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Haemophagocytic lymphohistiocytosis is a common and often fatal complication of severe liver dysfunction
300A
AASLD ABSTRACTS
HEPATOLOGY October 2001
511
512
REDUCED EXPRESSION OF THE ASTROCYTIC GLYCINE TRANSPORTER PROTEIN GLYT-1 IN FRONTAL CORTEX OF RATS W I T H ACUTE LIVER FAILURE. Claudia Zwingmann, Paul Desjardins, Adrianna Michalak, Alan S Hazell, Nicolas Chatauret, Roger F Butterworth, Neuroscience Research Unit, Hopital Saint-Lnc CHUM, Montreal, PQ Canada
HAEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS IS A COMMON AND OFTEN FATAL COMPLICATION OF SEVERE LIVER DYSFUNCTION. John Devlin, A Barnardo, G Auzinger, B Portmann, N Heaton, M Rela, J Wendon, King's College Hospital, London Umted Kingdom
Acute liver failure (ALF) results from severe inflammatory and/or necrotic liver disease. Hepatic encephalopathy (HE) is a serious neuropsychiatric complication of ALP whose pathogenesis remains incompletely understood. A leading hypothesis suggests that hyperammonemia and increased N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission may be central to the pathophysiology of HE in ALP. Endogenous ligands for the NMDA receptor in brain include glutamate (recognition site on the receptor) and glycine (modulatory site). Previous studies reveal a deficit in astrocytic clearance of glutamate in ALF (Hepatology 24, 908, 1996). Removal of glydne from brain extracellnlar space is mediated by a recently cloned and characterized high affinity astrocytic glycine transporter Glyt-1 which is expressed in brain regions with high NMDA receptor density; glycinergic neurons per se are restricted to spinal cord circuits in the mammalian central nervous system. The aim of the study was to measure Glyt-1 expression in relation to extracellular brain concentrations of glyeine during the progression of HE in ALP. Ischemic liver failure was induced in rats by portacaval anastomosis followed 24 h later by hepatic artery ligation. Appropriate sham-operated animals served as controls. Expression of Glyt-1 mRNA was studied by reverse transcription-polymerase chain reaction (RTPCR) and appropriate oligonucleotide primers. Immunohistochemical studies of 20 /xm sagittal sections and a polyclonal antibody to Glyt-I were used to investigate regional Glyt-1 protein expression. Brain extracellular fluid was collected by in vivo cerebral microdialysis using a 1 mm probe in frontal cortex at various times during the progression of encephalopathy in ALF rats. Extraceflular glycine concentrations were measured as their o-phthalaldehyde derivatives by HPLC using fluorescence detection. Expression of Glyt-1 mRNA was significantly decreased in the brain at coma stages of ALP to 51.58 +-9.61% of control (p<0.001). Immunohistochemical studies demonstrated a significant reduction in Glyt-1 immunolabelling of frontal cortex of ALF rats at coma stages of encephalopathy concomitant with significant 3-fold (p<0.01) increases of extracellular glycine. Increases of extracellular glycine paralleled the increases of extracellular glutamate and loss of glutamate transporter activity previously reported in brain in this model of ALP (Neurosci. Lett. 229,201, 1995). However, NMDA receptor densities per se (measured using quantitative receptor autoradiography and the selective ligand [3H]-MK801) were within normal limits in all brain structures of ALF rats. The finding of a loss of expression of the astrocytic glycine transporter Glyt-1 is consistent with the observed increase in extracellular glycine concentratiot~s. Impairment of the regulation of glycine concentrations at the synaptic level in the presence of unchanged NMDA receptor densities could contribute to an overactivation of the NMDA receptor in ALF. Modulation of astrocytic glycine transport and the use of NMDA receptor antagonists aimed specificallyat the glycine modulatory site, could offer novel approaches to the prevention and treatment of HE in ALF. [Funded by C1HR, Canada; CZ is recipient of a research award from the Quebec Ministry of Education]
An association between Haemophagocytic Lymphohistiocytosis (HLH) and liver disease is poorly defined. In this study, we review our experience of this condition when presenting in association with liver dysfunction including that following liver transplantation. 36 patients with hepatic dysfunction and histopathologically confirmed haemophagocytosis who fulfilled the Henter criteria were reviewed. In all cases a bone marrow aspirate / trephine was examined. This syndrome complicated three clinical groups: 1) Immunodeficient patients including liver transplant recipients, SLE etc 2) Haematological malignancy and 3) Acute liver failure (ALF) of unknown origin (NANB hepatitis). In the liver recipients, HLH complicated both early graft dysfunction and late cytomegalovirus infection and was associated with multi-organ failure and death in all cases. HLH was evident in 80% of adult transplant deaths in the last year (8 of 10). Fever was evident in the majority of the ALF cases with profound thrombocytopaenia invariable at presentation in all cases. Less than 10% of cases were diagnosed prior to referral. In groups 2 and 3, jaundice (mean 154 umoll), fever, splenomegaly and either pleural effusions or alveolar infiltrates were common at presentation. The AST activity (372 (iu/1) was modest. Haemophagocytosis was also present in post-mortem liver biopsies when available. Management protocols attempted in these patients included immunosuppression, chemotherapy, high-volume haemofihration, GCSF, intravenous immunoglobulin and anti-viral therapy. The response to these treatments was very poor with only four patients surviving. Disease progression was characterised by severe pancytopaenta, acute lung injury and circulatory failure. The association of HLH complicating acute liver disease merits urgent clinical and research attention. This report draws attention to the very unfavourable prognosis of this poorly recognised association and the difficulties encountered in management
513
514
UTILITY OF PROTON MAGNETIC RESONANCE SPECTROSCOPY (PMRS) IN CHILDREN W I T H LIVER FAILURE. Kathleen B Schwarz, Mary K Alford, Peter B Barker, Johns Hopkins Univ Sch of Medicine, Baltimore, MD
A COMPARISON OF MURINE HEPATIC AND RENAL DENDRITIC CELLS: PRE-TREATMENT W I T H TGF-I~ NOT IL-10 ABROGATES THE ALLOGENEIC T i l l RESPONSE IN VITRO. Matthias M Dollinger, Sarah E Howie, Jonathan R Lamb, Peter C Hayes, David J Harrison, University of Edinburgh, Edinburgh United Kingdom
PMRS is a new technique developed for delineation of the early diagnosis of metabolic disturbances in the brain which precede cerebral edema in patients with liver failure. Abnornlalities in adults with either fulminant or chronic liver failure include increased glutamine, and decreased myo-inositol and choline (Dig Dis Sci i996;i4:30-39). The purpose of our study was to report the application of this technique in six children. Sagittal Tl-weighted, axial T2weighted, axial flair images, axial diffusion-weighted scans and PRMS with TR/TE=2300/280 m/see were performed through the brain. Results were as follows: (See table) Clinical correlates: lethargy/poor school performance was the indication for the study in the chronic group; the 2 children with FHF recovered from hepatic coma. The 1-year had hyperammonemia x 1 week post-tx from primary non-function. Coma developed and re-tx was considered. After results of the above study, consistent with severe anoxia, re-ix was not performed and the infant has remained in a persistent vegetative state for months. Conclusion: PMRS is useful for diagnosing early cerebral metabolic disturbances in children with liver failure and also for distinguishing hepatic from anoxic encephalopathy. Peak Chronic AIH*
Age (y) 12
Byleds
7
Idopathic hepatitis Hepatitis 8
19
Acute FHF**
2
FHF
15
Post.tx * * *
1
5
Area Ratios (padetal gray padetal white matter/ matter) mltC CholCr NANCr NAA/Cho GLX/Cr MRI 0.10f 0.2tl 1.16t 5,58/ 0,711 Normat 0.14 635 1,60 4,52 0.96 0,71t 0.46/ 2.19t 3,40t 2.31/ Normal 0.49 0.91 2,09 4.42 2,14 0.47• 0,64I 2.t9t 3,40I 2.311 Heterolopic 0.61 0.47 2.09 4.42 2.14 suhependyma NIA N/A N/A N/A N/A Prominent 0,15 0.48 2.54 5.26 1,95 basal ganglia NtA NtA 0.701 0,71
0.36t 0.55 0.271 0.65
1.72t t.83 2.12/ 2.72
4.75• 335 7,84/ 4.20
2,46t 2,33 1,321 1,88
Normal Normal
Diffuse cerebral edema AIH*= autoimrnsne hepatitis; FHF**=fulminant hepatic failure; Post4x***=pest-transplant
Compared with other solid organs, the liver displays a unique tolerogenicity following transplantation. Dendritic cells (DC) might be implicated in this process and recently, we demonstrated a large resident population of lymphoid-related CD8o~+ DC within the tour/he liver, while renal DC were predominantly myeloid-related (J. Hepatol. 2001, 34:41). Functionally however, we found no differences between the DC populations in allogeneic T cell responses, raising the question if stimuli from the environment could influence the DC function. The cytokines IL-10 and TGF-/3 in particular have been implicated in tolerance induction and are produced by hepatic parenchymal ceils. Aim: To assess the effect of IL-10 and TGF-/3 on the ability of isolated hepatic and renal DC to stimulate primary allogeneic T cell responses. Methods: Primary murine DC (Balb/c) were isolated from liver, kidney and spleen by immuno-magnetic separation using the markers DEC-205 or CDllc. Following phenotypical analysis by flow cytometry (MHC II, B7-1/2, CD40 and CD8R ), DC were cultured for 48h with GM-CSF (100U/ml) alone or in combination with IL-10 (100U/ml) and TGF-beta (100U/ml). Stimulation of allogeneic splenic T cells (C3H; isolated by nylon wool) was assessed in a MLR by T cell proliferation ([3 H] thymidine incorporation) and INF-gamma release (ELISA). IL-10 and IL-12 p40 mRNA synthesis by DC was assessed using RT-PCR and quantitative Real-time PCR. Results: Resident hepatic (60% CD8c~+ ) and renal (<5% CD8ee+ ) DC differed in their lineage-related phenotype, but were both functionally immature with low expression of MHC class II and co-stimulatory signals (CD40, CD80 and CD86). Following 48h of culture with GM-CSF, both underwent functional maturation, up-regulated IL-10 and IL-12 mRNA synthesis and became as efficient as splenic DC in priming an allogeneic Thl response characterised by INF- T release. When exposed to TGF-I3 during their maturation process, both hepatic and renal DC lost their ability to induce a Th 1 response as demonstrated by T cell proliferation without significant INF-T release. In contrast, exposure of the DC to 1L-10 during the 48h of maturation resuhed only in a delayed development of the subsequent Thl-response. Analysis of the IL-10 and IL-12 mRNA synthesis revealed, that both cytokines were able to abrogated IL-12 mRNA production by the DC, however TGF-13 , but not IL-10, additionally induced a 6-fold increase of IL-10 mRNA synthesis as compared with stimulation by GM-CSF alone. Conclusion: Functionally, murine hepatic DC do not appear to behave inherently different in allogeneic DC-T cell interactions when compared with DC from other solid organs or lymphoid tissue. Environmental signals such as IL-10 and TGF-/3 are able to influence the DC function, however our results indicate that IL-10 has to be present during the antigen presentation to counter maturation signals provided by the T lymphocytes. TGFq3 in contrast seems to have a more lasting effect on DC function including up-regulation of IL-10 mRNA synthesis. Cytokines such as TGFq3 could therefore not only provide an explanation for organ-specific differences in DC function, but allow therapeutic approaches based on the functional similarity of organ-specific DC.
AASLD ABSTRACTS
HEPATOLOGY October 2001
511
512
REDUCED EXPRESSION OF THE ASTROCYTIC GLYCINE TRANSPORTER PROTEIN GLYT-1 IN FRONTAL CORTEX OF RATS W I T H ACUTE LIVER FAILURE. Claudia Zwingmann, Paul Desjardins, Adrianna Michalak, Alan S Hazell, Nicolas Chatauret, Roger F Butterworth, Neuroscience Research Unit, Hopital Saint-Lnc CHUM, Montreal, PQ Canada
HAEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS IS A COMMON AND OFTEN FATAL COMPLICATION OF SEVERE LIVER DYSFUNCTION. John Devlin, A Barnardo, G Auzinger, B Portmann, N Heaton, M Rela, J Wendon, King's College Hospital, London Umted Kingdom
Acute liver failure (ALF) results from severe inflammatory and/or necrotic liver disease. Hepatic encephalopathy (HE) is a serious neuropsychiatric complication of ALP whose pathogenesis remains incompletely understood. A leading hypothesis suggests that hyperammonemia and increased N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission may be central to the pathophysiology of HE in ALP. Endogenous ligands for the NMDA receptor in brain include glutamate (recognition site on the receptor) and glycine (modulatory site). Previous studies reveal a deficit in astrocytic clearance of glutamate in ALF (Hepatology 24, 908, 1996). Removal of glydne from brain extracellnlar space is mediated by a recently cloned and characterized high affinity astrocytic glycine transporter Glyt-1 which is expressed in brain regions with high NMDA receptor density; glycinergic neurons per se are restricted to spinal cord circuits in the mammalian central nervous system. The aim of the study was to measure Glyt-1 expression in relation to extracellular brain concentrations of glyeine during the progression of HE in ALP. Ischemic liver failure was induced in rats by portacaval anastomosis followed 24 h later by hepatic artery ligation. Appropriate sham-operated animals served as controls. Expression of Glyt-1 mRNA was studied by reverse transcription-polymerase chain reaction (RTPCR) and appropriate oligonucleotide primers. Immunohistochemical studies of 20 /xm sagittal sections and a polyclonal antibody to Glyt-I were used to investigate regional Glyt-1 protein expression. Brain extracellular fluid was collected by in vivo cerebral microdialysis using a 1 mm probe in frontal cortex at various times during the progression of encephalopathy in ALF rats. Extraceflular glycine concentrations were measured as their o-phthalaldehyde derivatives by HPLC using fluorescence detection. Expression of Glyt-1 mRNA was significantly decreased in the brain at coma stages of ALP to 51.58 +-9.61% of control (p<0.001). Immunohistochemical studies demonstrated a significant reduction in Glyt-1 immunolabelling of frontal cortex of ALF rats at coma stages of encephalopathy concomitant with significant 3-fold (p<0.01) increases of extracellular glycine. Increases of extracellular glycine paralleled the increases of extracellular glutamate and loss of glutamate transporter activity previously reported in brain in this model of ALP (Neurosci. Lett. 229,201, 1995). However, NMDA receptor densities per se (measured using quantitative receptor autoradiography and the selective ligand [3H]-MK801) were within normal limits in all brain structures of ALF rats. The finding of a loss of expression of the astrocytic glycine transporter Glyt-1 is consistent with the observed increase in extracellular glycine concentratiot~s. Impairment of the regulation of glycine concentrations at the synaptic level in the presence of unchanged NMDA receptor densities could contribute to an overactivation of the NMDA receptor in ALF. Modulation of astrocytic glycine transport and the use of NMDA receptor antagonists aimed specificallyat the glycine modulatory site, could offer novel approaches to the prevention and treatment of HE in ALF. [Funded by C1HR, Canada; CZ is recipient of a research award from the Quebec Ministry of Education]
An association between Haemophagocytic Lymphohistiocytosis (HLH) and liver disease is poorly defined. In this study, we review our experience of this condition when presenting in association with liver dysfunction including that following liver transplantation. 36 patients with hepatic dysfunction and histopathologically confirmed haemophagocytosis who fulfilled the Henter criteria were reviewed. In all cases a bone marrow aspirate / trephine was examined. This syndrome complicated three clinical groups: 1) Immunodeficient patients including liver transplant recipients, SLE etc 2) Haematological malignancy and 3) Acute liver failure (ALF) of unknown origin (NANB hepatitis). In the liver recipients, HLH complicated both early graft dysfunction and late cytomegalovirus infection and was associated with multi-organ failure and death in all cases. HLH was evident in 80% of adult transplant deaths in the last year (8 of 10). Fever was evident in the majority of the ALF cases with profound thrombocytopaenia invariable at presentation in all cases. Less than 10% of cases were diagnosed prior to referral. In groups 2 and 3, jaundice (mean 154 umoll), fever, splenomegaly and either pleural effusions or alveolar infiltrates were common at presentation. The AST activity (372 (iu/1) was modest. Haemophagocytosis was also present in post-mortem liver biopsies when available. Management protocols attempted in these patients included immunosuppression, chemotherapy, high-volume haemofihration, GCSF, intravenous immunoglobulin and anti-viral therapy. The response to these treatments was very poor with only four patients surviving. Disease progression was characterised by severe pancytopaenta, acute lung injury and circulatory failure. The association of HLH complicating acute liver disease merits urgent clinical and research attention. This report draws attention to the very unfavourable prognosis of this poorly recognised association and the difficulties encountered in management
513
514
UTILITY OF PROTON MAGNETIC RESONANCE SPECTROSCOPY (PMRS) IN CHILDREN W I T H LIVER FAILURE. Kathleen B Schwarz, Mary K Alford, Peter B Barker, Johns Hopkins Univ Sch of Medicine, Baltimore, MD
A COMPARISON OF MURINE HEPATIC AND RENAL DENDRITIC CELLS: PRE-TREATMENT W I T H TGF-I~ NOT IL-10 ABROGATES THE ALLOGENEIC T i l l RESPONSE IN VITRO. Matthias M Dollinger, Sarah E Howie, Jonathan R Lamb, Peter C Hayes, David J Harrison, University of Edinburgh, Edinburgh United Kingdom
PMRS is a new technique developed for delineation of the early diagnosis of metabolic disturbances in the brain which precede cerebral edema in patients with liver failure. Abnornlalities in adults with either fulminant or chronic liver failure include increased glutamine, and decreased myo-inositol and choline (Dig Dis Sci i996;i4:30-39). The purpose of our study was to report the application of this technique in six children. Sagittal Tl-weighted, axial T2weighted, axial flair images, axial diffusion-weighted scans and PRMS with TR/TE=2300/280 m/see were performed through the brain. Results were as follows: (See table) Clinical correlates: lethargy/poor school performance was the indication for the study in the chronic group; the 2 children with FHF recovered from hepatic coma. The 1-year had hyperammonemia x 1 week post-tx from primary non-function. Coma developed and re-tx was considered. After results of the above study, consistent with severe anoxia, re-ix was not performed and the infant has remained in a persistent vegetative state for months. Conclusion: PMRS is useful for diagnosing early cerebral metabolic disturbances in children with liver failure and also for distinguishing hepatic from anoxic encephalopathy. Peak Chronic AIH*
Age (y) 12
Byleds
7
Idopathic hepatitis Hepatitis 8
19
Acute FHF**
2
FHF
15
Post.tx * * *
1
5
Area Ratios (padetal gray padetal white matter/ matter) mltC CholCr NANCr NAA/Cho GLX/Cr MRI 0.10f 0.2tl 1.16t 5,58/ 0,711 Normat 0.14 635 1,60 4,52 0.96 0,71t 0.46/ 2.19t 3,40t 2.31/ Normal 0.49 0.91 2,09 4.42 2,14 0.47• 0,64I 2.t9t 3,40I 2.311 Heterolopic 0.61 0.47 2.09 4.42 2.14 suhependyma NIA N/A N/A N/A N/A Prominent 0,15 0.48 2.54 5.26 1,95 basal ganglia NtA NtA 0.701 0,71
0.36t 0.55 0.271 0.65
1.72t t.83 2.12/ 2.72
4.75• 335 7,84/ 4.20
2,46t 2,33 1,321 1,88
Normal Normal
Diffuse cerebral edema AIH*= autoimrnsne hepatitis; FHF**=fulminant hepatic failure; Post4x***=pest-transplant
Compared with other solid organs, the liver displays a unique tolerogenicity following transplantation. Dendritic cells (DC) might be implicated in this process and recently, we demonstrated a large resident population of lymphoid-related CD8o~+ DC within the tour/he liver, while renal DC were predominantly myeloid-related (J. Hepatol. 2001, 34:41). Functionally however, we found no differences between the DC populations in allogeneic T cell responses, raising the question if stimuli from the environment could influence the DC function. The cytokines IL-10 and TGF-/3 in particular have been implicated in tolerance induction and are produced by hepatic parenchymal ceils. Aim: To assess the effect of IL-10 and TGF-/3 on the ability of isolated hepatic and renal DC to stimulate primary allogeneic T cell responses. Methods: Primary murine DC (Balb/c) were isolated from liver, kidney and spleen by immuno-magnetic separation using the markers DEC-205 or CDllc. Following phenotypical analysis by flow cytometry (MHC II, B7-1/2, CD40 and CD8R ), DC were cultured for 48h with GM-CSF (100U/ml) alone or in combination with IL-10 (100U/ml) and TGF-beta (100U/ml). Stimulation of allogeneic splenic T cells (C3H; isolated by nylon wool) was assessed in a MLR by T cell proliferation ([3 H] thymidine incorporation) and INF-gamma release (ELISA). IL-10 and IL-12 p40 mRNA synthesis by DC was assessed using RT-PCR and quantitative Real-time PCR. Results: Resident hepatic (60% CD8c~+ ) and renal (<5% CD8ee+ ) DC differed in their lineage-related phenotype, but were both functionally immature with low expression of MHC class II and co-stimulatory signals (CD40, CD80 and CD86). Following 48h of culture with GM-CSF, both underwent functional maturation, up-regulated IL-10 and IL-12 mRNA synthesis and became as efficient as splenic DC in priming an allogeneic Thl response characterised by INF- T release. When exposed to TGF-I3 during their maturation process, both hepatic and renal DC lost their ability to induce a Th 1 response as demonstrated by T cell proliferation without significant INF-T release. In contrast, exposure of the DC to 1L-10 during the 48h of maturation resuhed only in a delayed development of the subsequent Thl-response. Analysis of the IL-10 and IL-12 mRNA synthesis revealed, that both cytokines were able to abrogated IL-12 mRNA production by the DC, however TGF-13 , but not IL-10, additionally induced a 6-fold increase of IL-10 mRNA synthesis as compared with stimulation by GM-CSF alone. Conclusion: Functionally, murine hepatic DC do not appear to behave inherently different in allogeneic DC-T cell interactions when compared with DC from other solid organs or lymphoid tissue. Environmental signals such as IL-10 and TGF-/3 are able to influence the DC function, however our results indicate that IL-10 has to be present during the antigen presentation to counter maturation signals provided by the T lymphocytes. TGFq3 in contrast seems to have a more lasting effect on DC function including up-regulation of IL-10 mRNA synthesis. Cytokines such as TGFq3 could therefore not only provide an explanation for organ-specific differences in DC function, but allow therapeutic approaches based on the functional similarity of organ-specific DC.