Feline Interleukin-31 Shares Overlapping Epitopes with the Oncostatin M Receptor and IL-31RA.

Interleukin-31 (IL-31) is a major protein involved in severe inflammatory skin disorders. Its signaling pathway is mediated through two type I cytokine receptors, IL-31RA (also known as the gp130-like receptor) and the oncostatin M receptor (OSMR). Understanding molecular details in these interactions would be helpful for developing antagonist anti-IL-31 monoclonal antibodies (mAbs) as potential therapies. Previous studies suggest that human IL-31 binds to IL-31RA and then recruits OSMR to form a ternary complex. In this model, OSMR cannot interact with IL-31 in the absence of IL-31RA. In this work, we show that feline IL-31 (fIL-31) binds independently with feline OSMR using surface plasmon resonance, an enzyme-linked immunosorbent assay, and yeast surface display. Moreover, competition experiments suggest that OSMR shares a partially overlapping epitope with IL-31RA. We then used deep mutational scanning to map the binding sites of both receptors on fIL-31. In agreement with previous studies of the human homologue, the binding site for IL31-RA contains fIL-31 positions E20 and K82, while the binding site for OSMR comprises the "PADNFERK" motif (P103-K110) and position G38. However, our results also revealed a new overlapping site, composed of positions R69, R72, P73, D76, D81, and E97, between both receptors that we called the "shared site". The conformational epitope of an anti-feline IL-31 mAb that inhibits both OSMR and IL-31RA also mapped to this shared site. Combined, our results show that fIL-31 binds IL-31RA and OSMR independently through a partially shared epitope. These results suggest reexamination of the putative canonical mechanisms for IL-31 signaling in higher animals.

Comparative functional characterization of canine IgG subclasses.

To date, very little is known about the functional characteristics of the four published canine IgG subclasses. It is not clear how each subclass engages the immune system via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC), or how long each antibody may last in serum. Such information is critical for understanding canine immunology and for the discovery of canine therapeutic monoclonal antibodies. Through both in vitro and ex vivo experiments to evaluate canine Fc's for effector function, complement binding, FcRn binding, and ADCC, we are now able to categorize canine subclasses by function. The subclasses share functional properties with the four human IgG subclasses and are reported herein with their function-based human analog. Canine Fc fusions, canine chimeras, and caninized antibodies were characterized. Canine subclasses A and D appear effector-function negative while subclasses B and C bind canine Fc gamma receptors and are positive for ADCC. All canine subclasses bind the neonatal Fc receptor except subclass C. By understanding canine IgGs in this way, we can apply what is known of human immunology toward translational and veterinary medicine. Thus, this body of work lays the foundation for evaluating canine IgG subclasses for therapeutic antibody development and builds upon the fundamental scholarship of canine immunology.

Knowledge and perceptions of couples' voluntary counseling and testing in urban Rwanda and Zambia: a cross-sectional household survey.

BACKGROUND: Most incident HIV infections in sub-Saharan Africa occur between cohabiting, discordant, heterosexual couples. Though couples' voluntary HIV counseling and testing (CVCT) is an effective, well-studied intervention in Africa, <1% of couples have been jointly tested. METHODS: We conducted cross-sectional household surveys in Kigali, Rwanda (n = 600) and Lusaka, Zambia (n = 603) to ascertain knowledge, perceptions, and barriers to use of CVCT. RESULTS: Compared to Lusaka, Kigali respondents were significantly more aware of HIV testing sites (79% vs. 56%); had greater knowledge of HIV serodiscordance between couples (83% vs. 43%); believed CVCT is good (96% vs. 72%); and were willing to test jointly (91% vs. 47%). Stigma, fear of partner reaction, and distance/cost/logistics were CVCT barriers. CONCLUSIONS: Though most respondents had positive attitudes toward CVCT, the majority were unaware that serodiscordance between cohabiting couples is possible. Future messages should target gaps in knowledge about serodiscordance, provide logistical information about CVCT services, and aim to reduce stigma and fear.

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore.

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

Development of a liquid chromatography/mass spectrometry-based drug accumulation assay in Pseudomonas aeruginosa.

Bacterial resistance to antibiotic therapy remains a worldwide problem. In Pseudomonasaeruginosa, rates of efflux confer inherent resistance to many antimicrobial agents, including fluoroquinolones, due to a high level of expression and a relatively high turnover number of the efflux pumps in gram-negative bacteria. To understand the roles of efflux pumps in both the influx and efflux of compounds in P. aeruginosa and to aid the chemistry compound design by bridging in vitro enzymatic binding data (IC(50) values) with whole cell results (MIC numbers), a collaborative effort was put forward to validate a series of bacterial penetration/accumulation assays for assessment of intracellular drug concentration. Initially, using 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP) as the tracer, a 96-well fluorescence assay was established to measure the time-dependent accumulation of DMP in wild-type (PAO1), MexABOprM deletion (PAO200), and MexABOprM-MexCDOprJ-MexJKL:FRT deletion mutants (PAO314). At steady state, the order of DMP accumulation was PAO314>PAO200>PAO1. Subsequently, the established assay conditions were applied to a radiolabeled assay format using (3)H-labeled ciprofloxacin. At the concentration tested, the accumulation of [(3)H]ciprofloxacin approached a plateau after 15 min and the amount of accumulation in PAO314 was higher (~2- to 10-fold) than that in PAO1. Finally, with an additional step of cell lysis, a liquid chromatography/mass spectrometry-based assay was established with ciprofloxacin with (i) superior sensitivity (the detection limit can be as low as 0.24 ng/ml for ciprofloxacin) and (ii) the ability to monitor cold or nonfluorescent compounds in a drug discovery setting.

3-aminoquinazolinediones as a new class of antibacterial agents demonstrating excellent antibacterial activity against wild-type and multidrug resistant organisms.

The 3-aminoquinzolinediones represent a new series of antibacterial agents structurally related to the fluoroquinolones. They are inhibitors of bacterial gyrase and topoisomerase IV and demonstrate clinically useful antibacterial activity against fastidious Gram-negative and Gram-positive organisms, including multidrug- and fluoroquinolone-resistant organisms. These agents also demonstrate in vivo efficacy in murine systemic infection models.

Role of inhibitor aliphatic chain in the thermodynamics of inhibitor binding to Escherichia coli enoyl-ACP reductase and the Phe203Leu mutant: a proposed mechanism for drug resistance.

The antibacterial target enoyl-acyl carrier protein (ACP) reductase is a homotetrameric enzyme that catalyzes the last reductive step of fatty acid biosynthesis. In the present paper, four 2-(2-hydroxyphenoxy)phenol inhibitors, wherein the 4-position substituent varied from H to n-propyl, were studied to determine the contribution of the aliphatic chain to the binding to the wild-type (wt) enoyl-ACP reductase from Escherichia coli (FabI) and a drug-resistant mutant, (F203L)FabI, in which phenylalanine 203 is mutated to leucine. Thermodynamic parameters of ternary complex formation (enzyme-NAD(+)-inhibitor) were determined by isothermal titration calorimetry. The inhibitor affinity to wt FabI and (F203L)FabI increases with increasing aliphatic chain length, although the corresponding affinity for (F203L)FabI is lower, and also, it shows no detectable binding to the 4-H inhibitor. A distinguishing feature of inhibitor binding to either binary enzyme-NAD(+) complex is the apparent negative cooperativity for binding to the tetramer with half-site occupancy. For both enzymes, binding is enthalpy, DeltaH, driven. However, binding DeltaH becomes less favorable with increasing aliphatic chain length. Increases in affinity are found to be exclusively due to favorable changes in solvation entropy. Incremental changes in thermodynamic parameters within the series of inhibitors binding to wt FabI and (F203L)FabI are approximately the same. However, absolute differences between the two enzymes for binding to a given inhibitor are significant, suggesting different binding modes. This finding, coupled with a binding site conformation that is likely to be more rigid in the mutant, appears to result in the drug resistance of (F203L)FabI.