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Graft-vs-host disease induced by in utero hematopoietic cell transplantation - a preclinical evaluation of safety in the canine model
PEDIATRIC SURGERY II METHODS: Prior to treatment, wild type C57Bl/6 and congenic EP1-/- mice were gavaged with commensal ampicillin-resistant E. coli and FITC-dextran. Animals were subjected to cecal ligation and puncture (CLP) or i.p. injection of 40 mg/kg LPS. Control animals were sham operated or injected with saline. Additional groups of WT mice received specific EP1 antagonists GW84876X or SC19220, or vehicle only. Blood, lymphatic, splenic, and ileal samples were collected 16 h post CLP or LPS injection. Serum FITC-dextran levels and bacterial colony counts from homogenized lymphatic tissues were determined. Expression of COX-2 and IL-6 were evaluated using Western blots and ELISA, respectively.
Graft-vs-host disease induced by in utero hematopoietic cell transplantation - a preclinical evaluation of safety in the canine model Jesse D Vrecenak, MD*, Erik G Pearson, MD, Carlyn A Todorow, BA, Haiying Li, BS, Antoneta Radu, BS, Mark P Johnson, MD, Alan W Flake, MD, FACS Children’s Hospital of Philadelphia, Philadelphia, PA INTRODUCTION: As we have previously shown in a canine model, in utero hematopoietic cell transplantation (IUHCT) allows stable alloengraftment and donor specific tolerance without toxic conditioning. Graft versus host disease (GVHD) represents its greatest potential risk. We aimed to establish a threshold T-cell dose for IUHCT-induced GVHD in the haploidentical canine model and to define the GVHD phenotype.
RESULTS: EP1 deficiency significantly protected the barrier during LPS-induced peritonitis but not during CLP-induced polymicrobial sepsis (Table). WT and EP1-/- animals did not differ in mucosal levels of COX-2 or IL-6. At the doses examined, EP1specific antagonists did not provide significant barrier protection.
METHODS: Intracardiac injections of maternal bone marrow (BM) cells were performed under ultrasound guidance at gestational age 38-43 days following CD3+ T-cell depletion and addback to achieve 1%, 3%, 5%, 7.5% and 16% (Whole BM) CD3 content. Surviving puppies were monitored daily for skin lesions, diarrhea/hematochezia, jaundice, weight loss and lethargy. Chimerism was assessed monthly or at necropsy.
NS
WT NS
LPS
p
FITC (ug/ml) 3.56.4 198159 12.321 290125 0.01 E. Coli (CFU/mg) 00 6181948 00 34945065 <0.01 Sham CLP Sham CLP p FITC (ug/ml) 6619 26571840 3582 1830640 0.35
RESULTS: Though prenatal survival was not significantly different than baseline (75% vs 76%) and pups were not runted at birth, severe GVHD was observed in all live-born recipients of 5%, 7.5% and 16% grafts, with onset between 1 and 14 days. Weight loss was the earliest and most consistent clinical feature of IUHCT-induced GVHD, and additional features included increased respiratory rate/effort, hepatic dysfunction, bloody diarrhea and skin sloughing/ulceration. Chimerism in all groups was similar, averaging 12.3% at 1 month for 1% CD3.
CONCLUSIONS: EP1 signaling contributes to the pathogenesis of gut barrier failure during endotoxemia, but not during polymicrobial sepsis. The two EP1 antagonists may have in vivo effects unrelated to blockade of EP1 signaling.
CONCLUSIONS: In the preclinical haploidentical canine model of IUHCT, the threshold for induction of GVHD appears to be between 3% and 5% graft CD3+ cell content. Given the complete absence of GVHD at CD3 doses of 1-3% and the excellent engraftment with the lowest dose, there is a safe therapeutic index for a clinical trial of IUHCT.
Corticotropin-releasing factor promotes necrotizing enterocolitis in formula fed neonatal rats Robert L Bell, MD, Aditi Bhargava, PhD, Min Liao, MD, Ginger Withers, PhD, Frans Kuypers, PhD, Wolfgang Stehr, MD, FACS Children’s Hospital Oakland Research Institute, Oakland, CA INTRODUCTION: The mechanism underlying necrotizing enterocolitis (NEC) remains unknown. The stress hormone corticotropinreleasing factor (CRF) impacts intestinal bacterial colonization patterns, barrier function, innate immune system activation, and vasoconstrictiondNEC’s key pathophysiologic processes. We therefore hypothesized that CRF plays a pivotal role during NEC’s pathogenesis. In this proof-of-concept study, we aimed: (1) to characterize CRF- and CRF-receptor (CRFR) expression patterns in the setting of experimental NEC, and (2) to determine the influence of CRFRagonist and antagonist administration on the disease’s incidence.
Role of prostaglandin E receptor (Ep1) in inflammatory gut barrier failure Scott S Short, MD, Michael Zobel, BA, BS, Henri A Ford, Jin Wang, MS, Stephanie Papillon, MD, Anatoly Grishin, PhD, Henri R Ford, MD, MHA, FACS Children’s Hospital Los Angeles, Los Angeles, CA INTRODUCTION: Systemic inflammation during peritonitis and sepsis is associated with induction of cyclooxygenase-2 (COX-2) and increased production of its product, PGE2. Our previous invivo and in-vitro studies demonstrate that high levels of PGE2 contribute to gut barrier breakdown, presumably via the PGE receptor EP1. We sought to define the role of EP1 in inflammatory gut barrier failure. We hypothesized that genetic ablation or pharmacologic inhibition of the EP1 receptor would limit inflammatory gut barrier failure.
ª 2013 by the American College of Surgeons Published by Elsevier Inc.
EP1-/LPS
METHODS: NEC was induced in newborn rats using formulafeeding and stress exposure (hypoxia/hypothermia). Formula-fed animals were randomized to stress (n¼12), no stress (n¼11), no stress with subcutaneous CRF-administration (n¼11), or stress after subcutaneous administration of the CRFR-antagonist Astressin
S80
ISSN 1072-7515/13/$36.00 http://dx.doi.org/10.1016/j.jamcollsurg.2013.07.175
Graft-vs-host disease induced by in utero hematopoietic cell transplantation - a preclinical evaluation of safety in the canine model Jesse D Vrecenak, MD*, Erik G Pearson, MD, Carlyn A Todorow, BA, Haiying Li, BS, Antoneta Radu, BS, Mark P Johnson, MD, Alan W Flake, MD, FACS Children’s Hospital of Philadelphia, Philadelphia, PA INTRODUCTION: As we have previously shown in a canine model, in utero hematopoietic cell transplantation (IUHCT) allows stable alloengraftment and donor specific tolerance without toxic conditioning. Graft versus host disease (GVHD) represents its greatest potential risk. We aimed to establish a threshold T-cell dose for IUHCT-induced GVHD in the haploidentical canine model and to define the GVHD phenotype.
RESULTS: EP1 deficiency significantly protected the barrier during LPS-induced peritonitis but not during CLP-induced polymicrobial sepsis (Table). WT and EP1-/- animals did not differ in mucosal levels of COX-2 or IL-6. At the doses examined, EP1specific antagonists did not provide significant barrier protection.
METHODS: Intracardiac injections of maternal bone marrow (BM) cells were performed under ultrasound guidance at gestational age 38-43 days following CD3+ T-cell depletion and addback to achieve 1%, 3%, 5%, 7.5% and 16% (Whole BM) CD3 content. Surviving puppies were monitored daily for skin lesions, diarrhea/hematochezia, jaundice, weight loss and lethargy. Chimerism was assessed monthly or at necropsy.
NS
WT NS
LPS
p
FITC (ug/ml) 3.56.4 198159 12.321 290125 0.01 E. Coli (CFU/mg) 00 6181948 00 34945065 <0.01 Sham CLP Sham CLP p FITC (ug/ml) 6619 26571840 3582 1830640 0.35
RESULTS: Though prenatal survival was not significantly different than baseline (75% vs 76%) and pups were not runted at birth, severe GVHD was observed in all live-born recipients of 5%, 7.5% and 16% grafts, with onset between 1 and 14 days. Weight loss was the earliest and most consistent clinical feature of IUHCT-induced GVHD, and additional features included increased respiratory rate/effort, hepatic dysfunction, bloody diarrhea and skin sloughing/ulceration. Chimerism in all groups was similar, averaging 12.3% at 1 month for 1% CD3.
CONCLUSIONS: EP1 signaling contributes to the pathogenesis of gut barrier failure during endotoxemia, but not during polymicrobial sepsis. The two EP1 antagonists may have in vivo effects unrelated to blockade of EP1 signaling.
CONCLUSIONS: In the preclinical haploidentical canine model of IUHCT, the threshold for induction of GVHD appears to be between 3% and 5% graft CD3+ cell content. Given the complete absence of GVHD at CD3 doses of 1-3% and the excellent engraftment with the lowest dose, there is a safe therapeutic index for a clinical trial of IUHCT.
Corticotropin-releasing factor promotes necrotizing enterocolitis in formula fed neonatal rats Robert L Bell, MD, Aditi Bhargava, PhD, Min Liao, MD, Ginger Withers, PhD, Frans Kuypers, PhD, Wolfgang Stehr, MD, FACS Children’s Hospital Oakland Research Institute, Oakland, CA INTRODUCTION: The mechanism underlying necrotizing enterocolitis (NEC) remains unknown. The stress hormone corticotropinreleasing factor (CRF) impacts intestinal bacterial colonization patterns, barrier function, innate immune system activation, and vasoconstrictiondNEC’s key pathophysiologic processes. We therefore hypothesized that CRF plays a pivotal role during NEC’s pathogenesis. In this proof-of-concept study, we aimed: (1) to characterize CRF- and CRF-receptor (CRFR) expression patterns in the setting of experimental NEC, and (2) to determine the influence of CRFRagonist and antagonist administration on the disease’s incidence.
Role of prostaglandin E receptor (Ep1) in inflammatory gut barrier failure Scott S Short, MD, Michael Zobel, BA, BS, Henri A Ford, Jin Wang, MS, Stephanie Papillon, MD, Anatoly Grishin, PhD, Henri R Ford, MD, MHA, FACS Children’s Hospital Los Angeles, Los Angeles, CA INTRODUCTION: Systemic inflammation during peritonitis and sepsis is associated with induction of cyclooxygenase-2 (COX-2) and increased production of its product, PGE2. Our previous invivo and in-vitro studies demonstrate that high levels of PGE2 contribute to gut barrier breakdown, presumably via the PGE receptor EP1. We sought to define the role of EP1 in inflammatory gut barrier failure. We hypothesized that genetic ablation or pharmacologic inhibition of the EP1 receptor would limit inflammatory gut barrier failure.
ª 2013 by the American College of Surgeons Published by Elsevier Inc.
EP1-/LPS
METHODS: NEC was induced in newborn rats using formulafeeding and stress exposure (hypoxia/hypothermia). Formula-fed animals were randomized to stress (n¼12), no stress (n¼11), no stress with subcutaneous CRF-administration (n¼11), or stress after subcutaneous administration of the CRFR-antagonist Astressin
S80
ISSN 1072-7515/13/$36.00 http://dx.doi.org/10.1016/j.jamcollsurg.2013.07.175