Tolerance to rat liver allografts. III. Donor cell migration and tolerance-associated cytokine production in peripheral lymphoid tissues.

The aim of this work was to investigate the mechanism of spontaneous rat liver allograft tolerance. Liver allografts from a LEW donor into DA recipient (LEW-->DA) or of PVG-->DA were spontaneously tolerated (TOL) across a complete MHC mismatch. In contrast, DA-->LEW or PVG-->LEW liver allografts were rejected in 10 to 15 days (REJ). We examined whether donor cell migration to recipient lymphoid tissues might be associated with TOL. Many donor cells were observed in draining (celiac) lymph nodes (LN) and spleen, reaching a peak on day 1 and then decreasing rapidly thereafter. Irradiation of liver donors, which we have previously shown to delete tolerance, significantly reduced the number of donor leukocytes in recipient lymphoid tissues. While this suggested an association between donor cell migration and tolerance, the number, distribution, and type of donor cells in recipient lymphoid tissues of REJ was similar to those of TOL. Expression of cytokine mRNA in LN and spleen showed an early increase in the expression of IL-2 and IFN-gamma mRNA on day 1 and then a rapid decrease to constitutive levels. Spleen and LN levels of IL-6, IL-10, TNF-alpha, or TGF-beta mRNA showed much less up-regulation than IL-2 or IFN-gamma. Paradoxically, there was greater expression of IL-2 and IFN-gamma mRNA in TOL lymphoid tissues than in REJ, and this superinduction was partially prevented by donor irradiation. Superinduction of IL-2 and IFN-gamma was, therefore, more closely associated with TOL than was donor cell migration. This was confirmed by treatment of TOL recipients with a short course of methylprednisolone, which reduced survival of subsequent donor strain skin grafts. This finding has implications for treatment of human liver transplants and is evidence for a novel pathway of transplant tolerance.

Deletion of spontaneous rat liver allograft acceptance by donor irradiation.

Liver transplants in rodents or pigs are often spontaneously accepted across a complete MHC mismatch. They induce tolerance to grafts of other organs or skin of liver donor strain and can even suppress ongoing rejection of heart grafts. It has not been established whether liver-induced tolerance is due to components of the liver or to passenger leukocytes within the liver. We depleted populations of passenger leukocytes from the transplanted liver by irradiation of the donor with 10 Gy, followed after 7 days by transplantation of the liver. Recipients of livers from irradiated donors had a median survival of 16 days compared with > 100 days for recipients of livers from normal donors. Examination of recipients of irradiated donor livers showed that allograft rejection was the cause of death. Syngeneic transplants of irradiated PVG donor to PVG recipient or of irradiated DA donor to DA recipient survived indefinitely. Parking of livers from irradiated PVG donors in normal PVG animals for 36 hr reconstituted tolerance when the livers were retransplanted to DA recipients. Livers from irradiated donors had greatly reduced passenger leukocyte numbers compared with normal or parked livers, with virtually complete loss of lymphocytes. These results show that spontaneous liver allograft acceptance is associated with populations of passenger leukocytes that are depleted by donor irradiation.

Tolerance to rat liver allografts. I. Differences between tolerance and rejection are more marked in the B cell compared with the T cell or cytokine response.

Liver allografts in the fully allogenic combination of LEW donor liver to DA recipient (LEW-->DA) are spontaneously tolerated (TOL) with no requirement for immunosuppression, while DA-->LEW allografts are rejected in 12-15 days (REJ). We investigated the mechanism of tolerance induction by identifying differences between TOL and REJ grafts from day 1 to day 9 after transplantation and in normal livers and syngeneic liver graft controls. Infiltrating cell populations were counted after immunohistochemical staining of liver graft sections. There were occasional minor differences between TOL and REJ grafts in the T cell or CD11b/c+ (monocyte/macrophage/granulocyte) infiltrate. In contrast, there was a major difference in B cell infiltrate between TOL and REJ liver grafts. Membrane IgD+ cells were significantly greater in TOL (1796 +/- 225) versus REJ (569 +/- 281) (P = 0.004) portal tracts, as were B220+ cells (1086 +/- 100 vs. 181 +/- 105, P = 0.0004) and CD45RC+ cells (2317 +/- 456 vs. 597 +/- 194, P = 0.004). IgG1, IgG2a, IgM, and IgD deposition in liver allografts, identified by immunohistochemical staining of tissue sections, revealed no IgG or IgD in normal rat liver and low levels of IgM. Deposition of IgG1 was observed in REJ but not in TOL liver on days 7 and 9. IgM was increased in both TOL and REJ liver and appeared to be associated mainly with hepatocytes in REJ and with infiltrate in TOL liver. There was a parallel increase in IgG1-expressing plasma cells in the spleen and lymph nodes of REJ but not TOL animals. Cytokine mRNA was analyzed by reverse transcription and semiquantitative polymerase chain reaction amplification of liver RNA. Increased levels of IL-2, IL-4, IL-6, IL-10, TNF-alpha, transforming growth factor-beta, and IFN-gamma were observed, with similar levels of expression in TOL compared with REJ liver. Cytokine mRNA in syngeneic grafts was not different from normal except for IL-6 and transforming growth factor-beta, which were increased. There is no major difference in the T cell component of the infiltrate or in the extent of upregulation of cytokine mRNA between TOL and REJ grafts. There is a major difference in the B cell compartment, with more B cells in TOL livers and deposition of IgG1 in REJ grafts.

Transjugular intrahepatic portosystemic stent-shunt (TIPSS) for variceal haemorrhage: initial results in 28 patients.

BACKGROUND: Endoscopic sclerotherapy is an effective form of treatment of bleeding varices in patients with cirrhosis. However, the mortality in patients who rebleed is high. Recently, transjugular intrahepatic portosystemic stent-shunt (TIPSS) has been developed as an alternative to surgical shunt formation in patients who have failed sclerotherapy. AIM: To review the early experience with TIPSS at a teaching hospital. METHODS: Twenty-eight patients underwent TIPSS on 30 occasions between September 1991 and June 1993 for bleeding oesophageal or gastric varices. The majority had alcoholic liver disease. RESULTS: TIPSS was performed successfully in all patients. Immediate control of bleeding was achieved, but one patient rebled within 24 hours. Complications related to the procedure occurred in 30%, but no patient died from these. Thirty-day mortality was 11% (three of 28), two patients dying from progressive liver failure and one from sepsis. A further three patients died from six weeks to two months following TIPSS, due to liver failure in one, spontaneous bacterial peritonitis in the second and in the third after a fall. This represents an overall mortality of 21%. Three patients have rebled at mean follow-up of 11.3 months. One of these had repeat TIPSS while the other two had balloon dilatation of the stent with control of bleeding. Four patients developed mild chronic encephalopathy which was readily controlled with medical therapy. CONCLUSIONS: TIPSS is an effective means for control of bleeding from oesophageal and/or gastric varices not responding to other methods. Further follow-up is required with regard to rates of rebleeding, encephalopathy and survival.

Exon eight APC mutations account for a disproportionate number of familial adenomatous polyposis families.

The familial adenomatous polyposis gene, APC, has recently been identified. Detection of APC mutations will facilitate genetic screening in family members at risk for this disease. The length of APC makes it impractical to examine the entire coding sequence in each new family encountered. Identification of mutation cluster regions within the gene has therefore become a priority. Initial reports suggested that exon eight might contain a disproportionate number of mutations. This study describes direct sequencing of exon eight in 21 unrelated Australians with familial adenomatous polyposis. Mutations were detected in three of the 21 subjects (14%). Two were previously described point mutations changing an arginine to a stop codon. The third was a novel two base-pair deletion producing a frameshift and downstream stop codon. All three mutations segregated with the disease gene in their respective families. Three at risk children from two of these families were studied and shown not to have inherited the disease producing mutation. These results confirm that exon eight is a frequent site of mutation in familial adenomatous polyposis and should be examined routinely in families requesting genetic screening.

Liver transplantation for primary sclerosing cholangitis versus primary biliary cirrhosis: a comparison of complications and outcome.

Primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC) are the most common cholestatic disorders in adulthood requiring hepatic transplantation. Although they run similar courses, they may have different problems before and after transplantation. The aim of this study was to compare pre- and post-transplant complications and outcomes in these two similar but distinct patient groups. One hundred and seventeen adult patients underwent liver transplantation at our institution over a 6 year period, including 19 with PSC and 20 with PBC. Pre-transplant there were no significant differences in age, liver biochemistry, haematology or Child-Pugh scores between the two groups. The mean duration of disease before transplant was longer in PSC patients (11.7 vs 6.5 years; P < 0.05). The prevalence of septic cholangitis was greater in PSC (58 vs 5%; P < 0.01) as was the requirement for surgical or radiological interventional procedures, excluding cholecystectomy (53 vs 0%; P < 0.01). At transplantation, four patients with PSC had previously unrecognized cholangiocarcinoma. In the pre-transplant period these four patients had uncontrolled biliary sepsis at the time of transplant vs five of 15 PSC patients without cholangiocarcinoma. Postoperatively, PSC patients had a greater prevalence of intra-abdominal sepsis requiring surgical or radiological intervention (42 vs 5%; P < 0.05). In comparison, patients with PBC had a high prevalence of skeletal complications (30 vs 10%; P < 0.05) particularly avascular necrosis (15 vs 0%). The prevalence of chronic rejection was similar in both groups (15%). Overall survival was higher in PBC patients (85 vs 63%; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Sclerosing cholangitis and biliary tract calculi--primary or secondary?

The clinical features of 61 patients with sclerosing cholangitis were reviewed. This group included 23 patients with biliary tract calculi, commonly considered as excluding the diagnosis of primary sclerosing cholangitis. The aim of this study was to compare these 23 patients (group A) with 38 patients with sclerosing cholangitis free of calculi (group B). Both groups had the following features in common: (i) age at presentation, (ii) incidence of inflammatory bowel disease, (iii) extent of radiological disease, (iv) prevalence of HLA-B8 and DR3 haplotype, (v) incidence of cholangiocarcinoma, and (vi) progression to hepatic transplantation (mean follow up 49.9 months). All patients in group A were symptomatic at diagnosis compared with 23 of the 38 patients (61%) in group B. Recurrent ascending cholangitis occurred in 12 patients in group A (52%) and two patients (5%) in group B. The similarity between the two groups was maintained when the nine patients in group A who developed calculi after sclerosing cholangitis was diagnosed were excluded. It is concluded that choledocholithiasis is part of the spectrum of primary sclerosing cholangitis and that it is not necessary to invoke choledocholithiasis as the initial lesion of the bile ducts in such patients.

Thrombocytopenia post liver transplantation. Correlations with pre-operative platelet count, blood transfusion requirements, allograft function and outcome.

This study reports that thrombocytopenia is a universal phenomenon post hepatic transplantation. In 53 consecutive adult patients undergoing liver transplantation the platelet count fell by a mean of 63% (157 x 10(9)/l to 50 x 10(9)/l). The platelet count reached a nadir at Day 5 post-transplant but returned to pre-operative levels by Day 14. Non-parametric regression analysis found that pre-operative platelet count, blood transfusion requirements and maximum post-operative ALT values were independent predictors of the percentage fall in platelet count. No correlation was seen with length of graft cold ischaemic time or the use of University of Wisconsin (UW) solution. The nadir day correlated with maximum post-operative bilirubin and ALT, graft ischaemic time and use of UW solution. Maximum post-operative ALT was also an independent predictor of nadir platelet count. It was observed that patients who did not survive the hospital admission had lower post-operative platelet counts and these did not return to pre-operative levels by Day 14. The percentage fall in platelet count was an independent predictor of survival. Severe thrombocytopenia was associated with cerebral haemorrhage in 3 patients. This report provides evidence that allograft dysfunction (maximum post-operative bilirubin and/or AST/ALT) was the most consistent independent predictor of the nadir platelet count, nadir day and percentage fall in platelet count post liver transplantation although the exact mechanism(s) of the platelet changes remain uncertain.

Striking variability of hepatic copper levels in fulminant hepatic failure.

Two cases of acute hepatic failure are reported in which the diagnosis of Wilson's disease was considered because of low serum ceruloplasmin, low serum copper levels and high 24 h urinary copper. Case 1 had Kayser-Fleischer rings, haemolysis and a high 24 h urinary copper, and so Wilson's disease was confidently diagnosed. Case 2 had high urinary copper excretion, but [64Cu] study indicated a 24:2 h ratio of 0.7 and made the diagnosis of Wilson's disease uncertain. Both patients underwent orthotopic hepatic transplantation, and multiple biopsies were taken from the resected specimen in order to estimate hepatic copper levels. In both cases, hepatic copper levels revealed considerable variation: 0.8-5.2 mumol/g dry wt (case 1) vs 0.02-12.65 mumol/g dry wt (case 2). In case 1, only two of 14 levels were within the diagnostic range for Wilson's disease (greater than 4 mumol/g dry wt), whereas hepatic copper levels in case 2 were in the Wilsonian disease range in three of 16 specimens. These results were in contrast to uniformly high hepatic copper levels in one patient with established cirrhosis secondary to Wilson's disease and two cases of primary biliary cirrhosis. This report indicates that hepatic copper levels vary greatly in acute liver failure, and that estimates from a single biopsy specimen may be misleading as to the cause of the underlying liver disease.

Risk estimation in familial adenomatous polyposis using DNA probes linked to the familial adenomatous polyposis gene.

The familial adenomatous polyposis gene has recently been assigned to the long arm of chromosome five through linkage to several 5q DNA probes. These probes can now be used to trace inheritance of the disease gene in affected families. In this study, DNA samples from 152 members of 10 Australian familial adenomatous polyposis families have been examined for restriction fragment length polymorphisms detected by DNA probes C11P11, ECB27, and YN5.48. Linkage analysis confirmed linkage between the familial adenomatous polyposis gene and each probe with a maximum combined LOD score of 2.82 for C11P11, 2.90 for ECB27 and 5.49 for YN5.48 all at a recombination fraction of zero. Risk estimates were determined for the 51 at risk individuals in these families based on their restriction fragment length polymorphism data alone or in addition by including the effect of age dependent penetrance. Thirty two of those at risk (63%) could be assigned specific high (greater than or equal to 95%) or low (less than or equal to 5%) risks of developing familial adenomatous polyposis on the basis of their probe results. When the effect of age dependent penetrance was included, 26 (51%) fell at the extremes of risk (greater than or equal to 99% or less than or equal to 1%). Such estimates provide a sound basis for planning sigmoidoscopic screening of at risk family members and will thus facilitate surveillance in familial adenomatous polyposis families.

Inflammatory pseudotumor of the liver causing biliary obstruction. Treatment by biliary stenting with 5-year follow-up.

Inflammatory pseudotumors of the liver are rare lesions that are becoming increasingly recognized, possibly because of frequent use of abdominal computed tomography (CT) and ultrasonography. Most previously reported cases have been treated by hepatic resection. For this reason, we describe a patient with obstructive jaundice secondary to an inflammatory hepatic pseudotumor in the porta hepatis. Bilateral internal biliary stents were inserted, and the patient remains well 5 years later. The clinicopathologic features of inflammatory pseudotumors of the liver are described, as are the implications of accurate diagnosis. There have been no previous reports of biliary stenting in management of inflammatory hepatic pseudotumors.

A quantitative analysis of T lymphocyte populations in human liver allografts undergoing rejection: the use of monoclonal antibodies and double immunolabeling.

The aim of this study was to quantitate T-cell populations infiltrating portal tracts, bile ducts and hepatic lobules in 82 biopsy specimens from 25 patients after orthotopic liver transplantation. Biopsy specimens taken immediately after revascularization of the graft were used as controls. Patients studied include 18 with initial rejection episodes, 11 with unresolved rejection, five with vanishing bile duct syndrome and eight patients with other forms of liver injury. Quantitation was done in a blinded fashion for the first 20 biopsy specimens. A double immunolabeling technique was used to simultaneously immunolabel bile duct structures (with anti-major histocompatibility complex class II or antikeratins) and lymphoid populations (with anti-CD2, anti-CD4 or anti-CD8). This facilitated the accurate quantitation of intraepithelial lymphocytes within bile ducts. This technique also enabled simultaneous detection of CD4 and CD8 antigens on lymphocytes in portal tracts. The predominant lymphocyte subtype within biliary epithelium during acute and chronic rejection was of the CD2+/CD8+ phenotype. CD8+/CD4+ ratio in bile ducts was approximately 5:1 in acute, unresolved and chronic rejection. In vanishing bile duct syndrome, double immunolabeling enabled the detection of destroyed interlobular bile duct remnants that were not apparent on routine hematoxylin and eosin staining. Attached to some of these structures were CD8+ lymphocytes. Lobular CD8+ cells were not prominent in acute rejection but increased significantly in biopsy specimens from patients with unresolved and chronic rejection. In chronic rejection, a selective increase was seen in these CD8+ cells in centrizonal regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Nodular regenerative hyperplasia mimicking cirrhosis of the liver.

Nodular regenerative hyperplasia of the liver usually presents with signs of portal hypertension with little evidence of obvious liver disease. We report a 47 year old man who presented with clinical signs of decompensated cirrhosis, recurrent encephalopathy, and tense ascites but at liver transplant was found to have nodular regenerative hyperplasia associated with a portal vein thrombosis.