Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity.

CD4(+) T helper lymphocytes that express interleukin-17 (T(H)17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in T(H)17 cell differentiation and function with susceptibility to Crohn's disease, rheumatoid arthritis and psoriasis. Thus, the pathway towards differentiation of T(H)17 cells and, perhaps, of related innate lymphoid cells with similar effector functions, is an attractive target for therapeutic applications. Mouse and human T(H)17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORγt, which is required for induction of IL-17 transcription and for the manifestation of T(H)17-dependent autoimmune disease in mice. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORγt transcriptional activity. Digoxin inhibited murine T(H)17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene specifically inhibited induction of IL-17 in human CD4(+) T cells. Using these small-molecule compounds, we demonstrate that RORγt is important for the maintenance of IL-17 expression in mouse and human effector T cells. These data indicate that derivatives of digoxin can be used as chemical templates for the development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease.

In-situ Reverse Phased HPLC Analysis of Intact Antibody-Drug Conjugates.

The field of oncology has recently seen an exponential growth in antibody-drug conjugates (ADCs) as a biopharmaceutical class with seven ADCs being launched onto the market in the last ten years. Despite the increase in the industrial research and development of these compounds, their structural complexity and heterogeneity continue to present various challenges regarding their analysis including reaction monitoring. Robust and simple reaction monitoring analysis are in demand in the view of at-line in-process monitoring, and can instill control, confidence and reliability in the ADC manufacturing process. Aiming at providing chromatographic methods for conjugation monitoring, we evaluated herein the potential of utilizing reverse phase HPLC analysis, without sample pretreatment, for characterization of traditional cysteine-based ADCs. This analysis can be used for estimation of drug antibody ratio (DAR), which has shown the same trends and results as other well-established HPLC techniques. This methodology was also applied to three ADCs derived from three different antibodies. Additionally, we analyzed unpurified ADC samples existing in a complex reaction matrix and separated ADC species and payload compounds. This investigation was conducted using three different ADCs based on different payloads. The results described herein indicate the potential application of this RP-HPLC methodology in reaction monitoring studies.

A Purification Strategy Utilizing Hydrophobic Interaction Chromatography to Obtain Homogeneous Species from a Site-Specific Antibody Drug Conjugate Produced by AJICAP™ First Generation.

In recent years, site-specific antibody drug conjugates (ADC)s have been in great demand because they have an expanded therapeutic index compared with conventional ADCs. AJICAP™ technology is a chemical conjugation platform to obtain site-specific ADCs through the use of a class of Fc-affinity compounds. Promising results from early technology development studies led to further investigation of AJICAP™ ADC materials to obtain site-specific and homogeneous drug antibody ratio (DAR) ADCs. Here we report site-specific conjugation followed by a preparative hydrophobic interaction chromatography (HIC) purification strategy to obtain purified "DAR = 1.0" and "DAR = 2.0" AJICAP™ ADC materials. Optimization of the mobile phase conditions and resin achieved a high recovery rate. In vitro biological assay demonstrated the target selective activity for purified homogeneous DAR ADCs. These results indicate the ability of a HIC purification strategy to provide "DAR = 1.0" and "DAR = 2.0" AJICAP™ ADCs with considerable potency and target selectivity.

Analytical Comparison of Antibody-drug Conjugates Based on Good Manufacturing Practice Strategies.

The production of antibody-drug conjugates (ADCs) has been in great demand in the field of cancer therapeutics. Although cysteine-based conjugation is the most common and well known process for producing ADCs, multiple analytical methods are required for accurate drug-antibody ratio (DAR) determination due to the heterogeneity of the ADCs. Here we report various analytical methods for DAR analysis of traditional cysteine-based ADCs; additionally, apply a good manufacturing practice (GMP) strategy to produce a four hundred milligram ADC batch for use in good laboratory practice (GLP) studies. The work described herein not only evaluates several analytical performances but also provides guidance for future phase appropriate ADC production while establishing a unique analytical strategy.

TCR-dependent differentiation of thymic Foxp3+ cells is limited to small clonal sizes.

Numerous studies have highlighted the importance of high-affinity interactions between T cell receptors (TCRs) and their ligands in the selection of Foxp3(+) regulatory T cells (T reg cells). To determine the role of the TCR in directing T cells into the Foxp3(+) lineage, we generated transgenic (Tg) mice expressing TCRs from Foxp3(+) cells. Initial analyses of the TCR Tg mice crossed with RAG-deficient mice showed that the percentage of Foxp3(+) cells was very low. However, intrathymic injection and bone marrow chimera experiments showed a saturable increase of the Foxp3(+) population when T reg TCR Tg cells were present in low numbers. Furthermore, when analyzing whole thymi of T reg TCR Tg RAG-deficient mice, we found significantly more Foxp3(+) cells than in conventional T cell TCR Tg mice. Our results indicate that although the TCR has an instructive role in determining Foxp3 expression, selection of Foxp3(+) individual clones in the thymus is limited by a very small niche.

The value-action gap in waste recycling: the case of undergraduates in Hong Kong.

The discrepancy between verbal and actual commitment in waste recycling and environmental behavior is thought to have attenuated the effectiveness of many environmental policy and measures. Studies purport to show the existence of such a value-action gap in environmental issues has been largely based on matching the verbal commitment to environmental value through self-reported environmental behavioral data. Therefore, there is a lack of direct evidence to prove that such a discrepancy exists. This study demonstrates a methodology (contrasting on-site observation with self-reported results) to measure the gap between verbal commitment and actual recycling behavior and provides an explanation on the recycling behavior of students at Hong Kong Baptist University in the hope that the lessons learnt can be generalized to a wider context. Our findings indicate that a gap between verbal recycling commitment and corresponding action does exist in waste recycling on this university campus. By using multiple linear regression analysis, we found that the self-reported recycling behavior of undergraduates cannot be meaningfully explained by most variables previously suggested in the general value-action model.

Unique maturation program of the IgE response in vivo.

A key event in the pathogenesis of asthma and allergies is the production of IgE antibodies. We show here that IgE(+) cells were exceptional because they were largely found outside germinal centers and expressed, from very early on, a genetic program of plasma cells. In spite of their extragerminal center localization, IgE(+) cells showed signs of somatic hypermutation and affinity maturation. We demonstrated that high-affinity IgE(+) cells could be generated through a unique differentiation program that involved two phases: a pre-IgE phase in which somatic hypermutation and affinity maturation take place in IgG1(+) cells, and a post-IgE-switching phase in which IgE cells differentiate swiftly into plasma cells. Our results have implications for the understanding of IgE memory responses in allergy.

Effects of E4031 and quinidine on atrial and ventricular refractoriness in rabbits in vivo.

This study assessed the effects of E4031 and quinidine on refractoriness (ERP) in a new in vivo model in rabbits. Following sinoatrial (SAN) and atrioventricular node (AVN) ablation ERP was determined in atria and ventricles with the shortest S1-S2 interval eliciting a second electrogram defined as the ERP. The effects of E4031 and quinidine (dose ranges 1-8 micromol/kg) were compared. E4031 dose-dependently increased ERP. The maximum change from pre-drug values with E4031 was 27+/-8 msec (a 36+/-12% increase) at 2 Hz in atria and 51+/-9 msec (27+/-5%) at 2 Hz in ventricles. Negative frequency-dependence was observed only in ventricles. Quinidine dose-dependently increased ERP. The maximum increase for quinidine was 23+/-3 msec (28+/-4%) at 2 Hz in atria and 25+/-10 msec (22+/-10%) at 6 Hz in ventricles, but without frequency-dependence in either tissue. In comparison to E4031, quinidine produced smaller changes in ERP and showed minimal frequency dependence. Thus, the added presence of sodium blocking actions with quinidine did not produce greater effects on ERP than I(Kr) blockade alone with E4031. However, quinidine also blocks other potassium currents, such as Ito, and the degree of I(Kr) blockade with E4031 was probably greater than that with the same dose of quinidine. This model may have clinical utility for testing multi-ion channel blocking drugs.

Effects of flecainide on atrial and ventricular refractoriness in rabbits in vitro and in vivo.

This study compared the in vitro versus in vivo effects of flecainide on effective refractory period (ERP) in atrial and ventricular tissue in rabbits. Flecainide (a class 1c agent) was chosen, on the basis of its known pharmacological profile and antiarrhythmic actions, to provide a reference compound for investigating models that suitably predict the clinical effects of antiarrhythmics. The rabbit models used were those previously described by Lowe et al. (2002) and Leung et al. (2003). ERP was measured as the shortest S1-S2 interval that elicited a second contraction (in vitro) or electrogram (in vivo). Flecainide (1-10 microM) in vitro produced a concentration-dependent increase in ERP. The greatest drug-induced change from pre-drug values in vitro occurred with the highest concentration in atria and ventricles at 4 Hz. The change was 30+/-4 msec (33+/-7%) in atria versus 53+/-8 msec (46+/-10%) in ventricles. In vivo, flecainide (1 - 4 micromol/kg) dose-dependently increased atrial ERP at 2 and 6 Hz. The biggest change was 28+/-17 msec (29+/-16%). However, there was no effect at 4 Hz. In the ventricles, a dose-related increase in ERP was only seen at 4 Hz (26+/-6 msec). Flecainide showed no frequency dependence of action on ERP in any preparation. Flecainide produced adverse effects both in vitro and in vivo. A concentration and frequency-dependent negative inotropic effect was seen in vitro, and dose-related hypotension in vivo. The highest dose (8 micromol/kg i.v.) of flecainide was lethal. Flecainide produced the expected electrophysiological and toxicity profile, both in vitro and in vivo. Despite such findings, the drug is used to terminate and prevent atrial arrhythmias clinically. In conclusion our rabbit models for determining ERP may not be useful in predicting the clinical usefulness of a drug like flecainide.