The epidemiology of inborn errors of immunity (IEI) in the Republic of Ireland was first published in 2005 but has not been updated since. IEI prevalence data from Northern Ireland was last published in 2018. Using data from the United Kingdom Primary Immune Deficiency (UKPID) and European Society for Immunodeficiencies (ESID) registries, we reviewed all registered cases of IEI affecting adult patients ≥ 18 years of age from the two largest immunology specialist centres in Northern Ireland and the Republic of Ireland, respectively and calculated the combined minimum adult prevalence of IEI on the island of Ireland for the first time. We also recorded data pertaining to presenting symptoms of IEI, diagnostic delay, immunoglobulin data, and genetic testing, as well as briefly reporting data pertaining to secondary immunodeficiency in both countries. As of 1 May 2020, we identified a minimum adult IEI prevalence in Ireland of 8.85/100,000 population.

OPINION article Front. Nutr., 12 January 2021 | https://doi.org/10.3389/fnut.2020.626712

Celiac disease (CD) affects a growing number of individuals worldwide. To elucidate the causes for this increase, future multidisciplinary collaboration is key to understanding the interactions between immunoreactive components in gluten-containing cereals and the human gastrointestinal tract and immune system and to devise strategies for CD prevention and treatment beyond the gluten-free diet. During the last meetings, the Working Group on Prolamin Analysis and Toxicity (Prolamin Working Group, PWG) discussed recent progress in the field together with key stakeholders from celiac disease societies, academia, industry and regulatory bodies. Based on the current state of knowledge, this perspective from the PWG members provides recommendations regarding clinical, analytical and legal aspects of CD. The selected key topics that require future multidisciplinary collaborative efforts in the clinical field are to collect robust data on the increasing prevalence of CD, to evaluate what is special about gluten-specific T cells, to study their kinetics and transcriptomics and to put some attention to the identification of the environmental agents that facilitate the breaking of tolerance to gluten. In the field of gluten analysis, the key topics are the precise assessment of gluten immunoreactive components in wheat, rye and barley to understand how these are affected by genetic and environmental factors, the comparison of different methods for compliance monitoring of gluten-free products and the development of improved reference materials for gluten analysis.

Celiac disease (CD) affects a growing number of individuals worldwide. To elucidate the causes for this increase, future multidisciplinary collaboration is key to understanding the interactions between immunoreactive components in gluten-containing cereals and the human gastrointestinal tract and immune system and to devise strategies for CD prevention and treatment beyond the gluten-free diet. During the last meetings, the Working Group on Prolamin Analysis and Toxicity (Prolamin Working Group, PWG) discussed recent progress in the field together with key stakeholders from celiac disease societies, academia, industry and regulatory bodies. Based on the current state of knowledge, this perspective from the PWG members provides recommendations regarding clinical, analytical and legal aspects of CD. The selected key topics that require future multidisciplinary collaborative efforts in the clinical field are to collect robust data on the increasing prevalence of CD, to evaluate what is special about gluten-specific T cells, to study their kinetics and transcriptomics and to put some attention to the identification of the environmental agents that facilitate the breaking of tolerance to gluten. In the field of gluten analysis, the key topics are the precise assessment of gluten immunoreactive components in wheat, rye and barley to understand how these are affected by genetic and environmental factors, the comparison of different methods for compliance monitoring of gluten-free products and the development of improved reference materials for gluten analysis.

Coeliac disease is a common small bowel enteropathy arising in genetically predisposed individuals and caused by ingestion of gluten in the diet. Great advances have been made in understanding the role of the adaptive immune system in response to gluten peptides. Despite detailed knowledge of these adaptive immune mechanisms, the complete series of pathogenic events responsible for development of the tissue lesion remains less certain. This review contributes to the field by discussing additional mechanisms which may also contribute to pathogenesis. These include the production of cytokines such as interleukin-15 by intestinal epithelial cells and local antigen presenting cells as a pivotal event in the disease process. A subset of unconventional T cells called gamma/delta T cells are also persistently expanded in the coeliac disease (CD) small intestinal epithelium and recent analysis has shown that these cells contribute to pathogenic inflammation. Other unconventional T cell subsets may play a local immunoregulatory role and require further study. It has also been suggested that, in addition to activation of pathogenic T helper cells by gluten peptides, other peptides may directly interact with the intestinal mucosa, further contributing to the disease process. We also discuss how myofibroblasts, a major source of tissue transglutaminase and metalloproteases, may play a key role in intestinal tissue remodeling. Contribution of each of these factors to pathogenesis is discussed to enhance our view of this complex disorder and to contribute to a wider understanding of chronic immune-mediated disease.

Common variable immunodeficiency (CVID) is a primary immunoglobulin deficiency characterized by recurrent infections and complications, including autoimmunity, enteropathy, polyclonal lymphocytic infiltration or lymphoid malignancy. Innate T cells can support B cell maturation and antibody production. We investigated the numbers, phenotypes and functions of circulating B cell, γδ T cell, invariant natural killer T (iNKT) cell and mucosal-associated invariant T (MAIT) cell subsets in 23 CVID patients and 27 healthy controls. Switched-memory B cells and plasmablasts were depleted in CVID patients (p<0.0001). γδ T cells were found at normal numbers, but iNKT and MAIT cells were depleted (p<0.0001 and p<0.002). MAIT cells were especially low in patients with complicated CVID (p<0.05). MAIT cells from patients appeared more activated and more frequently produced interleukin-17A, interleukin-22 and tumor necrosis factor-α than MAIT cells from healthy subjects in vitro. Thus, MAIT cell depletion and activation may contribute to immunodeficiency and complications associated with CVID.

Granulomatosis with polyangiitis is an uncommon condition characterised by vasculitis and associated granuloma formation with a highly specific autoantibody, namely proteinase 3-anti-neutrophil cytoplasmic antibody (ANCA). The respiratory tract and kidneys are the organ systems most often involved. Symptoms can be non-specific, and isolated hearing loss can predate other symptoms by months, leading to lengthy delays in diagnosis and treatment. Left untreated, hearing loss can be irreversible, and therefore early diagnosis is crucial. We present a case study of severe hearing impairment in an attempt to raise awareness of ear involvement as an early feature of this unusual condition.

A specific biomarker that can separate active renal vasculitis from other causes of renal dysfunction is lacking, with a kidney biopsy often being required. Soluble CD163 (sCD163), shed by monocytes and macrophages, has been reported as a potential biomarker in diseases associated with excessive macrophage activation. Thus, we hypothesized that urinary sCD163 shed by crescent macrophages correlates with active glomerular inflammation. We detected sCD163 in rat urine early in the disease course of experimental vasculitis. Moreover, microdissected glomeruli from patients with small vessel vasculitis (SVV) had markedly higher levels of CD163 mRNA than did those from patients with lupus nephritis, diabetic nephropathy, or nephrotic syndrome. Both glomeruli and interstitium of patients with SVV strongly expressed CD163 protein. In 479 individuals, including patients with SVV, disease controls, and healthy controls, serum levels of sCD163 did not differ between the groups. However, in an inception cohort, including 177 patients with SVV, patients with active renal vasculitis had markedly higher urinary sCD163 levels than did patients in remission, disease controls, or healthy controls. Analyses in both internal and external validation cohorts confirmed these results. Setting a derived optimum cutoff for urinary sCD163 of 0.3 ng/mmol creatinine for detection of active renal vasculitis resulted in a sensitivity of 83%, specificity of 96%, and a positive likelihood ratio of 20.8. These data indicate that urinary sCD163 level associates very tightly with active renal vasculitis, and assessing this level may be a noninvasive method for diagnosing renal flare in the setting of a known diagnosis of SVV.

BACKGROUND:Activated phosphoinositide 3-kinase δ syndrome (APDS) is a recently described combined immunodeficiency resulting from gain-of-function mutations in PIK3CD, the gene encoding the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). OBJECTIVE:We sought to review the clinical, immunologic, histopathologic, and radiologic features of APDS in a large genetically defined international cohort. METHODS:We applied a clinical questionnaire and performed review of medical notes, radiology, histopathology, and laboratory investigations of 53 patients with APDS. RESULTS:Recurrent sinopulmonary infections (98%) and nonneoplastic lymphoproliferation (75%) were common, often from childhood. Other significant complications included herpesvirus infections (49%), autoinflammatory disease (34%), and lymphoma (13%). Unexpectedly, neurodevelopmental delay occurred in 19% of the cohort, suggesting a role for PI3Kδ in the central nervous system; consistent with this, PI3Kδ is broadly expressed in the developing murine central nervous system. Thoracic imaging revealed high rates of mosaic attenuation (90%) and bronchiectasis (60%). Increased IgM levels (78%), IgG deficiency (43%), and CD4 lymphopenia (84%) were significant immunologic features. No immunologic marker reliably predicted clinical severity, which ranged from asymptomatic to death in early childhood. The majority of patients received immunoglobulin replacement and antibiotic prophylaxis, and 5 patients underwent hematopoietic stem cell transplantation. Five patients died from complications of APDS. CONCLUSION:APDS is a combined immunodeficiency with multiple clinical manifestations, many with incomplete penetrance and others with variable expressivity. The severity of complications in some patients supports consideration of hematopoietic stem cell transplantation for severe childhood disease. Clinical trials of selective PI3Kδ inhibitors offer new prospects for APDS treatment.

Aim Myocardial involvement in patients with systemic vasculitis portends a poorer prognosis. We sought to determine the extent of myocardial dysfunction in Granulomatosis with polyangitis and Churg Strauss disease using speckle tracking and strain imaging. Methods 20 patients and 22 controls with no prior cardiac history were recruited from December 2015 to April 2016. Baseline demographics recorded included age, sex, cardiovascular risk factors, time since diagnosis, immunosuppressive therapy and vasculitis damage index score (VDI). We performed transthoracic echocardiography using Vivid 7 GE machine along with twelve lead electrocardiograms (ECG) and biochemical analysis at the study visit. A single blinded operator utilising Echo Pac 11 software performed offline analysis of strain and speckle tracking measurements. Results Mean age of the vasculitis group was 56 ± 10 years with 50% being male, while mean age of controls was 53 ± 9 years with 55% male. There were no significant differences between groups in age, incidence of hypertension (p = 0.3) or diabetes (p = 0.6). Radial strain was significantly reduced in the vasculitis cohort (P = 0.05). The vasculitis cohort also showed a non-significant reduction in longitudinal strain and abnormal diastology as reflected in a reduction in tissue Doppler E’ velocities, decreased transmittal E/A ratio and increased E/E’ ratio when compared to controls (Table 1). There were negative correlations between increasing VDI score and reduction in mitral inflow velocities r = −0.5 (p = 0.02), number of clinical relapses and tissue Doppler E’ r = −0.6 (p = 0.009) and length of cyclophosphamide use with tissue Doppler E’ r = −0.5 (p = 0.02). Conclusion This is one of the largest studies investigating myocardial dysfunction in ANCA positive vasculitis. Patients demonstrated a reduction in radial strain and evidence of abnormal left ventricular relaxation. This may be indicative of underlying cardiac involvement.

BACKGROUND:There is an increasing demand for specialist public allergy services across Ireland. Little data exist on the patterns of allergic disease in Irish adults. The limited resources available require innovative strategies to ensure quality care delivery.AIMS:This study aimed to review the types of allergy referrals and diagnostic outcomes at a major Irish centre, and to establish an efficient method of communication with non-specialist practitioners.METHODS:Demographic data, referral characteristics and diagnostic outcomes from one hundred consecutive new allergy referrals were identified. Additionally, communications to a pilot email service were reviewed over a 12-month period and user satisfaction assessed.RESULTS:Requests for the investigation of food allergy accounted for 71% of referrals. Despite this, the main diagnostic outcome in this cohort was a non-allergic condition, chronic spontaneous urticaria (56%). immunoglobulin E (IgE)-mediated food allergy was definitively diagnosed in only 9% of patients, with the majority of these presenting with anaphylaxis. The allergy advice email service received 43 requests for assistance over 12 months, mainly for help in the interpretation of an allergy clinical history. Feedback on the email service was universally positive.CONCLUSIONS:The majority of patients in this cohort did not have IgE-mediated allergic disease. Increased awareness of the features that differentiate allergy from non-allergic conditions such as food intolerance or chronic spontaneous urticaria is required. The allergy advice email service should be developed further to play a key role in education and care delivery in partnership primary care.

Historically the diagnosis of celiac disease has relied upon clinical, serological, and histological evidence. In recent years the use of sensitive serological methods has meant an increase in the diagnosis of celiac disease. The heterogeneous nature of the disorder presents a challenge in the study and diagnosis of the disease with patients varying from subclinical or latent disease to patients with overt symptoms. Furthermore the related gluten-sensitive disease dermatitis herpetiformis, while distinct in some respects, shares clinical and serological features with celiac disease. Here we summarize current best practice for the diagnosis of celiac disease and briefly discuss newer approaches. The advent of next-generation assays for diagnosis and newer clinical protocols may result in more sensitive screening and ultimately the possible replacement of the intestinal biopsy as the gold standard for celiac disease diagnosis.

Antibodies of the IgA class directed against the enzyme tissue transglutaminase (tTG) are highly specific for coeliac disease (CD). IgG antibodies to tTG also occur in CD, and have also been reported in autoimmune diseases such as type 1 diabetes mellitus and Crohn's disease. In comparison to the IgA anti-tTG response, little is known of the IgG anti-tTG response in terms of epitope specificity and IgG subclass usage. The aim of this study was to investigate and compare epitopes recognised by CD and non-CD IgG anti-tTG antibodies, and determine the relative proportions of the IgG subclasses comprising this response. IgG anti-tTG positive individuals who did not have CD were identified by screening groups of patients with type I diabetes mellitus, Crohn's disease and granulomatosis with polyangiitis. Results from ELISA blocking experiments and mutant tTG antigens demonstrate that non-CD IgG anti-tTG bind different epitopic determinants to CD IgG anti-tTG. The IgG subclass usage of coeliac disease and type 1 diabetes was dominated by IgG1, whereas this IgG subclass was infrequently a component of the IgG anti-tTG response in diseases such as granulomatosis with polyangiitis and Crohn's disease. The differences in epitope specificity and subclass usage of IgG anti-tTG observed between CD and non-CD individuals may be due to the differing mechanisms underlying tTG autoimmunity.

The IN Cell Analyzer 1000 possesses several distinguishing features that make it a valuable tool in research today. This fully automated high content screening (HCS) system introduced quantitative fluorescent microscopy with computerized image analysis for use in cell-based analysis. Previous studies have focused on live cell assays, where it has proven to be a powerful and robust method capable of providing reproducible, quantitative data. Using HCS as a tool to investigate antigen expression in duodenal biopsies, we developed a novel approach to tissue positioning and mapping. We adapted IN Cell Analyzer 1000's image acquisition and analysis software for the investigation of tissue transglutaminase (tTG) and smooth muscle alpha-actin (SM α-actin) staining in paraffin-embedded duodenal tissue sections from celiac patients and healthy controls. These innovations allowed a quantitative analysis of cellular structure and protein expression. The results from routine biopsy material indicated the intensity of protein expression was altered in celiac disease compared to normal biopsy material.

Recently, Xing et al1Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (511) Google Scholar demonstrated the role of cytotoxic T lymphocytes in alopecia areata (AA) and provided important mechanistic information on the pathogenic T-cell inflammatory pathways in patients with this autoimmune condition. They described 3 patients with AA successfully treated with the oral Janus kinase (JAK) family protein tyrosine kinase inhibitor ruxolitinib. The inhibitory effectiveness of this small molecule in vitro was also reported in a novel interferonopathy coined stimulator of interferon genes (STING)–associated vasculopathy, which is caused by a gain-of-function (GOF) mutation in the TMEM173 gene encoding STING, resulting in hyperactivation of the signal transducer and activator of transcription (STAT) 1/STAT2 signaling pathways.2Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Montealegre Sanchez G.A. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (762) Google ScholarWe now demonstrate the potential utility of this drug for AA and other symptoms occurring in the wider context of a genetic immunodeficiency syndrome associated with autoimmunity. We recently treated AA associated with chronic mucocutaneous candidiasis (CMC; Online Mendelian Inheritance in Man #614162)3Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (82) Google Scholar, 4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (486) Google Scholar in a 28-year-old woman also using ruxolitinib. Our patient had a sporadic autosomal dominant heterozygous GOF mutation, c.1159A>G p.Thr387Ala in exon 14 of STAT1 and had a 2-year history of AA. The AA had progressed despite previous intralesional corticosteroid injections, with greater than 40% scalp involvement (Fig 1, left panel). Our patient had recalcitrant oral candidiasis (CMC) since childhood, with frequent flares despite daily oral fluconazole, frequent throat and chest infections requiring antibiotics, a prior history of autoimmune hepatitis, and an episode in adolescence of severe laryngeal erythema multiforme with alopecia and full hair regrowth.STAT1 GOF caused by these mutations is mediated through enhanced phosphorylation caused by impaired nuclear dephosphorylation, resulting in increased expression of interferon-stimulated genes.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar Interestingly, patients with GOF STAT1 mutations have decreased expression of STAT3-stimulated genes (unpublished data), which might explain downstream reduction of IL-17 expression and consequent susceptibility to mucocutaneous Candida species infections.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar In canonical IFN-γ–JAK–STAT1 signaling, ligand engagement of the IFN-γ receptor leads to activation of receptor-associated JAK1 and JAK2. Therefore we hypothesized that JAK1/2 inhibition would target this pathway and could ameliorate both the CMC and the associated autoimmune AA phenotype in our patient. Ruxolitinib, a JAK1/2 inhibitor that is US Food and Drug Administration (FDA) approved for the treatment of myelofibrosis, targets JAK1/2 pathway signaling and activation of transcription (JAK-STAT) pathways.6Harrison C. Kiladjian J.J. Al-Ali H.K. Gisslinger H. Waltzman R. Stalbovskaya V. et al.JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis.N Engl J Med. 2012; 366: 787-798Crossref PubMed Scopus (1299) Google ScholarAfter 2 weeks of treatment with oral ruxolitinib (20 mg twice daily), our patient experienced dramatic hair regrowth in all affected areas of alopecia. The hair regrowth, which was uniform and thick, continued over the treatment course. Full hair regrowth was noted at 12 weeks and was sustained 6 months after completion of therapy (Fig 1, right panel). Unexpectedly, the patient also reported complete resolution of the oral candidiasis while taking ruxolitinib. The CMC returned within 2 weeks of discontinuing the treatment, despite oral fluconazole. The 3 patients with AA described by Xing et al1Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (511) Google Scholar demonstrated impressive clinical responses to ruxolitinib therapy. Our patient with AA demonstrated similar results with no adverse effects and also reported great relief of CMC symptoms.The heterozygous mutation reported here, c.1159A>G p.Thr387Ala in the DNA-binding domain of STAT1 in exon 14, has not been previously reported, although it has been previously found in patients with CMC (Anne Puel, personal communications). We confirmed GOF by demonstrating hyperphosphorylation of STAT1 (Tyr701) after stimulation with IFN-α (Fig 2), as previously demonstrated in other patients with CMC with GOF STAT1 mutations.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar GOF STAT1 mutations in the DNA-binding domain were first reported by Takezaki et al3Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (82) Google Scholar and subsequently repeatedly confirmed,7Soltesz B. Toth B. Shabashova N. Bondarenko A. Okada S. Cypowyj S. et al.New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.J Med Genet. 2013; 50: 567-578Crossref PubMed Scopus (79) Google Scholar although different STAT1 mutations in the DNA-binding domain have also been reported to cause loss of function.8Sharfe N. Nahum A. Newell A. Dadi H. Ngan B. Pereira S.L. et al.Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.J Allergy Clin Immunol. 2014; 133: 807-817Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Patients with CMC caused by underlying GOF STAT1 mutations have chronic Candida species infection of the skin and mucous membranes and are thought to account for more than half of patients presenting with CMC.9Puel A. Cypowyj S. Marodi L. Abel L. Picard C. Casanova J.L. Inborn errors of human IL-17 immunity underlie chronic mucocutaneous candidiasis.Curr Opin Allergy Clin Immunol. 2012; 12: 616-622Crossref PubMed Scopus (220) Google Scholar The increased susceptibility to fungal infections is likely linked to reduced L17A and IL-22 production, as documented by us and others10Ng W.F. von Delwig A. Carmichael A.J. Arkwright P.D. Abinun M. Cant A.J. et al.Impaired T(H)17 responses in patients with chronic mucocutaneous candidiasis with and without autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.J Allergy Clin Immunol. 2010; 126 (e1-4): 1006-1015Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar and also demonstrated for our current patient (see Fig E1 in this article's Online Repository at www.jacionline.org). However, the mechanisms through which STAT1 GOF mutations lead to fungal susceptibility are still unclear, although inhibition of STAT3 activation and the consequent reduction in RORC gene transcription resulting in reduced levels of TH17 cytokines are likely to play a role. Additional inhibition of IL-17 production by CD4+ T cells might be mediated by IL-27 activation of STAT1, leading to overexpression of the inhibitory molecule programmed death ligand (PDL)-1 on naive T cells, preventing their commitment to the TH17 lineage.11Romberg N. Morbach H. Lawrence M.G. Kim S. Kang I. Holland S.M. et al.Gain-of-function STAT1 mutations are associated with PD-L1 overexpression and a defect in B-cell survival.J Allergy Clin Immunol. 2013; 131: 1691-1693Abstract Full Text Full Text PDF PubMed Scopus (63) Google ScholarFig 2Western blot of PBMC lysates from healthy control subjects (HC) and our patient with CMC (CMC pt). Cells were left unstimulated or stimulated with IFN-α (1 × 103 IU/mL) for 30 minutes in the presence or absence of ruxolitinib at 1 μmol/L. GAPDH, Glyceraldehgyde-3-phosphate dehydrogenase; pSTAT1, phosphorylated STAT1; pSTAT3, phosphorylated STAT3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We provide data demonstrating hyperphosphorylation of STAT1 consistent with a GOF STAT1 mutation in addition to the patient's clinical presentation of CMC. Western blotting (Fig 2) demonstrates hyperphosphorylation of the patient's STAT1 after stimulation with IFN-α, as well as complete inhibition of phosphorylation with the addition of ruxolitinib in doses that correspond to serum levels achieved in patients receiving 20 mg twice daily, as also shown by using flow cytometry (see Fig E2 in this article's Online Repository at www.jacionline.org). Importantly, in our and other patients with CMC, we do not see constitutive STAT1 phosphorylation, as has been reported in patients with STING-associated vasculopathy syndrome caused by an underlying GOF mutation in the gene encoding STING.2Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Montealegre Sanchez G.A. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (762) Google Scholar In addition, STAT3 phosphorylation is not reduced in this or other patients with CMC due to an underlying GOF STAT1 mutation (unpublished data), suggesting that the decreased IL-17A and IL-22 production repeatedly reported in these patients has a cause downstream of STAT3 activation. Although we did not demonstrate improvement of IL-17 production in vitro by addition of ruxolitinib, the clinical improvement in the patient's CMC was remarkable. Ruxolitinib might have differential effects on STAT1 and STAT3 in vivo, perhaps more so at the lower concentrations seen in tissues. Alternatively, a far more complex interaction between STAT1 and STAT3 must be envisaged, confirming that the underlying mechanisms remain unclear.Our case suggests the potential therapeutic benefit of ruxolitinib in the GOF STAT1 genetic immunodeficiency/autoimmunity syndrome, which can present with additional severe and often insufficiently appreciated problems for which there is no current treatment, such as debilitating superficial eye disease leading to blindness, esophageal dysmotility, and esophageal/oral cancer. Although further evaluation on additional patients is clearly needed, this therapy might represent an ideal personalized treatment for patients with GOF mutations in STAT1.For detailed methods, please see the Methods section in this article's Online Repository at www.jacionline.org. Recently, Xing et al1Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (511) Google Scholar demonstrated the role of cytotoxic T lymphocytes in alopecia areata (AA) and provided important mechanistic information on the pathogenic T-cell inflammatory pathways in patients with this autoimmune condition. They described 3 patients with AA successfully treated with the oral Janus kinase (JAK) family protein tyrosine kinase inhibitor ruxolitinib. The inhibitory effectiveness of this small molecule in vitro was also reported in a novel interferonopathy coined stimulator of interferon genes (STING)–associated vasculopathy, which is caused by a gain-of-function (GOF) mutation in the TMEM173 gene encoding STING, resulting in hyperactivation of the signal transducer and activator of transcription (STAT) 1/STAT2 signaling pathways.2Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Montealegre Sanchez G.A. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (762) Google Scholar We now demonstrate the potential utility of this drug for AA and other symptoms occurring in the wider context of a genetic immunodeficiency syndrome associated with autoimmunity. We recently treated AA associated with chronic mucocutaneous candidiasis (CMC; Online Mendelian Inheritance in Man #614162)3Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (82) Google Scholar, 4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (486) Google Scholar in a 28-year-old woman also using ruxolitinib. Our patient had a sporadic autosomal dominant heterozygous GOF mutation, c.1159A>G p.Thr387Ala in exon 14 of STAT1 and had a 2-year history of AA. The AA had progressed despite previous intralesional corticosteroid injections, with greater than 40% scalp involvement (Fig 1, left panel). Our patient had recalcitrant oral candidiasis (CMC) since childhood, with frequent flares despite daily oral fluconazole, frequent throat and chest infections requiring antibiotics, a prior history of autoimmune hepatitis, and an episode in adolescence of severe laryngeal erythema multiforme with alopecia and full hair regrowth. STAT1 GOF caused by these mutations is mediated through enhanced phosphorylation caused by impaired nuclear dephosphorylation, resulting in increased expression of interferon-stimulated genes.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar Interestingly, patients with GOF STAT1 mutations have decreased expression of STAT3-stimulated genes (unpublished data), which might explain downstream reduction of IL-17 expression and consequent susceptibility to mucocutaneous Candida species infections.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar In canonical IFN-γ–JAK–STAT1 signaling, ligand engagement of the IFN-γ receptor leads to activation of receptor-associated JAK1 and JAK2. Therefore we hypothesized that JAK1/2 inhibition would target this pathway and could ameliorate both the CMC and the associated autoimmune AA phenotype in our patient. Ruxolitinib, a JAK1/2 inhibitor that is US Food and Drug Administration (FDA) approved for the treatment of myelofibrosis, targets JAK1/2 pathway signaling and activation of transcription (JAK-STAT) pathways.6Harrison C. Kiladjian J.J. Al-Ali H.K. Gisslinger H. Waltzman R. Stalbovskaya V. et al.JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis.N Engl J Med. 2012; 366: 787-798Crossref PubMed Scopus (1299) Google Scholar After 2 weeks of treatment with oral ruxolitinib (20 mg twice daily), our patient experienced dramatic hair regrowth in all affected areas of alopecia. The hair regrowth, which was uniform and thick, continued over the treatment course. Full hair regrowth was noted at 12 weeks and was sustained 6 months after completion of therapy (Fig 1, right panel). Unexpectedly, the patient also reported complete resolution of the oral candidiasis while taking ruxolitinib. The CMC returned within 2 weeks of discontinuing the treatment, despite oral fluconazole. The 3 patients with AA described by Xing et al1Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (511) Google Scholar demonstrated impressive clinical responses to ruxolitinib therapy. Our patient with AA demonstrated similar results with no adverse effects and also reported great relief of CMC symptoms. The heterozygous mutation reported here, c.1159A>G p.Thr387Ala in the DNA-binding domain of STAT1 in exon 14, has not been previously reported, although it has been previously found in patients with CMC (Anne Puel, personal communications). We confirmed GOF by demonstrating hyperphosphorylation of STAT1 (Tyr701) after stimulation with IFN-α (Fig 2), as previously demonstrated in other patients with CMC with GOF STAT1 mutations.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar GOF STAT1 mutations in the DNA-binding domain were first reported by Takezaki et al3Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (82) Google Scholar and subsequently repeatedly confirmed,7Soltesz B. Toth B. Shabashova N. Bondarenko A. Okada S. Cypowyj S. et al.New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.J Med Genet. 2013; 50: 567-578Crossref PubMed Scopus (79) Google Scholar although different STAT1 mutations in the DNA-binding domain have also been reported to cause loss of function.8Sharfe N. Nahum A. Newell A. Dadi H. Ngan B. Pereira S.L. et al.Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.J Allergy Clin Immunol. 2014; 133: 807-817Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Patients with CMC caused by underlying GOF STAT1 mutations have chronic Candida species infection of the skin and mucous membranes and are thought to account for more than half of patients presenting with CMC.9Puel A. Cypowyj S. Marodi L. Abel L. Picard C. Casanova J.L. Inborn errors of human IL-17 immunity underlie chronic mucocutaneous candidiasis.Curr Opin Allergy Clin Immunol. 2012; 12: 616-622Crossref PubMed Scopus (220) Google Scholar The increased susceptibility to fungal infections is likely linked to reduced L17A and IL-22 production, as documented by us and others10Ng W.F. von Delwig A. Carmichael A.J. Arkwright P.D. Abinun M. Cant A.J. et al.Impaired T(H)17 responses in patients with chronic mucocutaneous candidiasis with and without autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.J Allergy Clin Immunol. 2010; 126 (e1-4): 1006-1015Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar and also demonstrated for our current patient (see Fig E1 in this article's Online Repository at www.jacionline.org). However, the mechanisms through which STAT1 GOF mutations lead to fungal susceptibility are still unclear, although inhibition of STAT3 activation and the consequent reduction in RORC gene transcription resulting in reduced levels of TH17 cytokines are likely to play a role. Additional inhibition of IL-17 production by CD4+ T cells might be mediated by IL-27 activation of STAT1, leading to overexpression of the inhibitory molecule programmed death ligand (PDL)-1 on naive T cells, preventing their commitment to the TH17 lineage.11Romberg N. Morbach H. Lawrence M.G. Kim S. Kang I. Holland S.M. et al.Gain-of-function STAT1 mutations are associated with PD-L1 overexpression and a defect in B-cell survival.J Allergy Clin Immunol. 2013; 131: 1691-1693Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar We provide data demonstrating hyperphosphorylation of STAT1 consistent with a GOF STAT1 mutation in addition to the patient's clinical presentation of CMC. Western blotting (Fig 2) demonstrates hyperphosphorylation of the patient's STAT1 after stimulation with IFN-α, as well as complete inhibition of phosphorylation with the addition of ruxolitinib in doses that correspond to serum levels achieved in patients receiving 20 mg twice daily, as also shown by using flow cytometry (see Fig E2 in this article's Online Repository at www.jacionline.org). Importantly, in our and other patients with CMC, we do not see constitutive STAT1 phosphorylation, as has been reported in patients with STING-associated vasculopathy syndrome caused by an underlying GOF mutation in the gene encoding STING.2Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Montealegre Sanchez G.A. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (762) Google Scholar In addition, STAT3 phosphorylation is not reduced in this or other patients with CMC due to an underlying GOF STAT1 mutation (unpublished data), suggesting that the decreased IL-17A and IL-22 production repeatedly reported in these patients has a cause downstream of STAT3 activation. Although we did not demonstrate improvement of IL-17 production in vitro by addition of ruxolitinib, the clinical improvement in the patient's CMC was remarkable. Ruxolitinib might have differential effects on STAT1 and STAT3 in vivo, perhaps more so at the lower concentrations seen in tissues. Alternatively, a far more complex interaction between STAT1 and STAT3 must be envisaged, confirming that the underlying mechanisms remain unclear. Our case suggests the potential therapeutic benefit of ruxolitinib in the GOF STAT1 genetic immunodeficiency/autoimmunity syndrome, which can present with additional severe and often insufficiently appreciated problems for which there is no current treatment, such as debilitating superficial eye disease leading to blindness, esophageal dysmotility, and esophageal/oral cancer. Although further evaluation on additional patients is clearly needed, this therapy might represent an ideal personalized treatment for patients with GOF mutations in STAT1. For detailed methods, please see the Methods section in this article's Online Repository at www.jacionline.org. We thank Lisa Turnbull, Clinical Scientist in our Northern Molecular Genetics Service, Newcastle, United Kingdom, for assistance with genetic analysis of our patient. MethodsWestern blottingEqual amounts of protein were separated on an SDS-PAGE gel, transferred to membranes, and probed with antibodies against STAT1 (9172), phosphorylated STAT1 (Tyr701/9171), STAT3 (4904), phosphorylated STAT3 (Tyr705/9145), and glyceraldehgyde-3-phosphate dehydrogenase (GAPDH 4134; all from Cell Signaling, Danvers, Mass) overnight at 4°C, followed by horseradish peroxidase–conjugated goat anti-rabbit antibody (P0448; DAKO, Glostrup, Denmark). Proteins were visualized with the Immobilon Western detection system (Millipore, Temecula, Calif).Flow cytometryPBMCs were fixed (Lyse/Fix buffer; BD Biosciences, San Jose, Calif) and permeablized (Phosflow Perm buffer III, BD Biosciences), according to the manufacturer's instructions. Cells were stained with antibodies to phosphorylated STAT1 (pSTAT1-Alexa Fluor 647, pY701) phosphorylated STAT3 (pSTAT3-Alexa Fluor 488, pY705), CD3– Pacific Blue, and CD4-Bv510 (all from BD Biosciences) in PBS containing 0.2% BSA for 1 hour. Data were collected with a FACSCaliber (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, Ore).Cytokine productionPBMCs from healthy control subjects and our patient with CMC were stimulated with 1:15,000 live Candida albicans (18804; ATCC, Manassas, Va) or PHA (2 mg/mL) or left unstimulated in the presence or absence of 1 μmol/L ruxolitinib (Selleckchem, Houston, Tex) for 5 days in RPMI 10% FCS and amphotericin (2.5 μg/mL; Sigma, St Louis, Mo) added on days 0 and 3. Supernatants were collected, and cytokine levels were measured as per the manufacturer's instructions with Ready-Set-Go kits (eBioscience, San Diego, Calif).Ethical approvalFig E2Flow cytometric trace of phosphorylated STAT1 (pSTAT1) gated on CD3+ lymphocytes. Whole blood from a patient with CMC was stimulated with IFN-γ (1 × 103 IU/mL) for 15 minutes with and without ruxolitinib (Rx) or left unstimulated and then stained with antibodies to CD3–Pacific Blue and pSTAT1–Alexa Fluor 647 antibody by using the BD Biosciences Phosphlow kit.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western blottingEqual amounts of protein were separated on an SDS-PAGE gel, transferred to membranes, and probed with antibodies against STAT1 (9172), phosphorylated STAT1 (Tyr701/9171), STAT3 (4904), phosphorylated STAT3 (Tyr705/9145), and glyceraldehgyde-3-phosphate dehydrogenase (GAPDH 4134; all from Cell Signaling, Danvers, Mass) overnight at 4°C, followed by horseradish peroxidase–conjugated goat anti-rabbit antibody (P0448; DAKO, Glostrup, Denmark). Proteins were visualized with the Immobilon Western detection system (Millipore, Temecula, Calif). Equal amounts of protein were separated on an SDS-PAGE gel, transferred to membranes, and probed with antibodies against STAT1 (9172), phosphorylated STAT1 (Tyr701/9171), STAT3 (4904), phosphorylated STAT3 (Tyr705/9145), and glyceraldehgyde-3-phosphate dehydrogenase (GAPDH 4134; all from Cell Signaling, Danvers, Mass) overnight at 4°C, followed by horseradish peroxidase–conjugated goat anti-rabbit antibody (P0448; DAKO, Glostrup, Denmark). Proteins were visualized with the Immobilon Western detection system (Millipore, Temecula, Calif). Flow cytometryPBMCs were fixed (Lyse/Fix buffer; BD Biosciences, San Jose, Calif) and permeablized (Phosflow Perm buffer III, BD Biosciences), according to the manufacturer's instructions. Cells were stained with antibodies to phosphorylated STAT1 (pSTAT1-Alexa Fluor 647, pY701) phosphorylated STAT3 (pSTAT3-Alexa Fluor 488, pY705), CD3– Pacific Blue, and CD4-Bv510 (all from BD Biosciences) in PBS containing 0.2% BSA for 1 hour. Data were collected with a FACSCaliber (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, Ore). PBMCs were fixed (Lyse/Fix buffer; BD Biosciences, San Jose, Calif) and permeablized (Phosflow Perm buffer III, BD Biosciences), according to the manufacturer's instructions. Cells were stained with antibodies to phosphorylated STAT1 (pSTAT1-Alexa Fluor 647, pY701) phosphorylated STAT3 (pSTAT3-Alexa Fluor 488, pY705), CD3– Pacific Blue, and CD4-Bv510 (all from BD Biosciences) in PBS containing 0.2% BSA for 1 hour. Data were collected with a FACSCaliber (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, Ore). Cytokine productionPBMCs from healthy control subjects and our patient with CMC were stimulated with 1:15,000 live Candida albicans (18804; ATCC, Manassas, Va) or PHA (2 mg/mL) or left unstimulated in the presence or absence of 1 μmol/L ruxolitinib (Selleckchem, Houston, Tex) for 5 days in RPMI 10% FCS and amphotericin (2.5 μg/mL; Sigma, St Louis, Mo) added on days 0 and 3. Supernatants were collected, and cytokine levels were measured as per the manufacturer's instructions with Ready-Set-Go kits (eBioscience, San Diego, Calif). PBMCs from healthy control subjects and our patient with CMC were stimulated with 1:15,000 live Candida albicans (18804; ATCC, Manassas, Va) or PHA (2 mg/mL) or left unstimulated in the presence or absence of 1 μmol/L ruxolitinib (Selleckchem, Houston, Tex) for 5 days in RPMI 10% FCS and amphotericin (2.5 μg/mL; Sigma, St Louis, Mo) added on days 0 and 3. Supernatants were collected, and cytokine levels were measured as per the manufacturer's instructions with Ready-Set-Go kits (eBioscience, San Diego, Calif). Ethical approval

Organ culture is a valuable technique in celiac disease research. It provides the opportunity to examine interactions between different cell types during the disease process without the need for invasive in vivo studies. Biopsies are maintained in an oxygen-rich environment, in contact with, but not submerged in, culture medium. A very straightforward and successful method of organ culture is described here.

The autoinflammatory syndromes are a family of disorders of dysregulated innate immunity that result in the overproduction of pro-inflammatory cytokines, in particular IL-1β [1]. The protean clinical manifestations of these diseases include: recurrent episodic fever, myalgia, arthralgia and arthritis, general malaise, and atypical urticaria or cutaneous features resembling a number of neutrophilic dermatoses, mainly pyoderma gangrenosum [2, 3]. Autoantibodies are characteristically absent. Symptom [...]