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Objective: Leptin (LEP) deficiency results in major metabolic perturbations, including obesity, dyslipidemia, and diabetes. Although LEP deficiency can be treated with daily injections of a recombinant LEP, generation of an antibody activating the LEP receptor (LEPR) that has both an intrinsically long half-life and low immunogenicity could be useful in the treatment of this condition., Methods: Phage display technology coupled with flow cytometry and cell-based in vitro assays were employed to identify an allosteric agonist of the mouse LEPR. LEP-deficient Lep(ob) /Lep(ob) mice were used to compare in vivo effects of LEP to antibody administration. To evaluate hypothalamic effects of treatment, changes in mRNA levels of neuropeptide Y and proopiomelanocortin were measured., Results: XPA.80.037 is a monoclonal antibody that demonstrates allosteric agonism of the mouse LEPR. Treatment of Lep(ob) /Lep(ob) mice with XPA.80.037 markedly reduced hyperphagia and body weight, normalized blood glucose and plasma insulin levels, and corrected dyslipidemia. These metabolic alterations correlated with changes in mRNA levels of neuropeptide Y and proopiomelanocortin, suggesting that XPA.80.037 had hypothalamic effects., Conclusions: Agonist allosteric monoclonal antibodies to the LEPR can correct metabolic effects associated with LEP deficiency in vivo and thereby have the potential to treat conditions of LEP deficiency., (© 2016 The Obesity Society.)

Background: Inpatient teams commonly track their tasks using paper checklists that are not shared between team members. Team members frequently communicate redundantly in order to prevent errors., Methods: We created a mobile, collaborative, real-time task list application on the iOS platform. The application listed tasks for each patient, allowed users to check them off as completed, and transmitted that information to all other team members. In this report, we qualitatively describe our experience designing and piloting the application with an inpatient pediatric ward team at an academic pediatric hospital., Results: We successfully created the tasklist application, however team members showed limited usage., Conclusion: Physicians described that they preferred the immediacy and familiarity of paper, and did not experience an efficiency benefit when using the electronic tasklist.

It is widely understood that selenite can be biologically reduced to elemental selenium. Limited studies have shown that selenite can also be immobilized through abiotic precipitation with sulfide, a product of biological sulfate reduction. We demonstrate that both pathways significantly contribute to selenite immobilization in a microfluidic flow cell having a transverse mixing zone between propionate and selenite that mimics the reaction zone along the margins of a selenite plume undergoing bioremediation in the presence of background sulfate. The experiment showed that red particles of amorphous elemental selenium precipitate on the selenite-rich side of the mixing zone, while long crystals of selenium sulfides precipitate on the propionate-rich side of the mixing zone. We developed a continuum-scale reactive transport model that includes both pathways. The simulated results are consistent with the experimental results, and indicate that spatial segregation of the two selenium precipitates is due to the segregation of the more thermodynamic favorable selenite reduction and the less thermodynamically favorable sulfate reduction. The improved understanding of selenite immobilization and the improved model can help to better design in situ bioremediation processes for groundwater contaminated by selenite or other contaminants (e.g., uranium(IV)) that can be immobilized via similar pathways.

Previously we reported studies of XMetA, an agonist antibody to the insulin receptor (INSR). We have now utilized phage display to identify XMetS, a novel monoclonal antibody to the INSR. Biophysical studies demonstrated that XMetS bound to the human and mouse INSR with picomolar affinity. Unlike monoclonal antibody XMetA, XMetS alone had little or no agonist effect on the INSR. However, XMetS was a strong positive allosteric modulator of the INSR that increased the binding affinity for insulin nearly 20-fold. XMetS potentiated insulin-stimulated INSR signaling ∼15-fold or greater including; autophosphorylation of the INSR, phosphorylation of Akt, a major enzyme in the metabolic pathway, and phosphorylation of Erk, a major enzyme in the growth pathway. The enhanced signaling effects of XMetS were more pronounced with Akt than with Erk. In cultured cells, XMetS also enhanced insulin-stimulated glucose transport. In contrast to its effects on the INSR, XMetS did not potentiate IGF-1 activation of the IGF-1 receptor. We studied the effect of XMetS treatment in two mouse models of insulin resistance and diabetes. The first was the diet induced obesity mouse, a hyperinsulinemic, insulin resistant animal, and the second was the multi-low dose streptozotocin/high-fat diet mouse, an insulinopenic, insulin resistant animal. In both models, XMetS normalized fasting blood glucose levels and glucose tolerance. In concert with its ability to potentiate insulin action at the INSR, XMetS reduced insulin and C-peptide levels in both mouse models. XMetS improved the response to exogenous insulin without causing hypoglycemia. These data indicate that an allosteric monoclonal antibody can be generated that markedly enhances the binding affinity of insulin to the INSR. These data also suggest that an INSR monoclonal antibody with these characteristics may have the potential to both improve glucose metabolism in insulinopenic type 2 diabetes mellitus and correct compensatory hyperinsulinism in insulin resistant conditions.

Novel therapies are needed for the treatment of hypoglycemia resulting from both endogenous and exogenous hyperinsulinema. To provide a potential new treatment option, we identified XMetD, an allosteric monoclonal antibody to the insulin receptor (INSR) that was isolated from a human antibody phage display library. To selectively obtain antibodies directed at allosteric sites, panning of the phage display library was conducted using the insulin-INSR complex. Studies indicated that XMetD bound to the INSR with nanomolar affinity. Addition of insulin reduced the affinity of XMetD to the INSR by 3-fold, and XMetD reduced the affinity of the INSR for insulin 3-fold. In addition to inhibiting INSR binding, XMetD also inhibited insulin-induced INSR signaling by 20- to 100-fold. These signaling functions included INSR autophosphorylation, Akt activation and glucose transport. These data indicated that XMetD was an allosteric antagonist of the INSR because, in addition to inhibiting the INSR via modulation of binding affinity, it also inhibited the INSR via modulation of signaling efficacy. Intraperitoneal injection of XMetD at 10 mg/kg twice weekly into normal mice induced insulin resistance. When sustained-release insulin implants were placed into normal mice, they developed fasting hypoglycemia in the range of 50 mg/dl. This hypoglycemia was reversed by XMetD treatment. These studies demonstrate that allosteric monoclonal antibodies, such as XMetD, can antagonize INSR signaling both in vitro and in vivo. They also suggest that this class of allosteric monoclonal antibodies has the potential to treat hyperinsulinemic hypoglycemia resulting from conditions such as insulinoma, congenital hyperinsulinism and insulin overdose.

Objective: Atherosclerosis is a condition that is increasingly contributing to worldwide mortality through complications such as stroke and myocardial infarction. IL-1β plays multiple direct, local roles in the formation and stability of the atheroma by eliciting the production of additional cytokines and proteolytic enzymes from macrophages, endothelial cells (EC) and smooth muscle cells (SMC). We therefore tested whether an anti-IL-1β antibody, XOMA 052, might inhibit the secretion of pro-atherogenic cytokines from macrophages in vitro and affect a positive outcome in the Apolipoprotein E-deficient mouse (ApoE(-/-)) model of atherosclerosis in vivo., Methods and Results: In an in vitro co-culture model, XOMA 052 inhibited macrophage-induced secretion of key atherogenic cytokines from EC and SMC, including IL-6, IL-8, MCP-1 and TNFα. The release of degradative enzymes, such as the matrix metalloproteinases MMP-3 and MMP-9, was also decreased by XOMA 052. In addition, XOMA 052 inhibited the secretion of IL-7 from EC and IL-4 from SMC, cytokines not previously reported to be driven by IL-1β in this context. In vivo, XMA052 MG1K, a chimeric murine version of XOMA 052, inhibited the formation of atherosclerotic lesions in the ApoE(-/-) model at all three doses tested. This effect was comparable to that reported for complete genetic ablation of IL-1β or IL-1R1 on an ApoE(-/-) background and was associated with decreases in plasma non-HDL/HDL cholesterol ratio and plaque lipid content and macrophage infiltration., Conclusions: These results demonstrate for the first time that an antibody targeting IL-1β can inhibit the progression of atherosclerosis in vivo, highlighting the importance of this key cytokine in cardiovascular disease., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In Brazil, where Leishmania chagasi causes endemic American visceral leishmaniasis (AVL), the spread and maintenance of human disease are attributed to canine reservoirs. However, despite measures directed toward the elimination of infected canines, the incidence of human disease continues to increase. To evaluate the role of infected canines in the acquisition of AVL by humans, we undertook a controlled intervention study in three similar, but isolated, valleys of Pancas, Espírito Santo, Brazil. In the two experimental (intervention) valleys, infected dogs were eliminated whereas in the control valley, seropositive canines remained untouched. During the 12-month study period, human seropositivity rates, as measured by dot enzyme-linked immunosorbent assay, increased from 15% to 54% in the intervention valleys and from 14% to 54% in the control valley. The elimination of infected canines in the intervention valleys did not result in a statistically significant difference between the incidences of human serological conversion in the intervention and control valleys at either 6 (20% and 22%, respectively; P = .5961) or 12 months (26% and 27%, respectively; P = .9442). The role of humans as a significant reservoir for AVL is proposed as an explanation for the study results.

Purpose: A major pathway for intracellular signaling from cell surface receptors, such as integrins, involves intracellular phosphorylation. In corneal fibroblasts, the authors have investigated the role of tyrosine phosphorylation in integrin-dependent cell adhesion to extracellular matrix., Methods: Antibodies were used to detect phosphotyrosine-containing proteins, including focal adhesion kinase in lysates and immunoprecipitates of corneal fibroblasts. The authors used anti-phosphotyrosine antibodies to localize phosphotyrosines in fixed cultured corneal fibroblasts. Similarly, immunocytochemical detection of vinculin was used to identify focal adhesions, the subcellular structures in which integrins organize attachment to matrix extracellularly and to cytoskeletal components intracellularly., Results: Suspension of corneal fibroblasts produced a dramatic decrease in detectable phosphotyrosines. During integrin-dependent fibroblast attachment to exogenously supplied fibronectin, the cytoplasmic phosphotyrosine kinase, focal adhesion kinase (FAK), pp125FAK, became tyrosine phosphorylated. However, FAK was not phosphorylated during fibroblast attachment to vitronectin or polylysine or when cells were kept in suspension. In addition, the treatment of suspended cells with antibody to the extracellular domain of fibronectin receptor caused FAK phosphorylation. Phosphotyrosine was colocalized with vinculin in newly formed focal adhesions. Focal adhesion formation was prevented by herbimycin A, an inhibitor of tyrosine kinases., Conclusions: In corneal fibroblasts, fibronectin receptor-specific signal transduction from extracellular matrix during the formation of focal adhesions requires tyrosine kinase activation, including phosphorylation of FAK. This underscores a role for the fibronectin receptor in signaling from the extracellular matrix in corneal fibroblasts.