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Studies were conducted to develop a new DPPIV–/–/Rag2–/– mouse model for hepatocyte transplantation by allogeneic and xenogeneic cells and to compare the proliferative capacity of p27 null hepatocytes versus normal hepatocytes in this system. Dipeptidyl peptidase IV (DPPIV) gene knockout mice, in which wild-type (wt) DPPIV+ donor hepatocytes can be readily identified by enzyme histochemistry, were bred with Rag2 null mice to prepare immunotolerant DPPIV–/–/Rag2–/– double knockout mice. DPPIV–/–/Rag–/– mice were transplanted with wt hepatocytes or p27 null mouse hepatocytes, which show enhanced cell cycle activity due to disruption of the Kip1 cyclin kinase inhibitor gene, and liver repopulation was assessed under nonproliferative versus proliferative experimental conditions. After their initial engraftment, transplanted wt hepatocytes did not proliferate in untreated livers or increase significantly in response to an acute liver regenerative stimulus. p27 null hepatocytes engrafted with the same efficiency as wt hepatocytes, but showed a noticeable, although not statistically significant, increase in proliferation in response to partial hepatectomy or acute CCl4 administration. Repeated treatments with CCl4 substantially increased proliferation and liver repopulation by p27 null hepatocytes but not by wt hepatocytes. These results suggest that p27 gene inactivation does not overcome proliferative restrictions imposed on hepatocytes by the normal liver, but that after repeated episodes of toxic liver injury, the augmented proliferative capacity of p27 null hepatocytes leads to significant liver repopulation compared with wt hepatocytes. These properties of p27-deficient hepatocytes could prove useful as a target for liver repopulation in patients with intermittent or a low level of chronic liver injury.

O ver the years, substantial evidence has accumulated demonstrating the existence of adult hepatic progenitor cells, also termed oval cells (OCs). When the regenerative capacity of terminally differentiated hepatocytes is exhausted or blocked, these cells are activated to proliferate and differentiate into hepatocytes and cholangiocytes. OCs sprout from the putative stem cell niche (canals of Hering), forming tortuous pseudoducts and invading the liver lobule. 1-6 These pseudoducts are in close proximity to desmin-positive stellate cells. 7 Recently, we found a population of activated mesenchymal cells, thymus cell antigen-1 (Thy-1)-expressing cells, surrounding OCs. These cells partially overlapped with the desmin-positive cells and produce inductive signals (growth factors and cytokines) in the OC niche. 8,9 Thy-1 is a cell surface glycophosphatidylinositollinked glycoprotein with a molecular mass of 35 kDa and is an adhesion molecule of the immunoglobulin super-

Major histocompatibility (MHC) class II antigen expression by thyroid epithelial cells has been widely implicated in the pathogenesis of autoimmune thyroid disease. We have examined rat MHC (RT1) class II antigen gene regulation in 1B-6 cells, cloned in our laboratory from the Fisher rat thyroid line FRT-L5. 1B-6 cells are TSH dependent for growth and proliferation, and are responsive to more than 5 microU/ml bovine TSH (bTSH) in terms of extracellular cAMP accumulation. Recombinant rat gamma interferon (gamma IF; 1-1000 U/ml) treatment for 5 days together with bTSH (10(3) microU/ml) was able to induce class II antigen expression in up to 90% of 1B-6 cells, as detected by a murine monoclonal antibody to rat MHC class II antigen (FITC-OX-6). Total cellular RNA was examined in Northern blot analyses using an HLA-DR alpha-chain-specific RNA probe, which shares 78% sequence homology with the rat RT1.D alpha-chain gene. A 1.4-kilobase mRNA transcript was detected in gamma IF-stimulated cells, but not in untreated cells. Dose-response studies of MHC class II gene expression, using a cytoplasmic RNA slot blot technique for alpha-chain mRNA levels and FITC-OX 6 monoclonal antibody for detection of MHC class II antigen expression, indicated 1B-6 sensitivity to gamma IF in the range of 10-100 U/ml (in the presence of 10(3) microU/ml bTSH) and to bTSH in the range of 10-100 microU/ml (in the presence of 10(2) U/ml gamma IF). These data demonstrate dual control of MHC class II antigen gene expression by gamma IF and bTSH in a differentiated rat thyroid cell. Clone 1B-6 represents a powerful means of analyzing reproducibly and in long term studies the regulation of thyroid cell MHC class II gene expression.

We have previously demonstrated that cultured rat thyroid cells do not exhibit constitutive expression of major histocompatibility (MHC) class II antigens. Using reovirus types 1 and 3, we infected 1B-6 cells (a cloned derivative of the Fisher rat cell line FRTL-5), and found a dose-dependent induction of thyroid cell MHC class II antigen expression as determined by laser flow cytometry and FITC-labelled OX-6 anti-RT1.B. As the number of viral particles/cell used for the infections increased from 5 to 100, the number of antigen positive cells increased, in reovirus type 3 infections to 50%, and in reovirus type 1 infections to 15%. Kinetic studies indicated that MHC class II antigen expression continues to increase three days after infection. Viral infection and the resulting MHC class II antigen expression may allow presentation of thyroid antigen to the immune system and participate in the initiation of autoimmune thyroid disease.

Graves' disease encompasses hyperthyroidism and a diffuse goiter associated with autoantibodies to the TSH receptor (TRAb). Although the cause of the goiter formation has been attributed to TRAb, the limited growth pattern of human adult thyroid cells in vitro has caused such a conclusion to be based on studies of nonhuman thyroid cell growth. We have recently characterized a predictable and precise technique for the measurement of human thyroid cell proliferation and function using fetal thyroid cells and have used this system to examine the influence of TRAb on human thyroid cell growth. Highly purified human immunoglobulin G (hIgG) preparations from normal individuals (n = 5) had no significant influence on human thyroid cell growth. However, hIgG from patients with detectable TRAb (TRAb-hIgG) (n = 13) induced a dose-related increase in extracellular cAMP (maximum effect at 0.1 mg/ml) and a 3-fold increase in human thyroid cell growth over a 4-day period (maximum effect at 1.5 mg/ml). Under basal thyroid cell culture conditions there were detectable, but low, levels of mRNA specific for the protooncogene c-fos, and this was markedly, and rapidly, induced by the addition of TRAb-hIgG but not normal hIgG. These data demonstrate induction of cellular growth by TRAb-hIgG in an homologous human thyroid cell culture system. Such observations support the hypothesis that goiter formation in patients with Graves' disease is, at least in part, secondary to the growth stimulating activity of TRAb-hIgG.