Liposomal vaccines incorporating molecular adjuvants and intrastructural T-cell help promote the immunogenicity of HIV membrane-proximal external region peptides

An HIV vaccine capable of inducing high and durable levels of broadly neutralizing antibodies has thus far proven elusive. A promising antigen is the membrane-proximal external region (MPER) from gp41, a segment of the viral envelope recognized by a number of broadly neutralizing antibodies. Though an attractive vaccine target due to the linear nature of the epitope and its highly conserved sequence, MPER peptides are poorly immunogenic and may require display on membranes to achieve a physiological conformation matching the native virus. Here we systematically explored how the structure and composition of liposomes displaying MPER peptides impacts the strength and durability of humoral responses to this antigen as well as helper T-cell responses in mice. Administration of MPER peptides anchored to the surface of liposomes induced MPER-specific antibodies whereas MPER administered in oil-based emulsion adjuvants or alum did not, even when combined with Toll-like receptor agonists. High-titer IgG responses to liposomal MPER required the inclusion of molecular adjuvants such as monophosphoryl lipid A. Anti-MPER humoral responses were further enhanced by incorporating high-Tm lipids in the vesicle bilayer and optimizing the MPER density to a mean distance of ∼10–15 nm between peptides on the liposomes' surfaces. Encapsulation of helper epitopes within the vesicles allowed efficient “intrastructural” T-cell help, which promoted IgG responses to MPER while minimizing competing B-cell responses against the helper sequence. These results define several key properties of liposome formulations that promote durable, high-titer antibody responses against MPER peptides, which will be a prerequisite for a successful MPER-targeting vaccine.Bill & Melinda Gates FoundationNational Institutes of Health (U.S.) (AI091693

Direct Visualization of Peptide/MHC Complexes at the Surface and in the Intracellular Compartments of Cells Infected In Vivo by Leishmania major

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo

To automate or not to automate: this is the question

New protocols and instrumentation significantly boost the outcome of structural biology, which has resulted in significant growth in the number of deposited Protein Data Bank structures. However, even an enormous increase of the productivity of a single step of the structure determination process may not significantly shorten the time between clone and deposition or publication. For example, in a medium size laboratory equipped with the LabDB and HKL-3000 systems, we show that automation of some (and integration of all) steps of the X-ray structure determination pathway is critical for laboratory productivity. Moreover, we show that the lag period after which the impact of a technology change is observed is longer than expected

Fibroadipogenic progenitors are responsible for muscle loss in limb girdle muscular dystrophy 2B

Altres ajuts: This work is supported by a MDA Career Development Award (MDA477331) to M.W.H. Additional financial support was provided by NIAMS (R01AR055686) and MDA (MDA277389) to J.K.J. and NIH (K26OD011171; R24HD050846, P50AR060836) grants to K.N. The authors acknowledge Carsten Bönnemann's contribution to this study. Microscopy imaging was performed at the CRI Cellular Imaging Core, which is supported by funds from CRI and NICHD (U54HD090257).Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients

Fibroadipogenic progenitors are responsible for muscle loss in limb girdle muscular dystrophy 2B.

Altres ajuts: This work is supported by a MDA Career Development Award (MDA477331) to M.W.H. Additional financial support was provided by NIAMS (R01AR055686) and MDA (MDA277389) to J.K.J. and NIH (K26OD011171; R24HD050846, P50AR060836) grants to K.N. The authors acknowledge Carsten Bönnemann's contribution to this study. Microscopy imaging was performed at the CRI Cellular Imaging Core, which is supported by funds from CRI and NICHD (U54HD090257).Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients

Different forms of copper and kinetin impacted element accumulation and macromolecule contents in kidney bean (Phaseolus vulgaris) seeds

The relationship between engineered nanomaterials and plant biostimulants is unclear. In this study, kidney bean (Phaseolus vulgaris) plantswere grown to maturity (90 days) in soil amendedwith nano copper (nCu), bulk copper (bCu), or copper chloride (CuCl2) at 0, 50, or 100mg kg−1, thenwatered with 0, 10, or 100 μMof kinetin (KN). Seeds were harvested and analyzed via ICP-OES and biochemical assays. While seed production was largely unaffected, nutritional valuewas significantly impacted. Accumulation of Cuwas enhanced by 5–10% fromcontrols by Cu-based treatments. Fe was the only macro/microelement significantly altered by nCu, which was ~29% lower than seeds fromuntreated plants. All forms of Cu combinedwith 10 μMKN reducedMg from9 to 12%. Application of KN plus bCu or CuCl2 elevated concentrations of Mn (31–41%) and S (19–22%), respectively. Protein content of seeds was stimulated (11–12%) by bCu, on average, and depressed by CuCl2 + KN (up to 22%). Variations in sugar and starch contentwere insignificant, compared to controls. Our results indicate that the interaction Cu × KN significantly altered the nutritional value of common beans, which has potential implications to agricultural practices incorporating Cu as either a pesticide or fertilizer