Epstein-Barr Virus LMP2A Reduces Hyperactivation Induced by LMP1 to Restore Normal B Cell Phenotype in Transgenic Mice

Epstein-Barr virus (EBV) latently infects most of the human population and is strongly associated with lymphoproliferative disorders. EBV encodes several latency proteins affecting B cell proliferation and survival, including latent membrane protein 2A (LMP2A) and the EBV oncoprotein LMP1. LMP1 and LMP2A signaling mimics CD40 and BCR signaling, respectively, and has been proposed to alter B cell functions including the ability of latently-infected B cells to access and transit the germinal center. In addition, several studies suggested a role for LMP2A modulation of LMP1 signaling in cell lines by alteration of TRAFs, signaling molecules used by LMP1. In this study, we investigated whether LMP1 and LMP2A co-expression in a transgenic mouse model alters B cell maturation and the response to antigen, and whether LMP2A modulates LMP1 function. Naïve LMP1/2A mice had similar lymphocyte populations and antibody production by flow cytometry and ELISA compared to controls. In the response to antigen, LMP2A expression in LMP1/2A animals rescued the impairment in germinal center generation promoted by LMP1. LMP1/2A animals produced high-affinity, class-switched antibody and plasma cells at levels similar to controls. In vitro, LMP1 upregulated activation markers and promoted B cell hyperproliferation, and co-expression of LMP2A restored a wild-type phenotype. By RT-PCR and immunoblot, LMP1 B cells demonstrated TRAF2 levels four-fold higher than non-transgenic controls, and co-expression of LMP2A restored TRAF2 levels to wild-type levels. No difference in TRAF3 levels was detected. While modulation of other TRAF family members remains to be assessed, normalization of the LMP1-induced B cell phenotype through LMP2A modulation of TRAF2 may be a pathway by which LMP2A controls B cell function. These findings identify an advance in the understanding of how Epstein-Barr virus can access the germinal center in vivo, a site critical for both the genesis of immunological memory and of virus-associated tumors

A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models

In the past decade, our increased elucidation of the molecular basis of cancer has led to the development of novel targeted strategies for specific inhibition of cancer signaling pathways that control growth, proliferation, apoptosis, and angiogenesis. Several monoclonal-antibody-based therapeutics and small-molecule drugs have received clearance for use as human therapeutics [1]. However, among these successes are many candidate drugs that have failed in clinical trials despite promising preclinical results [2]. The development of targeted therapeutics is expensive and time consuming. In their Critical Path Initiative, the United States Food and Drug Administration emphasized the need for more effective tools to facilitate the rapid development of improved cancer therapeutics. One such tool is the use of targeted molecular optical imaging probes or contrast agents to visualize the underlying processes in cancer. Optical imaging, also known as molecular imaging, is a rapidly developing field of research aimed at noninvasively interrogating animals for disease progression, evaluating the effects of a drug, assessing the pharmacokinetic behavior of a drug, or identifying molecular biomarkers of disease. A prerequisite of molecular imaging is the development of specific, targeted imaging contrast agents to assess these biological processes. Several optical aids have shown great utility in animal studies, including bioluminescence, fluorescent proteins, and fluorochrome-labeled agents. However, only the latter have the advantage of being potentially relevant to human clinical applications. The complexity of developing robust fluorochrome-labeled optical agents is often underestimated. Many studies describe the use of these agents, but guidelines for their development and testing are not readily available. The purpose of this review is to outline some of the considerations for developing and using fluorochrome-labeled optical contrast agents in animals. For simplicity, we have focused on the use of organic fluorochromes as labeling agents. These types of probes are generally the most straightforward to develop and have the greatest potential for translation to human clinical use. Nanoparticles such as quantum dots, while useful for some animal studies, are hampered by clearance issues and toxicity and will not be specifically discussed. However, the principles described here are generally applicable to any fluorescent optical imaging agent