Uof q2011final

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.

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

1Department of Environmental and Molecular ToxicologyOregon State University

2Department of Microbiology, University of Tennessee

Funding Acknowledgement: US National Institute of Allergy and Infectious Disease

(NIH)

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.

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;

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.

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

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

Ulcerative form of M. ulcerans infection

Sequelae

PathogenesisInfection

NoduleNon-ulcerative Forms

Necrotic Phase (Ulceration, Local Immunosuppression, Impaired T cell Homing)

Several months to years?

Reactive Phase (+ve DTH Response)

Spontaneous Healing?

Months to years?

Ulcer?

Ulcer That Does Not Heal

Plaque/edematous forms

Stellate Scar

KM. George et al (1999), Science, 238: 584

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

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

Experiment 1

Group UV-B Treatment

0 kJ Sham irradiated

3 kJ 1 kJ/m2/day for 3 days

30 kJ 10 kJ/m2/day for 3 days

0

0.5

1

1.5

2

2.5

3

3.5

290 300 310 320 330 340 350 360 370 380 390 400

Wavelength (nm)

Log

Rela

tive

Inte

nsity

Cou

nt

UVR Source

Solar Light

Relative Irradiance of the Source Versus Solar Radiation(Normalized to 1 at 300 nm).

Experiment 1

Days 1-3: irradiate

72 hours

S/C infect with 3 x 104 CFU in 0.1 ml PBS in left dorsal (irradiated) flankS/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 flankSham challenge with I/D 0.1 ml PBS in right flank

24 hours

Read DTH responses; kill and collect samples for histopathology

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.

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25

Days Post Injection

0 kJ

3 kJ

30 kJ

p =

0.0

097

p =

0.0

09

Pro

babi

lity

of

Wou

nd S

urvi

val

Effect of UV-B Pre-Exposure on the Healing of M. ulcerans Contaminated Injection Wounds

0

2

4

6

8

10

12

14

16

Day 7 Day 10 Day 14 Day 21

Days Post-Infection

Mea

n M

axim

um N

odul

e D

iam

eter

, mm

+/-

SEM

, n

= 5

0 kJ

3 kJ UV

30 kJ UV

p < 0.004

p < 0.026p < 0.009

p < 0.021p < 0.010

p < 0.195

p < 0.004

p < 0.079

Effect of UV-B Pre-Exposure on the Size of M. ulcerans Induced Skin Nodules

0

2

4

6

8

10

12

Mea

n D

iam

eter

of

Ind

ura

tio

n,

mm

+/-

SE

M, n =

5

0 kJ

3 kJ

30 kJ

p < 0.001

p = 0.002

p = 0.035

Effect of UV-B Pre-Exposure on DTH Responses to Burulin-S at 21 Days Post-Infection

Experiment 2

Group UV-B Treatment

0 kJ Sham irradiated

3 kJ 1 kJ/m2/day for 3 days

24 kJ 8 kJ/m2/day for 3 days

Experiment 2Days 1-3: irradiate

72 hours

I/D infect with 2 x 106 CFU in 0.1 ml PBS in left dorsal (irradiated) flankI/D inject with 0.1 ml PBS in right dorsal (irradiated) flank

Measure lesions at 4, 7, 10, 18, 23, 30 days post-infection

Day 21:I/D challenge with 0.02 μg Burulin S in 0.1 ml PBS in left flankSham 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

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.

Day 15

0

2

4

6

8

10

12

14

16

18

20

Mea

n U

lcer

Dev

elop

men

t Ti

me,

Day

s +/-

SEM

n

= 6

0 kJ

3 kJ

24 kJ

p = 0.024

The Effects of UV-B Pre-Exposure on M. ulcerans Ulcer Development Time

0

5

10

15

20

25

Day 4 Day 7 Day 10 Day 14 Day 18 Day 23 Day 30

Days Post-Infection

Mea

n L

esio

n (I

ndur

atio

n +

Ulc

er)

Dia

met

er +

/- S

EM

, n =

6

0 kJ

3 kJ

24 kJ

p = 0.154

p = 0.338

p < 0.035 p < 0.035

p ≤ 0.035 p ≤ 0.035

p ≤ 0.05

p = 0.028 p = 0.031

p = 0.094

p = 0.023

Effect of UV-B Pre-Exposure on the Size of M. ulcerans Induced Skin Lesions

UV

NH2

L-Histidine

Histidase

Protein (particularly filaggrin: histidine-rich protein that is a cementing substance for the tonofibrils in the keratin complex of the stratum corneum)

Catabolism

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

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 flankI/D inject with 0.1 ml PBS in right dorsal flank

Measure lesions at 5, 10, 15, and 21 days post-infection

Day 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

0

50

100

150

200

250

300

Neg

ativ

e C

ontr

ol

Veh

icle

Onl

y

0.1

mg

Tra

ns

0.5

mg

Tra

ns

1 m

g T

rans

0.1

mg

Cis

0.5

mg

Cis

1 m

g C

is

Neg

ativ

e C

ontr

ol

Veh

icle

Onl

y

0.1

mg

Tra

ns

0.5

mg

Tra

ns

1 m

g T

rans

0.1

mg

Cis

0.5

mg

Cis

1 m

g C

is

Neg

ativ

e C

ontr

ol

Veh

icle

Onl

y

0.1

mg

Tra

ns

0.5

mg

Tra

ns

1 m

g T

rans

0.1

mg

Cis

0.5

mg

Cis

1 m

g C

is

Neg

ativ

e C

ontr

ol

Veh

icle

Onl

y

0.1

mg

Tra

ns

0.5

mg

Tra

ns

1 m

g T

rans

0.1

mg

Cis

0.5

mg

Cis

1 m

g C

is

Pro

duct

of

Les

ion

Per

pend

icul

ar D

iam

eter

s m

m2

Mea

n +/

- SE

M;

n =

6

P > 0.6

P > 0.4

P > 0.001 P > 0.8

P > 0.4

P > 0.001

P > 0.8

P > 0.4

P > 0.001

P > 0.8

P > 0.4

P > 0.001

Day 5 Day 10 Day 15 Day 21

24 Hours Post-Challenge 48 Hours Post-Challenge

P > 0.9

P > 0.9

P < 0.001

P > 0.9

P > 0.6

P < 0.001

Effect of Topical Treatments on DTH Responses to M. ulcerans Cell Wall Fragments at 21 Days Post-Infection

Variable Nodular Disease Ulcerative Disease

UV

↑ Nodule size

Delayed healing of injection sites

↓ DTH

↑ Lesion size

No effect on DTH

Cis-Urocanic Acid --

↑ Overall lesion size

↓ DTH

Summary

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Trans-UCA cis-UCADermal non-myelinated sensory

c-type nerve fibers Calcitonin gene related peptide

Dermal mast cell degranulation

Histamine

Cyclooxygenase/Prostanoid dependent pathway

Photoimmunosuppression

UV Keratinocytes Nerve growth factor

Green = fast pathway ( minutes to hours)Blue = slow pathway (8-12 hours)

UV-cUCA- Neuropeptide-Histamine-Prostanoid Photoimmunosuppression Pathway.

Tumor necrosis factor alpha

Proposed Mode of Action

Questions?

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Current Research Project: An Equine Model of Neurolathyrism

Safety Assessment of Low Toxin Grass Pea (Lathyrus sativus)

Collaborators: Prof. Peter Spencer and Dr Valerie Palmer, OHSU

Funding Acknowledgement: Third World Medical Research Foundation

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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

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CH2

NH

CO

COOH

CH

NH2

β-ODAP

COOH CH2 CH COOH

CH2

COOH

NH2

L-Glutamate

40

Cellular target is the Ca2+ alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPA receptor)

Methionine and cysteine are protective

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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• India, • Pakistan, • France • Italy• Germany• Romania• Spain

• Romania• Spain• Ukraine• Afghanistan• China• Algeria• Ethiopia• Eritrea • South Sudan Gracias a la almorta.

Francisco Goya

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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

43Afghanistan 2005

44Bangladesh 2006

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Current Prevalence

• Ethiopia 6 per 1000

• India 5.3 per 1000

• Bangladesh 1.4 per 1000

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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

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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

48

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

49

Risk factors

• Heavy physical activity

• Male gender

• Young age (15-25 years)

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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)

51

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

53

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

54

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

55

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)

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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)

57

Research Opportunities

• Low β-ODAP seeds: NO SAFETY ASSESSMENT –

ARE THESE GRAINS REALLY SAFE?

(the mysterious “other neurotoxic nitriles”)

• CURRENTLY NO GOOD ANIMAL MODELS

58

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?)

59

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

60

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

61

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

63

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

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Other Research Interests

• Chemoprevention of ethanol-induced hepatotoxicity

• Computational/in silico toxicology/alternatives to in vivo testing

• In vitro alternatives to OECD toxicology testing

Questions?

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Clinical/Diagnostic Toxicology Fun

What is your diagnosis?

(Unlikely to occur in Australia)

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Timber (Canebrake) Rattlesnake (Crotalus horridus)

71A gentle reminder that in companion animals, many snake bites occur on the head!