Gennaro Ciliberto

Cis- and trans-acting elements responsible for the cell-specific expression of the human alpha 1-antitrypsin gene.

The 5′ flanking region of the human alpha 1‐antitrypsin (alpha 1‐AT) gene contains cis‐acting signals for liver‐specific expression and, when fused to a reporter gene, is able to drive the expression of this gene specifically in liver cells. Here we report the results of a functional dissection of the alpha 1‐AT regulatory region. The expression of the bacterial chloramphenicol‐transacetylase (CAT) gene, fused to a set of alpha 1‐AT 5′ flanking regions shortened by progressive deletions or mutated by base pair substitutions, has been compared by transfection in HepG2 (hepatocyte) and HeLa (non‐hepatocyte) human cell lines. A minimal tissue‐specific element has been identified between the nucleotides −137 and −37 (from the transcriptional start site). This DNA segment activates the heterologous SV40 promoter in hepatoma cell lines but not in HeLa cells. This element contains at least two regions referred to as the A (‐125/‐100) and B (‐84/‐70) domains, both essential for transcription. There are at least two other regulatory domains located upstream of the ‘minimal element’; the most active of these is located between positions −261 and −210 from the cap site. These upstream elements activate the heterologous SV40 early promoter both in hepatoma cell lines and in HeLa cells. Upon fractionation of rat liver nuclear extracts two proteins have been identified, alpha 1TF‐A and alpha 1TF‐B, which bind specifically to the A and B domains respectively. Transcriptionally inactive A and B domain mutants are not able to bind these proteins.

Transcriptional Control of Gene Expression in Hepatic Cells

In this chapter, we review the regulation of gene expression in hepatic cells, with emphasis on the cis-acting elements located in the proximal promoter region and on the properties of the transcriptional factors active in liver.

Harnessing the Immune System to Fight Cancer: The Promise of Genetic Cancer Vaccines

In spite of significant progress in recent years towards the development of new targeted therapies Cancer is still a largely unmet medical need and the leading cause of death in industrialized countries (Globocan Project, 2008). Cancer is continuously increasing and is associated with a variety of factors, including genetic predisposition, infectious agents, exposure to mutagens, as well as lifestyle factors (Minamoto et al, 1999). Cancer is linked to the occurrence of genetic and epigenetic changes (Heng et al, 2010) and indeed tumour cells harbor hundreds of these modifications as also witnessed by the recent results of genome wide analyses of cancer genomes (Sastre, 2011). This feature of cancer cells implies that they can be recognized as foreign entities and eliminated by our immune system, and is at the basis of the theory of immunosurveillance (Dunn et al, 2004). Several studies have shown that it is possible to establish clear correlates between the nature, density and location of immune cells within distinct tumour regions and the risk of disease relapse (reviewed in Mleknic et al, 2011). Compelling data have recently led to propose that an immune classification of patients, based on the density and the immune location within the tumour may have a prognostic value even superior to the standard TNM classification (Bindea et al, 2011; Fridman et al, 2011). In recent years a better knowledge of the immune system has led to an evolution of the initial concept of immunosurveillance into a more articulated theory of immunoediting (Schreiber et al, 2011). Cancer immunoediting acts as an intrinsic tumour suppressor mechanism that engages after cellular transformation has occurred and intrinsic tumour suppressor mechanisms have failed. One can envisage the existence of three sequential steps during clinical tumour evolution: elimination, equilibrium, and escape. In the first step, innate and adaptive immunity are capable of destroying transformed cells before they give rise to tumour masses. If this process is maximally efficient, then the host remains tumour free. If, however, cancer cell variants are not destroyed, they can enter into an equilibrium phase, in which their outgrowth is held in check by immunological mechanisms, which are principally due to the activation of IL12/IFN-dependent adaptive immunity, mainly driven by antigen-specific CD8+ and CD4+

A Universal Influenza B Peptide Vaccine

Paolo Ingallinella, Elisabetta Bianchi, Xiaoping Liang, Marco Finotto, Michael Chastain, Jiang Fan, Tong-Ming Fu, Hong Chang Song, Melanie Horton, Daniel Freed, Walter Manger, Emily Wen, Li Shi, Roxana Ionescu, Colleen Price, Marc Wenger, Emilio Emini, Riccardo Cortese, Gennaro Ciliberto, John Shiver and Antonello Pessi IRBM P.Angeletti, Pomezia (Rome), Italy; Merck Research Laboratories, West Point, PA, USA

Identification of a Novel HIV-1 Neutralizing Antibody Using Synthetic Peptides that Mimic a GP41 Fusion Intermediate

Introduction HIV-entry into cells is mediated by the envelope glycoprotein receptor-binding gp120 and fusogenic gp41 subunits. During the fusion process gp41 undergoes a series of conformational changes that culminate in formation of the fusogenic structure: a 6-helix bundle, where three α-helices formed by the heptad repeat region 2 (HR2) pack in an antiparallel manner against a central three-stranded coiled coil formed by the heptad repeat region 1 (HR1). Viral fusion progresses via formation of an intermediate, which transiently exposes the HR1 coiled coil and the HR2 peptides. By targeting these regions with peptides derived from HR1, HR2, it is possible to prevent formation of the 6-helix bundle, and block viral infectivity. We investigated if antibodies could also block HIV-1 entry by the same mechanism, since this would open the pathway to a vaccine targeting the fusion intermediate.