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Deprivation of enteral nutrition and changes in epithelial barrier function in humans
S72
Surgical Forum Abstracts
CONCLUSIONS: A macrophage T-cell suppressive factor is secreted following TLR stimulation that inhibits adaptive T-cell responses. This factor will need identification and targeting for optimizing tumor vaccine strategies.
Histone deacetylase inhibitors: Friend or foe in preventing neuroblastoma drug resistance Timothy B Lautz MD, Fei Chu MD, PhD, Sandra Clark BS, Mary Beth Madonna MD Children’s Memorial Hospital/Northwestern University, Chicago, IL INTRODUCTION: Drug resistance poses a major obstacle in the clinical treatment of neuroblastoma. Recent work from our laboratory has demonstrated that class II histone deacetylases (HDACs) are upregulated in drug resistant neuroblastoma cells. We further showed that HDAC stabilization of hypoxia inducible factor-1 alpha (HIF-1a) may contribute to the resistant phenotype. The aim of this study is to investigate the effect of HDAC inhibition on the development of drug resistance. METHODS: Two neuroblastoma cell lines (SK-N-SH and Be(2)C) were incubated with progressively increasing doses of doxorubicin to generate drug resistance. Parallel groups of cells received pretreatment with the pan-class II HDAC inhibitor vorinostat (1 M for SK-N-SH cells and 0.5 M for Be(2)C cells) with each logarithmic increase in doxorubicin concentration. Cell viability and expression of related target proteins were determined by MTT assay and western blot, respectively. RESULTS: Vorinostat is initially synergistic with doxorubicin, producing 80% more cell death in the wild-type cells than doxorubicin alone. Intermittent vorinostat treatment delays the expression of p-glycoprotein and enhances drug sensitivity in SK-N-SH cells. Interestingly, lower dose vorinostat treatment in the Be(2)C cells did not delay p-glycoprotein expression nor enhance drug sensitivity but promoted the drug resistance phenotype. Rebound overexpression of HDAC6 combined with upregulation of HIF-1a were found in these Be(2)C cells. CONCLUSIONS: HDAC inhibition with vorinostat can slow the development of drug resistance by preventing emergence of p-glycoprotein. Subtherapeutic vorinostat dosing may cause rebound HDAC6 overexpression, HIF-1a stabilization and a paradoxical increase in neuroblastoma drug resistance.
J Am Coll Surg
METHODS: Human small bowel was obtained during intestinal resection procedures. EBF was measured by transepithelial resistance (TER, Ohms*cm2) using an Ussing chamber, and by junctional protein (JP) abundance using real-time PCR (normalized to beta-actin); including: E-cadherin, claudin-2, occludin and tight JP-1 (TJP-1) from mucosal scrapings. Data are reported as mean⫾standard deviation. RESULTS: Nine patients (age 55 days to 18 years) were studied, and categorized as NPO the night before surgery (Preop) vs. NPO for 5 or more days (⬎5 day). Mean TER was not statistically different between groups by unpaired t-test (Table). However, examination of specimens taken from non-used ileum or from patients without enteral exposure for ⬎1 month demonstrated the greatest loss of TER (251⫾39). Analysis of junctional proteins failed to demonstrate a correlation between their abundance and duration of NPO (Table). Linear regression analysis showed correlation between TER and TJP-1 (p⫽0.04), TER and E-cadherin (p⫽0.09). Regression analysis did not show correlation between age versus TER or JP abundance. Transepithelial resistance E-cadherin Occludin Claudin-2 Tight JP-1
Preop
> 5 days
316⫾67 0.20⫾0.05 0.005⫾0.002 0.014⫾0.005 0.067⫾0.02
309⫾66 0.33⫾0.07 0.011⫾0.002 0.028⫾0.009 0.081⫾0.02
CONCLUSIONS: This study shows that despite dramatic loss of EBF in rodents on TPN, EBF loss takes far longer to occur in humans. The fairly tight correlation of TER and JP abundance suggests accurate measurement of EBF values. Further work will be needed to better assess EBF changes due to loss of enteral nutrition in humans.
Genetic variability influences susceptibility to Enterobacter sakazakii (ES)-induced necrotizing enterocolitis (NEC) in mice Shannon L Castle MD, Claudia Emami MD, Ashanti Franklin BS, Monica Williams BS, Jin Wang MS, Anatoly Grishin PhD, Henri R Ford MD, MHA, FACS Saban Research Institute/Childrens Hospital Los Angeles, Los Angeles, CA
Eiichi A Miyasaka MD, Manabu Okawada MD, Daniel H Teitelbaum MD University of Michigan, Ann Arbor, MI
INTRODUCTION: NEC is an inflammatory intestinal disorder that affects up to 10% of premature neonates. The genetic factors that determine susceptibility to this disease are not well-characterized. NEC presumably involves colonization of the gut with opportunistic pathogens. One such pathogen, Enterobacter sakazakii (ES) has been associated with hospital outbreaks of NEC. We have shown that ES induces NEC in rats. The purpose of this study was to determine the susceptibility of two genetically different strains of mice commonly used in disease models, FVB and C57BL/6, to ES-induced NEC.
INTRODUCTION: Rodent data shows a loss of epithelial barrier function (EBF) with total parenteral nutrition (TPN), however, little human data exists regarding EBF changes after varying time periods without enteral nutrition (NPO). We hypothesized that longer NPO periods lead to greater EBF decline.
METHODS: FVB and C57BL/6 mice were inoculated with 106 or 107 cfu ES or equivalent volume of PBS on day of life 1 and allowed to nurse with their mothers until onset of disease or day of life 4. Degree of intestinal inflammation was assessed by a pathologist blinded to the groups using H&E-stained ileal samples.
Deprivation of enteral nutrition and changes in epithelial barrier function in humans
Surgical Forum Abstracts
CONCLUSIONS: A macrophage T-cell suppressive factor is secreted following TLR stimulation that inhibits adaptive T-cell responses. This factor will need identification and targeting for optimizing tumor vaccine strategies.
Histone deacetylase inhibitors: Friend or foe in preventing neuroblastoma drug resistance Timothy B Lautz MD, Fei Chu MD, PhD, Sandra Clark BS, Mary Beth Madonna MD Children’s Memorial Hospital/Northwestern University, Chicago, IL INTRODUCTION: Drug resistance poses a major obstacle in the clinical treatment of neuroblastoma. Recent work from our laboratory has demonstrated that class II histone deacetylases (HDACs) are upregulated in drug resistant neuroblastoma cells. We further showed that HDAC stabilization of hypoxia inducible factor-1 alpha (HIF-1a) may contribute to the resistant phenotype. The aim of this study is to investigate the effect of HDAC inhibition on the development of drug resistance. METHODS: Two neuroblastoma cell lines (SK-N-SH and Be(2)C) were incubated with progressively increasing doses of doxorubicin to generate drug resistance. Parallel groups of cells received pretreatment with the pan-class II HDAC inhibitor vorinostat (1 M for SK-N-SH cells and 0.5 M for Be(2)C cells) with each logarithmic increase in doxorubicin concentration. Cell viability and expression of related target proteins were determined by MTT assay and western blot, respectively. RESULTS: Vorinostat is initially synergistic with doxorubicin, producing 80% more cell death in the wild-type cells than doxorubicin alone. Intermittent vorinostat treatment delays the expression of p-glycoprotein and enhances drug sensitivity in SK-N-SH cells. Interestingly, lower dose vorinostat treatment in the Be(2)C cells did not delay p-glycoprotein expression nor enhance drug sensitivity but promoted the drug resistance phenotype. Rebound overexpression of HDAC6 combined with upregulation of HIF-1a were found in these Be(2)C cells. CONCLUSIONS: HDAC inhibition with vorinostat can slow the development of drug resistance by preventing emergence of p-glycoprotein. Subtherapeutic vorinostat dosing may cause rebound HDAC6 overexpression, HIF-1a stabilization and a paradoxical increase in neuroblastoma drug resistance.
J Am Coll Surg
METHODS: Human small bowel was obtained during intestinal resection procedures. EBF was measured by transepithelial resistance (TER, Ohms*cm2) using an Ussing chamber, and by junctional protein (JP) abundance using real-time PCR (normalized to beta-actin); including: E-cadherin, claudin-2, occludin and tight JP-1 (TJP-1) from mucosal scrapings. Data are reported as mean⫾standard deviation. RESULTS: Nine patients (age 55 days to 18 years) were studied, and categorized as NPO the night before surgery (Preop) vs. NPO for 5 or more days (⬎5 day). Mean TER was not statistically different between groups by unpaired t-test (Table). However, examination of specimens taken from non-used ileum or from patients without enteral exposure for ⬎1 month demonstrated the greatest loss of TER (251⫾39). Analysis of junctional proteins failed to demonstrate a correlation between their abundance and duration of NPO (Table). Linear regression analysis showed correlation between TER and TJP-1 (p⫽0.04), TER and E-cadherin (p⫽0.09). Regression analysis did not show correlation between age versus TER or JP abundance. Transepithelial resistance E-cadherin Occludin Claudin-2 Tight JP-1
Preop
> 5 days
316⫾67 0.20⫾0.05 0.005⫾0.002 0.014⫾0.005 0.067⫾0.02
309⫾66 0.33⫾0.07 0.011⫾0.002 0.028⫾0.009 0.081⫾0.02
CONCLUSIONS: This study shows that despite dramatic loss of EBF in rodents on TPN, EBF loss takes far longer to occur in humans. The fairly tight correlation of TER and JP abundance suggests accurate measurement of EBF values. Further work will be needed to better assess EBF changes due to loss of enteral nutrition in humans.
Genetic variability influences susceptibility to Enterobacter sakazakii (ES)-induced necrotizing enterocolitis (NEC) in mice Shannon L Castle MD, Claudia Emami MD, Ashanti Franklin BS, Monica Williams BS, Jin Wang MS, Anatoly Grishin PhD, Henri R Ford MD, MHA, FACS Saban Research Institute/Childrens Hospital Los Angeles, Los Angeles, CA
Eiichi A Miyasaka MD, Manabu Okawada MD, Daniel H Teitelbaum MD University of Michigan, Ann Arbor, MI
INTRODUCTION: NEC is an inflammatory intestinal disorder that affects up to 10% of premature neonates. The genetic factors that determine susceptibility to this disease are not well-characterized. NEC presumably involves colonization of the gut with opportunistic pathogens. One such pathogen, Enterobacter sakazakii (ES) has been associated with hospital outbreaks of NEC. We have shown that ES induces NEC in rats. The purpose of this study was to determine the susceptibility of two genetically different strains of mice commonly used in disease models, FVB and C57BL/6, to ES-induced NEC.
INTRODUCTION: Rodent data shows a loss of epithelial barrier function (EBF) with total parenteral nutrition (TPN), however, little human data exists regarding EBF changes after varying time periods without enteral nutrition (NPO). We hypothesized that longer NPO periods lead to greater EBF decline.
METHODS: FVB and C57BL/6 mice were inoculated with 106 or 107 cfu ES or equivalent volume of PBS on day of life 1 and allowed to nurse with their mothers until onset of disease or day of life 4. Degree of intestinal inflammation was assessed by a pathologist blinded to the groups using H&E-stained ileal samples.
Deprivation of enteral nutrition and changes in epithelial barrier function in humans