Gianni Garotta

System for High-Level Production in Escherichia coli and Rapid Purification of Recombinant Proteins: Application to Epitope Mapping, Preparation of Antibodies, and Structure—Function Analysis

Publisher Summary This chapter presents an application of the system for high-level production in Escherichia coli and rapid purification of recombinant proteins. The advent of gene cloning, the engineering of vectors for efficient expression, and the application of fast and high-flux methods for protein purification made available many recombinant proteins of biological interest. This represented a breakthrough for the structure–function analysis of bioactive proteins and cell receptors and facilitated X-ray crystallographic studies for definition of the three-dimensional structures of these proteins. The E. coli expression system allows the high-level production of recombinant proteins in authentic form, as fusion proteins with the [His] 6 affinity tail and with mouse DHFR and the [His] 6 tail. Because of the presence of the affinity tail, proteins that are produced in a soluble form or can be solubilized with GuHCl or urea can be purified almost to homogeneity in one step by nickel chelate affinity chromatography. The purified recombinant proteins simplify the production of monoclonal and polyclonal antibodies directed against defined regions of the native proteins.

Role of interferon-gamma in mediating the antitumor efficacy of interleukin-12.

Although interleukin-12 (IL-12) has marked antitumor activity against the murine Renca renal cell carcinoma in vivo, no antiproliferative activity with IL-12 was observed against these tumor cells in vitro; in contrast, interferon-gamma (IFN-gamma) had growth inhibitory activity. Since one of the properties of IL-12 is its ability to stimulate production of IFN-gamma, the role of IFN-gamma in mediating the antitumor activity of IL-12 was evaluated. Substantially diminished antitumor activity was observed in mice injected with IL-12 and neutralizing antibody to murine IFN-gamma compared with mice receiving IL-12 alone, indicating that IFN-gamma was required for the optimal antitumor efficacy of IL-12. However, several lines of investigation suggest that the antitumor effect of IL-12 is not mediated solely through the induction of IFN-gamma. Exogenous administration of IFN-gamma to Renca tumor-bearing euthymic mice resulted in less antitumor efficacy than that which could be obtained with IL-12. In addition, the antitumor effect of IL-12 was reduced in nude mice compared with euthymic mice, but an approximately 10-fold higher level of serum IFN-gamma was induced in nude than in euthymic mice. Thus, these results indicate that induction of high serum levels of IFN-gamma is not sufficient to mediate the antitumor efficacy of IL-12.

Interaction between the components of the interferon gamma receptor complex

Interferon γ (IFN-γ) signals through a multimeric receptor complex consisting of two different chains: the IFN-γ receptor binding subunit (IFN-γR, IFN-γR1), and a transmembrane accessory factor (AF-1, IFN-γR2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-γ receptor complex, we examined the function of the receptor components in signal transduction upon IFN-γ treatment. A specific IFN-γR2:IFN-γ cross-linked complex was observed in cells expressing both IFN-γR1 and IFN-γR2 indicating that IFN-γR2 (AF-1) interacts with IFN-γ and is closely associated with IFN-γR1. We show that the intracellular domain of IFN-γR2 is necessary for signaling. Cells coexpressing IFN-γR1 and truncated IFN-γR2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-γR1 alone were unresponsive to IFN-γ treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1α were activated, and IFN-γR1 was phosphorylated only in cells expressing both IFN-γR1 and IFN-γR2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-γR2.

The interferon gamma (IFN-γ) receptor: a paradigm for the multichain cytokine receptor

Abstract With the purification and cloning of the interferon gamma (IFN-γ) receptor chains the mechanism of IFN-γ action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-γ) receptor complex consists of two chains: IFN-γR1, the ligand-binding chain, and IFN-γR2, the accessory chain. Binding of IFN-γ causes oligomerization of the two IFN-γ receptor subunits, IFN-γR1 and IFN-γR2, which initiates the signal transduction events: activation of Jakl and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-γR1 intracellular domain on Tyr 440 followed by phosphorylation and activation of Stat1α, the latent transcriptional factor. With all these steps established, the IFN-γ receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.

Tumor Rejection and Immune Memory Elicited by Locally Released LEC Chemokine Are Associated with an Impressive Recruitment of APCs, Lymphocytes, and Granulocytes

The human β chemokine known as LEC (also called NCC-4, HCC-4, or LMC) displays chemotactic activity for monocytes and dendritic cells. The possibility that its local presence increases tumor immunogenicity is addressed in this paper. TSA parental cells (TSA-pc) are poorly immunogenic adenocarcinoma cells that grow progressively, kill both nu/nu and syngeneic BALB/c mice, and give rise to lung metastases. TSA cells engineered to release LEC (TSA-LEC) are still able to grow in nu/nu mice, but are promptly rejected and display a marginal metastatic phenotype in BALB/c mice. Rejection is associated with a marked T lymphocyte and granulocyte infiltration, along with extensive macrophage and dendritic cell recruitment. NK cells and CD4+ T lymphocytes are uninfluential in TSA-LEC cell rejection, whereas both CD8+ lymphocytes and polymorphonuclear leukocytes play a major role. An antitumor immune memory is established very quickly after rejection, since 6 days later 75% of BALB/c mice were already resistant to a TSA-pc challenge. Spleen cells from rejecting mice display specific cytotoxic activity against TSA-pc and secrete IFN-γ and IL-2 when restimulated by TSA-pc. The ability of LEC to markedly improve recognition of poorly immunogenic cells by promoting APC-T cell cross-talk suggests that it could be an effective component of antitumor vaccines.

Prevention of neuron and oligodendrocyte degeneration by interleukin-6 (IL-6) and IL-6 receptor/IL-6 fusion protein in organotypic hippocampal slices

We investigated the effects of IL-6 and a chimeric derivative of IL-6 and soluble IL-6 receptor (IL6RIL6 chimera) on excitotoxic injury in rat organotypic hippocampal slices. Brief application of N-methyl-d-aspartate (NMDA) induced astrocyte reactivity, neuron cell death, and oligodendrocyte degeneration, the latter caused by secondary activation of AMPA/kainate receptors. Both these cytokines rescued neurons and oligodendrocytes, albeit the chimeric compound was much more potent and efficient than IL-6. No change was produced on reactive astrocytosis. The cytokines preserved myelin basic protein (MBP) production in slices exposed to excitotoxic insult, and when applied singularly for a week, they also enhanced both MBP and proteolipid protein expression. These effects occurred through activating the signal transducer gp130 and were associated with stimulation of transcription factors STAT1 and STAT3. Our results suggest that IL-6 and IL6RIL6 may prove to be valuable in treating neurodegenerative and demyelinating diseases.

Soluble Interleukin-6 (IL-6) Receptor/IL-6 Fusion Protein Enhances in Vitro Differentiation of Purified Rat Oligodendroglial Lineage Cells

We investigated the effects of a chimeric protein (IL6RIL6 chimera) containing interleukin-6 (IL-6) fused to its soluble receptor (sIL-6R) on the proliferation and/or differentiation of rat oligodendrocyte progenitor cells (OPCs) and on oligodendrocyte survival. Exposure of OPCs to IL6RIL6 chimera for 48 h induced a dose-dependent decrease of bromodeoxyuridine (BrdU) incorporation. IL6RIL6 chimera treatment for 48 h also strongly increased the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) by mitochondrial enzymes and enhanced oligodendrocyte staining with a mitochondrial fluorescent dye. A strong, dose-dependent increase in the number and length of processes immunostained for early (galactocerebroside) or late (myelin basic protein) oligodendrocyte differentiation markers was revealed after OPC treatment with IL6RIL6 chimera for 2-7 days, respectively. Moreover, treatment with IL6RIL6 chimera improved oligodendrocyte survival. The chimera-induced increase of oligodendrocyte arborization was mimicked, although with lower efficacy, by ciliary neurotrophic factor (CNTF) but not by IL-6 and was reduced in the presence of a gp130 soluble peptide which is able to inhibit the gp130-mediated signals of the IL-6/sIL-6R complex. Oligodendrocyte treatment with IL6RIL6 chimera for 30 min induced both signal transducer and the activator of transcription-1 (STAT-1) and STAT-3 phosphorylation and nuclear translocation. We conclude that, by interacting with membrane gp130 and possibly by activating Janus kinase/STAT pathways, IL6RIL6 chimera induces OPCs to differentiate into mature oligodendrocytes, promotes their survival, and could deserve investigation as a therapeutic strategy for enhancing remyelination.

Anticancer properties of the novel nitric oxide-donating compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid-nitric oxide in vitro and in vivo.

Preclinical studies have shown that nitric oxide (NO)–donating nonsteroidal anti-inflammatory drugs possess anticancer activities. Here, we report in vitro and in vivo studies showing the antitumor effect of the NO-donating isoxazole derivative (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid (GIT-27NO). GIT-27NO, but not the NO-deprived parental compound VGX-1027, significantly affected viability of both rodent (L929, B16, and C6) and human (U251, BT20, HeLa, and LS174) tumor cell lines. GIT-27NO triggered either apoptotic cell death (e.g., L929 cells) or autophagic cell death (C6 and B16 cells). Moreover, GIT-27NO hampered the viability of cisplatin-resistant B16 cells. NO scavenger hemoglobin completely prevented GIT-27NO-induced death, indicating that NO release mediated the tumoricidal effect of the compound. Increase in intracellular NO upon on the treatment was associated with intensified production of reactive oxygen species, whereas their neutralization by antioxidant N-acetylcysteine resulted in partial recovery of cell viability. The antitumor activity of the drug was mediated by the selective activation of mitogen-activated protein kinases in a cell-specific manner and was neutralized by their specific inhibitors. In vivo treatment with GIT-27NO significantly reduced the B16 melanoma growth in syngeneic C57BL/6 mice. The therapeutic effect occurred at dose (0.5 mg/mouse) up to 160 times lower than those needed to induce acute lethality (80 mg/mouse). In addition, a dose of GIT-27NO five times higher than that found effective in the melanoma model was well tolerated by the mice when administered for 4 consecutive weeks. These data warrant additional studies to evaluate the possible translation of these findings to the clinical setting. [Mol Cancer Ther 2008;7(3):510–20]

First trimester human trophoblast expresses both interferon-γ and interferon-γ-receptor

Interferon-gamma (IFN-gamma) is a lymphokine, produced by activated T lymphocytes, which plays a key regulatory role in the host immunological responses. In addition, IFN-gamma is expressed by human and porcine trophoblast. As IFN-gamma exerts its biological functions through specific cell surface receptors and a great number of IFN-gamma receptors (IFN-gamma R) have been purified from human placenta, we have examined the relative distribution of IFN-gamma and IFN-gamma R in human placental tissues at different stages of pregnancy. By using immunohistochemical analysis and monoclonal antibodies, it was found that IFN-gamma expression is intense in the first trimester but almost imperceptible at term, whereas the expression of IFN-gamma R is present at both stages of pregnancy. For both lymphokine and receptor, the most intense expression was observed in villous syncytiotrophoblast and in extravillous interstitial trophoblast. From these results it appears that the expression of IFN-gamma R in trophoblast is related to the presence of the lymphokine in the early phase of gestation but not later. On this basis, it can be argued that IFN-gamma exerts its functional role via an autocrine and/or a paracrine loop mainly during the first trimester.

CKβ8, a novel CC chemokine that predominantly acts on monocytes

We have studied the biological properties of a new human CC chemokine, CKβ8, consisting of 99 amino acids including six cysteines. CKβ8 mRNA transcripts were induced in monocytes by IL‐1β and, to a lesser extent, by IFNγ, and were detected in RNA extracted from normal human liver and gastrointestinal tract. CKβ8 is chemotactic for monocytes, but is inactive on IL‐2 conditioned T lymphocytes, eosinophils and neutrophils. Desensitization experiments indicate that CKβ8 and MIP‐1β completely share receptors on monocytes and that the CKβ8 receptor, which appears to differ from the known ones, is also recognized by MCP‐1, MCP‐2, MCP‐3, MCP‐4, MIP‐1α and RANTES.