Protection Mechanism against Prion Infection through Inhalation - Assignment Example

APPLICATION FOR A PROJECT GRANT Details of applicant: Sur Fore s Position Q2 and address of employing Q3 Type of grantrequested: Research Q4 Period for which support is sought: 24 months Q5 Proposed Start Date: 02/05/2014 Q6 Title of project: Depletion of receptor activator of NF-κB (RANKL) as a possible protection mechanism against prion infection through inhalation. Q7 SUMMARIES OF PROPOSED RESEARCH (both parts combined should be no more than 400 words). (a) For scientifically qualified assessors: Prion infections and their pathogenesis are dependent much on the agents crossing the epithelial coverings of the body cavities through to the recipient nervous system. Classified among the most fatal neurodegenerative disorders, prion infections are not only self-propagating, but are transmissible through a PrPSc, a misfolded conformation of the normal prion protein [PrPC] (Makarava, et al., 2010). Prion infections enter the hosts via several routes; the nasal cavity being among the most recent route demonstrated experimentally in hamsters, mice, and sheep (Bessen, et al., 2009). The infections are, however, not considered airborne, but are characterized by agent replication in lymphoreticular system (LRS) right before their entry into the central nervous system [CNS]. Notably, the microfold cells (M cells) within the follicle-associated epithelium (FAE), which is the main medium of transport for the infectious prion agents into the intestinal epithelium lies at the very heart of the study. The mechanism through which prions are picked up by respiratory M cells found both in the upper and lower respiratory tracts, into CNS is, however, uncertain; the ability of pulmonary M cells to transport prions across the respiratory epithelium has not been established. Accordingly, the study proposes the administration of RANKL (member of the tumor necrosis factor (TNF) in the Peyer’s Patches to initiate the process of M cell-differentiation, in effect, blocking neuroinvasion by the prion infectious agents. These experiments suggest the hypothesis that depleting M cells located in the follicle-associated epithelium (FAE) that overlies the NALT and other respiratory epitheliums by RANKL neutralization to prevent infection. (b) For readers who are not scientifically qualified: Prion infections are caused by a misfolding shape of cellular prion proteins PrPc to infectious PrPsc . The aggregation of PrPsc forms an amyloid plaque within the CNS, causing spongy architecture (holes) of the brain. Prion proteins are infectious, and can infect one animal to the next. It has long been believed that that prion diseases are acquired mainly through the oral route [ingestion]. Recent experimental evidence, however, points to possible infection through the nasal cavity, thus, informing the suggestion of infection through inhalation. Additional studies have also found M cells in the gut, giving clear indication for prion uptake by the oral route. The existence of M cells in the respiratory tracts suggests the ability to neutralize prions uptake across respiratory epitheliums, hence, the proposed project to confirm the same. Q8 DETAILS OF RESEARCH PROJECT (a) Aims: I. To identify how M cells located in the respiratory epithelium and the transcytosis of prion agents across the FAE in vivo, can be depleted through RANKL [receptor activator of NF-κB] neutralization before disease exposure. II. To determine the effect of M cell-depletion on the uptake of prions into the respiratory tract and disease susceptibly. Hypothesis: Depletion of RANKL decreases pulmonary M cell differentiation and protects against prion infection by the inhaled route. (b) Background: What causes prion disease? Prion disease represents a group of conditions that affect the nervous system in humans and animals. In people, these conditions impair brain function, causing changes in memory, personality, and behavior; a decline in intellectual function (dementia); and abnormal movements, particularly difficulty with coordinating movements (ataxia). The signs and symptoms of prion disease typically begin in adulthood and worsen with time, leading to death within a few months to several years. Prion diseases, otherwise known as Transmissible spongiform encephalopathies (TSEs) represent a group of neurodegenerative disorders affecting both animals and humans. The diseases are associated with the build of misfolded/abnormal or ‘rogue’ form of a naturally occurring cellular protein, prion protein, designated PrPc. The abnormal protein results from the abnormalization of normal prion protein; once in the body system, the abnormal prion proteins initiates he recruitment and subsequent conversion of of more normal prion proteins into abnormal shapes/forms, setting a a kind of precedence, leading to accumulation of the former in the system. The extracellular aggregation of the abnormal prion proteins within the CNS forms amyloid plaques, ultimately damaging the brain tissues, thus, the spongy architecture in Figure 1. Figure 1: This is a micrograph of the damaged brain tissues indicating the cytoarchitectural histopathologic changes found in bovine spongiform encephalopathy. The presence of vacuoles [i.e. microscopic “holes”] in the gray matter, gives the brain of BSE-affected cows a sponge-like appearances when examined in the laboratory (Wikipedia, 2003) Under normal circumstances, prion proteins are broken down by enzymes in the body once their purpose is served.  The abnormal prion proteins, however, cannot be broken down, thus, the reason behind their accumulation, subsequently causing damage to the brain tissues, in effect, interfering with normal functioning of the brain cells. The replication of the proon protein within the body system is descriptively a clinical silent phase. Though abnormal prion diseases can be transmitted horizontally between animals, the source of disease causing prion agents spreading the TSEs in free-ranging animals is not known (Miller, 2004). Prion diseases are rare in human, but are evidently increasing in animals, with chronic wasting disease (CWD) reported in a range of states in the United States, Canadians provinces as well as in the South Korea (Kim, et al., 2005; Sohn et al., 2002). The massive spread of CWD has called into attention of a possible spread beyond the limits of species barriers, for even the function of the normal prion protein remains largely unclear. Additionally, the infectious agents are generally stable and are infectious even after many years (Johnson, et al., 2006) The routes of transmission The oral route has long been established in the spread of prion diseases; consumption of foods contaminated with infectious prions is but one cause of infection. An example is the outbreak of the mad cow diseases in the United Kingdom in the recent past, the discovery of the variant Creutzfeldt–Jakob diseases (vCJD) in humans, and the transmissible milk encephalopathy (TME) found in ranch-raised mink in Canada, Finland, German and parts of the former USSR (Marsh and Hadlow, 1992). While the oral route of transmission of disease in animals, other route of prion may exist. Recent evidence adduced from controlled experiments with immunodeficient mice indicates that the diseases can be transmitted through air (Haybaeck et al., 2011). To be sure, Nathaniel D. Denkers (2013) notes that the inoculation of TME and CWD prions via the nasal passages in hamsters and mice has produced a transmission rates ranging between 10 to 100 times more efficient than oral route. Irrespective of the route of infection, whether through the mucosal contact in the gut or the nasal cavity, the infectious agents of prion diseases must be transported across the epithelial covering of the body cavity to gain entry into the body nervous system, where it replicates by recruiting normal prion proteins into disease causing prion proteins (Anthony and Kincaid, 2012) M cells: Portals for prions across the intestinal epithelium and respiratory epithelium M cells are located in the follicle related epithelium of the Peyer’s patch and the Bronchus-related lymphoid tissues such as the nasopharynx-associated lymphoid tissue (NALT) (Kiyono and Fukuyama, 2004). The depletion of these cells in the Peyer’s patches by RANKL neutralization (Figure 2) suggests that the M cells are the important sites of prion uptake from the gut lumen into the body system. (D S Donaldson, 2012). Figure 2: The number of M cells in the FAE was reduced significantly after anti-RANKL [mAb] treatment (P=0.0079). Data are representative of 3–5 Peyers patches from each of four control and four anti-RANKL mAb-treated mice (Donaldson, 2012). The successful transmission of disease causing prion proteins by aerosols, demonstrated in the study by Haybaeck, et al. (2011), supports the idea of nasal mucosal transmission mechanism. As alluded to in the preliminary analysis, the ability of M cells to transport prion causing diseases across the respiratory epithelium has, however, not been established. The location of the M cells within the surrounding of the respiratory tract [M and NALT M cells in the upper respiratory tract and M cells in the lower respiratory tract] (Kim et al., 2011), as shown in Figure 3, gives indications of a possible route, in effect calling for an investigative study for a possible remedy. Fig.3 Fig.3 shows microscopic analysis of respiratory M cells. Images A and B, shows that the M cells (B, arrow) in the nasal epithelium are distinct from the adjacent respiratory epithelial cells by their depressed and relatively darker brush borders. A is an enlargement of the image in shown in B. C–E shows the TEM respiratory M cells, which are comparatively shorter and more irregular microvilli with definite UEA-1+ signals (D), unlike the cilia of neighboring epithelial cells (Kim et al., 2011) These experiments suggest the very possibility of testing the hypothesis that M cells located in the follicle-associated epithelium (FAE) and those overlying the other respiratory epitheliums could be the basis of the respiratory prion disease infection. To be certain, prions transmission by air is a novel route highlighting the previously unrecognized risk factor for industrial workers such as those working mostly in the meat processing industry. The inference that aerosolization might facilitate the horizontal transmission of prions in free ranging animals only adds the very importance of having solid evidence on the same. The characterization of pulmonary M cells in prion uptake in other epithelial surfaces accelerates our understanding of prion infectious diseases, and that the respiratory route should thus be exception. Their manipulation may improve the efficacy of mucosal vaccines or help develop strategies to block transmission of prion infections. (C) Experimental Design and Methods: The proposed experiment involves treatments with anti-RANKL nAb, nonspecific rat mAb and a control vehicle to 1 group of 4 mice for checking the number of M cells; 3 groups of 12 mice: (i) wildtype (ii) non-infected brain homogenate (iii) infected brain homogenate. These sample sizes have been shown to yield statistically significant data using power analysis at 95%. Mice: C57BL/6 mice (6–8 weeks old) will be used and will be maintained under specific pathogen free (SPF) conditions throughout the project. The license to use the animals mentioned will sought and carried out under the ‘Animals (scientific procedures) Act 1986 and authority of a UK Home Office respectively; the mice in use will be humanely culled in accordance with the UK Home Office Schedule One method. Treatment with anti-RANKL mAb: To neutralize the activity of RANKL in vivo, mice will be intraperitoneally injected with 250μg of the IK22-5 rat anti-mouse RANKL mAb every 2 days for 8 days. A similar number of mice will be injected with an isotype-matched nonspecific rat mAb as a control (control Ig). M cells staining: Using the hard palate as a guide, we will use a large scalpel to remove the snout with a transverse cut behind the back molars. After removing the skin and any excess soft tissue, we will flush the external nares with PBS to wash out any blood within the nasal cavity before snap-freeze at the temperature of liquid nitrogen. For whole-mount staining, BALT, NALT and respiratory epithelium will be dissected from respiratory tract and fixed with BD Cytofix/Cytoperm. The tissues will subsequently be immunostained with rat anti-mouse GP2 mAb (GP2+) and subsequently stained with Alexa Fluor 488-conjugated anti-rat IgG Ab (green), rhodamine-conjugated UEA-1 (red), and Alexa Fluor 647-conjugated phalloidin (blue). Prion exposure: The inhalation chamber will be fitted with a nebulizer device to produce prion aerosols with brain homogenates at concentrations of 20%, driven into the chamber harboring terminally scrapie or healthy mice. The nebulizer will run with a pressure of 1.5 bar, generating 100% particles below 10 μm with 60% of the particles below 2.5 μm and 52% below 1.2 μm; such particle sizes are believed to be able to penetrate through to both the upper and lower airways (Haybaeck et al., 2011). 6 groups of mice (C57BL/6, n=10) will be exposed to prion aerosols from infectious or healthy brain homogenates (IBH and HBH) at 20% concentration of 10 minutes. RML6 (LD50 scrapie prions) from the infectious CD1 mice will be used throughout this experiment. Immunohistochemistry and immunofluorescent analyses: To establish the locations and boundaries of the various types of epithelia found within the nasal cavity, including the location of M cells, tissue sections from untreated animals or from animals exposed to IBH or HBH aerosols will be cleared, dehydrated, and stained with periodic acid-Schiff stain (PAS) or hematoxylin and eosin (H&E). IHC has been performed to detect PrPd as reported previously (Kincaid and Bartz, 2007); in the study, tissue sections were deparaffinized and were subjected to antigen retrieval in formic acid (95% concentration) for 10 min at room temperature. All subsequent steps were carried out at room temperature; incubations were separated by 3 rinses with 0.05% (vol/vol) Tween in Tris-buffered saline (TTBS). Endogenous peroxidase and nonspecific antibody binding were inhibited by incubating the tissue sections in 0.3% H2O2-methanol for 20 min, followed by incubation in 10% normal horse serum in TTBS for 30 min (Anthony and Kincaid, 2012). The project intends to follow the procedure where necessary. For the detection of disease specific prion (PrPd) in NALT, BALT, spleen and brain, tissues will be fixed in periodate-lysine-paraformaldehyde fixative and embedded in paraffin wax. PET immunoblot analysis will be used to confirm the PrPd detected by immunohistochemistry, which is PK-resistant PrPSc (blue/back). As noted in Donaldson (2012). Cellular PrPc can be detected by using the PrP-specific antibody (pAb), as such, astrocytes expressing glial fibrillary acidic protein (GFAP) (brown, fourth column), and active microglia expressing Iba-1 (brown, right-hand column) will be utilized to detect the brains of all clinically scrapie-affected control mice. The prion proteins will be visualized under the light microscope using the avidin-biotin complex. For the fluorescent microscopy, following the addition of primary antibody, streptavidin-conjugated or species-specific secondary antibodies together with Alexa Fluor 488 (green), Alexa Fluor 594 (red), or Alexa Fluor 647 (blue) dyes (Invitrogen) will be used. Diseases monitoring: Following exposure, mice will be coded and assessed for the signs of clinical prion disease and culled at a standard end point. Incubation period will be recorded for mice that do not develop clinical signs. Clinical signs will be observed by behavioral tests: water maze and rotarod, 54 mice (n=6) will be tested every 2 months until the end of the experiment scheduled to last 300 days. Brain tissues will be collected from clinically scrapie-affected mice and mice free of the clinical signs of prion disease at the end of the experiment, and the neuropathology within each brain tissues collected compared with reference to the condition discussed herein. Scapie diagnosis will be confirmed by hisopathological assessment of spongy vaculation in the brain tissues. 5 anatomic brain regions (hippocampus, cerebellum, olfactory bulb, frontal white matter, temporal white matter) will be selected for all investigations as the per experimental group (n=8). The tissues will be evaluated on a scale of 0-4 (not detectable, mild, moderate, severe and status spongiosus). The Investigator will be blinded to animal identification performed on histological analyses. Statistic evaluation: Results will be expressed as mean + standard error of the mean (SEM) or standard deviation (SD). Statistical significance between experiment groups will be assessed using an unpaired 2-sample student’s t-Test (G3* power), and accepted when p Read More