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Cytokine-chemokine signiture and severity of chronic hepatitis C
Forakolin(0 1 - 10 micmmolar), an activator of adenylate cyclase, increased nAChR desensitization but this effect was mimicked by dideoxyforskolin, an adenylate cyclase inactive forskolinanalog. These data indicate that simultaneous activation of nAChP,s and muscarinic receptors increases nAChR desensitization. This effect may involve activation of a PKCdependent pathway. These data also suggest that nAChRs and muscarinic receptors are coupled functionally, through an intracellnlar signaling pathway in myenteric neurons.
F3 and moderate HAl and 77 had fibrosis score F4 and high HAl on their biopsy. Serum cytokmes were measured by enzyme-linked-immunosorbent-assay. Student t test with Bonferoni correction was used to determine the significance between the different groups. Differences among groups were determined by the use of confidence intervals and analysis of variance. The X2 test or Fisher's exact test was used to compare frequency data between groups. A good correlation was seen between the HAl and TNF-a levels (r = 0.92, p
595
Divergent Role of The Peripheral Crfl and Crf2 Receptor in The Modulation of Visceral Pain in The Freely Moving Rat Marjoteen Nijsen, Nicolas Ongenae, Ann Meulemans, Bernard Coulie BACKGROUND:Stress plays a modulatory role in visceral pain perception. A likely interactive co-medrator is corticntropin-releasing factor (CRF). This study was aimed to elucidate the role of peripheral CRF and its receptors in the modulation of vasceral pain in freely moving rats. Both nociceptive and antinociceptive effects of systemically administered ERE have been reported previously. In the present study this dual action of CRF was unraveled by seIective activation of the peripheral CRFI and CRF2 receptor. METHODS: A telemetry trammitter, consisting of a bipolar dectrode pair, was chronically implanted into the rat to registerabdominal electromyography (EMG). A balloon catheter was chronically implanted m the duodenum to deliver volume-fixed staircase distensions (0.1 to 0.6 ml). Rats were studied 14 days after surgery. Behavioral responses and changes in area under the curve (AUC) of the EMG signal to duodenal distension were calculated to evaluate the degree ol visceral nocieeption. RESULTS: Distension-induced discomfort (behavioral activation) occurred at a volume of 0.2-0.3 ml and pain (stretching) at a volume of 0.4 ml. In addition, duodenaldistension significantly increased AUC of baseline EMG (p < 0.05). Intraperitoneal (ip) injection of CRF (n = 6; 50 t~g/kg) decreased the perception thresholds for discomfort (0.1 ml; p < 0.01) and pain (0.2 ml; p < 0.005) as compared to vehicle treatment (n= 6). Thisresponse could be inhibited by ip CP-154,526 (n = 6; 10 mg/kg, p < 0.fl5), a selective CRFI receptor antagonist. Furthermore, ip CRF increased the abdominal EMG response to duodenal distension (p < 0.05) as compared to vehicle treatment, an effect which was augmented by ip pre-treatment of the selective CRF2 antagonist anti-sauvagine30 (n=6; 100 ~g/kg; p < 0.001). In addition, ip injection of selective CRF2 agonists, stresscopin (n=4; 120 I*g/kg) or stresscopin-related peptide (n = 4; 25 v,g/kg), increased the perception thresholds ~or discomfort ( 0 4 ml; p < 0.005) and pain behavior (0.5-0.6 ml; p < 0.005) as compared to vehicle treatment (n = 5). Furthermore, both CRF2 agonists decreased the abdominal EMG response to vasceral pain (p< 0.05) in comparison to vehicle-treated rats. CONCLUSIONS: Our findings demonstrate that activation of the peripheral CRF1 receptor leadsto a facilitation of afferent transmission of wsceral nociceptive stimuli, whereas activation of the CRF2 receptor leads to antinociceptive action.
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HAlt:SO 1.9+ 0.2 1,~ 0.1. 5.~ 0.2** 8.0i-02. 11.8 28+4.2 ~ 8 138:1:24.* 346-J:24.* TNFa 49+9 199+_20.* 348:L15. 3565:26 IL2 125~15 210-J:10. 620.~20. 1850-a:50~ IL12 69~10 72.-1:12 19~15. 23~15 IL 18 130-J:10 190-a:15. 250"J:10. 41~15. TGF[3 - ng/mL 40-J:2 79+7* 180-~15.* 90.2:10.** ALT (81+8E) 7~20 79~10 99-~5 86i20 CytokJnes(pg/rnL.l:SE) ,.p<0.05and **0<0.00't higheras comparedto the previous group; ***p<0.001 loweras comparedto the previousgroup
598 Bmdykinin B2 Receptors Mediate Contraction in the Normal and Inflamed Human Gallbladder in Vitro Roberto De Giorgio, Marcello Trevisani, Silvia Amadesi, Fabien Schmidlin, Maria Poblete, Selena Harrison, Carlos Figneroa, Michele Tognetto, Giovanni Barbara, Vincenzo Stanghellini, Giuseppe Navarra, Nigel Bunnett, Roberto Corinaldesi, Pierangelo Geppetti Bradykinin (BK) produces inflammatory responses via B2 receptor activation, and expression of the components of the BK system is increased during inflammation. Here, we investigated the motor effects induced by BK and the expression of the B2 receptor mRNA along with kininogen and kallikrein immunoreactivity in human gallbladders. Specimens were obtained from patients undergoing cholecystectomy either for acute cholecystitis due to gallstone disease or during elective abdominal surgery for uncomplicated gastrointestinal, liver or pancreatic tumors (control group). BK produced a robust contraction of isolated human gallbladder strips. BK showed potency and efficacy higher than carhachol and similar to those of cholecystokmin. BK efficacy was increased in gallbladders obtained from acute cholecystins as compared to control patiems. The response to BK in acute cholecystitis gallbladders was inhibited by the B2 receptor antagonist, HOE140, but not by the B1 receptor antagonist, R-715, and it was not affected by indomethacin, atropine and a combination of NK1, NK2 and NK3 tachykinins receptor antagonists. A product of 525 bp was observed on a 1.5% agarose gel after RT-PCR amplification of RNA of gallbladder tissue taken from control patients. However, in tissues obtained from acute cholecystitis patients the amount of this PCR product was greatly increased, lmmunostaining for kininogen and kallikrein were present in the human gallbladder and were more intense at sites of inflammation. Conclusions: All the components of the BK system (kininogen, kallikrein and B2 receptors) are present in the human gallbladder, and their expression is more pronounced in inflamed gallbladders. IlK is a powerful spasmogen via B2 receptor activation in the normal and, particularly, in the inflamed human gallbladder.
596 Elevated Expression Of Caveolin-1 At Protein And Gene Levels In Human Cirrhotic Liver - Relationship To Nitric Oxide HiroakiYokomori, Masaya Oda, Hiromasa Ishii Backgroundand Aims: Nitric oxide is synthesized in diverse mammalian tissues by calmodulie-dependent nitric oxide synthases (NOS). Caveofin, the principle structural protein in caveolae, interacts with endothelial NOS (eNOS) to inhibit the enzyme by a reversible process modulated by Ca++-calmodulin. The present study examined the localization of eNOSand caveolin-1 at protein and mRNA levels in non-cirrhotic human liver tissue, and how the expressions are altered in cirrhotic liver Methods: Normal portions resected from casesof metastatic liver carcinoma were used as controls, and cirrhotic portions resected from cases of bepatocellular carcinoma with hepatitis C-related cirrhosis were used as circhoticspecimens. Anti-eNOS and anti-caveofin-1 antibodies were used in immunohistochemistryand Western blot. lmmuncelectron microscopy was conducted using immunoglobulin-goldcombined with silver staining. Morphometric analysis was performed on immunodectronmicrographs. For in situ hybridization (ISH), human eNOS and caveolin-1 peptide nucleic acid probes were used with the catalyzed signal amplification system. Results: In non-cirrhotic liver tissue, immunohistochemical and immuoelectron microscopy localized covet[in-1 predominately on portal veins and vascular walls and at low level on hepatic sinusoidal lining cells, while eNOS was observed scantily on sinusoidal lining cells. In urrhotic liver tissue, however, caveolin-1 expression was significantly increased white eNOS expressionwas slightly increased on sinusoidal fining cells. Western blot confirmed marked 0verexpression of caveolin-1 and only slight increase of eNOS in cirrhotic specimens. By ISH, eNOS mRNA was localized on portal veins and hepatic lining cells and caveolin-1 mRNAwas almost undetectable in non-cirrhotic tissue. In cirrhotic liver tissue, caveolin-1 mRNAwas overexpressed on sinusoidal lining cells, while eNOS mRNA expression was similarcompared to non-cirrhotic tissue. Conclusion: In cirrhotic human liver, markedly overexpression of caveolin-1 in sinusoidal ceils may promote caveolin-eNOS binding and reducethe activity of eNOS despite a slight increase of eNOS expression, leading to impaired NO production and increased hepatic microvascular tone.
599 Increased Plasma Levels and More Pronounced Vasoconstrictive Action of Ncuropeptide Y in the Splanchnic Circulation in Portal Hypertension Reiner Wiest, Lars Jurzik, Christoph Paetzel, Stefan Feuerbach, Juergen Schoelmerich, Rainer Straub Arterial vasodihtion and vascular hyporeactivity occur predominantly in the splanchnic circulation in portal hypertension. Compensatory stimulation of the sympathetic nervous system has been evidenced by increased serum levels of norepinephrine (NE). NeuropeptideY (NPY) is co-stored pre-synaptically with NE, is released upon sympathetic nerve stimulation and functions as co-transmitter. Alterations in release and vasoacnve effects of NPY in the splanchnic circulation in portal hypertension have not been investigated so far. Methods: We studied peripheral plasma levels of NPY (radio-immunoassay) in cirrhotic patients (n = 48) and healthy controls (n = 50) as well as portal and hepatic venous levels in cirrhoucs undergoing TIPS insertion (n= 15). Moreover, the effect of NPY (10-8 M) on vascular reactivity to methoxamine (MT: a 1-adrenergac agonist) was evaluated in the in-vitro perfused mesenteric vascular bed of portal-vein-figated (PVL) and sham operated (sham) rats. Results: Cirrhotics presented with significantly elevated plasma levels of NPY as compared to controLs (52,4+/-3,9 vs. 20,2+/-1,2 pmol, p<0.001), being not significantly different between patients child class A, B or C. Portal venous NPY levels were significandy higher than hepaticvenous levels (46,1 +/-5,9 vs. 31,5 +/-3,8 pmol, p<0.05) and decreased immediately after TIPS-insertion. The known marked vascular hyporeactivity to MT in the mesenteric vascular bed of PVL rats was corrected by NPY (Fig. IA,B). NPY increased vascular sensitivity in both study groups equally but enhanced vascular contractility only in PVL rats. Percemage change in maximal pressure response to MT induced by NPY was significantly more pronounced in PVI. than in sham rats (Fig.lC). Conclusions: Plasma levels of NPY are increased in cirrhotic patients being most predominant in the splanchic circulation. The vasoconstrictive effect of NPYin the mesenteric vascnlature is markedly more pronounced in PVL rots restoring vascular responsiveness and making NPY a superior vasoeonstrictive therapeutic candidate.
597 Cytokine-Chemokine Signiture and Severity of Chronic Hepatitis C ManuelaG. Neuman, Patrick Marcellni, Jean-Pierre Bonfiamou, Hemda SchmilowitzWeiss,lzabella Malkiewicz, Ran Tur-Kaspa, Tarik Asselah, Ziv Beu-Ari, Mark Bourliere, DominiqueValla, Ross Cameron Theaims of our study were to measure serum and cellular expression of intedeukins (ILs), tumornecrosis factor-alpha (TNFe0 and transforming growth factor-beta (TGF [3) levels at innon-treated chronic hepatitis C patients with various degrees of inflammation and fibrosis and to determine the correlation between these cytokines with the degree of inflammation and fibrosis. We studied 476 patients; 59 had Iow Knodell fibrosis score (F0-F1) and low Knodellhistological activity index (HAl); 170 had F2 fibrosis and low HAl; 170 had fibrosis
A-75
AGA
Abstracts
F3 and moderate HAl and 77 had fibrosis score F4 and high HAl on their biopsy. Serum cytokmes were measured by enzyme-linked-immunosorbent-assay. Student t test with Bonferoni correction was used to determine the significance between the different groups. Differences among groups were determined by the use of confidence intervals and analysis of variance. The X2 test or Fisher's exact test was used to compare frequency data between groups. A good correlation was seen between the HAl and TNF-a levels (r = 0.92, p
595
Divergent Role of The Peripheral Crfl and Crf2 Receptor in The Modulation of Visceral Pain in The Freely Moving Rat Marjoteen Nijsen, Nicolas Ongenae, Ann Meulemans, Bernard Coulie BACKGROUND:Stress plays a modulatory role in visceral pain perception. A likely interactive co-medrator is corticntropin-releasing factor (CRF). This study was aimed to elucidate the role of peripheral CRF and its receptors in the modulation of vasceral pain in freely moving rats. Both nociceptive and antinociceptive effects of systemically administered ERE have been reported previously. In the present study this dual action of CRF was unraveled by seIective activation of the peripheral CRFI and CRF2 receptor. METHODS: A telemetry trammitter, consisting of a bipolar dectrode pair, was chronically implanted into the rat to registerabdominal electromyography (EMG). A balloon catheter was chronically implanted m the duodenum to deliver volume-fixed staircase distensions (0.1 to 0.6 ml). Rats were studied 14 days after surgery. Behavioral responses and changes in area under the curve (AUC) of the EMG signal to duodenal distension were calculated to evaluate the degree ol visceral nocieeption. RESULTS: Distension-induced discomfort (behavioral activation) occurred at a volume of 0.2-0.3 ml and pain (stretching) at a volume of 0.4 ml. In addition, duodenaldistension significantly increased AUC of baseline EMG (p < 0.05). Intraperitoneal (ip) injection of CRF (n = 6; 50 t~g/kg) decreased the perception thresholds for discomfort (0.1 ml; p < 0.01) and pain (0.2 ml; p < 0.005) as compared to vehicle treatment (n= 6). Thisresponse could be inhibited by ip CP-154,526 (n = 6; 10 mg/kg, p < 0.fl5), a selective CRFI receptor antagonist. Furthermore, ip CRF increased the abdominal EMG response to duodenal distension (p < 0.05) as compared to vehicle treatment, an effect which was augmented by ip pre-treatment of the selective CRF2 antagonist anti-sauvagine30 (n=6; 100 ~g/kg; p < 0.001). In addition, ip injection of selective CRF2 agonists, stresscopin (n=4; 120 I*g/kg) or stresscopin-related peptide (n = 4; 25 v,g/kg), increased the perception thresholds ~or discomfort ( 0 4 ml; p < 0.005) and pain behavior (0.5-0.6 ml; p < 0.005) as compared to vehicle treatment (n = 5). Furthermore, both CRF2 agonists decreased the abdominal EMG response to vasceral pain (p< 0.05) in comparison to vehicle-treated rats. CONCLUSIONS: Our findings demonstrate that activation of the peripheral CRF1 receptor leadsto a facilitation of afferent transmission of wsceral nociceptive stimuli, whereas activation of the CRF2 receptor leads to antinociceptive action.
pnaeter
F0.Ft {~)
F2OTO}
F4fn)
F30to)
HAlt:SO 1.9+ 0.2 1,~ 0.1. 5.~ 0.2** 8.0i-02. 11.8 28+4.2 ~ 8 138:1:24.* 346-J:24.* TNFa 49+9 199+_20.* 348:L15. 3565:26 IL2 125~15 210-J:10. 620.~20. 1850-a:50~ IL12 69~10 72.-1:12 19~15. 23~15 IL 18 130-J:10 190-a:15. 250"J:10. 41~15. TGF[3 - ng/mL 40-J:2 79+7* 180-~15.* 90.2:10.** ALT (81+8E) 7~20 79~10 99-~5 86i20 CytokJnes(pg/rnL.l:SE) ,.p<0.05and **0<0.00't higheras comparedto the previous group; ***p<0.001 loweras comparedto the previousgroup
598 Bmdykinin B2 Receptors Mediate Contraction in the Normal and Inflamed Human Gallbladder in Vitro Roberto De Giorgio, Marcello Trevisani, Silvia Amadesi, Fabien Schmidlin, Maria Poblete, Selena Harrison, Carlos Figneroa, Michele Tognetto, Giovanni Barbara, Vincenzo Stanghellini, Giuseppe Navarra, Nigel Bunnett, Roberto Corinaldesi, Pierangelo Geppetti Bradykinin (BK) produces inflammatory responses via B2 receptor activation, and expression of the components of the BK system is increased during inflammation. Here, we investigated the motor effects induced by BK and the expression of the B2 receptor mRNA along with kininogen and kallikrein immunoreactivity in human gallbladders. Specimens were obtained from patients undergoing cholecystectomy either for acute cholecystitis due to gallstone disease or during elective abdominal surgery for uncomplicated gastrointestinal, liver or pancreatic tumors (control group). BK produced a robust contraction of isolated human gallbladder strips. BK showed potency and efficacy higher than carhachol and similar to those of cholecystokmin. BK efficacy was increased in gallbladders obtained from acute cholecystins as compared to control patiems. The response to BK in acute cholecystitis gallbladders was inhibited by the B2 receptor antagonist, HOE140, but not by the B1 receptor antagonist, R-715, and it was not affected by indomethacin, atropine and a combination of NK1, NK2 and NK3 tachykinins receptor antagonists. A product of 525 bp was observed on a 1.5% agarose gel after RT-PCR amplification of RNA of gallbladder tissue taken from control patients. However, in tissues obtained from acute cholecystitis patients the amount of this PCR product was greatly increased, lmmunostaining for kininogen and kallikrein were present in the human gallbladder and were more intense at sites of inflammation. Conclusions: All the components of the BK system (kininogen, kallikrein and B2 receptors) are present in the human gallbladder, and their expression is more pronounced in inflamed gallbladders. IlK is a powerful spasmogen via B2 receptor activation in the normal and, particularly, in the inflamed human gallbladder.
596 Elevated Expression Of Caveolin-1 At Protein And Gene Levels In Human Cirrhotic Liver - Relationship To Nitric Oxide HiroakiYokomori, Masaya Oda, Hiromasa Ishii Backgroundand Aims: Nitric oxide is synthesized in diverse mammalian tissues by calmodulie-dependent nitric oxide synthases (NOS). Caveofin, the principle structural protein in caveolae, interacts with endothelial NOS (eNOS) to inhibit the enzyme by a reversible process modulated by Ca++-calmodulin. The present study examined the localization of eNOSand caveolin-1 at protein and mRNA levels in non-cirrhotic human liver tissue, and how the expressions are altered in cirrhotic liver Methods: Normal portions resected from casesof metastatic liver carcinoma were used as controls, and cirrhotic portions resected from cases of bepatocellular carcinoma with hepatitis C-related cirrhosis were used as circhoticspecimens. Anti-eNOS and anti-caveofin-1 antibodies were used in immunohistochemistryand Western blot. lmmuncelectron microscopy was conducted using immunoglobulin-goldcombined with silver staining. Morphometric analysis was performed on immunodectronmicrographs. For in situ hybridization (ISH), human eNOS and caveolin-1 peptide nucleic acid probes were used with the catalyzed signal amplification system. Results: In non-cirrhotic liver tissue, immunohistochemical and immuoelectron microscopy localized covet[in-1 predominately on portal veins and vascular walls and at low level on hepatic sinusoidal lining cells, while eNOS was observed scantily on sinusoidal lining cells. In urrhotic liver tissue, however, caveolin-1 expression was significantly increased white eNOS expressionwas slightly increased on sinusoidal fining cells. Western blot confirmed marked 0verexpression of caveolin-1 and only slight increase of eNOS in cirrhotic specimens. By ISH, eNOS mRNA was localized on portal veins and hepatic lining cells and caveolin-1 mRNAwas almost undetectable in non-cirrhotic tissue. In cirrhotic liver tissue, caveolin-1 mRNAwas overexpressed on sinusoidal lining cells, while eNOS mRNA expression was similarcompared to non-cirrhotic tissue. Conclusion: In cirrhotic human liver, markedly overexpression of caveolin-1 in sinusoidal ceils may promote caveolin-eNOS binding and reducethe activity of eNOS despite a slight increase of eNOS expression, leading to impaired NO production and increased hepatic microvascular tone.
599 Increased Plasma Levels and More Pronounced Vasoconstrictive Action of Ncuropeptide Y in the Splanchnic Circulation in Portal Hypertension Reiner Wiest, Lars Jurzik, Christoph Paetzel, Stefan Feuerbach, Juergen Schoelmerich, Rainer Straub Arterial vasodihtion and vascular hyporeactivity occur predominantly in the splanchnic circulation in portal hypertension. Compensatory stimulation of the sympathetic nervous system has been evidenced by increased serum levels of norepinephrine (NE). NeuropeptideY (NPY) is co-stored pre-synaptically with NE, is released upon sympathetic nerve stimulation and functions as co-transmitter. Alterations in release and vasoacnve effects of NPY in the splanchnic circulation in portal hypertension have not been investigated so far. Methods: We studied peripheral plasma levels of NPY (radio-immunoassay) in cirrhotic patients (n = 48) and healthy controls (n = 50) as well as portal and hepatic venous levels in cirrhoucs undergoing TIPS insertion (n= 15). Moreover, the effect of NPY (10-8 M) on vascular reactivity to methoxamine (MT: a 1-adrenergac agonist) was evaluated in the in-vitro perfused mesenteric vascular bed of portal-vein-figated (PVL) and sham operated (sham) rats. Results: Cirrhotics presented with significantly elevated plasma levels of NPY as compared to controLs (52,4+/-3,9 vs. 20,2+/-1,2 pmol, p<0.001), being not significantly different between patients child class A, B or C. Portal venous NPY levels were significandy higher than hepaticvenous levels (46,1 +/-5,9 vs. 31,5 +/-3,8 pmol, p<0.05) and decreased immediately after TIPS-insertion. The known marked vascular hyporeactivity to MT in the mesenteric vascular bed of PVL rats was corrected by NPY (Fig. IA,B). NPY increased vascular sensitivity in both study groups equally but enhanced vascular contractility only in PVL rats. Percemage change in maximal pressure response to MT induced by NPY was significantly more pronounced in PVI. than in sham rats (Fig.lC). Conclusions: Plasma levels of NPY are increased in cirrhotic patients being most predominant in the splanchic circulation. The vasoconstrictive effect of NPYin the mesenteric vascnlature is markedly more pronounced in PVL rots restoring vascular responsiveness and making NPY a superior vasoeonstrictive therapeutic candidate.
597 Cytokine-Chemokine Signiture and Severity of Chronic Hepatitis C ManuelaG. Neuman, Patrick Marcellni, Jean-Pierre Bonfiamou, Hemda SchmilowitzWeiss,lzabella Malkiewicz, Ran Tur-Kaspa, Tarik Asselah, Ziv Beu-Ari, Mark Bourliere, DominiqueValla, Ross Cameron Theaims of our study were to measure serum and cellular expression of intedeukins (ILs), tumornecrosis factor-alpha (TNFe0 and transforming growth factor-beta (TGF [3) levels at innon-treated chronic hepatitis C patients with various degrees of inflammation and fibrosis and to determine the correlation between these cytokines with the degree of inflammation and fibrosis. We studied 476 patients; 59 had Iow Knodell fibrosis score (F0-F1) and low Knodellhistological activity index (HAl); 170 had F2 fibrosis and low HAl; 170 had fibrosis
A-75
AGA
Abstracts