Glucocorticoid-induced adrenal insufficiency after therapy with intravenous methylprednisolone in patients with moderate-to-severe and active Graves' orbitopathy: assessment with a low-dose corticotropin test.

PURPOSE: We aimed to assess adrenal function following treatment of moderate-to-severe and active Graves' orbitopathy (GO) with intravenous methylprednisolone (IVMP) in weekly pulses in a cumulative dose of 4.5 or 7.5 g. We evaluated the impact of IVMP pulses on adrenal reserve using a low-dose (1 µg) ACTH stimulation test (LDT) for the first time. METHODS: In this prospective study we evaluated adrenal function in 21 patients with moderate-to-severe and active GO treated with 12 weekly IVMP pulses according to the European Group on Graves' Orbitopathy (EUGOGO) recommendations. We assessed serum cortisol, plasma adrenocorticotropic hormone (ACTH), and dehydroepiandrosterone sulfate (DHEA-S) levels before the 1st and 12th IVMP pulse. We performed dynamic testing using LDT before the 12th IVMP pulse in all patients. In those who failed LDT, adrenal function was reassessed with LDT and the overnight metyrapone test after 4-7 weeks. RESULTS: Two patients failed to achieve serum cortisol levels ≥ 18.1 µg/dL at 30 and 60 min in LDT and were diagnosed with glucocorticoid-induced adrenal insufficiency (GC-induced AI). They were recommended to take hydrocortisone in situations of acute stress. Both patients were reassessed within 4-7 weeks after treatment cessation and showed an adequate response in LDT and overnight metyrapone test. We observed a statistically significant decrease in DHEA-S levels (p = 0.004) before the 12th IVMP pulse compared to baseline in all patients. CONCLUSION: For the first time, our research shows that administering IVMP in 12 weekly pulses can result in GC-induced AI. We suggest that patients should undergo careful evaluation for GC-induced AI, including LDT, after therapy with IVMP according to EUGOGO guidelines. Screening for altered adrenal reserve could prevent life-threatening complications, particularly during acute stress situations.

Dysthyroid optic neuropathy: emerging treatment strategies.

PURPOSE: Dysthyroid optic neuropathy (DON) is a rare sight-threatening complication of Graves' disease. First-line treatment for DON consists of high-dose intravenous methylprednisolone (ivMP), followed by immediate orbital decompression (OD) if the response is poor or absent as recommended by the 2021 European Group on Graves' orbitopathy guidelines. The safety and efficacy of the proposed therapy have been proven. However, consensus regarding possible therapeutic options for patients with contraindications to ivMP/OD or resistant form of disease is missing. This paper aims to provide and summarize all available data regarding possible alternative treatment strategies for DON. METHODS: A comprehensive literature search within an electronic database was performed including data published until December 2022. RESULTS: Overall, 52 articles describing use of emerging therapeutic strategies for DON were identified. Collected evidence indicates that biologics, including teprotumumab and tocilizumab, may be considered as an important possible treatment option for DON patients. Rituximab should be avoided in DON due to conflicting data and risk of adverse events. Orbital radiotherapy could be beneficial for patients with restricted ocular motility classified as poor surgical candidates. CONCLUSION: Only a limited number of studies have been dedicated to the therapy of DON, mostly retrospective with a small sample size. Clear criteria regarding diagnosis and resolution of DON do not exist, which restricts comparison of therapeutic outcomes. Randomized clinical trials and comparison studies with long-term follow-ups are necessary to verify the safety and efficacy of each therapeutic option for DON.

High-dose intravenous methylprednisolone therapy in patients with Graves' orbitopathy is associated with the increased activity of factor VIII.

PURPOSE: Venous thromboembolic events (VTE), with their life-threatening manifestation as pulmonary embolism, are potential adverse effects of intravenous methylprednisolone (IVMP) pulse therapy, partially due to a hypercoagulable state. The aim of the study was to analyze the influence of IVMP on selected hemostatic parameters in patients with moderate-to-severe Graves' orbitopathy (GO). METHODS: 26 euthyroid patients with GO were treated with 12 pulses of IVMP (6 × 0.5, 6 × 0.25 g every week). Hemostatic variables [factor (F) II, FV, FVII, FVIII, fibrinogen, antithrombin, activated partial thromboplastin time (aPTT), prothrombin time, international normalized ratio of prothrombin time, platelets and D-dimer] were analysed before, 24 and 48 h after 1st, 6th and 12th pulse. RESULTS: A constant, transient trend in changes of some hemostatic variables was observed after all assessed pulses. We discovered an increase in median activity of FVIII 24 and 48 h after pulses, with a shortening of aPTT 24 h after each IVMP pulse (p < 0.00005). FVII decreased 24 h after each pulse (p < 0.0005 after 1st and 12th, p < 0.00005 after 6th). Fibrinogen level decreased 48 h after each pulse (P < 0.00005). We did not observe any statistically significant changes in hemostatic parameters in the long-term evaluation. Therapy was concluded in one patient after the 9th pulse due to pulmonary embolism. CONCLUSIONS: The increase of FVIII activity is a consequence of treatment with IVMP and occurs after each pulse. In patients with additional risk factors of VTE, anticoagulation prophylaxis should be considered.

rs3827440, a nonsynonymous single nucleotide polymorphism within GPR174 gene in X chromosome, is associated with Graves' disease in Polish Caucasian population.

Recently Chu et al. conducted a two-stage genome wide association study in Chinese that identified a novel X-linked Graves' disease (GD) susceptibility marker at rs3827440 - a nonsynonymous (P162S) nucleotide transition (519C

Plasticity of Drosophila paired function.

The Drosophila Paired (Prd) transcription factor has homeodomain (HD) and paired domain (PD) DNA-binding activities required for in vivo function. Correspondingly, Prd activation of late even-skipped (eve) expression occurs through a conserved target sequence (PTE) with HD and PD half sites, both of which are required for activation. To investigate the relationship between the HD and PD, and their roles in conferring specificity to Prd function, we tested altered versions of the Prd protein and of the PTE target site using in vivo assays in embryos. We found that function through PTE was constrained by the targeting specifications of both the HD and PD as well as the spatial relationship between these two domains. PTE function was also constrained by the spacing between the target half sites for the PD and HD, although surprisingly, late eve activation was retained when PTE was replaced by in vitro optimized binding sites for either the PD alone or for an HD dimer. In contrast to late eve regulation, other Prd targets tolerated more changes in the Prd protein, suggesting that their target sequences may be qualitatively different from PTE.

Drosophila Paired regulates late even-skipped expression through a composite binding site for the paired domain and the homeodomain.

The even-skipped (eve) pair-rule gene plays a key role in the establishment of the anterior-posterior segmental pattern of the Drosophila embryo. The continuously changing pattern of eve expression can be resolved into two phases. Early expression consists of seven broad stripes in the blastoderm embryo, while late expression, which occurs after cellularization, consists of narrow stripes with sharp anterior borders that coincide with the odd-numbered parasegment boundaries. Previous studies have shown that these two phases are controlled by separate classes of cis elements in the eve promoter. Early stripes are expressed by multiple stripe-specific elements under the control of maternal-effect genes and gap genes, while late stripes are expressed by a single regulatory element, the 'late element', under the control of pair-rule genes including eve itself. We report here that paired (prd), a pair-rule gene which had been considered to be below eve in the regulatory hierarchy of pair-rule genes, in fact plays a critical role in the regulation of late eve expression. Transgenic analysis shows that this regulation is largely mediated by an evolutionarily conserved sequence within the late element termed PTE (Paired Target Element). In vitro analysis shows that the Prd protein binds strongly to this sequence. Interestingly, PTE contains juxtaposed binding sites for the two DNA-binding domains of the Prd protein, the paired domain and the homeodomain. Mutagenesis of either binding site leads to significant reduction in the activity of the late element, indicating that both DNA-binding domains in the Paired protein are required for regulation.

Both the paired domain and homeodomain are required for in vivo function of Drosophila Paired.

Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNA-binding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNA-binding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.