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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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slide1

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
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
HUS: Glomerular microangiopathic lesion in a human patient(Inward, et al., Pediatric Nephrology, 1997)
globotriaosyl ceramide gb3
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
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
Published animal models of Stx toxicity
  • Mouse
  • Rat
  • Ferret
  • Rabbit
  • Dog
  • Piglet
  • Monkey
  • Baboon
mouse model
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
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
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
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
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
slide12
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
Other animal models
  • Rat
  • Ferret
  • Bonnet macaque (Macaca radiata)
baboon model
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
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
slide16
Piglet: Thrombotic microangiopathic lesions in pre-glomerular arterioles (Gunzer et al., Am. J. Clin. Pathol., 2002)
piglet model continued
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 continued18
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 thrombotic microangiopathic lesion in renal cortex pohlenz et al infect immun 2005
Piglet: Thrombotic microangiopathic lesion in renal cortex (Pohlenz et al., Infect. Immun., 2005)
piglet summary
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
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
slide23
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
baboon glomerular thrombotic microangiopathic lesion taylor et al am j pathol 1999
Baboon: Glomerular thrombotic microangiopathic lesion (Taylor et al., Am. J. Pathol., 1999)
baboon renal tubular lesion taylor et al am j pathol 1999
Baboon: Renal tubular lesion (Taylor et al., Am. J. Pathol., 1999)

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