Case Study MICR 420 Emerging and Re-Emerging Infectious Diseases S2010

1. Case StudyMICR 420 Emerging and Re-Emerging Infectious DiseasesS2010 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 Borreliaburgdorferi and Rocky Mountain Spotted Fever were negative • Cultures were normal, and he was negative for venereal disease

4. Key information pointing to diagnosisPart 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 subacutesclerosingpanencephalitis, 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 Source: CDC

6. Normal conformer Rogue conformer http://www.cmpharm.ucsf.edu/cohen/pages/welcome.html

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 • The abnormal prion form PrPsccauses the conformation change of normal prionsPrPc 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 • PrPC 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 • PrPsc 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 PrPc upon exposure to its abnormal isoformPrPsc converts to the abnormal form, which aggregates and accumulates in the brain causing degeneration and amyloid buildup • How PrPsccauses the conformation, invade the CNS, and causes degeneration is not well understood • Scrapie-infected transgenic mice with a soluble PrPc did not develop prion disease • Neither PrPc nor PrPsc are very immunogenic in the species normally used to prepare antibodies

9. Primary Research Article Contributing to the Understanding of this Disease • ReinaldPamlona, Alba Naudi, et. al, 2008, Increased oxidation, glycoxidation, and lipoxidation of brain proteins in prion disease, Free Radical Biology & Medicine, 45: 1159-1166 • PrPsc may be directly involved in the pathogenesis of because it is temporally and anatomically correlated with the neuropathology • However, PrPsc may be involved in more complex effects involving other molecules • Neurodegeneration may result as a consequence of loss of function of PrPc or a toxic gain of function from PrPsc • 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 scrapieprions • Gas chromatography-mass spectrometry was used to analyze protein oxidation products and fatty acids • SDS-PAGE was used to determine ERK1/2 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 ERK1/2 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 ERK1/2 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 PrPscfollowed by immunodetection • Protein-misfolding cyclic amplification (PCMA): works similarly like PCR, it allows conversion of PrPc to PrPsc • 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 PrPsc and lower affinity to PrPc 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%) Source: CDC • 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

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 PrPsc. • 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 PrPsc because they are protease resistant and similar to PrPc. Hence it is hard to know the pathology of prions disease. • Various strains of prions cause different morphology in the brain.

16. References • http://health.nih.gov/topic/CreutzfeldtJakobDisease • http://www.cdc.gov/ncidod/dvrd/vcjd/index.htm • http://web.uct.ac.za/depts/mmi/jmoodie/neurol2.html#Retrovirus 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, 106-120 • Maurizio Polano, AlpanBek, Federico Benetti, Marco Lazzarino, and Giuseppe Legname. (2009) Structural Insights into Alternate Aggregated Prion Protein Forms. J. Mol. Biol., 393, 1033-1042 • Nuvolone, M., Aguzzi A., and Heikenwalder, M. (2009) Cells and prions: A license to replicate. FEBS Letters, 583, 2674-2684 • 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, 1159-1166 • Willey, Joanne et al. 2008. Prescott, Harley, & Klein’s Microbiology, 7th ed. McGraw Hill, NY