Characterization and application of two RANK-specific antibodies with different biological activities.

Antibodies play an important role in therapy and investigative biomedical research. The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation and epithelial cell activation. However, the study of RANK biology has been hampered by missing or insufficient characterization of high affinity tools that recognize RANK. Here, we present a careful description and comparison of two antibodies, RANK-02 obtained by phage display (Newa, 2014 [1]) and R12-31 generated by immunization (Kamijo, 2006 [2]). We found that both antibodies recognized mouse RANK with high affinity, while RANK-02 and R12-31 recognized human RANK with high and lower affinities, respectively. Using a cell apoptosis assay based on stimulation of a RANK:Fas fusion protein, and a cellular NF-κB signaling assay, we showed that R12-31 was agonist for both species. R12-31 interfered little or not at all with the binding of RANKL to RANK, in contrast to RANK-02 that efficiently prevented this interaction. Depending on the assay and species, RANK-02 was either a weak agonist or a partial antagonist of RANK. Both antibodies recognized human Langerhans cells, previously shown to express RANK, while dermal dendritic cells were poorly labeled. In vivo R12-31 agonist activity was demonstrated by its ability to induce the formation of intestinal villous microfold cells in mice. This characterization of two monoclonal antibodies should now allow better evaluation of their application as therapeutic reagents and investigative tools.

Secretory IgA's complex roles in immunity and mucosal homeostasis in the gut.

Secretory IgA (SIgA) serves as the first line of defense in protecting the intestinal epithelium from enteric toxins and pathogenic microorganisms. Through a process known as immune exclusion, SIgA promotes the clearance of antigens and pathogenic microorganisms from the intestinal lumen by blocking their access to epithelial receptors, entrapping them in mucus, and facilitating their removal by peristaltic and mucociliary activities. In addition, SIgA functions in mucosal immunity and intestinal homeostasis through mechanisms that have only recently been revealed. In just the past several years, SIgA has been identified as having the capacity to directly quench bacterial virulence factors, influence composition of the intestinal microbiota by Fab-dependent and Fab-independent mechanisms, promote retro-transport of antigens across the intestinal epithelium to dendritic cell subsets in gut-associated lymphoid tissue, and, finally, to downregulate proinflammatory responses normally associated with the uptake of highly pathogenic bacteria and potentially allergenic antigens. This review summarizes the intrinsic biological activities now associated with SIgA and their relationships with immunity and intestinal homeostasis.

Malaria vaccine candidate: design of a multivalent subunit α-helical coiled coil poly-epitope.

A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal.

A new class of isothiocyanate-based irreversible inhibitors of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.

Male mutation bias and possible long-term effects of human activities.

The ability of a population to adapt to changing environments depends critically on the amount and kind of genetic variability it possesses. Mutations are an important source of new genetic variability and may lead to new adaptations, especially if the population size is large. Mutation rates are extremely variable between and within species, and males usually have higher mutation rates as a result of elevated rates of male germ cell division. This male bias affects the overall mutation rate. We examined the factors that influence male mutation bias, and focused on the effects of classical life-history parameters, such as the average age at reproduction and elevated rates of sperm production in response to sexual selection and sperm competition. We argue that human-induced changes in age at reproduction or in sexual selection will affect male mutation biases and hence overall mutation rates. Depending on the effective population size, these changes are likely to influence the long-term persistence of a population.