Liver Transplantation and Immunologic Tolerance

Liver Transplantation and Liver Transplantation and Immunologic ToleranceImmunologic Tolerance

C. Andrew Bonham, M.D.

Associate Professor of Surgery

Stanford University Medical School

First Documented Liver Surgery

1950 1960 1970 1980 1990 2000 2010

First humanliver transplant

Collins solutionViaspan solution

Azathioprine

Antilymphocyteantisera

Steroids

OKT3

Cyclosporin

Tacrolimus

MycophenolateMofetil

IL-2RaRAPA

First split liver transplantFirst split liver transplant

First large animalFirst large animalliver transplantliver transplant

First adult-to-First adult-to-child transplantchild transplant

First domino First domino liver transplantliver transplant

First adult-to-adultFirst adult-to-adultliver transplantliver transplant

First liver First liver xenotransplantxenotransplant

First auxiliaryFirst auxiliary liver transplantliver transplant

First reduced size liver transplantFirst reduced size liver transplant

First liver geneFirst liver genetherapy experimenttherapy experiment

Veno-Veno-venousvenousbypassbypass

Patients Awaiting Organ Transplantation

• Waiting = 87,700 (as of March 11, 2005)Kidney 61,067Pancreas 1,683Kidney/Pancreas 2437Liver 17,263*Intestine 186Heart 3199Lung 3779Heart/Lung 169

*1/5 dies while waiting.

Countries Performing Liver Transplantation

1. Argentina2. Australia3. Austria4. Belgium5. Brazil6. Canada7. Chile8. China 9. Columbia10. Croatia11. Czech Republic12. England13. Egypt14. Finland15. France16. Germany

17. Greece18. Hong Kong19. India20. Iran21. Ireland22. Israel23. Italy24. Japan25. Korea26. Malaysia27. Mexico28. Netherlands29. New Zealand30. Norway31. Peru32. Philippines

33. Poland

34. Portugal

35. Russia

36. Saudi Arabia

37. Singapore

38. South Africa

39. Spain

40. Sweden

41. Switzerland

42. Taiwan

43. Turkey

44. Ukraine

45. United States

46. Venezuela

Years Post Transplantation

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1981 - 1985

1986 - 1990

1991 - 1998Log Rank: p = 0.0001

4000 Consecutive Liver Transplants

Patient Survival by Era

Causes Number (%)

Infection 464 28.4

Malignancy 190 11.6

Cardiovascular 135 8.3

Respiratory 114 7.0

Technical 113 6.9

Multiorgan system failure 109 6.7

Liver failure 96 5.9


Most Common Causes of Death ( n = 1633)

Complications of Immunosuppression

• Infection• Lymphoma• Hypertension• Renal failure• Ulcers• Seizures• Diabetes

• Osteopenia• Hirsutism• Impaired healing• Electrolyte abnormalities• Accelerated disease

recurrence

Evolution of Immunosuppression

• 1959 - First successful kidney transplants using TBI – Functioned 20 and 26 years without immunosuppression– Clusters of patients treated with azathioprine became drug-free

after treatment of rejection

• 1963 – Switch to heavy prophylactic immunosuppression with steroids prompted by 20-35% graft loss rate from rejection– No clusters of drug-free kidney recipients were ever described

again

• 1965 – Liver recognized as more tolerogenic


• 1966 – Spontaneous liver engraftment described– Tolerance to donor-derived skin or kidney grafts

• 1990s – Role of chimerism in solid-organ transplantation re-examined– Owen’s freemartin cattle

– Microchimerism in recipients of liver or kidney from opposite sex donor detected by PCR

• Role of residual antigen to maintain immunity or tolerance (Zinkernagel and Starzl)


• Heavy multi-drug immunosuppression may erode the tolerance mechanism of clonal deletion/exhaustion– Long-term immunosuppression required to prevent

emergence of reactive clones

• Pretreatment and minimal post-transplant immunosuppression may be conducive to tolerance

WEANING OF IMMUNOSUPPRESSION IN LIVER TRANSPLANT RECIPIENTS

Mazariegos, George V.3; Reyes, Jorge; Marino, Ignazio R.; Demetris, Anthony J.; Flynn, Bridget; Irish, William; McMichael, John; Fung, John J.; Starzl, Thomas E

95 patients weaned from immunosuppression

19% drug free for 10 months to 5 years

Pittsburgh Tolerance Protocol

• Pretreatment with ATG

• Post-transplant monotherapy immunosuppression

• Dose weaning at 6 months

Current immunosuppression of 38 patients pretreated with ALG before liver transplantation between July 2001 and March 2002

Dosing regimen n

Daily multidrug therapy 1

Daily monotherapy 8

Every other day 2

Three a week 6

Two a week 4

One a week 10

Every 2 weeks 2

Off immunosuppression 5

Total 38 *In most cases, tacrolimus

PNAS, Oct. 2004, Starzl.

Total Lymphoid Irradiation

• Is TLI conducive to tolerance induction?– Preconditioning regimen depletes immunoreactive

clones– Antigen presentation and immune activation impaired

Animals and Liver transplantsAllogeneic donor ACI (RT1a) livers were transplanted into Lewis (RT1l) recipients.

Treatment• ATG : 10 mg/kg of ATG IP on day 0 and 2, and 5 mg/kg on day 4, 6, and 8.• TLI : 8 treatments of 240 cGy each over 10 days starting on day 1.

TLI fields exposed the abdomen, peripheral lymph nodes, thymus, and spleen, but shielded the liver graft, skull, lungs, limbs, pelvis, and tail.

• MC: MC were injected intravenously into recipients on day 10 after completion of TLI.

Experimental Groups(1) Untreated allogeneic control(2) TLI alone(3) ATG alone(4) TLI and ATG(5) TLI and MC injection

Experimental Design

OLTxTLI x 8

LIMC, PBMC i.v.(day 10)

ATG x 5

• Outcome of ACI to Lewis Liver Transplants with or without TLI +/- Donor cell Infusion.

Treatment Median surv (d) % ACR % surv > 100d

Liver tx +:

None (6) 12.5 100 0

TLI (27) 109.1 14.8 40.7

TLI + MC(6) 92.8 33.3 33.3

ATG (6) 15.8 50.0 0

Cadaveric Liver Transplantation Protocol

Withdraw immunosuppression if:-no evidence of rejection-no GVHD

rATG (2.0 mg/kg)

LiverTransplant

Day 0

TLI

Tacrolimus wean month 3-6

Future Directions

• Xenotransplantation

• Artificial liver– Stem-cells and liver regeneration

• Treatment and prevention of hepatitis

This three-year-old boy was born with biliary atresia and had been explored previously.

The child was anesthetized with flouothane anesthesia and a large bore needle placed in a vein of the arm. A thoraco-abdominal incision was then made through the eighth right intercostals space and extending transversely down across the abdomen and then back up to the left costal margin. The operative wound was exceedingly difficult to keep dry during the deepening of the incision because of numerous collateral veins secondary to the portal hypertension.

At this time, word was passed from the donor room that the pumping system for perfusion of the liver had failed and that it would be necessary to act with the utmost expediency to remove the child’s liver. The triangular ligaments were then cut and the liver removed with considerably less than pains-taking care for hemostasis.

It should be mentioned that just before removing the liver, it was noticed that the needle had become disengaged from its position in the arm, and that the transfusions which had been given up to this point had all been passing out upon the table.

Upon release of the clamps, the liver immediately assumed a good color, and despite the hectic circumstances of the transplant, it was initially thought that a good result would be obtained. However, after about 15 or 20 minutes, it was noticed that all raw surfaces were profusely bleeding. A sample was sent and the report came back that there were extremely high titres of fibrinolysin and low titres of fibrinogen in the submitted blood. The next 5 or 6 hours were spent in a desperate effort to control hemorrhage, using literally thousands of sutures and ties, but the hemorrhagic diathesis could never be satisfactorily controlled.

Excerpts from the autopsy report:

Discussion: This three year old boy was first noted to be jaundiced at age one day. At the age of five months, an exploratory laparotomy revealed the absence of an extrahepatic biliary system and a liver biopsy was obtained. This showed the presence of early biliary cirrhosis.

During the next two years the child was hospitalized several times for various infections. His weight gain was poor but growth in height was normal. He was admitted at his mother’s request so that some research procedure might be carried out in an effort to prevent this illness in other children.

Autopsy examination showed that patent anastomoses of hepatic artery, portal vein and hepatic vein had been surgically effected. …The most significant findings were those of small hemorrhages in the adrenal cortex and lung and advanced, near total necrosis or autolysis of liver parenchyma.

A review of current literature does not reveal a report of attempted liver transplant in humans, although there is great activity with experimental animals. Moore, Wheeler, et al, carried out well controlled series of liver and spleen transplants on dogs. They did not attempt to block the immunological response, but only to delineate the technical and mechanical problems. One of 31 homotransplants survived 12 days but more distressing is that none of 21 autotransplants survived more than 14 days. The causes of death in the two groups were similar and mostly unassociated with liver failure. Also five of thirteen “sham” dissections failed to survive. (The entire procedure except removal and replacement of liver was effected.) These two latter groups would seem to point out the tremendous technical difficulties attending this procedure above and beyond the immunological problems involved. These authors remark “If the transfusion volume Is large (during surgery), the dog will be found to have a coagulation defect that makes hemostasis difficult.” This observation may be of some significance in this case.

UNOS Regions

Indications for liver transplantation in adult and pediatric patients

Biliary diseases Parenchymal diseasesPrimary biliary cirrhosis Viral hepatitisSecondary biliary cirrhosis Alcoholic cirrhosisSclerosing cholangitis Neonatal hepatitisBiliary atresia Congenital Hepatic Fibrosis

Autoimmune hepatitisInborn errors of metabolism Acute liver failure

Alpha 1-antitrypsin deficiency Severe hepatic traumaHemochromatosisByler’s disease Hepatic tumors Familial cholestasis Benign hepatic tumors Type I glycogen storage disease Primary hepatic malignancyHemophiliaCystic fibrosis

Contraindications for liver transplantation

Absolute• Advanced HCC - Stage IV• Liver metastases - except neuroendocrine tumors• Cholangiocarcinoma - except for in situ lesions• Active alcoholism• Active drug abuse• Active tuberculosis• AIDS• Refractory extra-hepatic sepsis• Extensive mesenteric venous thrombosis• Severe cardiopulmonary disease

Contraindications for liver transplantation

Relative

• HCC - Stage III

• Alcohol abstinence < 6 months

• Severe debilitation

• Metastatic neuroendocrine tumors

• HIV positive

Liver Transplantation: Pittsburgh Experience

Adults Children Total

Liver Transplant 4426 1163 5589

Liver + Intestine 108 83 191

Liver + Kidney 46 9 55

Liver + Pancreas 29 1 30

Liver + Islets 14 2 16

Liver + Heart 5 3 8

Total Transplant Activity(including re-transplants)


• 4000 primary liver transplants were performed between February 1981-April 1998

• Follow-up to March 2000

• Mean follow-up was 9.4 + 3.8 years (median 9.6 years, range 2-18 years)

Demographics


Era Time Frame Number of Patients

A (Cyclosporine) 1981 – 1985 478

B (UW; OKT3) 1986 – 1990 1382

C (FK506) 1991 – 1998 2140

Demographics

N (%)N (%)

HCV/NANB 680 (21.3)

Alcoholic 567 (17.8)

PBC 409 (12.8)

Cryptogenic 279 (8.7)

PSC 253 (7.9)

Malignancy 234 (7.3)

HBV 217 (6.8)

Autoimmune 147 (4.6)

N (%)N (%)

Metabolic Disease 100 (3.1)

Acute Liver Failure 76 (2.4)

Budd-Chiari 39 (1.2)

SBC 38 (1.2)

Carcinoid 22 (0.7)

Cystic fibrosis 11 (0.3)

Other 120 (3.8)

Total 3192

Indications -- Adults



HCV

ETOH

PBCPNC-C

PSC

Malig

HBV

PNC-AI

Other

N (%)N (%)

Biliary Atresia 416 (51.4)

Metabolic Disorders 109 (13.5)A-1-A 62 (7.6)

Wilson’s Disease 16 (2.0)

Tyrosinemia 13 (1.6)

GSD 13 (1.6)

Hemochromatosis 2 (0.2)

Other 3 (0.4)

Acute Liver Failure 48 (5.9)

N (%)N (%) Cryptogenic 30 (3.7)NANB/HCV 35 (4.3)Familial Cholestasis 27 (3.3)Malignancy 23 (2.8)Neonatal hepatitis 19 (2.3)SBC 17 (2.1)Hepatic Fibrosis 15 (1.8)Autoimmune 8 (1.0)Other 61 (7.5)Total 808

Indications -- Children



BA

Metabolic

Acute failure

PNC-C

Other


0

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Overall Survival


Overall Patient Survival

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Age <2 yrs

Age 3 to 18 yrs

Age 19 to 60 yrs

Age >60 yrs


Log Rank: p = 0.0001


Patient Survival by Age

0

5

10

15

20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18



Survival Attrition

%

0

20

40

60

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Overall Graft Survival



Overall Graft Survival


0

20

40

60

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0 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1981 to 1985

1986 to 1990

1991 to 1998 Log Rank: p = 0.0001


Graft Survival by Era

Re-Transplant NumberRe-Transplant Number NN %%

0 3226 80.6

1 774 19.4

2 148 3.7

3 20 0.5

>3 5 0.13


Patient Survival Following 1, 2, 3 and >3 LTx

0102030405060708090

100

0 1 Year 2 Year 5 Year 10 Year 15 Year 18 Year

1st LTx2nd LTx3rd LTx>3rd LTx


Years Post Retransplantation

Patient Survival by Diagnosis

0

20

40

60

80

100

0 1 3 5 7 9 11 13 15 17

Alcoholic

HCV+NANB

Malignancy

PBC+PSC+AI

HBV

Metabolic

Biliary Atresia

Fulminant Failure

Other


Years Post Retransplantation

Causes Number (%) Mean Interval (mo) Currently Alive n (%)

Primary Non Function 249 (32.2) 0.3+0.4 97 (38.8)Hepatic Artery Thrombosis 214 (27.6) 5.5+14 85 (39.7)Chronic Rejection 113 (14.5) 25.1+28.3 48 (42.4)Recurrent Disease 44 (5.5) 31.9+32.2 15 (34.8)Acute Rejection 38 (4.9) 5.4+20.4 13 (34.2)Biliary Complications 22 (2.8) 16.8+24 8 (36.3)Technical 12 (1.5) 34.9+35.2 6 (50)Miscellaneous 48 (6.2) 16.4+28 12 (25)Unknown 34 (4.3) 28+47 14 (40)Total 774 (19.4) 10.7+24 298 (38.5)


Causes of Retransplantation

Organ Shortage

Liver Transplant Status In the United States

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998

No. Transplants Waiting List Deaths Waiting

Operative Technique

Standard Procedure:Total Hepatectomy

The standard approach for orthotopic liver transplantation consists of a transverse upper abdominal incision with an upper midline extension up and usually including the xiphoid.


Because of the portal hypertension and vascularization of all hepatic ligaments, total hepatectomy usually starts with the dissection of the hepatoduodenal ligament in order to gain access to the portal vein, which allows the institution of venovenous bypass at any time. The dissection line (dotted line) should be close to the hilum of the liver to leave long stumps of the hepatic artery and common bile duct.


The wall of the portal vein is completely freed from all surrounding tissue. The surgeon now decides whether the venovenous bypass system should be used.

Implantation of the Graft

Upper caval anastomosis


Infrahepatic vena caval anastomosis.


Portal vein anastomosis

Implantation of the GraftImplantation of the Graft

Post Reperfusion

Implantation of the GraftImplantation of the Graft

Before and After


Hepatic arterial and Biliary anastomoses (choledochocholedochostomy)


End-to-end anastomosisEnd-to-end anastomosisRoux-en Y Roux-en Y hepaticojejunostomyhepaticojejunostomy

Postoperative cholangiography

Living Donor Liver Transplantation

Liver - Segmental/Lobar DonationLiver - Segmental/Lobar Donation– Adult-to-child - usually left lateral segments/left lobeAdult-to-child - usually left lateral segments/left lobe

– Adult-to-adult - usually right lobeAdult-to-adult - usually right lobe

Overall risk approximately 6/1200 transplantsOverall risk approximately 6/1200 transplants Overall early morbidity approximately 15%Overall early morbidity approximately 15% Unknown late morbidityUnknown late morbidity Related and unrelated liver donorsRelated and unrelated liver donors

– Should be an altruistic relationshipShould be an altruistic relationship

– Benefits to both donor and recipientBenefits to both donor and recipient

– Ethical issuesEthical issues

Liver Segments

Caudate lobe

II

IIIIV

V

VI

VIII

VII

II

III

I

IV

VII

V

VI

Right Lobe Left Lobe Right Lobe Left Lobe

From Netter

IVC

Right lobectomy Left lobectomy

Living Liver Donor Right Hepatectomy

Right lobe Left lobe

Living Liver Donor Transplant (right lobe)

Roux Y Hepatico-jejunostomy (R. ant. & post. ducts)

Accessory HV re-implanted

Thomas E. Starzl, M.D., Ph.D.Transplant Pioneer

Oldest living living transplant recipient with her husband 31 years s/p transplant for biliary atresia.

http://www.sciencemag.org/content/vol295/issue5557/images/large/se0420168001.jpeg

Quantitative Distribution of Liver Cell Type

05

10152025303540455055606570

Hepatocyte Endothelialcell

Kupffer cell Stellate cell Pit cell

Rela

tive c

ell

num

be r

(%)

CELL TYPES & ARRANGEMENT of LIVER

MicrovilliHepatocytes in plates

Bile canaliculus

Space of Disse

Fenestrated Endothelial cells

SINUSOIDKupffer cell

Stellate cell

HEPATOCYTE ENDOTHELIAL CELL KUPFFER CELL STELLATE CELL

Function of Liver Cells

Hepatocytes are the primary cells of the liver

Hepatocytes

- secrete bile salts to solubilize dietary fats

- heavily involved in glucose homeostasis and fat metabolism

- primary site of detoxification

- synthesis and degradation of blood proteins

Endothelial cells

- line the sinusoids of the liver vascular system

Stellate cells and Fibroblasts

- maintain the ECM and connective tissue that hold the liver together

Macrophages-Kupffer cells

- left the blood and taken up residence

- blood cell turnover

http://www.annalsnyas.org/content/vol979/issue1/images/large/4ff3.jpeg

Cellular Transplantation

• Cellular transplantation is a potential alternative of whole transplantation. However, cellular hepatocyte transplantation is limited by

• The ability to maintain and expand liver cells in vitro

• Limited numbers of transplanted cells• Low survival rate• Disappointed outcome of clinical trials

• Reasons:Lack of interaction of cell-cell, cell-matix,and cell-cytokines/hormones in the transplanted cells.Cells are trapped in sinusoid.Lack of capillary structures.

Potential Sources of Hepatocytes

Non-liver Source:• Embryonic stem cells• Bone marrow cells• Plasticity of other stem cells/progenitors, such as

umbilical cord blood• Difficult to control differentiation processing and

efficiency

Liver source:• Adult hepatocytes---difficult to handle• Adult liver progenitor cells • Immortalized hepatocytes and Xenogenic hepatocytes• Fetal liver cells

Human Fetal Liver Cells as Hepatocyte Source

•Fetal liver cells:– Rich in progenitors

• Differentiate into mature hepatocytes and biliary cells

• Minimal immunogenicity and nontumorigenicity

Isolation of Hepatocytes

Sites for Hepatocyte transplantation

Into Liver:– Portal vein (transjugular and transcutaneous)– Splenic pulp (translocation)– Injectable directly liver (matrigel)

Extrahepatocyte:– Renal capsule (microcarrier)– Peritoneal cavity (Dorsal fat pad)– Pulmonary artery bed

Intrahepatic infusion of hepatocytes. Hepatocytes are engrafted into the liver by transhepatic portal vein catheterization. Here, the left portal vein is punctured percutaneously using ultrasound guidance. A 5-French Kumpe catheter (Cook) is manipulated into the main portal vein and the position of the catheter is confirmed by injection of contrast material before hepatocytes are infused. Fox & Chowdhury, Hepatocyte transplantation. 2004. American Journal of Transplantation.

Human liver disease Animal model ReferencesClinically attempted

Hypoalbuminaemia Nagase analbuminemic rat

Moscioni et al. (1996)Lilja et al. (1997)Oren et al. (1999)

No

Crigler-Najjar syndrome

Gunn rat Matas et al. (1976)Groth et al. (1977)Tada et al. (1998)

Yes

Hypercholestrolemia Watanabe hereditary hyperlipidemic rabbitmouse model

Wiederkehr et al. (1990)Gunsalus et al. (1997)Mitchell et al. (2000)

*Yes

Wilson’s disease Long-Evans cinnamon rat

Wu et al. (1994)Yoshida et al. (1996)

No

Acute liver failure Lewis rats F344 rats

Kobayashi et al. (2000)Gupta et al. (2000)

Yes

Hepatocyte Transplantation-Preclinical and Clinical Studies

Hepatocytes in tissue engineering

• Hepatocyte viability and function is affected by a number of factors– Extracellular matrix– Presence of other cells– Oxygen concentration

Tissue Engineering

• Liver sinusoidal architecture is mimicked by creation of biopolymer scaffold– Micro-electro-mechanical system (MEMS) fabrication

techniques – Micro-etched wafer serves as mold for pattern transfer

to biocompatible polymer– Scaffold seeded with cells and provided with

continuous culture medium flow

Microcirculation Bioreactor System


Micro-etched Wafers



Microfabricated bioreactor

Three-dimensional tissue structure with viable hepatocytes created by

stereolithography

Microfabrication and cell co-culture

• Coculture of hepatocytes and fibroblasts on collagen coated microfabricated matrix

• Hepatocyte phenotype and function is preserved by coculture

“Smart” biomaterials

• 3 dimensional, porous, interconnected network• Maintain proper mechanical properties• Biocompatible• Elicit a controlled physiologic reaction

– Induce cell adhesion, migration, or function

– RGD signal peptide

– Nanoscale clustering of molecules on surface

– Gene-activated matrix (plasmid DNA)

Cell adhesion to RGD peptides nanoclustered on polymer surface

Plasmid expression in vivo from polymer matrix sponge

• Canine fibroblasts express plasmid -gal 3 weeks after polymer implantation

Hepatocyte metabolic activity is associated with oxygen zonation

Liver Engineering: Summary

• Cell source– Heterogeneous, viable, renewable

• Stem cells, cell lines, individuals

• Matrix– Biocompatible, interactive

• Smart materials interactive physiologically– Gene implantation, protein expression, protein degradation

• Host interaction– Blood flow, oxygenation, secretion, removal of excretions,

immunologic reactivity• Architecture, pore size, cell distribution and presentation

Dr. Starzl with a group of liver transplant recipients he transplanted as children in Pittsburgh

Todd McNeely as he was at the time of transplant at age 2 and as he is now as he sees Dr. Starzl for the first time in 18 years. Todd was the first pediatric liver recipient in Pittsburgh.

“Nurse, get on the internet, go to SURGERY.COM, scroll

down and click on the ‘Are you totally lost?’ icon.”

Liver Transplantation and Immunologic Tolerance

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