Remy Robert

Evidence that asthma is a developmental origin disease influenced by maternal diet and bacterial metabolites

Asthma is prevalent in Western countries, and recent explanations have evoked the actions of the gut microbiota. Here we show that feeding mice a high-fibre diet yields a distinctive gut microbiota, which increases the levels of the short-chain fatty acid, acetate. High-fibre or acetate-feeding led to marked suppression of allergic airways disease (AAD, a model for human asthma), by enhancing T-regulatory cell numbers and function. Acetate increases acetylation at the Foxp3 promoter, likely through HDAC9 inhibition. Epigenetic effects of fibre/acetate in adult mice led us to examine the influence of maternal intake of fibre/acetate. High-fibre/acetate feeding of pregnant mice imparts on their adult offspring an inability to develop robust AAD. High fibre/acetate suppresses expression of certain genes in the mouse fetal lung linked to both human asthma and mouse AAD. Thus, diet acting on the gut microbiota profoundly influences airway responses, and may represent an approach to prevent asthma, including during pregnancy.

Engineered antibody intervention strategies for Alzheimer's disease and related dementias by targeting amyloid and toxic oligomers

Most neurodegenerative disorders, such as Alzheimers (AD), Parkinsons, Huntingtons and Creutzfeldt-Jakob disease, are characterised by the accumulation of insoluble filamentous aggregates known as amyloid. These pathologies share common pathways involving protein aggregation which can lead to fibril formation and amyloid plaques. The 4 kDa Abeta peptide (39-43 amino acids) derived from the proteolysis of the amyloid precursor protein is currently a validated target for therapy in AD. Both active and passive immunisation studies against Abeta are being trialled as potential AD therapeutic approaches. In this study, we have characterised engineered antibody fragments derived from the monoclonal antibody, WO-2 which recognises an epitope in the N-terminal region of Abeta (amino acids 2-8 of Abeta). A chimeric recombinant Fab (rFab) and single chain fragments (scFvs) of WO-2 were constructed and expressed in Escherichia coli. Rationally designed mutants to improve the stability of antibody fragments were also constructed. All antibody formats retained high affinity (K(D) approximately 8 x 10(-9) M) for the Abeta peptide, comparable with the intact parental IgG as measured by surface plasmon resonance. Likewise, all engineered fragments were able to: (i) prevent amyloid fibrillisation, (ii) disaggregate preformed Abeta(1-42) fibrils and (iii) inhibit Abeta(1-42) oligomer-mediated neurotoxicity in vitro as efficiently as the whole IgG molecule. These data indicate that the WO-2 antibody and its fragments have immunotherapeutic potential. The perceived advantages of using small Fab and scFv engineered antibody formats which lack the effector function include more efficient passage across the blood-brain barrier and minimising the risk of triggering inflammatory side reactions. Hence, these recombinant antibody fragments represent attractive candidates and safer formulations of passive immunotherapy for AD.

MRI of inducible P‐selectin expression in human activated platelets involved in the early stages of atherosclerosis

The noninvasive imaging of atherosclerotic plaques at an early stage of atherogenesis remains a major challenge for the evaluation of the pathologic state of patients at high risk of acute coronary syndromes. Recent studies have emphasized the importance of platelet–endothelial cell interactions in atherosclerosis‐prone arteries at early stages, and the prominent role of P‐selectin in the initial loose contact between platelets and diseased vessel walls. A specific MR contrast agent was developed here for the targeting, with high affinity, of P‐selectin expressed in large amounts on activated platelets and endothelial cells. For this purpose, PEGylated dextran/iron oxide nanoparticles [PEG, poly(ethylene glycol)], named versatile ultrasmall superparamagnetic iron oxide (VUSPIO) particles, labeled with rhodamine were coupled to an anti‐human P‐selectin antibody (VH10). Flow cytometry and microscopy experiments on human activated platelets were highly correlated with MRI (performed at 4.7 and 0.2 T), with a 50% signal decrease in T2 and T1 values corresponding to the strong labeling of activated vs resting platelets. The number of 1000 VH10–VUSPIO nanoparticles attained per activated platelet appeared to be optimal for the detection of hypo‐ and hyper‐signals in the platelet pellet on T2‐ and T1‐weighted MRI. Furthermore, in vivo imaging of atherosclerotic plaques in ApoE mice at 4.7 T showed a spatial resolution adapted to the imaging of intimal thickening and a hypo‐signal at 4.7 T, as a result of the accumulation of VH10–VUSPIO nanoparticles in the plaque. Our work provides support for the further assessment of the use of VH10–VUSPIO nanoparticles as a promising imaging modality able to identify the early stages of atherosclerosis with regard to the pertinence of both the target and the antibody‐conjugated contrast agent used. Copyright

Engineered antibody approaches for Alzheimer’s disease immunotherapy

The accumulation of amyloid-β-peptide (Aβ or A-beta) in the brain is considered to be a key event in the pathogenesis of Alzheimers disease (AD). Over the last decade, antibody strategies aimed at reducing high levels of Aβ in the brain and or neutralizing its toxic effects have emerged as one of the most promising treatments for AD. Early approaches using conventional antibody formats demonstrated the potential of immunotherapy, but also caused a range of undesirable side effects such meningoencephalitis, vasogenic edema or cerebral microhemorrhages in both murine and humans. This prompted the exploration of alternative approaches using engineered antibodies to avoid adverse immunological responses and provide a safer and more effective therapy. Encouraging results have been obtained using a range of recombinant antibody formats including, single chain antibodies, antibody domains, intrabodies, bispecific antibodies as well as Fc-engineered antibodies in transgenic AD mouse and primate models. This review will address recent progress using these recombinant antibodies against Aβ, highlighting their advantages over conventional monoclonal antibodies and delivery methods.

Cytotoxicity and internalization of polymer hydrogel capsules by mammalian cells.

Polymer hydrogel capsules comprised of poly(methacrylic acid) chains and cross-linked via disulfide linkages were investigated for their cytotoxicity and mechanism of internalization in a variety of mammalian cells. The capsules were internalized by all the tested cell lines which differed in their morphology and function and over short to medium term (24 h) revealed no reduction in viability and metabolic activity of cells. The mechanism of capsule uptake was analyzed using inhibitors of various cellular entry pathways. Of these, blocking the clathrin-mediated endocytotic pathway resulted in a statistically significant reduction in capsule uptake, suggesting this was the predominant pathway of capsule entry in these cell lines. The uptake of solid particles with similar surface chemistry was not significantly decreased by the inhibitor of the clathrin-mediated pathway, which suggested that softness and concomitant flexibility of the hydrogel capsules were factors governing the entry mechanism. This work represents the first systematic study of the interaction of polymer hydrogel capsules with mammalian cells and provides essential information for the application of these capsules in biomedicine.

Identification of human scFVs targeting atherosclerotic lesions: Selection by single round in vivo phage-display

Our aim was to investigate by in vivo biopanning the lesions developed early in atherosclerosis and identify human antibodies that home to diseased regions. We have designed a two-step approach for a rapid isolation of human Monoclonal phage-display single-chain antibodies (MoPhabs) reactive with proteins found in lesions developed in an animal model of atherosclerosis. After a single round of in vivo biopanning, the MoPhabs were eluted from diseased sections of rabbit aorta identified by histology and NMR microscopy. MoPhabs expressed in situ were selected by subtractive colony filter screening for their capacity to recognize atherosclerotic but not normal aorta. MoPhabs selected by our method predominantly bind atherosclerotic lesions. Two of them, B3.3G and B3.GER, produced as scFv fragments, recognized an epitope present on the surface in early atherosclerotic lesions and within the intimal thickness in more complex plaques. These human MoPhabs homed to atherosclerotic lesions in ApoE-/- mice after in vivo injection. A protein of ∼56 kDa recognized by B3.3G was affinity-purified and identified by mass spectrometry analysis as vitronectin. This is the first time that single round in vivo biopanning has been used to select human antibodies as candidates for diagnostic imaging and for obtaining insight into targets displayed in atherosclerotic plaques.

Germline humanization of a murine Aβ antibody and crystal structure of the humanized recombinant Fab fragment

Alzheimers disease is the most common form of dementia, affecting 26 million people worldwide. The Aβ peptide (39–43 amino acids) derived from the proteolytic cleavage of the amyloid precursor protein is one of the main constituents of amyloid plaques associated with disease pathogenesis and therefore a validated target for therapy. Recently, we characterized antibody fragments (Fab and scFvs) derived from the murine monoclonal antibody WO‐2, which bind the immunodominant epitope (3EFRH6) in the Aβ peptide at the N‐terminus. In vitro, these fragments are able to inhibit fibril formation, disaggregate preformed amyloid fibrils, and protect neuroblastoma cells against oligomer‐mediated toxicity. In this study, we describe the humanization of WO‐2 using complementary determining region loop grafting onto the human germline gene and the determination of the three‐dimensional structure by X‐ray crystallography. This humanized version retains a high affinity for the Aβ peptide and therefore is a potential candidate for passive immunotherapy of Alzheimers disease.

Restricted V gene usage and VH/VL pairing of mouse humoral response against the N-terminal immunodominant epitope of the amyloid β peptide.

Over the last decade, the potential of antibodies as therapeutic strategies to treat Alzheimers disease (AD) has been growing, based on successful experimental and clinical trials in transgenic mice. Despite, undesirable side effects in humans using an active immunization approach, immunotherapy still remains one of the most promising treatments for AD. In this study, we analyzed the V genes of twelve independently isolated monoclonal antibodies raised against the N-terminal immunodominant epitope of the amyloid β peptide (Aβ or A beta). Surprisingly, we found a high and unusual level of restriction in the VH/VL pairing of these antibodies. Moreover, these antibodies mostly differ in their heavy chain complementary determining region 3 (HCDR3) and the residues in the antibodies which contact Aβ are already present in the germline V-genes. Based on these observations and or co-crystal structures of antibodies with Aβ, the aim of the current study was to better understand the role of antibody V-domains, HCDR3 regions, key contact residue (H58) and germline encoded residues in Aβ recognition. For that purpose, we designed and produced a range of recombinant Fab constructs. All the Fabs were tested and compared by surface plasmon resonance on Aβ(1-16), Aβ(1-42) high molecular weight and Aβ(1-42) low molecular weight soluble oligomers. Although all the Fabs recognized the Aβ(1-16) peptide and the Aβ(1-42) high molecular weight soluble oligomers, they did not bind the Aβ(1-42) low molecular weight soluble oligomers. Furthermore, we demonstrated that: (1) an aromatic residue at position H58 in the antibody is essential in the recognition of Aβ and (2) Fabs based on germline V-genes bind to Aβ monomers with a low affinity. These findings may have important implications in designing more effective therapeutic antibodies against Aβ.

The role of follicular helper T cell molecules and environmental influences in autoantibody production and progression to inflammatory arthritis in mice

Antibody‐mediated autoimmunity involves cognate interactions between self‐reactive T cells and B cells during germinal center (GC) reactions. The aim of this study was to determine the role of essential follicular helper T (Tfh) cell molecules (CXCR5, signaling lymphocytic activation molecule–associated protein) on autoreactive CD4+ cells and the role of certain environmental influences that may determine GC‐driven autoantibody production and arthritis development.

Structural studies of the tethered N-terminus of the Alzheimer's disease amyloid-beta peptide.

Alzheimers disease is the most common form of dementia in humans and is related to the accumulation of the amyloid‐β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal‐binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1–16 fused to the N‐terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti‐Aβ N‐terminal antibody WO2. The structure demonstrates that Aβ residues 10–16, which are not in complex with the antibody, adopt a mixture of local polyproline II‐helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10–16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13‐metal‐His14 coordination in the Aβ1–16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimers disease therapeutics aimed at stabilizing the N‐terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal‐binding‐induced neurotoxicity.Proteins 2013; 81:1748–1758.