PRP Antibody Research Articles

Preparation of PrP-specific Polyclonal Antibody via Immunization of PRNP-knockout Mice with Recombinant Human PrP Protein

ObjectiveThe definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrPSc in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification. MethodsWe prepared a PrP-specific polyclonal antibody (pAb P54) in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23–231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays. ResultsWestern blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrPC from healthy rodents and humans, and pathological PrPSc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrPC and PrPSc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei. ConclusionThe newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Immunogenicity and safety of two doses of catch-up immunization with Haemophilus influenzae type b conjugate vaccine in Indian children living with HIV

BackgroundChildren living with HIV are at increased risk of disease from Haemophilus influenzae type b (Hib). Data are limited on the immunogenicity of a two-dose, catch-up schedule for Hib conjugate vaccine (HibCV) among HIV-infected children accessing antiretroviral therapy (ART) late. ObjectivesThe objectives of the study were to: (1) evaluate baseline immunity to Hib and the immunogenicity and safety of two doses of HibCV among HIV-infected Indian children; and (2) document the threshold antibody level required to prevent Hib colonization among HIV-infected children following immunization. MethodsWe conducted a prospective cohort study among HIV-infected children 2–15 years of age and HIV-uninfected children 2–5 years of age. HIV-infected children received two doses of HibCV and uninfected children received one. Serum anti-Hib PRP IgG antibodies were measured at baseline and two months after immunization in the HIV-infected children. Nasopharyngeal (NP) swabs were collected at baseline and follow-up. Results125 HIV-infected and 44 uninfected children participated. 40% of HIV-infected children were receiving ART and 26% had a viral load >100,000 copies/mL. The geometric mean concentration of serum anti-Hib PRP antibody increased from 0.25μg/mL at baseline to 2.65μg/mL after two doses of HibCV, representing a 10.6-fold increase (p<0.0001). 76% percent of HIV-infected children mounted an immune response. Moderate or severe immune suppression, trimethoprim/sulfamethoxazole prophylaxis, and lower baseline antibody levels were associated with lower post-vaccine serum anti-Hib PRP IgG antibodies. A serum anti-Hib PRP IgG antibody level ≥3.3μg/mL was protective against Hib NP colonization. There were no differences in adverse events between HIV-infected and uninfected children. ConclusionIncluding a catch-up immunization schedule for older HIV infected children in countries introducing Hib vaccines is important. Older HIV-infected children with delayed access to ART and without suppressed viral loads mounted an adequate immune response following two doses of HibCV.

Microglia convert aggregated amyloid-β into neurotoxic forms through the shedding of microvesicles

Alzheimer's disease (AD) is characterized by extracellular amyloid-β (Aβ) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aβ species, rather than insoluble fibrils, are the most toxic forms of Aβ. Preventing soluble Aβ formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aβ species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aβ forms trafficked to MVs after Aβ internalization into microglia. MV neurotoxicity was neutralized by the Aβ-interacting protein PrP and anti-Aβ antibodies, which prevented binding to neurons of neurotoxic soluble Aβ species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.

Naturally Acquired and Conjugate Vaccine-Induced Antibody to Haemophilus influenzae Type b (Hib) Polysaccharide in Malian Children: Serological Assessment of the Hib Immunization Program in Mali

Haemophilus influenzae type b (Hib) conjugate vaccine for infants (6, 10, and 14 weeks of age) was introduced into the Malian Expanded Program on Immunization in July 2005, to diminish invasive Hib disease in young children. Antibodies to Hib capsular polysaccharide (PRP) were measured in infants and toddlers from an area already served by the Hib immunization program (Bamako) and in unimmunized children of the same age in a district (Kangaba) where Hib immunization had not yet begun. Among vaccinated Bamako children 6-23 months of age, 77-93% exhibited PRP titers ≥ 1.0 μg/mL, indicating long-term protection, versus only 10-23% of Kangaba children of that age. High PRP antibody titers in immunized children persisted through 2 years of age. Moreover, ∼50% of Bamako children exhibited anti-PRP titers ≥ 5.0 μg/mL; a level that impedes Hib upper respiratory carriage, and may thereby diminish the Hib transmission to the unimmunized susceptible population (i.e., providing indirect protection).

Immune Responses in Wild-type Mice Against Prion Proteins Induced Using a DNA Prime-Protein Boost Strategy

Objective To break immune tolerance to prion (PrP) proteins using DNA vaccines. Methods Four different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector: PrP-WT expressing wild-type PrP, Ubiq-PrP expressing PrP fused to ubiquitin, PrP-LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome-targeting signal, and PrP-ER expressing PrP locating the ER. Using a prime-boost strategy, three-doses of DNA vaccine were injected intramuscularly into Balb/c mice, followed by two doses of PrP protein. Two weeks after the last immunization, sera and spleens were collected and PrP-specific humoral and cellular immune responses evaluated by ELISA and ELISPOT tests. Results Higher levels of serum PrP antibodies were detected in mice vaccinated using the strategy of DNA priming followed by protein boosting. Of these, WT-PrP, Ubiq-PrP, and PrP-LII induced significantly higher humoral responses. ELISPOT tests showed markedly increased numbers of IFN-γ-secreting T cells in mice vaccinated using the strategy of DNA priming followed by protein boosting after stimulation with recombinant PrP23-90 and PrP23-231. PrP-ER induced the strongest T-cell response. Conclusion Prion vaccines can break tolerance to PrP proteins and induce PrP-specific humoral and cellular immune responses.

PrPSc-Specific Antibodies with the Ability to Immunodetect Prion Oligomers

The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs) derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p0/0 cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aβ and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids.

Codon 129 polymorphism and the E200K mutation do not affect the cellular prion protein isoform composition in the cerebrospinal fluid from patients with Creutzfeldt–Jakob disease

The cellular prion protein (PrP(c)) is a multifunctional, highly conserved and ubiquitously expressed protein. It undergoes a number of modifications during its post-translational processing, resulting in different PrP(c) glycoforms and truncated PrP(c) fragments. Limited data are available in humans on the expression and cleavage of PrP(c). In this study we investigated the PrP(c) isoform composition in the cerebrospinal fluid from patients with different human prion diseases. The first group of patients was affected by sporadic Creutzfeldt-Jakob disease exhibiting different PrP codon 129 genotypes. The second group contained patients with a genetic form of Creutzfeldt-Jakob disease (E200K). The third group consisted of patients with fatal familial insomnia and the last group comprised cases with the Gerstmann-Sträussler-Scheinker syndrome. We examined whether the PrP codon 129 polymorphism in sporadic Creutzfeldt-Jakob disease as well as the type of prion disease in human patients has an impact on the glycosylation and processing of PrP(c). Immunoblotting analyses using different monoclonal PrP(c) antibodies directed against various epitopes of PrP(c) revealed, for all examined groups of patients, a consistent predominance of the glycosylated PrP(c) isoforms as compared with the unglycosylated form. In addition, the antibody SAF70 recognized a variety of PrP(c) fragments with sizes of 21, 18, 13 and 12 kDa. Our findings indicate that the polymorphisms at PrP codon 129, the E200K mutation at codon 200 or the examined types of human transmissible spongiform encephalopathies do not exert a measurable effect on the glycosylation and processing of PrP(c) in human prion diseases.

A Randomized, Multicenter, Open-Label Clinical Trial to Assess the Immunogenicity of a Meningococcal C Vaccine Booster Dose Administered to Children Aged 14 to 18 Months

A booster meningococcal C (MenC) vaccine dose is recommended after the first year of life. The objective of this study was to assess its immunogenicity and factors that modify the immunoresponse. An open label study in which 389 children 14 to 18 months of age, previously primed with 3 doses of a MenC vaccine conjugated with CRM197 (MenC-CRM) or with 2 doses of a MenC vaccine conjugated with tetanus toxoid (MenC-TT), were randomized to be boosted with either of these vaccines and a DTaP-IPV-Hib vaccine at the same time. Immunogenicity against MenC and Haemophilus influenzae type b was assessed before and 1 month after the booster dose. Before the second year booster, 44.9% of the studied children had MenC bactericidal (SBA) seroprotection rate of > or =1:8, with no differences related to the vaccine used for priming, whereas the anti Hib antibody concentration was higher in children primed with the MenC-TT (0.59; 95% CI: 0.49-0.71 vs. 0.39; 95% CI: 0.32-0.48).One month after the MenC vaccine booster 99.5% of the children had SBA > or =1:128. Children primed with MenC-TT reached higher SBA titers: 6520 (95% CI: 5359-7932) than those primed with MenC-CRM: 1903 (95% CI: 1600-2262). Children primed with MenC-CRM had SBA titers of 2061 (95% CI: 1599-2627) when boosted with MenC-TT and 1746 (95% CI: 1378-2213) when boosted with MenC-CRM. Children primed with MenC-TT had SBA titers of 6786 (95% CI: 5023-9167) and 6278 (95% CI: 4841-8144) when boosted with MenC-TT or MenC-CRM. There was no difference in the PRP antibody concentration after boosting. A booster MenC dose induces high SBA and anti Hib response with over 99% of children seroprotected. Children primed with a MenC-TT vaccine reached SBA titers 3.5 times higher no matter which vaccine was used for boosting.

High prevalence of scrapie in a dairy goat herd: tissue distribution of disease-associated PrP and effect ofPRNPgenotype and age

Following a severe outbreak of clinical scrapie in 2006-2007, a large dairy goat herd was culled and 200 animals were selected for post-mortem examinations in order to ascertain the prevalence of infection, the effect of age, breed and PRNP genotype on the susceptibility to scrapie, the tissue distribution of diseaseassociated PrP (PrP(d)), and the comparative efficiency of different diagnostic methods. As determined by immunohistochemical (IHC) examinations with Bar224 PrP antibody, the prevalence of preclinical infection was very high (72/200; 36.0%), with most infected animals being positive for PrP(d) in lymphoreticular system (LRS) tissues (68/72; 94.4%) compared to those that were positive in brain samples (38/72; 52.8%). The retropharyngeal lymph node and the palatine tonsil showed the highest frequency of PrP(d) accumulation (87.3% and 84.5%, respectively), while the recto-anal mucosa-associated lymphoid tissue (RAMALT) was positive in only 30 (41.7%) of the infected goats. However, the efficiency of rectal and palatine tonsil biopsies taken shortly before necropsy was similar. The probability of brain and RAMALT being positive directly correlated with the spread of PrP(d) within the LRS. The prevalence of infection was influenced by PRNP genetics at codon 142 and by the age of the goats: methionine carriers older than 60 months showed a much lower prevalence of infection (12/78; 15.4%) than those younger than 60 months (20/42; 47.6%); these last showed prevalence values similar to isoleucine homozygotes of any age (40/80; 50.0%). Two of seven goats with definite signs of scrapie were negative for PrP(d) in brain but positive in LRS tissues, and one goat showed biochemical and IHC features of PrP(d) different from all other infected goats. The results of this study have implications for surveillance and control policies for scrapie in goats.

Occurrence and cellular localization of PrPd in kidneys of scrapie‐affected sheep in the absence of inflammation

Following a preliminary description of disease-associated prion protein (PrPd) deposition in the kidneys of scrapie-affected sheep, detailed studies have been undertaken in order to evaluate the factors that could account for such PrPd accumulation and to determine the precise location of PrPd in the renal papillae. Immunohistochemical (IHC) examinations for PrPd were conducted in kidneys collected at post-mortem from 30 naturally and 37 experimentally infected sheep. In addition, PrPd detection by western blot analysis (WB) and ultrastructural examination was carried out in a selection of kidneys. PrPd-specific, multifocal IHC labelling with antibody R145 was achieved in the kidneys of 44% and 51% of the naturally and experimentally infected sheep, respectively. The specificity of these results was confirmed by further IHC and WB using several PrP antibodies raised to different amino acid sequences, and by examination of control tissues. PrPd was shown to accumulate in the interstitium of the renal papillae, in association with the cell membrane and lysosomes of fibroblast-like cells, or extracellularly, in close contact with collagen and basal membranes. These deposits were unrelated to inflammatory changes in the kidney as shown by routine histology and by IHC for different immune cell markers. PrPd accumulated in the kidney of sheep that showed widespread PrPd deposition in the lymphoreticular system and had long incubation periods; these findings argue for a haematogenous origin of renal PrPd, although the precise site and mechanism-glomerular filtration and reabsorption at Henle's loop, or extravasation from vasa recta capillaries, or both-by which PrPd leaves the blood to accumulate in the interstitium of renal papillae remain to be determined. Either of these pathogenetic mechanisms could lead to environmental contamination via urine.

Toxic Effects of Intracerebral PrP Antibody Administration During the Course of BSE Infection in Mice

The absence of specific immune response is a hallmark of prion diseases. However, in vitro and in vivo experiments have provided evidence that an anti-PrP humoral response could have beneficial effects. Prophylactic passive immunization performed at the time of infection delayed or prevented disease. Nonetheless, the potential therapeutic effect of PrP antibodies administered shortly before the clinical signs has never been tested in vivo. Moreover, a recent study showed the potential toxicity of PrP antibodies administered intracerebrally. We aimed at evaluating the effect of a prolonged intracerebral anti-PrP antibody administration at the time of neuroinvasion in BSE infected Tg20 mice. Unexpectedly, despite a good penetration of the antibodies in the brain parenchyma, the treatment was not protective against the development of BSE. Instead, it led to an extensive neuronal loss, strong astrogliosis and microglial activation. Since this effect was observed after injection of anti-PrP antibodies as whole IgGs, F(ab')2 or Fab fragments, the toxicity was directly related to the ability of the antibodies to recognize native PrP and to the intracerebral concentration achieved, and not to the Fc portion or the divalence of the antibodies. This experiment shows that a prolonged treatment with anti-PrP antibodies by the intracerebral route can induce severe side-effects and calls for caution with regard to the use of similar approaches for late therapeutic interventions in humans.

Antibody prsistence after Haemophilus influenzae type b (Hib) primary vaccination and response to boosters in Korean children

Purpose : Antibody persistence after primary series of Haemophilus influenzae type b (Hib) vaccine and responses to boosters are seldom studied in Korean children. We performed this study to evaluate the antibody titer in relation to booster immunization of Hib vaccine in Korean children. Method : One hundred forty four children aged 12-23 months were enrolled in three university hospitals. The immunogenicity of boosters with Hib vaccine was assessed in children previously primed with Hib vaccine. Antibody persistence was also assessed in children who had received 3 doses of Hib vaccine without a booster. Anti-polyribosylribitol phosphate (PRP) IgG antibody levels and bactericidal titers were determined by enzyme immunoassay and bactericidal assay at the Center for Vaccine Evaluation and Study, Medical Research Institute, Ewha Womans University. Results : Prior to a booster in the second year of life, geometric mean antibody concentrations were 2.39μg/mL and the percent of subjects who had a anti-PRP antibody level 1μg/mL was 68.6%. After boosting, antibody concentration was 19.09μg/mL and the percent of subjects who had a anti- PRP antibody level 1μg/mL was 96.5%, which reflects previous immune priming. In subjects who had finished primary immunization only, the bactericidal titer was 3,946 and in subjects who had a booster, it was 11,205. Anti-PRP antibody level was correlated with serum bactericidal titer. Conclusion : Many children aged 12-23 month old still had protective antibodies after recommended primary immunization only. A booster dose seemed to induce good anamnestic antibody responses in Korean children.

Amplified Immunohistochemical Detection of PrPsc in Animal Transmissible Spongiform Encephalopathies Using Streptomycin

Due to its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used to study experimental and natural cases of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease in humans or scrapie and bovine spongiform encephalopathy (BSE) in animals. The limits of detection are particularly critical when PrPsc IHC is used for diagnostic purposes. In this article, we describe for the first time the use of streptomycin sulfate in IHC, providing a novel original and easy way to amplify specifically PrPsc immunohistochemical detection in natural cases of BSE and scrapie, as well as in experimental TSEs in mice models using two different PrP antibodies.

The Effect of Zinc Supplementation During Pregnancy on Immune Response to Hib and BCG Vaccines in Bangladesh

An essential role for zinc in development of the fetal immune system has been documented. However, the effect of antenatal zinc supplementation on infants' postnatal immune response to vaccinations is unknown. The objective of this study was to evaluate the effect of zinc supplementation during pregnancy on immune response to the Bacillus Calmette-Guerin (BCG) vaccine and the Haemophilus influenzae type b (Hib) component of the combined diphtheria, tetanus toxoid and pertussis (DTP)-Haemophilus influenzae type-b (Hib)- conjugate vaccine in poor Bangladeshi infants. We immunized 405 infants whose mothers were supplemented daily with 30 mg elemental zinc or placebo beginning at 12-16 weeks gestation with the standard BCG vaccine at birth. A subcohort of 203 infants were in addition immunized at 1-month intervals with three doses of DTP-Hib vaccine starting at 9 weeks of age. The delayed hypersensitivity (PPD) skin test was performed in 345 infants at 24 weeks of age. Hib polysaccharide (PRP) antibodies were assessed for 91 infants at 4 and 24 weeks of age. In infants born with low birth weight (LBW) a lower proportion of negative responses to PPD skin test were observed in the zinc (66.2%) compared to placebo (78.5%) group (p = 0.07). No differences were observed in normal birth weight infants. There were no differences in proportion of infants above the protective thresholds for anti-PRP antibodies between zinc (81%) and placebo (89%) group. Geometric mean PRP antibody titres at 4 and 24 weeks of age were not different between groups. Zinc supplementation during pregnancy did not enhance immune response to Hib-conjugate vaccine but there was a suggestion of improved delayed hypersensitivity immune responses to BCG-vaccine in Bangladeshi LBW infants.

Expression of Prion Protein in the Gut of Mice Infected Orally with the 301V Murine Strain of the Bovine Spongiform Encephalopathy Agent

Transmissible spongiform encephalopathies (TSEs) are characterized by the accumulation of an abnormal, disease-associated prion protein (PrP(d)). Expression of its normal cellular counterpart (PrP(c)) by the host is a pre-requisite for the spread of infection to the central nervous system and the development of disease. Moreover, cells expressing PrP(c) at specific sites such as the gastrointestinal tract might be regarded as the initial point of PrP(c)-PrP(d) conversion after infection by the oral route. In this study, inbred mice of the I/M strain were infected orally with the 301V murine strain of the bovine spongiform encephalopathy agent. The expression of PrP(c) and the accumulation of PrP(d) in the intestine was then investigated immunohistochemically, together with the variations in immunoreactivity that resulted from different pretreatments of the tissue. After proteinase K (PK) pretreatment, abnormal PrP was still detectable only in the gut-associated lymphoid tissue (GALT) of clinically affected mice and, to a much more limited degree, in the enteric nervous system (ENS). Cellular PrP that disappeared after PK treatment was particularly conspicuous in the ENS and present to a lesser extent in the GALT of all mice examined after inoculation with 301V or with normal brain homogenates, as well as in uninoculated controls. These findings suggested that not all PrP found in infected mice was PrP(d) and that part of the PrP(d) was sensitive to PK treatment. Reactivity to PrP antibody 1A8 was consistently found in the absorptive epithelium of the intestinal villi, with or without PK pretreatment. However, epithelial immunolabelling was comparable in inoculated and uninoculated mice and was also consistently seen in PrP "knockout" mice used as controls. It is therefore concluded that immunohistochemically detectable accumulation of PrP(d) in the gut of mice is a relatively late event in the pathogenesis of experimental infection in this model and that the immunoreactivity observed in the intestinal epithelium does not correspond to PrP expression. While enterocytes may still play a role in the uptake of infection from the intestinal lumen, the results do not suggest that these cells are a site of initial accumulation of PrP(d).

Immunohistochemical characteristics of disease-associated PrP are not altered by host genotype or route of inoculation following infection of sheep with bovine spongiform encephalopathy

It has previously been reported that disease-associated prion protein (PrP(d)) derived from natural scrapie and from sheep infected experimentally with bovine spongiform encephalopathy (BSE) differed in respect of their immunohistochemical and immunoblotting properties. For BSE, however, these initial observations were restricted to orally challenged sheep of the ARQ/ARQ PrP genotype. Here, extended examinations were performed on 28 sheep that developed neurological signs after BSE experimental infection by one of three routes. Intracerebrally infected ARQ/ARQ sheep showed more widespread and abundant accumulations of PrP(d) in tissues of the lymphoreticular system (LRS) than VRQ/VRQ animals, whereas no peripheral PrP(d) was detected in ARR/ARR sheep. The intensity and dissemination of PrP(d) accumulation in LRS tissues were less than those found previously in orally dosed sheep. AHQ/AHQ sheep challenged orally and ARQ/AHQ and ARQ/ARQ animals infected intravenously showed similar LRS-tissue PrP(d) distributions and levels to those of ARQ/ARQ sheep infected intracerebrally. The patterns of intra- and extracellular immunoreactivity to different PrP antibodies in brain and LRS tissues and the immunoblotting characteristics of PrP(res) from brain samples remained constant, irrespective of the route of inoculation and the PrP genotype, and were the same as described previously for ARQ/ARQ sheep dosed orally with BSE. These results suggest that the intracellular truncation of BSE PrP(d) and the proteinase K cleavage site of BSE PrP(res) are not altered by PrP genotype or by route of inoculation and that, therefore, screening tests based on these properties can be applied to identify potential sheep BSE cases occurring naturally.

Modulation of Serotonergic Receptor Signaling and Cross-talk by Prion Protein*

The inducible serotonergic 1C115-HT cell line expresses a defined set of serotonergic receptors of the 5-HT2B, 5-HT1B/D, and 5-HT2A subtypes, which sustain a regulation of serotonergic associated functions through G-protein-dependent signaling. 1C115-HT cells have been instrumental to assign a signaling function to the cellular prion protein PrPC. Here, we establish that antibody-mediated ligation of PrPC concomitant to agonist stimulation of 5-HT receptors modulates the couplings of all three serotonergic receptors present on 1C115-HT cells. Specific impacts of PrP antibodies were monitored depending on the receptor and pathway considered. PrPC ligation selectively cancels the 5-HT2A-PLC response, decreases the 5-HT1B/D negative coupling to adenylate cyclase, and potentiates the 5-HT2B-PLA2 coupling. As a result, PrPC ligation disturbs the functional interactions occurring between the signaling pathways of the three receptor subtypes. In 1C115-HT cells, antagonizing cross-talks arising from 5-HT2B and 5-HT2A receptors control the 5-HT1B/D function. PrPC ligation reinforces the negative regulation exerted by 5-HT2B on 5-HT1B/D receptors. On the other hand it abrogates the blocking action of 5-HT2A on the regulatory loop linking 5-HT1B/D receptors. We propose that the ligation of PrPC affects the potency or dynamics of G-protein activation by agonist-bound serotonergic receptors. Finally, the PrPC-dependent modulation of 5-HT receptor couplings is restricted to 1C115-HT cells expressing a complete serotonergic phenotype. It critically involves a PrPC-caveolin platform implemented on the neurites of 1C115-HT cells during differentiation. Our findings define PrPC as a modulator of 5-HT receptor coupling to G-proteins and thereby as a protagonist contributing to the homeostasis of serotonergic neurons. They provide a foundation for uncovering the impact of prion infection on serotonergic functions.

Distinct profiles of PrP(d) immunoreactivity in the brain of scrapie- and BSE-infected sheep: implications for differential cell targeting and PrP processing.

Previous studies have shown that the patterns of disease-specific prion protein (PrP(d)) accumulation in the brain (the 'PrP(d) profile') of scrapie-affected sheep are mainly influenced by the source of scrapie agent. We have now extended those studies to investigate the effect of different PrP antibodies on the PrP(d) profile of scrapie- and bovine spongiform encephalopathy (BSE)-affected sheep. Immunohistochemical examination of brains of 20 sheep was performed with four different PrP antibodies (P4, 521.7, 505.2 and R486), and the animals were allocated to four groups of five sheep each depending on the transmissible spongiform encephalopathy (TSE) agent source (two natural scrapie sources, SSBP/1 and BSE). Although the PrP(d) profiles depended on the antibody used, the four TSE sources could always be differentiated. Natural Suffolk scrapie showed the highest levels of glia-associated PrP(d), natural Welsh Mountain scrapie uniquely had consistent vascular PrP(d) plaques, SSBP/1 produced the highest intracellular accumulations of PrP(d) and BSE led to moderate accumulation of all PrP(d) patterns except for vascular plaques. The variations in PrP(d) profile between TSE sources appeared to be the result of variations in cell tropism and in PrP processing. These processing differences are possibly associated with changes in PrP(d) conformation, and are manifest as differences in intracellular truncation and in release to the extracellular space of the abnormal protein. Moreover, variations in PrP(d) conformation would appear to be also influenced by the cell type supporting infection, arguing that it is modulated by the interaction between the infectious agent and the host.

G1-dependent prion protein expression in human glioblastoma cell line T98G.

Human glioblastoma cell line T98G produced a cellular form of prion protein (PrP(C)), and we confirmed expression of PrP mRNA by RT-PCR. Immunoblot analysis of whole cell lysate revealed one major (35 kDa) and two faint bands (31, 25 kDa) that reacted with monoclonal anti-human PrP antibody 3F4. Cells treated with tunicamycin produced only a 25 kDa band, representing a deglycosylated form of PrP. Similarly, peptide: N-glycosidase F treatment of whole cell lysate altered the Asn-linked form to the deglycosylated form. When T98G cells were cultured for a longer period, the amount of PrP(C) per cell increased on Day 4 to 16 in a time-dependent manner. When the cells were cultured at high cell-density, the cells on Day 4 produced the same amount of PrP(C) as those on Day 16 of the usual culture. Moreover, in a serum-free medium, cells cultured at a low cell-density produced the same amount of PrP(C) as those cultured at the high cell-density. These results demonstrate that PrP(C) production in T98G cells was dependent on the phase of the cell cycle, probably the G1 phase.

A randomised, double-blind, controlled trial of the immunogenicity and tolerability of a meningococcal group C conjugate vaccine in young British infants

A double-blind, randomised, controlled trial was conducted in 248 British infants to assess the immunogenicity and tolerability of three doses of a meningococcal group C/CRM 197 conjugate vaccine (Lederle Laboratories, USA) given at 2, 3 and 4 months. Control children received three doses of Hepatitis B vaccine (Engerix B®; SmithKline Beecham). At 5 months of age, 100% of children receiving the conjugate vaccine had specific immunoglobulin G concentrations >2.0 μg/ml ( n=116) compared with only 4% of control children ( n=121). Those receiving the conjugate also had 2.5- and 1.6-fold higher geometric mean concentrations of PRP and diphtheria antibodies, respectively. The vaccine was well tolerated.