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Background The most common cause of primary adrenal failure (Addison’s disease) in the Western world is autoimmunity characterized by autoantibodies against the steroidogenic enzyme 21-hydroxylase (CYP21A2, 21OH). Detection of 21OH-autoantibodies is currently used for aetiological diagnosis, but how levels of 21OH-autoantibodies vary over time is not known. Setting Samples from the national Norwegian Addison’s Registry and Biobank established in 1996 (n = 711). Multi-parameter modelling of the course of 21OH-autoantibody indices over time. Results 21OH-autoantibody positivity is remarkably stable, and >90% of the patients are still positive 30 years after diagnosis. Even though the antibody levels decline with disease duration, it is only rarely that this downturn reaches negativity. 21OH-autoantibody indices are affected by age at diagnosis, sex, type of Addison’s disease (isolated vs autoimmune polyendocrine syndrome type I or II) and HLA genotype. Conclusion 21OH-autoantibodies are reliable and robust markers for autoimmune Addison’s disease, linked to HLA risk genotype. However, a negative test in patients with long disease duration does not exclude autoimmune aetiology.

Background Glioblastoma (GBM) is an aggressive malignant brain tumor where median survival is approximately 15 months after best available multimodal treatment. Recurrence is inevitable, largely due to O6 methylguanine DNA methyltransferase (MGMT) that renders the tumors resistant to temozolomide (TMZ). We hypothesized that pretreatment with bortezomib (BTZ) 48 hours prior to TMZ to deplete MGMT levels would be safe and tolerated by patients with recurrent GBM harboring unmethylated MGMT promoter. The secondary objective was to investigate whether 26S proteasome blockade may enhance differentiation of cytotoxic immune subsets to impact treatment responses measured by radiological criteria and clinical outcomes. Methods Ten patients received intravenous BTZ 1.3 mg/m2 on days 1, 4, and 7 during each 4th weekly TMZ‐chemotherapy starting on day 3 and escalated from 150 mg/m2 per oral 5 days/wk via 175 to 200 mg/m2 in cycles 1, 2, and 3, respectively. Adverse events and quality of life were evaluated by CTCAE and EQ‐5D‐5L questionnaire, and immunological biomarkers evaluated by flow cytometry and Luminex enzyme‐linked immunosorbent assay. Results Sequential BTZ + TMZ therapy was safe and well tolerated. Pain and performance of daily activities had greatest impact on patients' self‐reported quality of life and were inversely correlated with Karnofsky performance status. Patients segregated a priori into three groups, where group 1 displayed stable clinical symptoms and/or slower magnetic resonance imaging radiological progression, expanded CD4+ effector T‐cells that attenuated cytotoxic T‐lymphocyte associated protein‐4 and PD‐1 expression and secreted interferon γ and tumor necrosis factor α in situ and ex vivo upon stimulation with PMA/ionomycin. In contrast, rapidly progressing group 2 patients exhibited tolerised T‐cell phenotypes characterized by fourfold to sixfold higher interleukin 4 (IL‐4) and IL‐10 Th‐2 cytokines after BTZ + TMZ treatment, where group 3 patients exhibited intermediate clinical/radiological responses. Conclusion Sequential BTZ + TMZ treatment is safe and promotes Th1‐driven immunological responses in selected patients with improved clinical outcomes (Clinicaltrial.gov (NCT03643549))., Schematic of trial schedule. A, Timeline showing BORTEM‐17 treatment regimen. Bortezomib administered intravenous at days 1, 4, and 7 (48 hours pretreatment to deplete MGMT protein) before target TMZ 200 mg/m2 dose for 5 days (from and including days 3‐7), repeated in six cycles. In n = 3 patients per each dose 150 vs 175 vs 200 mg/m2 TMZ in dose pathfinding, safety evaluation. Clinical chemistry for renal, hepatic, and bone marrow monitoring for toxicity based on CTCAE v. 4.03. MRI tumor monitoring radiological response assessment based on RANO criteria. Rationale for sequential treatment schedule based on preclinical data. B, sequential administration in BORTEM‐17 clinical trial vs (C) previous studies where BTZ on days 1, 4, 8, and 11 was given concomitantly with TMZ from day 1 to 5, when MGMT levels were high. Dashed boxes mark days when TMZ doses might be more effective, (B) all five doses vs (C) three doses every month. BTZ, bortezomib; CBC Diff, complete blood count with differential test; CMP, comprehensive metabolic panel; KPS, Karnofsky performance score; LFT, liver function test; MGMT, O6‐methyl guanine DNA methyltransferase; MRI, magnetic resonance imaging; NANO, neurologic assessment in neuro‐oncology; QoL, quality of life; TMZ, temozolomide.

KARS1 encodes a lysyl-transfer RNA synthetase (LysRS) that links lysine to its cognate tRNA. Two different KARS1 isoforms exert functional effects in cytosol and mitochondria. Bi-allelic pathogenic variants in KARS1 have been associated to sensorineural hearing and visual loss, neuropathy, seizures, and leukodystrophy. We report the clinical, biochemical and neuroradiological features of nine individuals with KARS1-related disorder carrying 12 different variants with nine of them being novel. The consequences of these variants on the cytosol and/or mitochondrial LysRS were functionally validated in yeast mutants. Most cases presented with severe neurological features including congenital and progressive microcephaly, seizures, developmental delay/intellectual disability, and cerebral atrophy. Oculo-motor dysfunction and immuno-hematological problems were present in six and three cases, respectively. A yeast growth defect of variable severity was detected for most variants on both cytosolic and mitochondrial isoforms. The detrimental effects of two variants on yeast growth were partially rescued by lysine supplementation. Congenital progressive microcephaly, oculo-motor dysfunction and immuno-hematological problems are emerging phenotypes in KARS1-related disorders. The data in yeast emphasize the role of both mitochondrial and cytosolic isoforms in the pathogenesis of KARS1-related disorder and supports the therapeutic potential of lysine supplementation at least in a subset of patients. This article is protected by copyright. All rights reserved.

Background No reliable biomarkers exist to guide glucocorticoid (GC) replacement treatment in autoimmune Addison’s disease (AAD), leading to overtreatment with alarming and persistent side effects or undertreatment, which could be fatal. Objective To explore changes in gene expression following different GC replacement doses as a means of identifying candidate transcriptional biomarkers to guide GC replacement in AAD. Methods Step 1: Global microarray expression analysis on RNA from whole blood before and after intravenous infusion of 100 mg hydrocortisone (HC) in 10 patients with AAD. In 3 of the most highly upregulated genes, we performed real-time PCR (rt-PCR) to compare gene expression levels before and 3, 4, and 6 hours after the HC infusion. Step 2: Rt-PCR to compare expression levels of 93 GC-regulated genes in normal versus very low morning cortisol levels in 27 patients with AAD. Results Step 1: Two hours after infusion of 100 mg HC, there was a marked increase in FKBP5, MMP9, and DSIPI expression levels. MMP9 and DSIPI expression levels correlated with serum cortisol. Step 2: Expression levels of CEBPB, DDIT4, FKBP5, DSIPI, and VDR were increased and levels of ADARB1, ARIDB5, and POU2F1 decreased in normal versus very low morning cortisol. Normal serum cortisol levels positively correlated with DSIPI, DDIT4, and FKBP5 expression. Conclusions We introduce gene expression as a novel approach to guide GC replacement in AAD. We suggest that gene expression of DSIPI, DDIT4, and FKBP5 are particularly promising candidate biomarkers of GC replacement, followed by MMP9, CEBPB, VDR, ADARB1, ARID5B, and POU2F1.

Addison's disease is a prototypic organ-specific autoimmune disease affecting the adrenal cortex. The CXC chemokine ligand 10 (CXCL10) is expressed early in viral infections, and is produced by primary adrenocortical cells stimulated by certain cytokines. CXCL10 is also elevated in the serum of Addison's disease patients. We therefore investigated if the viral RNA substitute polyinosine-polycytidylic acid (poly (I:C)) could influence the cytokine induced production of CXCL10 by adrenocortical cells. We found that poly (I:C) could induce CXCL10 in NCI-H295R adrenocortical carcinoma cells, either alone or synergistically along with cytokines interferon-γ and tumor necrosis factor-α. This effect was found to be mediated by toll-like receptor 3 and both nuclear factor κB (NFκB) and signal transducer and activator of transcription-1 (STAT1), but not type I interferons, seemed to be involved. We propose that the combination of environmental and endogenous factors presented here, could contribute to the multifactorial pathogenesis of autoimmune Addison's disease.

Context: Autoimmune polyendocrine syndrome type 1 (APS1) is a childhood-onset monogenic disease defined by the presence of two of the three major components: hypoparathyroidism, primary adrenocortical insufficiency, and chronic mucocutaneous candidiasis (CMC). Information on longitudinal follow-up of APS1 is sparse. Objective: To describe the phenotypes of APS1 and correlate the clinical features with autoantibody profiles and autoimmune regulator (AIRE) mutations during extended follow-up (1996–2016). Patients: All known Norwegian patients with APS1. Results: Fifty-two patients from 34 families were identified. The majority presented with one of the major disease components during childhood. Enamel hypoplasia, hypoparathyroidism, and CMC were the most frequent components. With age, most patients presented three to five disease manifestations, although some had milder phenotypes diagnosed in adulthood. Fifteen of the patients died during follow-up (median age at death, 34 years) or were deceased siblings with a high probability of undisclosed APS1. All except three had interferon-ω) autoantibodies, and all had organ-specific autoantibodies. The most common AIRE mutation was c.967_979del13, found in homozygosity in 15 patients. A mild phenotype was associated with the splice mutation c.879+1G>A. Primary adrenocortical insufficiency and type 1 diabetes were associated with protective human leucocyte antigen genotypes. Conclusions: Multiple presumable autoimmune manifestations, in particular hypoparathyroidism, CMC, and enamel hypoplasia, should prompt further diagnostic workup using autoantibody analyses (eg, interferon-ω) and AIRE sequencing to reveal APS1, even in adults. Treatment is complicated, and mortality is high. Structured follow-up should be performed in a specialized center., Extended follow-up (1996–2016) of 52 Norwegian APS-1 patients describing clinical manifestations, natural course including mortality, autoantibody profiles and Autoimmune Regulator (AIRE) mutations.

Autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) is a monogenic autoimmune disease caused by mutations in the AIRE gene. Although mainly an endocrine disease, a substantial fraction of patients have gastrointestinal manifestations. In this study, we have examined the role of anticommensal responses and their regulation. APECED patients had increased levels of Abs against Saccharomyces cerevisiae (p < 0.0001) and against several species of commensal gut bacteria, but not against species predominantly associated with other locations. The anticommensal Ab levels did not correlate with gastrointestinal autoantibodies, neutralizing anti–IL-17 or –IL-22 Abs, or gastrointestinal symptoms, although scarcity of the available clinical data suggests that further study is required. However, the anti–S. cerevisiae Ab levels showed a significant inverse correlation with FOXP3 expression levels in regulatory T cells (Treg), previously shown to be dysfunctional in APECED. The correlation was strongest in the activated CD45RO+ population (ρ = −0.706; p < 0.01). APECED patients also had decreased numbers of FOXP3+ cells in gut biopsies. These results show that APECED patients develop early and sustained responses to gut microbial Ags in a pattern reminiscent of Crohn’s disease. This abnormal immune recognition of gut commensals is linked to a systemic Treg defect, which is also reflected as a local decrease of gut-associated Treg. To our knowledge, these data are the first to show dysregulated responses to non-self commensal Ags in APECED and indicate that AIRE contributes to the regulation of gut homeostasis, at least indirectly. The data also raise the possibility of persistent microbial stimulation as a contributing factor in the pathogenesis of APECED.

In primary adrenocortical failure (Addison's disease) caused by autoimmunity, autoantibodies to the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are detected in the majority of patients. It is currently uncertain whether the autoantibodies themselves participate in the pathogenesis, or if they merely reflect an on-going T cell mediated response. The identification of T cells reactive with 21OH, if any, has been hampered by the lack of a high-quality antigen. In the current study recombinant human 21OH has been expressed in Spodoptera frugiperda insect cells using a baculovirus expression system. Recombinant enzymatically active 21OH was purified to apparent homogeneity by immobilized metal ion affinity chromatography. The purified enzyme was highly immunogenic in immunized SJL/J mice, and immune responses to 21OH-derived peptides assayed as T cell proliferation and interferon gamma production could be invoked after priming with the recombinant protein. Furthermore, purified 21OH was recognized by sera from patients with autoimmune Addison's disease, and it could block the binding of radiolabeled in vitro translated 21OH in a sensitive fluid-phase radioimmunoassay. We conclude that the recombinant preparation of 21OH presented here is of sufficient purity and quality to be used for studies of cellular and humoral immunity in autoimmune Addison's disease.

Autoimmune Addison's disease is thought to result from T cell mediated autoimmunity. Autoantibodies against the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are found in most patients, and 21OH is therefore a likely target for antigen-specific T cells.The aim was to study cellular immunity to 21OH and its associations with 21OH autoantibodies and human leukocyte antigen alleles in autoimmune Addison's disease.Peripheral blood mononuclear cells were collected from 33 patients with autoimmune Addison's disease and 21 controls. Cellular proliferation and production of cytokines in response to stimulation with 21OH or 21OH-derived peptides were tested.Cellular proliferation (P = 0.0009) and secretion of interferon-gamma (P0.0001) in response to 21OH was significantly higher in patients compared to healthy controls and associated with the presence of 21OH autoantibodies (P = 0.0052). Furthermore, the 21OH-specific production of interferon-gamma was enhanced in the presence of 21OH autoantibodies. This effect was partially inhibited by antibodies against the Fc receptor for IgG, CD32. Moreover, mature dendritic cells proved superior to the other antigen-presenting cells in invoking cellular responses to 21OH. An association between cellular immunity to 21OH and the high-risk HLA genotype for Addison's disease, DRB1*0301-DQ2/DRB1*0404-DQ8, was observed (P = 0.0089). Finally, a significant association between the DRB1*0404-DQ8 haplotype and cellular responses to a 21OH-derived peptide predicted to bind to DRB1*0404 was detected (P = 0.0055).Patients with autoimmune Addison's disease have circulating 21OH-specific T cells, with amino acids 342-361 of 21OH possibly constituting a disease-specific epitope presented by HLA-DRB1*0404.

SummaryThe autoimmune regulator (AIRE) gene is crucial for establishing central immunological tolerance and preventing autoimmunity. Mutations in AIRE cause a rare autosomal-recessive disease, autoimmune polyendocrine syndrome type 1 (APS-1), distinguished by multi-organ autoimmunity. We have identified multiple cases and families with mono-allelic mutations in the first plant homeodomain (PHD1) zinc finger of AIRE that followed dominant inheritance, typically characterized by later onset, milder phenotypes, and reduced penetrance compared to classical APS-1. These missense PHD1 mutations suppressed gene expression driven by wild-type AIRE in a dominant-negative manner, unlike CARD or truncated AIRE mutants that lacked such dominant capacity. Exome array analysis revealed that the PHD1 dominant mutants were found with relatively high frequency (>0.0008) in mixed populations. Our results provide insight into the molecular action of AIRE and demonstrate that disease-causing mutations in the AIRE locus are more common than previously appreciated and cause more variable autoimmune phenotypes.

Autoimmune Addison's disease (AAD) is a disorder caused by an immunological attack on the adrenal cortex. The interferon (IFN)-inducible chemokine CXCL10 is elevated in serum of AAD patients, suggesting a peripheral IFN signature. However, CXCL10 can also be induced in adrenocortical cells stimulated with IFNs, cytokines, or microbial components. We therefore investigated whether peripheral blood mononuclear cells (PBMCs) from AAD patients display an enhanced propensity to produce CXCL10 and the related chemokine CXCL9, after stimulation with type I or II IFNs or the IFN inducer poly (I:C). Although serum levels of CXCL10 and CXCL9 were significantly elevated in patients compared with controls, IFN stimulated patient PBMC produced significantly less CXCL10/CXCL9 than control PBMC. Low CXCL10 production was not significantly associated with medication, disease duration, or comorbidities, but the low production of poly (I:C)-induced CXCL10 among patients was associated with an AAD risk allele in the phosphatase nonreceptor type 22 (PTPN22) gene. PBMC levels of total STAT1 and -2, and IFN-induced phosphorylated STAT1 and -2, were not significantly different between patients and controls. We conclude that PBMC from patients with AAD are deficient in their response to IFNs, and that the adrenal cortex itself may be responsible for the increased serum levels of CXCL10. publishedVersion

Background Autoimmune Addison’s disease (AAD) is caused by multiple genetic and environmental factors. Variants of genes encoding immunologically important proteins such as the HLA molecules are strongly associated with AAD, but any environmental risk factors have yet to be defined. We hypothesized that primary or reactivating infections with cytomegalovirus (CMV) could represent an environmental risk factor in AAD, and that CMV specific CD8+ T cell responses may be dysregulated, possibly leading to a suboptimal control of CMV. In particular, the objective was to assess the HLA-B8 restricted CD8+ T cell response to CMV since this HLA class I variant is a genetic risk factor for AAD. Methods To examine the CD8+ T cell response in detail, we analyzed the HLA-A2 and HLA-B8 restricted responses in AAD patients and healthy controls seropositive for CMV antibodies using HLA multimer technology, IFN-γ ELISpot and a CD107a based degranulation assay. Results No differences between patients and controls were found in functions or frequencies of CMV-specific T cells, regardless if the analyses were performed ex vivo or after in vitro stimulation and expansion. However, individual patients showed signs of reactivating CMV infection correlating with poor CD8+ T cell responses to the virus, and a concomitant upregulation of interferon regulated genes in peripheral blood cells. Several recently diagnosed AAD patients also showed serological signs of ongoing primary CMV infection. Conclusions CMV infection does not appear to be a major environmental risk factor in AAD, but may represent a precipitating factor in individual patients. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0822-z) contains supplementary material, which is available to authorized users.