C. Guerriero

TPA and cycloheximide modulate the activation of NF‐κB and the induction and stability of nitric oxide synthase transcript in primary neonatal rat hepatocytes

12‐O‐Tetradecanoylphorbol 13‐acetate (TPA) elicited a transient increase in the transcription of the inducible nitric oxide synthase (iNOS) gene coupled with a shortening of the half‐life of its mRNA in primary neonatal rat hepatocytes. These effects of TPA were preceded by a surge in nuclear translocation of the transcription factor NF‐κB, and followed by a mounting accumulation of NO2 − in the growth medium. Even cycloheximide (CHX) added by itself elicited an early, sustained activation of NF‐κB followed by an intense induction of iNOS gene expression, irrespective of what degree of protein synthesis inhibition was brought about by the several concentrations tested. When given together, TPA and CHX exerted additive effects on hepatocellular iNOS mRNA levels. These results suggest the likelihood of an ordered sequence of events by which an activated NF‐κB mediates the induction of iNOS gene expression in TPA‐ and/or CHX‐treated primary hepatocytes.

Induction of an antitumour adaptive immune response elicited by tumour cells expressing de novo B7‐1 mainly depends on the anatomical site of their delivery: the dose applied regulates the expansion of the response

De novo expression of costimulatory molecules in tumours generally increases their immunogenicity, but does not always induce a protective response against the parental tumour. This issue was addressed in the mouse Sp6 hybridoma model, comparing different immunization routes (subcutaneous, intraperitoneal and intravenous) and doses (0·5 × 106 and 5 × 106 cells) of Sp6 cells expressing de novo B7‐1 (Sp6/B7). The results can be summarized as follows. First, de novo expression of B7‐1 rendered Sp6 immunogenic, as it significantly reduced the tumour incidence to ≤15% with all delivery routes and doses tested, whereas wild‐type Sp6 was invariably tumorigenic (100% tumour incidence). Second, long‐lasting protection against wild‐type Sp6 was mainly achieved when immunization with Sp6/B7 was subcutaneous: a dose of 0·5 × 106 Sp6/B7 cells elicited protection that was confined to sites in the same anatomical quarter as the immunizing injection. Repeated injections of the same dose extended protection against wild‐type Sp6 to other anatomical districts, as well as a single injection of a 10‐fold higher dose (5 × 106 cells). Finally, Sp6‐specific cytotoxic T‐lymphocyte activity was detected in draining lymph nodes, and the splenic expansion of Sp6‐specific cytotoxic T‐lymphocyte precursors quantitatively correlated with the dose of antigen. We conclude that activation of a protective immune response against Sp6 depends on the local environment where the immunogenic form of the ‘whole tumour cell antigen’ is delivered. The antigen dose regulates the anatomical extent of the protective response.

Myelin basic protein epitopes secreted by human T cells encounter natural autoantibodies in the serum

A previously isolated and characterized IgM monoclonal antibody (mAb 1H6.2) specific to myelin basic protein (MBP) and to MBP epitopes expressed by nonneural cells was used to immunoprecipitate and investigate the expression of MBP epitopes by human T cells. Peripheral T lymphocytes secreted MBP epitopes, and secretion increased in time after mitogen stimulation. Conversely, thymocytes secreted these proteins independently on mitogen stimulation. Specific antibody reactivity (primarily due to IgG3) towards immunoprecipitated MBP epitopes was found in all tested sera from healthy donors and from multiple sclerosis patients as well as in sera from normal human cord blood. Collectively, these data provide insights into the immunological mechanisms leading to central and peripheral tolerance to MBP products.

Morphology, growth, and gene expression in five newly isolated murine hepatocellular tumor cell lines

Five murine hepatocellular tumor cell lines (HepM-1-5) were isolated and grown in a synthetic medium added with hormones, growth factors and/or serum. The morphology of these lines ranged from a nearly homogeneous epithelial-like shape (HepM-2) to a stromal appearance (HepM-1). The remaining lines displayed a mixed morphology. For their proliferation all of the cell lines retained a clear dependence on the extracellular calcium level and hormonal and/or serum growth factors and, rather homogeneously, they did not express the albumin, alpha-fetoprotein (with the exception of HepM-2 cells), tyrosine aminotransferase, and ornithine transcarbamylase genes, whereas they all exhibited discrete levels of the ornithine aminotransferase mRNA. Only HepM-3 and HepM-5 lines expressed the procollagen type I gene.