Patients with STAT1 Gain-of-function Mutations Display Increased Apoptosis which is Reversed by the JAK Inhibitor Ruxolitinib.

INTRODUCTION: The signal transducer and activator of transcription (STAT1) gain-of-function (GOF) syndrome accounts for most cases of chronic mucocutaneous candidiasis but is characterized by a broader clinical phenotype that may include bacterial, viral, or invasive fungal infections, autoimmunity, autoinflammatory manifestations, vascular complications, or malignancies. The severity of lymphopenia may vary and influence the infectious morbidity. METHODS: In our cohort of seven STAT1-GOF patients, we investigated the mechanisms that may determine T lymphopenia, we characterized the interferon gene signature (IGS) and analyzed the effect of ruxolitinib in reverting the immune dysregulation. RESULTS: STAT1-GOF patients exhibited increased T lymphocyte apoptosis that was significantly augmented in both resting conditions and following stimulation with mitogens and IFNα, as evaluated by flow cytometry by Annexin V/ Propidium iodide assay. The JAK inhibitor ruxolitinib significantly reduced the IFNα-induced hyperphosphorylation of STAT1 and reverted the stimulation-induced T-cell apoptosis, in vitro. In two adult STAT1-GOF patients, the JAKinib treatment ameliorated chronic mucocutaneous candidiasis and lymphopenia. Most STAT1-GOF patients, particularly those who had autoimmunity, presented increased IGS that significantly decreased in the two patients during ruxolitinib treatment. CONCLUSION: In STAT1-GOF patients, T lymphocyte apoptosis is increased, and T lymphopenia may determine higher risk of severe infections. The JAKinib target therapy should be evaluated to treat severe chronic candidiasis and lymphopenia, and to downregulate the IFNs in patients with autoinflammatory or autoimmune manifestations.

Treatment response to Janus kinase inhibitor in a child affected by Aicardi-Goutières syndrome.

Key Clinical Message: Baricitinib, a Janus kinase inhibitor (JAK-inhibitor), seems to contribute to an improvement of a child affected by Aicardi-Goutières syndrome (AGS), reducing the interferon score and determining a recovery of cognitive, communicative, and relational dysfunctions, while the gross motor deficit persisted. Abstract: We report the treatment response to baricitinib, a JAK-inhibitor, in a 4-year-old girl affected by Aicardi-Goutières syndrome (AGS2, RNASEH2B mutation). Using quantitative measures, we detected a significant amelioration characterized by a complete recovery of cognitive, communicative, and relational skills after 8 and 16 months from the beginning of therapy.

Aerosol Jet® Printing of Poly(3,4-Ethylenedioxythiophene): Poly(Styrenesulfonate) onto Micropatterned Substrates for Neural Cells In Vitro Stimulation.

In neural tissue engineering (NTE), topographical, electrical, mechanical and/or biochemical stimulations are established methods to regulate neural cell activities in in vitro cultures. Aerosol Jet® Printing is here proposed as enabling technology to develop NTE integrated devices for electrically combined stimulations. The printability of a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT: PSS) commercial ink onto a reference substrate was firstly investigated and the effect of the process parameters on the quality of printed lines was analyzed. The study was then extended for printing thick electrodes and interconnects; the print strategy was finally transferred to a silicon-based wafer with patterned microchannels of proven cellular adhesion and topographical guidance. The results showed values of electrical resistance equal to ~16 Ω for printed electrodes which are ~33 µm thick and ~2 mm wide. The electrical impedance of the final circuit in saline solution was detected in the range of 1 - 2 kΩ at 1 kHz, which is in line with the expectations for bioelectronic neural interfaces. However, cells viability assays on the commercial PEDOT: PSS ink demonstrated a dose dependent cytotoxic behavior. The potential cause is associated with the presence of a cytotoxic co-solvent in the ink's formulation, which is released in the medium culture, even after a post-sintering process on the printed electrodes. This work is a first step to develop innovative in vitro NTE devices via a printed electronic approach. It also sheds new insights the transfer of AJ® print strategies across different substrates, and biocompatibility of commercial PEDOT: PSS inks.

Case Report: The JAK-Inhibitor Ruxolitinib Use in Aicardi-Goutieres Syndrome Due to ADAR1 Mutation.

Type I Interferonopathies comprise inherited inflammatory diseases associated with perturbation of the type I IFN response. Use of Janus kinase (JAK) inhibitors has been recently reported as possible tools for treating some of those rare diseases. We describe herein the clinical picture and treatment response to the JAK-inhibitor ruxolitinib in a 5-year-old girl affected by Aicardi-Goutières Syndrome type 6 (AGS6) due to ADAR1 mutation. The girl's interferon score (IS) was compared with that of her older brother, suffering from the same disorder, who was not treated. We observed a limited, but distinct neurological improvement (Gross Motor Function and Griffiths Mental Development Scales). Analysis of IS values of the two siblings during the treatment showed several changes, especially related to infections; the IS values of the child treated with ruxolitinib were consistently lower than those measured in her brother. Based on these observations we suggest that the use of ruxolitinib in children with the same condition might be effective in inhibiting type I interferon response and that starting this therapy at early age in children with AGS could mitigate the detrimental effects of type I interferon hyperproduction.

Establishment of three Joubert syndrome-derived induced pluripotent stem cell (iPSC) lines harbouring compound heterozygous mutations in CC2D2A gene.

We have developed Joubert syndrome (JS)-derived induced pluripotent stem cell (iPSC) lines from dermal fibroblasts biopsied from a female patient harbouring novel compound heterozygous mutations in CC2D2A gene. The newly established iPSC lines provide tremendous promises for development of JS-derived neuronal cell lines to uncover the molecular and cellular mechanisms underlying the pathogenesis of JS and to develop therapeutic interventions for treatment of JS.

Selective Laser Melting and Electron Beam Melting of Ti6Al4V for Orthopedic Applications: A Comparative Study on the Applied Building Direction.

The 3D printing process offers several advantages to the medical industry by producing complex and bespoke devices that accurately reproduce customized patient geometries. Despite the recent developments that strongly enhanced the dominance of additive manufacturing (AM) techniques over conventional methods, processes need to be continually optimized and controlled to obtain implants that can fulfill all the requirements of the surgical procedure and the anatomical district of interest. The best outcomes of an implant derive from optimal compromise and balance between a good interaction with the surrounding tissue through cell attachment and reduced inflammatory response mainly caused by a weak interface with the native tissue or bacteria colonization of the implant surface. For these reasons, the chemical, morphological, and mechanical properties of a device need to be designed in order to assure the best performances considering the in vivo environment components. In particular, complex 3D geometries can be produced with high dimensional accuracy but inadequate surface properties due to the layer manufacturing process that always entails the use of post-processing techniques to improve the surface quality, increasing the lead times of the whole process despite the reduction of the supply chain. The goal of this work was to provide a comparison between Ti6Al4V samples fabricated by selective laser melting (SLM) and electron beam melting (EBM) with different building directions in relation to the building plate. The results highlighted the influence of the process technique on osteoblast attachment and mineralization compared with the building orientation that showed a limited effect in promoting a proper osseointegration over a long-term period.

Generation of induced pluripotent stem cell (iPSC) lines from a Joubert syndrome patient with compound heterozygous mutations in C5orf42 gene.

We have generated new disease-specific induced pluripotent stem cell (iPSC) lines from skin fibroblasts obtained from a female patient with Joubert syndrome (JS) caused by compound heterozygous mutations in C5orf42 gene. The generated iPSCs offer an unprecedented opportunity to obtain iPSC-derived neurons to investigate the pathogenesis of JS in vitro and to develop therapeutic strategies.

Biomarkers and Precision Therapy for Primary Immunodeficiencies: An In Vitro Study Based on Induced Pluripotent Stem Cells From Patients.

Ataxia telangiectasia (AT) and Aicardi-Goutières syndrome (AGS) are inherited disorders of immunity with prevalent neurological phenotype. Available treatments are only partially effective, and the prognosis is poor. Induced pluripotent stem cells (iPSCs) are obtained by reprogramming patient somatic cells, preserving the donor individual genetic heritage and creating patient-specific disease models, useful to investigate pathogenesis and drug effects and to develop precision therapies. The aim is to investigate the cytotoxicity of a panel of immunomodulators using iPSCs of patients with AT or different forms of AGS (AGS1, AGS2, and AGS7). iPSCs were obtained by reprogramming AT and AGS patients' cells and, as a control, the BJ normal human fibroblast line, using Sendai virus. Cytotoxic effects of two drugs proposed to treat respectively AT and AGS (dexamethasone and mepacrine) were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 72 hours' exposure. Data were obtained also for other immunomodulatory drugs (thioguanine, mercaptopurine, thalidomide, and lenalidomide). Relative expression of genes involved in the tested drug pathways was analyzed. AGS7-derived iPSCs displayed altered viability when treated with a low dose of mepacrine and higher expression of cyclic guanosine monophosphate-adenosine monophosphate synthase, which is the main target for mepacrine action. AGS7-derived iPSCs were also more sensitive to thioguanine, while AGS2 and AT iPSCs were less sensitive to this medication than the BJ-iPSC. All iPSCs were equally sensitive to mercaptopurine and resistant to dexamethasone, thalidomide, and lenalidomide. This work establishes an innovative in vitro model that is useful to investigate the mechanisms of drugs potentially effective in AT and AGS.

Nebulized jet-based printing of bio-electrical scaffolds for neural tissue engineering: a feasibility study.

In this paper we investigate the application of a direct writing technique for printing conductive patterns onto a biocompatible electrospun-pyrolysed carbon-fibre-based substrate. The result is a first study towards the production of bio-electrical scaffolds that could be used to enhance the promotion of efficient connections among neurons for in vitro studies in the field of neural tissue engineering. An electrospinning process is employed for production of the materials derived from the precursor polyacrylonitrile, in which the embedding of carbon nanotubes (CNTs) is also investigated. Subsequently, the methodology of research into suitable parameters for the printed electronics, using a commercial silver nanoparticle (Øavg,particle size âˆ¼ 100 nm) ink, is described. The results show values of 2 Ω cm for the resistivity of the carbon-fibre materials and conductive printed lines of resistance ∼50 Ω on glass and less than ∼140 Ω on carbon-fibre samples. Biocompatibility results demonstrate the possibility of using electrospun-pyrolysed mats, also with embedded CNTs, as potential neural substrates for spatially localized electrical stimulation across a tissue. In addition, the data concerning the potential toxicity of silver suspensions are in accordance with the literature, showing a dose-dependent behaviour. This work is a pioneering feasibility study of the use of the flexible and versatile printed electronic approach, combined with engineered biocompatible substrates, to realize integrated bio-electrical scaffolds for in vitro neural tissue engineering applications.

Generation of 3 clones of induced pluripotent stem cells (iPSCs) from a patient affected by Autosomal Recessive Osteopetrosis due to mutations in TCIRG1 gene.

Autosomal recessive osteopetrosis (ARO) is a rare inherited disorder leading to increased bone density with impairment in bone resorption. Among the genes responsible for ARO, the TCIRG1 gene, coding for the a3 subunit of the osteoclast proton pump, is mutated in more than 50% of the cases, increasing the importance of TCIRG1-iPSCs as disease model. We generated 3 iPSC clones derived from Peripheral Blood Mononuclear Cells (PBMCs) of a patient carrying the heterozygous mutations p.Y512X and c.2236 + 1G > A. A Sendai virus-based vector was used and the iPSCs were characterized for genetic identity to parental cells, genomic integrity, pluripotency, and differentiation ability.

Generation of three isogenic induced Pluripotent Stem Cell lines (iPSCs) from fibroblasts of a patient with Aicardi Goutières Syndrome carrying a c.2471G>A dominant mutation in IFIH1 gene.

Aicardi-Goutières syndrome (AGS) is an early-onset monogenic encephalopathy characterized by intracranial calcification, leukodystrophy and cerebrospinal fluid lymphocytosis. To date, seven genes have been related to AGS. Among these, IFIH1 encodes for MDA5, a cytosolic double-stranded RNA receptor, and is responsible for AGS type 7. We generated three isogenic iPSC clones, using a Sendai virus-based vector, starting from fibroblasts of a patient carrying a dominant mutation in IFIH1. All lines were characterized for genomic integrity, genetic uniqueness, pluripotency, and differentiation capability. Our clones might offer a good model to investigate AGS7 pathophysiological mechanism and to discover new biomarkers for this condition treatment.

Establishment of three iPSC lines from fibroblasts of a patient with Aicardi Goutières syndrome mutated in RNaseH2B.

We report the generation of three isogenic iPSC clones (UNIBSi007-A, UNIBSi007-B, and UNIBSi007-C) obtained from fibroblasts of a patient with Aicardi Goutières Syndrome (AGS) carrying a homozygous mutation in RNaseH2B. Cells were transduced using a Sendai virus based system, delivering the human OCT4, SOX2, c-MYC and KLF4 transcription factors. The resulting transgene-free iPSC lines retained the disease-causing DNA mutation, showed normal karyotype, expressed pluripotent markers and could differentiate in vitro toward cells of the three embryonic germ layers.

Generation of three iPSC lines from fibroblasts of a patient with Aicardi Goutières Syndrome mutated in TREX1.

Fibroblasts from a patient with Aicardi Goutières Syndrome (AGS) carrying a compound heterozygous mutation in TREX1, were reprogrammed into induced pluripotent stem cells (iPSCs) to establish isogenic clonal stem cell lines: UNIBSi006-A, UNIBSi006-B, and UNIBSi006-C. Cells were transduced using the episomal Sendai viral vectors, containing human OCT4, SOX2, c-MYC and KLF4 transcription factors. The transgene-free iPSC lines showed normal karyotype, expressed pluripotent markers and displayed in vitro differentiation potential toward cells of the three embryonic germ layers.

Generation of 3 clones of induced pluripotent stem cells (iPSCs) from a patient affected by Crohn's disease.

Crohn's disease is a debilitating and incurable chronic inflammatory bowel disease, affecting millions of individuals worldwide, with an increasing frequency. Surgery must be applicable in half of the cases often with a disabling course, and pharmacological treatments may have adverse complications. We generated three isogenic clones of iPSCs from peripheral blood mononuclear cells (PBMCs) of a patient with Crohn's Disease under pharmacological treatment without adverse effects. Sendai virus based vector was used and the iPSCs were characterized for genetic uniqueness, genomic integrity, pluripotency, and differentiation ability. These iPSCs will be a powerful tool to develop tailored therapies.

Generation of induced pluripotent stem cells (iPSCs) from patient with Cri du Chat Syndrome.

The Cri du Chat Syndrome (CdCS) is a genetic disease resulting from variable size deletion occurring on the short arm of chromosome 5. The main clinical features are a high-pitched monochromatic cry, microcephaly, severe psychomotor and mental retardation with characteristics of autism spectrum disorders such as hand flapping, obsessive attachments to objects, twirling objects, repetitive movements, and rocking. We reprogrammed to pluripotency peripheral blood mononuclear cells derived from a patient carrying large deletion on the short arm of chromosome 5, using a commercially available non-integrating expression system. The iPSCs expressed pluripotency markers and differentiated in the three embryonic germ layers.

Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations.

BACKGROUND: Gain-of-function (GOF) mutations affecting the coiled-coil domain or the DNA-binding domain of signal transducer and activator of transcription 1 (STAT1) cause chronic mucocutaneous candidiasis disease. This condition is characterized by fungal and bacterial infections caused by impaired generation of TH17 cells; meanwhile, some patients with chronic mucocutaneous candidiasis disease might also have viral or intracellular pathogen infections. OBJECTIVE: We sought to investigate the effect of STAT1 GOF mutations on the functioning of natural killer (NK) cells. METHODS: Because STAT1 is involved in the signaling response to several cytokines, we studied NK cell functional activities and STAT1 signaling in 8 patients with STAT1 GOF mutations. RESULTS: Functional analysis of NK cells shows a significant impairment of cytolytic and degranulation activities in patients with STAT1 GOF mutations. Moreover, NK cells from these patients display lower production of IFN-γ in response to IL-15 and reduced proliferation after stimulation with IL-2 or IL-15, suggesting that STAT5 signaling is affected. In addition, signaling studies demonstrate that the increased phosphorylation of STAT1 in response to IFN-α is associated with detectable activation of STAT1 and increased STAT1 binding to the interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) promoter in response to IL-15, whereas STAT5 phosphorylation and DNA binding to IL-2 receptor α (IL2RA) are reduced or not affected in response to the same cytokine. CONCLUSION: These observations suggest that persistent activation of STAT1 might affect NK cell proliferation and functional activities.