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Nerve growth factor participates in long-term alterations of colonic sensitivity and mucosal barrier induced by neonatal maternal deprivation in rats
examinations, BrdU incorporation, mucin immunohistochemical staining, growth factors (VEGF, EGF, TGF-ct, COX-2 and pS2). cDNA microarray was done. Results: Prominent decreases in ulcer site were noted in DA30 and DA100 treated groups compared to famotidine treated group. On histologtc examination, remodeling and regeneration were pronounced in DA-9601 treated groups BrdU labeling index was significantly increased in DA30 and DA]00 treated groups compared to control and famotidine treated groups. Mucin expressions (PAS staining & HLK immunohistochemical staining) in also increased in DA30 and DA100 groups compared to control and famotidine group, cDNA microarray showed that clustered genes in cell proliferation and anti-proliferation were largely involved in DA-9601 treated ulcer healing. Remodeling of ulcerated mucins, comprised of thin mucosal thickness and decreased number of dilated gastric glands was the main feature of DA-9601 treated group. Conclusion: Cytoprotective drug is prerequisited for QOUH and the efficacy was proved by high throughput technology.
$972 Prenatal Development of the Intestinal and Extraintestinal T Cell Receptor 6 Repertoire: Identical, Dominant TCRDV3 Transcripts are Present in Different Fetal Piglets Wolfgang Hohmeier, Wiebke Geisel, John Butler, Marek Sinkora, Jiri Sinkora, Wolfgang F Caspary "//B T cells are likely to play an important role in autoimmune conditions such as Crohns disease and ulcerative colitis. However the functions and the antigens recognized by ~/B T cells are largely unknown. It is thought that they recognize antigens expressed by damaged or stressed epithelial cells. In order to establish a large animal model for analyzing the effects of age and microbial factors we have previously characterized the porcine "r TCR repertoire in multiple intestinal and extraintestinal sites. We could demonstrate that the TCR B repertoire is highly compartmentalized not only within differetu organs but also within the intestinal tract. In the present study we analyzed the prenatal development of the porcine TCR 8 repertoire to gain further insight into the nature and development of these cells. Methods: Fetal ttssues from the jejunum, colon, spleen, thymus, liver, bone marrow and PBMC were obtained from fetal piglets between 38 and 114 days of gestation. TCRDV1-DV5 transcripts were amplified by RT-PCR after which complementarity-determining region (CDR) 3 spectratyping was performed. Individual bands were excised from the gels, cloned and sequenced. Results: 1) At 38 days gestation (DG) hardly any TCR 5 transcripts could be detected and only TCRDV1 and DV3 transcripts could be amplified from the thynuc samples. Thus, the ~//B TCR is rarely expressed at this early age. 2) At DG57 TCRDVI-DV5 transcripts could be amplified from all organs and spectratyping showed a significant increase in the CRD3 length during fetal life. 3) In comrast to the other DV families, the TCRDV3 repertoire was highly oligoclonal at DG57 and a dominant band of identical CDR3 length was present in all organs. This dominant band was still present, although less dominant, in the older piglets. 4) Sequence analysis of these dominant bands revealed one identical TCRDV3 transcript which lacked N region diversity. 5) This TCRDV3 transcript was infrequently found during the first weeks after birth but never in adult pigs. Conclusions: The porcine TCR 8 repertoire diversifies during gestation and is polyclonal at birth The dominam TCRDV3 transcript, which is present in all fetus at mid gestation, indicates an important function of this particular ",//'6T cell subset during gestation. The increase of CDR3 length during fetal life indicates increasing TdT activity. In contrast to adult pigs there is no compartmentalization of the TCR 5 repertoire between different organs.
$975 Nerve Growth Factor Participates in Long-Term Alterations of Colonic Sensitivity and Mucosal Barrier Induced by Neonatal Maternal Deprivation in Rats Frederick Barreau, Jean Fioramonti, Lionel Bueno Background/Aims: Previous studies have shown that neonatal maternal deprivation alters the adult brain neuronal circuitry, and visceral sensitivity. Recently, maternal deprivation has been reported to increase Nerve Growth Factor (NGF) expression, and neonatal NGF treatment to induce thermal hyperalgesia in adult rats. The present study aimed to establish whether NGF released during maternal deprivation is involved in altered visceral sensitivity and gut permeability in the adult rat. Methods: Two groups of 10 male Wistar rat pups were submitted to a maternal deprivation procedure (3 hours daily during postnatal days 2 to 14) and two groups were left undisturbed with their dam. During maternal separation, pups were treated with 10~l of anti-NGF antisera intraperitoneally or inactive (boiled) antiNGF antibodies. Concomitamly, non-deprived rats were treated intraperitoneally by NGF (lmg/kg in lop.1 saline) or saline solution (lOp.l). Visceral sensitivity was assessed in 10week adult rats by the number of abdominal comractions (AC) induced by progressive (0.41.2 ml) rectal distensions. Gut paracdhilar permeability was assessed by oral administration of S~Cr-EDTA (lp.Ci/rat), and expressed as the percentage of radioactivity collected in the urine for 24 hours. Then rats were sacrificed, and myeloperoxidase activity (MPO, U/g) (mean_+ SEM for each group) assayed in colonic tissue. Results: In comparison with controls, maternally deprived and NGF treated non-deprived rats had a sigmflcantly increased visceral sensitivity for 0.8-1.2 ml distensions (29 _+3, 26 + 2 and 36-+ 6, 33 + 3 AC/5min, respectively; vs. 18 + 1 and 22 _+2 AC/5min in controls). Ami-NGF treatment in deprived rats restored a normal visceral sensitivity for 0.4-0.8-1.2 ml, (6-+ 2; 19 + 3; 23 _+2, respectively). In nondeprived rats, in comparison with controls, NGF treatment increased gut permeability (3.5+0.3 vs. 2.3+0.2%; p<0.05) and colonic MPO activity (349+62 vs. l12_+27U/g protein; p
S973 Intestine Gena-Regulation by Glutamine (Gln) Deprivation in E. Coli Lipopolysaccharide (LPS)-Treated, Artificially Fed Rat Pups Nan Li, Kellym Liboni, Hanry Baker, Beth J. Lassman, Andrea Fredridcks, Amy D. Mackey, Josef Neu Background: Gin is reported to be an important amino acid for the developing small intestine because of its role as an energy substrate. Recent literature supports that ghitamine may play several other important roles in the intestine. Whether glutamine has nutrient-gene regulatory effects has not previously been evaluated. The effects of ghitamine on the proinflammatory response in the developing small intestine have also not been previously evaluated. Hypothesis: Gln deprivation or supplementation will significamly alter several genes mvolved in small intestinal function. Furthermore, we hypothesize that ghitamine will regulate the pro-inflammatory response in the small intestine exposed to LPS. Methods: Using an artificially reared "pup-in-a-cup" rat model, the effects of supplemental Gln and were examined in the presence of LPS. Four groups of 6-7d old pups were fed a rat milk substitute (RMS) via a gastrostomy tube providing 100 or 25% protein (pro) with and without additional Gln or glutamate (Glu) for another 6d. Microarray analyses were done on intesunal total RNA using Affymetrix gene chips. Intestinal rat IL-8 (cytokine-induced neutrophil chemoattractant) was evaluated by RT-PCR as an indicator of the pro-inflammatory response. Results: LPS treatment decreased pup body weights and these were not statistically different among the LPS-treated groups. LPS-treated pups receiving supplemental Gln had a lower expression level of rat interleukin-8 mRNA than pups receiving 25% pro +/- Ghi or 100% pro RMS. Microarray analysis of intestinal RNA showed that Gln supplememation upregulated the protein turnover genes cathepsin, p95, and catpain and downregulated sucrase-isomahase, arginase If, fatty acid transporter, vascular endothelial growth factor, and caspase-3 more than five fold. Conclusion: These results demonstrated that Gln supplementation decreases the LPS-induced rat IL-8 inflammatory response in infant rat intestine. It also suggested an important role for Gin in the developing, stressed intestine which includes regulation of genes involved with inflammation, apoptosis, protein turnover, and nutriem absorption. Acknowledgement: This study was supported by N1H grant ROIHD38954.
$976 principal Components Analysis Exposes a Genomic Basis for Microsatellite Instability Status Florin M. Selaru, Yuriko Mori, Fumaki Sato, Jing Yin, Yan Xu, Suna Wang, Andreea Olaru, Elena Deacu, Thomas C. Liu, John M. Abraham, David Shthata, Stephen J. Meltzer Background: Frequent microsatellite instability (MSI-H), due to defective DNA mismatch repair, identifies a unique clinico-molecular group of tumors. However, this group has never been characterized on a global gene expression basis. Moreover, tumors with infrequent microsatelhie instability (MSI-L) do not share uniform clinico-molecular features, hence their existence as a distinct group has been challenged. We analyzed global gene expression profiles of colon cancers using an nnsupervtsed bioinformatics approach, principal components analysis (PCA). PCA reorganizes complex data into several independent components, revealing multiple layers of sample grouping hidden in the data. Methods: RNAs from 41 primary colon cancers (12 MSI-H, 14 MSI-L and 15 microsatellite-stable (MSS) minors) were hybridized to microarrays containing 8,064 cDNA clones, and these data were analyzed using PCA. The resulting components were tested for correlations with clinicopathologic and molecular features of tumors, including MSI status. Results (Table): MSI-H tumors were defined by PCA component 3. This was the most influential biologic component affecting expression profile diversity, consistent with the known uniqueness of MSI-H tumors. Notably, another PCA component defined MSI-L tumors, a group which has eluded precise molecular definition and whose existence has been controversial. Additional features correlated with PCA components, including anatomic location and lymph node metastasis. Genes with impact on each component were also identified. Conclusion: Our study supports the hypothesis that MS[-L tumors form a distinct category, and that a biological basis for this category may exist.
$974 Genomic Profiles and Achievement of QOUH by The Administration of Da-9601, a Novel Mucoprotectant Tae Young Oh, Ju Mi Kim, Seul Min Choi, Byoung Ok A.hn, Won Bae Kim, Young Bae Kim, Ki Mypng Lee, Jin Hong Kim, Sung Won Cho, Ki Baik Hahm Background: Quality of ulcer healing (QOUH) is defined as ideal ulcer healing, showing fine granular ulcer scar, high functional maturation, and resistance to ulcer recurrence. Acid suppressants alone are insufficient to achieve QOUH since they lack actions of regeneration and differentiation. Therefore, cytoprotectants are under clinical use to achieve QOUH. Aims: The exact fundamental genomic profiles of QOUH are not known. DA-9601 is a novel mucosal protectant originated from the ethanol extract ofArtemisia asiatica. The current study was designed to develop the appropriate animal model for QOUH and to evaluate the effect of DA-9601 on molecular and functional genomic profile on QOUH. Method: Acetic acid was injected into the serosal surface of SD rats to induce gastric ulcers. The rats were grouped to further treatment as follows: control, H2-RA (famotidine, 10 mg/kg) treated, DA-9601 30 mg/kg (DA30) and DA-9601 100 mg/kg (DAIO0). The rats were sacrificed after 8 weeks of treatment. The resected stomach tissues were studied; gross and hLstologic
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AGA
Abstracts
$972 Prenatal Development of the Intestinal and Extraintestinal T Cell Receptor 6 Repertoire: Identical, Dominant TCRDV3 Transcripts are Present in Different Fetal Piglets Wolfgang Hohmeier, Wiebke Geisel, John Butler, Marek Sinkora, Jiri Sinkora, Wolfgang F Caspary "//B T cells are likely to play an important role in autoimmune conditions such as Crohns disease and ulcerative colitis. However the functions and the antigens recognized by ~/B T cells are largely unknown. It is thought that they recognize antigens expressed by damaged or stressed epithelial cells. In order to establish a large animal model for analyzing the effects of age and microbial factors we have previously characterized the porcine "r TCR repertoire in multiple intestinal and extraintestinal sites. We could demonstrate that the TCR B repertoire is highly compartmentalized not only within differetu organs but also within the intestinal tract. In the present study we analyzed the prenatal development of the porcine TCR 8 repertoire to gain further insight into the nature and development of these cells. Methods: Fetal ttssues from the jejunum, colon, spleen, thymus, liver, bone marrow and PBMC were obtained from fetal piglets between 38 and 114 days of gestation. TCRDV1-DV5 transcripts were amplified by RT-PCR after which complementarity-determining region (CDR) 3 spectratyping was performed. Individual bands were excised from the gels, cloned and sequenced. Results: 1) At 38 days gestation (DG) hardly any TCR 5 transcripts could be detected and only TCRDV1 and DV3 transcripts could be amplified from the thynuc samples. Thus, the ~//B TCR is rarely expressed at this early age. 2) At DG57 TCRDVI-DV5 transcripts could be amplified from all organs and spectratyping showed a significant increase in the CRD3 length during fetal life. 3) In comrast to the other DV families, the TCRDV3 repertoire was highly oligoclonal at DG57 and a dominant band of identical CDR3 length was present in all organs. This dominant band was still present, although less dominant, in the older piglets. 4) Sequence analysis of these dominant bands revealed one identical TCRDV3 transcript which lacked N region diversity. 5) This TCRDV3 transcript was infrequently found during the first weeks after birth but never in adult pigs. Conclusions: The porcine TCR 8 repertoire diversifies during gestation and is polyclonal at birth The dominam TCRDV3 transcript, which is present in all fetus at mid gestation, indicates an important function of this particular ",//'6T cell subset during gestation. The increase of CDR3 length during fetal life indicates increasing TdT activity. In contrast to adult pigs there is no compartmentalization of the TCR 5 repertoire between different organs.
$975 Nerve Growth Factor Participates in Long-Term Alterations of Colonic Sensitivity and Mucosal Barrier Induced by Neonatal Maternal Deprivation in Rats Frederick Barreau, Jean Fioramonti, Lionel Bueno Background/Aims: Previous studies have shown that neonatal maternal deprivation alters the adult brain neuronal circuitry, and visceral sensitivity. Recently, maternal deprivation has been reported to increase Nerve Growth Factor (NGF) expression, and neonatal NGF treatment to induce thermal hyperalgesia in adult rats. The present study aimed to establish whether NGF released during maternal deprivation is involved in altered visceral sensitivity and gut permeability in the adult rat. Methods: Two groups of 10 male Wistar rat pups were submitted to a maternal deprivation procedure (3 hours daily during postnatal days 2 to 14) and two groups were left undisturbed with their dam. During maternal separation, pups were treated with 10~l of anti-NGF antisera intraperitoneally or inactive (boiled) antiNGF antibodies. Concomitamly, non-deprived rats were treated intraperitoneally by NGF (lmg/kg in lop.1 saline) or saline solution (lOp.l). Visceral sensitivity was assessed in 10week adult rats by the number of abdominal comractions (AC) induced by progressive (0.41.2 ml) rectal distensions. Gut paracdhilar permeability was assessed by oral administration of S~Cr-EDTA (lp.Ci/rat), and expressed as the percentage of radioactivity collected in the urine for 24 hours. Then rats were sacrificed, and myeloperoxidase activity (MPO, U/g) (mean_+ SEM for each group) assayed in colonic tissue. Results: In comparison with controls, maternally deprived and NGF treated non-deprived rats had a sigmflcantly increased visceral sensitivity for 0.8-1.2 ml distensions (29 _+3, 26 + 2 and 36-+ 6, 33 + 3 AC/5min, respectively; vs. 18 + 1 and 22 _+2 AC/5min in controls). Ami-NGF treatment in deprived rats restored a normal visceral sensitivity for 0.4-0.8-1.2 ml, (6-+ 2; 19 + 3; 23 _+2, respectively). In nondeprived rats, in comparison with controls, NGF treatment increased gut permeability (3.5+0.3 vs. 2.3+0.2%; p<0.05) and colonic MPO activity (349+62 vs. l12_+27U/g protein; p
S973 Intestine Gena-Regulation by Glutamine (Gln) Deprivation in E. Coli Lipopolysaccharide (LPS)-Treated, Artificially Fed Rat Pups Nan Li, Kellym Liboni, Hanry Baker, Beth J. Lassman, Andrea Fredridcks, Amy D. Mackey, Josef Neu Background: Gin is reported to be an important amino acid for the developing small intestine because of its role as an energy substrate. Recent literature supports that ghitamine may play several other important roles in the intestine. Whether glutamine has nutrient-gene regulatory effects has not previously been evaluated. The effects of ghitamine on the proinflammatory response in the developing small intestine have also not been previously evaluated. Hypothesis: Gln deprivation or supplementation will significamly alter several genes mvolved in small intestinal function. Furthermore, we hypothesize that ghitamine will regulate the pro-inflammatory response in the small intestine exposed to LPS. Methods: Using an artificially reared "pup-in-a-cup" rat model, the effects of supplemental Gln and were examined in the presence of LPS. Four groups of 6-7d old pups were fed a rat milk substitute (RMS) via a gastrostomy tube providing 100 or 25% protein (pro) with and without additional Gln or glutamate (Glu) for another 6d. Microarray analyses were done on intesunal total RNA using Affymetrix gene chips. Intestinal rat IL-8 (cytokine-induced neutrophil chemoattractant) was evaluated by RT-PCR as an indicator of the pro-inflammatory response. Results: LPS treatment decreased pup body weights and these were not statistically different among the LPS-treated groups. LPS-treated pups receiving supplemental Gln had a lower expression level of rat interleukin-8 mRNA than pups receiving 25% pro +/- Ghi or 100% pro RMS. Microarray analysis of intestinal RNA showed that Gln supplememation upregulated the protein turnover genes cathepsin, p95, and catpain and downregulated sucrase-isomahase, arginase If, fatty acid transporter, vascular endothelial growth factor, and caspase-3 more than five fold. Conclusion: These results demonstrated that Gln supplementation decreases the LPS-induced rat IL-8 inflammatory response in infant rat intestine. It also suggested an important role for Gin in the developing, stressed intestine which includes regulation of genes involved with inflammation, apoptosis, protein turnover, and nutriem absorption. Acknowledgement: This study was supported by N1H grant ROIHD38954.
$976 principal Components Analysis Exposes a Genomic Basis for Microsatellite Instability Status Florin M. Selaru, Yuriko Mori, Fumaki Sato, Jing Yin, Yan Xu, Suna Wang, Andreea Olaru, Elena Deacu, Thomas C. Liu, John M. Abraham, David Shthata, Stephen J. Meltzer Background: Frequent microsatellite instability (MSI-H), due to defective DNA mismatch repair, identifies a unique clinico-molecular group of tumors. However, this group has never been characterized on a global gene expression basis. Moreover, tumors with infrequent microsatelhie instability (MSI-L) do not share uniform clinico-molecular features, hence their existence as a distinct group has been challenged. We analyzed global gene expression profiles of colon cancers using an nnsupervtsed bioinformatics approach, principal components analysis (PCA). PCA reorganizes complex data into several independent components, revealing multiple layers of sample grouping hidden in the data. Methods: RNAs from 41 primary colon cancers (12 MSI-H, 14 MSI-L and 15 microsatellite-stable (MSS) minors) were hybridized to microarrays containing 8,064 cDNA clones, and these data were analyzed using PCA. The resulting components were tested for correlations with clinicopathologic and molecular features of tumors, including MSI status. Results (Table): MSI-H tumors were defined by PCA component 3. This was the most influential biologic component affecting expression profile diversity, consistent with the known uniqueness of MSI-H tumors. Notably, another PCA component defined MSI-L tumors, a group which has eluded precise molecular definition and whose existence has been controversial. Additional features correlated with PCA components, including anatomic location and lymph node metastasis. Genes with impact on each component were also identified. Conclusion: Our study supports the hypothesis that MS[-L tumors form a distinct category, and that a biological basis for this category may exist.
$974 Genomic Profiles and Achievement of QOUH by The Administration of Da-9601, a Novel Mucoprotectant Tae Young Oh, Ju Mi Kim, Seul Min Choi, Byoung Ok A.hn, Won Bae Kim, Young Bae Kim, Ki Mypng Lee, Jin Hong Kim, Sung Won Cho, Ki Baik Hahm Background: Quality of ulcer healing (QOUH) is defined as ideal ulcer healing, showing fine granular ulcer scar, high functional maturation, and resistance to ulcer recurrence. Acid suppressants alone are insufficient to achieve QOUH since they lack actions of regeneration and differentiation. Therefore, cytoprotectants are under clinical use to achieve QOUH. Aims: The exact fundamental genomic profiles of QOUH are not known. DA-9601 is a novel mucosal protectant originated from the ethanol extract ofArtemisia asiatica. The current study was designed to develop the appropriate animal model for QOUH and to evaluate the effect of DA-9601 on molecular and functional genomic profile on QOUH. Method: Acetic acid was injected into the serosal surface of SD rats to induce gastric ulcers. The rats were grouped to further treatment as follows: control, H2-RA (famotidine, 10 mg/kg) treated, DA-9601 30 mg/kg (DA30) and DA-9601 100 mg/kg (DAIO0). The rats were sacrificed after 8 weeks of treatment. The resected stomach tissues were studied; gross and hLstologic
A - 12 9
AGA
Abstracts