Hepatocyte isolation from pig livers after warm ischaemic injury

Abstract Hepatocyte cultures have been used extensively for a wide variety of physiological, pharmacological and experimental studies. The warm ischaemic period before isolation is kept to a minimum to achieve a high yield of cells isolated and a good viability for culture. We have recently introduced a new concept of liver resuscitation after warm ischaemia that is based on a 3-h reperfusion period with an improved perfusate and simultaneous dialysis. In this study, we applied the new technique for hepatocyte isolation from livers subjected to 80 min of complete ischaemia at 37 °C. Cell yield was improved by a resuscitating perfusion from 58% to 73% and viability from 39% to 76%.     

MRL/lpr-->severe combined immunodeficiency mouse allografts produce autoantibodies, acute graft-versus-host disease or a wasting syndrome depending on the source of cells.

MRL/lpr (lpr) mice spontaneously develop a lupus-like illness as well as massive lymphadenopathy. Attempts to transfer autoimmunity by adoptive transfer or radiation bone marrow chimeras have been unsuccessful. Since severe combined immunodeficiency (SCID) mice have been engrafted with human and rat xenografts without apparent graft-versus-host disease (GVHD), we subjected SCID mice to low-dose irradiation and reconstituted the mice with spleen cells from young or old lpr mice or with lpr bone marrow. Fourteen out of twenty (70%) of SCID mice engrafted with spleen cells from old lpr mice produced autoantibodies (anti-DNA and anti-Sm) without evidence of the severe lymphoid atrophy previously described for lpr spleen-->+/+ chimeras. SCID mice engrafted with spleen cells from young lpr mice developed acute GVHD and 5/6 (83%) died within 4 weeks post-transfer. Although 8/11 (73%) of lpr-->SCID bone marrow allografts survived for at least 4 months, these mice developed a wasting disease characterized by lymphoid atrophy and fibrosis without the production of autoantibodies. None of the lpr-->SCID grafts resulted in the transfer of double negative T cells or the lymphoproliferative syndrome characteristic of MRL/lpr mice. These findings indicate that SCID mice can be engrafted with splenocytes from old MRL/lpr mice and that B cells continue to secrete autoantibodies for several months in the SCID recipients. This study also demonstrates that, unlike i.p. transplant of xenogeneic cells, acute GVHD is a consistent feature of i.p. transplants of normal allogeneic mononuclear cells into SCID mice.     

Growth inhibition and transformation of a human fetal tracheal epithelial cell line by long-term exposure to diethylnitrosamine

In order to obtain more information on the in vitro transformationof human cells, a human fetal tracheal epithelial cell line(FHET16/5) was exposed for a long time to diethylnitrosamine(DEN). In 20 passages, this cell line (diploid, male) maintainedstrong immunohistochemical reactivity for carcino-embryonnicantigen and wool merokeratin; it was negative for vimentin.The cells contained PAS-positive mucous substances and ultrastructurallywere found to have desmosomelike attachments. Treatment of thecells was with 0.3% dimethyl sulphoxide (DMSO), or DMSO with150, 450, 1000 or 2000 µg/ml of DEN. It was started atthe ninth passage and continued for six passages over 9 weeksfor the control (DMSO) and the three lowest control doses ofDEN, and for three passages over 9 weeks for the 2000 µg/mlDEN group. Cells grown for 13 days after the end of treatmentwere plated in soft agar and injected subcutaneously in nudemice. The frequency of anchorage-independent colonies grownin soft agar was directly related to DEN dose. Colony-formingefficiency, as an expression of toxic effect, was also dosedependent. Autoradiographically detected unscheduled DNA synthesisindicated an association between anchorage-independent transformationand DNA alterations induced by DEN. Cells injected into nudemice did not produce tumours during a 6-month period, but invasivenesswas observed when cells from the 2000 µg/ml DEN groupwere transplanted on the dermis of cultured chick embryo skin.The results indicate that DEN causes anchorage-independent transformationaccompanied by unscheduled DNA synthesis in a fetal human trachealepithelial cell line.     

The Fas protein is expressed at high levels on CD4+CD8+ thymocytes and activated mature lymphocytes in normal mice but not in the lupus-prone strain, MRL lpr/lpr.

The lymphoproliferation (lpr) mutation in the MRL strain of mice is caused by the insertion of the early transposable element ETn in the Fas gene. The insertion causes a striking decrease in Fas mRNA expression and is associated clinically with marked acceleration of the lupus-like disease. To further explore the role of the Fas protein in T-cell selection in the thymus and tolerance in the peripheral immune system, we produced a monospecific polyclonal anti-murine Fas antibody that binds to a polymorphic region of the protein. Fas protein expression was detected on approximately 90% of BALB/c and MRL +/+ thymocytes, and the expression was highest on CD4+CD8+ thymocytes, the stage at which most thymocytes die by apoptosis. In contrast to the high level of expression of Fas on thymocytes, Fas was detected on < 10% of normal splenic T cells. After activation of splenic T cells with Con A or anti-CD3 and interleukin 2, Fas expression increased approximately 10-fold. Fas expression on splenic B cells was also markedly up-regulated after activation with lipopolysaccharide or anti-mu antibodies. The Fas protein was not detected on resting or activated lymphocytes obtained from MRL lpr/lpr mice. Together, these findings suggest that Fas plays a role in both thymic selection and T-cell survival in the periphery and that the accelerated autoimmunity in MRL lpr/lpr mice results from a defect in both of these pathways.