Slit2 May Underlie Divergent Induction by Thyrotropin of IL-23 and IL-12 in Human Fibrocytes.

IL-23 and IL-12, two structurally related heterodimeric cytokines sharing a common subunit, divergently promote Th cell development and expansion. Both cytokines have been implicated in the pathogenesis of thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves disease. In TAO, CD34+ fibrocytes, putatively derived from bone marrow, can be identified in the orbit. There they masquerade as CD34+ orbital fibroblasts (OF) (CD34+ OF) and cohabitate with CD34- OF in a mixed fibroblast population (GD-OF). Slit2, a neural axon repellent, is expressed and released by CD34- OF and dampens the inflammatory phenotype of fibrocytes and CD34+ OF. In this study we report that thyrotropin (TSH) and the pathogenic, GD-specific monoclonal autoantibody, M22, robustly induce IL-23 in human fibrocytes; however, IL-12 expression is essentially undetectable in these cells under basal conditions or following TSH-stimulation. In contrast, IL-12 is considerably more inducible in GD-OF, cells failing to express IL-23. This divergent expression and induction of cytokines appears to result from cell type-specific regulation of both gene transcription and mRNA stabilities. It appears that the JNK pathway activity divergently attenuates IL-23p19 expression while enhancing that of IL-12p35. The shift from IL-23p19 expression in fibrocytes to that of IL-12p35 in their derivative CD34+ OF results from the actions of Slit2. Thus, Slit2 might represent a molecular determinant of balance between IL-23 and IL-12 expression, potentially governing immune responses in TAO.

Reliability and Validity of a Method for the Assessment of Sport Rock Climbers' Isometric Finger Strength.

ABSTRACT: Torr, O, Randall, T, Knowles, R, Giles, D, and Atkins, S. The reliability and validity of a method for the assessment of sport rock climbers' isometric finger strength. J Strength Cond Res 36(8): 2277-2282, 2022-Isometric strength of the finger flexors is considered to be one of the main physical determinants of sport rock climbing performance. We set out to determine the test-retest reliability and criterion validity of a low resource maximal isometric finger strength (MIFS) testing protocol that uses a pulley system to add or remove weight to/from a climber's body. To determine test-retest reliability, 15 subjects' MIFS was assessed on 2 occasions, separated by a minimum of 48 hours. Body mass and maximum load were recorded on both occasions. Intra-class correlation coefficients (ICCs) between visits for all variables were very good (ICC > 0.91), with small bias and effect sizes-particularly when expressed as a percentage of body mass (ICC = 0.98, 95% confidence interval 0.93-0.99). To determine the criterion validity of MIFS and climbing ability, data of 229 intermediate to higher elite climbers were compared. Pearson's product moment correlations demonstrated good agreement, again particularly between total load when expressed as a percentage of body mass and climbing performance ( r = 0.421-0.503). The results illustrate the sensitivity of a simple test for the determination of MIFS in intermediate to height elite climbers from an ecologically valid, climbing specific test that only requires equipment found at most climbing walls. This low resource test protocol for the assessment of isometric finger strength has wide-reaching utility, for instance when assessing strength before and after a training intervention or when prescribing load intensities for exercises aimed at improving maximal finger strength.

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.

De novo triiodothyronine formation from thyrocytes activated by thyroid-stimulating hormone.

The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Under normal physiological circumstances, only one-fifth of circulating T3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating hormone receptors (TSHRs), patients develop a syndrome of relative T3 toxicosis. Thyroidal T4 production results from iodination of thyroglobulin (TG) at residues Tyr5 and Tyr130, whereas thyroidal T3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T3 is formed de novo independently of deiodination from T4 We found that upon iodination in vitro, de novo T3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibody. We present data suggesting that TSH-stimulated TG phosphorylation contributes to enhanced de novo T3 formation. These effects were reversed within a few days after removal of the hyperstimulating conditions. Indeed, direct exposure of PCCL3 cells to human serum from two patients with Graves' disease, but not control sera, led to secretion of TG with an increased intrinsic ability to form T3 upon in vitro iodination. Furthermore, TG secreted from human thyrocyte cultures hyperstimulated with TSH also showed an increased intrinsic ability to form T3 Our data support the hypothesis that TG processing in the secretory pathway of TSHR-hyperstimulated thyrocytes alters the structure of the iodination substrate in a way that enhances de novo T3 formation, contributing to the relative T3 toxicosis of Graves' disease.

Influence of wheel size on muscle activity and tri-axial accelerations during cross-country mountain biking.

This study aimed to investigate the influence of different mountain bike wheel diameters on muscle activity and whether larger diameter wheels attenuate muscle vibrations during cross-country riding. Nine male competitive mountain bikers (age 34.7 ± 10.7 years; stature 177.7 ± 5.6 cm; body mass 73.2 ± 8.6 kg) participated in the study. Riders performed one lap at race pace on 26, 27.5 and 29 inch wheeled mountain bikes. sEMG and acceleration (RMS) were recorded for the full lap and during ascent and descent phases at the gastrocnemius, vastus lateralis, biceps brachii and triceps brachii. No significant main effects were found by wheel size for each of the four muscle groups for sEMG or acceleration during the full lap and for ascent and descent (P > .05). When data were analysed between muscle groups, significant differences were found between biceps brachii and triceps brachii (P < .05) for all wheel sizes and all phases of the lap with the exception of for the 26 inch wheel during the descent. Findings suggest wheel diameter has no influence on muscle activity and vibration during mountain biking. However, more activity was observed in the biceps brachii during 26 inch wheel descending. This is possibly due to an increased need to manoeuvre the front wheel over obstacles.

The effect of mountain bike wheel size on cross-country performance.

The purpose of this study was to determine the influence of different wheel size diameters on indicators of cross-country mountain bike time trial performance. Nine competitive male mountain bikers (age 34.7 ± 10.7 years; stature 177.7 ± 5.6 cm; body mass 73.2 ± 8.6 kg) performed 1 lap of a 3.48 km mountain bike (MTB) course as fast as possible on 26″, 27.5″ and 29″ wheeled MTB. Time (s), mean power (W), cadence (revs · min-1) and velocity (km · h-1) were recorded for the whole lap and during ascent and descent sections. One-way repeated measure ANOVA was used to determine significant differences. Results revealed no significant main effects for any variables by wheel size during all trials, with the exception of cadence during the descent (F(2, 16) = 8.96; P = .002; P2 = .53). Post hoc comparisons revealed differences lay between the 26″ and 29″ wheels (P = .02). The findings indicate that wheel size does not significantly influence performance during cross-country when ridden by trained mountain bikers, and that wheel choice is likely due to personal choice or sponsorship commitments.

The Presence of Bilateral Imbalance of the Lower Limbs in Elite Youth Soccer Players of Different Ages.

The aim of this study was to examine bilateral differences in ground reaction forces (GRF), measured during a deep squat (DS) exercise, in a population of elite youth soccer players. Bilateral muscle balance is a key component in promoting musculoskeletal health of performers, yet there is a limited evidence base investigating such imbalances in youth. Seventy-four subjects were assigned to performance groups according to chronological age (younger than 13, 14, 15, 16, 17 years). Analysis of physical maturity status revealed that very few players were classified as "early" or "late" maturers. Players completed an overhead DS exercise, as part of preseason functional movement screening. Peak GRF were assessed using a twin force plate system. Significant differences (p ≤ 0.05) were identified between right and left side peak GRF for all groups except the youngest (U13) and oldest (U17). Nondominant "sides" showed the highest levels of PGRF across all groups. The magnitude of PGRF was not significantly different both within and between groups, except for the left side in the U13 to U15 groups (p = 0.04). Results from this study show that performance asymmetry is marked in adolescence. There seems a "trigger point" during the early stage of adolescence, when bilateral imbalances become marked. These differences do seem to reduce during the later stages of adolescence. Correct attention to focussed training, designed to remediate any imbalance, is warranted in adolescent groups. This is important with respect of the key associations between bilateral asymmetry and risk of injury.

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, (125)iodine and [(35)S]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.

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.

Electromyographic response of global abdominal stabilizers in response to stable- and unstable-base isometric exercise.

Core stability training traditionally uses stable-base techniques. Less is known as to the use of unstable-base techniques, such as suspension training, to activate core musculature. This study sought to assess the neuromuscular activation of global core stabilizers when using suspension training techniques, compared with more traditional forms of isometric exercise. Eighteen elite level, male youth swimmers (age, 15.5 ± 2.3 years; stature, 163.3 ± 12.7 cm; body mass, 62.2 ± 11.9 kg) participated in this study. Surface electromyography (sEMG) was used to determine the rate of muscle contraction in postural musculature, associated with core stability and torso bracing (rectus abdominus [RA], external obliques [EO], erector spinae [ES]). A maximal voluntary contraction test was used to determine peak amplitude for all muscles. Static bracing of the core was achieved using a modified "plank" position, with and without a Swiss ball, and held for 30 seconds. A mechanically similar "plank" was then held using suspension straps. Analysis of sEMG revealed that suspension produced higher peak amplitude in the RA than using a prone or Swiss ball "plank" (p = 0.04). This difference was not replicated in either the EO or ES musculature. We conclude that suspension training noticeably improves engagement of anterior core musculature when compared with both lateral and posterior muscles. Further research is required to determine how best to activate both posterior and lateral musculature when using all forms of core stability training.

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.

The Effects of Sprint vs. Resisted Sled-Based Training; an 8-Week in-Season Randomized Control Intervention in Elite Rugby League Players.

The aim of the current study was to examine the efficacy of resisted sled-based training compared to traditional unresisted sprint training in terms of mediating improvements in speed, agility, and power during an eight-week period of in-season training in elite rugby league players. Participants were randomly separated into either resisted sled or traditional sprint-based training groups and they completed an eight-week in-season training block with training prescribed based on the group to which they were assigned. Measures of 5 m, 10 m, and 20 m sprint times in addition to countermovement jump height and 505-agility test time were measured at baseline, four-weeks and eight-weeks. For sprint-based outcomes, although both groups improved significantly, there were no statistical differences between the two training methods. However, at the eight-week time point there were significant improvements in 505-agility test (sprint group: baseline = 2.45 and eight-weeks = 2.42 s/sled group: baseline = 2.43 and eight-weeks = 2.37 s) and countermovement jump (sprint group: baseline = 39.18 and eight-weeks = 39.49 cm/sled group: baseline = 40.43 and eight-weeks = 43.07 cm) performance in the sled training group. Therefore, the findings from this investigation may be important to strength and conditioning coaches working in an elite rugby league in that resisted sled training may represent a more effective method of sprint training prescription.

What happens inside a fuel cell? Developing an experimental functional map of fuel cell performance.

Fuel cell performance is determined by the complex interplay of mass transport, energy transfer and electrochemical processes. The convolution of these processes leads to spatial heterogeneity in the way that fuel cells perform, particularly due to reactant consumption, water management and the design of fluid-flow plates. It is therefore unlikely that any bulk measurement made on a fuel cell will accurately represent performance at all parts of the cell. The ability to make spatially resolved measurements in a fuel cell provides one of the most useful ways in which to monitor and optimise performance. This Minireview explores a range of in situ techniques being used to study fuel cells and describes the use of novel experimental techniques that the authors have used to develop an 'experimental functional map' of fuel cell performance. These techniques include the mapping of current density, electrochemical impedance, electrolyte conductivity, contact resistance and CO poisoning distribution within working PEFCs, as well as mapping the flow of reactant in gas channels using laser Doppler anemometry (LDA). For the high-temperature solid oxide fuel cell (SOFC), temperature mapping, reference electrode placement and the use of Raman spectroscopy are described along with methods to map the microstructural features of electrodes. The combination of these techniques, applied across a range of fuel cell operating conditions, allows a unique picture of the internal workings of fuel cells to be obtained and have been used to validate both numerical and analytical models.

Capture and light-induced release of antibiotics by an azo dye polymer.

The isomerisation of azo dyes can induce conformational changes which have potential applications in medicine and environmental protection. We developed an agar diffusion assay to test the capture and release of biologically active molecules from an azo electro-optic polymer, Poly (Disperse Red 1 methacrylate) (DR1/PMMA). The assay monitors the growth of bacteria placed in soft agar under a glass coverslip. Antibiotics can then be applied on the coverslip resulting in the clearance of the area under the coverslip due to growth inhibition. This assay demonstrates that DR1/PMMA is able to capture either tetracycline or ampicillin and the relative amount of DR1/PMMA required for capture was determined. Finally, the active antibiotics can be released from DR1/PMMA by exposure to green laser light. Exposure to white light from a torch or to heat does not release the antibiotic.

HIF2A-LOX Pathway Promotes Fibrotic Tissue Remodeling in Thyroid-Associated Orbitopathy.

Thyroid-associated orbitopathy (TAO) is a disfiguring periocular connective tissue disease associated with autoimmune thyroid disorders. It is a potentially blinding condition, for which no effective pharmacological treatment has been established. Despite a suggested role played by autoimmune thyrotropin receptor activation in the pathogenesis of TAO, the cellular and molecular events contributing to the fibrotic and inflammatory disease process of TAO are not fully defined. By developing a three-dimensional organoid culture of human orbital fibroblasts (OFs), we sought to determine the molecular mechanism underlying the fibrotic disease process of TAO. In this ex vivo model, we have demonstrated that hypoxia-inducible factor (HIF) 2α (HIF2A), but not its paralog HIF1A, accelerates extracellular matrix (ECM) deposition by inducing a collagen-cross-linking enzyme, lysyl oxidase (LOX). Inhibiting HIF2A and LOX with short hairpin RNA or small molecular antagonists effectively ameliorated fibrotic disease process within TAO organoids. Conversely, the overexpression of a constitutively active HIF2A in mouse OFs was sufficient to initiate LOX-dependent fibrotic tissue remodeling in OF organoids. Consistent with these findings, HIF2A and LOX were highly expressed in human TAO tissues paralleling excess ECM deposition. We propose that the HIF2A-LOX pathway can be a potential therapeutic target for the prevention and treatment of TAO.

The magnitude of translational and rotational head accelerations experienced by riders during downhill mountain biking.

OBJECTIVES: To determine the magnitude of translational and rotational head accelerations during downhill mountain biking. DESIGN: Observational study. METHODS: Sixteen male downhill cyclists (age 26.4±8.4years; stature 179.4±7.2cm; mass 75.3±5.9kg) were monitored during two rounds of the British Downhill Series. Riders performed two runs on each course wearing a triaxial accelerometer behind the right ear. The means of the two runs for each course were used to determine differences between courses for mean and maximum peak translational (g) and rotational accelerations (rad/s2) and impact duration for each course. RESULTS: Significant differences (p<0.05) were revealed for the mean number of impacts (>10g), FW=12.5±7.6, RYF=42.8±27.4 (t(22.96)=-4.70; p<0.001; 95% CI=17.00 to 43.64); maximum peak rotational acceleration, FW=6805.4±3073.8rad/s2, RYF=9799.9±3381.7rad/s2 (t(32)=-2.636; p=0.01; 95% CI=680.31 to 5308.38); mean acceleration duration FW=4.7±1.2ms, RYF=6.5±1.4ms (t(32)=-4.05; p<0.001; 95% CI=0.91 to 2.76) and maximum acceleration duration, FW=11.6±4.5ms, RYF=21.2±9.1 (t(29.51)=-4.06; p=0.001; 95% CI=4.21 to 14.94). No other significant differences were found. CONCLUSIONS: Findings indicate that downhill riders may be at risk of sustaining traumatic brain injuries and course design influences the number and magnitude of accelerations.

Profiling of translational and rotational head accelerations in youth BMX with and without neck brace.

OBJECTIVES: To investigate the influence of BMX helmets and neck braces on translational and rotational accelerations in youth riders. DESIGN: Mixed model, repeated measure and correlation. METHODS: Twenty three competitive youth BMX riders classified by age group (6-9 years, 10-13 years and 14-18 years) completed 6 laps of an indoor BMX track at race pace, 3 laps without a neck brace (NB) and 3 without brace (WB). A triaxial accelerometer with gyroscope was placed behind the right ear to determine the mean number of accelerations, translational and rotational, of the head between conditions and by age group. RESULTS: Significant reductions by condition (p=0.02) and by age (p=0.04) were found for the number of accelerations, though no interactions (condition×age) were revealed. Significant increases by age (p=0.01) were revealed for translational accelerations, whilst significant increases by condition (p=0.02) were found for rotational accelerations. In addition, significant correlations were revealed between relative helmet mass and age (r=0.83; p=0.001) and relative helmet mass and number of accelerations (r=0.46; p=0.03). CONCLUSIONS: Accelerations at the head decreased with increased age, possibly due to the influence of greater stabilising musculature. Additionally, neck braces also significantly reduced the number of accelerations. However, the magnitude of accelerations may be influenced by riding dynamics. Therefore, the use of neck braces combined with strength work to develop neck strength, could aid in the reduction of head accelerations in youth BMX riders.

CD34- Orbital Fibroblasts From Patients With Thyroid-Associated Ophthalmopathy Modulate TNF-α Expression in CD34+ Fibroblasts and Fibrocytes.

Purpose: Orbital fibroblasts from patients with Graves' disease (GD-OF) express many different cytokines when treated with bovine thyrotropin (bTSH). The present study aimed to determine why TNF-α cannot be induced by bTSH in GD-OF. Methods: Fibrocytes and GD-OFs were cultivated from donors who were patients in a busy academic medical center practice. Real-time PCR, Western blot analysis, reporter gene assays, cell transfections, mRNA stability assays, ELISA, and flow cytometry were performed. Results: We found that bTSH induces TNF-α dramatically in fibrocytes but is undetectable in GD-OF. The induction in fibrocytes is a consequence of increased TNF-α gene promoter activity and is independent of ongoing protein synthesis. It could be attenuated by dexamethasone and the IGF-1 receptor inhibiting antibody, teprotumumab. When separated into pure CD34+ OF and CD34- OF subsets, TNF-α mRNA became highly inducible by bTSH in CD34+ OF but remained undetectable in CD34- OF. Conditioned medium from CD34- OF inhibited induction of TNF-α in fibrocytes. Conclusions: Our data indicate that CD34- OF appear to release a soluble(s) factor that downregulates expression and induction by bTSH of TNF-α in fibrocytes and their derivative CD34+ OF. We proffer that CD34- OF produce an unidentified modulatory factor that attenuates TNF-α expression in GD-OF and may do so in the TAO orbit.

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.

Elevated Serum Tetrac in Graves Disease: Potential Pathogenic Role in Thyroid-Associated Ophthalmopathy.

Context: The sources and biological impact of 3,3',5,5' tetraiodothyroacetic acid (TA4) are uncertain. CD34+ fibrocytes express several proteins involved in the production of thyroid hormones. They infiltrate the orbit in Graves disease (GD), an autoimmune process known as thyroid-associated ophthalmopathy. It appears that the thyrotropin receptor plays an important role in the pathogenesis of thyroid-associated ophthalmopathy. Objective: To quantify levels of TA4 in healthy participants and those with GD, determine whether fibrocytes generate this thyroid hormone analogue, and determine whether TA4 influences the actions of thyroid-stimulating hormone and thyroid-stimulating immunoglobulins in orbital fibroblasts. Design/Setting/Participants: Patients with GD and healthy donors in an academic medical center clinical practice were recruited. Main Outcome Measures: Liquid chromatography-tandem mass spectrometry, autoradiography, real-time polymerase chain reaction, hyaluronan immunoassay. Results: Serum levels of TA4 are elevated in GD. TA4 levels are positively correlated with those of thyroxine and negatively correlated with serum levels of triiodothyronine. Several cell types in culture generate TA4 from ambient thyroxine, including fibrocytes, HELA cells, human Müller stem cells, and retinal pigmented epithelial cells. Propylthiouracil inhibits TA4 generation. TA4 enhances the induction by thyrotropin and thyroid-stimulating immunoglobulins of several participants in the pathogenesis of thyroid-associated ophthalmopathy, including interleukin 6, hyaluronan synthase 1, prostaglandin endoperoxide H synthase 2, and haluronan production. Conclusion: TA4 may be ubiquitously generated in many tissues and enhances the biological impact of thyrotropin and thyroid-stimulating immunoglobulins in orbital connective tissue. These findings may identify a physiologically important determinant of extrathyroidal thyroid-stimulating hormone action.