Characterisation of MRI Indeterminate Breast Lesions Using Dedicated Breast PET and Prone FDG PET-CT in Patients with Breast Cancer-A Proof-of-Concept Study.

Magnetic resonance imaging (MRI) in patients with breast cancer to assess extent of disease or multifocal disease can demonstrate indeterminate lesions requiring second-look ultrasound and ultrasound or MRI-guided biopsies. Prone positron emission tomography-computed tomography (PET-CT) is a dedicated acquisition performed with a breast-supporting device on a standard PET-CT scanner. The MAMmography with Molecular Imaging (MAMMI, Oncovision, Valencia, Spain) PET system (PET-MAMMI) is a true tomographic ring scanner for the breast. We investigated if PET-MAMMI and prone PET-CT were able to characterise these MRI- indeterminate lesions further. A total of 10 patients with breast cancer and indeterminate lesions on breast MRI were included. Patients underwent prone PET-MAMMI and prone PET-CT after injection of FDG subsequently on the same day. Patients then resumed their normal pathway, with the clinicians blinded to the results of the PET-MAMMI and prone PET-CT. Of the MRI-indeterminate lesions, eight were histopathologically proven to be malignant and two were benign. PET-MAMMI and prone PET-CT only were able to demonstrate increased FDG uptake in 1/8 and 0/8 of the MRI-indeterminate malignant lesions, respectively. Of the MRI-indeterminate benign lesions, both PET-MAMMI and prone PET-CT demonstrated avidity in 1/2 of these lesions. Our findings do not support the use of PET-MAMMI to characterise indeterminate breast MRI lesions requiring a second look ultrasound.

Evolving role of adiponectin in cancer-controversies and update.

Adiponectin (APN), an adipokine produced by adipocytes, has been shown to have a critical role in the pathogenesis of obesity-associated malignancies. Through its receptor interactions, APN may exert its anti-carcinogenic effects including regulating cell survival, apoptosis and metastasis via a plethora of signalling pathways. Despite the strong evidence supporting this notion, some work may indicate otherwise. Our review addresses all controversies critically. On the whole, hypoadiponectinaemia is associated with increased risk of several malignancies and poor prognosis. In addition, various genetic polymorphisms may predispose individuals to increased risk of obesity-associated malignancies. We also provide an updated summary on therapeutic interventions to increase APN levels that are of key interest in this field. To date efforts to manipulate APN levels have been promising, but much work remains to be done.

Adiponectin and its receptor signaling: an anti-cancer therapeutic target and its implications for anti-tumor immunity.

INTRODUCTION: Adiponectin (APN), produced by adipocytes, has direct anti-diabetic, anti-atherogenic and anti-inflammatory properties. Circulating APN levels are lower in obesity, a disease state that is often associated with several malignancies. AREA COVERED: Increasingly, clinical data suggests that serum APN may have an important protective role in carcinogenesis. Certain cancer cell types express APN receptors and their downstream signaling pathways may influence cancer biology, possibly by regulating cell proliferation and inducing apoptosis. However, APN's role in the immune system, in particular to the anti-tumor response, remains elusive. Therefore, this review critically addresses all controversies associated with the effect of APN on the immune system. EXPERT OPINION: Currently, the promise of interfering with APN and its receptor axis as a novel anti-cancer therapeutic target is rather encouraging. Greater understanding of the immunological side effects following this interference is crucial for the development of effective therapeutic strategies against obesity-associated malignancies. APN receptor signaling on immune cells can blunt anti-tumor immunity and induce tumor-specific tolerance. This may also have far-reaching consequences on the application of APN as an anti-cancer agent.

Adiponectin receptor signaling on dendritic cells blunts antitumor immunity.

Immune escape is a fundamental trait of cancer. Dendritic cells (DC) that interact with T cells represent a crucial site for the development of tolerance to tumor antigens, but there remains incomplete knowledge about how DC-tolerizing signals evolve during tumorigenesis. In this study, we show that DCs isolated from patients with metastatic or locally advanced breast cancer express high levels of the adiponectin receptors AdipoR1 and AdipoR2, which are sufficient to blunt antitumor immunity. Mechanistic investigations of ligand-receptor interactions on DCs revealed novel signaling pathways for each receptor. AdipoR1 stimulated IL10 production by activating the AMPK and MAPKp38 pathways, whereas AdipoR2 modified inflammatory processes by activating the COX-2 and PPARγ pathways. Stimulation of these pathways was sufficient to block activation of NF-κB in DC, thereby attenuating their ability to stimulate antigen-specific T-cell responses. Together, our findings reveal novel insights into how DC-tolerizing signals evolve in cancer to promote immune escape. Furthermore, by defining a critical role for adiponectin signaling in this process, our work suggests new and broadly applicable strategies for immunometabolic therapy in patients with cancer.

Vascular endothelial growth factor receptors and the therapeutic targeting of angiogenesis in cancer: where do we go from here?

Abstract Vascular Endothelial Growth Factor receptors (VEGFRs), the interactions with their ligands and the subsequent signalling pathways are known to play a vital role in tumour angiogenesis. Initial clinical trials of VEGFR inhibitors were disappointing but over the past decade some therapies have been successfully brought to market. At present, VEGFR inhibitors appear to be most promising as adjuvants to conventional chemotherapy. However, several interacting signalling molecules and downstream pathways have recently been shown to interact with VEGFR signalling and provide promising novel targets, such as the platelet-derived growth factor (PDGF), epithelial growth factor (EGF), human epithelial receptor-2, (HER-2) Tie-2 and oestrogen receptors. Elucidation of this web of signalling pathways may identify new therapeutic strategies which may be used in combination with VEGFR inhibitors to augment the efficacy of anti-angiogenic cancer treatments. This review assesses the role of modulating VEGFR activity in cancer and systematically examines current evidence and trials in this area.

Blocking the interaction of vascular endothelial growth factor receptors with their ligands and their effector signaling as a novel therapeutic target for cancer: time for a new look?

INTRODUCTION: The interaction between the VEGFRs and their ligands plays an important role in tumor angiogenesis. Despite a series of problems encountered during early work on blocking growth factors, current evidence injects further vigor into researching the modulation of VEGFR activity. Emerging preclinical and clinical studies suggest that attenuating receptor activity can synergistically promote antitumor action if utilized concurrently with conventional therapies. AREAS COVERED: This review presents an up-to-date assessment of the potential role of modulating receptor activities in various cancers. The sentinel work on the proof of principles in various animal models, and the current translational research on these small molecule inhibitors and receptor blocking antibodies, from Phase I to Phase III trials, has been systematically examined with an emphasis on agents in earlier stages of development. EXPERT OPINION: Many clinical trials are ongoing, but early phase trials show promising results. Recently, there has been a huge explosion of research activity either in the development of new drugs or in the understanding its biology. Many current trials lend support to the rationale behind these therapies, which can function as adjuvants to conventional treatments. It has been argued that normalization of tumor-induced vasculature can promote better drug delivery and prevent resistance to radiotherapy. However, strategies involving the inhibition of the interaction of VEGFRs with ligands and their downstream pathways are not, in general, at a stage where it will be directly useful in clinical cancer treatment. A deeper understanding of these biologic therapies will help to improve the efficacy of conventional treatments and furthermore reduce dose-dependent cytotoxic activity.

Induction of pathogenic cytotoxic T lymphocyte tolerance by dendritic cells: a novel therapeutic target.

IMPORTANCE OF THE FIELD: Dendritic cells (DCs) have an important role, both direct and indirect, in controlling the expansion and function of T cells. Of the different subsets of T cells, cytotoxic T lymphocytes (CTLs/CD8(+) T cells) have been implicated in the pathogenesis and development of many diseases, including various forms of autoimmunity and transplant rejection. It may therefore be of therapeutic benefit to control the function of CTL in order to modulate disease processes and to ameliorate disease symptoms. Currently, pharmacological approaches have been employed to either directly or indirectly modulate the function of T cells. However, these treatment strategies have many limitations. Many experimental data have suggested that it is possible to alter CTL activity through manipulation of DC. AREAS COVERED IN THIS REVIEW: Novel strategies that condition DCs to influence disease outcome through manipulation of CTL activity, both directly and indirectly. This includes the modulation of co-stimulation, negative co-stimulation, as well as manipulation of the cytokine milieu during CTL generation. Furthermore, DCs may also impact CTL activity through effects on effector and regulatory cells, along with manipulation of bioenergetic regulation, apoptotic-cell mediated tolerance and through the generation of exosomes. The implications of related interventions in the clinical arena are in turn considered. WHAT THE READER WILL GAIN: Insight into such indirect methods of controlling CTL activity allows for an understanding of how disease-specific T cells may be regulated, while also sparing other aspects of adaptive immunity for normal physiological function. Such an approach towards the treatment of disease represents an innovative therapeutic target in the clinical arena. TAKE HOME MESSAGE: There are numerous innovative methods for using DCs to control CTL responses. Manipulation of this interaction is thus an attractive avenue for the treatment of disease, particularly those of immune dysregulation, such as seen in autoimmunity and transplantation. With the number of studies moving into clinical stages constantly increasing, further advances and successes in this area are inevitable.

Manipulation of indoleamine 2,3-dioxygenase (IDO) for clinical transplantation: promises and challenges.

BACKGROUND: Since the discovery that indoleamine 2,3-dioxygenase (IDO) is a modulator for maintenance of fetomaternal immuno-privilege state, it has been implicated in tumour tolerance, autoimmune diseases and asthma. IDO is an IFN-gamma-inducible, intracellular enzyme that catalyzes the initial and rate-limiting step in the degradation of tryptophan. It has been suggested that IDO can regulate the immune system either through deprivation of tryptophan that is essential for T cell proliferation or via cytotoxic effects of kynurenine pathway metabolites on T cell survival. METHODS: The sources of information used were obtained through Pubmed/Medline. RESULTS/CONCLUSION: While IDO emerges as a regulator of immunity, its role in controlling allo-response is unfolding. IDO can control T cell responses to allo-antigens and induce generation of allo-specific regulatory T cells. Exploiting IDO as a modulator of transplant rejection, many groups have manipulated its activity to prolong allograft survival in transplantation models. Despite the initial promise, its application to clinical transplantation may be limited. We therefore examine the potentials and limitations associated with clinical translation of IDO into a therapeutic.

Interaction of current cancer treatments and the immune system: implications for breast cancer therapeutics.

Early diagnosis and treatment of breast cancer may account for the current improvement in the mortality of breast cancer. However, achieving a complete 'cure' is the holy grail of cancer medicine and, in many cases, cancer patients still succumb to their ultimate fate. There is therefore a need to devise innovative therapies to overcome this problem. To this end, many emerging therapies utilizing the immune system to eradicate the residues of disease have been described in the preclinical and clinical arenas. However, there is very little work examining the impact of immunotherapy on the existing natural immunity. The relationship between antitumor immunity, in the form of immunotherapy (either passive or active), and current strategies of treatment also needs to be explored. If we are to improve the success of cancer treatment, we must understand how current therapies interact with the immune system and with the emerging immunotherapies. For breast-cancer treatment to be successful, therapeutics should be tailored towards antitumor immunity; they should also avoid tumor-specific tolerance. The sources of information used to prepare this paper were obtained through published work on Pubmed/Medline and materials published on the US/UK governmental agencies' websites.

Changing viral tropism using immunoliposomes alters the stability of gene expression: implications for viral vector design.

Many strategies for redirecting the tropism of murine Moloney leukemia virus (MMLV) have been described. Preformed virion-liposome complexes, termed virosomes, have been reported to be relatively stable. Virosomes mediate envelope-independent transduction that allows efficient superinfection of resistant cell lines; however, virosome-mediated transduction behaves in a non-target-specific manner. We developed a novel method using antibodies to direct MMLV to vascular endothelium. We have given the term immunovirosomes to the complexes formed between viruses, liposomes, and antibodies. These immunovirosomes improve the transduction efficiency of the viruses and alter their tropism. We have shown improved transduction when immunovirosomes were targeted at the endocytic receptors CD71 and CD62E/P and rather less good delivery when targeted at CD106. The enhancement of the transduction efficiency was transient, however, suggesting that rerouting the entry pathway of viruses alters the expression properties of the viruses.

Gene delivery to vascular endothelium using chemical vectors: implications for cardiovascular gene therapy.

The vascular endothelium is an attractive target for gene therapy because of its accessibility and its importance in the pathophysiology of a wide range of cardiovascular conditions. In general, viral methods have been shown to be very effective at delivering genes to endothelium. The immunogenicity and pathogenicity associated with viral vectors have led increased efforts to seek alternative means of 'ferrying' therapeutic genes to endothelium or to decrease the short-comings of viral vectors. This paper reviews developments in non-viral technology. In addition, discussion also covers the mechanisms whereby existing chemical vectors deliver DNA to cells. Understanding the pathways of vector internalisation and intracellular traffic is important in developing strategies to improve vector technology. The authors propose that the chemical vector may represent a robust and versatile technology to 'ferry' therapeutic genes to vascular endothelium in order to modify the endothelial dysfunction associated with many cardiovascular diseases.

Knockdown of mouse VCAM-1 by vector-based siRNA.

Graft rejection is critically dependent on the recruitment of leukocytes via adhesion molecules on the endothelium, and inhibition of these interactions can prolong graft survival. We have therefore developed an approach using siRNA to inhibit the expression of VCAM-1 in endothelial cells. We transfected siRNA constructs into murine corneal and vascular endothelium and looked at expression of VCAM-1 and other surface molecules by flow cytometry. Adhesion assays (both static and under flow) were used to determine the effect of VCAM-1 inhibition. The activation of cellular stress responses was assessed by RT-PCR. Constructs encoding siRNA can block expression of VCAM-1 in both corneal and vascular endothelial cells (in the latter case after cytokine stimulation). Inhibition of VCAM-1 expression reduced the ability of T cells to adhere to endothelium. However, there were non-specific effects of siRNA expression, including upregulation of (Programmed Death Ligand 1) PDL1 and decreased cell growth. Analysis of stress pathways showed that the endothelial cells transfected with siRNA had upregulated molecules associated with cell stress. While these data are supportive of a potential therapeutic role for siRNA constructs in blocking the expression of adhesion molecules, they also highlight potential non-specific effects of siRNA that must be carefully considered in any application of this technology.

Gene therapy for transplantation with viral vectors--how much of the promise has been realised?

Gene therapy holds promise in preventing the development of many diseases. One of the possible applications is the management of organ transplantation. Over the years, advances in vector development have allowed the clinical progression of this form of therapy to become more attainable. Viral vector technology has proved to be better than non-viral vectors at ferrying therapeutic genes to cells. However, many deficiencies in viral vectors hinder the full realisation of gene-based therapy in transplantation. Here, these deficiencies and their ramifications for the future of viral vector development are fully analysed. The authors propose that the slow progress of gene therapy in transplantation may be related to the deficiencies in viral vectors.

Function of indoleamine 2,3-dioxygenase in corneal allograft rejection and prolongation of allograft survival by over-expression.

Indoleamine 2,3-dioxygenase (IDO) suppresses T cell responses by its action in catabolising tryptophan. It is important in maintenance of immune privilege in the placenta. We investigated the activity of IDO in the cornea, following corneal transplantation and the effect of IDO over-expression in donor corneal endothelium on the survival of corneal allografts. IDO expression was analysed and functional activity was quantified in normal murine cornea and in corneas following transplantation as allografts. Low levels of IDO, at both mRNA and protein levels, was detected in the normal cornea, up-regulated by IFN-gamma and TNF. Expression of IDO in cornea was significantly increased following corneal transplantation. However, inhibition of IDO activity in vivo had no effect on graft survival. Following IDO cDNA transfer, murine corneal endothelial cells expressed functional IDO, which was effective at inhibiting allogeneic T cell proliferation. Over-expression of IDO in donor corneal allografts resulted in prolonged graft survival. While, on one hand, our data indicate that IDO may augment corneal immune privilege, up-regulated IDO activity following cytokine stimulation may serve to inhibit inflammatory cellular responses. While increasing IDO mRNA expression was found in allogeneic corneas at rejection, over-expression in donor cornea was found to significantly extend survival of allografts.

Effect of vectors on human endothelial cell signal transduction: implications for cardiovascular gene therapy.

OBJECTIVE: Endothelium is an important target for gene therapy. We have investigated the effect of viral and nonviral vectors on the phenotype and function of endothelial cells (ECs) and developed methods to block any activation caused by these vectors. METHODS AND RESULTS: Transduction of ECs with viral vectors, including adenovirus, lentiviruses, and Moloney murine leukemia virus, can induce a pro-inflammatory phenotype. This activation was reduced when nonviral vectors were used. We demonstrate that after transduction there is upregulation of dsRNA-triggered antiviral and PI3K/Akt signaling pathway. Blockade of the NFkappaB, PI3-K, or PKR signaling pathways all operated to inhibit partially virally induced activation, and inhibition of both PKR and PI3-K pathways totally blocked EC activation. Furthermore, inhibition of IFN-alpha/beta in addition to PI3-K was effective at preventing EC activation. CONCLUSIONS: Viral vectors, although efficient at transducing ECs, result in their activation. Blockade of the signaling pathways involved in viral activation may be used to prevent such activation.

Gene therapy for clinical transplantation: where do we stand in 2006?

Gene therapy holds a great promise to prevent allograft rejection or to induce transplant tolerance. Many achievements in vector development have allowed the progression of this therapy to become more attainable in clinical transplantation. In this articles, the authors examine the exciting development in various vector technologies that allows this form of therapy to take the central stage of clinical transplantation. Also highlighted are various therapeutic strategies that might ultimately result in the realization of gene-based treatment in clinical transplantation.

Delivery of oligodeoxynucleotides into human saphenous veins and the adjunct effect of ultrasound and microbubbles.

Therapy with naked oligodeoxynucleotides (ODNs, molecular weight: 3000 to 7500) provides an elegant means of modulating gene expression without the problems associated with conventional gene therapy, but the relatively low transfer efficiency on intravascular administration is a limitation to clinical application. Ultrasound, which can be potentiated by microbubbles, shows promise as a method of delivering macromolecules such as plasmid DNA and other transgenes into cells. Since uptake of molecules into cells depends on their molecular weight, it might be expected that the delivery of ODNs, which are relatively small, will be facilitated by ultrasound and microbubbles. In the present study, we delivered ODNs into veins using ultrasound and microbubbles. First, we quantified the uptake of fluorescent-labeled ODNs into intact ex vivo human saphenous veins and isolated smooth muscle cells from the veins, evaluating the effect of ultrasound and microbubbles on uptake. Ultrasound potentiated the delivery of ODN in cells, except at high concentrations. When intact veins were studied, we achieved nuclear localization of fluorescent-labeled ODNs in cells. This increased with increasing concentration and incubation time and was not potentiated by ultrasound, even when microbubbles were used. We then applied a therapeutic ODN (antisense to intercellular adhesion molecule 1, ICAM-1) to vein samples and documented a functional inhibition of gene expression in a sequence-specific manner at the protein level with immunohistochemistry and western blot analysis. Again, no significant difference was seen with adjunct ultrasound. These observations suggest high diffusion of ODNs into human saphenous veins in this ex vivo model, indicating potential applications to inhibition of vascular bypass graft occlusion and other vasculopathies. Although microbubble-ultrasound was of value with cells in culture, it was not beneficial with intact veins.

Creation of tolerogenic human dendritic cells via intracellular CTLA4: a novel strategy with potential in clinical immunosuppression.

Activation of T lymphocytes requires the recognition of peptide-major histocompatibility complexes (MHCs) and costimulatory signals provided by antigen-presenting cells (APCs). It has been shown that T-cell activation without costimulation can lead to anergy. In this study, we developed a novel strategy to inhibit expression of B7 molecules (CD80/86) by transfecting APCs with a gene construct encoding a modified cytotoxic T lymphocyte antigen 4 (CTLA4) molecule (CTLA4-KDEL) that is targeted to the endoplasmic reticulum (ER). APCs expressing this construct failed to express CD80/86 on their surface, were unable to stimulate allogeneic and peptide-specific T-cell responses, and induced antigen-specific anergy of the responding T cells. Cells expressing CTLA4-KDEL do not up-regulate the indoleamine 2, 3-dioxygenase enzyme, unlike cells treated with soluble CTLA4-immunoglobin (Ig). This gene-based strategy to knock out surface receptors is an attractive alternative to using immature dendritic cells for preventing transplant rejection and treating of autoimmune diseases.

Tolerant T cells display impaired trafficking ability.

Based on our previous observation that anergic T lymphocytes lose their migratory ability in vitro, we have proposed that anergic T cells are retained in the site where they have been generated to exert their regulatory function. In this study we have analyzed T lymphocyte trafficking and motility following the induction of tolerance in vivo. In a model of non-deletional negative vaccination to xenoantigens in which dendritic cells (DC) localize to specific lymphoid sites depending on the route of administration, tolerant T cells remained localized in the lymph nodes colonized by tolerogenic DC, while primed T cells could traffic efficiently. Using an oral tolerance model that enables the 'tracking' of ovalbumin-specific TCR-transgenic T cells, we confirmed that T cells lose the ability to migrate through syngeneic endothelial cell monolayers following tolerance induction in vivo. Finally, we show that tolerant T cells (both in vitro and ex vivo) can inhibit migration of responsive T cells in an antigen-independent manner. Thus, hyporesponsive T cells localize at the site of tolerance induction in vivo, where they exert their anti-inflammatory properties. In physiological terms, this effect is likely to render immunoregulation a more efficient and controllable event.

Inhibition of NF-kappa B and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells.

Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and alpha-tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA-regulated, eukaryotic translation initiation factor 2alpha, NF-kappaB, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4(+)CD45RO, CD4(+)CD45RA, and CD4(+)CD25(-) subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-beta. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens.