BACKGROUND Thyroid-associated ophthalmopathy, a condition commonly associated with Graves’ disease, remains inadequately treated. Current medical therapies, which primarily consist of glucocorticoids, have limited efficacy and present safety concerns. Inhibition of the insulin-like growth factor I receptor (IGF-IR) is a new therapeutic strategy to attenuate the underlying autoimmune pathogenesis of ophthalmopathy. METHODS We conducted a multicenter, double-masked, randomized, placebo-controlled trial to determine the efficacy and safety of teprotumumab, a human monoclonal antibody inhibitor of IGF-IR, in patients with active, moderate-to-severe ophthalmopathy. A total of 88 patients were randomly assigned to receive placebo or active drug administered intravenously once every 3 weeks for a total of eight infusions. The primary end point was the response in the study eye. This response was defined as a reduction of 2 points or more in the Clinical Activity Score (scores range from 0 to 7, with a score of ≥3 indicating active thyroid-associated ophthalmopathy) and a reduction of 2 mm or more in proptosis at week 24. Secondary end points, measured as continuous variables, included proptosis, the Clinical Activity Score, and results on the Graves’ ophthalmopathy–specific quality-of-life questionnaire. Adverse events were assessed. RESULTS In the intention-to-treat population, 29 of 42 patients who received teprotumumab (69%), as compared with 9 of 45 patients who received placebo (20%), had a response at week 24 (P<0.001). Therapeutic effects were rapid; at week 6, a total of 18 of 42 patients in the teprotumumab group (43%) and 2 of 45 patients in the placebo group (4%) had a response (P<0.001). Differences between the groups increased at subsequent time points. The only drug-related adverse event was hyperglycemia in patients with diabetes; this event was controlled by adjusting medication for diabetes. CONCLUSIONS In patients with active ophthalmopathy, teprotumumab was more effective than placebo in reducing proptosis and the Clinical Activity Score. (Funded by River Vision Development and others; ClinicalTrials.gov number, NCT01868997.)

Modes of sex determination are quite variable in vertebrates. The developmental decision to form a testis or an ovary can be influenced by one gene, several genes, environmental variables, or a combination of these factors. Nevertheless, certain morphogenetic aspects of sex determination appear to be conserved in amniotes. Here we clone fragments of nine candidate sex-determining genes from the snapping turtle Chelydra serpentina, a species with temperature-dependent sex determination (TSD). We then analyze expression of these genes during the thermosensitive period of gonad development. In particular, we compare gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature. Expression of Dmrt1 and Sox9 mRNA increased gradually at the male-producing temperature, but was suppressed at the female-producing temperature. This finding suggests that Dmrt1 and Sox9 play a role in testis development. In contrast, expression of aromatase, androgen receptor (Ar), and Foxl2 mRNA was constant at the male-producing temperature, but increased several-fold in embryos at the female-producing temperature. Aromatase, Ar, and Foxl2 may therefore play a role in ovary development. In addition, there was a small temperature effect on ERα expression with lower mRNA levels found in embryos at the female-producing temperature. Finally, Dax1, Fgf9, and SF-1 were not differentially expressed during the sex-determining period, suggesting these genes are not involved in sex determination in the snapping turtle. Comparison of gene expression profiles among amniotes indicates that Dmrt1 and Sox9 are part of a core testis-determining pathway and that Ar, aromatase, ERα, and Foxl2 are part of a core ovary-determining pathway.

y-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, is known to interact with two classes of GABA receptors denoted GABAA and GABAB. Using Xenopus oocytes, we compared the electrical and pharmacological properties of GABA receptors expressed by poly(A)+ RNA isolated from mammalian brain and retina. RNA from cerebral cortex expressed GABA responses with features characteristic of currents mediated by GABAA receptors. In contrast, RNA from retina expressed responses mediated by GABAA receptors and, in addition, GABA responses that were insensitive to the GABAA antagonist bicuculline and the GABAB agonist baclofen and showed no modulation by barbiturates or benzodiazepines. The bicuculline/baclofen-insensitive GABA response was a Cl current that was blocked by picrotoxin but showed little desensitization or outward rectification. Our results suggest that mmalian retina contains RNAs encoding GABA receptors with distinct pharmacology.In mammals, there are two well-characterized classes of receptors for the inhibitory neurotransmitter y-aminobutyric acid (GABA). GABAA receptors are ligand-gated Cl-channels that are competitively antagonized by bicuculline, noncompetitively blocked by picrotoxin, and allosterically modulated by barbiturates and benzodiazepines (1-3). Molecular cloning of cDNAs encoding GABAA receptor subunits indicates that the receptors are heteromeric and comprised of up to four different subunits, found in a variety of closely related subtypes (e.g., refs. 4-7). In contrast, GABAB receptors regulate K+ and Ca2+ channels through GTP-binding proteins and intracellular messenger pathways (8). These receptors have not been cloned but are presumed to belong to the superfamily of GTP-binding-protein-coupled receptors. GABAB receptors are selectively activated by baclofen, are antagonized by phaclofen and 2-hydroxysaclofen, and are not affected by bicuculline, picrotoxin, or any of the GABAA modulators (9,10).Xenopus oocytes are now widely used to study receptors and ion channels expressed after microinjection of either heterologous poly(A)+ RNA or RNAs transcribed from cloned cDNAs (for reviews, see refs. 11 and 12). GABAA subunits are readily expressed in oocytes and assemble to form receptors that have electrical and pharmacological properties similar to those reported for cells in situ (e.g., refs. 4-7, 13-15).Almost every neurotransmitter/neuromodulator identified in mammalian brain has also been found in retina (e.g., ref. 16). We used Xenopus oocytes to characterize neurotransmitter receptors expressed by retina RNAs, investigating whether there were any clear differences between the properties of brain and retina receptors. Initial studies, using poly(A)+ RNA isolated from bovine retina, showed that retina RNA primarily expressed receptors to excitatory amino acids, glycine, and substance P (ref. 17 and unpublished results). Herein we report on the GABA receptors encoded by retina RNAs. MATERIALS AND METHODSEleven poly(A)+ RNA preparations were made from ...

SUMMARY1. Catecholamines, adenosine, gonadotrophins, vasoactive intestinal peptide (VIP) and E-series prostaglandins all elicit K+ currents in follicle-enclosed Xenopus oocytes. Evidence suggests that cyclic nucleotides act as intracellular messengers in the activation of this K+ conductance. Muscarinic agonists and some divalent cations (e.g. Co2+, Mn2+, Ni2+ and Cd2+) elicit slow oscillatory Cl-currents, which are activated through hydrolysis of inositol phospholipids and mobilization of intracellular calcium by inositol phosphates.2. We investigated whether these membrane current responses were generated in the oocyte itself or in enveloping follicular cells which are coupled to the oocyte by gap junctions. Oocytes were defolliculated, either enzymatically using collagenase, or by manual dissection combined with rolling over poly-L-lysine-coated slides. Removal of follicular cells was checked using scanning electron microscopy. AIemnbrane current responses of defolliculated oocytes were compared with responses seen in follicle-enclosed oocytes taken from the same ovary.3. The K+ responses evoked by all the various hormones/neurotransmitters were either drastically reduced (> 90%) or abolished by defolliculation. K+ currents generated by the adenylate cyclase activator forskolin and by intraoocyte injection of adenosine 3',5'-cyclic monophosphate (cyclic AMP). or guanosine 3',5'-cyclic monophosphate were similarly reduced in defolliculated oocytes. In contrast, oscillatory Cl-currents to acetylcholine and divalent cations were selectively preserved through defolliculation.4. Injection of cyclic AMP (1-20 pmol) into defolliculated oocytes had little or no effect on oscillatory Cl-currents elicited by ACh. However, the calcium-dependent transient Cl-current, activated by depolarization of the oocyte membrane, was consistently potentiated (100-900%) by injections of cyclic AMP (1-10 pmol).5. These experiments suggest that cyclic nucleotide-activated K+ currents arise essentially in follicular cells and are monitored within the oocyte through electrical coupling by gap junctions. Oscillatory Cl-responses evoked by ACh and divalent cations are produced largely or wholly in the oocyte itself.

1 Caspases play a critical role in apoptosis, and are considered to be key targets for the design of cytoprotective drugs. As part of our antiapoptotic drug-discovery effort, we have synthesized and characterized Z-VD-fmk, MX1013, as a potent, irreversible dipeptide caspase inhibitor. 2 MX1013 inhibits caspases 1, 3, 6, 7, 8, and 9, with IC 50 values ranging from 5 to 20 nm. MX1013 is selective for caspases, and is a poor inhibitor of noncaspase proteases, such as cathepsin B, calpain I, or Factor Xa (IC 50 values 410 mm). 3 In several cell culture models of apoptosis, including caspase 3 processing, PARP cleavage, and DNA fragmentation, MX1013 is more active than tetrapeptide-and tripeptide-based caspase inhibitors, and blocked apoptosis at concentrations as low as 0.5 mm. 4 MX1013 is more aqueous soluble than tripeptide-based caspase inhibitors such as Z-VAD-fmk. 5 At a dose of 1 mg kg À1 i.v., MX1013 prevented liver damage and the lethality caused by Fas death receptor activation in the anti-Fas mouse-liver apoptosis model, a widely used model of liver failure. 6 At a dose of 20 mg kg À1 (i.v. bolus) followed by i.v. infusion for 6 or 12 h, MX1013 reduced cortical damage by approximately 50% in a model of brain ischemia/reperfusion injury. 7 At a dose of 20 mg kg À1 (i.v. bolus) followed by i.v. infusion for 12 h, MX1013 reduced heart damage by approximately 50% in a model of acute myocardial infarction. 8 Based on these studies, we conclude that MX1013, a dipeptide pan-caspase inhibitor, has a good combination of in vitro and in vivo properties. It has the ability to protect cells from a variety of apoptotic insults, and is systemically active in three animal models of apoptosis, including brain ischemia.