Stephen J. Atkins

Interaction of retinitis pigmentosa GTPase regulator (RPGR) with RAB8A GTPase: implications for cilia dysfunction and photoreceptor degeneration

Defects in biogenesis or function(s) of primary cilia are associated with numerous inherited disorders (called ciliopathies) that may include retinal degeneration phenotype. The cilia-expressed gene RPGR (retinitis pigmentosa GTPase regulator) is mutated in patients with X-linked retinitis pigmentosa (XLRP) and encodes multiple protein isoforms with a common N-terminal domain homologous to regulator of chromosome condensation 1 (RCC1), a guanine nucleotide exchange factor (GEF) for Ran GTPase. RPGR interacts with several ciliopathy proteins, such as RPGRIP1L and CEP290; however, its physiological role in cilia-associated functions has not been delineated. Here, we report that RPGR interacts with the small GTPase RAB8A, which participates in cilia biogenesis and maintenance. We show that RPGR primarily associates with the GDP-bound form of RAB8A and stimulates GDP/GTP nucleotide exchange. Disease-causing mutations in RPGR diminish its interaction with RAB8A and reduce the GEF activity. Depletion of RPGR in hTERT-RPE1 cells interferes with ciliary localization of RAB8A and results in shorter primary cilia. Our data suggest that RPGR modulates intracellular localization and function of RAB8A. We propose that perturbation of RPGR–RAB8A interaction, at least in part, underlies the pathogenesis of photoreceptor degeneration in XLRP caused by RPGR mutations.

Human fibrocytes coexpress thyroglobulin and thyrotropin receptor

Thyroglobulin (Tg) is the macromolecular precursor of thyroid hormones and is thought to be uniquely expressed by thyroid epithelial cells. Tg and the thyroid-stimulating hormone receptor (TSHR) are targets for autoantibody generation in the autoimmune disorder Graves disease (GD). Fully expressed GD is characterized by thyroid overactivity and orbital tissue inflammation and remodeling. This process is known as thyroid-associated ophthalmopathy (TAO). Early reports suggested that in TAO, both Tg and TSHR become overexpressed in orbital tissues. Previously, we found that CD34+ progenitor cells, known as fibrocytes, express functional TSHR, infiltrate the orbit, and comprise a large subset of orbital fibroblasts in TAO. We now report that fibrocytes also express Tg, which resolves as a 305-kDa protein on Western blots. It can be immunoprecipitated with anti-Tg Abs. Further, 125iodine and [35S]methionine are incorporated into Tg expressed by fibrocytes. De novo Tg synthesis is attenuated with a specific small interfering RNA targeting the protein. A fragment of the Tg gene promoter fused to a luciferase reporter exhibits substantial activity when transfected into fibrocytes. Unlike fibrocytes, GD orbital fibroblasts, which comprise a mixture of CD34+ and CD34− cells, express much lower levels of Tg and TSHR. When sorted into pure CD34+ and CD34− subsets, Tg and TSHR mRNA levels become substantially higher in CD34+ cells. These findings indicate that human fibrocytes express multiple “thyroid-specific” proteins, the levels of which are reduced after they infiltrate tissue. Our observations establish the basis for Tg accumulation in orbital GD.

Using Two‐Component Systems and Other Bacterial Regulatory Factors for the Fabrication of Synthetic Genetic Devices

Synthetic biology is an emerging field in which the procedures and methods of engineering are extended living organisms, with the long-term goal of producing novel cell types that aid human society. For example, engineered cell types may sense a particular environment and express gene products that serve as an indicator of that environment or affect a change in that environment. While we are still some way from producing cells with significant practical applications, the immediate goals of synthetic biology are to develop a quantitative understanding of genetic circuitry and its interactions with the environment and to develop modular genetic circuitry derived from standard, interoperable parts that can be introduced into cells and result in some desired input/output function. Using an engineering approach, the input/output function of each modular element is characterized independently, providing a toolkit of elements that can be linked in different ways to provide various circuit topologies. The principle of modularity, yet largely unproven for biological systems, suggests that modules will function appropriately based on their design characteristics when combined into larger synthetic genetic devices. This modularity concept is similar to that used to develop large computer programs, where independent software modules can be independently developed and later combined into the final program. This chapter begins by pointing out the potential usefulness of two-component signal transduction systems for synthetic biology applications and describes our use of the Escherichia coli NRI/NRII (NtrC/NtrB) two-component system for the construction of a synthetic genetic oscillator and toggle switch for E. coli. Procedures for conducting measurements of oscillatory behavior and toggle switch behavior of these synthetic genetic devices are described. It then presents a brief overview of device fabrication strategy and tactics and presents a useful vector system for the construction of synthetic genetic modules and positioning these modules onto the bacterial chromosome in defined locations.

Rituximab (Rituxan) Therapy for Severe Thyroid-Associated Ophthalmopathy Diminishes IGF-1R+ T Cells

CONTEXT Rituximab depletes CD20(+) B cells and has shown potential benefit in thyroid-associated ophthalmopathy (TAO). The impact of rituximab on T cell phenotype in TAO is unexplored. OBJECTIVE The objective of the study was to quantify the abundance of IGF-I receptor-positive (IGF-1R(+)) CD4 and CD8 T cells in active TAO before and after treatment with rituximab. DESIGN This was a retrospective case series assessing IGF-1R(+) T cells before and after treatment with rituximab with an 18-month follow-up. SETTING The study was conducted at a tertiary care medical center. PATIENTS Study participants included eight patients with severe TAO. INTERVENTIONS Two infusions of rituximab (1 g or 500 mg each) were administered 2 weeks apart. MAIN OUTCOME MEASURES Quantification of IGF-1R(+) T cells using flow cytometry was measured. RESULTS Eight patients with moderate to severe TAO [mean pretreatment clinical activity score (CAS) 5.1 ± 0.2 (SEM)] were treated. Four to 6 weeks after treatment, CAS improved to 1.5 ± 0.3, whereas the proportion of IGF-1R(+) CD3(+) T cells declined from 41.9% to 28.3% (P = .004). The proportion of IGF-1R(+) CD4(+) and IGF-1R(+) CD8(+) T cells declined 4-6 weeks after treatment (from 45.6% to 21.5% and from 32.0% to 15.8%, P = .003 and P = .001, respectively). In two patients, IGF-1R(+) CD4(+) and IGF-1R(+) CD8(+) subsets approximated pretreatment levels after 16 weeks. CONCLUSIONS Frequency of IGF-1R(+) T cells in patients with TAO declines within 4-6 weeks after rituximab treatment. This phenotypic shift coincides with clinical improvement. Thus, assessment of the abundance of IGF-1R(+) T cells in response to rituximab may provide a biomarker of clinical response. Our current findings further implicate the IGF-1R pathway in the pathogenesis of TAO.

Disrupted TSH Receptor Expression in Female Mouse Lung Fibroblasts Alters Subcellular IGF-1 Receptor Distribution

A relationship between the actions of TSH and IGF-1 was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR and IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the noncanonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunofluorescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without and with intact TSHR expression. IGF-1 treatment of TSHR(+/+) fibroblasts resulted in reduced nuclear and cytoplasmic staining for IGF-1Rα, whereas it enhanced the nuclear signal in TSHR(-/-) cells. In contrast, IGF-1 enhanced cytoplasmic IGF-1Rβ in TSHR(-/-) fibroblasts while increasing the nuclear signal in TSHR(+/+) cells. These findings indicate the intimate relationship between TSHR and IGF-1R found earlier in human orbital fibroblasts also exists in mouse lung fibroblasts. Furthermore, the presence of TSHR in these fibroblasts influenced not only the levels of IGF-1R protein but also its subcellular distribution and response to IGF-1. They suggest that the mouse might serve as a suitable model for delineating the molecular mechanisms overarching these two receptors.

A Synthetic Biology Approach to Understanding Biological Oscillations: Developing a Genetic Oscillator for Escherichia coli

Our goals are to construct a simple genetic clock that will stably oscillate in Escherichia coli and to identify the design principles and parameters responsible for oscillations. We previously described a simple genetic circuit of linked activator and repressor operons that produced damped oscillations. Here, we altered the repression of the activator operon and identified an oscillator that produces improved oscillations over our initial system. We also explored mathematical models of the oscillator. Toy models were used to investigate the behaviors that may be obtained from our clock circuitry. Depending on parameters, the circuitry produced a wide array of oscillatory systems, including sinusoidal and relaxation oscillators. We also attempted to explicitly model all known interactions that affect the oscillator, producing a 32-dimensional ODE model. This model can produce results similar to those obtained in experiments, and we have begun attempts to fit experimental data to the model.

Slit2 Modulates the Inflammatory Phenotype of Orbit-Infiltrating Fibrocytes in Graves’ Disease

Human CD34+ fibrocytes, circulating monocyte lineage progenitor cells, have recently been implicated in thyroid-associated ophthalmopathy (TAO), the ocular manifestation of Graves’ disease (GD). Fibrocytes express constitutive MHC class II (MHC-2) and, surprisingly, thyroglobulin (Tg) and functional thyrotropin (TSH) receptor (TSHR). Underlying expression of these thyroid proteins is the autoimmune regulator protein (AIRE). Fibrocytes respond robustly to TSH and thyroid-stimulating Igs by generating extremely high levels of inflammatory cytokines, such as IL-6. In TAO, they appear to infiltrate the orbit, where they transition to CD34+ orbital fibroblasts (OF). There, they coexist with CD34− OF as a mixed fibroblast population (GD-OF). In contrast to fibrocytes, GD-OF express vanishingly low levels of MHC-2, Tg, TSHR, and AIRE. Further, the amplitude of IL-6 induction by TSH in GD-OF is substantially lower. The molecular basis for this divergence between fibrocytes and CD34+ OF remains uncertain. In this article, we report that Slit2, an axon guidance glycoprotein, is constitutively expressed by the CD34− OF subset of GD-OF. Culture conditioned medium (CM) generated by incubating with GD-OF and CD34− OF substantially reduces levels of MHC-2, Tg, TSHR, and AIRE in fibrocytes. Expression can be restored by specifically depleting CM of Slit2. The effects of CD34− OF CM are mimicked by recombinant human Slit2. TSH induces Slit2 levels in GD-OF by enhancing both Slit2 gene transcription and mRNA stability. These findings suggest that Slit2 represents a TSH-inducible factor within the TAO orbit that can modulate the inflammatory phenotype of CD34+ OF and therefore may determine the activity and severity of the disease.

Increased CD40+ Fibrocytes in Patients With Idiopathic Orbital Inflammation.

Objective: To investigate the phenotypic and functional characteristics of peripheral and tissue-infiltrating stem cells called fibrocytes in patients with idiopathic orbital inflammation (IOI). Methods: Seven patients with IOI were studied. In the 3 patients requiring orbital biopsy, fibrocytes were identified in orbital tissue from patients with IOI compared with healthy controls using immunohistochemistry. Fibrocytes from the peripheral blood of all 7 patients and controls were quantified and phenotyped by flow cytometry and immunofluorescence for expression of CD34, alpha smooth muscle actin, CD40, and collagen 1. Quantitation of CD40-mediated interleukin-6 (IL-6) production was measured using enzyme-linked immunosorbent assay. Results: Orbital biopsy specimens from patients with IOI demonstrate tissue infiltration by fibrocytes (n = 3). Fibrocytes are present in the peripheral blood of IOI patients (n = 7) but are scarce in healthy donors (n = 19). Fibrocytes from IOI patients express substantial levels of CD40, and ligation of CD40 increases IL-6 expression. Conclusions: Fibrocytes are present in the peripheral blood and orbital tissues of patients with IOI and constitutively express CD40 and express IL-6 in response to ligation. This site-specific predilection of CD34+ fibrocytes to sites of orbital inflammation and fibrosis may suggest a role in IOI. Moreover, CD40-mediated activation cytokine production may contribute to the proinflammatory and profibrotic features of IOI and may provide a mechanism for future targeted therapy.