Studies Utilising a Transgenic Mouse Model to Investigate the Pathogenesis of HTLV-I Tax

In order to investigate the contribution that the HTLV-I (Human T-cell Leukaemia Virus Type I) tax oncogene contributes to the transformed phenotype we generated transgenic mice that expressed the HTLV-I tax transgene under the regulatory influence of the CD3- s promoter-enhancer elements. These mice developed a variety of different types of pathology, the most pertinent of which were tumours (overall incidence 28/57 (49%)). The principal tumours were mesenchymal tumours which developed at sites of wounding (ear and tail tips) (overall incidence 18/31 (58%); average latency 202.5 days), and salivary and mammary adenomas (overall incidence 10/26 (38%); average latency 399 days). Immunocytochemical analysis of these tumours revealed that subpopulations of tumour cells expressed high levels of Tax protein as well as Myc, Fos, Jun and p53 proteins. Analysis of these tumours using the TUNEL (Terminal dUTP Nick End Labelling) technique revealed a similar pattern of apoptosis. Dual immunofluorescent localisation of Tax expressing cells with cells undergoing apoptosis revealed a remarkable degree of coincidence between these processes indicating that Tax expression and apoptosis are intimately linked in vivo. When CD3-tax transgenic mice were bred onto a p53 null or hemizygous background the resultant tumours which emerged at ear and tail tips were identical to the original cohort of tumours indicating a lack of cooperation between tax and p53 (average latency for mesenchymal tumours emerging on a p53 wild type background: 277d (9.2months) and 128d (4.3 months) cf p53+/- background; 233d (7.8 months) and 156d (5.2 months)). A Tax expression system was also developed in vitro using a pBabe retroviral vector which transcribed a Tax-MER (Tax-modified oestrogen receptor) fusion product. This fusion protein was dependent upon functional activity by induction with 4-hydroxy tamoxifen. The results of these experiments using a CREB/ATF responsive reporter gene indicated that this system produced a functional fusion protein that was well regulated by 4-hydroxy tamoxifen. Functional mutants of Tax were made (Ml, M47 and C23S) based on published data (Smith and Greene, 1990; Semmes and Jeang, 1992) which were designed to segregate the functional domains of Tax. Analysis of these mutants indicated that M47 least efficiently induced apoptosis in vitro. In our hands this protein also least efficiently transactivated the CREB/ATF pathway (although transactivation of the NFkB pathway was difficult to determine in this assay system). These results may indicate the general importance of the CREB/ATF pathway for apoptotic induction. Similarly these results, together with the transgenic mouse experiments, may also indicate that low levels of Tax expression does not induce apoptosis

STRUCTURE AND FUNCTION ANALYSIS OF PAR-4

Par-4 is a leucine zipper domain protein that induces apoptosis on its own in certain cancer cells and in Ras-transformed cells, but not in normal or immortalized cells. Par-4 induces apoptosis by activation of the Fas death receptor pathway and co-parallel inhibition of NF-B transcription activity. Cells that are resistant to apoptosis by Par-4 alone, however, are greatly sensitized by Par-4 to the action of other pro-apoptotic insults such as growth factor withdrawal, TNF, ionizing radiation, intracellular calcium elevation, or those involved in neuronal degeneration such as Alzheimer\u27s, Parkinson\u27s, Huntington\u27s and Stroke. Previous studies have suggested that the apoptosis-sensitization potential of Par-4 is dependent upon inhibition of PKC or WT1 cell survival function by direct interaction between the leucine zipper domain at the carboxy-terminus of Par-4 and the zinc finger domains of PKC or WT1. In this study, I performed structure-function analysis using GFP-fusion proteins and deletion mutants to identify the functional localization and domains of Par-4 that are essential for apoptosis induction. My findings suggest that apoptosis by Par-4 is dependent on its translocation to the nucleus for induction of apoptosis. A bipartite nuclear localization signal sequence corresponding to amino acids 137-155 was necessary for nuclear translocation of Par-4. Importantly, the core residues 137-204 in the center part of Par-4 were necessary and sufficient to induce Fas pathway activation, inhibition of nuclear NF-B transcription activity and apoptosis. These findings imply that binding of Par-4 via its leucine zipper domain to other proteins is dispensable for apoptosis by Par-4

Recent Advances in HTLV Research 2015

The human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) were both discovered over three decades ago and infect millions people worldwide. HTLV-1 is associated with the adult T-cell leukemia/lymphoma (ATLL) in about 2% of individuals infected, and another 2 to 3% of individuals develop a neurologic disorder called HTLV-associated myelopathy (HAM). HTLV-2 causes HAM in approximately 1 to 2% of infected individuals, but does not cause ATLL. HTLV-1 and HTLV-2 have served as excellent models for the study of the epidemiology and pathogenesis of virus-associated cancers as well as autoimmune conditions such as multiple sclerosis. Recently, two new members—HTLV-3 and HTLV-4—have been discovered in bushmeat hunters from central Africa, which emphasizes the urgent need for continual surveillance for new human retroviruses and their capacity to cause disease. Important public health issues remain open issues to be addressed in spite of the basic epidemiology of HTLV-1 and HTLV-2 being reasonably well defined. Clinical research is needed in developing potential HTLV-1 and HTLV-2 vaccines, as well as development of treatment options for ATLL and HAM. This ‘Recent Advances Issue’ contains both reviews and updates on research that encompasses these areas

Functions of the vFLIP protein of KSHV.

KSHV infection is associated with both endothelial and B cell tumours. In KSHV the genes expressed in latency have been implicated in cell transformation. vFLIP is one of a small number of viral proteins expressed in latently infected tumour cells. In KSHV-infected primary effusion lymphoma (PEL) cells, vFLIP binds to, and persistently activates the IkB kinase complex, leading to constitutive activation of the canonical NF-kB pathway. We have previously shown in our lab that vFLIP directly interacts with the IKKy subunit of the IKK complex (Field, et al., 2003) to activate IKK. In this report, we demonstrate that vFLIP also activates the alternative NF-kB pathway, which involves processing of the p100 protein precursor and generation of the p52 subunit. Stable vFLIP expression in Jurkat cells stimulates expression of endogenous p100 and nuclear accumulation of p52 and RelB. Metabolic radiolabelling of transiently transferred 293T cells indicates that vFLIP promotes proteolysis of p100 and active generation of p52. Moreover, we show that vFLIP associates with p100 when over-expressed in Jurkat cells, or when endogenously expressed in PEL cells, and a region in the C-terminus of p100, which includes the p100 DD, is identified as the vFLIP binding region. Finally, inhibition of p100 and p52 production mediated by siRNA knockdown leads to the induction of apoptosis in PEL cells, inferring that vFLIP activation of the alternative NF-kB pathway contributes to PEL survival. These data demonstrate that vFLIP activates both canonical and alternative NF-kB pathways, a property shared with the Tax oncoprotein of HTLV-1 and LMP1 of EBV. In addition, we have examined the effect of vFLIP on primary human dermal microvascular endothelial cell (MVEC) survival, as vFLIP is expressed in the KSHV-infected cells within KS lesions. Stable vFLIP expression in MVECs induces the activation of the classical NF-kB pathway and the nuclear translocation of RelA/p65. vFLIP-mediated NF-kB activation prevents detachment-induced apoptosis (anoikis) of MVECs, but does not inhibit growth factor removal-induced apoptosis, by inducing the secretion of an additional paracrine survival factor(s). These data strongly support an important role for vFLIP in NF-kB activation, which may be crucial for cell transformation by KSHV, for the survival of infected cells, and for metastasis

HIV-Host Interactions

HIV remains the major global health threat, and neither vaccine nor cure is available. Increasing our knowledge on HIV infection will help overcome the challenge of HIV/AIDS. This book covers several aspects of HIV-host interactions in vitro and in vivo. The first section covers the interaction between cellular components and HIV proteins, Integrase, Tat, and Nef. It also discusses the clinical relevance of HIV superinfection. The next two chapters focus on the role of innate immunity including dendritic cells and defensins in HIV infection followed by the section on the impact of host factors on HIV pathogenesis. The section of co-infection includes the impact of Human herpesvirus 6 and Trichomonas vaginalis on HIV infection. The final section focuses on generation of HIV molecular clones that can be used in macaques and the potential use of cotton rats for HIV studies

Studies of the role of nf-κb in controlling osteoclast differentiation and bone loss

Increased osteoclast (OC) bone resorption and/or decreased osteoblast (OB) bone formation contribute to bone loss in osteoporosis and rheumatoid arthritis (RA). Findings of the basic and translational research presented in this thesis demonstrate a number of mechanisms by which cytokine-induced NF-κB activation controls bone resorption and formation: 1) Tumour necrosis factor-α (TNF) expands pool of OC precursors (OCPs) by promoting their proliferation through stimulation of the expression of macrophage colony stimulating factor (M-CSF) receptor, c-Fms, and switching M-CSF-induced resident (M2) to inflammatory (M1) macrophages with enhanced OC forming potential and increased production of inflammatory factors through induction of NF-κB RelB; 2) Similar to RANKL, TNF sequentially activates transcriptional factors NF-κB p50 and p52 followed by c-Fos and then NFATc1 to induce OC differentiation. However, TNF alone nduces very limited OC differentiation. In contrast, it pre-activates OCPs to express cFos which cooperates with interleukin-1 (IL-1) produced by these OCPs in an autocrine mechanism by interacting with bone matrix to mediate the OC terminal differentiation and bone resorption from these pre-activated OCPs. 3) TNF-induced OC formation is independent of RANKL but it also induces NF-κB2 p100 to limit OC formation and bone resorption, and thus p100 deletion accelerates joint destruction and systemic bone loss in TNF-induced RA; 4) TNF receptor associated factor-3 (TRAF3) limits OC differentiation by negatively regulating non-canonical NF-κB activation and RANKL induces TRAF3 ubiquitination and lysosomal degradation to promote OC differentiation. Importantly, a lysosomal inhibitor that inhibits TRAF3 degradation prevents ovariectomy-induced bone loss; 5) RelB and Notch NICD bind RUNX2 to inhibit OB differentiation and RelB:p52 dimer association with NICD inhibit OB differentiation by enhancing the binding of RBPjκ to Hes1. These findings suggest that non-canonical NF- κB signaling could be targets to develop new therapies for RA or osteoporosis. For example 1) Agents that degrade TNF-induced RelB could block M1 macrophage differentiation to inhibit inflammation and joint destruction for the therapy of RA; 2)Agents that prevent p100 processing or TRAF3 degradation could inhibit bone resorption and also stimulate bone formation simultaneously for the therapy of osteoporosis

Development of lentiviral vectors targeted to P38MAPK inhibition and Akt activation in motor neurons of a mouse model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by progressive loss of motor neurons in the cortex, brainstem and spinal cord. This leads to weakness and muscular atrophy that evolves to paralysis and death. Studies on patients and animal models of ALS suggest that the pathology is the consequence of the unbalance between pro-degenerative (p38MAPK) and pro-survival (Akt) pathways in motor neurons. In order to re-equilibrate this complex interplay, the major objective of this study was to develop a gene-based approach to target, in vivo, these two intracellular signaling proteins. We demonstrated for the first time that a construct derived from Hb9 promoter can be used in lentivectors to restrict transgene expression to motor neurons in vivo; moreover we found that induction of Akt pathway, through expression of constitutively activated Akt3 in motor neurons, prevents neuronal loss in SOD1G93A mice, although it does not influence their premature death. On the other hand, we used RNAi to downregulate p38MAPKalpha in a mouse model of ALS. We revealed that expression of p38MAPK-targeted shRNA in the spinal cord of presymptomatic mice reduces motor neuronal loss in the early phases of the pathology, but then it is not able to sustain neuronal survival during disease progression, eventually resulting in worsening of symptoms and shortening of life span. We found preliminary evidences that hyperactivation of microglial cells may be responsible for this more aggressive phenotype, suggesting that a fine tuning of microglial reactivity may be important to avoid pro-degenerative effects. Overall these data support the hypothesis that modulation of pro-degenerative and pro-survival pathways may help counteracting motor neuronal degeneration, and strengthen the evidence that preservation of neuronal perikaria is not sufficient to ameliorate disease progression in ALS. The peripheral compartment (axons, muscles) should be regarded as an additional target for potential therapeutic approaches

Lentivector based gene transfer for immunotherapy – application of integration deficient vectors and PDL1 knockdown as tools to manipulate immune responses

Lentiviral‐based vectors are effective and promising tools for the generation of cell mediated immunity. Multiple studies have demonstrated that subcutaneous injection of lentivectors encoding tumour antigens results in induction of strong CTL responses and often in tumour killing. However, integration of lentivectors into human genomic DNA poses a risk of insertional mutagenesis. Indeed, this possibility has been highlighted by gene therapy trials that resulted in the development of T cell leukaemia in several patients. For this reason, non‐integrating lentiviral vectors (NILVs) have been developed as a safer alternative for gene delivery. The first part of this thesis demonstrates that lentivectors carrying multiple mutations preventing integration are effective vaccines. Subcutaneous injection of these vectors resulted in induction of systemic dose‐dependant CD8+ T‐cell responses to the encoded antigen. The duration of the persistence of antigen presentation was measured using transfer of OT1 transgenic T cells into previously immunized mice. Measuring expansion of those cells revealed that the antigen was present and presented for at least 30 days. CD8+ T‐cell responses were further enhanced by addition of dendritic cell (DC) stimulators: p38 MAP kinase and NF‐κB stimulators. These activators led to a more rapid response peaking at day 7. Finally, NILVs expressing the antigen and DC activators were tested in a tumour therapy model and were found to be effective. The second part of this thesis focused on altering DC‐T cell interactions to enhance responses to immunization by lentivector‐mediated knockdown of PDL1 on DCs. The analysis of DCs infected with anti‐PDL1 shRNA showed that knocking down this molecule drives DCs towards a mature phenotype. The influence of PDL1 knockdown was assessed on co‐cultured T cells. The absence of PDL1 enhanced their proliferation and reduced antigenic stimulation induced TCR complex degradation. DCs transduced with lentivectors expressing PDL1 shRNA were also tested in vaccination and tumour therapy

Regulation of dendritic cell function by Dectin-1.

Innate pattern recognition receptors (PRRs) expressed on dendritic cells (DC) link direct recognition of pathogens to initiation of T cell responses. Here I describe evidence that Dectin-1 is a novel pattern recognition receptor involved the activation of dendritic cells by the yeast cell wall preparation, zymosan. Zymosan contains ligands for the known PRR Toll-like Receptor-2 (TLR2). Interestingly, recent work in macrophages has implicated the p-glucan receptor Dectin-1 in zymosan recognition. This thesis demonstrates that Dectin-1 can function as a PRR independently of the Toll-like Receptor system to induce DC cytokine production (IL-2, IL-10) and Notch-ligand upregulation (Jagged-1). My work has helped determine that Dectin-1 can signal via a novel HemlTAM motif to the tyrosine kinase Syk. DC stimulated with zymosan upregulate IL-10, IL-2 and Jagged-1 in a Syk-dependent manner. Indeed, IL-10 and Jagged-1 induction is independent of TLR-mediated recognition of zymosan. In addition, zymosan induced ERK activation is entirely dependent on signalling through Syk and is independent of TLR signalling. I demonstrate that this ERK activation is necessary for the induction of IL-2 and IL-10 in response to zymosan. Finally I present preliminary findings on how the unusual cytokine signature of zymosan-stimulated DCs may bias Thl and Thl7 differentiation induced in vitro