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Endocytosis is an essential step in endotoxin (LPS)-stimulated TNFα formation by Kupffer cells
448A 1365
AASLD
ABSTRACTS
ENDOCYTOSIS IS AN ESSENTIAL STEP IN E N D O T O X I N (LPS)-STIMULATED T N F a F O R M A T I O N BY KUPFFER CELLS. SN Liehtmanl, J Wan~l, _C_7,Jaal~l, E DeFlom 1, JJ Lemnsters2. Depts. of Ipediatrics and 2Cell Biology & Anatomy, University of North Carolina, Chapel Hill.
1366
Introduction - Kupffer cells (KC) clear bacterial products from the intestine by endocytic uptake. A major bacterial polymer, LPS, activates KCs to release proinflammatory mediators involved in shock/sepsis, alcoholic liver disease and livel' reperfusion injury. Our Aim was to study the role of endocytosis in LPS-stimulation of KCs.
GADOLINIUM PREVENTS H E P A T I C GRANULOMAS AND ARTHRITIS INDUCED BY STREPTOCOCCAL • PEPTIDOGLYCAN-POLYSACCHARIDE (PG-PS). SN Lichlman. J Wang. C Zhang. JH Schwab. Dept. of Pediatrics, University of North Carolina, Chapel Hill, NC. Introduction -PG-PS is a major component of bacterial cell walls and IP injection induces relapsing arthritis and granulomatous hepatitis. Previous studies indicated the importance of the liver in chronic arthritis, by providing a reservoir of PG-PS or releasing mediator. The role of Kupffer cells (KC) in the evolution of these lesions was studied using GdC13 treatment (Gad) which causes KC death.
Methods - KCs were purified from Sprague-Dawley rat livers using
centrifugal eluttiation and cultured for 48 hr prior to use. Inhibitors were added for 1 hr before addition of LPS and then incubated with LPS (E. eoli 0111:B4) for 4 hr. Supematants were assayed for TNFa using ELISA. RNA was extracted after 90 rain to assess TNFa mRNA by Northern blots.
Methods -PG-PS (2 ktg/gm) from group A streptococci was injected
Results - Cytoehalasin B (blocks endocytosis) and bafilomycin A (inhibits the FP-ATPase pump) prevented LPS-stimulated TNFa release by 95% and 94%, respectively, (p < 0.001) and also prevented TNFa mRNA synthesis. Incubation of LPS with KCs for 5, 10, 15, 20, 30, and 60 rain induced 0%, 8%, 38%, 57%, 83%, and 94% of maximum TNFa release showing that 15-20 min exposure to LPS is needed to signal half maximal TNFa production.
of liver granulomas and prevented arthritis (Table, * = p < .001). IA PG-PS and Gad did not prevent arthritis or its reactivation by endotoxin.
IP and rats were treated with buffer or IV Gad (20 mg/kg 2X/wk) and studied 21 days later. To study its local effect on joint macrophages, Gad was administered with PG-PS intra-articularly (IA). Results -Gad treatment significantly decreased liver weight, number
Group (n) Buffer (6) Gad (6)
Conclusions - Endoc.vtosis and acidification of endosomal vesicles are important early steps in KC activation by LPS. The 10-20 rain need-ed to signal LPS-stimulated TNFa release may represent the time required for endocytic uptake and processing of the bacterial cell wall product.
1367
HEPATOLOGY O c t o b e r 1995
ASSESSMENT OF LIVER FUNCTION BEFORE, DURING AND AFTER INTERFERON (IFN) TREATMENT FOR CHRONIC HEPAqTHS C. _F_ Lirussi. M Crovatto °. G Santini°. A Beecarello. L Bortolato. C Triehes. C Paleari§, L Okolicsanvi* and G Creoaldi. Institute of Internal Medicine, University of Padua, °Microbiology and Immunology Unit, Pordenone Regional Hospital, §Institute of Clinical Chemistry, University of Padua and *Chair of Gastroenterology, University of Parma, Italy. There is evidence that IFN depresses the drug metabolizing activity (DMA) (Eur J Clin Pharmaenl, 39, 365, 1990; Hepatology, 17, 65, 1993). Conversely, liver function might influence treatment outcome in addition to host-, viral- and drug-related factors. We therefore: i) studied the effect of IFN on DMA and on the functioning liver mass as assessed by monoethylglycinexylidide (MEGX) formation from lidocaine and galactose elimination capacity (GEC), respectively and ii) investigated if there was any relationship between these quantitative LFTs and the response to IFN, including HCV genotypes (Okamoto's classification). Twenty-one pts with biopsy proven CAH (n=13) or cirrhosis (n=8) received rIFN ct-2b (Intron-A°, 6 MU tiw for 4 mo followed by 3 MU tiw for 8 too). MEGX formation (45-mni sample) and GEC test were measured in lg pts at mo 0, 4 and 12 and in g pts 6 mo after 1FN withdrawal. Results: Three pts withdrew from treaanent in the first 2-3 mo for severe side effects; 12 pts (genotype II n=5) showed complete or partial response (R) while 9 (type II n=7) were non responders (NR). Pre-treaanent MEGX values were 63 ng/ml±6 SEM in R and 53±5 in NR O--m) and did not change significantly in both R (68±7 at mo 4, 65±8 at mo 12) and in NR (67±7 at mo 4). 1FN withdrawal caused no changes in MEGX values (from 63±6 to 70~7). Pretreatment GEC values were similar in R and NR (2.3 mM/min~0.2 vs 1.9~.2) and remained stable throughout the treatment period as well as after IFN withdrawal in both groups. In eoneinsion: i) IFN ct-2b, given at a dose of 9-18 MU/week, does not impair DMA and the functioning liver mass as assessed by MEGX and GEC respectively; ii) unlike genotype detection, pre-treatment assessment of liver function does not seem to help in the selection of pts most likely to respond to IFN treatment.
liverwt (tnn) 13_+2 9_+2*
granulomas (her LPF) 7+2 2_+1"
max joint (ram) 11_+2 7+.5*
A joint (mm~ 4.5_+1 .5_+.3*
Conclusions -IV Gad prevents liver granulomas and arthritis after IP PG-PS. Therefore, KCs are important in liver granuloma formation. Joint disease induced by IA PG-PS was not prevented by Gad treatment, indicating that Gad did not directly influence joint macrophages. This supports the hypothesis that KCs activated by PGPS mediate the recurrence of arthritis in this model.
1368
EFFECT OF UDCA ON LIVER IRON STORE IN RATS W I T H NORMAL OR IRON SUPPLEMENTED DIET. Robert. B Turlin. D Le Ouilleuc. G Lescoat. H. Mathiex,Fortanet*. Y
Deumaier. P Brissot. INSERM U49, University Hospital Pontchailiou, Rennes and * Laboratoire HOUDE,, Paris, France. Previous data suggest that umodeoxycbolic acid (UDCA) could facilitate iron biliery excretion (GastmenterOlogy,1991;101:1673). Conversely, bile salts may be important for iron absorption (Am J Physiol,1994;266:G-318). Therefore, if UDCA increases iron absorption this could expose patients under UDCA to the risk of developing liver iron excess. Aim. This work was performed to analyse the effect of U I ~ A on liver iron store in rats with normal or iron supplemented diet. Material and methods. 45 male Sprague-Dawiey rats were included in this study and divided in 9 groups of 5 rats. UDCA was administmd by garage 5 days a week at 25 mg/kg and iron overload was performed concomitantly using 5% carbonyl iron diet. Control groups were treated with UDCA solvant (PCB). Hepatic iron status was evaluated by liver iron concentration CLIC) measummunt and histologie assessment using the total iron score (TIS) (Gamoentemlogy,1992;102:2050). Results. Main results are ex ~ressedin the Table as mean:t: standard deviations. Tre~ment GROUP IJBW (%) TIS LIC (panole/g duration , of dry weight) 0 A= control 4.7+ 0.3 0 15.4+ 2.2 B= PCB 3.6.-+0.5 0 11.6:t: 1.5 1 month
C= U l n a D= PCB + iron
E= UDCA + iron F= PCB 2 months G=- UDCA H= PCB + iron I= UDCA + iron
3-5+ 0.2 3.2.-t:0.2
3.6:1:0.3 3.3+ 0.3 3.3+ 0.2 3.2:1:0.3 3.3+ 0.3
0.6£-0.9
12.8.~1.6 8.4.+2.1 0.6:i:0.6 0.2._+0.4 12.+.5.4 9.6+9.4
12.4+ 1.5
69.8+ 7.4 67.8+ 20.5 13.8+1.3 12.2.-I: 1.9 78.8+ 26 82.6+ 40.7
LIC was higher in iron treated groups compared with those without iron supplementation- Them was no significant modification of the liver/body weight ratio CL/BVOand LIC between rats treated or not with UDCA whatever the iron content of diet. In one month iron treated rats TIS was significantly lower in UDCA group (E) compare to PCB group (D) (p<0.02; MannWhitney's test). Condusions. (i) UDCA does not increase total liver iron stores in normal and iron overloaded rats as indicated by both histological and biochemical data; (ii) may, transiently, lower liver iron levels in iron overloaded rats as suggested by the histological trend. On the whole, UDCA is
not likely to be responsible for increased iron store in treated patients.
AASLD
ABSTRACTS
ENDOCYTOSIS IS AN ESSENTIAL STEP IN E N D O T O X I N (LPS)-STIMULATED T N F a F O R M A T I O N BY KUPFFER CELLS. SN Liehtmanl, J Wan~l, _C_7,Jaal~l, E DeFlom 1, JJ Lemnsters2. Depts. of Ipediatrics and 2Cell Biology & Anatomy, University of North Carolina, Chapel Hill.
1366
Introduction - Kupffer cells (KC) clear bacterial products from the intestine by endocytic uptake. A major bacterial polymer, LPS, activates KCs to release proinflammatory mediators involved in shock/sepsis, alcoholic liver disease and livel' reperfusion injury. Our Aim was to study the role of endocytosis in LPS-stimulation of KCs.
GADOLINIUM PREVENTS H E P A T I C GRANULOMAS AND ARTHRITIS INDUCED BY STREPTOCOCCAL • PEPTIDOGLYCAN-POLYSACCHARIDE (PG-PS). SN Lichlman. J Wang. C Zhang. JH Schwab. Dept. of Pediatrics, University of North Carolina, Chapel Hill, NC. Introduction -PG-PS is a major component of bacterial cell walls and IP injection induces relapsing arthritis and granulomatous hepatitis. Previous studies indicated the importance of the liver in chronic arthritis, by providing a reservoir of PG-PS or releasing mediator. The role of Kupffer cells (KC) in the evolution of these lesions was studied using GdC13 treatment (Gad) which causes KC death.
Methods - KCs were purified from Sprague-Dawley rat livers using
centrifugal eluttiation and cultured for 48 hr prior to use. Inhibitors were added for 1 hr before addition of LPS and then incubated with LPS (E. eoli 0111:B4) for 4 hr. Supematants were assayed for TNFa using ELISA. RNA was extracted after 90 rain to assess TNFa mRNA by Northern blots.
Methods -PG-PS (2 ktg/gm) from group A streptococci was injected
Results - Cytoehalasin B (blocks endocytosis) and bafilomycin A (inhibits the FP-ATPase pump) prevented LPS-stimulated TNFa release by 95% and 94%, respectively, (p < 0.001) and also prevented TNFa mRNA synthesis. Incubation of LPS with KCs for 5, 10, 15, 20, 30, and 60 rain induced 0%, 8%, 38%, 57%, 83%, and 94% of maximum TNFa release showing that 15-20 min exposure to LPS is needed to signal half maximal TNFa production.
of liver granulomas and prevented arthritis (Table, * = p < .001). IA PG-PS and Gad did not prevent arthritis or its reactivation by endotoxin.
IP and rats were treated with buffer or IV Gad (20 mg/kg 2X/wk) and studied 21 days later. To study its local effect on joint macrophages, Gad was administered with PG-PS intra-articularly (IA). Results -Gad treatment significantly decreased liver weight, number
Group (n) Buffer (6) Gad (6)
Conclusions - Endoc.vtosis and acidification of endosomal vesicles are important early steps in KC activation by LPS. The 10-20 rain need-ed to signal LPS-stimulated TNFa release may represent the time required for endocytic uptake and processing of the bacterial cell wall product.
1367
HEPATOLOGY O c t o b e r 1995
ASSESSMENT OF LIVER FUNCTION BEFORE, DURING AND AFTER INTERFERON (IFN) TREATMENT FOR CHRONIC HEPAqTHS C. _F_ Lirussi. M Crovatto °. G Santini°. A Beecarello. L Bortolato. C Triehes. C Paleari§, L Okolicsanvi* and G Creoaldi. Institute of Internal Medicine, University of Padua, °Microbiology and Immunology Unit, Pordenone Regional Hospital, §Institute of Clinical Chemistry, University of Padua and *Chair of Gastroenterology, University of Parma, Italy. There is evidence that IFN depresses the drug metabolizing activity (DMA) (Eur J Clin Pharmaenl, 39, 365, 1990; Hepatology, 17, 65, 1993). Conversely, liver function might influence treatment outcome in addition to host-, viral- and drug-related factors. We therefore: i) studied the effect of IFN on DMA and on the functioning liver mass as assessed by monoethylglycinexylidide (MEGX) formation from lidocaine and galactose elimination capacity (GEC), respectively and ii) investigated if there was any relationship between these quantitative LFTs and the response to IFN, including HCV genotypes (Okamoto's classification). Twenty-one pts with biopsy proven CAH (n=13) or cirrhosis (n=8) received rIFN ct-2b (Intron-A°, 6 MU tiw for 4 mo followed by 3 MU tiw for 8 too). MEGX formation (45-mni sample) and GEC test were measured in lg pts at mo 0, 4 and 12 and in g pts 6 mo after 1FN withdrawal. Results: Three pts withdrew from treaanent in the first 2-3 mo for severe side effects; 12 pts (genotype II n=5) showed complete or partial response (R) while 9 (type II n=7) were non responders (NR). Pre-treaanent MEGX values were 63 ng/ml±6 SEM in R and 53±5 in NR O--m) and did not change significantly in both R (68±7 at mo 4, 65±8 at mo 12) and in NR (67±7 at mo 4). 1FN withdrawal caused no changes in MEGX values (from 63±6 to 70~7). Pretreatment GEC values were similar in R and NR (2.3 mM/min~0.2 vs 1.9~.2) and remained stable throughout the treatment period as well as after IFN withdrawal in both groups. In eoneinsion: i) IFN ct-2b, given at a dose of 9-18 MU/week, does not impair DMA and the functioning liver mass as assessed by MEGX and GEC respectively; ii) unlike genotype detection, pre-treatment assessment of liver function does not seem to help in the selection of pts most likely to respond to IFN treatment.
liverwt (tnn) 13_+2 9_+2*
granulomas (her LPF) 7+2 2_+1"
max joint (ram) 11_+2 7+.5*
A joint (mm~ 4.5_+1 .5_+.3*
Conclusions -IV Gad prevents liver granulomas and arthritis after IP PG-PS. Therefore, KCs are important in liver granuloma formation. Joint disease induced by IA PG-PS was not prevented by Gad treatment, indicating that Gad did not directly influence joint macrophages. This supports the hypothesis that KCs activated by PGPS mediate the recurrence of arthritis in this model.
1368
EFFECT OF UDCA ON LIVER IRON STORE IN RATS W I T H NORMAL OR IRON SUPPLEMENTED DIET. Robert. B Turlin. D Le Ouilleuc. G Lescoat. H. Mathiex,Fortanet*. Y
Deumaier. P Brissot. INSERM U49, University Hospital Pontchailiou, Rennes and * Laboratoire HOUDE,, Paris, France. Previous data suggest that umodeoxycbolic acid (UDCA) could facilitate iron biliery excretion (GastmenterOlogy,1991;101:1673). Conversely, bile salts may be important for iron absorption (Am J Physiol,1994;266:G-318). Therefore, if UDCA increases iron absorption this could expose patients under UDCA to the risk of developing liver iron excess. Aim. This work was performed to analyse the effect of U I ~ A on liver iron store in rats with normal or iron supplemented diet. Material and methods. 45 male Sprague-Dawiey rats were included in this study and divided in 9 groups of 5 rats. UDCA was administmd by garage 5 days a week at 25 mg/kg and iron overload was performed concomitantly using 5% carbonyl iron diet. Control groups were treated with UDCA solvant (PCB). Hepatic iron status was evaluated by liver iron concentration CLIC) measummunt and histologie assessment using the total iron score (TIS) (Gamoentemlogy,1992;102:2050). Results. Main results are ex ~ressedin the Table as mean:t: standard deviations. Tre~ment GROUP IJBW (%) TIS LIC (panole/g duration , of dry weight) 0 A= control 4.7+ 0.3 0 15.4+ 2.2 B= PCB 3.6.-+0.5 0 11.6:t: 1.5 1 month
C= U l n a D= PCB + iron
E= UDCA + iron F= PCB 2 months G=- UDCA H= PCB + iron I= UDCA + iron
3-5+ 0.2 3.2.-t:0.2
3.6:1:0.3 3.3+ 0.3 3.3+ 0.2 3.2:1:0.3 3.3+ 0.3
0.6£-0.9
12.8.~1.6 8.4.+2.1 0.6:i:0.6 0.2._+0.4 12.+.5.4 9.6+9.4
12.4+ 1.5
69.8+ 7.4 67.8+ 20.5 13.8+1.3 12.2.-I: 1.9 78.8+ 26 82.6+ 40.7
LIC was higher in iron treated groups compared with those without iron supplementation- Them was no significant modification of the liver/body weight ratio CL/BVOand LIC between rats treated or not with UDCA whatever the iron content of diet. In one month iron treated rats TIS was significantly lower in UDCA group (E) compare to PCB group (D) (p<0.02; MannWhitney's test). Condusions. (i) UDCA does not increase total liver iron stores in normal and iron overloaded rats as indicated by both histological and biochemical data; (ii) may, transiently, lower liver iron levels in iron overloaded rats as suggested by the histological trend. On the whole, UDCA is
not likely to be responsible for increased iron store in treated patients.