Durable Immunity to Ricin Toxin Elicited by Intranasally Administered Monoclonal Antibody-Based Immune Complexes.

Inhalation of ricin toxin (RT) elicits profuse inflammation and cell death within the upper and lower airways, ultimately culminating in acute respiratory distress syndrome. We previously reported that the effects of pulmonary RT exposure in mice are nullified by intranasal administration of an mAb mixture consisting of PB10, directed against ricin's enzymatic subunit (RTA), and SylH3, directed against ricin's binding subunit (RTB). We now report that delivery of PB10 and SylH3 as an RT-mAb immune complex (RIC) to mice by the intranasal or i.p. routes stimulates the rapid onset of RT-specific serum IgG that persists for months. RIC administration also induced high-titer, toxin-neutralizing Abs. Moreover, RIC-treated mice were immune to a subsequent 5 × LD50 RT challenge on days 30 or 90. Intranasal RIC administration was more effective than i.p. delivery at rendering mice immune to intranasal RT exposure. Finally, we found that the onset of RT-specific serum IgG following RIC delivery was independent of FcγR engagement, as revealed through FcγR knockout mice and RICs generated with PB10/SylH3 LALA (leucine to alanine) derivatives. In conclusion, a single dose of RICs given intranasally to mice was sufficient to stimulate durable protective immunity to RT by an FcγR-independent pathway.

Structural Analysis of Toxin-Neutralizing, Single-Domain Antibodies that Bridge Ricin's A-B Subunit Interface.

Ricin toxin kills mammalian cells with notorious efficiency. The toxin's B subunit (RTB) is a Gal/GalNAc-specific lectin that attaches to cell surfaces and promotes retrograde transport of ricin's A subunit (RTA) to the trans Golgi network (TGN) and endoplasmic reticulum (ER). RTA is liberated from RTB in the ER and translocated into the cell cytoplasm, where it functions as a ribosome-inactivating protein. While antibodies against ricin's individual subunits have been reported, we now describe seven alpaca-derived, single-domain antibodies (VHHs) that span the RTA-RTB interface, including four Tier 1 VHHs with IC50 values <1 nM. Crystal structures of each VHH bound to native ricin holotoxin revealed three different binding modes, based on contact with RTA's F-G loop (mode 1), RTB's subdomain 2γ (mode 2) or both (mode 3). VHHs in modes 2 and 3 were highly effective at blocking ricin attachment to HeLa cells and immobilized asialofetuin, due to framework residues (FR3) that occupied the 2γ Gal/GalNAc-binding pocket and mimic ligand. The four Tier 1 VHHs also interfered with intracellular functions of RTB, as they neutralized ricin in a post-attachment cytotoxicity assay (e.g., the toxin was bound to cell surfaces before antibody addition) and reduced the efficiency of toxin transport to the TGN. We conclude that the RTA-RTB interface is a target of potent toxin-neutralizing antibodies that interfere with both extracellular and intracellular events in ricin's cytotoxic pathway.

Sensitization of Airway Epithelial Cells to Toxin-Induced Death by TNF Superfamily Cytokines.

The TNF superfamily of proinflammatory and proapoptotic cytokines influence tissue-wide responses to molecular insults such as small molecules, toxins, and viral infections that perturb cellular homeostasis at the level of DNA replication, transcription, and translation. In the context of acute lung injury, for example, TNF superfamily members like TNF-α and TRAIL can severely exacerbate disease pathophysiology. This chapter describes a systematic approach to optimization of mammalian cell viability assays and transcriptional profiling through nCounter® Technology to permit a detailed examination of how TNF-α and TRAIL modulate programmed cell death pathways in concert with ricin toxin, a ribosome-inactivating protein (RIP) and a potent inducer of acute respiratory distress. We compare two widely used luciferase- and colorimetric-based cell viability assays and provide optimization protocols for adherent and non-adherent cell lines. We provide a computational workflow to facilitate downstream analysis of datasets generated from nCounter® gene expression panels. While combined treatment with ricin toxin and TRAIL serves as the exemplar, the methodologies are applicable to any TNF superfamily member in combination with any biological agent of interest.

Heparan Sulfate Structure Affects Autophagy, Lifespan, Responses to Oxidative Stress, and Cell Degeneration in Drosophila parkin Mutants.

Autophagy is a catabolic process that provides cells with energy and molecular building blocks during nutritional stress. Autophagy also removes misfolded proteins and damaged organelles, a critical mechanism for cellular repair. Earlier work demonstrated that heparan sulfate proteoglycans, an abundant class of carbohydrate-modified proteins found on cell surfaces and in the extracellular matrix, suppress basal levels of autophagy in several cell types during development in Drosophila melanogaster In studies reported here, we examined the capacity of heparan sulfate synthesis to influence events affected by autophagy, including lifespan, resistance to reactive oxygen species (ROS) stress, and accumulation of ubiquitin-modified proteins in the brain. Compromising heparan sulfate synthesis increased autophagy-dependent processes, evident by extended lifespan, increased resistance to ROS, and reduced accumulation of ubiquitin-modified proteins in the brains of ROS exposed adults. The capacity of altering heparan sulfate biosynthesis to protect cells from injury was also evaluated in two different models of neurodegeneration, overexpression of Presenilin and parkin mutants. Presenilin overexpression in the retina produces cell loss, and compromising heparan sulfate biosynthesis rescued retinal patterning and size abnormalities in these animals. parkin is the fly homolog of human PARK2, one of the genes responsible for juvenile onset Parkinson's Disease. Parkin is involved in mitochondrial surveillance and compromising parkin function results in degeneration of both flight muscle and dopaminergic neurons in Drosophila Altering heparan sulfate biosynthesis suppressed flight muscle degeneration and mitochondrial dysmorphology, indicating that activation of autophagy-mediated removal of mitochondria (mitophagy) is potentiated in these animals. These findings provide in vivo evidence that altering the levels of heparan sulfate synthesis activates autophagy and can provide protection from a variety of cellular stressors.

Heparan sulfate proteoglycans regulate autophagy in Drosophila.

Heparan sulfate-modified proteoglycans (HSPGs) are important regulators of signaling and molecular recognition at the cell surface and in the extracellular space. Disruption of HSPG core proteins, HS-synthesis, or HS-degradation can have profound effects on growth, patterning, and cell survival. The Drosophila neuromuscular junction provides a tractable model for understanding the activities of HSPGs at a synapse that displays developmental and activity-dependent plasticity. Muscle cell-specific knockdown of HS biosynthesis disrupted the organization of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), and affected the number and morphology of mitochondria. We provide evidence that these changes result from a dysregulation of macroautophagy (hereafter referred to as autophagy). Cellular and molecular markers of autophagy are all consistent with an increase in the levels of autophagy in the absence of normal HS-chain biosynthesis and modification. HS production is also required for normal levels of autophagy in the fat body, the central energy storage and nutritional sensing organ in Drosophila. Genetic mosaic analysis indicates that HS-dependent regulation of autophagy occurs non-cell autonomously, consistent with HSPGs influencing this cellular process via signaling in the extracellular space. These findings demonstrate that HS biosynthesis has important regulatory effects on autophagy and that autophagy is critical for normal assembly of postsynaptic membrane specializations.