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Abstract Background In certain cases, anti-idiotypic antibodies that recognize an antigen-combining site of an antibody can mimic the structure and/or function of certain nominal antigens. This feature makes them particularly useful if conventional experimental approaches fail to fulfil expectations, especially when the molecule of interest is infectious, toxic or difficult to isolate and purify. We suggest the application of an anti-idiotype concept to the field of prion biology, with the aim of evoking a humoral immune response against the pathological isoform of the prion protein (PrPSc). Different ways to induce anti-idiotypic responses were studied in mice and chickens using various forms of V5B2, a PrPSc-specific monoclonal antibody we have described previously. Results The preparation of anti-idiotypic monoclonal antibodies was achieved with well-defined strategies of immunization, selection and subsequent characterization. Our results demonstrate that it is possible to induce a strong anti-idiotypic immune response against the V5B2 monoclonal antibody in both xenogeneic and syngeneic experimental systems. From the competition seen between polyclonal and monoclonal anti-idiotypic antibodies and the original immunogen, the P1 peptide, and even more importantly, the ultimate target antigen, PrPSc, we conclude that selected antibodies bind to the antigen-combining site of the V5B2 monoclonal antibody and might even resemble the PrPSc-specific epitope. The involvement of both antigen-combining sites in the interaction between V5B2 and the most promising monoclonal anti-idiotypic antibody was further supported by molecular docking. Conclusion The results of the present study not only provide an example of the successful production of Ab2 monoclonal antibodies based on a well planned strategy for selection, but should also provide a new experimental approach that is applicable to the field of prion diseases.

Cellular prion protein (PrP C ) is a glycosylphosphatidylinositol (GPI)-anchored protein most abundantly found in the outer membrane of neurons. Due to structural characteristics (a flexible tail and structured core), PrP C interacts with a wide range of partners. Although PrP C has been proposed to be involved in many physiological functions, only peripheral nerve myelination homeostasis has been confirmed as a bona fide function thus far. PrP C misfolding causes prion diseases and PrP C has been shown to mediate β-rich oligomer-induced neurotoxicity in Alzheimer's and Parkinson's disease as well as neuroprotection in ischemia. Upon proteolytic cleavage, PrP C is transformed into released and attached forms of PrP that can, depending on the contained structural characteristics of PrP C , display protective or toxic properties. In this review, we will outline prion protein and prion protein fragment properties as well as overview their involvement with interacting partners and signal pathways in myelination, neuroprotection and neurodegenerative diseases.

Prion protein (PrP) is a biomolecule that is involved in neuronal signaling, myelinization, and the development of neurodegenerative diseases. In the cell, PrP is shed by the ADAM10 protease. This process generates PrP molecules that lack glycophosphatidylinositol anchor, and these molecules incorporate into toxic aggregates and neutralize toxic oligomers. Due to this dual role, these molecules are important biomarkers for neurodegenerative diseases. In this review, we present shed PrP as a potential biomarker, with a focus on PrP226*, which may be the main biomarker for predicting neurodegenerative diseases in humans., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood-brain barrier (BBB) makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs), and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv) with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A "Trojan horse" method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT). Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs) are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs) and neural stem cells (NSCs) show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts), are making their way into glioma treatment as another type of cell-based therapy using the antibody to bind to the specific target(s). Finally, the current clinical trials are reviewed, showing the most recent progress of attractive approaches to deliver therapeutic antibodies across the BBB aiming at the specific antigen.

In the brain of patients with transmissible spongiform encephalopathies, besides PrP Sc aggregates, deposition of truncated PrP molecules was described. Jansen et al. reported two clinical cases with deposition of C-terminally truncated PrP, one of them ending with Tyr226. We have previously described the discovery of monoclonal antibody V5B2 that selectively recognizes this version of the prion protein, which we called PrP226*. Using monoclonal antibody V5B2 we showed that accumulation of PrP226* is characteristic for most types of human and animal TSEs. Its distribution correlates to the distribution of PrP Sc aggregates. To gain insight into the structural basis of its presence and distribution in PrP aggregates, we have determined the NMR structure of recombinant PrP226*. The structure of the protein consists of a disordered N-terminal part (residues 90-125) and a structured C-terminal part (residues 126-226). The C-terminal segment consists of four α-helices and a short antiparallel β-sheet. Our model predicts a break in the C-terminal helix and reorganized hydrophobic interactions between helix α 3 and β 2 -α 2 loop due to the shorter C-terminus. The structural model gives information on the possible role of the protein in the development of amyloid disease and can serve as a foundation to develop tools for prevention and treatment of prion diseases., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Prion diseases are a group of fatal neurodegenerative diseases caused by scrapie form of prion protein, PrP Sc . Prion protein (PrP) is bound to the cell via glycophosphatidylinositol (GPI) anchor. The role of GPI anchor in PrP Sc replication and propagation remains unclear. It has been shown that anchorless and truncated PrP accelerate the formation and propagation of prions in vivo and further increases the risk for transmission of prion diseases among species. To explain the role of anchorless forms of PrP in the development of prion diseases, we have prepared five C-terminal PrP truncated variants, determined their thermodynamic properties and analyzed the kinetics of conversion into amyloid fibrils. According to our results thermodynamic and kinetic properties are affected both by pH and truncation. We have shown that the shortest variant was the most destabilized and converted faster than other variants in acidic pH. Other variants converted with longer lag time of fibrillization than WT despite comparable or even decreased stability in acidic pH. Our results indicate that even the change in length for 1 amino acid residue can have a profound effect on in vitro conversion., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab) was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR) class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrP(C)). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrP(C) using a well-established methodology based on the immunization of Prnp (0/0) mice. Additionally, we show their ability to block prion (PrP(Sc)) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrP(C).

Putative pea bilin and cyclic tetrapyrrole transporter proteins were identified by means of an antibody raised against a bilirubin-interacting aminoacidic sequence of mammalian bilitranslocase (TC No. 2.A.65.1.1). The immunochemical approach showed the presence of several proteins mostly in leaf microsomal, chloroplast and tonoplast vesicles. In these membrane fractions, electrogenic bromosulfalein transport activity was also monitored, being specifically inhibited by anti-bilitranslocase sequence antibody. Moreover, the inhibition of transport activity in pea leaf chloroplast vesicles, by both the synthetic cyclic tetrapyrrole chlorophyllin and the heme catabolite biliverdin, supports the involvement of some of these proteins in the transport of linear/cyclic tetrapyrroles during chlorophyll metabolism. Immunochemical localization in chloroplast sub-compartments revealed that these putative bilitranslocase-like transporters are restricted to the thylakoids only, suggesting their preferential implication in the uptake of cyclic tetrapyrrolic intermediates from the stroma during chlorophyll biosynthesis. Finally, the presence of a conserved bilin-binding sequence in different proteins (enzymes and transporters) from divergent species is discussed in an evolutionary context.

It was shown previously that truncated molecules of prion protein can be found in brains of patients with some types of transmissible spongiform encephalopathy. One such molecule, PrP226*, is a fragment of prion protein, truncated at Tyr226. It was found to be present in aggregates, from which it can be released using chaotropic salts. In this study we investigated the distribution of PrP226* in Creutzfeldt-Jakob disease affected human brain, employing the mAb V5B2, specifically recognizing this fragment. The results show that PrP226* is not evenly distributed among different regions of human brain. Among brain regions analyzed, the fragment was found most likely to be accumulated in the cerebellum. Its distribution correlates with the distribution of PrP(Sc).

Background: The accumulation of the misfolded forms of cellular prion protein, i.e. prions (PrPSc), in the brain is one of the crucial characteristics of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). Cellular prion protein is normally linked to the cell surface by the glycosylphosphatidylinositol (GPI) anchor. There is accumulating evidence that the GPI-anchorless prion protein may act as an accelerator of formation and propagation of prions. In the TSE affected human brain we have previously discovered a novel GPI-anchorless prion protein fragment, named PrP226*, which ends with the tyrosine 226. This fragment can be labeled specifically by the monoclonal antibody V5B2., Methods: We developed a DELFIA based assay for quick and sensitive detection of the PrP226* fragment in human brain tissue homogenates. By calculating the ratio between the signals of native (N) and denatured (D) samples applied to the assay we were able to observe significant difference between 24 TSE affected brains and 10 control brains. The presence of PrP226* in brain tissue was confirmed by western blot., Results: Our results demonstrate that PrP226* is present in small quantities in healthy human brain, whereas in degenerated brain it accumulates in prion aggregates, proportionally to PrPSc. Samples with high D/N ratio generally comprised more proteinase K resistant PrP, while no correlation was found between the quantity of PrP226* and standard classification of Creutzfeldt-Jakob disease (CJD)., Conclusions: In the present study we show that the PrP226* fragment accumulates in prion aggregates and after being released from them by a denaturation procedure, could serve as a proteinase K digestion independent biomarker for human TSEs. The PrP226* assay described in this paper offers a tool to follow and study this unique anchorless PrP fragment in various parts of human brain and possibly also in other tissues and body fluids.