γδ T-cell Receptors Derived from Breast Cancer-Infiltrating T Lymphocytes Mediate Antitumor Reactivity.

γδ T cells in human solid tumors remain poorly defined. Here, we describe molecular and functional analyses of T-cell receptors (TCR) from tumor-infiltrating γδ T lymphocytes (γδ TIL) that were in direct contact with tumor cells in breast cancer lesions from archival material. We observed that the majority of γδ TILs harbored a proinflammatory phenotype and only a minority associated with the expression of IL17. We characterized TCRγ or TCRδ chains of γδ TILs and observed a higher proportion of Vδ2+ T cells compared with other tumor types. By reconstructing matched Vδ2- TCRγ and TCRδ pairs derived from single-cell sequencing, our data suggest that γδ TILs could be active against breast cancer and other tumor types. The reactivity pattern against tumor cells depended on both the TCRγ and TCRδ chains and was independent of additional costimulation through other innate immune receptors. We conclude that γδ TILs can mediate tumor reactivity through their individual γδ TCR pairs and that engineered T cells expressing TCRγ and δ chains derived from γδ TILs display potent antitumor reactivity against different cancer cell types and, thus, may be a valuable tool for engineering immune cells for adoptive cell therapies.

A silencer element in the first intron of the glutamine synthetase gene represses induction by glucocorticoids.

The enzyme glutamine synthetase (GS) ranks as one of the most remarkable glucocorticoid-inducible mammalian genes. In many tissues and cell lines, the synthetic glucocorticoid dexamethasone alone increases GS expression several fold. The direct response is mainly mediated by a cellular glucocorticoid receptor that, upon binding of the hormone, interacts with glucocorticoid responsive elements (GREs) of the gene. In cells of hepatocellular origin the response is mediated by a GRE located in the first intron of the gene. Surprisingly, hepatocytes do not respond to glucocorticoids with enhanced GS expression, despite the presence of an intact glucocorticoid receptor, which, in the same cells, stimulates expression of other genes such as tyrosine amino transferase. Reporter gene assays identified a sequence element downstream from the intronic GRE that inhibits the enhancement of expression by glucocorticoids. This silencer was designated GS silencer element of the rat. Gel mobility shift assays demonstrate the binding of a factor in hepatocyte nuclear extract. This yet unknown factor was designated GS silencer-binding protein. It is absent in FAO cells that respond to glucocorticoids with enhanced expression of GS and present in HepG2 cells that do not respond.

Gene transfer to trabecular meshwork endothelium via direct injection into the Schlemm canal and in vivo toxicity study.

PURPOSE: Our aim was to investigate the efficiency of adenoviral gene transfer via direct injection into the Schlemm canal ex vivo in human donor eyes and to examine the effect of human MMP-3 transgene expression in a rat model in vivo. METHODS: A viscocanalostomy-like operation was performed and adenoviral vector encoding for MMP-3 and green fluorescent protein was injected into human Schlemm canal or rat anterior chamber. RESULTS: Transgene expression was high in trabecular meshwork endothelium in human donor eyes. In vivo, adenovirus caused dose-dependent inflammation. CONCLUSIONS: Direct injection of adenoviral vectors into the Schlemm canal has potential in glaucoma treatment.

HTS compatible assay for antioxidative agents using primary cultured hepatocytes.

We have used primary cultured rat hepatocytes to establish a system that is compatible with HTS for screening substance libraries for biologically active compounds. The hepatocytes were treated with t-BHP to induce oxidative stress, leading to the formation ROS. The involvement of ROS in oxidative stress and pathological alterations has been of major interest in recent years, and there is great demand to identify new compounds with antioxidant potential. In most HTS programs each compound is tested in duplicate, and may only be tested once. Because of this it is important to develop assays that can identify candidate compounds accurately and with high confidence. Using newly available cell-based assay systems, we have developed a system that can detect active compounds (hits) with a high degree of confidence. As an example of an agent that can be detected from a substance library, we analyzed the effect of fisetin as an antioxidative compound using this system. All measurements were performed using the newly developed and highly versatile Multilabel-Reader Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany). The data presented show that all Z' factors determined were highly reliable. Although the protocol is primarily designed to screen for substances with antioxidative potential, it can easily be adapted to screen for other biologically active substances.

Dual luciferase assay for secreted luciferases based on Gaussia and NanoLuc.

Just recently, NanoLuc, a new engineered luciferase based on the small subunit of the luciferase from Oplophorus gracilirostris was introduced. Like the luciferase from Gaussia princeps, this luciferase is secreted into the medium. Both luciferases are the smallest and brightest luciferases known and well-suited for reporter assays. In our experiments, we demonstrate that both luciferases can be used together in a dual-reporter assay by solving the problem that NanoLuc produces a significant signal with coelenterazine, which is the substrate for Gaussia luciferase. We found that the background signal from NanoLuc with coelenterazine can be calculated from the determination of NanoLuc activity in the presence of its substrate furimazine. This in turn allows the precise determination of the activity of Gaussia which does not produce light in the presence of furimazine. Based on this observation, we developed a high sensitive dual secreted luciferase assay which allows the determination of both activities in a single cotransfection experiment. We demonstrate the versatility and robustness of the assay for the normalization of reporter gene activities. Since Gaussia luciferase and NanoLuc are nonhomologous reporters, the method to determine both luciferase activities may also be useful for coincidence reporter gene systems for high-throughput screening.

An intronic silencer element is responsible for specific zonal expression of glutamine synthetase in the rat liver.

The most striking phenomenon of glutamine synthetase (GS) expression in the liver is its unique restriction to cells surrounding the terminal hepatic venules. Expression is positively regulated by elements located in the 5'-upstream region and in the first intron of the gene. It was long believed that transcription factors present in GS-positive cells and absent in GS-negative cells are responsible for the phenomenon of zonal expression. However, strong enhancers are equally active in both types of cells. Therefore, the existence of a silencer mechanism in GS-negative hepatocytes was postulated. In the present study, a GS silencer element was investigated that was previously identified within the first intron and was shown to be able to prevent glucocorticoid-induced expression in cells negative for a transacting factor designated GS silencer element-binding protein. Reporter gene assays with the silencer element in combination with the most potent 5'-enhancer of the GS gene demonstrate that the silencer element is able to prevent enhancement mediated by the 5'-enhancer in combination with a heterologous as well as with the homologous promoter. More importantly, the effect of the silencer is shown to be restricted to GS-negative hepatocytes. In conclusion, the phenomenon of zonal expression of GS in the liver is caused by a protein present in GS-negative cells and absent in GS-positive cells that interacts with the silencer element in the first intron and not by a differential expression of enhancer-binding proteins.

Antiproliferative effect of mycophenolate mofetil on cultured human Tenon fibroblasts.

BACKGROUND: Wound healing after glaucoma filtering surgery is often complicated by exaggerated scarring of the subconjunctival Tenon's layer. Therefore, antiproliferatives are commonly employed. The immunosuppressive drug mycophenolate mofetil (MMF) is used to prevent graft rejection after kidney or liver transplantation. The effect is mediated by inhibition of lymphocyte proliferation. In this study we investigated the effect of MMF on human Tenon fibroblast proliferation in cell culture. METHODS: Human Tenon fibroblasts (HTF) were cultivated with 10% fetal calf serum. Cells were incubated with MMF concentrations of 0.1 microM to 3000 microM for up to 20 days. In a second set of experiments HTF were incubated for 10 min only in MMF solutions. Cell counts were performed to evaluate the proliferation rate. The proliferation was also assessed by Ki67 staining. Morphological changes were documented by vimentin staining. RESULTS: Growth inhibition of HTF by MMF was concentration dependent. IC(50) was 0.85+/-0.05 microM for 6 days of incubation. Brief exposure to MMF leads to a reversible growth arrest for up to 14 days with concentrations of 1000 microM or higher. Ki67 staining confirmed the concentration dependent proliferation rate. CONCLUSION: MMF has a concentration-dependent antiproliferative effect on HTF without any detected cytotoxicity in the applied concentration range. Brief incubation also leads to a growth arrest; therefore, intraoperative MMF application might prevent exaggerated scarring after glaucoma filtering surgery.

Glucocorticoid induced expression of glutamine synthetase in hepatoma cells.

The enzyme glutamine synthetase (GS) ranks as one of the most remarkable glucocorticoid-inducible vertebrate genes. However, little is known about the responsible DNA elements and the mode of glucocorticoid action. This is especially the case for the induction of GS in hepatoma cells. In the work presented, the rat hepatoma cell line FAO was used as a model to study the induction of GS under the influence of glucocorticoids. FAO cells do not show GS activity in the absence of glucocorticoids and are strongly responding to their presence. Analyzing sequences of several thousand base pairs upstream and downstream from the transcriptional start point of the GS gene, a glucocorticoid responsible element was identified within the first intron of the gene. However, evidence is presented that aside from a primary effect on transcription glucocorticoids mediate their effect on the expression of GS also at the posttranscriptional level.