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

1. 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

2. Features of Shiga toxin-producing infections • Hemorrhagic colitis • Neurological • Hemolytic Uremic Syndrome (HUS) • Thrombocytopenia • Hemolytic anemia • Thrombotic microangiopathy - glomerulus

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

4. 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

5. Endpoints examined in published animal studies • Mortality • Intestinal • Neurological • Renal – HUS • In vitro – e.g. effects on cytokines and inflammatory mediators

6. Published animal models of Stx toxicity • Mouse • Rat • Ferret • Rabbit • Dog • Piglet • Monkey • Baboon

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. Other animal models • Rat • Ferret • Bonnet macaque (Macaca radiata)

14. 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

15. 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

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

17. 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

18. 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.

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

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

21. 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

22. 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

23. 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

25. Extra slides

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

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

28. Baboon: Renal tubular lesion (Taylor et al., Am. J. Pathol., 1999) Return to Main Menu.