Corrigendum: Abnormalities of thymic stroma may contribute to immune dysregulation in murine models of leaky severe combined immunodeficiency.

[This corrects the article DOI: 10.3389/fimmu.2011.00015.].

Progesterone receptor is constitutively expressed in induced Pluripotent Stem Cells (iPSCs).

Induced Pluripotent Stem Cells (iPSCs) are nowadays a common starting point for wide-ranging applications including 3D disease modeling (i.e. organoids) and in future regenerative medicine. Physiological processes like homeostasis, cell differentiation, development and reproduction are tightly regulated by hormones through binding to their transmembrane or nuclear receptors of target cells. Considering their pleiotropic effect, take into account also their expression in an iPSCs-based disease modeling would better recapitulate the molecular events leading to 3D organoid development and disease study. Here we reported the expression pattern of estrogen receptor (ERα) and progesterone receptor (PR) in four different iPSCs, obtained from CD34 + progenitor cells and skin fibroblasts with four different methods. Expression of ERα and PR mRNA were significantly downregulated in iPSCs as well as fibroblasts compared to MCF7 positive control. Immunofluorescence (IF) staining detected only the expression of PR protein in all the different iPSCs cell lines, while ERα was not detectable. By flow cytometry analysis we observed that the ~ 65% of the total population of iPSCs cells expressed only PR, with 100% fold increase compared to HSPCs and fibroblasts, while ERα was not expressed. Our results collectively demonstrated for the first time that the reprogramming of somatic cells into iPSCs leads to the expression of PR receptor.

Plasma p-tau181 and amyloid markers in Alzheimer's disease: A comparison between Lumipulse and SIMOA.

Aim of the project was to evaluate the technical and clinical validity of plasma Lumipulse p-tau, Aß42 and Aß40 species and their correlation with CSF core Alzheimer's Disease (AD) markers; a method comparison with SIMOA was also performed. One-hundred-thirthy-three participants, namely 55 A+T+N+ AD, 28 Neurodegenerative disorders (NDD) and 50 controls were enrolled for the study. Lumipulse technical validity showed high stability for p-tau181, Aß42, and Aß40, with higher stability of p-tau to repeated freezing thaw cycles. p-tau181 levels detected by both techniques were higher in AD compared to both NDD/controls and exhibited a similar correlation with CSF p-tau levels, whereas Aß42 levels were slightly lower in AD with both methods. In the comparison between SIMOA and Lumipulse plasma markers, both techniques exhibited similar diagnostic accuracy for AD for p-tau181 (0.87; 95 %CI 0.81-0.94, vs 0.85; 95 %CI 0.78-0.93), whereas the best performance was reached by p-tau181/ Aß42 Lumipulse ratio (ROC AUC 0.915, 95 %CI 0.86-0.97). The study thus confirmed the construct validity of both Lumipulse and SIMOA techniques for the identification of CSF AD pattern in clinical settings.

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.

Joubert syndrome-derived induced pluripotent stem cells show altered neuronal differentiation in vitro.

Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability, and a peculiar cerebellar and brainstem malformation, the "molar tooth sign." Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper, we developed an in vitro neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (AHI1, CPLANE1, TMEM67, and CC2D2A) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31 days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context.

Rituximab Monotherapy Is Effective as First-Line Treatment for Granulomatous Lymphocytic Interstitial Lung Disease (GLILD) in CVID Patients.

Granulomatous lymphocytic interstitial lung disease (GLILD) represents a fatal immune dysregulatory complication in common variable immunodeficiency (CVID). Evidence-based diagnostic guidelines are lacking, and GLILD treatment consists in immunosuppressive drugs; nonetheless, therapeutical strategies are heterogeneous and essentially based on experts' opinions and data from small case series or case reports.We aimed to evaluate the efficacy and safety of first-line Rituximab monotherapy for CVID-related GLILD, by assessing symptoms and quality of life alterations, immunological parameters, pulmonary function tests, and lung computed tomography.All six GLILD patients received Rituximab infusions as a first-line treatment. Rituximab was administered at 375 mg/m2 monthly for six infusions followed by maintenance every 3 months; none of the patients experienced severe adverse events. Symptom burden and quality of life significantly improved in treated patients compared to a control group of CVID patients without GLILD. Rituximab treatment indirectly caused a trend toward reduced T-cell activation and exhaustion markers sCD25 and sTIM-3. Lung function improved in treated patients, with statistically significant increases in TLC and DLCO. Lung CT scan findings expressed by means of Baumann scoring system displayed a reduction in the entire cohort.In conclusion, first-line monotherapy with Rituximab displayed high efficacy in disease remission in all treated patients, with improvement of symptoms and amelioration of quality of life, as well as restoration of PFTs and lung CT scan findings.

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.

Ischaemic cerebral small vessel disease caused by adenosine deaminase 2 deficiency syndrome.

BACKGROUND AND PURPOSE: Only a small proportion of cerebral small vessel disease (cSVD), a frequent cause of stroke and cognitive or motor disability in adults, is attributable to monogenic conditions. The hereditary nature of a patient's cSVD may be masked by a mild or non-informative phenotype, as single-gene disorders have a variable mode of presentation, penetrance and disease severity. CASE DESCRIPTION: An adult patient is here described with recurrent acute ischaemic strokes due to cSVD with no other phenotypic manifestation, in whom the pathogenic c.139G>A (p.G47R) missense variant in ADA2 (NM_001282225.2), consistent with the diagnosis of adenosine deaminase 2 deficiency syndrome, was detected by targeted next-generation sequencing. CONCLUSIONS: Clinical suspicion of adenosine deaminase 2 deficiency syndrome may be overlooked in stroke patients in whom other specific disease features are lacking. This case enlarges the mode of presentation of the syndrome and highlights the diagnostic potential of next-generation sequencing of known cSVD genes in young adults with recurrent small subcortical infarcts presenting with a lacunar syndrome.

Pathological 25 kDa C-Terminal Fragments of TDP-43 Are Present in Lymphoblastoid Cell Lines and Extracellular Vesicles from Patients Affected by Frontotemporal Lobar Degeneration and Neuronal Ceroidolipofuscinosis Carrying a GRN Mutation.

Frontotemporal lobar degeneration (FTLD) is a complex disease, characterized by progressive degeneration of frontal and temporal lobes. Mutations in progranulin (GRN) gene have been found in up to 50% of patients with familial FTLD. Abnormal deposits of post-translationally-modified TAR DNA-binding protein of 43 kDa (TDP-43) represent one of the main hallmarks of the brain pathology. To investigate in peripheral cells the presence of the different TDP-43 forms, especially the toxic 25 kDa fragments, we analyzed lymphoblastoid cell lines (LCLs) and the derived extracellular vesicles (EVs) from patients carrying a GRN mutation, together with wild-type (WT) healthy controls. After characterizing EV sizes and concentrations by nanoparticle tracking analysis, we investigated the levels of different forms of the TDP-43 protein in LCLs and respective EVs by Western blot. Our results showed a trend of concentration decreasing in EVs derived from GRN-mutated LCLs, although not reaching statistical significance. A general increase in p-TDP-43 levels in GRN-mutated LCLs and EVs was observed. In particular, the toxic 25 kDa fragments of p-TDP-43 were only present in GRN-mutated LCLs and were absent in the WT controls. Furthermore, these fragments appeared to be more concentrated in EVs than in LCLs, suggesting a relevant role of EVs in spreading pathological molecules between cells.

Differences Between Plasma and Cerebrospinal Fluid p-tau181 and p-tau231 in Early Alzheimer's Disease.

Plasma phosphorylated tau species have been recently proposed as peripheral markers of Alzheimer's disease (AD) pathology. In this cross-sectional study including 91 subjects, plasma and cerebrospinal fluid (CSF) p-tau181 and p-tau231 levels were elevated in the early symptomatic stages of AD. Plasma p-tau231 and p-tau181 were strongly related to CSF phosphorylated tau, total tau and amyloid and exhibited a high accuracy-close to CSF p-tau231 and p-tau181-to identify AD already in the early stage of the disease. The findings might support the use as diagnostic and prognostic peripheral AD biomarkers in both research and clinical settings.

DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières syndrome astrocytes.

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.

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.

When a Nontuberculous Mycobacterial Infection Reveals an Error of Immunity: A Single Center's Experience.

We present an algorithm that may be applied in case of a diagnosis of pediatric nontuberculous mycobacterial disease to identify the patients who may require an immunologic assessment to discover a possible underlying immune system defect predisposing to their nontuberculous mycobacterial infections.

Omenn Syndrome due to RAG1 Mutation Presenting With Nonimmune Hydrops Fetalis in Two Siblings.

Omenn syndrome (OS) is a rare variant of severe combined immunodeficiency characterized by susceptibility to severe opportunistic infections and peculiar manifestations, such as protein-losing erythroderma, alopecia, hepatosplenomegaly, lymphadenopathies, and severe diarrhea. The typical form of the disease is caused by hypomorphic mutation of the recombination-activating genes (RAG1 and RAG2), which are critical in initiating the molecular processes leading to lymphocyte and immunoglobulin receptor formation. Affected patients lack B cells, whereas autoreactive oligoclonal T cells infiltrate the skin, gut, spleen, and liver. In the absence of hematopoietic stem cell transplantation, patients with OS usually succumb early in life because of opportunistic infections. The incidence of OS is estimated to be <1 per 1 000 000; however, the actual frequency is difficult to ascertain. We report 2 siblings affected by OS due to a homozygous frameshift mutation (NM_000448.3:c.519delT, p.E174Sfs*26) in the RAG1 gene presenting with nonimmune hydrops fetalis (NIHF). To the best of our knowledge, this is the first reported association between OS and NIHF. NIHF specifically refers to the presence of ≥2 abnormal fluid collections in the fetus, without red blood cell alloimmunization. A broad spectrum of pathologies is associated with NIHF; however, in ∼20% of the cases, the primary cause remains unclear. Understanding the etiology of NIHF is essential for guiding clinical management, determining prognosis, and informing parents regarding recurrence risk. Our case contributes to expanding the spectrum of OS presentation and highlights the importance of a complete immunologic and genetic workup in otherwise unexplained cases of NIHF.

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.

Thymic Epithelial Cell Alterations and Defective Thymopoiesis Lead to Central and Peripheral Tolerance Perturbation in MHCII Deficiency.

Major Histocompatibility Complex (MHC) class II (MHCII) deficiency (MHCII-D), also known as Bare Lymphocyte Syndrome (BLS), is a rare combined immunodeficiency due to mutations in genes regulating expression of MHCII molecules. MHCII deficiency results in impaired cellular and humoral immune responses, leading to severe infections and autoimmunity. Abnormal cross-talk with developing T cells due to the absence of MHCII expression likely leads to defects in thymic epithelial cells (TEC). However, the contribution of TEC alterations to the pathogenesis of this primary immunodeficiency has not been well characterized to date, in particular in regard to immune dysregulation. To this aim, we have performed an in-depth cellular and molecular characterization of TEC in this disease. We observed an overall perturbation of thymic structure and function in both MHCII-/- mice and patients. Transcriptomic and proteomic profiling of murine TEC revealed several alterations. In particular, we demonstrated that impairment of lymphostromal cross-talk in the thymus of MHCII-/- mice affects mTEC maturation and promiscuous gene expression and causes defects of central tolerance. Furthermore, we observed peripheral tolerance impairment, likely due to defective Treg cell generation and/or function and B cell tolerance breakdown. Overall, our findings reveal disease-specific TEC defects resulting in perturbation of central tolerance and limiting the potential benefits of hematopoietic stem cell transplantation in MHCII deficiency.

E-Cadherin Expression and Blunted Interferon Response in Blastic Plasmacytoid Dendritic Cell Neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive neoplasm derived from plasmacytoid dendritic cells (pDCs). In this study, we investigated by immunohistochemical analysis the expression of E-cadherin (EC) on pDCs in reactive lymph nodes and tonsils, bone marrow, and in BPDCN. We compared the expression of EC in BPDCN to that in leukemia cutis (LC) and cutaneous lupus erythematosus (CLE), the latter typically featuring pDC activation. In BPDCN, we also assessed the immunomodulatory activity of malignant pDCs through the expression of several type I interferon (IFN-I) signaling effectors and downstream targets, PD-L1/CD274, and determined the extent of tumor infiltration by CD8-expressing T cells. In reactive lymph nodes and tonsils, pDCs expressed EC, whereas no reactivity was observed in bone marrow pDCs. BPDCN showed EC expression in the malignant pDCs in the vast majority of cutaneous (31/33 cases, 94%), nodal, and spleen localizations (3/3 cases, 100%), whereas it was more variable in the bone marrow (5/13, 38,5%), where tumor cells expressed EC similarly to the skin counterpart in 4 cases and differently in other 4. Notably, EC was undetectable in LC (n=30) and in juxta-epidermal pDCs in CLE (n=31). Contrary to CLE showing robust expression of IFN-I-induced proteins MX1 and ISG5 in 20/23 cases (87%), and STAT1 phosphorylation, BPDCN biopsies showed inconsistent levels of these proteins in most cases (85%). Expression of IFN-I-induced genes, IFI27, IFIT1, ISG15, RSAD2, and SIGLEC1, was also significantly (P<0.05) lower in BPDCN as compared with CLE. In BPDCN, a significantly blunted IFN-I response correlated with a poor CD8+T-cell infiltration and the lack of PD-L1/CD274 expression by the tumor cells. This study identifies EC as a novel pDC marker of diagnostic relevance in BPDCN. The results propose a scenario whereby malignant pDCs through EC-driven signaling promote the blunting of IFN-I signaling and, thereby, the establishment of a poorly immunogenic tumor microenvironment.

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.

Case Report: Hypomorphic Function and Somatic Reversion in DOCK8 Deficiency in One Patient With Two Novel Variants and Sclerosing Cholangitis.

DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokinesis 8 (DOCK8) gene. The disease has a wide clinical spectrum encompassing recurrent infections (candidiasis, viral and bacterial infections), virally driven malignancies and immune dysregulatory features, including autoimmune (cytopenia and vasculitis) as well as allergic disorders (eczema, asthma, and food allergy). Hypomorphic function and somatic reversion of DOCK8 has been reported to result in incomplete phenotype without IgE overproduction. Here we describe a case of DOCK8 deficiency in a 8-year-old Caucasian girl. The patient's disease was initially classified as autoimmune thrombocytopenia, which then evolved toward a combined immunodeficiency phenotype with recurrent infections, persistent EBV infection and lymphoproliferation. Two novel variants (one deletion and one premature stop codon) were characterized, resulting in markedly reduced, but not absent, DOCK8 expression. Somatic reversion of the DOCK8 deletion was identified in T cells. Hypomorphic function and somatic reversion were associated with restricted T cell repertoire, decreased STAT5 phosphorylation and impaired immune synapse functioning in T cells. Although the patient presented with incomplete phenotype (absence of markedly increase IgE and eosinophil count), sclerosing cholangitis was incidentally detected, thus indicating that hypomorphic function and somatic reversion of DOCK8 may delay disease progression but do not necessarily prevent from severe complications.