We report the case of an ultimately successful liver transplant recipient whose post-transplant course was complicated by the early development of multiple abscesses in the graft. Post-transplant cholangiography identified multiple shear injuries to the second and third order intrahepatic bile ducts, originating from blunt trauma to the donor liver. Treatment was non-operative following recent reports of the successful management of intrahepatic bile duct injury in the stable trauma patient. This discussion adds to the limited literature available on the transplantation of injured donor livers, despite this being a relatively common practice. Further experience is needed in determining the appropriate criteria for the use of traumatized donor livers. Cholangiography carried out on the back table may help to determine if such injured livers are suitable for transplantation.

Alopecia areata is an autoimmune disease targeted at hair follicles with infiltrated T lymphocytes probably playing an important role in the pathogenesis. It was reported in 1985 that mechlorethamine was effective on alopecia areata patients. This has never been confirmed since. The aims of the study were to investigate the effects of mechlorethamine on balding C3H/HeJ mice affected with an alopecia-areata-like disease and to study the underlying mechanisms. Mice were treated on half of the dorsal skin with mechlorethamine and the contralateral side was treated with the vehicle ointment. After 10 wk of mechlorethamine therapy, a full pelage of hair covered the treated side in all the mice and was maintained during the study, whereas the vehicle-treated sides showed either no change or continued hair loss. Immunohistochemistry revealed that infiltrated CD4+ and CD8+ lymphocytes were eliminated from the treated side. In vitro cell viability assay showed that lymphocytes were much more sensitive to the cytotoxic effects of mechlorethamine than skin and hair follicular cells. RNase protection assay and real-time reverse transcription polymerase chain reaction showed that tumor necrosis factor alpha/beta, interleukin-12, and interferon-gamma were inhibited by mechlorethamine upon successful treatment. Our findings support that mechlorethamine restores follicular activity by selectively targeting infiltrated lymphocytes in vivo in alopecia-areata-affected mice.

A 15-year-old female with carbamyl phosphate synthetase deficiency, cystic fibrosis, and cystic fibrosis-related diabetes underwent orthotopic cadaveric liver transplantation. Metabolic control was maintained during the procedure with nutritional support and the use of intravenous sodium phenylacetate and benzoate. Her postoperative course was complicated by seizures and a transient decline in her pulmonary function tests, which returned to preoperative levels within one year of the transplant. Now, four years post-transplant, her quality of life has dramatically improved. There are only four Canadian centres with paediatric liver transplantation programs. However, expert medical care for adults with inborn error of metabolism is even more limited, suggesting that access to adult medical care is one of the many factors to be considered when liver transplantation is contemplated for patients with metabolically unstable conditions.

OBJECTIVES:To study the indications for liver transplantation among British Columbia's First Nation population.MATERIALS AND METHODS:A retrospective analysis of the British Columbia Transplant Society's database of Aboriginal and non-Aboriginal liver transplant recipients from 1989 to 1998 was undertaken. For primary biliary cirrhosis (PBC), the transplant assessment database (patients with and without transplants) was analyzed using a binomial distribution and compared with published census data regarding British Columbia's proportion of Aboriginal people.RESULTS:Between 1989 and 1998, 203 transplantations were performed in 189 recipients. Fifteen recipients were Aboriginal (n=15; 7.9%). Among all recipients, the four most frequent indications for liver transplantation were hepatitis C virus (HCV) infection (n=57; 30.2%), PBC (n=34; 18.0%), alcohol (n=22; 11.6%) and autoimmune hepatitis (n=14; 7.4%). Indications for liver transplantation among Aboriginal people were PBC (n=8; 53.3%; P<0.001 compared with non-Aboriginal people), autoimmune hepatitis (n=4; 26.67%; P=0.017), acute failure (n=2; 13.3%) and HCV (n=1). Among all patients referred for liver transplantation with PBC (n=43), 29 (67.44%) were white and 11 (25.6%) were Aboriginal. A significant difference was found between the proportion of Aboriginal people referred for liver transplantation and the proportion of Aboriginal people in British Columbia (139,655 of 3,698,755 [3.8%]; 1996 Census, Statistics Canada) (P<0.001).CONCLUSIONS:Aboriginal people in British Columbia are more likely to be referred for liver transplantation with a diagnosis of PBC but are less likely to receive a liver transplant because of HCV or alcohol than are non-Aboriginal people.

UNLABELLED:De novo HBV infection post-liver transplantation (LT) from an anti-HBc seropositive donor rarely presents as acute failure. We report a 42-year-old Caucasian female, HBsAg and anti-HBc seronegative, with primary biliary cirrhosis who received an allograft from a HBsAg negative, anti-HBc seropositive donor. The patient, previously vaccinated years pre-LT, was re-vaccinated against HBV and 1 year post-LT had an anti-HBs titre of 256 IU/l. Two years post-LT, elevated serum aminotransferases and worsening liver function with an INR of 2.0 developed. The HBsAg became positive, anti-HBs undetectable and serum HBV-DNA >2000 pg/ml by hybridisation assay. Liver biopsy revealed significant ballooning degeneration, piecemeal necrosis and positive immunostaining for HBsAg. Progressive liver failure developed followed by sepsis and terminal multi-organ failure. Subsequent analysis of the predominant HBV strain revealed mutations in the "a" determinant: Met 133 Thr (codon change ATG to ACG) and Asn 131 Thr.CONCLUSION:' Acute de novo HBV infection from an anti-HBc sero-positive donor may occur long after LT despite protective anti-HBs titres post-vaccination secondary to the emergence of "a" determinant mutated strains of HBV.

ObjectiveTo determine whether there is a role for assessing peripheral blood mononuclear cell (PBMC) cytokine patterns as a means of measuring the immunologic and clinical status of liver transplant recipients.Summary Background DataThe role of assessing cytokine patterns in the prediction of clinical graft rejection or acceptance remains unclear. The purpose of this study was to examine the cytokine profiles of PBMC stimulated in vitro with donor alloantigen and to correlate prospectively the data with clinical assessment of graft status in orthotopic liver transplant (OLT) recipients.MethodsPBMCs from OLT recipients were examined for proliferation and cytokine mRNA expression after stimulation by donor alloantigen, third-party alloantigen, or phytohemagglutinin (PHA), mRNA extracted from PBMC was amplified by reverse transcriptase-polymerase chain reaction with oligospecific primer pairs for interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN) gamma, tumor necrosis factor (TNF) alpha and transforming growth factor (TGF) beta. Results were prospectively correlated with each patient's allograft status.ResultsIncreased IL-4 and TGF-beta and decreased IL-2, IFN gamma, and TNF-alpha mRNA expression by PBMCs in response to donor alloantigen stimulation predicted immunologic graft stability over a minimum 60-day interval compared with mRNA expression of PBMCs from patients with established rejection or those who experienced a rejection episode within a 30-day period (p < 0.05). Stimulation of recipient PBMCs with third-party alloantigens or PHA yielded similar but less specific results. PBMC proliferation to varying antigenic stimulation did not correlate with clinical graft status, nor did cytokine production by unstimulated PBMC.ConclusionsProspective assessment of cytokine expression by PBMC from OLT recipients in response to stimulation by donor alloantigen is helpful for predicting the clinical status of the allograft and may be useful in the development of more precise immunologic monitoring protocols.

Current protocols for prophylaxis against allograft reinfection after liver transplantation for chronic hepatitis B virus (HBV) infection include the administration of large doses of hepatitis B immune globulin (HBIG), with considerable associated economic costs. Monotherapeutic prophylaxis with lamivudine has been complicated by the development of resistant strains of HBV. We studied the effectiveness of a posttransplantation prophylaxis protocol using combination lamivudine and low-dose HBIG in 7 consecutive patients with chronic HBV infection, 4 of whom were serum HBV DNA positive before pretransplantation lamivudine therapy. All patients were serum HBV DNA negative at transplantation and received lamivudine, 100 mg/d, posttransplantation. HBIG, 2170 IU, was administered intramuscularly intraoperatively and daily for 14 days. Maintenance HBIG therapy consisted of 2170 IU intramuscularly twice weekly, tapered to every 2 to 4 weeks by 12 months posttransplantation. Target serum HBIG (HBV surface antibody) titers were less than 500 IU/L for 6 months, then greater than 300 IU/L until 12 months posttransplantation. Induction serum HBIG titers were determined daily in 5 patients, and both serum HBIG and hepatitis B surface antigen were determined every 4 weeks in all patients. One patient died 61 days posttransplantation; the surviving patients (n = 6) were followed up for a mean of 532 days (range, 395 to 648 days). No patient has developed allograft reinfection. In the induction period, a target HBIG titer of greater than 500 IU/L was not achieved until a mean of 6.8 days (range, 5 to 10 days). In the maintenance period, all patients achieved the target HBIG titer. This suggests combination lamivudine and low-dose HBIG is effective in preventing allograft reinfection by HBV.

Donor-specific increased renal graft survival after portal vein (pv) immunization is associated with decreased production of cytotoxic T lymphocytes and altered cytokine production (decreased IL-2 production; increased IL-4, IL-10 and TGF beta). In mice with surviving renal grafts donor antigen, with host dendritic cells (DC), recalled cytotoxicity from C57BL/6 immune C3H cells, and TL-4 not IL-2 production, despite decreased cytotoxicity seen in the transplant recipients themselves. Fresh donor DC induced IL-2 not IL-4 production. Blocking expression of ICAM-1 on donor grafts further increased survival. C3H responder spleen cells, incubated with C57BL/6 DC and C3H hepatic cells, transferred tolerance to C57BL/6 renal grafts in vivo. Taken together these data suggest a role for ICAM-1: persistent donor antigen (on host DC), and accessory hepatic monocytes, in the induction/maintenance of tolerance after pv immunization.

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the α subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein γ subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different G β subunits reveals specific interaction between RGS11 and G β5 . The expression of mRNA for RGS11 and G β5 in human tissues overlaps. The G β5 /RGS11 heterodimer acts as a GAP on G αo , apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for G α proteins and form complexes with specific G β subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.

BALB/c, DBA/2 and C57BL/6 mice of different ages (ranging from 8 to 110 weeks of age) were used as spleen cell donors to assay cytokine production from ConA activated spleen and Peyer's Patch (PP) lymphocytes. As reported in an earlier publication, there was an age-related decline in IL-2 production in all strains, with a general increase in IL-4 and IL-10 production with age, this being particularly marked for PP cell preparations. Similar conclusions were reached from independent analysis of CD44hi and CD44lo cell populations in these groups (memory vs. naive cells, respectively). Interestingly, IL-6 production was dramatically increased (some 4-5-fold in the different strains) and significantly increased levels of IL-6 were detected in the serum of aged mice. A previously described sheep fetal liver extract was able to reverse, to varying degrees, these cytokine changes associated with aging. Interestingly, when cells from aged mice were adoptively transferred to lethally irradiated young (8 week) recipients, the cytokine production phenotype of cells harvested from recipient mice 3 weeks later was that of the aged donor, unless recipients were treated continually with extract. Treatment of the donor alone produced minimal changes in cytokine production 3 weeks following adoptive transfer. The effect of extract was reversed in treated aged mice by concomitant daily intravenous infusion of the competitive inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine (NMMA)), which also decreased the increased serum nitrate levels in mice treated with extract. Our data suggest an important role for reactive nitrogen products, themselves induced by fetal liver extract, in age-associated changes in cytokine production.

BACKGROUND:A replication-defective retrovirus BAG vector was tested for in situ delivery of the beta-galactosidase gene to auxiliary liver transplant in a rat model.METHODS:The BAG vector, which was shown to be effective in genetic transduction of cultured NIH/3T3 cells, was produced in a psi2 packaging cell and later amplified in a selected PA317 clone. Hepatocyte replication was induced by one-third hepatectomy of the donor liver, and the procedure was followed by auxiliary partial liver transplantation. Twenty-four hours after hepatic induction or transplantation, viral supernatant at 37 degrees C was perfused into the liver graft via the portal vein during a temporary occlusion of the graft portal vein.RESULTS:All animals survived the transplantation procedures and were killed at specified time intervals. Histochemical staining of the liver graft specimens indicated the expression of beta-galactosidase in the gene transferred group but not in the control animals. As demonstrated by polymerase chain reaction assay, the proviral beta-galactosidase sequence was present in the graft specimens, but absent from all other tissues tested.CONCLUSIONS:In short, the retrovirus BAG vector can be useful for in situ delivery of foreign genes to liver graft in transplantation and other clinical settings, providing a simple, consistent, and reliable alternative in hepatic gene therapy experiments.

The liver of anaesthetized adult ( > 350 g) Lewis rats was perfused in vitro after cannulation of both the hepatic and portal vein, with clamping of the hepatic artery. Heparinized blood (400 microliters) was withdrawn at 0, 6, 12, 18, 24, 30 and 36 h from each site, and serum and peripheral blood lymphocytes (PBL) isolated after ficoll/hypaque separation. Serum was tested in bioassays for cytokines known to modulate lymphocyte:endothelial interactions in vivo and in vitro (IFN gamma, TGF beta, TNF alpha, IL-6, IL-1). In some experiments rats received, via portal venous infusion, a sterile inoculum of 150 x 10(6) semi-allogeneic (LBN F1) spleen cells immediately or 12 h after the start of the study. In animals which were unchallenged with cells via the portal vein, subtle differences in detectable cytokines were observed between hepatic and portal blood samples, as reported in earlier studies. Within 12 h the minor perturbations observed in cytokine profiles following surgical insult resolved, and the changes between hepatic and portal venous samples remained constant throughout the study in control rats. However, in rats treated with LBNF1 cells, changes in the cytokine profiles were seen compared with control animals, and as a function of time post F1 cell infusion. Changes in mRNAs for different cytokines were observed in PBL taken from portal/hepatic blood in these same animals. These data point to one possible mechanism whereby the liver may influence immunological processes following portal venous antigen challenge.

Naive or preimmunized (to B10.BR or BALB.k) C3H/HeJ mice received skin grafts from multiple minor histoincompatible B10.BR or BALB.k mice following antigen-specific portal venous (p.v.) pretransplant transfusion, a protocol known to produce prolongation of graft survival in naive animals. In addition, groups of mice received intravenous (i.v.) infusion following transplantation with a mixture of monoclonal antibodies (mAb) to vascular adhesion molecule-1L: very late activation antigen-4 (VCAM-1:VLA-4) or intracellular adhesion molecule-1:lymphocyte function-associated antigen-3 (ICAM-1:LFA-1). Cells were harvested from different tissues of the grafted mice at various times post grafting. RNA was extracted and analysed, using polymerase chain reaction, for expression of different cytokines potentially involved in the regulation of graft rejection [interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumour necrosis factor-alpha, interferon-gamma and transforming growth factor-beta]. In addition, using limiting dilution analysis, we investigated the frequency of allo-specific and third-party reactive cells producing IL-2 and IL-4 in vitro in different tissues of grafted mice following these treatments. The mAb treatment protocol which produced optimum increases in graft survival in naive versus immune mice was different, with anti-LFA-1:ICAM-1 superior for naive mice compared with anti-VLA-4:VCAM-1, and vice versa for immune animals. However, in each case, increased survival was associated with increases local to the graft in the frequency of occurrence of antigen-specific type-2 cytokine-producing cells.