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  • 1. Structure• Recent Research Projects: – UV or cis-Urocanic Acid Pre-exposure Increases the Severity of Mycobacterium ulcerans Infection in a Hairless Guinea Pig Model of Buruli Ulcer Disease; – An Equine Model of Neurolathyrism and Safety Assessment of Low Toxin Grass Pea (Lathyrus sativus); – Some Clinical Toxicology Fun (if there is time);• Other Research Interests. 1
  • 2. UV or cis-Urocanic Acid Pre-exposure Increases The Severity Of Mycobacterium ulcerans Infection In A Hairless Guinea Pig Model Of Buruli Ulcer Disease Cope R.B.1, P. Small2 1Departmentof Environmental and Molecular Toxicology Oregon State University 2Department of Microbiology, University of Tennessee Funding Acknowledgement:US National Institute of Allergy and Infectious Disease (NIH) 2
  • 3. Features of Buruli Ulcer Disease (Bairnsdale Ulcer, Daintree Ulcer)• Non-HIV associated, emergent, chronic, high morbidity, skin disease caused by Mycobacterium ulcerans infection;• A single Buruli ulcer can cover up to 15% of the body surface;• 92% of ulcers are on the limbs;• Antibiotic treatment in human cases is often ineffective except in the early nodular stages of the disease (rifampin, rifapentine and clarithromycin);• Mainstay of treatment is surgical removal at the nodular stage.
  • 4. Features of Buruli Ulcer Disease• Transmission: – The disease is associated with aquatic and swampy environments with the mycobacterium occurring in biofilms, soil, aquatic insects, fish and wildlife; – Acanthamoeba sp. appear to be the host in the environment; – M. ulcerans has been detected in mosquitoes and water bugs; – Epidemiology in West Africa favors insect-mediated transmission;
  • 5. Features of Buruli Ulcer Disease• Transmission: HOWEVER: – Case studies have demonstrated Buruli ulcers developing after wood splinter  gunshot/land-mine wounds and after exposure of minor skin wounds to swamp water/biofilms; – Key factor appears to be any form of skin break that allows penetration of Acanthamoeba sp. into the dermis.
  • 6. Features of Buruli Ulcer Disease• Optimal treatment is surgical excision and skin grafting but results are often poor – The rate of limb amputation, is about 36% of cases • 100% amputation rate if osteomyelitis is present – With optimal surgical treatment, approximately 60% of patients will have significant permanent reduction in motion of one or more joints • 63-76% reduction of motion if knees, elbows or wrists are involved • Marked restriction is the most common long-term outcome when ulceration of the hand is present
  • 7. Features of Buruli Ulcer Disease• Surgical outcomes: – Significant cosmetic deformity is the usual outcome; notably worse when the lesion is located on the head – Recurrence following surgical excision and multiple new ulcers possibly due to surgery-induced spread are common – Average number of operations per patient is 2.4 with a range of 2-5 – Average period of hospitalization is > 6 months
  • 8. Ulcerative form of M. ulcerans infection
  • 9. Sequelae
  • 10. Pathogenesis InfectionNon-ulcerative Forms Nodule Necrotic Phase (Ulceration, Local Immunosuppression, Impaired T cell Homing)Plaque/edematous forms Several months to years? Ulcer? Reactive Phase (+ve DTH Response) Months to years? Ulcer That Does Not Heal Spontaneous Healing? Stellate Scar
  • 11. KM. George et al (1999), Science, 238: 584
  • 12. Specific Aims• To test the hypothesis that UV-B pre-exposure enhances M. ulcerans infection in a Crl:IAF(HA)-hrBR hairless guinea pig model of human Buruli ulcer disease• To test the hypothesis that topical cis-urocanic acid pre-exposure enhances M. ulcerans infection in a Crl:IAF(HA)-hrBR hairless guinea pig model of human Buruli ulcer disease
  • 13. General Aspects of Experiments• Used Mycobacterium ulcerans MacCallum et al (ATCC 35840) – Virulent mycolactone producing wild-type strain – Prepared from log-phase growth broth cultures – Grown at 32OC
  • 14. Experiment 1Group UV-B Treatment0 kJ Sham irradiated3 kJ 1 kJ/m2/day for 3 days30 kJ 10 kJ/m2/day for 3 days
  • 15. 3.5 3Log Relative Intensity Count 2.5 2 1.5 1 UVR Source 0.5 Solar Light 0 290 300 310 320 330 340 350 360 370 380 390 400 Wavelength (nm) Relative Irradiance of the Source Versus Solar Radiation (Normalized to 1 at 300 nm).
  • 16. Experiment 1 Days 1-3: irradiate 72 hoursS/C infect with 3 x 104 CFU in 0.1 ml PBS in left dorsal (irradiated) flank S/C inject with 0.1 ml PBS in right dorsal (irradiated) flank Measure lesions at 7, 10, 14 and 21 days post-infection Day 21:I/D challenge with 0.02 μg burulin S in 0.1 ml PBS in left flank Sham challenge with I/D 0.1 ml PBS in right flank 24 hours Read DTH responses; kill and collect samples for histopathology
  • 17. Results• 30 kJ/m2 UV-B dose induced erythema/edema of the ear tips, but not the dorsal mid-line;• M. ulcerans contaminated injection sites showed delayed healing. No grossly detectable skin lesions were present at any of the PBS injected sites at 24 hours post-injection;• M. ulcerans infected sites developed distinct, clearly- demarcated, subcutaneously situated skin nodules by day 7 post-infection.
  • 18. Effect of UV-B Pre-Exposure on the Healing of M. ulcerans Contaminated Injection Wounds 1Probability of Wound Survival 0.8 0.6 p = 0.0097 0.4 p = 0.009 0 kJ 0.2 3 kJ 30 kJ 0 0 5 10 15 20 25 Days Post Injection
  • 19. Effect of UV-B Pre-Exposure on the Size of M. ulcerans Induced Skin Nodules 16 0 kJ p < 0.004 14 3 kJ UVDiameter, mm +/- SEM, n = 5 p < 0.009 Mean Maximum Nodule 30 kJ UV p < 0.026 12 p < 0.004 p < 0.021 p < 0.010 10 p < 0.079 p < 0.195 8 6 4 2 0 Day 7 Day 10 Day 14 Day 21 Days Post-Infection
  • 20. Effect of UV-B Pre-Exposure on DTH Responses to Burulin-S at 21 Days Post-Infection 12 p < 0.001 0 kJ 10 p = 0.002 3 kJ Mean Diameter of Induration, 30 kJ mm +/- SEM, n = 5 8 p = 0.035 6 4 2 0
  • 21. Experiment 2Group UV-B Treatment0 kJ Sham irradiated3 kJ 1 kJ/m2/day for 3 days24 kJ 8 kJ/m2/day for 3 days
  • 22. Experiment 2 Days 1-3: irradiate 72 hoursI/D infect with 2 x 106 CFU in 0.1 ml PBS in left dorsal (irradiated) flank I/D inject with 0.1 ml PBS in right dorsal (irradiated) flank Measure lesions at 4, 7, 10, 18, 23, 30 days post-infectionDay 21:I/D challenge with 0.02 μg Burulin S in 0.1 ml PBS in left flank Sham challenge with I/D 0.1 ml PBS in right flank 24 hours Day 22: Read DTH responses Day 30: Kill and collect histology samples
  • 23. Results• 24 kJ/m2 UV-B dose was sub-inflammatory;• M. ulcerans inoculated sites developed distinct, clearly- demarcated skin nodules by day 4 post-infection;• Injection sites did not heal;• All animals able to mount a DTH response to burulin-S @ 21 days post-infection. UV-B pre-exposure had no significant (p > 0.1) effect on DTH responses.
  • 24. Day 15
  • 25. The Effects of UV-B Pre-Exposure on M. ulcerans Ulcer Development Time 20 p = 0.024 18 0 kJ 16 3 kJ Time, Days +/- SEM n = 6 Mean Ulcer Development 24 kJ 14 12 10 8 6 4 2 0
  • 26. Effect of UV-B Pre-Exposure on the Size of M. ulcerans Induced Skin Lesions 25 p ≤ 0.035 p ≤ 0.035 0 kJ p < 0.035 p < 0.035 p ≤ 0.05 3 kJ Mean Lesion (Induration + Ulcer) 20 p = 0.028 p = 0.031 24 kJ Diameter +/- SEM, n = 6 15 p = 0.023 p = 0.338 p = 0.094 10 p = 0.154 5 0 Day 4 Day 7 Day 10 Day 14 Day 18 Day 23 Day 30 Days Post-Infection
  • 27. Protein Catabolism(particularly filaggrin: histidine-richprotein that is a cementing substancefor the tonofibrils in the keratincomplex of the stratum corneum) L-Histidine NH2 UV
  • 28. Experiment 3Group Topical Treatment 1 No treatment 2 Vehicle only 3 0.1 mg trans-urocanic acid 4 0.5 mg trans-urocanic acid 5 1 mg trans-urocanic acid 6 0.1 mg cis-urocanic acid 7 0.5 mg cis-urocanic acid 8 1 mg cis-urocanic acid
  • 29. Experiment 3 Days 1-3: topical treatments 72 hours I/D infect with 1.5 x 107 CFU in 0.1 ml PBS in left dorsal flank I/D inject with 0.1 ml PBS in right dorsal flank Measure lesions at 5, 10, 15, and 21 days post-infectionDay 21:I/D challenge with 2 μg M. ulcerans cell wall fragments in 0.1 ml PBS in left flank. Sham challenge with I/D 0.1 ml PBS in right flank. 24 hours Day 22: Read DTH responses Kill and collect histology samples
  • 30. 2 Product of Lesion Perpendicular Diameters mm Mean +/- SEM; n = 6 50 100 150 200 250 300 0 Negative Control Vehicle Only 0.1 mg Trans P > 0.6 0.5 mg Trans 1 mg TransDay 5 P > 0.001 0.1 mg Cis 0.5 mg Cis P > 0.4 1 mg Cis Negative Control Vehicle Only 0.1 mg Trans P > 0.8 0.5 mg Trans 1 mg TransDay 10 0.1 mg Cis P > 0.001 0.5 mg Cis P > 0.4 1 mg Cis Negative Control Vehicle Only 0.1 mg Trans P > 0.8 0.5 mg Trans 1 mg TransDay 15 0.1 mg Cis P > 0.001 0.5 mg Cis P > 0.4 1 mg Cis Negative Control Vehicle Only 0.1 mg Trans P > 0.8 0.5 mg Trans 1 mg Trans P > 0.001Day 21 0.1 mg Cis 0.5 mg Cis P > 0.4 1 mg Cis
  • 31. Maximum Diameter of Induration (mm) Mean +/- SEM; n = 6 10 12 14 16 0 2 4 6 8 Negative Control Vehicle Only 0.1 mg Trans P > 0.9 0.5 mg Trans 1 mg Trans P < 0.001 0.1 mg Cis24 Hours Post-Challenge 0.5 mg Cis P > 0.9 1 mg Cis Negative Control Vehicle Only 0.1 mg Trans P > 0.9 0.5 mg Trans 1 mg Trans P < 0.001 0.1 mg Cis48 Hours Post-Challenge 0.5 mg Cis P > 0.6 Effect of Topical Treatments on DTH Responses to M. 1 mg Cis ulcerans Cell Wall Fragments at 21 Days Post-Infection
  • 32. Summary Variable Nodular Disease Ulcerative Disease ↑ Nodule size ↑ Lesion size Delayed healing of UV injection sites No effect on DTH ↓ DTH ↑ Overall lesion sizeCis-Urocanic Acid -- ↓ DTH
  • 33. Proposed Mode of Action UV-cUCA- Neuropeptide-Histamine-Prostanoid Photoimmunosuppression Pathway. Dermal non-myelinated sensoryTrans-UCA cis-UCA c-type nerve fibers Calcitonin gene related peptide UV Keratinocytes Nerve growth factor Dermal mast cell degranulation Tumor necrosis factor alpha Histamine Green = fast pathway ( minutes to hours) Blue = slow pathway (8-12 hours) Cyclooxygenase/Prostanoid dependent pathway Photoimmunosuppression 35
  • 34. Questions? 36
  • 35. Current Research Project: An Equine Model of NeurolathyrismSafety Assessment of Low Toxin Grass Pea (Lathyrus sativus) Collaborators: Prof. Peter Spencer and Dr Valerie Palmer, OHSUFunding Acknowledgement: Third World Medical Research Foundation 37
  • 36. Neurolathyrism Neurolathyrism is a neurodegenerative spastic paralysis caused by consumption of grass pea (L. sativus) Toxin is β-N-oxalyl-α,β-diaminopropionic acid (β-ODAP) [Synonym β-oxalylaminoalanine (BOAA)] Rumors about other undefined neurotoxic nitriles 38
  • 37. CH2 CH COOH CH2 CH COOHNH NH2 CH2 NH2CO COOHCOOH β-ODAP L-Glutamate 39
  • 38. Glutamatergic input BOAA, SCN- 3-nitropropionic acid domoic acid Soma NMDA AMPA KA Axon Dendrite Mito Excito toxcity Soma Axon Na+ H2O Ca2+Cellular target is the Ca2+ alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPA receptor) Methionine and cysteine are protective 40
  • 39. History• Ancient disease: oldest definitively described neuro-toxic disease – The disease was known to ancient Hindus, to Hippocrates (460–377 BC), Pliny the Elder (23–79 AD), Pedanius Dioskurides (50 AD) and Galen (130–210 AD) – Outbreaks of neurolathyrism have been systematically reported since the 1700’s in: •Bangladesh •Romania •India, •Spain •Pakistan, •Ukraine •France •Afghanistan •Italy •China •Germany •Algeria •Romania •Ethiopia •Spain •Eritrea •South Sudan Gracias a la almorta. Francisco Goya 41
  • 40. Known Recent epidemics• 1931-1932: Ukraine (The Holodomor)• 1941-1946: Spain• 1973: China• 1976: Bangladesh• 1976: Ethiopia• 1998: Nepal (non published)• 1998: Afghanistan (non published)• 1997-99: Ethiopia/Eritrea• Present: Afghanistan• Present: Somalia (Darfur) and South Sudan 42
  • 41. Afghanistan 2005 43
  • 42. Bangladesh 2006 44
  • 43. Current Prevalence• Ethiopia 6 per 1000• India 5.3 per 1000• Bangladesh 1.4 per 1000 45
  • 44. Why Grass Pea?• Often the last source of food available during a drought• Often the first food available following the end of a drought• Starvation food source 46
  • 45. Why Grass Pea?• Particularly useful food crop if it can be detoxified• High protein: source of protein and essential amino acids in areas where protein malnutrition is comon and there are few protein sources• Resist both water shortage and water logging i.e. survives both droughts and floods• Easy to grow – Requires no fertilizers – Requires no insecticides – Requires no machinery – Improves soil fertility (nitrogen fixer) – Compatible with rice production 47
  • 46. Why Grass Pea?• High protein/energy supplement for ruminants: aid to goat and cattle production in arid/semi-arid areas• Detoxified grain may be suitable for poultry, hogs and fish: major source of protein in many areas in Africa and Asia 48
  • 47. Risk factors• Heavy physical activity• Male gender• Young age (15-25 years) 49
  • 48. Risk factors• Low cysteine and methionine intake• Consumption of 400 g of grass pea for more than 2 weeks (acute disease can occur with higher consumption for shorter period)• Notably, grass pea eaters appear relatively well- nourished when compared with the general population (starving) 50
  • 49. Clinical features• Permanent “non-progressive”* upper motor neuron disorder with symmetrical spastic paralysis – (*Evidence of sensorimotor neuropathy and signs of muscle denervation almost 30 years after the onset of the disease in 7% of Romanian Jews affected by neurolathyrism)• Largely pyramidal distribution – Leg weakness – Exaggerated thigh, adductor, patellar and ankle reflexes – Bilateral presence of ankle clonus and extensor plantar reflexes/Babinski responses – Scissor/cross-legged sitting posture and gait 51
  • 50. Clinical features• Sensation, sphincter, cerebellar, cranial nerve and cognitive/cortical functions are are spared• Changes are permanent• Does not appear to affect life-span• No treatment 53
  • 51. Problems/Research Opportunities• Little or no human pathology• Published reports are rare and mostly examined cases 30+ years after the onset of disease (predominantly from the epidemic in Spain in the 1940’s)• No systematic neuropathology studies 54
  • 52. Problems/Research Opportunities• Available neuropathology studies, which have focused on the spinal cord, show predominantly distal symmetrical degeneration of lateral and ventral corticospinal tracts, distal degeneration of spinocerebellar and gracile tracts and Hirano bodies in anterior horn cells with no reduction of the lower motor neuron pool• MRI has never been performed (Amazing!)• Pathology resembles primary lateral sclerosis (LMN pool is preserved) 55
  • 53. Research Opportunities• Rodent models: – Good models of osteolathyrism/vascular lathyrism (β-aminopropionitrile, Lathyrus odoratus, sweet pea) (Hydrazine compounds [semicarbazide], isoniazid) NOT RELEVENT TO NEUROLATHYRISM – Repeated injection of β-ODAP results in rats with paraparesis of the legs, though at a low incidence rate of 0.032. These paralyzed rats displayed severe atrophy of the ventral root of the lumbar cord as well as degeneration of lower motor neurons. PATHOLOGY DOES NOT MATCH THE HUMAN DISEASE (HUMAN DISEASE IS EXCLUSIVELY UMN) 56
  • 54. Research Opportunities• Low β-ODAP seeds: NO SAFETY ASSESSMENT – ARE THESE GRAINS REALLY SAFE? (the mysterious “other neurotoxic nitriles”)• CURRENTLY NO GOOD ANIMAL MODELS 57
  • 55. Research Opportunities• Potential animal models – Domestic animals: disease occurs in horses, cattle, sheep, pigs, elephants, ducks, geese, hens, and peacocks – Sheep are relatively resistant: tolerate a diet of 50% grass pea with no problems – Reports of neurolathyrism in species other than the horse and sheep are anecdotal, incomplete and old (more mythology than science?) 58
  • 56. Research Opportunities• Potential animal models: Macaque• Onset of disease depends on dose: can reproduce disease via sub-acute high dose or chronic low dose• Produces pyramidal disease that resembles the human disease 59
  • 57. Research Opportunities• Unlike humans, NEUROLOGICAL IMPROVEMENT OCCURRED after cessation of grass pea administration• Neuropathology studies showed no evidence of neuronal degeneration in motor cortex or spinal cord• Electrophysiology in macaques does not match the human disease 60
  • 58. Problems/Research Opportunities• Potential animal models: Horse• Horse is reputed to be the most susceptible species: Stockman (1929) • Diet made exclusively of L. sativus precipitated signs after 10 days. • Horses fed only 1–2 quarts per day succumbed after 2–3 months, and neurological manifestations appeared a month or more after the diet was withdrawn • Neurological changes were permanent• Natural cases of neurolathyrism occur sporadically in horses in eastern Oregon 61
  • 59. Current Research Objectives• Establish an equine model of neurolathyrism – Reproduce clinical neurolathyrism in miniature horses using high β-ODAP and derive the Bench Mark Dose – Systematic neuropathology examination of the equine disease – Systematic electrophysiological examination of the equine disease 63
  • 60. Other Research Interests• Chemoprevention of ethanol-induced hepatotoxicity• Computational/in silico toxicology/alternatives to in vivo testing• In vitro alternatives to OECD toxicology testing 64
  • 61. Questions? 65
  • 62. Clinical/Diagnostic Toxicology Fun What is your diagnosis? (Unlikely to occur in Australia) 66
  • 63. 67
  • 64. 68
  • 65. 69
  • 66. Timber (Canebrake) Rattlesnake (Crotalus horridus) 70
  • 67. A gentle reminder that in companion animals, 71 many snake bites occur on the head!