Assessment of Rapid Morphological Changes Associated with Elevated cAMP Levels in Human Orbital Fibroblasts

Orbital fibroblasts exhibit a phenotype distinct from that of other types of fibroblasts. Addition of prostaglandin E₂(PGE₂) to culture medium elicits a dramatic change in orbital fibroblast morphology. That response is mediated through the generation of cAMP. Orbital fibroblasts can generate high levels of PGE₂through induction by proinflammatory cytokines of prostaglandin endoperoxide H synthase-2 (PGHS-2). Here we compare the influence on fibroblast morphology of exogenous PGE₂, forskolin, and 8-br-cAMP to that mediated through PGHS-2 induction by a lymphocyte-derived cytokine. Within a few hours, orbital fibroblasts treated with any of these test compounds appear under phase-contrast microscopy to exhibit a stellate morphology. When these changes were assessed quantitatively by electric cell–substrate impedance sensing (ECIS), it became evident that 8-br-cAMP, forskolin, and PGE₂initiated shape changes within 30 min of addition to the culture medium, while effects of the cytokine were first evident after approximately 3.5 h. Dermal fibroblasts failed to respond to any of these compounds with regard to changes in cellular morphology. Analysis of micromotion, manifested as small impedance fluctuations, revealed that orbital fibroblasts treated with 8-br-cAMP exhibit less motion than did untreated cells. These results suggest that orbital fibroblast shape can be altered by several compounds known to alter intracellular cAMP levels. They demonstrate the utility of ECIS in the assessment of very rapid and dynamic cellular events associated with changes in cell morphology.     

The Incidence of Graves’ Hyperthyroidism Before and After COVID-19 Messenger RNA Vaccination

ObjectiveCase reports of postvaccine early-onset Graves’ hyperthyroidism (PVGD) after the administration of COVID-19 vaccination have emerged. Our aim was to investigate whether the incidence of Graves’ hyperthyroidism (GD) has increased after the introduction of COVID-19 vaccination.MethodsWe compared the incidence of new-onset GD at a single academic center during 2 periods: December 2017 to October 2019 and December 2020 to October 2022, ie, before and after the implementation of COVID-19 vaccinations. We defined PVGD as laboratory-confirmed hyperthyroidism and GD within 4 weeks after the vaccination or clear onset of symptoms of thyrotoxicosis within 4 weeks of vaccination with evidence of hyperthyroidism and GD within 3 months.ResultsDuring the prevaccination period, 803 patients carried diagnoses of GD, and of these, 131 were new. During the postvaccination period, 901 patients carried diagnoses of GD, and of these, 138 were new. There was no statistically significant difference in the incidence of GD (P = .52), age at onset, gender, or race between the 2 groups. Twenty-four of 138 newly diagnosed patients in the post–COVID-19 group met the criteria for PVGD. The median free T4 was higher, but this was not statistically significant (3.9 vs 2.5 ng/dL, P = .05). There were no differences in age, gender, race, antibody titers, or type of vaccination between PVGD and controls.ConclusionThere was no increase in new-onset GD after COVID-19 vaccination. Median free T4 was higher in patients with PVGD, but this was not statistically significant.     

促甲状腺素受体抗体及白细胞介素-2、白细胞介素-6在Graves病131I治疗中的变化及临床意义

目的:研究促甲状腺素受体抗体(TRAb)及白细胞介素-2(IL-2)、白细胞介素-6(IL-6)在Graves病~(131)I治疗中的变化并分析其临床意义。方法:选取2015年8月-2016年8月于我院接受~(131)I治疗的Graves病患者112例为研究对象,按照第6个月的甲状腺功能检查结果的不同分为治愈组(n=54),缓解组(n=21)和甲减组(n=37)。分别比较治疗前、治疗3个月、6个月后三组患者的甲状腺功能指标以及TRAb、IL-2、IL-6水平。结果:治疗前三组患者游离T3(FT3)、游离T4(FT4)、促甲状腺激素(TSH)水平比较差异无统计学意义(P0.05),治疗3个月、6个月后三组患者FT3、FT4水平均明显低于治疗前,而TSH水平明显高于治疗前(P0.05)。治疗3个月、6个月后甲减组FT3、FT4水平均明显低于治愈组、缓解组,治愈组又明显低于缓解组,TSH水平明显高于治愈组、缓解组,治愈组又明显高于缓解组(P0.05)。治疗前三组患者TRAb、IL-2、IL-6水平比较差异无统计学意义(P0.05)。治疗3个月后三组患者TRAb水平均明显高于治疗前(P0.05),治疗6个月后治愈组与甲减组患者TRAb水平与治疗前比较无统计学差异(P0.05),但缓解组患者TRAb水平高于甲减组,差异有统计学意义(P0.05)。治疗3个月、6个月后,治愈组与甲减组患者IL-2、IL-6水平均低于治疗前,而缓解组高于甲减组,差异均有统计学意义(P0.05)。结论:TRAb、IL-2、IL-6可作为评估Graves病~(131)I治疗后的指标,通过检测三项指标治疗前后水平变化情况,从而为临床预后评估提供指导作用。     

Graves病患者外周血中CD4+CD25+Treg细胞上的PD-1/PD-L1的表达及意义

目的:探究Graves病患者外周血中CD4+CD25+Treg细胞上的PD-1/P-L1的表达及意义。方法:收集2017年6月至2017年12月就诊于西南医科大学附属医院内分泌科的Graves病、Graves眼病及体检中心的健康者的外周血进行流式检测。结果:与正常对照组相比,Graves病及Graves眼病组CD4+CD25+Treg细胞比例明显降低(P0.05);PD-1+Treg、PD-L1+Treg、PD-1+/PD-L1+Treg细胞比例均较正常组明显降低(P0.05);此外,Graves眼病组的结果较Graves病组更低(P0.05)。结论:CD4+CD25+Treg的降低会导致Graves病和Graves眼病患者的免疫状态活化,甲状腺自身抗体的增加和活化,促进疾病的发生。此外于PD-1和PD-L1阳性细胞比例的减少对于其对免疫的负向调节有所减弱,从而导致患者体内免疫耐受的降低和免疫稳态的打破,可能导致机体对甲状腺抗原的耐受消失,导致GD及眼病的发生发展。     

Leptin receptor is a key gene involved in the immunopathogenesis of thyroid-associated ophthalmopathy

Thyroid-associated ophthalmopathy (TAO), the most common and severe manifestation of Graves' disease (GD), is a disfiguring and potentially blinding autoimmune disease. The high relapse rate (up to 20%) and substantial side effects of glucocorticoid treatment further decrease the life quality of TAO patients. To develop novel therapies, we amid to explore the immunopathogenesis of TAO. To identify the key immune-related genes (IRGs) in TAO, we integrated the IRG expression profiles in thyrocytes from a GD patient set (GD vs healthy control) and a TAO patient set (TAO vs GD). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) and receiver operating characteristic (ROC) curve analyses identified the leptin receptor (LEPR) gene as the key IRG in TAO immunopathogenesis. Gene set enrichment analysis (GSEA) suggested enrichment of the antigen presentation pathway in TAO patients with higher LEPR. Increased LEPR expression was validated in TAO orbital tissues, and weighted gene co-expression network analysis (WGCNA) showed that cell adhesion processes were positively correlated with LEPR. Our study revealed that LEPR is a key gene in TAO immunopathogenesis and plays different roles in thyrocytes and orbital tissues. Our findings provide new insights into diagnostic and therapeutic biomarkers for TAO.     

激素冲击治疗联合放疗对中重度Graves 眼病临床疗效分析

目的:探讨激素冲击治疗联合放疗用于中重度Graves眼病的临床疗效。方法:将2014年2月~2015年6月之间我院收治的109例中重度Graves眼病患者随机分为A组(n=37)、B组(n=35)、C组(n=37)。A组患者单纯采用抗甲状腺药物治疗,B组患者则在A组的基础上进行激素冲击治疗,C组患者则采用B组基础上联合球后放疗方案治疗,所有患者治疗后保持随访至少6个月,对比三组疗效,记录三组患者治疗前、治疗6个月时的临床活动评分(CAS)及眼球突出度,及随访期间不良反应情况。结果:三组有效率比较,C组B组A组,差异有统计学意义(P0.05)。治疗后三组CAS评分均显著优于治疗前(P0.05),且C组显著低于A、B组(P0.05),A、B组间比较无显著差异(P0.05)。治疗后B、C组眼球突出度显著低于治疗前(P0.05),且C组显著低于A、B组(P0.05)。A、B、C三组不良反应发生率分别为0、14.29%、5.41%,A、C组比较无显著差异(P0.05),B组不良反应发生率明显高于A、C组(P0.05)。结论:激素冲击联合放疗治疗中重度Graves眼病具有显著的疗效,能够有效降低患者的CAS评分及眼球突出度,效果明显优于单纯抗甲状腺治疗或激素冲击治疗,且安全性较好,值得在临床上推广和应用。     

Inhibition of adenylate cyclase activity of human thyroid membranes by gangliosides

Gangliosides inhibit basal, thyrotropin-induced and fluoride-induced adenylate cyclase activity of human thyroid membranes in physiological conditions. In contrast neutral glycolipids, phospholipids and neuraminic acid containing oligosaccharides show no effect. The efficacy of inhibition is more dependent upon the position of the sialic acid residues than upon their absolute number. In general gangliosides with disialyl groups are more inhibitory than those with single sialyl moieties. The inhibitory effects of the individual gangliosides on the two modes of stimulation are parallel. This parallelism suggests that the inhibitory effect is located at the postreceptor level and that the gangliosides interact directly with the adenylate cyclase system. A possible role of thyroid membrane gangliosides as suppressive cofactors of adenylate cyclase is discussed in relation to recent findings of stimulating anti-ganglioside antibodies in Graves' disease.     

TGF-beta induced hyaluronan synthesis in orbital fibroblasts involves protein kinase C betaII activation in vitro

Graves' ophthalmopathy is accompanied by hyaluronan (HA) accumulation in the orbital space and infiltration of immunocompetent cells and cytokines, including IFN-gamma, IL-1beta, and TGF-beta. We examined the signal transduction pathways by which TGF-beta induces HA synthesis in normal orbital fibroblasts, orbital fibroblasts from patients with Graves' ophthalmopathy, and abdominal fibroblasts. Calphostin C inhibited the stimulation of HA synthesis by TGF-beta. Phorbol 12-myristate 13-acetate (PMA) activation of PKC stimulated HA production. The effects of TGF-beta and PMA were not synergistic. Stimulation by TGF-beta and PMA were dependent on protein synthesis and their effects were inhibited by cycloheximide. Since TGF-beta-induced HA synthesis was inhibited by BAPTA or by PKC inhibitors, a calcium-dependent PKC was most likely involved. The PKA inhibitor H-89 enhanced TGF-beta- and PMA-induced HA synthesis, thus showing that communication between the PKA and PKC pathways was evident. TGF-beta stimulated the translocation of PKCbetaII to the cell membrane. PKCbetaII, a key enzyme in the regulation of HA synthesis by TGF-beta, might be an appropriate target for therapeutic compounds to be used to treat Graves' ophthalmopathy accompanied by inflammation.