Biomedical subjects

Raymond Bujdoso

Publications and source records attributed to Raymond Bujdoso.

14 recordsLinked to original sources

PubMed2007

Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay.

PrPSc [abnormal disease-specific conformation of PrP (prion-related protein)] accumulates in prion-affected individuals in the form of amorphous aggregates. Limited proteolysis of PrPSc results in a protease-resistant core of PrPSc of molecular mass of 27-30 kDa (PrP27-30). Aggregated forms of PrP co-purify with prion infectivity, although infectivity does not always correlate with the presence of PrP27-30. This suggests that discrimination between PrPC (normal cellular PrP) and PrPSc by proteolysis may underestimate the repertoire and quantity of PrPSc subtypes. We have developed a CDI (conformation-dependent immunoassay) utilizing time-resolved fluorescence to study the conformers of disease-associated PrP in natural cases of sheep scrapie, without using PK (proteinase K) treatment to discriminate between PrPC and PrPSc. The capture-detector CDI utilizes N-terminal- and C-terminal-specific anti-PrP monoclonal antibodies that recognize regions of the prion protein differentially buried or exposed depending on the extent of denaturation of the molecule. PrPSc was precipitated from scrapie-infected brain stem and cerebellum tissue following sarkosyl extraction, with or without the use of sodium phosphotungstic acid, and native and denatured PrPSc detected by CDI. PrPSc was detectable in brain tissue from homozygous VRQ (V136 R154 Q171) and ARQ (A136 R154 Q171) scrapie-infected sheep brains. The highest levels of PrPSc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPSc was significantly reduced, up to 90% in some cases, when samples were treated with PK prior to the CDI. Collectively, our results show that the level of PrPSc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is PK-sensitive.

Animals↗

PubMed2006

Ovine plasma prion protein levels show genotypic variation detected by C-terminal epitopes not exposed in cell-surface PrPC.

Ovine PBMCs (peripheral blood mononuclear cells) express PrP(C) [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrP(C) expression by PBMCs, together with plasma PrP(C) levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrP(C) expression on normal ovine PBMCs by FACS with the presence of PrP(C) in plasma measured by capture-detector immunoassay. FACS showed similar levels of cell-surface PrP(C) on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrP(C) showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrP(C) levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrP(C). Homozygous VRQ sheep showed the highest plasma PrP(C) level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrP(C) levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrP(C) expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrP(C) in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrP(C) was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrP(C).

Animals↗

PubMed2006

Genetic vaccination with Flt3-L and GM-CSF as adjuvants: Enhancement of cellular and humoral immune responses that results in protective immunity in a murine model of hepatitis C virus infection.

AIM: To investigate whether transfection of plasmid DNA encoding these cytokines enhances both humoral and cellular immune responses to hepatitis C virus (HCV) in a murine model. METHODS: We established a tumor model of HCV infection using syngenic mouse myeloma cells stably transfected with NS5. Co-vaccination of DNA encoding granulocyte macrophage colony-stimulating factor (GM-CSF) and Flt-3 ligand together with a plasmid encoding for the HCV NS5 protein was carried out. Mice were sacrificed 14 d after the last immunization event with collection of spleen cells and serum to determine humoral and cellular immune responses. RESULTS: Co-vaccination of DNA encoding GM-CSF and Flt-3 ligand together with a plasmid encoding for the HCV NS5 protein induced increased antibody responses and CD4+ T cell proliferation to this protein. Vaccination with DNA encoding GM-CSF and Flt-3L promoted protection against tumor formation and/or reduction in mice co-immunized with cytokine-encoding DNA constructs. This suggests this strategy is capable of generating cytotoxic T lymphocyte activity in vivo. Following inoculation with plasmid DNA encoding Flt-3L, no increase in spleen size or in dendritic cell (DC) and natural killer cell numbers was observed. This was in contrast to a dramatic increase of both cell types after administration of recombinant Flt3-L in vivo. This suggests that vaccination with plasmid DNA encoding cytokines that regulate DC generation and mobilization may not promote unwanted side effects, such as autoimmunity, splenic fibrosis or hematopoietic malignancies that may occur with administration of recombinant forms of these proteins. CONCLUSION: Our data support the view that plasmid DNA vaccination is a promising approach for HCV immunization, and may provide a general adjuvant vaccination strategy against malignancies and other pathogens.

Adjuvants, Immunologic↗

PubMed2006

Elevated PrPC expression predisposes to increased HSV-1 pathogenicity.

PrPC is a ubiquitously expressed glycophos-phatidylinositol-linked cell-surface glycoprotein found primarily in neural tissue. Although its normal function has not been established, there is evidence suggesting that PrPC is involved in cell signalling and cellular homeostasis. This suggests that variation in neuronal expression levels of this protein contributes towards pathogenicity induced by neurotropic agents. We have investigated the pathological response to infection with herpes simplex virus type-1 (HSV-1) in strains of mice that express different levels of PrPC. Prnp-/- mice fail to express PrPC due to an interruption in the open reading frame of the Prnp gene, whilst tg19 and tga20 mice express approximately 5 and 10 times more PrPC protein, respectively, than wild-type animals. Mice that express normal or increased levels of PrPC protein were more susceptible to acute HSV-1 infection than Prnp-/- mice. Following ear pinna inoculation with HSV-1 SC16, the order of susceptibility was tga20>tg19>wild-type>Prnp-/-. This trend was reversed when latent virus was assessed. Prnp-/- mice expressed significantly higher levels of latency-associated transcript-positive neurons in various tissues when compared with wild-type, tg19 and tga20 mice. Collectively, our data show that acute HSV-1 replication proceeds more efficiently in neuronal tissue that expresses PrPC protein and lends support to the view that this protein is involved in regulation of neurotropic viral pathogenesis. This suggests that interference of PrPC expression, or possible biochemical pathways associated with its function, may serve as an effective means of limiting the pathogenesis of acute HSV-1 infection.

Animals↗

PubMed2005

Structural differences between allelic variants of the ovine prion protein revealed by molecular dynamics simulations.

We have modeled ovine prion protein (residues 119-233) based on NMR structures of PrP from other mammalian species. Modeling of the C-terminal domain of ovine PrP predicts three helices: helix-1 (residues 147-155), flanked by two short beta-strands; helix-2 (residues 176-197), and helix-3 (residues 203-229). Molecular dynamics simulations on this model of ovine PrP have determined structural differences between allelic variants. At neutral pH, limited root mean-squared (RMS) fluctuations were seen in the region of helix-1; between beta-strand-2 and residue 171, and the loop connecting helix-2 and helix-3. At low pH, these RMS fluctuations increased and showed allelic variation. The extent of RMS fluctuation between beta-strand 2 and residue 171 was ARR > ARQ > VRQ. This order was reversed for the loop region connecting helix-2 and helix-3. Although all three variants have the potential to display an extended helix at the C-terminal region of helix-1, the major influence of the VRQ allele was to restrict the conformations of the Asn162 and Arg139 side-chains. Variations observed in the simulations in the vicinity of helix-1 correlated with reactivity of C-terminal specific anti-PrP monoclonal antibodies with peripheral blood cells from scrapie-susceptible and -resistant genotypes of sheep: cells from VRQ homozygous sheep showed uniform reactivity, while cells from ARQ and ARR homozygous sheep showed variable binding. Our data show that molecular dynamics simulations can be used to determine structural differences between allelic variants of ovine PrP. The binding of anti-PrP monoclonal antibodies to ovine blood cells may validate these structural predictions.

Animals↗

PubMed2005

Modification of blood cell PrP epitope exposure during prion disease.

PrPC [normal cellular PrP (prion-related protein)] is a glycosylphosphatidylinositol-linked cell-surface glycoprotein that is expressed primarily by cells of the central and peripheral nervous system and the lymphoreticular system. During prion disease, PrPC undergoes structural modification to PrPSc (abnormal disease-specific conformation of PrP). The appearance of prion infectivity and PrPSc within different peripheral lymphoid tissue sites during natural scrapie infection in sheep is suggestive of haematogenic dissemination. For this to occur, blood cells may harbour or carry disease-associated PrP and in doing so present altered conformations of PrP on their cell-surface. In the present study, we show that changes in PrP epitope expression, or accessibility, can be detected on peripheral blood mononuclear cells during the course of experimental scrapie in susceptible sheep. Peripheral blood mononuclear cells isolated from VRQ homozygous lambs inoculated orally with scrapie were probed with either N- or C-terminal-specific anti-PrP monoclonal antibodies and analysed by flow cytometry. During the progression of scrapie, significant alterations were seen in the exposure of particular cell-surface PrP epitopes. These modifications included increased accessibility to N-terminal regions of the PrP molecule, to the region between beta-strand-2 and residue 171, and to the C-terminal region of helix-3. Increased accessibility in the globular C-terminal domain of PrP occurred in the vicinity of tyrosine dimers, which are believed to have increased solvent exposure in disease-associated PrP. We suggest that the alterations in anti-PrP monoclonal antibody recognition of cell-surface PrP on blood cells from scrapie-infected sheep are indicative of structural changes within this molecule that may be relevant to prion disease.

Aging↗

PubMed2004

Conformational variation between allelic variants of cell-surface ovine prion protein.

The distribution of prion infectivity and PrPSc between peripheral lymphoid tissues suggests their possible haematogenic spread during the progression of natural scrapie in susceptible sheep. Since ovine PBMCs (peripheral blood mononuclear cells) express PrPC, they have the potential to carry or harbour disease-associated forms of PrP. To detect the possible presence of disease-associated PrP on the surface of blood cells, an understanding is required of the conformations that normal ovine cell-surface PrPC may adopt. In the present study, we have used monoclonal antibodies that recognize epitopes in either the N- or C-terminal portions of PrP to probe the conformations of PrPC on ovine PBMCs by flow cytometry. Although PBMCs from scrapie-susceptible and -resistant genotypes of sheep expressed similar levels of cell-surface PrPC, as judged by their reactivity with N-terminal-specific anti-PrP monoclonal antibodies, there was considerable genotypic heterogeneity in the region between helix-1 and residue 171. Cells from PrP-VRQ (V136R154Q171) sheep showed uniform reactivity with monoclonal antibodies that bound to epitopes around helix-1, whereas cells from PrP-ARQ (A136R154Q171) and PrP-ARR (A136R154R171) sheep showed variable binding. The region between b-strand-2 and residue 171, which includes a YYR motif, was buried or obscured in cell-surface PrPC on PBMCs from scrapie-susceptible and -resistant sheep. However, an epitope of PrPC that is influenced by residue 171 was more exposed on PBMCs from PrP-VRQ sheep than on PBMCs from the PrP-ARQ genotype. Our results highlight conformational variation between scrapie-susceptible and -resistant forms of cell-surface PrPC and also between allelic variants of susceptible genotypes.

Alleles↗

PubMed2004

Copper induces increased beta-sheet content in the scrapie-susceptible ovine prion protein PrPVRQ compared with the resistant allelic variant PrPARR.

Prion diseases are characterized by conformational change in the copper-binding protein PrP (prion protein). Polymorphisms in ovine PrP at amino acid residues 136, 154 and 171 are associated with variation in susceptibility to scrapie. PrPVRQ [PrP(Val136/Arg154/Gln171)] or PrPARQ [PrP(Ala136/Arg154/Gln171)] animals show susceptibility to scrapie, whereas those that express Ala136/Arg154/Arg171 (PrPARR) show resistance. Results are presented here that show PrPVRQ and PrPARR display different conformational responses to metal-ion interaction. At 37 degrees C copper induced different levels of b-sheet content in the allelic variants of ovine full-length prion protein (amino acid 25-232). PrPVRQ showed a significant increase in b-sheet content when exposed to copper at 37 degrees C, whereas PrPARR remained relatively unchanged. The conversion of a-helical PrPVRQ to b-sheet form was shown by CD spectroscopy and the decreased binding of C-terminal specific monoclonal anti-PrP antibodies. This conversion to an increased b-sheet form did not occur with truncated PrPVRQ (amino acids 89-233), which demonstrates that additional metal-binding sites outside of the N-terminus may not overtly influence the overall structure of ovine PrP. Despite the difference in b-sheet content, both the scrapie-susceptible and -resistant allelic forms of ovine PrP acquired resistance to proteinase K digestion following exposure to copper at 37 degrees C, suggesting the potential for disease-associated PrPARR to accumulate in vivo. Our present study demonstrates that allelic variants of ovine PrP differ in their structure and response to the interaction with copper. These observations will contribute to a better understanding of the mechanism of susceptibility and resistance to prion disease.

Alleles↗

PubMed2004

Accelerated prion disease in the absence of interleukin-10.

The identity of pro- and anti-inflammatory cytokines in the neuropathogenesis of prion diseases remains undefined. Here we have investigated the role of anti-inflammatory cytokines on the progression of prion disease through the use of mice that lack interleukin-4 (IL-4), IL-10, IL-13, or both IL-4 and IL-13. Collectively our data show that among these anti-inflammatory cytokines, IL-10 plays a prominent role in the regulation of prion disease. Mice deficient in IL-10 are highly susceptible to the development of prion disease and show a markedly shortened incubation time. In addition, we have correlated cytokine gene expression in prion-inoculated IL-10(-/-) mice to wild-type-inoculated animals. Our experiments show that in the absence of IL-10 there is an early expression of tumor necrosis factor alpha (TNF-alpha). In wild-type prion-inoculated mice, the expression of TNF-alpha mRNA occurs at a later time point that correlates with the extended incubation time for terminal disease development in these animals compared to those that lack IL-10. Elevated levels of IL-13 mRNA are found at early time points in the central nervous system of prion-inoculated IL-10(-/-) mice. At terminal disease, the brains of wild-type mice inoculated with RML or ME7 are characterized by elevated levels of mRNA for the proinflammatory cytokines TNF-alpha and IL-1beta, together with the anti-inflammatory cytokines IL-10, IL-13, and transforming growth factor beta. Our data are consistent with a role for proinflammatory cytokines in the initiation of pathology during prion disease and an attempt by anti-inflammatory cytokines to regulate the ensuing, invariably fatal pathology.

Animals↗

PubMed2003

Detection of bovine spongiform encephalopathy, ovine scrapie prion-related protein (PrPSc) and normal PrPc by monoclonal antibodies raised to copper-refolded prion protein.

Prion-related protein (PrP) is a glycosylphosphatidylinositol-linked cell-surface protein expressed by a wide variety of cells, including those of the nervous system and the immune system. Several functions of normal cellular PrP (PrPc) have been proposed that may be associated with the capacity of this protein to bind copper. In the present study, we describe the generation of a panel of monoclonal antibodies raised to copper-refolded PrP, which may be used to analyse the normal and disease-associated forms of this protein. The anti-PrP monoclonal antibodies were reactive by Western blot and ELISA with recombinant murine PrPc refolded in the presence or absence of either copper or manganese, and with the disease-susceptible allelic form V136R154Q171 ('VRQ'; where single-letter amino-acid notation has been used) and disease-resistant allelic form A136R154R171 ('ARR') of recombinant ovine PrPc. FACS analysis of lymphoid cells using these monoclonal antibodies showed that wild-type non-activated mouse lymphocytes expressed little, if any, PrPc. These monoclonal antibodies were shown to react with the unglycosylated and monoglycosylated forms of PrPSc (abnormal disease-specific conformation of PrP) in prion-infected tissue samples from all of the different species tested by Western blot. In addition, this analysis allowed one to make a distinction between bovine spongiform encephalopathy ('BSE') and scrapie PrPSc) isolates from experimentally infected sheep on the basis of their different electrophoretic mobilities.

Animals↗

PubMed2003

Subclinical prion disease induced by oral inoculation.

Natural transmission of prion disease is believed to occur by peripheral infection such as oral inoculation. Following this route of inoculation, both the peripheral nervous system and the lymphoreticular system may be involved in the subsequent neuroinvasion of the central nervous system by prions, which may not necessarily result in clinical signs of terminal disease. Subclinical prion disease, characterized by the presence of infectivity and PrP(Sc) in the absence of overt clinical signs, may occur. It is not known which host factors contribute to whether infection with prions culminates in a terminal or subclinical disease state. We have investigated whether the level of host PrP(c) protein expression is a factor in the development of subclinical prion disease. When RML prion inoculum was inoculated by either the i.c. or intraperitoneal route, wild-type and tga20 mice both succumbed to terminal prion disease. In contrast, orally inoculated tga20 mice succumbed to terminal prion disease, whereas wild-type mice showed no clinical signs. However, wild-type mice sacrificed 375 or 525 days after oral inoculation harbored significant levels of brain PrP(Sc) and infectivity. These data show that same-species transmission of prions by the oral route in animals that express normal levels of PrP(c) can result in subclinical prion disease. This indicates that the level of host PrP(c) protein expression is a contributing factor to the regulation of development of terminal prion disease. Events that increase PrP(c) expression may predispose a prion-infected animal to the more deleterious effects of prion pathology.

Administration, Oral↗

PubMed2002

Metal imbalance and compromised antioxidant function are early changes in prion disease.

The prion protein (PrP) has been shown to bind copper. In the present study we have investigated whether prion disease in a mouse scrapie model resulted in modification of metal concentrations. We found changes in the levels of copper and manganese in the brains of scrapie-infected mice prior to the onset of clinical symptoms. Interestingly, we noted a major increase in blood manganese in the early stages of disease. Analysis of purified PrP from the brains of scrapie-infected mice also showed a reduction in copper binding to the protein and a proportional decrease in antioxidant activity between 30 and 60 days post-inoculation. We postulate that alterations in trace-element metabolism as a result of changes in metal binding to PrP are central to the pathological modifications in prion disease.

Animals↗

PubMed2002

PrP(c) expression influences the establishment of herpes simplex virus type 1 latency.

PrP(c) is a glycophosphatidylinositol-linked cell-surface protein expressed principally by neural tissue. The normal function of this protein is unestablished, although a role in either transmembrane signaling, cell-cell adhesion, or copper metabolism has been proposed. In this study we have investigated the effect of the neurotropic virus herpes simplex virus type 1 (HSV-1) in strains of mice which express different levels of PrP(c). Viral gene expression under the control of the HSV-1 early promoter IE110, detected either by in situ hybridization for RNA transcripts or by beta-galactosidase (beta-Gal) activity from an inserted lacZ gene, showed that the magnitude of HSV replication was retarded in PrP-/- mice. This was reflected in the lower level of acute viral titers in tissues from these virus-inoculated mice. However, HSV-inoculated PrP-/- mice contained higher levels of latent virus in both peripheral and central nervous tissue than those seen in mice which express PrP(c). Our observations show that lack of PrP(c) expression favors the establishment of HSV latency whereas HSV replication proceeds more efficiently in neuronal tissue that expresses this protein. The data further suggest that PrP(c) may be involved in a metabolic pathway that culminates in apoptosis of neurons that have been infected by neurotropic viruses.

Animals↗

PubMed2002

Chronic subclinical prion disease induced by low-dose inoculum.

We have compared the transmission characteristics of the two mouse-adapted scrapie isolates, ME7 and Rocky Mountain Laboratory (RML), in tga20 mice. These mice express elevated levels of PrP protein compared to wild-type mice and display a relatively short disease incubation period following intracerebral prion inoculation. Terminal prion disease in tga20 mice induced by ME7 or RML was characterized by a distinct pattern of clinical signs and different incubation times. High-dose RML inoculated intracerebrally into tga20 mice induced the most rapid onset of clinical signs, with mice succumbing to terminal disease after only 58 +/- 3 days. In contrast, high-dose ME7 gave a mean time to terminal disease of 74 +/- 0 days. Histological examination of brain sections from prion-inoculated tga20 mice at terminal disease showed that ME7 gave rise to a more general and extensive pattern of vacuolation than RML. Low-dose inoculum failed to induce terminal disease but did cause preclinical symptoms, including the appearance of reversible clinical signs. Some mice oscillated between showing no clinical signs and early clinical signs for many months but never progressed to terminal disease. Brain tissue from these mice with chronic subclinical prion disease, sacrificed at >200 days postinoculation, contained high levels of infectivity and showed the presence of PrP(Sc). Parallel analysis of brain tissue from mice with terminal disease showed similar levels of infectivity and detectable PrP(Sc). These results show that high levels of infectivity and the presence of the abnormal isomer of PrP can be detected in mice with subclinical disease following low-dose prion inoculation.

Animals↗