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Dr. Dominic Beal will present “Clinically relevant positive controls validate the use of serial FACS analysis, and video endoscopy in two murine adoptive transfer models of colitis” and “Endoscopy and FACS analysis in classic and humanized murine models of GVHD”.
May 9th, 2015
Session Title: Cell Subsets, Cytokines, and Inflammation
Exhibit Hall B, Poster abbreviation HUM1P.315 and HUM1P.316
Clinically relevant positive controls validate the use of serial FACS analysis, and video endoscopy in two murine adoptive transfer models of colitis
Dominic R. Beal*, Sean M. Graham, Gregory D. Lyng, and Stephen T. Sonis | Biomodels LLC, Watertown, MA, USA
The adoptive transfer of naïve TH cells into immunodeficient mice is one of the best characterized immunological models of chronic colitis (CC). Traditional disease severity endpoints of weight loss and histopathology are often misleading since colitis may occur in the absence of weight loss, mice may not engraft, and weight loss and colitis may respond independently to established or experimental treatment.
To overcome these limitations, we analyzed peripheral blood to track engraftment, and performed serial endoscopies to assess onset and severity of CC in two murine models of adoptive transfer-induced colitis: naive T cells from C57Bl/6J males sorted as either CD4+, CD45RBHIGH (FACS), or CD4+, CD44-, CD62L+ (negative magnetic separation) were injected IP into RAG2-/- males.
Onset of colitis corresponded with the onset of weight loss, and day 14 peripheral blood CD4+ counts correlated well with both final weight loss (R2=0.74) and final endoscopy score (R2=0.79), indicating that day 14 CD4+ count was a reliable predictor of disease severity. Treatment with anti-TNFα had little effect on terminal weight loss, but significantly affected endoscopy score (2.0±0.15 vs. 1.1±0.14 at day 49 on a scale of 0-3). Together these observations suggest that endoscopy is a useful addition to weight change as a complete readout for assessing in-life disease severity in these models.
Endoscopy and FACS analysis in classic and humanized murine models of GVHD
Dominic R. Beal, Sean M. Graham, Brett Van Dam, Gregory D. Lyng, and Stephen T. Sonis | Biomodels LLC, Watertown, MA, USA
It is perplexing that standard murine GVHD model endpoints of weight loss (WL) and composite clinical changes (CCC) often fail to respond to standard forms of therapy (tacrolimus and anti-p40 monoclonal antibody). To improve on the translatability of GVHD models we evaluated serial endoscopy as a means to directly assess injury and serial blood counts to predict onset of severe disease. Two models were used. CD3+ cells were magnetically depleted from bone marrow harvested from male Balb/c mice. Varying numbers of (depleted) bone marrow and splenocytes were then injected IV into lethally irradiated (8Gy) C57Bl/6 hosts. Serial endoscopy performed after GVHD onset (~2 weeks post transfer) demonstrated changes which strongly correlated with WL and CCC (R2=-0.84 and 0.79).
Next we induced GVHD in a humanized model by the adoptive transfer of human PBMCs into severely immunocompromised mice (NSG). Serial blood draws demonstrated a strong correlation between peripheral blood human T cell (PBTC) numbers and WL and CCC (R2=-0.85 and 0.67 on day 42) suggesting that assessment of PBTC numbers may be applicable to the evaluation of therapies directly targeting these cell types. Day 7 human PBTC counts predicted final WL and CCC scores with high correlation (R2=-0.86 and 0.90) indicating that this metric can be used to stratify animals into groups prior to the onset of observable disease and treatment.