1,721,186 research outputs found
Visualizing Membranes : 3D Electron Microscopic Imaging of Cellular Structures
No full textCells are organized in a highly complex manner. And while there are many different types of cells - each organized in a different manner according to their function - they do share certain commonalities. Among these commonalities are membranes that functions not only as a barrier between the extra- and intra-cellular environment, but is also involved in many cell-biological processes like signaling and transport; and since each of these processes require a different set of components and organization, a large heterogeneity is expected within these membranes. Electron microscopic techniques have been invaluable to our understanding of the complex cellular architecture (and the corresponding dynamics). In recent years, 3D electron microscopic techniques like electron tomography and the focussed ion-beam scanning electron microscope have emerged and provided us with an exciting new perspective of cellular structures and their inter- and intra-organellar relationships. However, extracting relevant information from (3D) micrographs heavily relies on manual segmentation techniques and is therefore affected by the eye of the beholder. In the work presented in this thesis we have explored template matching as an approach to harvest biologically relevant information from tomographic volumes in a (semi-)objective manner. By using a stylized cuboid-shaped template we could extract membranes from the tomographic volumes and thus study the overall morphology of organelles. But more importantly, by comparing the matching results of slightly distinct templates, we were able to visualize ultra-structural details that are lost by manual segmentation. By applying this approach we up the ante of electron tomographic approaches and were able to bring it into a new realm where we can study and visualize in three dimensions the membrane-organization of biologically functional and significant events like lipid- and protein-domains
Llama VHH as immunotherapeutics in Alzheimer's disease
No full textAlzheimer's Disease (AD) is the most common form of dementia among elderly in the Western world. AD is a devastating neurodegenerative disease where patients starting with episodic memory problems end up completely bedridden and care dependent. At present there is no real therapy stopping or reversing AD progression. Roughly 100 years ago Alois Alzheimer described the deposition of amyloid-beta (Aβ) in post-mortem brains, one of the hallmarks of AD. Since this description AD has had great attention. However, the exact mechanism of AD remains unclear, a general consensus is reached over Aβ as a peptide involved in AD progression. Aβ is derived from amyloid-beta precursor protein (APP) by the proteolytic cleavage activity of beta-secretase BACE1. Interesting, next to AD patients, healthy individuals also produce Aβ in their brain. So next to the production and presence of Aβ, an extra mechanism is in place to exert toxicity and therefore AD progression. The hydrophobic nature of Aβ makes this peptide prone to aggregate. Overwhelming evidence point to an aggregated form of Aβ (oligomer) as the culprit of toxicity. This makes aggregation of Aβ the mechanism responsible for the generation of toxic Aβ species. Members from the Camelidae family possess, next to conventional IgGs, a heavy chain only antibody. The antigen-binding domain of this antibody (VHH) is fully function while being about ten times smaller compared to a conventional antibody. Next to size, VHH have several other benefits like stability, production costs and genetic modulation. Next to that, VHH are found that inhibit protein aggregation and enzyme activity. We exploited the capability of VHH to inhibit protein aggregation by selecting and generation VHH against Aβ. Llamas were immunized with patient material containing severe Aβ deposition. The subsequently generated VHH library yielded one VHH that inhibits aggregation, and reduced Aβ toxicity. Mutation studies and an additional crystal structure led to the hypothesis that this VHH binds to a small aggregated form of Aβ. A secondary strategy to prevent Aβ toxicity is to inhibit Aβ production by targeting BACE1. We selected a VHH that inhibits BACE1 activity in vitro and in vivo. The in vivo pilot experiment showed a reduced concentration of Aβ and therefore might be of therapeutical value. Problematic is the delivery of compounds to the brain due to the blood brain barrier (BBB). We provided a proof of principle to deliver compounds over cell layers that were BBB models. Transferrin Receptor (TfR) was used to piggy bag an anti-TfR covalently coupled to an anti-Aβ over the cell layer. The aim of this thesis was to find VHH against Ab targets and characterize these VHH. We made great progress in characterizing VHH G7 in vitro and explain characteristics needed for aggregation and toxicity inhibition. Besides this we found a VHH inhibiting BACE1 activity in vitro and in vivo providing a secondary therapeutic strategy for AD. Furthermore, proof of principle is provided to deliver compounds over cellular monolayers modeling the blood brain barrier
HIV-1, how llamas help us fight the AIDS pandemic
No full textHuman Immunodeficiency Virus type 1 (HIV-1) is one of the major health problems worldwide and has been for over thirty years. Most (67%) of the people infected with HIV-1 are living in sub-Saharan Africa. Here, the access to treatments is limited and most women are not in a position to protect themselves from getting infected. The aim of this study was to identify VHH that are both broad and potent HIV-1 neutralizers, which can be used in the fight against HIV-1. A VHH is an antibody fragment containing the antigen binding domain derived from a heavy chain only antibody. This special type of antibody can be found in members of the camel family. The advantage of VHH is that they are relatively small, stable, cheap to produce and easy to work with. In order to raise VHH against HIV-1, two llamas were immunized with a mix of its envelope proteins. After cloning of the llama’s VHH repertoire into a phagemid expression system, multiple different strategies were followed to identify neutralizing VHH. In total, twelve families of neutralizing VHH were found and characterized in more detail. These VHH target four independent areas on the HIV-1 envelope protein. The best VHH of them being J3, which neutralized 96 out of a 100 tested viruses with a median IC50 of 0.9 µg/ml and is targeting the CD4 binding site. The CD4 binding site on the envelope protein is considered an important area as the first interaction between virus and its target cell occurs via CD4. Previous studies have shown that linking two VHH together into so-called biheads may increase their breadth and potency. For this reason we have linked two different VHH, targeting independent epitopes, together to form bi-specific biheads. Several combinations of VHH have been tried, the best (J3 linked to 2E7) yielded upto 1400 times increase in potency compared to a mix of the individual VHH. Another bihead (J3 linked to 1F10) was capable of neutralizing a viral strain J3 alone could not neutralize, possibly improving the breadth of this molecule above 96%
Exploitation of the transcytotic pathway in polarised cells by camelid single domain antibodies
No full textDelivery of drugs to sites of disease can be hampered by the presence of cellular barriers in the human body. The presence of such barriers reduces the therapeutic activity of drugs on the disease target. The barrier present between the bloodstream and the brain for instance, stringently controls what goes into the brain and what not. Diseases that take place in such enclosed systems may be difficult to reach for pharmaceutical compounds because of these barriers and this can reduce their therapeutic effectivity. In this thesis we have described the development of molecules that interact with specific barriers in the human body and that mediate translocation across these barriers. As platform we used antibodies from the llama. Llama antibodies have several favourable properties as compared with other mammalian antibodies. The llama antibody platform was used in the described studies to make entities capable of translocating across cellular barriers. Part of the described studies has looked at the removal of compounds from the body by efflux via the intestinal wall (a cellular barrier). We developed molecules which can interact specifically with that barrier and mediate the passage of unwanted molecules. These molecules can be used for example for the removal of viruses that have infected the body. Fusion of the translocation molecules with compounds that bind viruses would enable the resulting compounds to both bind the virus and mediate its translocation across the intestinal wall. Such therapeutic strategies could tackle the infections of persistent viruses such as HIV. The other part of the described studies has looked at the delivery of blood borne compounds across the blood brain barrier to the central nervous system. Efficient platforms that mediate brain delivery from the bloodstream are scarce/ non-existent. We found molecules which have favourable properties in artificial model systems mimicking the blood-brain barrier. Application of these molecules to mice showed that there was a significant proportion of the injected dose able to accumulate in the brain, which indicates that our molecules indeed can target the brain. Further genetic adaptation of the antibodies might improve the translocation process to the brain environment and deliver a bigger pool of therapeutics to the site of disease. Development of these molecules might yield entities capable of delivering therapeutically relevant compounds to the brain. At the moment, brain delivery of pharmaceuticals is still a feature that is hardly possible without invasive procedures. Due to this aberrant delivery, there is no proper treatment possible of diseases like Alzheimer’s, Parkinson’s and brain cancer. We hope that with further development, our molecules will be able to give a solution to the brain delivery problem and thereby aid in the treatment of brain afflictions. This thesis has given examples of how antibodies from the llama can be used as retargeting agents. We have shown that they can be designed to translocate across cellular barriers and to take along cargo in this process. Development of the antibodies may yield molecules which can help in the diagnosis and treatment of several diseases
Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide
No full textHeavy-chain antibodies are present in llamas next to conventional immunoglobulins. Their variable domain, also known as VHH is a small, single-chain globular protein with full antigen-binding capacity. Its single-domain nature and some structural differences from conventional immunoglobulins make it a tool with high potential for a number of biotechnological and medical applications. The HIV pandemic represents since three decades a dramatic health problem throughout the world and especially in resource-limited countries. As an alternative to vaccination, prevention is regarded as the best way to control the pandemic, both from a healthcare and economic point of view. The development of microbicides that help preventing sexually transmitted infections of HIV (the most common way the virus is transmitted) is therefore highly desirable. In this thesis, several aspects of the development of llama heavy-chain antibody fragments as active compound of topical microbicides are investigated. In chapter one the 3D structure of the HIV neutralizing VHH D7 was determined. The extension, flexibility and amino acid-composition of its CDRs were compared to other known (human) neutralizing antibodies in order to define the way D7 neutralizes the virus by binding gp120. Structural comparison suggested diverse modes of interaction. Mutational analysis identified CDR3 as key area of the interaction with gp120, whose conformational flexibility is likely to accommodate multiple modes of antibody binding inside the CD4 binding site. Comparison of the related VHH A12 and D7 revealed broad and narrow cross-clade neutralization phenotypes respectively. In chapter two a family-specific phage library was generated and a number of homologous VHH was selected with varying neutralizing profiles. Analysis of their sequences and their recognition of a panel of different HIV-1 subtypes, allowed the understanding of the molecular basis of the different neutralization behaviors. In particular the important role played by the last two C-terminal amino acids of CDR3 was demonstrated. Secretion efficiency of VHH is a particularly important factor when large amounts are needed at low production cost. In chapter three we analyzed databases of structures and sequences to find sequence-related factors that influence the yield of VHH when produced in the host S. cerevisiae. We showed how the combined presence of five key residues and specific J segments is fundamental for proper folding. We also suggested that interactions with ER-resident chaperones are necessary to achieve high secretion efficiency. In the last chapter, VHH A12 and D7 were used as models to study their availability, activity and stability in a VHH-based topical microbicide. We systematically analyzed likely challenges that VHH will have to face in case of use in sub-tropical settings of resource-limited countries, as HIV preventative drug. VHH proved to be readily available when formulated as tablets or aqueous gel and very stable after extended incubation at physiological temperature and at low pH. We also investigated the tissue-penetration kinetics of VHH in order to assess whether the drug would be present in the sub-mucosal layers of the vagina and once there, carry out its protective action
Research data supporting "In Vivo Biomolecular Imaging of Zebrafish Embryos using Confocal Raman Spectroscopy"
Get PDFResearch raw data supporting Hogset et al., "In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy", 2020, Nature Communications
VHH Activators and Inhibitors for Protein Kinase C Epsilon
No full textProtein kinase C epsilon (PKCε), which is one of the novel PKC isozymes, is widely expressed throughout the body and has important roles in the function of the nervous, cardiovascular and immune systems. In order to better understand PKCε regulated pathways, isozyme specific activity modulators are desperately needed. Such compounds could also be developed into drugs for diseases such as diabetes, cancer and Alzheimer’s disease, where PKCε dysregulation is implicated. This thesis describes the selection and characterization of PKCε specific llama single chain antibodies, known as VHHs. VHHs have several advantages compared to the conventional antibodies consisting of two heavy and two light chains. VHHs are small, easy to clone, convenient to genetically modify, remarkably stable, and tend to be highly specific for the target antigen. PKCε specific VHHs were selected in one round from an immune phage-VHH library using phage display. The VHHs that showed the strongest binding to human PKCε were produced in E. coli as monovalent proteins and tested for their ability to influence PKCε activity in in vitro kinase activity assays. Three VHHs (A10, C1 and D1) were found to increase the activity of PKCε, whereas three other VHHs (C7, E6 and G8) inhibited PKCε kinase activity. These effects were PKCε specific, since the VHHs had no effect on the activity of PKCδ, PKCθ or protein kinase A. A more detailed kinetic analysis revealed that the activators increased PKCε activity by increasing the maximum rate of the reaction and/or by speeding up the reaction. Furthermore, VHHs E6 and G8 were found to be non-competitive enzyme inhibitors. Interestingly, the binding site of all of the tested VHHs was in the catalytic domain of PKCε. Moreover, the binding of the VHHs was found to be conformation dependent and species specific. In order to study whether the VHHs could also influence PKCε activity in a cellular context, the VHHs were cloned into a mammalian expression vector with a C-terminal mCherry tag, and the VHH-mCherry proteins were transiently expressed in HeLa cells. Several cellular effects, such as changes in cell morphology, increases in cytotoxicity, and inhibition of PKCε downregulation, were observed for some of the VHH-mCherry constructs. The most direct indication that the VHHs can also influence PKCε activity in cells came from PKCε-EGFP translocation studies, where the activator A10-mCherry increased the rate of PKCε-EGFP translocation to the membranes in response to phorbol ester stimulation. In contrast, the inhibitors C7-mCherry and G8-mCherry decreased the rate of translocation. The results presented in this thesis demonstrate that highly specific VHHs against an intracellular antigen can be selected in one round from an immune phage-VHH library. Furthermore, it was shown that the selected VHHs can act as activators or inhibitors of PKCε activity in vitro and in cells. In addition to the potential therapeutic applications of the VHHs for diseases with aberrant PKCε signalling, these PKCε specific activity modulators will be valuable tools in research into PKCε regulated pathway
Sol-gel transitions and liquid crystal phase transitions in concentrated aqueous suspensions of colloidal gibbsite platelets
No full textIn this paper, we present a comprehensive study of the sol-gel transitions and liquid crystal phase transitions in aqueous suspensions of positively charged colloidal gibbsite platelets at pH 4-5 over a wide range of particle concentrations (50-600 g/L) and salt concentrations (10-4-10-1 M NaCl). A detailed sol-gel diagram was established by oscillatory rheological experiments. These demonstrate the presence of kinetically arrested states both at high and at low salt concentrations, enclosing a sol region. Birefringence and iridescence show that in the sol state nematic and hexagonal columnar liquid crystal phases are formed. The gel and liquid crystal structures are studied in further detail using small-angle X-ray scattering (SAXS) and cryo-focused ion beam/scanning electron microscopy (cryo-FIB-SEM). The gel formed at high salt concentration shows signatures of a sponge-like structure and does not display birefringence. In the sol region, by lowering the salt concentration and/or increasing the gibbsite concentration, the nematic phase gradually transforms from the discotic nematic (ND) into the columnar nematic (NC) with much stronger side-to-side interparticle correlations. Subsequently, this NC structure can be either transformed into the hexagonal columnar phase or arrested into a birefringent repulsive gel state with NC structure
Novel contrasting and labeling procedures for correlative microscopy of thawed cryosections
No full textOne of the major challenges for correlative microscopy is the preparation of the sample; the protocols for transmission electron microscopy (TEM) and fluorescence microscopy (FM) often prove to be incompatible. Here, we introduce 2+Staining: an improved contrasting procedure for Tokuyasu sections that yields both excellent positive membrane contrast in the TEM and bright fluorescence of the probe labeled on the section. 2+Staining involves the contrasting of the immunolabeled sections with 1% osmium tetroxide, 2% uranyl acetate and lead citrate in sequential steps, followed by embedding in 1.8% methyl cellulose. In addition, we demonstrate an amplification of the fluorescent signal by introducing additional antibody incubation steps to the immunolabeling procedure. The methods were validated using the integrated laser and electron microscope (iLEM), a novel tool for correlative microscopy combining FM and TEM in a single setup. The approaches were tested on HL-60 cells labeled for lysosomal-associated membrane protein 2 (LAMP-2) and on sections of muscle from a facioscapulohumeral dystrophy mouse model. Yielding excellent results and greatly expediting the workflow, the methods are of great value for those working in the field of correlative microscopy and indispensible for future users of integrated correlative microscopy
A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition
No full textThe membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10
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