Evicting hitchhiker antigens from purified antibodies

Antibodies that target common cellular structures may have a propensity to bind those very same antigens as they become exposed in dead or dying cells during production in a bioreactor. Those tendencies can be accentuated if the targeted epitope is highly conserved across species. While attention to contaminants such as endotoxin, viral particles, cellular DNA and even prions has grown coincident with the emergence of the monoclonal antibody industry, it is surprising how little attention has been focused on hitchhiker antigens that may co-elute while bound to the supposedly pure antibody. In this case study, we will focus on anti-histone antibodies and the measures we have taken to eliminate stowaways, such as histone–DNA complexes. These simple measures include the addition of a quartenary amine guard column to the protein A, adjusting the ionic strength of the cell culture supernatant to 400 mM sodium chloride, and establishing a mobile phase gradient from 400 mM to 2 M during protein A chromatography. Initially adjusting the cell culture to 600 mM can compromise the quartenary amine guard column. Also, we demonstrate the applicability of these techniques in both the R&D lab and the manufacturing plant, particularly in improving the apparent potency of antibodies destined for the clinic. Given the prominence of anti-histone antibodies in chromatin immunoprecipitation (ChIP), the implications of hitchhiker antigens interferring with the results of an experiment are far-reaching, indeed, we detect them in some popularly used antibodies. Moreover, a wide variety of monoclonals that may target antigens expressed by the producer cell line may face similar problems, resulting in a decreased production yield, as well as a diminished apparent binding potency.     

Structure of the antiviral assembly inhibitor CAP-1 complex with the HIV-1 CA protein

The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CA^N) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CA^N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N′-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.     

Point mutations in the HIV-1 matrix protein turn off the myristyl switch

During the late phase of human immunodeficiency virus type-1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P(2)) and Gag's N-terminally myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P(2) binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P(2) is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P(2) or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr(s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5)P(2) binding. This mutant binds PI(4,5)P(2) with twofold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding.     

Quantitative Circulatory Physiology: an integrative mathematical model of human physiology for medical education

We have developed Quantitative Circulatory Physiology (QCP), a mathematical model of integrative human physiology containing over 4,000 variables of biological interactions. This model provides a teaching environment that mimics clinical problems encountered in the practice of medicine. The model structure is based on documented physiological responses within peer-reviewed literature and serves as a dynamic compendium of physiological knowledge. The model is solved using a desktop, Windows-based program, allowing students to calculate time-dependent solutions and interactively alter over 750 parameters that modify physiological function. The model can be used to understand proposed mechanisms of physiological function and the interactions among physiological variables that may not be otherwise intuitively evident. In addition to open-ended or unstructured simulations, we have developed 30 physiological simulations, including heart failure, anemia, diabetes, and hemorrhage. Additional stimulations include 29 patients in which students are challenged to diagnose the pathophysiology based on their understanding of integrative physiology. In summary, QCP allows students to examine, integrate, and understand a host of physiological factors without causing harm to patients. This model is available as a free download for Windows computers at http://physiology.umc.edu/themodelingworkshop.     

A review of the subspecies status of the Icelandic Purple Sandpiper Calidris maritima littoralis

The Icelandic Purple Sandpiper Calidris maritima littoralis (C.L. Brehm, 1831) represents one member of a poorly understood subspecies complex. Currently, differences in size define two other subspecies: Calidris maritima belcheri Engelmoer & Roselaar, 1998, which breeds in north‐eastern Canada along the Hudson Bay and James Bay, and Calidris maritima maritima (Brunnich, 1764), which breeds along the Arctic coasts elsewhere in northern Canada, Greenland, Svalbard, Scotland, and Fennoscandia, to northern central Siberia. There are large size differences amongst populations of C. m. maritima, however. As an Arctic/Alpine breeding bird, C. m. littoralis could provide an interesting perspective on the evolutionary changes following a northwards expansion of a species after glacial retreat. Considering the extent of the ice sheet in the northern hemisphere during the last glaciation, and the short period of time since it ended, the correct attribution of subspecies status for C. m. maritima may reflect either rapid diversification from a single population or ancestral splits of distinct evolutionary lineages that survived in isolation at southern latitudes. We applied morphometric subspecies criteria, diagnosability by Amadon's rule, and genetic analysis of five nuclear introns, and the mitochondrial DNA markers cytochrome oxidase c subunit I (COI) and NADH dehydrogenase subunit 2 (ND2), to geographically separate breeding populations in order to examine the subspecies status of the Icelandic population. The results do not provide support for the subspecies status of the Icelandic population because the nominate and Icelandic subspecies fail to uphold Amadon's rule, and genetic analyses indicate that the study populations derive from a single shared refugium. © 2015 The Linnean Society of London     

Rh-I-UEA-1 polymerized liposomes target and image adenomatous polyps in the APC(Min/+) mouse using optical colonography

Mutated adenomatous polyposis coli (APC) genes predispose transformations to neoplasia, progressing to colorectal carcinoma. Early detection facilitates clinical management and therapy. Novel lectin-mediated polymerized targeted liposomes (Rh-I-UEA-1), with polyp specificity and incorporated imaging agents were fabricated to locate and image adenomatous polyps in APC(Min/+) mice. The biomarker α-L-fucose covalently joins the liposomal conjugated lectin Ulexeuropaeus agglutinin (UEA-1), via glycosidic linkage to the polyp mucin layer. Multispectral optical imaging (MSI) corroborated a global perspective of specific binding (rhodamine B 532 nm emission, 590-620 nm excitation) of targeted Rh-I-UEA-1 polymerized liposomes to polyps with 1.4-fold labeling efficiency. High-resolution coregistered optical coherence tomography (OCT) and fluorescence molecular imaging (FMI) reveal the spatial correlation of contrast distribution and tissue morphology. Freshly excised APC(Min) bowels were incubated with targeted liposomes (UEA-1 lectin), control liposomes (no lectin), or iohexol (Omnipaque) and imaged by the three techniques. Computed tomographic quantitative analyses did not confirm that targeted liposomes more strongly bound polyps than nontargeted liposomes or iohexol (Omnipaque) alone. OCT, with anatomic depth capabilities, along with the coregistered FMI, substantiated Rh-I-UEA-1 liposome binding along the mucinous polyp surface. UEA-1 lectin denotes α-l-fucose biomarker carbohydrate expression at the mucin glycoprotein layer; Rh-I-UEA-1 polymerized liposomes target and image adenomatous polyps in APC(Min) mice.     

Dissecting the serotonergic food signal stimulating sensory-mediated aversive behavior in C. elegans

Nutritional state often modulates olfaction and in Caenorhabditis elegans food stimulates aversive responses mediated by the nociceptive ASH sensory neurons. In the present study, we have characterized the role of key serotonergic neurons that differentially modulate aversive behavior in response to changing nutritional status. The serotonergic NSM and ADF neurons play antagonistic roles in food stimulation. NSM 5-HT activates SER-5 on the ASHs and SER-1 on the RIA interneurons and stimulates aversive responses, suggesting that food-dependent serotonergic stimulation involves local changes in 5-HT levels mediated by extrasynaptic 5-HT receptors. In contrast, ADF 5-HT activates SER-1 on the octopaminergic RIC interneurons to inhibit food-stimulation, suggesting neuron-specific stimulatory and inhibitory roles for SER-1 signaling. Both the NSMs and ADFs express INS-1, an insulin-like peptide, that appears to cell autonomously inhibit serotonergic signaling. Food also modulates directional decisions after reversal is complete, through the same serotonergic neurons and receptors involved in the initiation of reversal, and the decision to continue forward or change direction after reversal is dictated entirely by nutritional state. These results highlight the complexity of the "food signal" and serotonergic signaling in the modulation of sensory-mediated aversive behaviors.