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Assess incidence, severity, and glucose excursion outcomes in thyroid eye disease (TED) patients receiving the insulin-like growth factor-1 receptor inhibitor teprotumumab from 3 clinical trials. Analysis of pooled glycemic data over time. Eighty-four teprotumumab- and 86 placebo-treated active TED patients from the phase 2 and phase 3 (OPTIC) controlled clinical trials and 51 teprotumumab-treated patients from the OPTIC extension (OPTIC-X) trial. Eight intravenous infusions were given over 21 weeks. Phase 2 serum glucose was measured at weeks 1, 4, 15, and 21, with fasting measurements at weeks 1 and 4. Serum glucose was measured at each study visit in OPTIC and OPTIC-X, with fasting measurements at weeks 1 and 4 (in patients without diabetes) or all visits (in patients with diabetes). In all studies, hemoglobin A1c (HbA1c) was measured at baseline, 12, and 24 weeks plus weeks 36 and 48 in OPTIC-X. Serum glucose and HbA1c. In the phase 2 and 3 studies, 9 hyperglycemic episodes occurred in 8 teprotumumab patients; mean HbA1c level increased 0.22% from baseline to week 24 (to 5.8%; range, 5.0%–7.9%) versus 0.04% in patients receiving the placebo (to 5.6%; range, 4.6%–8.1%). At study end, 78% (59/76) of teprotumumab patients and 87% (67/77) of patients receiving placebo had normoglycemic findings. Normoglycemia was maintained in 84% (57/68) of patients receiving teprotumumab and 93% (64/69) of patients receiving placebo. Among baseline prediabetic patients, 43% (3/7) remained prediabetic in both groups, and 29% (2/7) of teprotumumab patients and 14% (1/7) of patients receiving placebo had diabetic findings at week 24. OPTIC-X patients trended toward increased fasting glucose and HbA1c whether initially treated or retreated with teprotumumab. Fasting glucose commonly rose after 2 or 3 infusions and stabilized thereafter. Most hyperglycemic incidents occurred in patients with baseline prediabetes/diabetes but were controlled with medication. No evidence was found for progression or increased incidence of hyperglycemia with subsequent doses. Serious glycemic excursions are uncommon in patients with normoglycemia before teprotumumab therapy. Patients with controlled diabetes or impaired glucose tolerance can be treated safely if baseline screening, regular monitoring of glycemic control, and timely treatment of hyperglycemia are practiced. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article. [ABSTRACT FROM AUTHOR]

The etiology of Graves’ orbitopathy (GO) remains enigmatic. Optimal therapeutic choices for the hyperthyroidism associated with Graves’ disease (GD) in the presence of GO remain controversial. Whether antithyroid drugs (ATDs), radioiodine (RAI), or thyroidectomy should be favored in such patients remains debated. Pre-therapy variables such as ethnicity, sex, age, thyroid function, level of TSH-receptor antibodies and smoking behavior influence response to therapy. Among the most important management goals are restoring euthyroidism and abstaining from smoking. On average, ATDs and thyroidectomy – independent of extent – do not influence the natural course of GO. RAI can cause de novo development or progression of GO, which is largely preventable with oral steroid prophylaxis. In patients with mild GO, the thyroid treatment is largely independent of GO. Moderate to severe GO should be treated promptly. Deciding whether, in the latter, GD is better treated with ATDs, RAI, or surgery, is based more on expert opinion than on evidence. It is clear that in the individual patient a number of factors, not addressed in any trial, influence the final choice of therapy for GD, including concern of developing or negatively affecting pre-existing GO. Evidently, there is room for improving therapy of GO. Progress using novel drugs such as rituximab, which might potentially influence positively both GD and GO, are impatiently awaited. [ABSTRACT FROM AUTHOR]

The etiology of Graves’ orbitopathy (GO) remains enigmatic and thus controversy surrounds its pathogenesis. The role of the thyroid stimulating hormone receptor (TSHR) and activating antibodies directed against it in the hyperthyroidism of Graves’ disease (GD) is firmly established. Less well elucidated is what part the TSHR pathway might play in the development of GO. Also uncertain is the participation of other cell surface receptors in the disease. Elevated levels of insulin-like growth factor-1 receptor (IGF-1R) have been found in orbital fibroblasts as well as B and T cells from patients with GD. These abnormal patterns of IGF-1R display are also found in rheumatoid arthritis and carry functional consequences. In addition, activating IgGs capable of displacing IGF-1 from IGF-1R have also been detected in patients with these diseases. IGF-1R forms a complex with TSHR which is necessary for at least some of the non-canonical signaling observed following TSHR activation. Functional TSHR and IGF-1R have also been found on fibrocytes, CD34+ bone marrow-derived cells from the monocyte lineage. Levels of TSHR on fibrocytes greatly exceed those found on orbital fibroblasts. When ligated by TSH or M22, a TSHR-activating monoclonal antibody, fibrocytes produce extremely high levels of several cytokines and chemokines. Moreover, fibrocytes infiltrate both the orbit and thyroid in GD. In sum, based on current evidence, IGF-1R and TSHR can be thought of as “partners in crime”. Involvement of the former probably transcends disease boundaries, while TSHR may not. [ABSTRACT FROM AUTHOR]

The propensity of the skin of the lower anterior leg to be involved in Graves' dermopathy prompted an examination of the specific protein synthesis and response to interferon gamma in cultured fibroblasts from this area. Confluent cultures from normal skin of the lower leg and from the abdomen of the same three donors were pulse labeled with [35S]methionine for 3 h and subjected to two-dimensional protein gel electrophoresis and fluorography. Protein spots were mapped using a computer-driven program and the relative densities of the resolvable spots analyzed. Fibroblasts from the two anatomic sites display distinct patterns of de novo protein synthesis. Of the 157 abundant spots arbitrarily chosen for analysis, 31% varied substantially in levels of expression between the sites. A number of proteins appear to be expressed only in cultures derived from one of the two anatomic sites. Interferon gamma (100 U/ml) present in the culture medium for 48 h influenced the abundance of a number of proteins in a site-specific manner. Among them, plasminogen activator inhibitor type-I was induced three to five times in the leg cultures, whereas this same polypeptide was down-regulated in abdominal fibroblasts. A 54-kD protein was induced in interferon-treated cultures from both sites at least 50 times. It appears that fibroblasts from different regions of the integument are intrinsically distinct in terms of both their protein synthetic programs and their responses to cytokines. [ABSTRACT FROM AUTHOR]

Thyroid-associated ophthalmopathy (TAO) remains a vexing autoimmune component of Graves' disease that can diminish the quality of life as a consequence of its impact on visual function, physical appearance and emotional well-being. Because of its relative rarity and variable presentation, the development of highly effective and well-tolerated medical therapies for TAO has been slow relative to other autoimmune diseases. Contributing to the barriers of greater insight into TAO has been the historical absence of high-fidelity preclinical animal models. Despite these challenges, several agents, most developed for treatment of other diseases, have found their way into consideration for use in active TAO through repurposing. Among these, teprotumumab is a fully human inhibitory monoclonal antibody against the insulin-like growth factor I receptor. It has shown remarkable effectiveness in moderate to severe, active TAO in two completed multicenter, double masked, and placebo controlled clinical trials. The drug exhibits a favorable safety profile. Teprotumumab has recently been approved by the U.S. F.D.A, and may rapidly become the first line therapy for this disfiguring and potentially blinding condition. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to investigate the effects of K+ channel blockade on the uterorelaxant effects of nifedipine in human myometrium during pregnancy. Study Design: Biopsies of human myometrium were obtained at elective cesarean section (n = 24). Dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were subjected to K+ channel blockade using tetraethylammonium (TEA) or iberiotoxin (IbTX) followed by cumulative additions of nifedipine (1 nmol/L–10 μmol/L). Control experiments were run simultaneously. Integrals of contractile activity were measured using the PowerLab hardware unit and Chart v3.6 software. Data were analyzed using one-way analysis of variance (ANOVA) followed by post hoc analysis. Results: Nifedipine exerted a potent and cumulative inhibitory effect on spontaneous contractions and oxytocin-induced contractions in human myometrium in vitro, in comparison to control measurements (P < .05, n = 6). Incubation of strips with TEA or IbTX, prior to addition of nifedipine, significantly attenuated the relaxant effect exerted by nifedipine (P < .05, n = 6). Conclusion: This study demonstrates that the uterorelaxant effect of nifedipine is attenuated by potassium channel (K+) blockade. This suggests that K+ channel conductance, and particularly the BKCa channel, plays a role in the potent relaxant effect of nifedipine, hitherto presumed to act solely through L-gated calcium channels. [Copyright &y& Elsevier]

Abstract: Identification of the short DNA sequence motifs that serve as binding targets for transcription factors is an important challenge in bioinformatics. Unsupervised techniques from the statistical learning theory literature have often been applied to motif discovery, but effective solutions for large genomic datasets have yet to be found. We present here three self-organizing neural networks that have applicability to the motif-finding problem. The core system in this study is a previously described SOM-based motif-finder named SOMBRERO. The motif-finder is integrated in this work with a SOM-based method that automatically constructs generalized models for structurally related motifs and initializes SOMBRERO with relevant biological knowledge. A self-organizing tree method that displays the relationships between various motifs is also presented, and it is shown that such a method can act as an effective structural classifier of novel motifs. The performance of the three self-organizing neural networks is evaluated here using various datasets. [Copyright &y& Elsevier]

The purpose of this study was to develop an enzyme-linked immunospot assay (ELISpot assay) that can be used with human adherent cells. While standard enzyme-linked immunosorbent assays (ELISAs) are available and widely used and ELISpot assays are used for nonadherent lymphocytes, no ELISpot assay has been developed for adherent cells. We used primary human fibroblasts from four different tissues (myometrium, lung, gingiva, and orbit), either unstimulated or interleukin (IL)-1β-activated, to evaluate an ELISpot assay. Antibody pairs for IL-6 and IL-8 were used and results were compared to a standard ELISA. We found that we could reliably detect IL-6 and IL-8 spots with as few as 10 fibroblasts. Optimal cell numbers were 50 cells per well incubated for 8 h, although spots appeared as early as 2 h after incubation. Spots were absent when cells, primary, or secondary anti-cytokine antibodies were omitted from the protocol. Spot number and size can be ascertained using current automated ELISpot reader technology. The frequency of IL-6 and IL-8-producing human fibroblasts could also be determined. For example, 60% of the lung fibroblasts express IL-6, but IL-8 can be detected from only 40% of the cells. Approximately 80% of the human orbital fibroblasts make IL-6, whereas approximately 50% generate IL-8 following IL-1β stimulation. These new findings show that fibroblasts from different human tissues display different frequencies of cytokine production and this further supports the concept of fibroblast diversity. The sensitivity of this new ELISpot assay is adequate for cytokine detection in just a few cells, unlike the standard ELISA. It should permit ascertaining the frequency of fibroblasts and other adherent cells that produce cytokines and, if desired, can be used in tandem with a standard ELISA to determine total cytokine produced. Moreover, the assay is suitable for normal human adherent cells that are often short-lived and difficult to cultivate. [Copyright &y& Elsevier]

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 novoT3 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 formT3 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. [ABSTRACT FROM AUTHOR]

We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae . The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 10 2 –10 4 cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range. [ABSTRACT FROM AUTHOR]

Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae , Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae , N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (< 60 min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans. [ABSTRACT FROM AUTHOR]

Haemophilus influenzae is recognised as an important human pathogen associated with invasive infections, including bloodstream infection and meningitis. Currently used molecular-based diagnostic assays lack specificity in correctly detecting and identifying H. influenzae . As such, there is a need to develop novel diagnostic assays for the specific identification of H. influenzae . Whole genome comparative analysis was performed to identify putative diagnostic targets, which are unique in nucleotide sequence to H. influenzae . From this analysis, we identified 2 H. influenzae putative diagnostic targets, phoB and pstA , for use in real-time PCR diagnostic assays. Real-time PCR diagnostic assays using these targets were designed and optimised to specifically detect and identify all 55 H. influenzae strains tested. These novel rapid assays can be applied to the specific detection and identification of H. influenzae for use in epidemiological studies and could also enable improved monitoring of invasive disease caused by these bacteria. [ABSTRACT FROM AUTHOR]

Contractility of the myometrial smooth muscle cells during the estrous cycle and pregnancy is modulated by estrogen but the temporal expression of estrogen (relative to progesterone) and the type of contraction involved are distinctly different in pregnancy and estrous. This in vitro cell culture study investigated the global gene expression profile of human uterine smooth muscle cells (hUtSMCs) following 17β-estradiol (E2) treatment. In response to E2 treatment 540 genes, many of which have not been previously described as estrogen responsive, were identified as significantly differentially expressed. These genes are involved in biological processes that include muscle contraction, cell migration and adhesion, apoptosis and phosphorylation. Evidence from this study suggests that 17β-estradiol may have effects that are contrary to an overall contraction phenotype. The hUtSMC in vitro culture system is a useful model to investigate steroid effects on smooth muscle cells and may provide additional clues as to how smooth muscle cells behave in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Pregnant women in Nigeria use plant preparations to facilitate childbirth and to reduce associated pain. The rationale for this is not known and requires pharmacological validation. Aim of study: Obtain primary information regarding the traditional use of plants and analyze their uterine contractility at cellular level. Materials and methods: Semi-structured, open interviews using questionnaires of traditional healthcare professionals and other informants triggered the collection and identification of medicinal plant species. The relative traditional importance of each medicinal plant was determined by its use-mention index. Extracts of these plants were analyzed for their uterotonic properties on an in vitro human uterine cell collagen model. Result: The plants Calotropis procera, Commelina africana, Duranta repens, Hyptis suaveolens, Ocimum gratissimum, Saba comorensis, Sclerocarya birrea, Sida corymbosa and Vernonia amygdalina were documented and characterized. Aqueous extracts from these nine plants induced significant sustained increases in human myometrial smooth muscle cell contractility, with varying efficiencies, depending upon time and dose of exposure. Conclusion: The folkloric use of several plant species during childbirth in Nigeria has been validated. Seven plants were for the first time characterized to have contractile properties on uterine myometrial cells. The results serve as ideal starting points in the search for safe, longer lasting, effective and tolerable uterotonic drug leads. [Copyright &y& Elsevier]

UDP-glucose dehydrogenase (UGDH) catalyzes the formation of UDP-glucuronate. Glucuronate represents an integral component of the glycosaminoglycan, hyaluronan, which accumulates in orbital Graves disease. Here we report that orbital fibroblasts express higher levels of UGDH than do those from skin. This is a consequence of greater UGDH gene promoter activity and more abundant steady-state UGDH mRNA. Six Sp1 sites located in the proximal 550 bp of the UGDH gene promoter appear to determine basal promoter activity, as does a previously unrecognized 49-bp sequence spanning -1436 nucleotides (nt) and -1388 nt that negatively affects activity. Nuclear Sp1 protein is more abundant in orbital fibroblasts, and its binding to specific sites on DNA is greater than that in dermal fibroblasts. Mutating each of these Sp1 sites in a UGDH gene promoter fragment, extending from -1387 to +71 nt and fused to a luciferase reporter, results in divergent activities when transfected in orbital and dermal fibroblasts. Reducing Sp1 attenuated UGDH gene promoter activity, lowered steady-state UGDH mRNA levels, and reduced UGDH enzyme activity. Targeting Sp1 and UGDH with specific siRNAs also lowered hyaluronan synthase-1 (HAS-1) and HAS-2 levels and reduced hyaluronan accumulation in orbital fibroblasts. These findings suggest that orbital fibroblasts express high levels of UGDH in an anatomic-specific manner, apparently the result of greater constitutive Sp1. These high UGDH levels may underlie susceptibility of the orbit to localized overproduction of hyaluronan in Graves disease. [ABSTRACT FROM AUTHOR]

Abstract: Graves disease represents a systemic autoimmune process targeting the thyroid, orbit, and pretibial skin. The thyroid dysfunction is treatable, but no consistently effective medical therapy has yet been described for the orbital manifestations of Graves disease, also known as thyroid-associated ophthalmopathy or thyroid eye disease. Several autoantigens are potentially relevant to the pathogenesis of thyroid eye disease. Activating antibodies generated against the thyrotropin receptor can be detected in a majority of patients, and these drive hyperthyroidism. However, stimulating antibodies against the insulin-like growth factor-1 receptor (IGF-1R) may also play a role in the extra-thyroid manifestations of Graves disease. IGF-1R is overexpressed by orbital fibroblasts derived from patients with thyroid eye disease, whereas IGF-1R+ T and IGF-1R+ B cells are considerably more frequent in Graves disease. Actions of several cytokines and the molecular interplay peculiar to the orbit appear to provoke the inflammation, fat expansion, and deposition of excessive extracellular matrix molecules in thyroid eye disease. Based upon these new insights, several therapeutic strategies can now be proposed that, for the first time, might specifically interrupt its pathogenesis. [Copyright &y& Elsevier]

Orbital fibroblasts orchestrate tissue remodeling in Graves disease, at least in part, because they exhibit exaggerated responses to proinflammatory cytokines. A hallmark of late stage orbital disease is vision-threatening fibrosis, the molecular basis of which remains uncertain. We report here that the Th2 cytokines, interleukin (IL)-4 and IL-13, can induce in these cells the expression of 15-lipoxygenase-1 (15- LOX-1) and in so doing up-regulate the production of 15-hydroxyeicosatetraenoic acid. IL-4 increases 15-LOX-1 protein levels through pretranslational actions. The increased steady-state 15-LOX-1 mRNA is independent of ongoing protein synthesis and involves very modestly increased gene promoter activity. Importantly, IL-4 substantially enhances 15-LOX-1 transcript stability, activity that localizes to a 293-bp sequence of the 3′-untranslated region. IL-4 activates Jak2 in orbital fibroblasts. Interrupting signaling through that pathway, either with the specific chemical inhibitor, AG490, or by transiently transfecting the cells with a Jak2 dominant negative mutant kinase, attenuates the 15-LOX-1 induction. Interferon γ, a Th1 cytokine, could block this induction by attenuating IL-4-dependent mRNA stabilization. 15-LOX-1 protein and its mRNA were undetectable in IL-4-treated dermal fibroblasts, despite comparable levels of cell surface IL-4 receptor and phosphorylated Jak2 and STAT6. Our findings suggest that orbital connective tissues may represent a site of localized 15-hydroxyeicosatetraenoic acid generation resulting from cell type-specific 15-LOX-1 mRNA stabilization by IL-4. These results may have relevance to the pathogenesis of orbital Graves disease, an inflammatory autoimmune condition that gives way to extensive fibrosis associated with a Th2 response. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]