Cross-Linking Cellular Prion Protein Induces Neuronal Type 2-Like Hypersensitivity.

Background: Previous reports identified proteins associated with 'apoptosis' following cross-linking PrPC with motif-specific anti-PrP antibodies in vivo and in vitro. The molecular mechanisms underlying this IgG-mediated neurotoxicity and the role of the activated proteins in the apoptotic pathways leading to neuronal death has not been properly defined. Previous reports implicated a number of proteins, including apolipoprotein E, cytoplasmic phospholipase A2, prostaglandin and calpain with anti-PrP antibody-mediated 'apoptosis', however, these proteins are also known to play an important role in allergy. In this study, we investigated whether cross-linking PrPC with anti-PrP antibodies stimulates a neuronal allergenic response. Methods: Initially, we predicted the allergenicity of the epitope sequences associated with 'neurotoxic' anti-PrP antibodies using allergenicity prediction servers. We then investigated whether anti-PrP antibody treatment of mouse primary neurons (MPN), neuroblastoma cells (N2a) and microglia (N11) cell lines lead to a neuronal allergenic response. Results: In-Silico studies showed that both tail- and globular-epitopes were allergenic. Specifically, binding regions that contain epitopes for previously reported 'neurotoxic' antibodies such as ICSM18 (146-159), ICSM35 (91-110), POM 1 (138-147) and POM 3 (95-100) lead to activation of allergenic related proteins. Following direct application of anti-PrPC antibodies on N2a cells, we identified 4 neuronal allergenic-related proteins when compared with untreated cells. Furthermore, we identified 8 neuronal allergenic-related proteins following treatment of N11 cells with anti-PrPC antibodies prior to co-culture with N2a cells when compared with untreated cells. Antibody treatment of MPN or MPN co-cultured with antibody-treated N11 led to identifying 10 and 7 allergenic-related proteins when compared with untreated cells. However, comparison with 3F4 antibody treatment revealed 5 and 4 allergenic-related proteins respectively. Of importance, we showed that the allergenic effects triggered by the anti-PrP antibodies were more potent when antibody-treated microglia were co-cultured with the neuroblastoma cell line. Finally, co-culture of N2a or MPN with N11-treated with anti-PrP antibodies resulted in significant accumulation of NO and IL6 but not TNF-α in the cell culture media supernatant. Conclusions: This study showed for the first time that anti-PrP antibody binding to PrPC triggers a neuronal hypersensitivity response and highlights the important role of microglia in triggering an IgG-mediated neuronal hypersensitivity response. Moreover, this study provides an important impetus for including allergenic assessment of therapeutic antibodies for neurodegenerative disorders to derive safe and targeted biotherapeutics.

Demyelinating polyneuropathy in goats lacking prion protein.

Studies in mice with ablation of Prnp, the gene that encodes the cellular prion protein (PrPC ), have led to the hypothesis that PrPC is important for peripheral nerve myelin maintenance. Here, we have used a nontransgenic animal model to put this idea to the test; namely, goats that, due to a naturally occurring nonsense mutation, lack PrPC . Teased nerve fiber preparation revealed a demyelinating pathology in goats without PrPC . Affected nerves were invaded by macrophages and T cells and displayed vacuolated fibers, shrunken axons, and onion bulbs. Peripheral nerve lipid composition was similar in young goats with or without PrPC , but markedly different between corresponding groups of adult goats, reflecting the progressive nature of the neuropathy. This is the first report of a subclinical demyelinating polyneuropathy caused by loss of PrPC function in a nontransgenic mammal.

Roles of Prion Protein in Virus Infections.

The normal cellular prion protein, designated PrPC, is a membrane glycoprotein expressed most abundantly in brains, particularly by neurons, and to a lesser extent in non-neuronal tissues including lungs. Conformational conversion of PrPC into the amyloidogenic isoform is a key pathogenic event in prion diseases. We recently found that PrPC has a protective role against infection with influenza A viruses (IAVs) in mice by reducing reactive oxygen species in the lungs after infection with IAVs. The antioxidative activity of PrPC is probably attributable to its function to activate antioxidative enzyme Cu/Zn-superoxide dismutase, or SOD1, through regulating Cu content in lungs infected with IAVs. Oxidative stress could play a pivotal role in the pathogenesis of a wide range of viral infections. Here, we introduce our and others' studies on the role of PrPC in viral infections, and raise the attractive possibility that PrPC might be a novel target molecule for development of antioxidative therapeutics against not only IAV infection but also other viral infections.

Immunization of cervidized transgenic mice with multimeric deer prion protein induces self-antibodies that antagonize chronic wasting disease infectivity in vitro.

Chronic wasting disease (CWD) is the most contagious prion disease. It is expanding rapidly in North America, was found recently in Europe, and the potential for transmission to humans cannot be excluded yet. We hypothesized that it is possible to prevent peripheral CWD infection and CWD prion shedding by inducing auto-antibodies against the cellular prion protein (PrPC) by active vaccination. Our objective is to overcome self-tolerance against PrP by using a multimeric recombinant PrP (recPrP) as an immunogen. We expressed in E. coli, purified and refolded four immunogens: cervid and murine recPrP in monomeric and dimeric form. Testing immunogenicity in sera of the vaccinated transgenic mice expressing cervid PrP revealed that all four immunogens effectively overcame self-tolerance against the prion protein as shown by high antibody titers. Confocal microscopy analysis revealed effective binding of post-immune sera to surface-located PrPC in both murine and cervid PrP expressing cultured cells. Remarkably, the post-immune auto-antibodies effectively inhibited CWD-induced prion conversion in RT-QuIC assay when incubated with either PrP substrate or CWD seed. Furthermore, they mitigated prion propagation in CWD-infected cervid-PrP expressing RK13 cells. Together, multimeric recombinant cervid PrP effectively overcomes self-tolerance to PrP and induces auto-antibodies that interfere with CWD conversion in vitro.

A roadmap for investigating the role of the prion protein in depression associated with neurodegenerative disease.

The physiological properties of the native, endogenous prion protein (PrP(C)) is a matter of concern, due to its pleiotropic functions and links to neurodegenerative disorders and cancer. In line with our hypothesis that the basic function of PrP(C) is to serve as a cell surface scaffold for the assembly of signaling modules, multiple interactions have been identified of PrP(C) with signaling molecules, including neurotransmitter receptors. We recently reported evidence that PrP(C) may modulate monoaminergic neurotransmission, as well as depressive-like behavior in mice. Here, we discuss how those results, together with a number of other studies, including our previous demonstration that both inflammatory and behavioral stress modulate PrP(C) content in neutrophils, suggest a distributed role of PrP(C) in clinical depression and inflammation associated with neurodegenerative diseases. An overarching understanding of the multiple interventions of PrP(C) upon physiological events may both shed light on the pathogenesis of, as well as help the identification of novel therapeutic targets for clinical depression, Prion and Alzheimer's Diseases.

Comparison of the anti-prion mechanism of four different anti-prion compounds, anti-PrP monoclonal antibody 44B1, pentosan polysulfate, chlorpromazine, and U18666A, in prion-infected mouse neuroblastoma cells.

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP(Sc)) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP(Sc) formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP(C)) and PrP(Sc) in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP(Sc) levels without altering intracellular distribution of PrP(Sc). PPS did not change the distribution and levels of PrP(C), whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP(C) to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP(Sc) formation. In contrast, CPZ and U18666A initiated the redistribution of PrP(Sc) from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP(C). The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP(Sc) redistribution by CPZ or U18666A partly antagonized PrP(Sc) degradation, suggesting that the transfer of PrP(Sc) to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP(Sc) degradation. This study revealed that precise analysis of the intracellular dynamics of PrP(C) and PrP(Sc) provides important information for understanding the mechanism of anti-prion agents.

An overview of the diagnostic tools.

Prions are unconventional infectious agents that cause fatal neurological illnesses such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy, and scrapie. Variant CJD can occur via blood transfusions. However, as no screening assay is available, uncertainties remain over the prevalence of vCJD in asymptomatic blood donors. Development of a diagnostic assay is therefore a primary objective. Little is known about the nature, distribution and level of infectivity in human blood and we have to rely on assumptions made from animal models. Ideally, two types of assays are required: a rapid high-throughput assay to routinely screen all blood donations and a confirmatory assay to ensure that all positive results from initial screening are true positives. Key event in prion disease is thought to be the conversion of normal cellular prion protein PrPc to a misfolded aggregated form termed PrP(TSE). This specific characteristic has been exploited to develop some tests.

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein.

Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.

CD44-positive cancer stem cells expressing cellular prion protein contribute to metastatic capacity in colorectal cancer.

Cancer stem cells are implicated in tumor progression, metastasis, and recurrence, although the exact mechanisms remain poorly understood. Here, we show that the expression of cellular prion protein (PrPc, PRNP) is positively correlated with an increased risk of metastasis in colorectal cancer. PrPc defines a subpopulation of CD44-positive cancer stem cells that contributes to metastatic capacity. PrPc(+)CD44(+) colorectal cancer stem cells displayed high liver metastatic capability, unlike PrPc(-)CD44(+) stem cells, that was inhibited by RNAi-mediated attenuation of PrPc. Notably, administration of PrPc monoclonal antibodies significantly inhibited tumorigenicity and metastasis of colorectal cancer stem cells in mouse models of orthotopic metastasis. PrPc promoted epithelial to mesenchymal transition (EMT) via the ERK2 (MAPK1) pathway, thereby conferring high metastatic capacity. Our findings reveal the function of PrPc in regulating EMT in cancer stem cells, and they identify PrPc as candidate therapeutic target in metastatic colorectal cancer.

Neuroimmunoendocrine regulation of the prion protein in neutrophils.

The prion protein (PrP(C)) is a cell surface protein expressed mainly in the nervous system. In addition to the role of its abnormal conformer in transmissible spongiform encephalopathies, normal PrP(C) may be implicated in other degenerative conditions often associated with inflammation. PrP(C) is also present in cells of hematopoietic origin, including T cells, dendritic cells, and macrophages, and it has been shown to modulate their functions. Here, we investigated the impact of inflammation and stress on the expression and function of PrP(C) in neutrophils, a cell type critically involved in both acute and chronic inflammation. We found that systemic injection of LPS induced transcription and translation of PrP(C) in mouse neutrophils. Up-regulation of PrP(C) was dependent on the serum content of TGF-ß and glucocorticoids (GC), which, in turn, are contingent on the activation of the hypothalamic-pituitary-adrenal axis in response to systemic inflammation. GC and TGF-ß, either alone or in combination, directly up-regulated PrP(C) in neutrophils, and accordingly, the blockade of GC receptors in vivo curtailed the LPS-induced increase in the content of PrP(C). Moreover, GC also mediated up-regulation of PrP(C) in neutrophils following noninflammatory restraint stress. Finally, neutrophils with up-regulated PrP(C) presented enhanced peroxide-dependent cytotoxicity to endothelial cells. The data demonstrate a novel interplay of the nervous, endocrine, and immune systems upon both the expression and function of PrP(C) in neutrophils, which may have a broad impact upon the physiology and pathology of various organs and systems.

Prolongation of prion disease-associated symptomatic phase relates to CD3+ T cell recruitment into the CNS in murine scrapie-infected mice.

Prion diseases are caused by the transconformation of the host cellular prion protein PrP(c) into an infectious neurotoxic isoform called PrP(Sc). While vaccine-induced PrP-specific CD4(+) T cells and antibodies partially protect scrapie-infected mice from disease, the potential autoreactivity of CD8(+) cytotoxic T lymphocytes (CTLs) received little attention. Beneficial or pathogenic influence of PrP(c)-specific CTL was evaluated by stimulating a CD8(+) T-cell-only response against PrP in scrapie-infected C57BL/6 mice. To circumvent immune tolerance to PrP, five PrP-derived nonamer peptides identified using prediction algorithms were anchored-optimized to improve binding affinity for H-2D(b) and immunogenicity (NP-peptides). All of the NP-peptides elicited a significant number of IFNγ secreting CD8(+) T cells that better recognized the NP-peptides than the natives; three of them induced T cells that were lytic in vivo for NP-peptide-loaded target cells. Peptides 168 and 192 were naturally processed and presented by the 1C11 neuronal cell line. Minigenes encoding immunogenic NP-peptides inserted into adenovirus (rAds) vectors enhanced the specific CD8(+) T-cell responses. Immunization with rAd encoding 168NP before scrapie inoculation significantly prolonged the survival of infected mice. This effect was attributable to a significant lengthening of the symptomatic phase and was associated with enhanced CD3(+) T cell recruitment to the CNS. However, immunization with Ad168NP in scrapie-incubating mice induced IFNγ-secreting CD8(+) T cells that were not cytolytic in vivo and did not influence disease progression nor infiltrated the brain. In conclusion, the data suggest that vaccine-induced PrP-specific CD8(+) T cells interact with prions into the CNS during the clinical phase of the disease.

Features of follicular dendritic cells in ovine pharyngeal tonsil: an in vivo and in vitro study in the context of scrapie pathogenesis.

Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal tonsils serve as a natural portal of entry for scrapie. This study describes for the first time the broad cell populations in the lymphoid compartment of pharyngeal tonsils and more specifically inside the lymphoid follicles where the scrapie agent accumulates during the period of latency. Follicular dendritic cells (FDCs), stromal cells located in the light zone of the germinal centre of lymphoid follicles, seem to be the principal causal factor in the accumulation of the infectious agent in transmissible spongiform encephalopathy (TSE) diseases. Knowing that efficient lymphoreticular prion propagation requires PrPc expression, we analysed the expression of PrPc with the mouse monoclonal antibody Pri 909 both in situ and on FDC-cluster-enriched cell suspensions. In situ, a positive staining was observed in the germinal centre of pharyngeal lymph follicles. The germinal centre labelling was due to the presence of a follicular dendritic network as revealed after immunogold staining of isolated FDC clusters. Our results suggest that the pharyngeal lymphoreticular system and more specifically PrPc expressing follicular dendritic cells could serve as a prion "reservoir" during the latency phase, thus playing a key role during the scrapie lymphoinvasion.

Underestimation of the expression of cellular prion protein on human red blood cells.

BACKGROUND: Recent transmissions of variant Creutzfeldt-Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrP(C) ) in both plasma and blood cells. According to published studies, most of PrP(C) in blood cells resides in platelets (PLTs) and white blood cells. STUDY DESIGN AND METHODS: To clarify conflicting reports about the quantity of PrP(C) associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RESULTS: RBCs expressed 290 ± 140 molecules of PrP(C) per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrP(C). Binding of alternate PrP(C) MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrP(C) per cell on RBCs to be just four times lower than in PLTs. A similar level of PrP(C) was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrP(C) conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. CONCLUSIONS: Taken together, human RBCs express low but significant amounts of PrP(C) /cell, which makes them, due to high RBC numbers, major contributors to the pool of cell-associated PrP(C) in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrP(C) in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrP(C).

Monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection.

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection. In vitro and in vivo studies have also observed prion retention by dendritic cells, although their role in trafficking remains unclear. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection. These prior studies also suffer from the problem of differentiating between endogenous PrP(C) and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

[New knowledge about Creutzfeldt-Jakob disease can yield therapeutic possibilities]. / Ny kunnskap om Creutzfeldt-Jakobs sykdom kan gi terapeutiske muligheter.

BACKGROUND: Creutzfeldt-Jakob disease (CJD) is a rare neurological disease which is always terminal, often within months. The disease attracted attention in the 1990 s, when reports appeared about people acquiring the disease by eating meat from cattle infected with BSE (bovine spongiform encephalopathy). Recent progress within research on CJD has raised hopes about finding a treatment. The most promising results of this research are presented. MATERIAL AND METHODS: The review is based on articles (and references in them) identified through non-systematic searches in PubMed. RESULTS: CJD is a disease that emerges when the cellular form of the prion protein PrPC is transformed into the disease-associated PrPSc. Transgenic mice that did not express PrPC did not develop clinical signs of the disease after being exposed to prions. Both administration of antibodies towards PrPC and use of RNA interference technique to reduce PrPC gene expression increased the survival time in mice. Pentosan polysulphate (a drug against interstitial cystitis) infused intraventriculary in mice, prolonged the incubation time from 51 to 123 days. The drug has been tested in patients with CJD with promising results with respect to survival time. INTERPRETATION: New treatment modalities have shown results that raise hopes about finding curative treatment in the future.

Simple method of monoclonal antibody production against mammalian cellular prion protein.

Monoclonal antibodies (MAbs) against prion protein (PrP) are powerful tools for diagnosis and research in transmissible spongiform encephalopathies. Ten MAbs to recombinant/native cellular PrP (PrPc) in mammals were prepared with a simple method and identified in detail. Normal BALB/c mice were immunized with the recombinant bovine mature PrP (rbomPrP) and PrP27-30 (rboPrP27-30) expressed in Escherichia coli. The immunized splenocytes were fused with SP2/0 mouse myeloma cells, and positive hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA). The characterizations of these MAbs, such as Ig, Ig subclass, titer, affinity index, specificity, epitopes recognized, and binding to recombinant/native PrPc of cattle, sheep, or human beings, were evaluated by Western blotting and indirect or sandwich ELISA. Ten MAbs could be divided into five groups depending on the results of indirect ELISA additivity test and their reaction to E. coli-expressed truncated-PrPs. Isotyping of the MAbs revealed that they belong to IgG1, IgG2a, and IgG2b subclass. Their indirect ELISA titers were between 10(3) and 10(6). Affinity constants were between 10(9) and 10(12) M(-1). Ten MAbs specifically reacted with the rbomPrP, without binding to prion-like protein Doppel and the lysates of E. coli. These MAbs could also respond to the recombinant mature PrP (rmPrP) of sheep and human beings. Also of interest was the ability of the MAbs to bind with dimer of rmPrP and PrP extracted from the brain tissue of cattle or sheep. We conclude that anti-PrP MAbs successfully prepared with a simple method could potentially be useful in mammalian prion research.

[Systematic review of the therapeutics for prion diseases].

Prion diseases are fatal infectious neurodegenerative disorders; examples include the Creutzfeldt-Jakob disease affecting humans and bovine spongiform encephalopathy in cattle. The causative agents of these diseases--the prions--are thought to consist of the pathogenic isoform of the prion protein PrP(Sc), which is produced by the conformational conversion of the normal isoform PrP(c). Many lines of evidence indicate that the constitutive conversion of PrP(c) to PrP(Sc), resulting in a marked accumulation of PrP(Sc) in the brain, is a central event in the pathogenesis of prion diseases. A large number of compounds have been identified as anti-prion agents and capable of reducing the PrP(Sc) levels in infected cells. Some of these compounds have been found to be partially effective in infected animals, thus resulting in the prolongation of the incubation or survival times and a few of these compounds were or are under clinical trials. However, none of these compounds have proven to be therapeutically effective against this group of diseases. This is probably because (1) these compounds fail to cross the blood-brain barrier and (2) their effectiveness is reduced because they are administered only to patients with clinically advanced disease owing to a lack of diagnostic indicators for presymptomatic individuals. In this communication, we systematically list these anti-prion compounds and summarize their effectiveness and possible mechanisms of action.

Role of cellular prion proteins in the function of macrophages and dendritic cells.

The cellular isoform of prion proteins (PrPC) is expressed in hematopoietic stem cells, granulocytes, T and B lymphocyte natural killer cells, platelets, monocytes, dendritic cells, and follicular dendritic cells, which may act as carrier cells for the spread of its abnormal isoform (PrPSc) before manifesting transmissible spongiform encephalopathies (TSEs). In particular, macrophages and dendritic cells seem to be involved in the replication of PrPSc after ingestion. In addition, information on the role of PrPC during phagocytotic activity in these cells has been obtained. A recent study showed that resident macrophages from ZrchI PrP gene (Prnp)-deficient (Prnp-/-) mice show augmented phagocytotic activity compared to Prnp+/+ counterparts. In contrast, our study suggests that Rikn Prnp-/- peritoneal macrophages show pseudopodium extension arrest and up-regulation of phagocytotic activity compared to Prnp+/+ cells. Although reports regarding phagocytotic activity in resident and peritoneal macrophages are inconsistent between ZrchI and Rikn Prnp-/- mice, it seems plausible that PrPC in macrophages could contribute to maintain the immunological environment. This review will introduce the recent progress in understanding the functions of PrPC in macrophages and dendritic cells under physiological conditions and its involvement in the pathogenesis of prion diseases.

Curative properties of antibodies against prion protein: a comparative in vitro study of monovalent fragments and divalent antibodies.

Prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans, are a group of devastating neurodegenerative disorders for which no therapy is yet available. However, passive immunotherapy appears to be a promising therapeutic approach, given that antibodies against the cellular prion protein (PrPc) have been shown in vitro to antagonize deposition of the disease-associated prion protein (PrPSc). Nevertheless, in vivo deleterious side effects of injected anti-PrP antibodies have been reported, mainly due to their Fc fragments and divalence. In this context, we examined here the ability of five Fabs (monovalent fragments devoid of the Fc part), prepared from antibodies already characterized in the laboratory, to inhibit prion replication in infected neuronal cells. We show that all Fabs (which all retain the same apparent affinity for PrPc as their whole antibody counterpart, as measured in EIA experiments) recognize quite well membrane bound-PrP in neuronal cells (as shown by flow cytometry analysis) and inhibit PrPSc formation in infected cells in a dose-dependent manner, most of them (four out of five) exhibiting a similar efficiency as whole antibodies. From a fundamental point of view, this report indicates that the in vitro curative effect of antibodies i) is epitope independent and only related to the efficiency of recognizing the native, membrane-inserted form of neuronal PrP and ii) probably occurs by directly or indirectly masking the PrPc epitopes involved in PrPSc interaction, rather than by cross-linking membrane bound PrPc. From a practical point of view, i.e. in the context of a possible immunotherapy of prion diseases, our data promote the use of monovalent antibodies (either Fabs or engineered recombinant fragments) for further in vivo studies.

Production and characterization of a panel of monoclonal antibodies against native human cellular prion protein.

The human prion diseases, such as variant Creutzfeldt-Jakob disease (vCJD), are characterized by the conversion of the normal cellular prion protein (PrP(C)) into an abnormal disease associated form (PrP(Sc)). Monoclonal antibodies (MAbs) that recognize these different PrP isoforms are valuable reagents both in the diagnosis of these diseases and in prion disease research in general but we know of no attempts to raise MAbs against native human PrP(C). We immunized prion protein gene ablated (PrP(-/-)) mice with native human PrP(C) purified from platelets (pHuPrP) generating a predominantly IgG isotype anti-pHuPrP polyclonal antibody response in all mice. Following fusion of splenocytes from the immunized mice with SP2/0 myeloma cells, we were able to identify single cell clone and cryopreserve 14 stable hybridoma cell lines producing MAbs that reacted with pHuPrP. The properties of these MAbs (such as isotype, binding to native/denatured pHuPrP, and HuPrP epitopes recognized) are described. Furthermore, several of these MAbs showed a selectivity in their ability to immunoprecipitate disease associated PrP(Sc) and its corresponding protease resistant core (PrP(res)).