# Presentation: 656691

## Experimental Animal Models for the Evaluation of Therapeutic Products Indicated for Shiga-toxin Producing Infections

- Amy C. Nostrandt, D.V.M., Ph.D.
- Pharmacologist
- DAIOP, CDER, FDA

## Features of Shiga toxin-producing infections 

- Hemorrhagic colitis
- Neurological
- Hemolytic Uremic Syndrome (HUS)
- Thrombocytopenia
- Hemolytic anemia
- Thrombotic microangiopathy - glomerulus

## HUS: Glomerular microangiopathic lesion in a human patient(Inward, et al., Pediatric Nephrology, 1997)

## Globotriaosyl ceramide (Gb3)

- Receptor
- Distribution
- Glomerular in infant
- Renal cortical tubules in adult
- Factors influencing the cytotoxic effects of Stx
- Fatty acid chain length appears to determine intracellular trafficking of the Gb3:Stx complex
- Interaction with macrophages and monocytes resulting in cytokine production that may amplify effects

## Endpoints examined in published animal studies

- Mortality
- Intestinal
- Neurological
- Renal – HUS
*In vitro *– e.g. effects on cytokines and inflammatory mediators

## Published animal models of Stx toxicity

- Mouse
- Rat
- Ferret
- Rabbit
- Dog
- Piglet
- Monkey
- Baboon

## Mouse model

- STEC or enteric *E. coli* transfected with genes for Stx PO or Stx IV
- Acute renal tubular necrosis
- Subset of medullary and cortical tubules
- Distribution of Gb3 receptors differs from human
- No effect on glomerulus
- No diarrhea or intestinal lesions consistent with those seen in humans
- CNS
- Mortality

## Mouse model (continued)

- Barrett et al. (1989)
- Stx-2 with and without endotoxin (LPS)
- LPS either enhanced toxicity or protected from mortality, depending on timing of administration relative to Stx
- Keepers et al. (2006)
- IP injection of Stx-2 and LPS
- Neutrophilia, thrombocytopenia, hemolysis
- Increased serum creatinine and BUN
- Glomerular fibrin deposition, thrombosis
- Tesh et al. (1994)
- Macrophage/monocyte responses to Stx

## Summary:  Mouse model

- Adult animals only
- Renal lesions localized to tubules
- No lesions seen in the glomerulus
- Do not develop hemorrhagic diarrhea
- CNS lesions
- Macrophages and monocytes do not respond to Stx with cytokine production as they do in human
- Mortality is the main endpoint examined

## Rabbit model

- O157:H7, O153, or Stx PO
- Diarrhea and cecal lesions
- Renal effects - not seen in NZW rabbits (Richardson et al., 1992); seen in Dutch Belted (DB) rabbits (Garcia et al., 2006)
- Stx IV
- Intestinal lesions
- CNS lesions
- Gb3 receptors in brain
- Renal lesions different from human
- Modulation by LPS
- Stx intrathecally - CNS

## Summary:  Rabbit

- Develop diarrhea, not always hemorrhagic
- Develop hemorrhagic lesions and thrombotic microangiopathy in cecum
- Develop CNS lesions – main endpoint examined
- Develop characteristic renal lesions in some studies, but not in others (NZW vs. DB)
- Limited information in juvenile or neonatal animals is available
- Exquisitely sensitive to disruption in balance of microflora of the gut

## Dog

- Hertzke et al. (1995), Raife et al. (2004)
- Idiopathic cutaneous and renal glomerular vasculopathy in greyhounds
- Thrombocytopenia
- Acute renal failure
- Renal lesions similar to HUS in humans
- No data describing Gb3 receptor location and density in tissues

## Other animal models

- Rat
- Ferret
- Bonnet macaque (*Macaca radiata*)

## Baboon model

- Stx1 or Stx2 IV
- Effect of dose
- Thrombocytopenia
- Hemolytic anemia
- Azotemia
- Melena
- Pathology
- Glomerular thrombotic microangiopathy
- Renal proximal tubule epithelial necrosis
- Intestinal mucosal epithelial necrosis

## Piglet model

- Susceptible to natural and experimental disease due to STEC
- Gunzer et al. (2002)
- Gnotobiotic piglets
- O157:H7 or O26:H11 STEC PO
- Watery diarrhea, locomotor disorders, increased serum creatinine
- Kidney pathology
- Glomerular thrombotic microangiopathy
- Fragmented RBC in blood vessels
- Intestinal pathology – mesenteric petechiae, erosive colitis, attaching and effacing lesions in the colon
- CNS

## Piglet:  Thrombotic microangiopathic lesions in pre-glomerular arterioles (Gunzer et al., Am. J. Clin. Pathol., 2002)

## Piglet model (continued)

- Dean-Nystrom et al. (2000, 2003)
- O157:H7 and other STEC strains PO to cesarean-derived, colostrum-deprived neonatal piglets or to suckling piglets
- Intestinal and CNS signs and lesions
- Noted vascular lesions in the kidney
- Winter et al. (2004)
- Stx1 and Stx2 binding in kidney, ileum, cerebellum, liver, colon and cecum from piglets
- Stx binding and Gb3 demonstrated in alveolar macrophages from piglets and peripheral blood leukocyte smears from adult pigs
- Binding to PMNs and monocytes as in humans

## Piglet model (continued)

- Pohlenz et al. (2005)
- Histopathological examination of renal tissue from multiple studies of Stx in piglets orally infected with O157:H7 or other STEC strains
- Found characteristic renal lesions of HUS, including glomerular thrombotic microangiopathy in most infected piglets from multiple studies
- Renal lesions were consistent with sites that were shown to stain for Gb3 receptors.

## Piglet:  Glomerular microthrombi in renal cortex (Pohlenz et al., Infect. Immun., 2005)

## Piglet:  Thrombotic microangiopathic lesion in renal cortex (Pohlenz et al., Infect. Immun., 2005)

## Piglet:  Summary

- Juvenile or neonatal animals
- Anatomical and physiological similarity to human
- Oral inoculation of STEC
- Colitis / diarrhea
- CNS
- HUS - thrombocytopenia, fragmented erythrocytes, glomerular thrombotic microangiopathy

## Conclusions

- Several animal species have been used to investigate the effects of STEC or Stx
- Some species have been used to investigate individual effects, while  others have been used to characterize a spectrum of findings
- Variable in similarity to human disease

- The ideal model should:
- exhibit disease similar to that seen in human patients
- be representative of the age and physiological status of the patient population
- be repeatable in multiple laboratories (validated)
- lend itself to investigation of therapeutic modalities to treat one or more aspects of the disease

## Extra slides

## Dutch Belted Rabbit:(Garcia et al., J. Inf. Dis., 2006)

## Baboon:  Glomerular thrombotic microangiopathic lesion (Taylor et al., Am. J. Pathol., 1999)

## Baboon:  Renal tubular lesion (Taylor et al., Am. J. Pathol., 1999)

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