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RT-PCR demonstration of retinoid X receptor mRNA expression in rat Ito cells
At136
AASLD ABSTRACTS
A NOVEL METHOD FOR SIMULTANEOUS QUANTITATION OF VESICLE AGGREGATION AND FUSION: RELATION TO BILIARY LIPID SECRETION AND BILE METASTABIMTY H Ochi, S Tazuma, T Ohya, Y Hattori, K Horikawa, K Teramen, N Hirano, H Miura, D Tsuchimoto, H Miyake, T Kajihara, T Nishioka, S Hatsushika, G Kajiyama, K Itoh*. First Dept. Int. Med. & Dept. Radiology, Hiroshima Univ., 1-2-3, Kasumi, Minami-ku, Hiroshima 734, Japan. Lecithin-cholesterol vesicle is present in hepato-biliary systems, and its aggregation and fusion regulate hepatic lipid secretion and bile cholesterol metastabilify. Our previous reports suggest that such transformations are modulated by certain proteins (8BA 1166:25,1993) and fatty acid composition of lecithin (BBA 1167:142,1993 & BBA 1215:74,1994). Thus, the quantitative assessment of vesicle transformations would clarify the mechanistic role of factors affecting biliary lipid secretion and cholesterol metastabiUty in bile. AIM: To develope the method that simultaneously quantitates vesicle aggregation and fusion, and thereby to characterize mechanism(s) of action of effector substances on bile cholesterol metastability. METHODS: 1. Lipid vesicle solution was prepared using 100 uM phosphatidylserine (PS, bovine brain). 2. Octadecylrhodamine B (R18) was incorporated into vesicle membranes for fusion measurement. Vesicle aggregation was quantitatively assessed from the light scattered intensity (SLI) at 276 nm, and vesicle fusion was estimated by reduction in self-quenching of R18 fluorescence, simultaneously (incubation time, 30 min). 3. Divalent cations, Ca2+ (1-5 mM) and Mg2+ (1-5 mM), were added respectively to vesicle solutions, and effects on vesicle transformations were studied, RESULTS: 1. Aggregation and fusion of PS vesicles were simultaneously estimated in the present system. 2. Ca2+ was revealed to accelerate both vesicle aggregation and fusion, whereas Mg2+ accelerated vesicle aggregation but not fusion. Control Ca (raM) Mg (mM) 1 5 1 5 SLI (%) 100 145 160 150 165 R18 (%) 100 65 55 98 97 SUMMARY AND CONCLUSIONS: 1. A method for simultaneous quantitation of vesicle aggregation and fusion was developed. 2. Using this method, mechanistic roles of effector substances of cholesterol nucleation processes can be precisely clarified. Especially, actions of biliary proteins which bind to vesicles may be able to be further characterized.
PCR-DETECTION OF HBV AND HCV IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF HEMODIALYSIS PATIENTS. Ch~ _Oesterreicher, J. Hammer, U. Koch, F. Pfeffel, D. Petermann, G. SunderPlassmann*, Ch. MOiler. Depts. of Gastroenterology & Hepatology and Nephrology*, University of Vienna, AUSTRIA Patients undergoing chronic hemodialysis have an increased risk of hepatitis B- (HBV) and hepatitis C (HCV)-infection also in low endemic countries. Impaired cell-mediated and humoral immunity in patients with end-stage renal disease can result in undetectable levels of antibodies against HBV or HCV. Therefore, direct detection of virus genoms by polymerase chain reaction (PCR) is preferable but viral titer in serum can fall below the dete6tion limit of PER-based assays. Peripheral blood mononuclear cells (PBMC) are known to be susceptible to infections of both HBV and HCV. HYPOTHESIS: Detection of HBV and HCVgenomes in PMNC might uncover otherwise hidden infections in patients with end-stage renal disease. AIM: To determine the presence of HBV and HCV genomes in PMNC in patients with end-stage renal disease. METHODS: 67 patients (35 M, 32 F; range: 22,2 - 82,7 years; mean age 54,3 years; SD + 17) undergoing chronic hemodialysis (mean duration: 36,3 months; SD + 27,5; range: 5,5 - 185,2 months) were investigated for serologic markers of HBV and HCV as well as for the presence of HBV and HCV genomes in serum and in PBMC. Standard nested PCR analysis was used to detect HBV-DNA and HCV-RNA, respectively. RESULTS: None of the 67 patients was I-IBsAg positive "or showed positive HBVDNA in serum. But in 5 patients HBV-DNA in PBMC was detectable as marker for infection with hepatitis B virus; in addition those patients were also antiHBc negative. In 10 patients HCV-RNA was positive in serum by PCR; in 5 of those patients it was also positive in PBMC. 1 patient was positive for HCV-RNA in PBMC but was negative for HCV-RNA in serum. Of the 11 patients who had HCV-RNA present either in serum and/or in PBMC, 4 patients were negative for anti-HCV. Thus ,in 8,9°/, of patients undergoing chronic hemodialysis we found evidence of infection with I-IBV or HCV by detection of viral genomes in PBMC without the presence of detectable viremia, antigenemia or specific viral antibodies in serum. In SUMMARY. direct detection of viral genoms in PBMC is able to uncover HBV- or HCV-infections in hemodialysis patients which are no_Atdetectable by today's routinely performed hepatitis screening.
GASTROENTEROLOGY, Vol. 108, No. 4
HETEROGENOUS EXPRESSIONS OF CYTOKERATIN
SUBCLASSES
IN
INTRAHEPATIC BILIARY T R A C T IMPLICATIONS FOR A SPECIFIC BILE DUCT ANTIGEN IN PRIMARY BILIARY CIRRHOSIS. M. Oda, Y. Kamegaya) H. Kaneko) H. Yokomori, S. Kazemoto, T. Akiba) M. Nakamura, H. Ishii. Dept. of Internal Medicine) School of Medicine) Keio University) Tokyo) Japan We have reported that high titers of antibody against cytokeratin subclass CKI reflect bile duct destruction in primary biliary cirrhosis (PBC). A certain subeellular co mponent of bile duct epithelium is considered to become antigenic and to mediate bile duct damage through cellular and humoral immune responses. However, this antigenic component is still unknown. The aim of the present study is to provide evidence that CKI shows specific expressions in the biliary tract in PBC compared with another eytokeratin subclass. Surgical liver biopsy specimens from 6 patients with PBC (stage I~II) and from 6 unicteric patientswith gallstones were subjected to the indirect immunoperoxidase method using the monoelonal anti-cytokeratin subclass antibodies. The monoelonal anti-CKl antibody was produced by immunizing a Bal b/c mouse with CKI purified from Ptk2 cells, and identified as molecular weight 52kD by SDS-PAGE. Another monoclonal anti-CK M630 antibody (DAKO) was produced in the same manner against CK M630 (66 and 57kD) purified from human stratum corneum. CKI was most strongly expressed in the interlohular and septal bile duct epithelium in PBC. These expressions of CKI were most intense in th e degenerated epithelial cells of bile ducts characterized by chronic non-suppuratlve destructive eholangitis (CNSDC). On the other hand CKI was hardly expressed throughout the biliary tract in cholelithiasis. In contrast CK-M630 Was homogeneously evident throughout the epithelial cells of the biliary tract from bile ductules to septal bile ducts not only in PBC but also in cholelithiasis. These expressions of CK M630 showed no significant differences between PBC and cholelithiasis. In conclusion cytokeratin subclass CKI is aberrantly expressed particularly in the epithelial cells of bile ducts showing CNSDC, implying that the antigenecity of CKI may trigger the cellular and humoral immune responses in the process of bile duct destruction in PBC.
RT-PCR DEMONSTRATION OF RETINOID X RECEPTOR mRNA EXPRESSION IN RAT ITO CELLS. M. Ohata. M. Lin, H. Tsukamoto. USC Center for Liver Diseases. USC School of Med, Los Angeles, CA. Retinoid X receptors (RXR) which have high affinity for 9-cis retinoic acid, function as a transcription factor to regulate target genes such as CRBPII. They also work as an auxiliary protein to form a heterodimer with receptors for all-trans retinoic acid, vitamin D or thyroid hormone. the ligands shown to modulate proliferation and matrix gene expression by Ito cells. Thus the involvement of RXR as a co-regulator of biological responses by Ito cells can be inferred. However, RXR expression by Ito cells has not been reported. The presenl study has examined mRNA expression of three RXR isotypes by rat Ito cells by RT-PCR on RNA obtained from freshly isolated Ito cells. Livers of male Wistar rats were perfused with pronase and collagenase, followed by arabinogalactan gradient centrifugation to isolate Ito cells (purity>98%). RNA was immediately extracted and used for RT-PCR. After reverse transcription of Ito cell mRNA, cDNA was amplified using specific primers based on mouse cDNA sequences as shown below: RXRtx sense CAA TGG CGT CCT CAA GGT TC r1531-550 antisense ACT CCA CCT CGT TCT CAT TC nl856837 RXRI~ sense AGA TCA ACT CCA CAG TGT CG nt221-240 antisense TCT CCA TCC CCG TCT TrG TC nlflJ8-589 RXRT sense CAT CAC TTC TGC CAT GGG TC nl338-357 antisense ACT GGC ACA TfC TGC CTC AC hiS(O-787 Agarose gel electrophoresis demonstrated single bands of PCR product with expected sizes for RXRo~ (325b), RXRI~ (387b), and RXR? (468b). The identity of PCR products was verified by restriction enzyme cleavage. Using an amplified [~-actin PCR product which served as a control for each reaction, the relative mRNA expression for RXR isotypes was shown to be in order of 13>o~>y, [~ being slightly more abundant than ct, and T being barely detectable. In activated Ito ceils cultured on plastic dish for 2 wk, mRNA expression of RXR7 was prominently induced while that of RXRct and RXR~ were moderately increased. These results demonstrate the first detection of RXR mRNA expression in rat Ito cells and suggest possible involvement of the receptors in regulation of Ito cell biology. (Supported by a PHS grant AA06603 and Dept Vet Affairs Med Res)
AASLD ABSTRACTS
A NOVEL METHOD FOR SIMULTANEOUS QUANTITATION OF VESICLE AGGREGATION AND FUSION: RELATION TO BILIARY LIPID SECRETION AND BILE METASTABIMTY H Ochi, S Tazuma, T Ohya, Y Hattori, K Horikawa, K Teramen, N Hirano, H Miura, D Tsuchimoto, H Miyake, T Kajihara, T Nishioka, S Hatsushika, G Kajiyama, K Itoh*. First Dept. Int. Med. & Dept. Radiology, Hiroshima Univ., 1-2-3, Kasumi, Minami-ku, Hiroshima 734, Japan. Lecithin-cholesterol vesicle is present in hepato-biliary systems, and its aggregation and fusion regulate hepatic lipid secretion and bile cholesterol metastabilify. Our previous reports suggest that such transformations are modulated by certain proteins (8BA 1166:25,1993) and fatty acid composition of lecithin (BBA 1167:142,1993 & BBA 1215:74,1994). Thus, the quantitative assessment of vesicle transformations would clarify the mechanistic role of factors affecting biliary lipid secretion and cholesterol metastabiUty in bile. AIM: To develope the method that simultaneously quantitates vesicle aggregation and fusion, and thereby to characterize mechanism(s) of action of effector substances on bile cholesterol metastability. METHODS: 1. Lipid vesicle solution was prepared using 100 uM phosphatidylserine (PS, bovine brain). 2. Octadecylrhodamine B (R18) was incorporated into vesicle membranes for fusion measurement. Vesicle aggregation was quantitatively assessed from the light scattered intensity (SLI) at 276 nm, and vesicle fusion was estimated by reduction in self-quenching of R18 fluorescence, simultaneously (incubation time, 30 min). 3. Divalent cations, Ca2+ (1-5 mM) and Mg2+ (1-5 mM), were added respectively to vesicle solutions, and effects on vesicle transformations were studied, RESULTS: 1. Aggregation and fusion of PS vesicles were simultaneously estimated in the present system. 2. Ca2+ was revealed to accelerate both vesicle aggregation and fusion, whereas Mg2+ accelerated vesicle aggregation but not fusion. Control Ca (raM) Mg (mM) 1 5 1 5 SLI (%) 100 145 160 150 165 R18 (%) 100 65 55 98 97 SUMMARY AND CONCLUSIONS: 1. A method for simultaneous quantitation of vesicle aggregation and fusion was developed. 2. Using this method, mechanistic roles of effector substances of cholesterol nucleation processes can be precisely clarified. Especially, actions of biliary proteins which bind to vesicles may be able to be further characterized.
PCR-DETECTION OF HBV AND HCV IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF HEMODIALYSIS PATIENTS. Ch~ _Oesterreicher, J. Hammer, U. Koch, F. Pfeffel, D. Petermann, G. SunderPlassmann*, Ch. MOiler. Depts. of Gastroenterology & Hepatology and Nephrology*, University of Vienna, AUSTRIA Patients undergoing chronic hemodialysis have an increased risk of hepatitis B- (HBV) and hepatitis C (HCV)-infection also in low endemic countries. Impaired cell-mediated and humoral immunity in patients with end-stage renal disease can result in undetectable levels of antibodies against HBV or HCV. Therefore, direct detection of virus genoms by polymerase chain reaction (PCR) is preferable but viral titer in serum can fall below the dete6tion limit of PER-based assays. Peripheral blood mononuclear cells (PBMC) are known to be susceptible to infections of both HBV and HCV. HYPOTHESIS: Detection of HBV and HCVgenomes in PMNC might uncover otherwise hidden infections in patients with end-stage renal disease. AIM: To determine the presence of HBV and HCV genomes in PMNC in patients with end-stage renal disease. METHODS: 67 patients (35 M, 32 F; range: 22,2 - 82,7 years; mean age 54,3 years; SD + 17) undergoing chronic hemodialysis (mean duration: 36,3 months; SD + 27,5; range: 5,5 - 185,2 months) were investigated for serologic markers of HBV and HCV as well as for the presence of HBV and HCV genomes in serum and in PBMC. Standard nested PCR analysis was used to detect HBV-DNA and HCV-RNA, respectively. RESULTS: None of the 67 patients was I-IBsAg positive "or showed positive HBVDNA in serum. But in 5 patients HBV-DNA in PBMC was detectable as marker for infection with hepatitis B virus; in addition those patients were also antiHBc negative. In 10 patients HCV-RNA was positive in serum by PCR; in 5 of those patients it was also positive in PBMC. 1 patient was positive for HCV-RNA in PBMC but was negative for HCV-RNA in serum. Of the 11 patients who had HCV-RNA present either in serum and/or in PBMC, 4 patients were negative for anti-HCV. Thus ,in 8,9°/, of patients undergoing chronic hemodialysis we found evidence of infection with I-IBV or HCV by detection of viral genomes in PBMC without the presence of detectable viremia, antigenemia or specific viral antibodies in serum. In SUMMARY. direct detection of viral genoms in PBMC is able to uncover HBV- or HCV-infections in hemodialysis patients which are no_Atdetectable by today's routinely performed hepatitis screening.
GASTROENTEROLOGY, Vol. 108, No. 4
HETEROGENOUS EXPRESSIONS OF CYTOKERATIN
SUBCLASSES
IN
INTRAHEPATIC BILIARY T R A C T IMPLICATIONS FOR A SPECIFIC BILE DUCT ANTIGEN IN PRIMARY BILIARY CIRRHOSIS. M. Oda, Y. Kamegaya) H. Kaneko) H. Yokomori, S. Kazemoto, T. Akiba) M. Nakamura, H. Ishii. Dept. of Internal Medicine) School of Medicine) Keio University) Tokyo) Japan We have reported that high titers of antibody against cytokeratin subclass CKI reflect bile duct destruction in primary biliary cirrhosis (PBC). A certain subeellular co mponent of bile duct epithelium is considered to become antigenic and to mediate bile duct damage through cellular and humoral immune responses. However, this antigenic component is still unknown. The aim of the present study is to provide evidence that CKI shows specific expressions in the biliary tract in PBC compared with another eytokeratin subclass. Surgical liver biopsy specimens from 6 patients with PBC (stage I~II) and from 6 unicteric patientswith gallstones were subjected to the indirect immunoperoxidase method using the monoelonal anti-cytokeratin subclass antibodies. The monoelonal anti-CKl antibody was produced by immunizing a Bal b/c mouse with CKI purified from Ptk2 cells, and identified as molecular weight 52kD by SDS-PAGE. Another monoclonal anti-CK M630 antibody (DAKO) was produced in the same manner against CK M630 (66 and 57kD) purified from human stratum corneum. CKI was most strongly expressed in the interlohular and septal bile duct epithelium in PBC. These expressions of CKI were most intense in th e degenerated epithelial cells of bile ducts characterized by chronic non-suppuratlve destructive eholangitis (CNSDC). On the other hand CKI was hardly expressed throughout the biliary tract in cholelithiasis. In contrast CK-M630 Was homogeneously evident throughout the epithelial cells of the biliary tract from bile ductules to septal bile ducts not only in PBC but also in cholelithiasis. These expressions of CK M630 showed no significant differences between PBC and cholelithiasis. In conclusion cytokeratin subclass CKI is aberrantly expressed particularly in the epithelial cells of bile ducts showing CNSDC, implying that the antigenecity of CKI may trigger the cellular and humoral immune responses in the process of bile duct destruction in PBC.
RT-PCR DEMONSTRATION OF RETINOID X RECEPTOR mRNA EXPRESSION IN RAT ITO CELLS. M. Ohata. M. Lin, H. Tsukamoto. USC Center for Liver Diseases. USC School of Med, Los Angeles, CA. Retinoid X receptors (RXR) which have high affinity for 9-cis retinoic acid, function as a transcription factor to regulate target genes such as CRBPII. They also work as an auxiliary protein to form a heterodimer with receptors for all-trans retinoic acid, vitamin D or thyroid hormone. the ligands shown to modulate proliferation and matrix gene expression by Ito cells. Thus the involvement of RXR as a co-regulator of biological responses by Ito cells can be inferred. However, RXR expression by Ito cells has not been reported. The presenl study has examined mRNA expression of three RXR isotypes by rat Ito cells by RT-PCR on RNA obtained from freshly isolated Ito cells. Livers of male Wistar rats were perfused with pronase and collagenase, followed by arabinogalactan gradient centrifugation to isolate Ito cells (purity>98%). RNA was immediately extracted and used for RT-PCR. After reverse transcription of Ito cell mRNA, cDNA was amplified using specific primers based on mouse cDNA sequences as shown below: RXRtx sense CAA TGG CGT CCT CAA GGT TC r1531-550 antisense ACT CCA CCT CGT TCT CAT TC nl856837 RXRI~ sense AGA TCA ACT CCA CAG TGT CG nt221-240 antisense TCT CCA TCC CCG TCT TrG TC nlflJ8-589 RXRT sense CAT CAC TTC TGC CAT GGG TC nl338-357 antisense ACT GGC ACA TfC TGC CTC AC hiS(O-787 Agarose gel electrophoresis demonstrated single bands of PCR product with expected sizes for RXRo~ (325b), RXRI~ (387b), and RXR? (468b). The identity of PCR products was verified by restriction enzyme cleavage. Using an amplified [~-actin PCR product which served as a control for each reaction, the relative mRNA expression for RXR isotypes was shown to be in order of 13>o~>y, [~ being slightly more abundant than ct, and T being barely detectable. In activated Ito ceils cultured on plastic dish for 2 wk, mRNA expression of RXR7 was prominently induced while that of RXRct and RXR~ were moderately increased. These results demonstrate the first detection of RXR mRNA expression in rat Ito cells and suggest possible involvement of the receptors in regulation of Ito cell biology. (Supported by a PHS grant AA06603 and Dept Vet Affairs Med Res)