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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

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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 l.jpg
Features of Shiga toxin-producing infections Products Indicated for 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 l.jpg
HUS: Glomerular microangiopathic lesion in a human patient Products Indicated for Shiga-toxin Producing Infections(Inward, et al., Pediatric Nephrology, 1997)


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Globotriaosyl ceramide (Gb3) Products Indicated for Shiga-toxin Producing Infections

  • 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


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Endpoints examined in published animal studies Products Indicated for Shiga-toxin Producing Infections

  • Mortality

  • Intestinal

  • Neurological

  • Renal – HUS

  • In vitro – e.g. effects on cytokines and inflammatory mediators


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Published animal models of Stx toxicity Products Indicated for Shiga-toxin Producing Infections

  • Mouse

  • Rat

  • Ferret

  • Rabbit

  • Dog

  • Piglet

  • Monkey

  • Baboon


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Mouse model Products Indicated for Shiga-toxin Producing Infections

  • 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


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Mouse model (continued) Products Indicated for Shiga-toxin Producing Infections

  • 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


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Summary: Mouse model Products Indicated for Shiga-toxin Producing Infections

  • 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


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Rabbit model Products Indicated for Shiga-toxin Producing Infections

  • 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


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Summary: Rabbit Products Indicated for Shiga-toxin Producing Infections

  • 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


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Dog Products Indicated for Shiga-toxin Producing Infections

  • 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


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Other animal models Products Indicated for Shiga-toxin Producing Infections

  • Rat

  • Ferret

  • Bonnet macaque (Macaca radiata)


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Baboon model Products Indicated for Shiga-toxin Producing Infections

  • 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


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Piglet model Products Indicated for Shiga-toxin Producing Infections

  • 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


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


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Piglet model (continued) pre-glomerular arterioles (Gunzer et al., Am. J. Clin. Pathol., 2002)

  • 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


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Piglet model (continued) pre-glomerular arterioles (Gunzer et al., Am. J. Clin. Pathol., 2002)

  • 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.



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


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Piglet: Summary (Pohlenz et al., Infect. Immun., 2005)

  • Juvenile or neonatal animals

  • Anatomical and physiological similarity to human

  • Oral inoculation of STEC

    • Colitis / diarrhea

    • CNS

    • HUS - thrombocytopenia, fragmented erythrocytes, glomerular thrombotic microangiopathy


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Conclusions (Pohlenz et al., Infect. Immun., 2005)

  • 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


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  • The ideal model should: (Pohlenz et al., Infect. Immun., 2005)

    • 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


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Extra slides (Pohlenz et al., Infect. Immun., 2005)


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Dutch Belted Rabbit: (Pohlenz et al., Infect. Immun., 2005)(Garcia et al., J. Inf. Dis., 2006)


Baboon glomerular thrombotic microangiopathic lesion taylor et al am j pathol 1999 l.jpg
Baboon: Glomerular thrombotic microangiopathic lesion (Taylor et al., Am. J. Pathol., 1999)


Baboon renal tubular lesion taylor et al am j pathol 1999 l.jpg
Baboon: Renal tubular lesion (Taylor et al., Am. J. Pathol., 1999)

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