- Email: [email protected]
Differential gene expression profiles of HCV infected patients at the time of liver transplant (LT)
HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2 0 0 1
AASLD ABSTRACTS
445A
1091
1092
A TARGET DOMAIN IN THE NEWLY-DISCOVERED HCV ALTERNATE READING FRAME PROTEIN IDENTIFIED VgITH A N O V E L S E Q U E N C E
D Y N A M I C S O F H C V HYPERVARIABLE R E G I O N 1 (HVR1) QUASISPECIES I N C H I M P A N Z E E S DURING CHRONIC HCV I N F E C T I O N A N D EFF E C T OF PASSIVE TRANSFER O F A N T I - H C V I M M U N O G L O B U L I N . Saleem Kamili, Xiaofang Li, R o o h i Abubaker, D i v i s i o n o f Viral Hepatitis, NCID, Centers for Disease C o n t r o l a n d Prevention, Atlanta, GA; Patrizia Farci, Univ e r s i t y o f Cagliari, Cagliari Italy; J o h n Spelbring, Dorrie Carson, D i v i s i o n of Viral Hepatitis, NCID, Centers for Disease C o n t r o l a n d Prevention, Atlanta, GA; All Fattom~ Robert Naso, Nabi, Rockville, MD; Kris Krawczynski, D i v i s i o n o f Viral Hepatitis, NCID, Centers for Disease C o n t r o l a n d Prevention, Atlanta, GA
ANALYSIS P A C K A G E . Jose L W a l e w s k i , Toby R Keller, D e c h e r d D Stump, J u l i o A Gutierrez, W e s t y n B r a n c h - E l l i m a n , Alfredo Rodriguez, Gary Benson, Andrea D Branch, M o u n t Sinai School o f Medicine, N e w York, NY BACKGROUND.HCV was originally thought to express a single polyprotein translated from one open reading frame. Recently, immune responses to a new protein translated from an alternate reading frame, ARFP, were reported (Walewski et aL RNA, 2001; Fig). Efforts to develop vaccines and anti-HCV pharmaceuticals would benefit from more complete information about the full spectrum of HCV proteins and from improved methods for identifying features present in HCV proteins of all genotypes. METHODS. A novel sequence analysis package was developed to map conserved domains in ARFP and in conventional HCV proteins. This package provides a visual display of the site~ frequency, and character of point mutations and allows hundreds of protein sequences to be individually compared to standardized consensus sequences. The consensus sequences were constmcted so as to provide equal representat/on of genotypes from all parts of the world, eliminating the bias caused by overrepresentation in GenBank of certain genotypes, such as genotype 1. Analysiswas carried out on 255 full-length sequences of the HCV core protein and 234 sequences of ARFPs which contain at least 124 codons. As a point of reference, conventional homology analysis was also performed on a set of eight highly divergent full-length sequences. RESULTS. The average percent homology of the conventional proteins ranged from a high of approximately 90% among core proteins to a low of about 60% among E1 and NS2 proteins. The average percent homology of the ARFPs was about 63% and thus fell within the range of the HCV proteins of the standard reading frame. In the core protein, the new sequence analysis package revealed two short variable regions, one near the center of the protein (amino acids 70-78), and one at the carboxyl terminus (amino acids 185-191). At most positions (84%), the core amino acids are semi-variant or invariant. At these positions, 90% or more of the 255 sequences have the same amino acid as the core reference sequence. Like the core protein, ARFP contains many invariant and semi-variant amino acids. Within the 234 ARFP sequences, 43 amino acid positions (34% of the total) are identical in 90% or more of the sequences. A highly conserved, and potentially antigenic, domain spans positions 6-47 of ARFP. CONCLUSIONS.A novel sequence analysis package has been developed and used to analyze HCV proteins. It quickly maps conserved and variable domains, and identified a section of ARFP which may be a useful component of vaccines and diagnostic tests. Supported by grants: NSF CCR0073081 (GB) and NIH NIDDK R01 DK52071
(ADB).
CORE I Et I E~ I NS2 I NS3 I NS4a I nS4h I NSSa I NSSb gQS~ 61% 70% 62% 82% 69% 75% 64% 76%
ARFP
s3~
H C V G e n o m i c M a p S h o w i n g A R F P , and the A v e r a g e A m i n o Acid H o m o l o g y of H C V Proteins
Viralquasispeciesheterogeneity,whichmayhe influencedby both viraland host-immunefactors, has been shown to have clinicaland pathobiologlcalimplicationsin chronic HCV infection.The present study was designed to determine the dynamics of HVRI quasispeeies in chronically HCV-infected chimpanzees, which remain the onlyexperimentalmodelof HCVinfection.Three chimpanzees (CH1439, CH1483,and CH1556), inoculatedwithan HCVUS1 straingenotype Ia and havinga durationof chroincityrangingfrom 3 to 8 years, were studied. In addition,H'qR1quasispecieswere also evaluatedduring a 3-month period of anti-HCV infusions. Molecular analysis of the HVR1 region was performed in the inoeulum and serial serum samplescollectedpost-inoculation(p.i.) at 8-10month intervals( 16 timepointsduring 1-90months and 1-74 months in CH1439 and CH1483, respectively; and I1 time points during 1-41 months in CH1556). Intravenousinfusionsof anti-HCV immunoglobuBn(HCIg) [CivaeirTM, Nabi, Rockvifle,MD, USAI,virus-inactivated5% IgG,negativefor HCVRNA,were administered(100ragof lg/kgbodyweight2x per week for 15 weeks) to all three chimpanzees. HVR1 quasispecieswere determined by cloning and sequencing (an averageof 28 tones were sequenced fromeach timepoint sample).The geneticdiversityof HVRI during HCIginfusionswas measured by mean Hammingdistance. Overall, a total of 105 distinct variants, based on deduced amino acid sequences, were found in the analyzed samples. The master sequence (represented by > 50% clones) present in the inoculumwas maintainedas a dominantstrain in CH1439, CHI483, and CH1556until 64, 24, and 8 months p.i., respectively.A minor variantcharacterized by a singleamino acid change from hisridineto argininepresent at baselinein CH1439graduallyincreased in proportion over time and emerged as the dominantstrain from 68 to 90 months p.i. (the last sample analyzed). The same variant aLsoemerged as the predominant strain in CH1556 from 13 mondls p.i. Another variantalso characterized by a singleamino acid change fromserine to asparagine emergedas the dominant strain in CH1483 from 29-74 months p.i (last sample analyzed). Minor variants, often represented by singleclones, were detected in all the samples throughout the duration of chronicity, however, identical variantswere rarely detected in the subsequent sampleswithin any animal. Duringthe period of HCIginfusions,which was marked by a significantincrease in the levelsof anti-E2 antibody, reduced rote of HCV replicationin two of three chimpanzees, and decrease in the level of alanine aminotmrLsferase activityin all three animals, there was a significantdecrease in the proportion of the dominantsequence present at that time in all the three animals and multiple new minor variantsemerged. Mean Hamming distance, determined in 4 timepoints (2 before, I each during,and at the end of HCIginfusions),increased in all three animals after HCIg infusions. Molecularanalysis of HVRI in serial s~rum specimens from chronically infected chimpanzees indicated that the natural evolutionof chronic HCV infectionin chimpanzees is associatedwith a continuous change of genetic compositionof HVRland the occurrence of the newly emerged dominatingviral quasispecies. Although the original dominant sequence present in the inoculum persisted throughout the course of infection, it graduallydecreased in proportion and finally disappeared in CH1439and CH1483. Passivetransferof anti-HCVwas concordant withprominentchanges in the patterns ofHVRI quasispeeiesin serum and influencedthe geneticdiversityof the variantsduringthe treatment, suggestingpotentialpathobiologicsignificanceof immunepressure in chronic HCVinfection.
1093
1094
D I F F E R E N T I A L G E N E EXPRESSION PROFILES OF HCV INFECTED PATIENTS AT THE T I M E O F LIVER TRANSPLANT (LT). C h e n Liu, A n a n d Patel, H a i z h e n Zhu, H o n g s h a n Zhao, C h r i s t i n e Collins, Sarah Eckenrode, JinX i o n g She, Regino Gonzalez-Peralta, G a r y Davis, D a v i d Nelson, U n i v e r s i t y o f Florida, Gainesville, FL
PROGRESSION OF HEPATITIS C VIRUS (HCV) I N F E C T I O N : R E L A T I O N T O I N T R A H E P A T I C A N D PERIPHERAL HCV SPECIFIC C D 4 + T CELL
Background: The outcome of HCV infection is determined by the interaction between the virus and the host i m m u n e system. In its attempt to cIear the virus from the'. liver, the immune system contributes to the hepatocellular injury seen in chronically infected patients. Hypothesis: Genes encoding immune factors and cytokine signalling pathways are differentially expressed within the liver in HCV infection and play a role in diseaseprogression. Aim: To quantify the expression of mRNAs encoding host antiviral defense and immunoregulatory elements in liver tissue at the time of LT and determine if there is a role in the development of recurrent HCV infection. Methods: A eDNA microarray system that contains 832 cDNA clones corresponding to cytokines and related genes involved in immune regulation and signal transduction pathways was developed in our laboratories. Liver-derived RNA from controls (normal liver at the time of metastatic colon cancer resection) and explant tissue from HCV patients undergoing LT were used for array analysis. All LT patients had histologic follow-up after transplant and were categorized as: cholestatic HCV, minimal disease recurrence, or severe recurrence with at least bridging fibrosis. Results: To date, we have analyzed 15 explants from HCV cirrhotics who have at least 1-year post-transplant follow-up and 5 controls. While there is extensive variability among patients, s i g n i f c a n t up-regulation for certain inflammatory (TNF superfamily members, interferon inducible proteins, interferon gamma inducible protein) pathways exist in chronic HCV cirrhosis. Of interest, molecules such as MHC Class I and II subunits, beta-2 microglobulin and natural killer cell protein are uptegulated, while granzyme B, CD28 and CD37 are down-regulated. In addition to these overall changes, there is dramatic variability in the T h l f F h 2 cytokine expression between patients at the time of LT. For example, IL-12 expression was downregulated in 5 subjects (range - 0.2 to 0.5) and up-regulate in the 6 others (range +1.8 to 4.2), while IL-10 and IL-2 receptor alpha / beta expression are downregulated in. 10/15 of our patients, with levels as low as -0.16 compared to control. Similar wide variations are seen with IL 5, 7 and 13. In the two patients who developed a severe cholestatic hepatitis post-LT, there was preferential up-regulation of two small inducible cytokine subfamily members and three interferon-inducible proteins at the time of L2". Post-LT gene expression analysis is underway. Conclusions: (1) Despite the common final pathway of cirrhosis, unique and distinct differences exist in host i m m u n e gene expression at the time of transplant. (2) There is a marked up-regulation of genes involved in cellular immunity, implicating this pathway in the development of cirrhosis and (3) Host gene expression at the time of transplant may play a role in post-LT disease recurrence. Implications: These preliminary results indicate that differential gene expression may affect the pathogenic pathways in HCV infection. The potential contribution of these factors to the discordant outcomes seen after liver transplant is the focus of ongoing evaluation.
R E S P O N S E S A N D C Y T O K I N E P A T T E R N S . Sanaa M Kamal, H a r v a r d Insts of Medicine, Boston, M A ; J e n s W Rasenack, U n i v o f Freiburg, F r e i b u r g G e r m a n y ; C a m i l l a A G r a h a m , Beth Israel Deaconess Medical Ctr, Boston, MA; Leonardo Bianchi, U n i v of Basel, Basel Switzerland; Qe He, Beth Israel Deaconess Medical Ctr, Boston, MA; A h m e d A Tawil, A i n Shams Fac of Medicine, Cairo Egypt; M a h m o u d M Massoud, A i n S h a m s U n i v , Cairo Egypt; M a r g a r e t J Koziel, Beth Israel Deaconess Medical Ctr, Boston, MA BACKGROUND:Factors that influence disease progression and natural history of hepatitis C infection are to date not clearly defined. Patients co-infected with hepatitis C virus (HCV) and Schistosonm mansoni (S.mansoni), a parasitic disease characterized by T-helper 2 bias, show accelerated progression of liver disease compared to patients with HCV alone. This pattern of vira//parasitic co-infection thus offers a unique opportunity to study the human immune responses towards HCV in a relatively short time frame. We hypothesized that HCV specific immune responses in the early phase of HCV infection may account for the variability in the disease progression. OBJECTIVES:To determine the evolution and kinetics of intrahepatic and peripheral HCV specific CD4+ responses and cytokine patterns in a prospective study of 2 well characterized cohorts with either HCV alone or HCV & S.mansoni coinfection and correlate these parameters to the clinical outcome and progression of HCV infection. METHODS: 22 patients who failed to clear HCV vizemia after acute HCV infection (10 with HCV monoinfecrion: Group A and 12 with HCV & S.manoni coinfection: Group B) matched for age, gender, duration of HCV infection, HCV genotype, histological grading & staging at entry were enrolled and prospectively followed for a median of 72 months. Patients were clinically & virologically assessed at entry and semi-annually until the end of follow-up. Two liver biopsies were performed, at entry and at end of follow up, and evaluated (Ishak score) with estimation of the fibrosis progression rate/year lntrahepatfc and eripheral CD4+ T cell responses (proliferation assay) and IFN-T, TNF-a and IL-10 production Enzyme-linked immnnospot: ELISPOTassay) to a panel of HCV antigens were parallelly assessed at entry and end of follow-up. The mean number of spot-forming cells in 2 wells obtained without antigen (control) was subtracted from the mean of wells in the presence of HCV antigen. Results from both peripheral and intrahepatic compartments at entry and end of follow-up were compared for both groups and correlated to viral loads, histologic scores & rate of fibrosis progression. RESULTS:Although at baseline biopsies, both groups had no fibrosis, there was a significant difference in liver fibrosis scores at follow-up (4.3+/-0.9 in the coinfected group vs 0.8 +/-0.5 in monoinfected patients; P < 0.001); with fibrosis progression rate 0.58/year vs 0.08/year respectively (p<0.001). At baseline, HCV-specific CD4+ T cell responses with significant number of IFN--y and TNF-~ producing cells (Thl) in response to at least 2 HCV antigens were detected in both compartments with comparable vigor, frequency and breadth in 8/10 HCV monoinfected patients vs 5/12 coinfected patients, 2 of whom also produced IL-10 from intrahepatic cells (Th0/Th2). At end of follow-up, HCV CD4 + T cell responses were focused in the liver and showed significant difference in frequency, strength, breadth, cytokine pattern compared to the peripheral compartment. Intrahepatic CD4+ T cell responses were detected in 7/10 monoinfected patients (mainly IFN-7, TNF-a) & 4/12 coinfected patients (with only IL-10 production) vs 4/10 monoinfected (IFN-3,, TNF-a) and 1/12 coinfected patient (1L-10) in peripheral blood. The magnitude of the intrahepatic HCV-specificCD4+ response at baseline was inversely correlated with the fibrosis progression rate. CONCLUSION:HCV-specificCD4+ T cell responses compartmentalize to the liver once chronic infection is established. The marked qualitative and quantitative variations in the intrahepatic and peripheral HCV specific CD4+ responses and cytokine production detected in both {~roups suggest that failure to develop early significant Thl responses enhance disease progression.
~
AASLD ABSTRACTS
445A
1091
1092
A TARGET DOMAIN IN THE NEWLY-DISCOVERED HCV ALTERNATE READING FRAME PROTEIN IDENTIFIED VgITH A N O V E L S E Q U E N C E
D Y N A M I C S O F H C V HYPERVARIABLE R E G I O N 1 (HVR1) QUASISPECIES I N C H I M P A N Z E E S DURING CHRONIC HCV I N F E C T I O N A N D EFF E C T OF PASSIVE TRANSFER O F A N T I - H C V I M M U N O G L O B U L I N . Saleem Kamili, Xiaofang Li, R o o h i Abubaker, D i v i s i o n o f Viral Hepatitis, NCID, Centers for Disease C o n t r o l a n d Prevention, Atlanta, GA; Patrizia Farci, Univ e r s i t y o f Cagliari, Cagliari Italy; J o h n Spelbring, Dorrie Carson, D i v i s i o n of Viral Hepatitis, NCID, Centers for Disease C o n t r o l a n d Prevention, Atlanta, GA; All Fattom~ Robert Naso, Nabi, Rockville, MD; Kris Krawczynski, D i v i s i o n o f Viral Hepatitis, NCID, Centers for Disease C o n t r o l a n d Prevention, Atlanta, GA
ANALYSIS P A C K A G E . Jose L W a l e w s k i , Toby R Keller, D e c h e r d D Stump, J u l i o A Gutierrez, W e s t y n B r a n c h - E l l i m a n , Alfredo Rodriguez, Gary Benson, Andrea D Branch, M o u n t Sinai School o f Medicine, N e w York, NY BACKGROUND.HCV was originally thought to express a single polyprotein translated from one open reading frame. Recently, immune responses to a new protein translated from an alternate reading frame, ARFP, were reported (Walewski et aL RNA, 2001; Fig). Efforts to develop vaccines and anti-HCV pharmaceuticals would benefit from more complete information about the full spectrum of HCV proteins and from improved methods for identifying features present in HCV proteins of all genotypes. METHODS. A novel sequence analysis package was developed to map conserved domains in ARFP and in conventional HCV proteins. This package provides a visual display of the site~ frequency, and character of point mutations and allows hundreds of protein sequences to be individually compared to standardized consensus sequences. The consensus sequences were constmcted so as to provide equal representat/on of genotypes from all parts of the world, eliminating the bias caused by overrepresentation in GenBank of certain genotypes, such as genotype 1. Analysiswas carried out on 255 full-length sequences of the HCV core protein and 234 sequences of ARFPs which contain at least 124 codons. As a point of reference, conventional homology analysis was also performed on a set of eight highly divergent full-length sequences. RESULTS. The average percent homology of the conventional proteins ranged from a high of approximately 90% among core proteins to a low of about 60% among E1 and NS2 proteins. The average percent homology of the ARFPs was about 63% and thus fell within the range of the HCV proteins of the standard reading frame. In the core protein, the new sequence analysis package revealed two short variable regions, one near the center of the protein (amino acids 70-78), and one at the carboxyl terminus (amino acids 185-191). At most positions (84%), the core amino acids are semi-variant or invariant. At these positions, 90% or more of the 255 sequences have the same amino acid as the core reference sequence. Like the core protein, ARFP contains many invariant and semi-variant amino acids. Within the 234 ARFP sequences, 43 amino acid positions (34% of the total) are identical in 90% or more of the sequences. A highly conserved, and potentially antigenic, domain spans positions 6-47 of ARFP. CONCLUSIONS.A novel sequence analysis package has been developed and used to analyze HCV proteins. It quickly maps conserved and variable domains, and identified a section of ARFP which may be a useful component of vaccines and diagnostic tests. Supported by grants: NSF CCR0073081 (GB) and NIH NIDDK R01 DK52071
(ADB).
CORE I Et I E~ I NS2 I NS3 I NS4a I nS4h I NSSa I NSSb gQS~ 61% 70% 62% 82% 69% 75% 64% 76%
ARFP
s3~
H C V G e n o m i c M a p S h o w i n g A R F P , and the A v e r a g e A m i n o Acid H o m o l o g y of H C V Proteins
Viralquasispeciesheterogeneity,whichmayhe influencedby both viraland host-immunefactors, has been shown to have clinicaland pathobiologlcalimplicationsin chronic HCV infection.The present study was designed to determine the dynamics of HVRI quasispeeies in chronically HCV-infected chimpanzees, which remain the onlyexperimentalmodelof HCVinfection.Three chimpanzees (CH1439, CH1483,and CH1556), inoculatedwithan HCVUS1 straingenotype Ia and havinga durationof chroincityrangingfrom 3 to 8 years, were studied. In addition,H'qR1quasispecieswere also evaluatedduring a 3-month period of anti-HCV infusions. Molecular analysis of the HVR1 region was performed in the inoeulum and serial serum samplescollectedpost-inoculation(p.i.) at 8-10month intervals( 16 timepointsduring 1-90months and 1-74 months in CH1439 and CH1483, respectively; and I1 time points during 1-41 months in CH1556). Intravenousinfusionsof anti-HCV immunoglobuBn(HCIg) [CivaeirTM, Nabi, Rockvifle,MD, USAI,virus-inactivated5% IgG,negativefor HCVRNA,were administered(100ragof lg/kgbodyweight2x per week for 15 weeks) to all three chimpanzees. HVR1 quasispecieswere determined by cloning and sequencing (an averageof 28 tones were sequenced fromeach timepoint sample).The geneticdiversityof HVRI during HCIginfusionswas measured by mean Hammingdistance. Overall, a total of 105 distinct variants, based on deduced amino acid sequences, were found in the analyzed samples. The master sequence (represented by > 50% clones) present in the inoculumwas maintainedas a dominantstrain in CH1439, CHI483, and CH1556until 64, 24, and 8 months p.i., respectively.A minor variantcharacterized by a singleamino acid change from hisridineto argininepresent at baselinein CH1439graduallyincreased in proportion over time and emerged as the dominantstrain from 68 to 90 months p.i. (the last sample analyzed). The same variant aLsoemerged as the predominant strain in CH1556 from 13 mondls p.i. Another variantalso characterized by a singleamino acid change fromserine to asparagine emergedas the dominant strain in CH1483 from 29-74 months p.i (last sample analyzed). Minor variants, often represented by singleclones, were detected in all the samples throughout the duration of chronicity, however, identical variantswere rarely detected in the subsequent sampleswithin any animal. Duringthe period of HCIginfusions,which was marked by a significantincrease in the levelsof anti-E2 antibody, reduced rote of HCV replicationin two of three chimpanzees, and decrease in the level of alanine aminotmrLsferase activityin all three animals, there was a significantdecrease in the proportion of the dominantsequence present at that time in all the three animals and multiple new minor variantsemerged. Mean Hamming distance, determined in 4 timepoints (2 before, I each during,and at the end of HCIginfusions),increased in all three animals after HCIg infusions. Molecularanalysis of HVRI in serial s~rum specimens from chronically infected chimpanzees indicated that the natural evolutionof chronic HCV infectionin chimpanzees is associatedwith a continuous change of genetic compositionof HVRland the occurrence of the newly emerged dominatingviral quasispecies. Although the original dominant sequence present in the inoculum persisted throughout the course of infection, it graduallydecreased in proportion and finally disappeared in CH1439and CH1483. Passivetransferof anti-HCVwas concordant withprominentchanges in the patterns ofHVRI quasispeeiesin serum and influencedthe geneticdiversityof the variantsduringthe treatment, suggestingpotentialpathobiologicsignificanceof immunepressure in chronic HCVinfection.
1093
1094
D I F F E R E N T I A L G E N E EXPRESSION PROFILES OF HCV INFECTED PATIENTS AT THE T I M E O F LIVER TRANSPLANT (LT). C h e n Liu, A n a n d Patel, H a i z h e n Zhu, H o n g s h a n Zhao, C h r i s t i n e Collins, Sarah Eckenrode, JinX i o n g She, Regino Gonzalez-Peralta, G a r y Davis, D a v i d Nelson, U n i v e r s i t y o f Florida, Gainesville, FL
PROGRESSION OF HEPATITIS C VIRUS (HCV) I N F E C T I O N : R E L A T I O N T O I N T R A H E P A T I C A N D PERIPHERAL HCV SPECIFIC C D 4 + T CELL
Background: The outcome of HCV infection is determined by the interaction between the virus and the host i m m u n e system. In its attempt to cIear the virus from the'. liver, the immune system contributes to the hepatocellular injury seen in chronically infected patients. Hypothesis: Genes encoding immune factors and cytokine signalling pathways are differentially expressed within the liver in HCV infection and play a role in diseaseprogression. Aim: To quantify the expression of mRNAs encoding host antiviral defense and immunoregulatory elements in liver tissue at the time of LT and determine if there is a role in the development of recurrent HCV infection. Methods: A eDNA microarray system that contains 832 cDNA clones corresponding to cytokines and related genes involved in immune regulation and signal transduction pathways was developed in our laboratories. Liver-derived RNA from controls (normal liver at the time of metastatic colon cancer resection) and explant tissue from HCV patients undergoing LT were used for array analysis. All LT patients had histologic follow-up after transplant and were categorized as: cholestatic HCV, minimal disease recurrence, or severe recurrence with at least bridging fibrosis. Results: To date, we have analyzed 15 explants from HCV cirrhotics who have at least 1-year post-transplant follow-up and 5 controls. While there is extensive variability among patients, s i g n i f c a n t up-regulation for certain inflammatory (TNF superfamily members, interferon inducible proteins, interferon gamma inducible protein) pathways exist in chronic HCV cirrhosis. Of interest, molecules such as MHC Class I and II subunits, beta-2 microglobulin and natural killer cell protein are uptegulated, while granzyme B, CD28 and CD37 are down-regulated. In addition to these overall changes, there is dramatic variability in the T h l f F h 2 cytokine expression between patients at the time of LT. For example, IL-12 expression was downregulated in 5 subjects (range - 0.2 to 0.5) and up-regulate in the 6 others (range +1.8 to 4.2), while IL-10 and IL-2 receptor alpha / beta expression are downregulated in. 10/15 of our patients, with levels as low as -0.16 compared to control. Similar wide variations are seen with IL 5, 7 and 13. In the two patients who developed a severe cholestatic hepatitis post-LT, there was preferential up-regulation of two small inducible cytokine subfamily members and three interferon-inducible proteins at the time of L2". Post-LT gene expression analysis is underway. Conclusions: (1) Despite the common final pathway of cirrhosis, unique and distinct differences exist in host i m m u n e gene expression at the time of transplant. (2) There is a marked up-regulation of genes involved in cellular immunity, implicating this pathway in the development of cirrhosis and (3) Host gene expression at the time of transplant may play a role in post-LT disease recurrence. Implications: These preliminary results indicate that differential gene expression may affect the pathogenic pathways in HCV infection. The potential contribution of these factors to the discordant outcomes seen after liver transplant is the focus of ongoing evaluation.
R E S P O N S E S A N D C Y T O K I N E P A T T E R N S . Sanaa M Kamal, H a r v a r d Insts of Medicine, Boston, M A ; J e n s W Rasenack, U n i v o f Freiburg, F r e i b u r g G e r m a n y ; C a m i l l a A G r a h a m , Beth Israel Deaconess Medical Ctr, Boston, MA; Leonardo Bianchi, U n i v of Basel, Basel Switzerland; Qe He, Beth Israel Deaconess Medical Ctr, Boston, MA; A h m e d A Tawil, A i n Shams Fac of Medicine, Cairo Egypt; M a h m o u d M Massoud, A i n S h a m s U n i v , Cairo Egypt; M a r g a r e t J Koziel, Beth Israel Deaconess Medical Ctr, Boston, MA BACKGROUND:Factors that influence disease progression and natural history of hepatitis C infection are to date not clearly defined. Patients co-infected with hepatitis C virus (HCV) and Schistosonm mansoni (S.mansoni), a parasitic disease characterized by T-helper 2 bias, show accelerated progression of liver disease compared to patients with HCV alone. This pattern of vira//parasitic co-infection thus offers a unique opportunity to study the human immune responses towards HCV in a relatively short time frame. We hypothesized that HCV specific immune responses in the early phase of HCV infection may account for the variability in the disease progression. OBJECTIVES:To determine the evolution and kinetics of intrahepatic and peripheral HCV specific CD4+ responses and cytokine patterns in a prospective study of 2 well characterized cohorts with either HCV alone or HCV & S.mansoni coinfection and correlate these parameters to the clinical outcome and progression of HCV infection. METHODS: 22 patients who failed to clear HCV vizemia after acute HCV infection (10 with HCV monoinfecrion: Group A and 12 with HCV & S.manoni coinfection: Group B) matched for age, gender, duration of HCV infection, HCV genotype, histological grading & staging at entry were enrolled and prospectively followed for a median of 72 months. Patients were clinically & virologically assessed at entry and semi-annually until the end of follow-up. Two liver biopsies were performed, at entry and at end of follow up, and evaluated (Ishak score) with estimation of the fibrosis progression rate/year lntrahepatfc and eripheral CD4+ T cell responses (proliferation assay) and IFN-T, TNF-a and IL-10 production Enzyme-linked immnnospot: ELISPOTassay) to a panel of HCV antigens were parallelly assessed at entry and end of follow-up. The mean number of spot-forming cells in 2 wells obtained without antigen (control) was subtracted from the mean of wells in the presence of HCV antigen. Results from both peripheral and intrahepatic compartments at entry and end of follow-up were compared for both groups and correlated to viral loads, histologic scores & rate of fibrosis progression. RESULTS:Although at baseline biopsies, both groups had no fibrosis, there was a significant difference in liver fibrosis scores at follow-up (4.3+/-0.9 in the coinfected group vs 0.8 +/-0.5 in monoinfected patients; P < 0.001); with fibrosis progression rate 0.58/year vs 0.08/year respectively (p<0.001). At baseline, HCV-specific CD4+ T cell responses with significant number of IFN--y and TNF-~ producing cells (Thl) in response to at least 2 HCV antigens were detected in both compartments with comparable vigor, frequency and breadth in 8/10 HCV monoinfected patients vs 5/12 coinfected patients, 2 of whom also produced IL-10 from intrahepatic cells (Th0/Th2). At end of follow-up, HCV CD4 + T cell responses were focused in the liver and showed significant difference in frequency, strength, breadth, cytokine pattern compared to the peripheral compartment. Intrahepatic CD4+ T cell responses were detected in 7/10 monoinfected patients (mainly IFN-7, TNF-a) & 4/12 coinfected patients (with only IL-10 production) vs 4/10 monoinfected (IFN-3,, TNF-a) and 1/12 coinfected patient (1L-10) in peripheral blood. The magnitude of the intrahepatic HCV-specificCD4+ response at baseline was inversely correlated with the fibrosis progression rate. CONCLUSION:HCV-specificCD4+ T cell responses compartmentalize to the liver once chronic infection is established. The marked qualitative and quantitative variations in the intrahepatic and peripheral HCV specific CD4+ responses and cytokine production detected in both {~roups suggest that failure to develop early significant Thl responses enhance disease progression.
~