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C D 8 αα T lymphocytes in human adult liver — evidence for extrathymic T cell differentiation?
126A
AASLD ABSTRACTS
77 DOES LONG TERM CALCITONIN,
CALCIUM AND VIT D 3 ADMINISTRATION IN PRIMARY BILIARY CIRRHOSIS (PBC) PATIENTS PREVENTS BONE DBMINERALIZATION? E Mor.teiro, J Garcia-Costa, M Masearenhas. A Morbey, A Galv~o-Teles. Gas:to and Endocrine Units Sta. Maria University Hospital. Lisbon, Portugal.
Bone demineralization usually complicates PBC patients, mainly in post menopausal women. The aim of this prospective vertical study was to evaluate the bone mass density (BMD, g/cm~) in PBC patients o'~ calcium (Ca), salmon ealcitonin (CT) and Vit D3. Material and Methods: A group of 9 PBC caucasian women [treated with: Ca 500mg/day + Vit Ds: 200.000 U/month + CT: 100 U intranasal 15 days per month, longer than lyr (mean age + SO: 58.4 + 12.4 yr)] was compared with a control group of 9 healthy caucasian women (m _+ SD: 58.4 + 12.4) matched for age. BMD was evaluated by dual-energy x-ray absorptiometry at the lumbar spine (L2 -L4), femoral neck, troehanter and ward's triangle using a Norland densitometer. Body mass index (BMI, Kg/m2) was calculated. Data were evaluated by the One-Way Anova and the Spearman's correlation coefficient tests. Results: The mean age, BMI and BMD at the 4 sites were identical in both groups (see Table).
Age BMI BMD L2-L4 femoral neck trochanter ward's triangle
PBC Group
Control Group
p
58.4 + 12.4 27.8 + 3.4 0.839_+0.2 0.728 -+ 0.1 0.577-+0.1 0:503 + 0.1
58.4 -+ 12.4 27=2 + 4.0 0.865"_+0.1 0.741+0.1 0.615-+0.1 0.558-+0.1
NS NS NS NS NS NS
However, positive relationships BMI/BMD at the lumbar spine and at the trocbanter (r2>0.5) were detected in PBC group but not in the control group. Confusions: PBC patients treated for more than one year with CT, Ca and Vit D3 and control women have similar BMD. BMD increases with BMI in the PBC patients, showing the importance of nutrition in these patients.
79
RAGI, RAG2 AND T d T EXPRESSION IN ADULT HUMAN LIVER: EVIDENCE OF EXTRATHYMIC T CELL DIFFERENTIATION.
C. CollinsI , S. Norris1,2 G. McEntee3, O. Traynor3. J. He,arty2___& C. O'Farretlyl: ERC 1, Liver Unit2 & Dept. of Surgery3, St. Vincent's HOspital,Elm Park, Dublin 4, Ireland. INTRODUCTION: Animal studies have suggested that T lymphocyte maturation occurs in extrathymic sites including the liver. Expression of Recombination Activator Genes l & 2 (RAG 1 & 2) and Terminal Deoxynuchiotidyl Transferase (TdT) are required for T cell antigen receptor gene rearrangement and are used as markers of extrathymic T cell diffesentiadon. AIM: T o determine i f adult human liver is a site of R A G 1, R A G 2 and T d T gene expresssion. METHODS: Normal human liver tissue was obtaiued from five d o n o r organs at time of transplantation and total R N A isolated. R T - P C R was used to detect R A G I , R A G 2 and T d T m R N A in normal adult liver tissue, e D N A synthesis was carded out using A M V reverse transcriptase. Primers were specifically designed for P C R of haman RAG1, R A G 2 and T d T eDNA. R E S U L T S : Four adult bone marrow specimens were positive for R A G 1 , R A G 2 and T d T expression aud were used as positive controls. Peripheral blood mononuclear cell (PBMC) preparations from four individuals were negative for R A G and T d T specific m R N A . All liver specimens were positive for both R A G 1 and R A G 2 m R N A after nested P C R of liver cDNA. T d T expression was detected in two liver specimens after southern b lo ttin g of P C R product o n t o n y l o n m e m b r a n e and p r o b i n g with a digoxigenin labelled TdT specific oligonuleotide probe (Table 1.). Table 1
TISSUES LIVER 1 LIVER2 LIVERS LIVER4
RAG l + + + +
RAG 2 + + + ÷
Tdr NO NO ND
LIVER 5 PBMC l
+
+
+
+ + +
+ + +
+ + +
PBMC 2 PBMC 3 PBMC 4 PBMC S BONEMARROW 1 BONEMARROW2 BONEMARROWS BONEMARROW4
CONCLUSION: . Expression of R A G 1, 2 mid T d T ill adult huma n liver provides strong evidence that T cells can differentiate within this organ; these findings may have major implications for the generation of graft tolerance post liver transplantation.
78
HEPATOLOGYOctober 1995
Hypercoagulability in patients with Primary Biliary Cirrhosis (PBC) and Primary Sclerosing Cholangltis (PSC) evaluated by thromboelastography.
Z Ben4Ut, M Panagou, D Patch, S Bates', E Osman, AK Burroughs. Hepatobiliary and Liver Transplantation Unit, and ~Department of Anaesthesia, Royal Free Hospital, Pond Street, LONDON UK. Patients with PBC and PSC survive variceal bleeding better than alcoholic cirrhotics and have less bleeding at liver transplantation. We hypothesized that PBC and PSC patients may be hypercoagulable. As this is difficult to diagnose with routine laboratory tests, we used thromboelastography (TEG), which is a simple technique for evaluating whole blood clotting and fibrinolysis to establish if hypercoagulability was present, defined by TEG values greater than 2SD over controls: r < 19 mm (this reflects plasma clotting factors), maximum amplitude (ma) > 6 0 mm, and alpha angle > 43 ° (this reflects platelets and fibrinogen levels). We evaluated 48 PBC and 21 PSC patients and 40 noncholestatic cirrhotic patients as a control group with TEG, full blood count, prothrombin time (PT), partial thromboplastin time (PTTK), fibrinogen, protein S, C, anti thrombin III levels and activated protein C (APC) phenotype. Two or more TEG abnormalities indicating hypercoagulability were diagnosed in 14 of 48 (29%) PBC and in 9 of 21 (43%) PSC patients independent Of cirrhosis, but only in 2 of 40 (5%) of the control group, p
80 C D 8 a c t T
Lymphocytes in human adult liver evidence for extrathymic T cell differentiation? S~ Norris. C. Collins. I. H e , a r t y : C. O'Farrellg. Liver Unit a n d Education & Research Centre, St. Vincent's Hospital, Dublin, Ireland. The liver is not g e n e r a l l y t h o u g h t to be i m m u n o l o g i c a l l y i m p o r t a n t a n d l i t t l e is k n o w n a b o u t t h e p h e n o t y p i c p r o p e r t i e s of local T lymphocytes. The a i m of this s t u d y was to p e r f o r m flow cytometric analyses of hepatic T l y m p h o c y t e s u b p o p u l a t i o n s f r o m a d u l t n o r m a l liver. M a t e r i a l s a n d m e t h o d s : N o r m a l h e p a t i c t i s s u e (wet w e i g h t 2 0 0 g ) was o b t a i n e d from five d o n o r organs at time of t r a n s p l a n t , a n d single cell s u s p e n s i o n s were p r e p a r e d b y e n z y m a t i c a n d m e c h a n i c a l dissociation. Cells were s t a i n e d with m o n o c l o n a l antibodies specific for CD8a a n d CD8[~ chains, CD3, CD4, CDS, CD19, CD56, CD16, aISTCR, a n d y6TCR, a n d a n a l y z e d b y two colour and t h r e e colour flow c y t o m e t r y . Results: Cell yields r a n g e d from 0 . 4 - 3 . 7 x 1 0 6 / m l with a m e a n v i a b i l i t y of 79% (range 71-90%). O f the hepatic CD3+ cells, 72% were CD8+ and 23% were CD4+, with a CD4/8 ratio of 1:3 in c o n t r a s t to the p e r i p h e r a l CD4/8 ratio of 2:1. CD19+ cells a c c o u n t e d for 1.4% of the total CD3+ p o p u l a t i o n (range 1.1-1.7%), while 16.9% of the gate e x p r e s s e d NK s u r f a c e m a r k e r s CD3-CD56+CD16+ (range 8.5-20%). 18% of the h e p a t i c CD3+ cells e x p r e s s e d vSTCR (range 14-21.8%), a h i g h e r p e r c e n t a g e t h a n r e p o r t e d for any o t h e r tissue. Significantly, CD8act a c c o u n t e d for 27% (mean) of the total hepatic CD8+ p o p u l a t i o n . C o n c l u s i o n s : 1. The high CD8 count, reversed CD4/8 ratio, a n d i n c r e a s e d local p o p u l a t i o n of ySTCR+ cells suggest t h a t these cells m a y be u n i q u e to the liver and play a n i m p o r t a n t role in tissue s u r v e i l l a n c e . 2. Since m u r i n e s t u d i e s h a v e d e m o n s t r a t e d that CD8aa lymphocytes mature extrathymically, the s i g n i f i c a n t p o p u l a t i o n of C D 8 a a cells in the a d u l t h u m a n liver suggests t h a t this o r g a n is also a site of e x t r a t h y m i c differentiation. These findings may have important i m p l i c a t i o n s for the u n d e r s t a n d i n g of a u t o i m m u n i t y a n d graft tolerance.
AASLD ABSTRACTS
77 DOES LONG TERM CALCITONIN,
CALCIUM AND VIT D 3 ADMINISTRATION IN PRIMARY BILIARY CIRRHOSIS (PBC) PATIENTS PREVENTS BONE DBMINERALIZATION? E Mor.teiro, J Garcia-Costa, M Masearenhas. A Morbey, A Galv~o-Teles. Gas:to and Endocrine Units Sta. Maria University Hospital. Lisbon, Portugal.
Bone demineralization usually complicates PBC patients, mainly in post menopausal women. The aim of this prospective vertical study was to evaluate the bone mass density (BMD, g/cm~) in PBC patients o'~ calcium (Ca), salmon ealcitonin (CT) and Vit D3. Material and Methods: A group of 9 PBC caucasian women [treated with: Ca 500mg/day + Vit Ds: 200.000 U/month + CT: 100 U intranasal 15 days per month, longer than lyr (mean age + SO: 58.4 + 12.4 yr)] was compared with a control group of 9 healthy caucasian women (m _+ SD: 58.4 + 12.4) matched for age. BMD was evaluated by dual-energy x-ray absorptiometry at the lumbar spine (L2 -L4), femoral neck, troehanter and ward's triangle using a Norland densitometer. Body mass index (BMI, Kg/m2) was calculated. Data were evaluated by the One-Way Anova and the Spearman's correlation coefficient tests. Results: The mean age, BMI and BMD at the 4 sites were identical in both groups (see Table).
Age BMI BMD L2-L4 femoral neck trochanter ward's triangle
PBC Group
Control Group
p
58.4 + 12.4 27.8 + 3.4 0.839_+0.2 0.728 -+ 0.1 0.577-+0.1 0:503 + 0.1
58.4 -+ 12.4 27=2 + 4.0 0.865"_+0.1 0.741+0.1 0.615-+0.1 0.558-+0.1
NS NS NS NS NS NS
However, positive relationships BMI/BMD at the lumbar spine and at the trocbanter (r2>0.5) were detected in PBC group but not in the control group. Confusions: PBC patients treated for more than one year with CT, Ca and Vit D3 and control women have similar BMD. BMD increases with BMI in the PBC patients, showing the importance of nutrition in these patients.
79
RAGI, RAG2 AND T d T EXPRESSION IN ADULT HUMAN LIVER: EVIDENCE OF EXTRATHYMIC T CELL DIFFERENTIATION.
C. CollinsI , S. Norris1,2 G. McEntee3, O. Traynor3. J. He,arty2___& C. O'Farretlyl: ERC 1, Liver Unit2 & Dept. of Surgery3, St. Vincent's HOspital,Elm Park, Dublin 4, Ireland. INTRODUCTION: Animal studies have suggested that T lymphocyte maturation occurs in extrathymic sites including the liver. Expression of Recombination Activator Genes l & 2 (RAG 1 & 2) and Terminal Deoxynuchiotidyl Transferase (TdT) are required for T cell antigen receptor gene rearrangement and are used as markers of extrathymic T cell diffesentiadon. AIM: T o determine i f adult human liver is a site of R A G 1, R A G 2 and T d T gene expresssion. METHODS: Normal human liver tissue was obtaiued from five d o n o r organs at time of transplantation and total R N A isolated. R T - P C R was used to detect R A G I , R A G 2 and T d T m R N A in normal adult liver tissue, e D N A synthesis was carded out using A M V reverse transcriptase. Primers were specifically designed for P C R of haman RAG1, R A G 2 and T d T eDNA. R E S U L T S : Four adult bone marrow specimens were positive for R A G 1 , R A G 2 and T d T expression aud were used as positive controls. Peripheral blood mononuclear cell (PBMC) preparations from four individuals were negative for R A G and T d T specific m R N A . All liver specimens were positive for both R A G 1 and R A G 2 m R N A after nested P C R of liver cDNA. T d T expression was detected in two liver specimens after southern b lo ttin g of P C R product o n t o n y l o n m e m b r a n e and p r o b i n g with a digoxigenin labelled TdT specific oligonuleotide probe (Table 1.). Table 1
TISSUES LIVER 1 LIVER2 LIVERS LIVER4
RAG l + + + +
RAG 2 + + + ÷
Tdr NO NO ND
LIVER 5 PBMC l
+
+
+
+ + +
+ + +
+ + +
PBMC 2 PBMC 3 PBMC 4 PBMC S BONEMARROW 1 BONEMARROW2 BONEMARROWS BONEMARROW4
CONCLUSION: . Expression of R A G 1, 2 mid T d T ill adult huma n liver provides strong evidence that T cells can differentiate within this organ; these findings may have major implications for the generation of graft tolerance post liver transplantation.
78
HEPATOLOGYOctober 1995
Hypercoagulability in patients with Primary Biliary Cirrhosis (PBC) and Primary Sclerosing Cholangltis (PSC) evaluated by thromboelastography.
Z Ben4Ut, M Panagou, D Patch, S Bates', E Osman, AK Burroughs. Hepatobiliary and Liver Transplantation Unit, and ~Department of Anaesthesia, Royal Free Hospital, Pond Street, LONDON UK. Patients with PBC and PSC survive variceal bleeding better than alcoholic cirrhotics and have less bleeding at liver transplantation. We hypothesized that PBC and PSC patients may be hypercoagulable. As this is difficult to diagnose with routine laboratory tests, we used thromboelastography (TEG), which is a simple technique for evaluating whole blood clotting and fibrinolysis to establish if hypercoagulability was present, defined by TEG values greater than 2SD over controls: r < 19 mm (this reflects plasma clotting factors), maximum amplitude (ma) > 6 0 mm, and alpha angle > 43 ° (this reflects platelets and fibrinogen levels). We evaluated 48 PBC and 21 PSC patients and 40 noncholestatic cirrhotic patients as a control group with TEG, full blood count, prothrombin time (PT), partial thromboplastin time (PTTK), fibrinogen, protein S, C, anti thrombin III levels and activated protein C (APC) phenotype. Two or more TEG abnormalities indicating hypercoagulability were diagnosed in 14 of 48 (29%) PBC and in 9 of 21 (43%) PSC patients independent Of cirrhosis, but only in 2 of 40 (5%) of the control group, p
80 C D 8 a c t T
Lymphocytes in human adult liver evidence for extrathymic T cell differentiation? S~ Norris. C. Collins. I. H e , a r t y : C. O'Farrellg. Liver Unit a n d Education & Research Centre, St. Vincent's Hospital, Dublin, Ireland. The liver is not g e n e r a l l y t h o u g h t to be i m m u n o l o g i c a l l y i m p o r t a n t a n d l i t t l e is k n o w n a b o u t t h e p h e n o t y p i c p r o p e r t i e s of local T lymphocytes. The a i m of this s t u d y was to p e r f o r m flow cytometric analyses of hepatic T l y m p h o c y t e s u b p o p u l a t i o n s f r o m a d u l t n o r m a l liver. M a t e r i a l s a n d m e t h o d s : N o r m a l h e p a t i c t i s s u e (wet w e i g h t 2 0 0 g ) was o b t a i n e d from five d o n o r organs at time of t r a n s p l a n t , a n d single cell s u s p e n s i o n s were p r e p a r e d b y e n z y m a t i c a n d m e c h a n i c a l dissociation. Cells were s t a i n e d with m o n o c l o n a l antibodies specific for CD8a a n d CD8[~ chains, CD3, CD4, CDS, CD19, CD56, CD16, aISTCR, a n d y6TCR, a n d a n a l y z e d b y two colour and t h r e e colour flow c y t o m e t r y . Results: Cell yields r a n g e d from 0 . 4 - 3 . 7 x 1 0 6 / m l with a m e a n v i a b i l i t y of 79% (range 71-90%). O f the hepatic CD3+ cells, 72% were CD8+ and 23% were CD4+, with a CD4/8 ratio of 1:3 in c o n t r a s t to the p e r i p h e r a l CD4/8 ratio of 2:1. CD19+ cells a c c o u n t e d for 1.4% of the total CD3+ p o p u l a t i o n (range 1.1-1.7%), while 16.9% of the gate e x p r e s s e d NK s u r f a c e m a r k e r s CD3-CD56+CD16+ (range 8.5-20%). 18% of the h e p a t i c CD3+ cells e x p r e s s e d vSTCR (range 14-21.8%), a h i g h e r p e r c e n t a g e t h a n r e p o r t e d for any o t h e r tissue. Significantly, CD8act a c c o u n t e d for 27% (mean) of the total hepatic CD8+ p o p u l a t i o n . C o n c l u s i o n s : 1. The high CD8 count, reversed CD4/8 ratio, a n d i n c r e a s e d local p o p u l a t i o n of ySTCR+ cells suggest t h a t these cells m a y be u n i q u e to the liver and play a n i m p o r t a n t role in tissue s u r v e i l l a n c e . 2. Since m u r i n e s t u d i e s h a v e d e m o n s t r a t e d that CD8aa lymphocytes mature extrathymically, the s i g n i f i c a n t p o p u l a t i o n of C D 8 a a cells in the a d u l t h u m a n liver suggests t h a t this o r g a n is also a site of e x t r a t h y m i c differentiation. These findings may have important i m p l i c a t i o n s for the u n d e r s t a n d i n g of a u t o i m m u n i t y a n d graft tolerance.