Catherine J. Hwang

Evidence for an Association between Thyroid-Stimulating Hormone and Insulin-Like Growth Factor 1 Receptors: A Tale of Two Antigens Implicated in Graves’ Disease

Thyroid-stimulating hormone receptor (TSHR) plays a central role in regulating thyroid function and is targeted by IgGs in Graves’ disease (GD-IgG). Whether TSHR is involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the orbital manifestation of GD, remains uncertain. TSHR signaling overlaps with that of insulin-like grow factor 1 receptor (IGF-1R). GD-IgG can activate fibroblasts derived from donors with GD to synthesize T cell chemoattractants and hyaluronan, actions mediated through IGF-1R. In this study, we compare levels of IGF-1R and TSHR on the surfaces of TAO and control orbital fibroblasts and thyrocytes and explore the physical and functional relationship between the two receptors. TSHR levels are 11-fold higher on thyrocytes than on TAO or control fibroblasts. In contrast, IGF-1R levels are 3-fold higher on TAO vs control fibroblasts. In pull-down studies using fibroblasts, thyrocytes, and thyroid tissue, Abs directed specifically against either IGF-1Rβ or TSHR bring both proteins out of solution. Moreover, IGF-1Rβ and TSHR colocalize to the perinuclear and cytoplasmic compartments in fibroblasts and thyrocytes by confocal microscopy. Examination of orbital tissue from patients with TAO reveals similar colocalization to cell membranes. Treatment of primary thyrocytes with recombinant human TSH results in rapid ERK phosphorylation which can be blocked by an IGF-1R-blocking mAb. Our findings suggest that IGF-1R might mediate some TSH-provoked signaling. Furthermore, they indicate that TSHR levels on orbital fibroblasts are considerably lower than those on thyrocytes and that this receptor associates with IGF-1R in situ and together may comprise a functional complex in thyroid and orbital tissue.

Rituximab Treatment of Patients with Severe, Corticosteroid-Resistant Thyroid-Associated Ophthalmopathy

PURPOSE To study the effectiveness of anti-CD20 (rituximab [RTX]; Rituxan; Genentech, Inc., South San Francisco, CA) therapy in patients with severe, corticosteroid (CS)-resistant thyroid-associated ophthalmopathy (TAO). DESIGN Retrospective, interventional case series. PARTICIPANTS Six consecutive subjects with severe, progressive TAO unresponsive to CS. METHODS Electronic medical record review of consecutive patients receiving RTX during the previous 18 months. Responses to therapy were graded using standard clinical assessment and flow cytometric analysis of peripheral lymphocytes. MAIN OUTCOME MEASURES Clinical activity score (CAS), proptosis, strabismus, treatment side effects, and quantification of regulatory T cells. RESULTS Six patients were studied. Systemic CS failed to alter clinical activity in all patients (mean CAS+/-standard deviation, 5.3+/-1.0 before vs. 5.5+/-0.8 during therapy for 7.5+/-6.4 months; P = 1.0). However, after RTX treatment, CAS improved from 5.5+/-0.8 to 1.3+/-0.5 at 2 months after treatment (P<0.03) and remained quiescent in all patients (CAS, 0.7+/-0.8; P<0.0001) at a mean follow-up of 6.2+/-4.5 months. Vision improved bilaterally in all 4 patients with dysthyroid optic neuropathy (DON). None of the 6 patients experienced disease relapse after RTX infusion, and proptosis remained stable (Hertel measurement, 24+/-3.7 mm before therapy and 23.6+/-3.7 mm after therapy; P = 0.17). The abundance of T regulatory cells, assessed in 1 patient, increased within 1 week of RTX and remained elevated at 18 months of follow-up. CONCLUSIONS In progressive, CS-resistant TAO, rapid and sustained resolution of orbital inflammation and DON followed treatment with RTX. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Orbital fibroblasts from patients with thyroid-associated ophthalmopathy overexpress CD40: CD154 hyperinduces IL-6, IL-8, and MCP-1.

PURPOSE Fibroblast diversity represents an emerging concept critical to our understanding of tissue inflammation, repair, and remodeling. Orbital fibroblasts heterogeneously display Thy-1 and exhibit unique phenotypic attributes that may explain the susceptibility of the human orbit to thyroid-associated ophthalmopathy (TAO). In the present study the authors investigated the role of CD40 ligation on macrophage chemoattractant protein-1 (MCP-1), IL-6, and IL-8 expression in fibroblasts from patients with TAO. METHODS Human orbital fibroblasts were cultured from tissues obtained with informed consent from patients with TAO and from patients undergoing surgery for other noninflammatory conditions. The fibroblasts were then examined by flow cytometry, microscopy, and cytokine assays. RESULTS The authors report that orbital fibroblasts from patients with TAO expressed elevated levels of CD40. Surface CD40 could be further upregulated by IFN-gamma in TAO and control fibroblasts. This upregulation was mediated through Jak2 and could be blocked by dexamethasone and AG490, a powerful and specific inhibitor of tyrosine kinase. Treatment with CD154, the ligand for CD40, upregulated the expression of IL-6, IL-8, and MCP-1 in TAO fibroblasts but failed to do so in control cultures. Thy-1(+) fibroblasts displayed higher CD40 levels than did their Thy-1(-) counterparts and were largely responsible for this cytokine production. IL-1beta also induced MCP-1, IL-6, and IL-8 more vigorously in TAO-derived fibroblasts. CONCLUSIONS Characterization of orbital fibroblasts and their differential expression of cytokines and receptors should prove invaluable in understanding the site-specific nature of TAO and the development of specific therapies.

B Cells from Patients with Graves’ Disease Aberrantly Express the IGF-1 Receptor: Implications for Disease Pathogenesis

Graves’ disease (GD) is an autoimmune process involving the thyroid and connective tissues in the orbit and pretibial skin. Activating anti-thyrotropin receptor Abs are responsible for hyperthyroidism in GD. However, neither these autoAbs nor the receptor they are directed against have been convincingly implicated in the connective tissue manifestations. Insulin-like growth factor-1 receptor (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD and when ligated with IgGs from these patients, express the T cell chemoattractants, IL-16, and RANTES. Disproportionately large fractions of peripheral blood T cells also express IGF-1R in patients with GD and may account, at least in part, for expansion of IGF-1R+ memory T cells. We now report a similarly skewed B cell population exhibiting the IGF-1R+ phenotype from the blood, orbit, and bone marrow of patients with GD. This expression profile exhibits durability in culture and is maintained or increased with CpG activation. Moreover, IGF-1R+ B cells produce pathogenic Abs against the thyrotropin receptor. In lymphocytes from patients with GD, IGF-1 enhanced IgG production (p < 0.05) and increased B cell expansion (p < 0.02) in vitro while those from control donors failed to respond. These findings suggest a potentially important role for IGF-1R display by B lymphocytes in patients with GD in supporting their expansion and abnormal Ig production.

Aesthetic lateral canthoplasty.

Introduction: To report our technique and experience in using a minimally invasive approach for aesthetic lateral canthoplasty. Methods: Retrospective analysis of patients undergoing lateral canthoplasty through a minimally invasive, upper eyelid crease incision approach at Jules Stein Eye Institute by one surgeon (R.A.G.) between 2005 and 2008. Concomitant surgical procedures were recorded. Preoperative and postoperative photographs at the longest follow-up visit were analyzed and graded for functional and cosmetic outcomes. Results: A total of 600 patients (1,050 eyelids) underwent successful lateral canthoplasty through a small incision in the upper eyelid crease to correct lower eyelid malposition (laxity, ectropion, entropion, retraction) and/or lateral canthal dystopia, encompassing 806 reconstructive and 244 cosmetic lateral canthoplasties. There were 260 males and 340 females, with mean age of 55 years old (range, 4–92 years old). Minimum follow-up time was 3 months (mean, 6 months; maximum, 6 years). Complications were rare and minor, including transient postoperative chemosis. Eighteen patients underwent reoperation in the following 2 years for recurrent lower eyelid malposition and/or lateral canthal deformity. Conclusions: Lateral canthoplasty through a minimally invasive upper eyelid crease incision and resuspension technique can effectively address lower eyelid laxity and/or dystopia, resulting in an aesthetic lateral canthus.

Dihydrotestosterone stimulates proliferation and differentiation of fetal calvarial osteoblasts and dural cells and induces cranial suture fusion.

Background: The higher prevalence of metopic and sagittal suture synostosis in male infants suggests a role for androgens in early craniofacial development. These experiments characterize the influence of androgen stimulation on growth and differentiation of fetal dural and calvarial bone cells and on cranial suture fusion. Methods: Primary murine fetal (E18) dural cells and calvarial osteoblasts were isolated and cultured. Cells were treated for 48 hours with 5&agr;-dihydrotestosterone (0 to 1000 nM). Cell proliferation was examined by nonradioactive proliferation assay; mRNA expression of alkaline phosphatase, transforming growth factor (TGF)-&bgr;1, and the bone matrix proteins osteopontin, osteocalcin, and type 1 collagen was determined by reverse-transcriptase polymerase chain reaction. In separate experiments, intact fetal calvariae were grown in tissue culture with 10 nM 5&agr;-dihydrotestosterone for 7 and 14 days and then examined histologically. Results: Androgen stimulation at 5 nM increased proliferation of fetal dural cells by 46.0 percent and of fetal calvarial osteoblasts by 20.5 percent. Dural expression of osteopontin, osteocalcin, and type 1 collagen was enhanced by 5&agr;-dihydrotestosterone, as was that of TGF-&bgr;1 and alkaline phosphatase. Androgen stimulation increased calvarial osteoblast expression of alkaline phosphatase and TGF-&bgr;1 but induced little change in expression of osteocalcin, osteopontin, and type 1 collagen. In tissue culture, 5&agr;-dihydrotestosterone stimulated osteoid formation and fusion of sagittal sutures. Conclusions: Androgen stimulation of dural cells and osteoblasts isolated from fetal calvaria promotes cell proliferation and osteoblastic differentiation and can induce cranial suture fusion. These results suggest that sex steroid hormone signaling may stimulate sutural osteogenesis by means of osteodifferentiation of dural cells, thus explaining the male prevalence of nonsyndromic craniosynostosis.

Cosmetic Microdroplet Botulinum Toxin A Forehead Lift: A New Treatment Paradigm.

Purpose:To investigate the safety and efficacy of a microdroplet, cosmetic, periocular botulinum toxin A method that extensively treats the eyebrow depressors but leaves the brow elevators untreated. Methods:This is a 5-year retrospective, consecutive, nonrandomized series of botulinum toxin treatments. The study was reviewed by an institutional review board and complied with the Health Insurance Portability and Accountability Act (HIPAA). Patients were treated with 33 U onabotulinum toxin (BOTOX, Allergan, Inc., Irvine, CA, U.S.A.) injected in microdroplets of 10 to 20 &mgr;l. Sixty to 100 injections of microdroplets were needed to complete a treatment pattern concentrated at the brow, glabella, and crows feet area. The forehead was not treated. Patients who returned between 10 and 45 days were studied with image analysis. Results:There were 563 consecutive microdroplet treatments on 227 unique patients (female, n = 175, mean age 46 ± 4 years; male, n = 52, mean age 44 ± 8 years). The incidence of ptosis was 0.2% and transient. Forty-nine patients returned for a follow-up visit between 10 and 45 days and were included for image analysis to compare the before and after results of treatment. The average brow height was 24.6 mm before and 25 mm after treatment (p = 0.02). Photonumeric scales for forehead lines, brow ptosis, and brow furrow all showed statistically significant improvements (p < 0.0001). Conclusions:The microdroplet brow lift method safely concentrates cosmetic botulinum toxin treatment along the eyebrow, crows feet, and glabellar area, resulting in a brow lift effect that reduces forehead lines, elevates the eyebrow, and reduces the furrow along the brow. This new treatment paradigm results in an aesthetic improvement to the face and periocular area without the forehead paralysis associated with conventional treatment.

Adalimumab as steroid-sparing treatment of inflammatory-stage thyroid eye disease.

Purpose: Steroids are often used as medical therapy for active thyroid eye disease (TED). While high-dose steroids have been shown to be effective in reducing the severity of TED symptoms, the side effects of steroids can be severe. As the pathogenesis of TED is thought to involve the upregulation of proinflammatory cytokines, including tumor necrosis factor-&agr; (TNF-&agr;), it has been postulated that anti-TNF agents may be used as steroid-sparing agents in the treatment of TED. This retrospective study was conducted to examine the efficacy of adalimumab, a subcutaneously administered TNF-&agr; antagonist, in treating the inflammatory symptoms of active TED. Methods: All patients in the inflammatory phase of TED who were treated with adalimumab at the Jules Stein Eye Institute over a 2-year period were reviewed. Data concerning visual acuity, optic nerve function, extraocular motility restriction, binocular visual fields, and proptosis were extracted from patient charts. Clinical photographs from baseline and 3-month follow-up visits were reviewed by masked orbital specialists. Each photograph was graded on the severity of conjunctival injection, chemosis, eyelid erythema, and eyelid edema on a scale from 1 to 4. An inflammatory score was calculated as the sum of these 4 elements. Groups were compared using paired t tests. Results: Six of 10 patients showed a decrease in inflammatory score while on adalimumab, whereas 3 showed an increase and 1 stayed the same. One patient experienced a significant complication (hospital admission for sepsis). Eight patients received concomitant tapering steroids during the first 6 weeks of therapy as the adalimumab reached maximum efficacy. When data from all 10 subjects were analyzed together, there was no significant change in inflammatory index after 3 months of treatment with adalimumab. However, when the 5 patients with a high baseline inflammatory index (>4) were considered separately, there was a significant improvement (mean decrease of 5.2 ± 2.7; p < 0.01) after adalimumab treatment. Four of 5 patients also reported a subjective improvement in symptoms while on adalimumab. Conclusions: This study suggests that adalimumab may have a role in the treatment of active TED with prominent inflammatory symptoms. The use of adalimumab and other immunosuppressive agents in the treatment of TED may help to mitigate some of the metabolic and psychiatric side effects of pulsed steroid treatment. A future randomized controlled study will be necessary to determine the efficacy of adalimumab as a primary therapy for TED.

A Three-Dimensional Construct of the Aging Eyebrow: The Illusion of Volume Loss

BACKGROUND The eyebrows and eyebrow fat pads, key structures in upper facial aesthetics, are particularly vulnerable to age-related changes. OBJECTIVES In this study, the authors compare the impact of aging on the eyebrows and eyebrow fat pad volume in men and women through three-dimensional (3D) volumetric analysis. METHODS Electronic medical records of patients seen at the Jules Stein Eye Institute in the Division of Orbital and Ophthalmic Plastic Surgery between 2005 and 2010 were reviewed. Patients were included if they had undergone investigative imaging of the orbit for unilateral pathology. Computed tomography (CT) scans of patients with Graves disease diagnosis, extensive orbital trauma, and/or previous eyebrow surgery were excluded. A total of 52 CT scans (24 men and 28 women) were retained for analysis. A 3D reconstruction software was used to analyze the scans and calculate volumes of the retroorbicularis oculi fat (ROOF), galeal fat (ROOF and subcutaneous fat), and soft tissue muscles. RESULTS Galeal and brow fat volumes showed a significant positive trend toward enlargement in women (P values of .01 and .05, respectively). Although men showed a tendency toward fat enlargement with age, this was not statistically significant. Soft tissue-muscle volume decreased significantly in aging women (9.32 mm(3)/y) (P = .02). Data indicated that soft tissue volume in men tended to increase with age (3.92 mm(3)/y) but not significantly (P = .36). Neither total volume nor brow thickness appeared to change significantly in women (P = .56, P = .73). In men, total volume and brow thickness showed weak evidence of increasing with age (P = .12, P = .22). Linear regressions of Hertel measurements with and without sex interaction showed no statistically significant trend between the amount of proptosis and the galeal or brow fat. CONCLUSIONS Although overall eyebrow volume does not change with age, the relative contribution of fat and soft tissue to the total volume does seem to change. This pattern also differs between males and females. As women age, the fat volume increases and the soft tissue volume decreases. In men, the shift from soft tissue volume to fat volume is less pronounced. Although many clinicians have been drawn to the concept of fat volume deflation as a key element of facial aging, this study does not support this perspective in the eyebrow fat pad. An increasingly refined understanding of the dynamics of facial aging is mandatory for clinical diagnosis and will likely provide the framework from which to develop more innovative treatment options.

Immunolocalization of androgen receptor in the developing craniofacial skeleton.

Male predominance in metopic and sagittal craniosynostosis and in nonsynostotic plagiocephaly suggests a role for circulating androgens in early craniofacial development. Androgens have been documented to play an important role in postnatal skeletal growth, and the androgen receptor has been recently demonstrated in human and rat osteoblast-like cell lines and in human long bones. The purpose of this study was to describe the expression of androgen receptor in the fetal craniofacial skeleton. The heads of E18 fetal CD-1 male and female mice were fixed in 10% formalin, decalcified, and embedded in paraffin. Four- to 6-μm coronal and sagittal sections were stained with a monoclonal antibody specific to androgen receptor, which was detected by an avidinbiotin conjugate and peroxidase system. The sections were then examined for androgen receptor expression patterns. Strong androgen receptor immunoreactivity was observed in the dura mater of developing fetuses. Androgen receptor expression was also noted in cells lining the osteogenic fronts and in calvarial osteoblasts. Similar androgen receptor expression patterns were found in male and female mice. Androgen receptor is abundantly expressed in fetal dura mater and calvarial bone. This study confirms the presence of androgen receptor in the murine fetal craniofacial skeleton, suggesting a potential role for the anabolic effects of androgens in the developing craniofacial skeleton.