Transmission of hepatitis B infection from hepatitis B core antibody--positive liver allografts is prevented by lamivudine therapy.

Donor shortage has led to the use of hepatitis B core antibody (anti-HBc)--positive (anti-HBc(+)) liver allografts for patients in need of relatively urgent orthotopic liver transplantation (OLT). Because anti-HBc(+) allografts transmit hepatitis B virus (HBV) infection at a high rate, effective prophylaxis is required. We assessed the effectiveness of lamivudine in preventing HBV transmission by anti-HBc(+) allografts. Between March 1996 and March 2000 at Cedars-Sinai Medical Center (Los Angeles, CA), 15 of 169 patients (8.9%) received liver allografts from anti-HBc(+) donors. Six patients were hepatitis B surface antigen (HBsAg)(+) (group 1), and 9 patients were HBsAg negative (HBsAg(-); group 2) before OLT. All patients were administered lamivudine, 100 or 150 mg/d, orally after OLT. Patients who were HBsAg(+) before OLT also were administered hepatitis B immunoglobulin (HBIG) prophylaxis. Hepatitis B serological tests were performed on all patients, and HBV DNA was determined in liver tissues in 10 patients. All 15 patients remained HBsAg(-) at their last follow-up 2 to 40 months (mean, 17 months) post-OLT. All patients in group 1 had antibody to HBsAg (anti-HBs) titers greater than 250 mIU/mL post-OLT (mean follow-up, 20 months; range, 7 to 40 months). Of the 2 patients in group 1 who underwent liver biopsy after OLT, 1 patient had detectable hepatic HBV DNA despite being anti-HBs(+) and HBsAg(-). Among the patients in group 2, none acquired anti-HBc or HBsAg. Hepatic HBV DNA was undetectable in the 7 patients in group 2 who underwent liver biopsy after OLT. Anti-HBc(+) allografts can be safely used in patients who undergo OLT for chronic hepatitis B and susceptible transplant recipients if prophylaxis with combination HBIG and lamivudine or lamividine alone is administered after OLT, respectively. However, more data are needed to determine the efficacy of lamivudine monotherapy in preventing transmission of HBV infection from anti-HBc(+) liver allografts to susceptible recipients.

Hepatocarcinogenesis (Z#2)/mutagenesis during initiation stage.

Previously, we developed a model for high incidence, endogenously generated hepatocellular carcinoma (HCC), the human alpha-1-antitrypsin (alpha1AT) Z gene transgenic mouse (Z#2). We now examine the potential utility of a model for endogenous carcinogenesis utilizing the Z#2 mouse also transgenic for the lacI gene, a convenient target for in vivo mutagenesis studies. We crossed the Z#2 line and mice transgenic for lambda/lacI shuttle vector (Big Blue), for determination of lacI mutant frequency during initiation of endogenous carcinogenesis. Five month old double transgenic mice (Z#2+/lacI+) successfully displayed: (1) the expected post-inflammatory stage of Z#2 carcinogenesis; and (2) hepatic lacI mutants measured at frequencies (10(-5)-10(-4)) useful to mutagenesis studies. In this study, hepatic lacI mutation frequencies in Z#2 transgenic mice appeared to be only slightly increased (< 2x) when compared to age matched negative controls. In the future, it may be important to reconcile possibly limited lacI mutagenesis at the time of initiation and demonstrated high incidence of hepatocarcinogenesis.

Posttransplant lymphoproliferative disorder in liver allograft biopsies: a comparison of three methods for the demonstration of Epstein-Barr virus.

Posttransplant lymphoproliferative disorder (PTLD) is associated with Epstein-Barr virus (EBV), and may clinically resemble acute allograft rejection. Three methods to show EBV in tissue were evaluated in 15 liver allograft biopsies from 12 patients including four with PTLD: (1) semiquantitative polymerase chain reaction (PCR) for EBV DNA; (2) in situ hybridization for EBV RNA (EBER); and (3) immunoperoxidase for EBV latent membrane protein (LMP). Index cases had a PCR dot blot result of "positive" or "weak positive." Findings were correlated with histology, clinical data, therapy, and outcome. All four PTLD patients had a clinical diagnosis of acute rejection. All four showed EBV: PCR 4, EBER 4, LMP 3, Liver function tests were elevated in three, but EBV viral capsid antigen (VCA) IgM was not increased in three, but EBV viral capsid antigen (VCA) IgM was not increased in three. Immunosuppression was withdrawn and all four patients underwent a second transplantation. One died 4 days posttransplant with disseminated PTLD, two died of sepsis at 1.5 and 14 months, and one is well at 3 years without PTLD. Eleven biopsies without PTLD showed: acute rejection 7, acute rejection and hepatitis 1, hepatitis B 1, and non-inflammatory changes 2. In this group, EBV results included: PCR weak positive in 10 and 1+ in one, EBER negative in ten and rare positive cells in one, LMP negative in 11. Liver function tests were elevated in 10, whereas VCA IgM was not increased in three and increased in one. Patients with acute rejection were treated with increased immunosuppression: none developed PTLD, with follow-up of at least 6 months in nine cases. Two patients died within 4 months of biopsy. One patient with PTLD in tonsils had a liver biopsy showing both acute rejection and EBV (PCR 1+, rare EBER + small cells). Histological studies combined with special EBV detection methods, can be useful to evaluate atypical lymphoid infiltrates in liver allograft biopsies and confirmation of a diagnosis of PTLD. All three methods are useful; EBER and PCR are the most sensitive. EBER and LMP can use paraffin sections.

Hepatitis C virus is not recoverable from liver tissue in cryptogenic cirrhosis: failure to identify hepatitis C virus-RNA using reverse transcription-mediated polymerase chain reaction.

Polymerase chain reaction (PCR) has been used to study liver biopsy tissue in patients with known or suspected hepatitis C virus (HCV). Recent studies of cryptogenic cirrhosis using PCR have been based on study of sera, and HCV has not been shown. The failure to show HCV in patients so studied has left unanswered the question of whether or not patients with cryptogenic cirrhosis could still harbor the virus in the liver. The authors studied liver tissue, obtained at the time of orthopic liver transplantation from 10 patients clinically diagnosed as having end-stage liver disease without demonstrable origin, so-called cryptogenic cirrhosis, using reverse transcription (RT)-PCR to try to recover HCV-RNA. Formalin-fixed, paraffin-embedded tissue was used. For comparison, the authors also studied similarly obtained samples from 10 patients with typical hepatitis C-associated cirrhosis and 10 patients with end-stage liver disease resulting from autoimmune hepatitis. The authors recovered HCV-RNA from 9 of 10 livers from patients with cirrhosis resulting from HCV, and 3 of 10 livers from patients with autoimmune hepatitis. HCV-RNA was not recovered from any of the livers of the 10 patients designated as having cryptogenic cirrhosis.

Changes in tonsils and adenoids in children with posttransplant lymphoproliferative disorder: report of three cases with early involvement of Waldeyer's ring.

Posttransplant lymphoproliferative disorder (PTLD) is an infrequent complication of transplantation in children, and this report emphasizes the value of tonsil and adenoid biopsy in the early management of this potentially life threatening condition. In all three cases biopsy specimens of tonsils and adenoids were diagnostic of polymorphic diffuse B-cell hyperplasia (PBCH). Immunophenotyping showed no immunoglobulin (Ig) light chain restriction, although immunoglobulin heavy chain (IgH) gene rearrangement was monoclonal in two cases. Despite an absence of serological evidence for acute Epstein-Barr virus (EBV) infection, EBV was detected in all cases by semiquantitative polymerase chain reaction (PCR) for EBV DNA, by in situ hybridization for EBV mRNA (EBER), and by immunoperoxidase for EBV latent membrane protein (LMP). All three patients were treated with reduced immunosuppression and acyclovir and are well (19, 28, and 28 months' follow-up) with no recurrence. Children without previous EBV exposure may develop PTLD localized to the tonsils/adenoids, and biopsy specimens of these tissues may permit early diagnosis and clinical intervention. Despite monoclonal gene rearrangement in two cases, overall features were not indicative of malignancy. Strong association with EBV is helpful in confirming the diagnosis of PTLD and is consistent with initial presentation in the tonsils/adenoids.

Hepatocarcinogenesis is the sequel to hepatitis in Z#2 alpha 1-antitrypsin transgenic mice: histopathological and DNA ploidy studies.

Z mutant-associated alpha 1-antitrypsin deficiency in human beings leads to hepatitis and, in some cases, hepatocellular carcinoma. To begin to delineate the molecular basis for the development of hepatocellular carcinoma in alpha 1-antitrypsin deficiency, we previously developed transgenic mice using human alpha 1-antitrypsin M and Z genomic clones. High-copy Z lineage mice (12 gene copies/haploid mouse genome; "Z#2") had hepatocytes distended with human alpha 1-antitrypsin deficiency globules. Hepatitis was present, and the morphological changes mimicked those observed in human alpha 1-antitrypsin deficiency-related liver disease. The numbers of hepatocytes containing alpha 1-antitrypsin globules decreased with age, and alpha 1-antitrypsin-negative nodular aggregates of hepatocytes increased in number and size. Hepatocytic dysplasia occurred as early as 6 wk and was almost universally present at 1 yr. Nodules of dysplastic cells demonstrating aneuploidy were seen as early as 10 wks. These became persistent, proliferative lesions. Dysplasia and aneuploidy distinctly increased with time and advancing microscopic stage as lesions progressed to malignancy. Tumors were seen after 1 yr as adenomas, which are aneuploid and most likely well-differentiated hepatocellular carcinoma, and borderline malignant lesions; and, in 82% of Z#2 mice 16 to 20 mo old, as invasive hepatocellular carcinoma. These observations suggest but do not conclusively prove that hepatocellular carcinoma in alpha 1-antitrypsin deficiency and other hepatic disorders arises as a result of a common, endogenously stimulated pathway for hepatocellular carcinogenesis.

Epstein-Barr virus infection in liver transplantation patients: correlation of histopathology and semiquantitative Epstein-Barr virus-DNA recovery using polymerase chain reaction.

Epstein-Barr virus (EBV) infection may complicate orthotopic liver transplantation, and can lead to hepatitis with subsequent graft failure and to benign and malignant lymphoproliferative disorders. Early diagnosis allows for prevention or treatment of complications. Histopathologic features of EBV infection in the liver vary and may be difficult to recognize. To delineate the morphologic features that allow for recognition we studied 61 biopsy specimens from 37 patients, correlating the results of EBV-DNA demonstration after polymerase chain reaction with histopathology of formalin-fixed, hematoxylin-eosin-stained liver biopsy specimens. DNA was extracted from fresh liver biopsy samples, and polymerase chain reaction was carried out with EBV primers (capsid protein gp220) using standard techniques and 25-cycle amplification. Epstein-Barr virus-related sequences after polymerase chain reaction were detected by DNA blot assay. Histopathologic features were classified into three categories on the basis of the semiobjective determination of the number and distribution of immunoblasts and other immature lymphocytes in portal tracts and sinusoids: highly suggestive (three biopsy specimens), indeterminant (one biopsy specimen), and negative (57 biopsy specimens). Only the three highly suggestive biopsy specimens had high levels of EBV-DNA. We conclude that the histopathologic features of EBV infection after orthotopic liver transplantation can be relied on to establish the diagnosis.

Detection of Epstein-Barr virus by polymerase chain reaction in transbronchial biopsies of lung transplant recipients: evidence of infection?

Thirty-nine paraffin-embedded transbronchial biopsies from ten lung transplant recipients were evaluated by the polymerase chain reaction for the presence of Epstein-Barr virus (EBV). Six of these patients had positive EBV serology. Nine biopsies from nine non-transplant patients with nonspecific interstitial pneumonitis were studied as a control group. Eight biopsies from four of the ten transplant patients (40%) were positive for EBV by polymerase chain reaction, utilizing 40 cycles and two sets of primers (501/502) complementary to sequences within the Bam H1 W region of the viral genome. Five of these eight positive biopsies had been diagnosed as mild acute cellular rejection based on clinicopathologic criteria. Three of the four EBV-positive patients showed clinical improvement with antiviral agents prescribed for concomitant cytomegalovirus infection. Our data demonstrate the potential of polymerase chain reaction to detect small quantities of EBV in transbronchial biopsies from lung transplant recipients. Such findings should be interpreted cautiously in the evaluation of lung transplant recipients, since EBV can be detected in the absence of a lymphoproliferative disorder or an active pneumonitis caused by this virus.

Histopathology of alpha 1-antitrypsin liver disease in a transgenic mouse model.

Transgenic mice were constructed using human alpha 1-antitrypsin M and Z genomic clones. Livers of the M lineage mice showed slight cellular pleomorphism and immunohistochemically demonstrable finely granular alpha 1-antitrypsin material in hepatocytes. Z lineage mice with five gene copies per haploid mouse genome (Z#1) demonstrated fine granular alpha 1-antitrypsin material and a few large globules. In contrast, Z lineage mice with 12 gene copies per haploid mouse genome (Z#2) demonstrated hepatocytes filled with homogeneous, eosinophilic globules that were strongly reactive with diastase and periodic acid-Schiff and antibody to alpha 1-antitrypsin. Scattered microscopic polymorphonuclear leukocyte accumulations were seen that contained extracellular alpha 1-antitrypsin material, but there was neither histological nor serological evidence of mouse infectious hepatitis. In young animals, small clusters of hepatocytes lacking alpha 1-antitrypsin material were seen. These cells were the dominant population in older animals and formed nodular arrangements. Fibrosis was not demonstrable in neonatal and young animals or in any of the controls, but perisinusoidal fibrosis was seen in older Z#2 mice. Groups of hepatocytes without alpha 1-antitrypsin material showed dysplastic changes. We conclude that the transgenic mouse is a reliable and useful model in which to study the effects of alpha 1-antitrypsin in the liver because it demonstrates changes similar to those in the human disease.

Epstein--Barr virus nuclear antigen in a non-Hodgkin's lymphoma.

This paper describes the course of a patient with treated non-Hodgkin's lymphoma, which originally presented as a large nasopharyngeal mass. The tumor, though irradiated, recurred on two separate occasions in the right and left inguinal regions. At the time of tumor recurrence, markers were present which have been associated with past or recurrent Epstein-Barr virus (EBV) infection, namely, antibodies to the EBV viral capsid antigen (VCA) and the Epstein-Barr nuclear antigen (EBNA). EBNA was detected in approximately 10% of the neoplastic cells. An unusual immunoglobulin heavy and light chain switch within the tumor cells of the two separate inguinal node tumor recurrences was also observed. Whether EBV plays a role in the pathogenesis of lymphomas remains unclear.

Feline lymphocytes: observations on surface membrane concanavalin A receptor mobility.

The mechanics of concanavalin A receptor mobility of the feline lymphocyte surface membrane were investigated, utilizing fluorescein-labeled lectin to quantitate lymphocyte capping. The results of this study indicated that lectin concentration and buffer selection were critical for extensive receptor redistribution with cap formation of feline lymphocytes. Maximal capping was obtained with 50 microgram of concanavalin A/ml of minimal essential medium. The mean capping rate of peripheral blood lymphocytes increased significantly with colchicine exposure at 10(-7) M concentration. The mean values of capping increased slightly with advancing age of feline donors, although this difference was not statistically significant. Concurrent work has indicated that concanavalin A capping may be useful in the study of immunosuppression in feline leukemia virus-infected cats.

Immunosuppressive properties of a virion polypeptide, a 15,000-dalton protein, from feline leukemia virus.

The 15,000-molecular-weight polypeptide (p15) of feline leukemia virus (FeLV) was shown to impair normal lymphocyte function in vitro and to abrogate immunity to feline oncornavirus disease in vivo. FeLVp15 suppressed concanavalin A-induced blast transformation of normal feline lymphocytes by 68%, while other virion proteins had no effect. p15 suppression was not due to toxicity, nor was p15 a competitive inhibitor of concanavalin A binding. Capping of receptors for concanavalin A on normal feline lymphocytes also was inhibited by either inactivated FeLV or FeLV p15. Groups of cats were immunized with either killed feline oncornavirus-associated cell membrane antigen bearing tumor cells or tumor cells plus FeLV p15. After challenge with feline sarcoma virus, three of four p15-treated cats developed progressive fatal fibrosarcoma as compared to one of five non-p15-treated cats. The cats receiving p15 also had lower cytotoxic antibody titers against feline oncornavirus-associated cell membrane antigen (mean peak titer, 1:6) than did the non-p15 group (1:74). These data support the hypothesis that the immunosuppression in cats infected with FeLV is mediated by FeLV p15.

Mobility of lymphocyte surface membrane concanavalin A receptors of normal and feline leukemia virus-infected viremic felines.

The virus-associated depression of concanavalin A mitogenesis which accompanies feline leukemia virus-induced cat lymphoma was investigated by comparing lymphocyte surface receptor mobility of normal cats to that of viremic diseased animals. The mechanics of feline lymphocyte receptor mobility were studied using fluorescein-conjugated concanavalin A to quantitate lymphocyte capping. The results of a study of 21 disease-free animals showed that cat lymphocytes undergo appreciable concanavalin A capping, with a mean capping rate of 17% under conditions developed in this study. In contrast, morphologically normal peripheral blood lymphocytes of six feline leukemia virus-infected viremic cats, with or without lymphoma, exhibited a mean capping of only 7%, significantly less than that of the control animals (p less than 0.005). These findings suggest that a membrane-related lymphocyte deficiency accompanies the development of virus-induced lymphoma in the cat.

Inhibition of human lymphocyte mitogen and antigen response by a 15,000-dalton protein from feline leukemia virus.

Peripheral blood lymphocyte response of normal human subjects to mitogens and antigens was suppressed by a 15,000-dalton protein (p15) from a C-type feline leukemia virus. Four of six subjects were suppressed 70 to 96% when responding to concanavalin A or phytohemagglutinin in the presence of 5.0 microgram of p15. The three subjects who responded to streptokinase-streptodornase and Candida were suppressed 68 to 91% when cultured with 5.0 microgram of p15. The subviral protein did not appear to be cytotoxic at doses reported. Lymphocyte membrane studies with fluorescein isothiocyanate-concanavalin A revealed a reduction in concanavalin A-induced cap formation of 51 to 91% in the presence of the same dose of p15. These results demonstrate that in vitro immunological dysfunction in human lymphocytes can be induced by a C-type virion protein.