to abnormal stroma is the Nude/SCID syndrome, first described in mice in 1966. In man, the DiGeorge Syndrome (DGS) has long been considered the human prototype of a severe T-cell differentiation defect. More recently, the human equivalent of the murine Nude/SCID has been described, contributing to unravel important issues of the T-cell ontogeny in humans. Both mice and human diseases are due to alterations of the FOXN1, a developmentally regulated transcription factor selectively expressed in skin and thymic epithelia.

Social Network and Environment as Determinants of Disability and Quality of Life in Aging: Results From an Italian Study.

Background: The increase in life expectancy is leading to a worldwide increase in chronic diseases and disability, with significant concern about their management and long-term care. Investigating the aging process using a bio-psychosocial perspective is essential to understanding how to reduce disability and improve the quality of life of aging people. This study aims to explore the role of social networks and built environment as predictors of disability and quality of life in the Italian population aged over 50 years. Materials and Methods: The research protocol is composed of several tools: World Health Organization Disability Assessment Scale 2.0 (WHODAS 2.0), World Health Organization Quality of Life Assessment in Aging (WHOQOL-AGE), Social Network Index (SNI), the Courage Built Environment Self-Reported Questionnaire (CBE-SR), and collection of sociodemographic information and information on health system coverage. Results: A total of 431 people were administered the protocol, and among them, 209 were males and 222 were females, with a mean age of 70 years. The majority of the sample reported earning a middle or high school diploma, and 60.6% of the sample declared to have a good health status. The results showed that people with a good social support network have higher levels of functioning and quality of life. However, the built environment did not significantly predict either disability or quality of life. Conclusions: These results could provide elements for dialogue with institutions and policymakers. This is fundamental to develop active policies aimed at the implementation of services and systems to promote healthy aging process.

A Novel SPINK5 Gene Mutation Associated with Netherton Syndrome in an Omani Patient.

Netherton syndrome (NS) is an autosomal recessive primary immunodeficiency. It is characterised by substantial skin barrier defects and is often misdiagnosed as severe atopic dermatitis or hyper-immunoglobulin E syndrome. Although more than 80 NS-associated pathogenic mutations in the serine peptidase inhibitor kazal type 5 (SPINK5) gene have been reported worldwide, only one has been reported in the Arab population to date. We report the case of a novel association between the c.1887+1G>A mutation in the SPINK5 gene and NS in an Omani-Arab patient born in 2014 who was managed at a paediatric immunology clinic in Muscat, Oman. Accurate genetic diagnosis facilitated tailored clinical management of the index patient and enabled the provision of genetic counselling and offering of future reproductive options to the individuals related to the index patient.

BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation.

Type 1 regulatory T (Tr1) cells are subset of peripherally induced antigen-specific regulatory T cells. IL-10 signaling has been shown to be indispensable for polarization and function of Tr1 cells. However, the transcriptional machinery underlying human Tr1 cell differentiation and function is not yet elucidated. To this end, we performed RNA sequencing on ex vivo human CD49b+LAG3+ Tr1 cells. We identified the transcription factor, BHLHE40, to be highly expressed in Tr1 cells. Even though Tr1 cells characteristically produce high levels of IL-10, we found that BHLHE40 represses IL-10 and increases IFN-γ secretion in naïve CD4+ T cells. Through CRISPR/Cas9-mediated knockout, we determined that IL10 significantly increased in the sgBHLHE40-edited cells and BHLHE40 is dispensable for naïve CD4+ T cells to differentiate into Tr1 cells in vitro. Interestingly, BHLHE40 overexpression induces the surface expression of CD49b and LAG3, co-expressed surface molecules attributed to Tr1 cells, but promotes IFN-γ production. Our findings uncover a novel mechanism whereby BHLHE40 acts as a regulator of IL-10 and IFN-γ in human CD4+ T cells.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

Identification of preexisting adaptive immunity to Cas9 proteins in humans.

The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials.

Creating a Culture of Health in Planning and Implementing Innovative Strategies Addressing Non-communicable Chronic Diseases.

Ongoing demographic changes are challenging health systems worldwide especially in relation to increasing longevity and the resultant rise of non-communicable diseases (NCDs). To meet these challenges, a paradigm shift to a more proactive approach to health promotion, and maintenance is needed. This new paradigm focuses on creating and implementing an ecological model of Culture of Health. The conceptualization of the Culture of Health is defined as one where good health and well-being flourish across geographic, demographic, and social sectors; fostering healthy equitable communities where citizens have the opportunity to make choices and be co-producers of healthy lifestyles. Based on Antonovsky's Salutogenesis model which asserts that the experience of health moves along a continuum across the lifespan, we will identify the key drivers for achieving a Culture of Health. These include mindset/expectations, sense of community, and civic engagement. The present article discusses these drivers and identifies areas where policy and research actions are needed to advance positive change on population health and well-being. We highlight empirical evidence of drivers within the EU guided by the activities within the thematic Action Groups of the European Innovation Partnership on Active and Healthy Aging (EIP on AHA), focusing on Lifespan Health Promotion and Prevention of Age-Related Frailty and Disease (A3 Action Group). We will specifically focus on the effect of Culture on Health, highlighting cross-cutting drivers across domains such as innovations at the individual and community level, and in synergies with business, policy, and research entities. We will present examples of drivers for creating a Culture of Health, the barriers, the remaining gaps, and areas of future research to achieve an inclusive and sustainable asset-based community.

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency.

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)-related diseases. Three patients presented with EBV-associated Hodgkin's lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro-generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70-CD27 interactions therefore play a nonredundant role in T and B cell-mediated immunity, especially for protection against EBV and humoral immunity.

In this issue: FOX genes and the immune response.

In this issue of the journal, we host a topic focused on the roles of human Forkhead-box (FOX) gene family members, including FOXN1, FOXP genes, and FOXO1, in the immune system. In the first review of the issue, Palamaro et al. focused their attention on the pivotal role of FOXN1 in T-cell development and the clinical implications of its mutation in humans. Dr Fleskens and Dr van Boxtel detailed the role of FOXP members in immune regulation. Concerning the roles of FOXP members in immune response, Vent-Schmidt et al. focused the attention on FOXP3. Passerini et al. highlighted the current knowledge on the involvement of FOXP3 in the development and function of Treg cells. Eventually, in the last review of this special issue, Szydlowski et al. focused on the role of FOXO1 in B-cell development and differentiation, paying particular attention to the role of PI3K-AKT signaling activation in development, differentiation, function, and homeostasis of B cells. Notably, studies on the role of FOX family members in immune response could be helpful to further investigate pathogenesis of immunological disease and to develop a novel therapeutics approach for human diseases.

FOXN1 in organ development and human diseases.

FOXN1 gene belongs to the forkhead box gene family that comprises a diverse group of "winged-helix" transcription factors that have been implicated in a variety of biochemical and cellular processes, such as development, metabolism, aging and cancer. These transcription factors share the common property of being developmentally regulated and of directing tissue-specific transcription and cell-fate decisions. Foxn1 is selectively expressed in thymic and skin epithelial cells, where it acts through its molecular targets to regulate the balance between growth and differentiation. In particular, Foxn1 is required for thymic epithelial patterning and differentiation from the initial epithelial thymic anlage to a functional cortical and medullary thymic epithelial cells (TECs) meshwork necessary for the crosstalk with the lymphoid compartment. A mutation in FoxN1 generates alymphoid cystic thymic dysgenesis due to defective TECs, causing primary T-cell immunodeficiency, named Nude/SCID syndrome, and leads to a hairless "nude" phenotype in both mice and humans. This immune defect represents the first example of a Severe Combined Immunodeficiencies (SCID) phenotype not primarily related to an abnormality intrinsic of the hematopoietic cell, but rather to a peculiar alteration of the thymic epithelia cell. This review focuses on the key role of FOXN1 in cell development and its clinical implication in humans.

FOXN1: A Master Regulator Gene of Thymic Epithelial Development Program.

T cell ontogeny is a sophisticated process, which takes place within the thymus through a series of well-defined discrete stages. The process requires a proper lympho-stromal interaction. In particular, cortical and medullary thymic epithelial cells (cTECs, mTECs) drive T cell differentiation, education, and selection processes, while the thymocyte-dependent signals allow thymic epithelial cells (TECs) to maturate and provide an appropriate thymic microenvironment. Alterations in genes implicated in thymus organogenesis, including Tbx1, Pax1, Pax3, Pax9, Hoxa3, Eya1, and Six1, affect this well-orchestrated process, leading to disruption of thymic architecture. Of note, in both human and mice, the primordial TECs are yet unable to fully support T cell development and only after the transcriptional activation of the Forkhead-box n1 (FOXN1) gene in the thymic epithelium this essential function is acquired. FOXN1 is a master regulator in the TEC lineage specification in that it down-stream promotes transcription of genes, which, in turn, regulate TECs differentiation. In particular, FOXN1 mainly regulates TEC patterning in the fetal stage and TEC homeostasis in the post-natal thymus. An inborn null mutation in FOXN1 leads to Nude/severe combined immunodeficiency (SCID) phenotype in mouse, rat, and humans. In Foxn1 (-/-) nude animals, initial formation of the primordial organ is arrested and the primordium is not colonized by hematopoietic precursors, causing a severe primary T cell immunodeficiency. In humans, the Nude/SCID phenotype is characterized by congenital alopecia of the scalp, eyebrows, and eyelashes, nail dystrophy, and a severe T cell immunodeficiency, inherited as an autosomal recessive disorder. Aim of this review is to summarize all the scientific information so far available to better characterize the pivotal role of the master regulator FOXN1 transcription factor in the TEC lineage specifications and functionality.