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Case # 68 Shan Kuang Julian Lopez Maria De Leon. Case Summary Patient History: 54 year old retired US Air Force Pilot Spent several years in Guam and.

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Presentation on theme: "Case # 68 Shan Kuang Julian Lopez Maria De Leon. Case Summary Patient History: 54 year old retired US Air Force Pilot Spent several years in Guam and."— Presentation transcript:

1 Case # 68 Shan Kuang Julian Lopez Maria De Leon

2 Case Summary Patient History: 54 year old retired US Air Force Pilot Spent several years in Guam and Vietnam, and worked for 23 years as a pilot His eating habits were conventional, and he had no history of alcohol use He has never been transfused, had no history of tick bites, and had no known risk factors for HW The patient’s problem: Chronic neurologic disorder Initial symptoms: forgetfulness, subtle behavioral changes, headaches, and fatigue Later symptoms: disequilibrium, wide-based gait, double vision He died 6 months after the onset of symptoms

3 Key information pointing to diagnosis Part 1 The disease may not have a bacterial cause: He was never febrile, indicating he had no inflammatory immune response Treatment with antibiotics (penicillin and tetracycline) against lyme disease and Rocky Mountain Spotted Fever were ineffective Serology test for Borrelia burgdorferi and Rocky Mountain Spotted Fever were negative Cultures were normal, and he was negative for venereal disease

4 Key information pointing to diagnosis Part 2 Clues to the disease may be found in the neurological tests: Computed tomogram scan revealed atrophy in the cerebrum and cerebellum, which correlate with his behavioral, mental, and physical changes He had no mass lesions, ruling out tumors and stroke Tests were done on his cerebrospinal fluid (CSF) Some viruses can cause neurological disorders HIV with dementia, Measles/rubeola with subacute sclerosing panencephalitis, JC virus with progressive multifocal leukoencephalopathy (myelin degradation) The results of the tests were normal, and he was HIV negative No biopsy was done, but an autopsy showed extensive spongiform changes in the cerebrum, cerebellum, and basal ganglia

5 Diagnosis Sporadic occurrence of classic Creutzfeldt-Jakob Disease due to spontaneous transformation of normal prion proteins to abnormal prions Clinical and Pathologic Characteristics Distinguishing Classic CJD from Variant CJD CharacteristicClassic CJDVariant CJD Median age at death68 years28 years Median duration of illness4-5 months13-14 months Clinical signs and symptomsDementia; early neurologic signsProminent psychiatric/behavioral symptoms; painful dyesthesiasis; delayed neurologic signs Periodic sharp waves on electroencephalogram Often presentOften absent "Pulvinar sign" on MRI*Not reportedPresent in >75% of cases Presence of "florid plaques" on neuropathology Rare or absentPresent in large numbers Immunohitochemical analysis of brain tissue Variable accumulationMarked accumulation of protease-resistance prion protein Presence of agent in lymphoid tissueNot readily detectedReadily detected Increased glycoform ratio on immunoblot analysis of protease-resistance prion protein Not reportedMarked accumulation of protease-resistance prion protein Source: Adapted from Belay E., Schonberger L. Variant Creutzfeldt-Jakob Disease and Bovine Spongiform Encephalopathy. Clin Lab Med 2002;22: *An abnormal signal in the posterior thalami on T2- and diffusion-weighted images and fluid-attenuated inversion recovery sequences on brain magnetic resonance imaging (MRI); in the appropriate clinical context, this signal is highly specific for vCJD Source: CDC

6 Normal conformerRogue conformer

7 Microbiology of Prions Prion diseases (transmissible spongiform encephalopathies) are infectious and cause fatal neurodegenerative disorders in humans and animals The disease is caused by proteinaceous infectious particles called prions sc The abnormal prion form PrP sc causes the conformation change of normal prions PrP c in the host to the abnormal form The diseases can occur sporadically, through familial transmission, or horizontal transmission Prion diseases: Scrapie—sheep, goats Bovine spongiform encephalopathy—cattle Transmissable mink encephalopathy—mink Creutzfeldt—jakob disease—man, sporadic, familial, transmission; Gerstmann-Streussler disease—man, familial Fatal familial insomnia—man, familial Kuru—man, Fore people of New Guinea Chronic Wastings Disease—Elk, Deer

8 Pathogenesis of Prions Disease PrP C is a glycosylphosphatidylinositol-anchored glycoprotein present in all cell types It has an unstructured N-terminal portion and a mostly α -helical folded C- terminus It is expressed in all cell types, and is highly conserved among mammals Its function is not well known PrP sc consists of various strains with various incubation periods and lesion profiles The C-terminal is folded into a tightly packed highly protease resistant domain enriched in β -structure It is resistant to heat, ultra-violet light, and formaldehyde The secondary lymphoid organs play a major role in replication Normal PrP c upon exposure to its abnormal isoform PrP sc converts to the abnormal form, which aggregates and accumulates in the brain causing degeneration and amyloid buildup How PrP sc causes the conformation, invade the CNS, and causes degeneration is not well understood Scrapie-infected transgenic mice with a soluble PrP c did not develop prion disease Neither PrP c nor PrP sc are very immunogenic in the species normally used to prepare antibodies

9 Primary Research Article Contributing to the Understanding of this Disease Reinald Pamlona, Alba Naudi, et. al, 2008, Increased oxidation, glycoxidation, and lipoxidation of brain proteins in prion disease, Free Radical Biology & Medicine, 45: PrP sc may be directly involved in the pathogenesis of because it is temporally and anatomically correlated with the neuropathology However, PrP sc may be involved in more complex effects involving other molecules Neurodegeneration may result as a consequence of loss of function of PrP c or a toxic gain of function from PrP sc The study aims to see whether oxidative stress is a mechanism involved with neurodegeneration in prions disease Experimental Setup: Brains of 6 patients with sporadic CJD, age matched Brains of syrian hamsters inoculated with 263K scrapie prions Gas chromatography-mass spectrometry was used to analyze protein oxidation products and fatty acids SDS-PAGE was used to determine ERK 1/2 and p38 activation

10 Results Fig 1. Protein oxidation products are higher in brains affected by prion disease in humans. Similar results were shown in scrapie- affected Syrian hamsters. There is a positive correlation with GSA and MDAL and CML, and MDAL and CML (figure not shown). Fig 3. Modest yet significant changes in fatty acid composition is associated with prion disease in humans. Lower intensity changes were found in scrapie-infected hamsters Fig 5. There is significant negative correlation between protein oxidation product GSA and poly unsaturated fatty acids in the human brain. Similar correlations were found in PUFA and MDAL and PUFA and CML. Fig 6. There is increased phosphorylation of ERK 1/2 and p38 in scrapie-affected hamster brains.

11 Conclusion The amount of specific chemically defined oxidation, lipoxidtion, and glycoxidation products are higher in brains from both CJD affected humans and scrapie-infected hamsters The slight changes in fatty acids may be due to a homeostatic response to reduce oxidative damage, a loss of function in homeostatic systems, or a higher rate of lipid damage that cannot be fixed The correlation between different oxidation products suggest an elevated underlying oxidative stress that acts on the different possible oxidative routes and different available oxidizable substrates The negative correlation of oxidation products and PUFA suggests a wider association of fatty acid composition and protein oxidative damage Some studies suggest that oxidative modifications lead to loss of protein function, while other studies suggest that oxidative stress induces a cell signaling cascade that increases reactive oxygen species (ROS) and causes over activation of routes that lead to neurodegeneration The increased phosphorylation of ERK 1/2 and P38 suggests the latter, but more needs to be examined The results suggest that patients with compromised antioxidant defenses may have accelerated clinical progression of prions disease

12 Diagnostic Tests for Prion Disease Currently there is no single diagnostic test for CJD Neurological examinations, standard diagnostic tests, electroencephalogram, and magnetic resonance imaging are used to rule out treatable forms of dementia A biopsy or autopsy is used to confirm diagnosis of CJD Biopsy poses some risk to the surgeon and patient, and the results are dependent on the tissue obtained being affected Laboratory testing: Bioassays and cell assays: the subject is injected with the test sample Conformation-dependent immunoassay: selective precipitation of PrP sc followed by immunodetection Protein-misfolding cyclic amplification (PCMA): works similarly like PCR, it allows conversion of PrP c to PrP sc Western blotting: antibodies are added after treatment with protease Immunohistochemistry: look for characteristic deposition patterns Paraffin embedded tissue (PET): technique that combines WB and IHC New techniques are being developed Amyloid seeding assay (ASA): prions are capable to seed amyloid formation Monoclonal antibodies 1.5D7 and 1.6F4 have higher affinity to PrP sc and lower affinity to PrP c than current antibodies being used

13 Prevention of disease 85% cases of classical CJD is sporadic, 5-10% is genetic Prevention in a hospital setting: Wash hands and exposed skin, and cover cuts and wounds Wear surgical gloves and facial protection Soak instruments in undiluted chlorine bleach, use autoclave to sterilize Be careful of transmission modes Vertical transmission Surgery: dura mater grafts, exposure to brain tissue and CSF, and transplanted corneas, implantation of unsterilized electrodes in brain Contaminated human growth hormones and blood (people who reside in a country that is known for BSE for more than 3 months cannot donate blood) Prevention of BSE and vCJD The FDA has prohibited the slaughter of downer-cows (non-ambulatory) Beef is recalled from cattle slaughtered in plants confirmed with BSE positive cows In 2009 the FAD and CIFA (Canada) have enhanced a feed-ban on animal feeds, pet foods, and fertilizers

14 Epidemiology and Threats Approximately 1 in 1 million people are affected with CJD worldwide; recently there are fewer than 300 cases of CJD per year in the U.S. Most of classical CJD cases are sporadic due to spontaneous mutation of the prion protein Some cases were acquired through transmission (fewer than 1%) The first case of BSE was in the 1970s in the UK Cattle was fed meat-and-bone meal contaminated with BSE-infected products In 2008 there were more than 484,500 cases of BSE in the UK As of 2010 there were 21 cases of BSE in North America vCJD is hypothesized to occur due to ingestion of BSE prion infected meat The first case occurred in the UK in 1996 In 2006 there were 200 cases worldwide, 164 in the UK and 3 in the U.S. There are no treatments for prion disease, only drugs to alleviate symptoms Opiates, clonazepam and sodium valproate to relieve myoclonus Source: CDC

15 Take home message Sporadic CJD involves a chronic neurologic disorder with initial symptoms of forgetfulness, subtle behavioral changes, headaches, and fatigue. Later symptoms include disequilibrium, wide-based gait, and double vision. It is a terminal disease. The main theory is that classic CJD is caused by abnormal prions that convert the normal host prions to its abnormal isotype which accumulates in the brain and causes neurodegeneration. The main diagnostic tests for prions are PCMA, IHC, and WB. But these techniques are limited because the antibodies used are not specific for PrP sc. There is a lack of treatment in this disease. Banning contaminated food prevents vCJD and careful hospital procedures prevents iatrogenic transmission. However most of the disease is acquired genetically or sporadically. There are limitations in testing and knowing the structure of PrP sc because they are protease resistant and similar to PrP c. Hence it is hard to know the pathology of prions disease. Various strains of prions cause different morphology in the brain.

16 References Disease Cordes, H., Bergstrom, A.L., Ohm, J., Laursen, H., and Heegaard, P.M. (2008) Characterisation of new monoclonal antibodies reacting with prions from both human and animal brain tissues. Journal of Immunology Methods, 337, Maurizio Polano, Alpan Bek, Federico Benetti, Marco Lazzarino, and Giuseppe Legname. (2009) Structural Insights into Alternate Aggregated Prion Protein Forms. J. Mol. Biol., 393, Nuvolone, M., Aguzzi A., and Heikenwalder, M. (2009) Cells and prions: A license to replicate. FEBS Letters, 583, Pamplona, R., Naudi A., Gavin,R., Pastrana, M.A., Sajnani, G., Ilieva, E.V., Antonio del Rio, J., Portero-Otin,M., Ferrer, I., and Requena, J.R. (2008) Increased oxidation, glycoxidation, and lipoxidation of brain proteins in prion disease. Free Radical Biology & Medicine, 45, Willey, Joanne et al Prescott, Harley, & Klein’s Microbiology, 7 th ed. McGraw Hill, NY


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