High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies

As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.     

Virulence studies on chromosomal α-toxin and Θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as Θ-toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α-toxin or phospholipase C. The resultant mutants failed to produce detectable Θ-toxin or α-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α-toxin in gas gangrene or clostridial myonecrosis.     

Fuel of fear and force: gasoline's energetic power and its entanglement in composite ethics

Energy is far more than a resource exploited by states and corporations. Yet, at the level of consumption it is generally thought to be a difficult phenomenon to examine because it is so familiar that we barely notice its role in our lives; or at the very least, its production becomes obscured by this pragmatic daily engagement. The other side to the story – the one in which energy is a provision distributed to and experienced by people in intimate and unanticipated ways — is distinctly perceptible in locales like the Amazon rain forest where conventional energy provisions are absent. This essay explores how everyday encounters with gasoline offer insights into ethical judgements among the Sanema of Venezuelan Amazonia. The fuel is so pervasive that it is increasingly present in numerous facets of daily life, including gold mining, dilemmas of kinship, the animist world of vengeful spirit masters, and ethically infused rumours of disaster. Being a volatile substance – simultaneously vaporous, explosive, narcotic, and caustic – gasoline is also a vital entity that holds a particularly intriguing place in the Sanema's understanding of personhood and ethics. Indeed, its mysterious and unsettling qualities cause it to become entangled within a composite form of ethics that defines Sanema social worlds.     

Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.     

Synthesis and evaluation of alkoxy-phenylamides and alkoxy-phenylimidazoles as potent sphingosine-1-phosphate receptor subtype-1 agonists

In the design of potent and selective sphingosine-1-phosphate receptor agonists, we were able to identify two series of molecules based on phenylamide and phenylimidazole analogs of FTY-720. Several designed molecules in these scaffolds have demonstrated selectivity for S1P receptor subtype 1 versus 3 and excellent in vivo activity in mouse. Two molecules PPI-4621 (4b) and PPI-4691 (10a), demonstrated dose responsive lymphopenia, when administered orally.     

Bifidobacteria-mediated immune system imprinting early in life

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Alterations in high-affinity binding characteristics and levels of opioids in invertebrate ganglia during aging: Evidence for an opioid compensatory mechanism

In Mytilus and Leucophaea the high-affinity binding site density is significantly lower in old animals than in young animals, whereas the low-affinity site density remains unchanged. In Mytilus the estimated met-enkephalin and met-enkephalin-Arg6-Phe7 levels are significantly higher in old than in young animals. In Leucophaea only the met-enkephalin level can be determined, and it is also higher in old animals. The decrease in the high-affinity binding site density and the corresponding increase in endogenous enkephalin levels suggest the existence of an opioid compensatory mechanism associated with the aging process. In Mytilus there is a demonstrated decrease with age in intraganglionic dopamine levels in response to applied opiates. In addition, the inhibition of dopamine-stimulated adenylate cyclase activity by opiates also decreases in older animals. In Leucophaea the sex difference in opioid binding densities diminishes with age.