Validation of STAT1 variants found in patients with primary immunodeficiencies and evaluation of the effect of JAK inhibition using an in vitro model

Regulation of cellular responses to interferons, cytokines, growth factors and hormones is mediated by signal transducer and activator of transcription (STAT) proteins. In the immune system binding of a cytokine (e.g. IFN) to the corresponding surface receptor, Janus kinase (JAK) molecules are phosphorylated, resulting in the docking and phosphorylation of the associated STAT proteins. The STATs will form homo or heterodimers and translocate to regulate transcription of pro-inflammatory target genes. Mutations in STAT1 are known to result in immunodeficiency and/or immune dysregulation syndromes. In this project, the functional impact of variants in the STAT1 gain-of-function gene (STAT1 GOF) will be analyzed on a protein level. The effect of a directed treatment approach targeting the JAK-STAT pathway (JAK inhibitors) will be evaluated in an in vitro model. Freshly isolated PBMCs or whole blood samples from patients and healthy controls were obtained. The cells were stimulated with IFNg and the treated with the JAK inhibitor Ruxolitinib. Extra and intracellular staining with anti-human fluorochrome conjugated antibodies was performed in order to determine the expression of STAT1 and pSTAT1 on monocytes by means of flow cytometry. Two pediatric patients and one related adult patient were studied and the pathogenicity of the variants was confirmed as STAT1 and pSTAT1 levels in the patients at baseline as well as after IFNg stimulation were markedly increased, when compared with healthy controls. The in vitro administration of different concentrations of the JAK inhibitor Ruxolitinib resulted in the normalization of pSTAT1 levels in the cells obtained from patients with STAT1 GOF mutations. Patients with STAT1 GOF mutations show a severe clinical phenotype with recurrent bacterial and fungal infections. Currently no specific treatment options are available. However recent case reports suggested the benefit of JAK inhibition. Therefore we studied the effect of this drug using primary cells in an in vitro model on a molecular level. Importantly two patients have been started on this medication and have achieved a significant clinical response. We are currently evaluating the capacity of our protocol to identify patients with alterations of the JAK-STAT pathway eligible for this targeted treatment approach

Colchicine treatment in children with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome: A multicenter study in Spain

Objective: To evaluate the efficacy of colchicine therapy in pediatric patients with PFAPA syndrome who present with an incomplete response to the standard treatment or with frequent episodes (an interval of less than 14 days between two disease flares). Methods: A multicenter cohort study of children diagnosed with PFAPA syndrome and treated with colchicine was performed in three separate hospitals located in Spain. The patients clinical and laboratory data were reviewed by accessing their medical records. Response to colchicine was evaluated after 12 months of treatment for frequency, duration, and intensity of PFAPA episodes. Results: A total of 13 children were included in our study, 43% of whom were boys. Median age of the colchicine therapy initiation was 6 years (interquartile range (IQR)=3-9.5). Following a 12-month period of colchicine therapy (median dosage of 0.02 mg/kg/day; IQR=0.02-0.03), a significant decrease in the median number of flares (median 8; IQR=7-14 vs 3; IQR=2-4; p=0.005) and the duration of disease episodes (median 4 days; IQR=3.25-5.125 vs 1 day; IQR=1-2; p=0.003) was observed. Furthermore, the highest degree of fever during disease flares was reduced from median 40ºC (IQR=39.5-40) to 38.5ºC (IQR=37.7-38.9) (p=0.002). Conclusion: Colchicine therapy decreased the frequency and intensity of PFAPA. The use of colchicine could be an effective treatment in pediatric patients with PFAPA syndrome who present with frequent or severe relapses

Double Trouble: a patient with STAT1 GOF and Down Syndrome

Multiple biological processes are regulated by different cytokines through signal transducer and activator of transcription (STAT) factors. The type I interferons (IFN) activate the JAK-STAT pathway, that induces the tyrosine phosphorylation of STAT1 (pSTAT1) and its binding to the DNA to activate transcription of STAT-dependent genes. Gain of function (GOF) mutations in STAT1 result in hyperactivation of STAT1 and upregulation of downstream genes. Patients with STAT1 GOF present a wide spectrum of clinical manifestations characterized by a reduction of circulating Th17 cells and high susceptibility to chronic mucocutaneous candidiasis (CMC). A cluster of six interferon receptors (IFN-R) genes is required for the detection of the different types of IFN. Four of the six IFN-R genes are located on the human chromosome 21. Down syndrome (DS), or trisomy 21, is one of the most known diseases causing intellectual disability. In addition, DS patients are more frequent to present autoimmune phenotypes and fungal infections like CMC (56-76%). In common with STAT1 GOF, DS patients could suffer from similar autoimmune disorders due to hyperresponsiveness to IFNs. In this project, the functional impact of both diseases will be analysed in a patient with STAT1 GOF and DS. Freshly isolated PBMCs or whole blood were obtained from a STAT1 GOF and DS patient, her mother and healthy controls. Flow cytometry analysis was conducted on whole blood treated in vitro with IFNα, IFNγ and IL6. Extra and intracellular staining with different antibodies specific for monocytes, STAT1 and pSTAT1 was performed for the study of STAT1 phosphorylation. PBMCs were stimulated with cytokines and qPCR was conducted in order to measure the gene transcription of STAT1, CxCL10, SOCS1, SOCS3, PIAS1 and PIAS3. The study of the pathogenicity of both diseases, STAT1 and DS, were carried out in a pediatric patient with STAT1 GOF and DS that presented severe disease and her mother who shares the same mutation in STAT1 and mild symptoms. The analysis of the STAT1 and pSTAT1 levels, before and after stimulation, indicated an elevated level in both patients compared with healthy controls. However, the levels of total STAT1 in the pediatric patient showed a greater increase in comparison to her mother. These results could explain part of the severe phenotype in the pediatric patient. Importantly this patient has been receiving treatment for a year which has markedly improved her quality of life

Biallelic TRAF3IP2 variants causing chronic mucocutaneous candidiasis in a child harboring a STAT1 variant

[Background] Inherited chronic mucocutaneous candidiasis (CMC) is often caused by inborn errors of immunity, impairing the response to, or the production of IL-17A and IL-17F. About half of the cases carry STAT1 gain-of-function (GOF) mutations. Only few patients have been reported with mutations of TRAF3IP2, a gene encoding the adaptor ACT1 essential for IL-17 receptor(R) signaling. We investigated a 10-year-old girl with CMC, carrying a heterozygous variant of STAT1 and compound heterozygous variants of TRAF3IP2.[Methods] By flow cytometry, STAT1 levels and phosphorylation (CD14+) as well as IL-17A, IL-22, IFN-γ, and IL-4 production (memory CD4+ T cells) were determined. ACT1 expression and binding to IL-17RA were assessed by Western blot and co-immunoprecipitation in HEK-293T cells transfected with plasmids encoding wild-type or mutant HA-tagged ACT1 and Flag-IL-17RA. We evaluated IL-17A responses by measuring luciferase induction under a NF-κB-driven reporter system in HEK-293T cells and Gro-α secretion in fibroblasts. [Results] A STAT1 variant (c.1363G>A/p.V455I) was identified by next-generation sequencing and classified as likely non-pathogenic as functional testing revealed normal STAT1 expression and phosphorylation upon IFN-γ. We also found compound heterozygous variants (c.1325A>G/p.D451G and c.1335delA/p.K454fs11*) of TRAF3IP2. By overexpression, despite normal protein expression, and impaired (K454fs11*) or normal (D451G) interaction with IL-17RA, both mutant alleles resulted in impaired NF-κB activation. Patient's fibroblasts displayed abolished GRO-α secretion upon IL-17A stimulation. Finally, ex vivo CD4+ T cells showed increased IL-17A, IL-22, and IL-4 and normal low IFN-γ expression upon stimulation. [Conclusion] We identify novel compound heterozygous variants of TRAFP3IP2 causing autosomal recessive ACT1 deficiency in a child with CMC and provide a review of the current literature

No differences of immune activation and microbial translocation among HIV-infected children receiving combined antiretroviral therapy or protease inhibitor monotherapy.

This is a cross-sectional study of 15 aviremic chronic HIV-infected children revealing no differences in immune activation (IA; HLA-DRCD38 CD4 and CD8 T cells, and sCD14) and microbial translocation (MT; lipopolysaccharides (LPS) and 16S rDNA) among HIV-infected patients under combined antiretroviral treatment (cART; n = 10) or ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv; n = 5). In both cases, IA and MT were lower in healthy control children (n = 32). This observational study suggests that ritonavir boosted protease inhibitor monotherapy (mtPI/rtv) is not associated with an increased state of IA or MT as compared with children receiving cART

The Relationship Between the Site of Metastases and Outcome in Children With Stage IV Wilms Tumor: Data From 3 European Pediatric Cancer Institutions

The aim of this study was to analyze in detail the site of metastasis of stage 4 Wilms tumor (WT) and its correlation with outcome. The databases from 3 major European pediatric cancer institutions were screened for children with WT between 1994 and 2011. Of 208 children identified, 31 (14.9%) had metastases at diagnosis. The lung was affected in 29 children (93.5%) and the liver in 6 children (19.4%). Twenty-seven children (87.1%) had metastases isolated to 1 organ, with the lung being the most common site (80.7%). Five-year overall survival was significantly better in those children with distant disease in either lung or liver (95.8%) compared with those affected in both lung and liver (57.1%, P=0.028). Further, prognostic markers were the response of metastases to preoperative chemotherapy (P=0.0138), high-risk histology (P=0.024), and local stage (P=0.026). Five-year overall survival was 82.1% and 5-year event-free survival was 67.9%. The overall follow-up time was 74.1 and 87.2 (2 to 151) months among survivors, and the treatment-related complication rate was 16.7%. In conclusion, in our series of stage 4 WT, prognosis was excellent if histology was favorable, metastatic disease was isolated to either lungs or liver, and if metastases responded to preoperative chemotherapy

The Relationship Between the Site of Metastases and Outcome in Children With Stage IV Wilms Tumor: Data From 3 European Pediatric Cancer Institutions

The aim of this study was to analyze in detail the site of metastasis of stage 4 Wilms tumor (WT) and its correlation with outcome. The databases from 3 major European pediatric cancer institutions were screened for children with WT between 1994 and 2011. Of 208 children identified, 31 (14.9%) had metastases at diagnosis. The lung was affected in 29 children (93.5%) and the liver in 6 children (19.4%). Twenty-seven children (87.1%) had metastases isolated to 1 organ, with the lung being the most common site (80.7%). Five-year overall survival was significantly better in those children with distant disease in either lung or liver (95.8%) compared with those affected in both lung and liver (57.1%, P=0.028). Further, prognostic markers were the response of metastases to preoperative chemotherapy (P=0.0138), high-risk histology (P=0.024), and local stage (P=0.026). Five-year overall survival was 82.1% and 5-year event-free survival was 67.9%. The overall follow-up time was 74.1 and 87.2 (2 to 151) months among survivors, and the treatment-related complication rate was 16.7%. In conclusion, in our series of stage 4 WT, prognosis was excellent if histology was favorable, metastatic disease was isolated to either lungs or liver, and if metastases responded to preoperative chemotherapy

Imaging findings of multisystem inflammatory syndrome in children associated with COVID-19

[Background] A hyperinflammatory immune-mediated shock syndrome has been recognised in children exposed to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19).[Objective] To describe typical imaging findings in children with multisystem inflammatory syndrome associated with COVID-19.[Materials and methods] During the first wave of the COVID-19 pandemic, imaging studies and clinical data from children treated for multisystem inflammatory syndrome were collected from multiple centres. Standardised case templates including demographic, biochemical and imaging information were completed by participating centres and reviewed by paediatric radiologists and paediatricians.[Results] We included 37 children (21 boys; median age 8.0 years). Polymerase chain reaction (PCR) testing was positive for SARS-CoV-2 in 15/37 (41%) children and immunoglobulins in 13/19 children (68%). Common clinical presentations were fever (100%), abdominal pain (68%), rash (54%), conjunctivitis (38%) and cough (32%). Thirty-three children (89%) showed laboratory or imaging findings of cardiac involvement. Thirty of the 37 children (81%) required admission to the intensive care unit, with good recovery in all cases. Chest radiographs demonstrated cardiomegaly in 54% and signs of pulmonary venous hypertension/congestion in 73%. The most common chest CT abnormalities were ground-glass and interstitial opacities (83%), airspace consolidation (58%), pleural effusion (58%) and bronchial wall thickening (42%). Echocardiography revealed impaired cardiac function in half of cases (51%) and coronary artery abnormalities in 14%. Cardiac MRI showed myocardial oedema in 58%, pericardial effusion in 42% and decreased left ventricular function in 25%. Twenty children required imaging for abdominal symptoms, the commonest abnormalities being free fluid (71%) and terminal ileum wall thickening (57%). Twelve children underwent brain imaging, showing abnormalities in two cases.[Conclusion] Children with multisystem inflammatory syndrome showed pulmonary, cardiac, abdominal and brain imaging findings, reflecting the multisystem inflammatory disease. Awareness of the imaging features of this disease is important for early diagnosis and treatment.Peer reviewe

Primary Immune Regulatory Disorders With an Autoimmune Lymphoproliferative Syndrome-Like Phenotype: Immunologic Evaluation, Early Diagnosis and Management

Primary immune regulatory disorders (PIRD) are associated with autoimmunity, autoinflammation and/or dysregulation of lymphocyte homeostasis. Autoimmune lymphoproliferative syndrome (ALPS) is a PIRD due to an apoptotic defect in Fas-FasL pathway and characterized by benign and chronic lymphoproliferation, autoimmunity and increased risk of lymphoma. Clinical manifestations and typical laboratory biomarkers of ALPS have also been found in patients with a gene defect out of the Fas-FasL pathway (ALPS-like disorders). Following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA), we identified more than 600 patients suffering from 24 distinct genetic defects described in the literature with an autoimmune lymphoproliferative phenotype (ALPS-like syndromes) corresponding to phenocopies of primary immunodeficiency (PID) (NRAS, KRAS), susceptibility to EBV (MAGT1, PRKCD, XIAP, SH2D1A, RASGRP1, TNFRSF9), antibody deficiency (PIK3CD gain of function (GOF), PIK3R1 loss of function (LOF), CARD11 GOF), regulatory T-cells defects (CTLA4, LRBA, STAT3 GOF, IL2RA, IL2RB, DEF6), combined immunodeficiencies (ITK, STK4), defects in intrinsic and innate immunity and predisposition to infection (STAT1 GOF, IL12RB1) and autoimmunity/autoinflammation (ADA2, TNFAIP3,TPP2, TET2). CTLA4 and LRBA patients correspond around to 50% of total ALPS-like cases. However, only 100% of CTLA4, PRKCD, TET2 and NRAS/KRAS reported patients had an ALPS-like presentation, while the autoimmunity and lymphoproliferation combination resulted rare in other genetic defects. Recurrent infections, skin lesions, enteropathy and malignancy are the most common clinical manifestations. Some approaches available for the immunological study and identification of ALPS-like patients through flow cytometry and ALPS biomarkers are provided in this work. Protein expression assays for NKG2D, XIAP, SAP, CTLA4 and LRBA deficiencies and functional studies of AKT, STAT1 and STAT3 phosphorylation, are showed as useful tests. Patients suspected to suffer from one of these disorders require rapid and correct diagnosis allowing initiation of tailored specific therapeutic strategies and monitoring thereby improving the prognosis and their quality of life.his work was supported by grants from Fondo de Investigación Sanitaria (FIS-PI16/2053) to LA and LG-G. The project has been co-financed with FEDER funds. ML-N was co-financed by Fondo Social Europeo, Programa Operativo de empleo juvenil (YEI)

SARS-CoV-2 infection in a pediatrics STAT1 GOF patient under Ruxolitinib therapy-a matter of balance?

Recently, 94 inborn errors of immunity (IEI) patients suffering from COVID-19 have been described, overall demonstrating a mild phenotype [1] although more severe disease manifestations have been suggested for patients with alterations in the interferon (IFN) signaling pathway, including auto-antibodies against type I IFN [2]. Patients with STAT1 GOF mutations show a complex and often severe phenotype, combining an increased susceptibility of fungal, (myco-) bacterial and viral infections as well as autoimmune and autoinflammatory manifestations [3]. Characteristically, in response to type I and type II IFN stimulation, these patients show STAT1 hyperphosphorylation [3, 4]. Whether in the context of SARS-CoV-2 infection, the hyperactivation of the IFN-JAK1/2-STAT1 pathway would be protective (antiviral effect) or deleterious (hyperinflammation) is unclear. Ruxolitinib (a selective JAK1/2 inhibitor) has been successfully used in STAT1 GOF patients controlling many disease manifestations [5] and also resulted in improved pulmonary function and faster recovery from lymphopenia in previously healthy individuals suffering from severe COVID-19 [6].This work was supported by the Instituto de Salud Carlos III, Madrid (Spain) [Sara Borrell, CD20/00124 to P.B.L, Juan Rodés JR18/00042 to P.O, FIS PI19/01471 to O.N] and the Consejería de Salud, Junta Andalucía [SA0051/2020 to O.N]. A.G-V was supported by the Instituto de Salud Carlos III, cofinanced by the European Development Regional Fund (“A way to achieve Europe”), Subprograma Miguel Servet (CP19/00159).Peer reviewe