Michelle Pate

Antimicrobial Activities and Structures of Two Linear Cationic Peptide Families with Various Amphipathic β-Sheet and α-Helical Potentials

ABSTRACT Many naturally occurring antimicrobial peptides comprise cationic linear sequences with the potential to adopt an amphipathic α-helical conformation. We designed a linear 18-residue peptide that adopted an amphipathic β-sheet structure when it was bound to lipids. In comparison to a 21-residue amphipathic α-helical peptide of equal charge and hydrophobicity, this peptide possessed more similar antimicrobial activity and greater selectivity in binding to and inducing leakage in vesicles composed of bacterial membrane lipids than vesicles composed of mammalian membrane lipids (J. Blazyk, R. Weigand, J. Klein, J. Hammer, R. M. Epand, R. F. Epand, W. L. Maloy, and U. P. Kari, J. Biol. Chem. 276:27899-27906, 2001). Here, we compare two systematically designed families of linear cationic peptides to evaluate the importance of amphipathicity for determination of antimicrobial activity. Each peptide contains six lysine residues and is amidated at the carboxyl terminus. The first family consists of five peptides with various capacities to form amphipathic β-sheet structures. The second family consists of six peptides with various potentials to form amphipathic α helices. Only those peptides that can form a highly amphipathic structure (either a β sheet or an α helix) possessed significant antimicrobial activities. Striking differences in the abilities to bind to and induce leakage in membranes and lipid vesicles were observed for the two families. Overall, the amphipathic β-sheet peptides are less lytic than their amphipathic α-helical counterparts, particularly toward membranes containing phosphatidylcholine, a lipid commonly found in mammalian plasma membranes. Thus, it appears that antimicrobial peptides that can form an amphipathic β-sheet conformation may offer a selective advantage in targeting bacterial cells.

Dendritic Cells The Tumor Microenvironment and the Challenges for an Effective Antitumor Vaccination

Many clinical trials have been carried out or are in progress to assess the therapeutic potential of dendritic-cell- (DC-) based vaccines on cancer patients, and recently the first DC-based vaccine for human cancer was approved by the FDA. Herewith, we describe the general characteristics of DCs and different strategies to generate effective antitumor DC vaccines. In recent years, the relevance of the tumor microenvironment in the progression of cancer has been highlighted. It has been shown that the tumor microenvironment is capable of inactivating various components of the immune system responsible for tumor clearance. In particular, the effect of the tumor microenvironment on antigen-presenting cells, such as DCs, does not only render these immune cells unable to induce specific immune responses, but also turns them into promoters of tumor growth. We also describe strategies likely to increase the efficacy of DC vaccines by reprogramming the immunosuppressive nature of the tumor microenvironment.

The interplay between surfaces and soluble factors define the immunologic and angiogenic properties of myeloid dendritic cells

BackgroundDendritic cells (DCs) are antigen presenting cells capable of inducing specific immune responses against microbial infections, transplant antigens, or tumors. Interestingly, microenvironment conditions such as those present in tumor settings might induce a DC phenotype that is poorly immunogenic and with the capability of promoting angiogenesis. We hypothesize that this plasticity may be caused not only by the action of specific cytokines or growth factors but also by the properties of the surfaces with which they interact, such as extracellular matrix (ECM) components.ResultsHerewith we studied the effect of different surfaces and soluble factors on the biology of DCs. To accomplish this, we cultured murine myeloid(m) DCs on surfaces coated with fibronectin, collagen I, gelatin, and Matrigel using poly-D-lysine and polystyrene as non-biological surfaces. Further, we cultured these cells in the presence of regular DC medium (RPMI 10% FBS) or commercially available endothelial medium (EGM-2). We determined that mDCs could be kept in culture up to 3 weeks in these conditions, but only in the presence of GM-CSF. We were able to determine that long-term DC cultures produce an array of angiogenic factors, and that some of these cultures still retain the capability to induce T cell responses.ConclusionsAltogether these data indicate that in order to design DC-based vaccines or treatments focused on changing the phenotype of DCs associated with diseases such as cancer or atherosclerosis, it becomes necessary to fully investigate the microenvironment in which these cells are present or will be delivered.

Toll-Like Receptors as Novel Therapeutic Targets for Ovarian Cancer

Ovarian cancer (OC) is an aggressive disease that affects approximately 1 in 70 women and has a poor prognosis (<50%, 5-year survival rate), in part because it is often diagnosed at a late stage. There are three main types of OC: neoplasms of surface epithelial, germ cell, or stromal origin, with surface epithelial tumors comprising about 80% of all OCs. In addition to improving diagnostics, it is necessary to develop more effective treatments for epithelial-origin OC. Here, we describe the paradoxical roles of toll-like receptor (TLR) signaling in the progression of cancer and discuss how its modulation may result in decreased tumor growth and metastasis via the attenuation of proangiogenic cytokines and potentiation of proapoptotic factors. In particular, it has been found that TLR activity can behave like a “double-edged sword”, as its signaling pathways have been implicated as having both tumor-suppressive and tumor-promoting effects. With particular emphasis on OC, we discuss the need to consider the signaling details of TLRs and associated proteins in the multiple cell types present in the tumor milieu to achieve safe and effective design of TLR-based cancer therapies.

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyers patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4. Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Adhesion to substrates induces dendritic cell endothelization and decreases immunological response.

Dendritic cells (DCs) are antigen presenting cells capable of inducing specific immune responses against microbial infections, transplant antigens, or tumors. DCs have been shown to possess a high plasticity showing different phenotypes in response to their microenvironment. For example, tumor-associated DCs can acquire an angiogenic phenotype thus promoting tumor growth. Further, DCs cultured in vitro under different conditions are able to upregulate the expression of endothelial markers and to express angiogenic factors. Indeed, it has been shown that soluble factors such as VEGF of PGE-2, that are present in the microenvironment of several tumors, affect the biology of these cells. We hypothesize that in addition to soluble factors the adhesion to different substrates will also define the phenotype and function of DCs. Herewith we demonstrate that murine myeloid(m) DCs upregulate endothelial markers such as VE-Cadherin, and to a lesser extent TIE-2, and decrease their immune capabilities when cultured on solid surfaces as compared with the same cells cultured on ultra-low binding (ULB) surfaces. On the other hand, the expression of angiogenic molecules at the level of RNA was not different among these cultures. In order to further investigate this phenomenon we used the murine ID8 model of ovarian cancer which can generate solid tumors when cancer cells are injected subcutaneously or a malignant ascites when they are injected intraperitoneally. This model gave us the unique opportunity to investigate DCs in suspension or attached to solid surfaces under the influence of the same tumor cells. We were able to determine that DCs present in solid tumors showed higher levels of expression of endothelial markers and angiogenic molecules but were not able to respond to inflammatory stimuli at the same extent as DCs recovered from ascites. Moreover, mDCs cultured on ULB surfaces in the presence of tumor factors do not expressed endothelial markers. Taking into account all these data we consider that tumor factors might be responsible for inducing angiogenic properties in DCs, but that in some settings the expression of endothelial markers such as VE-Cadherin and TIE-2 might be a function of attachment to solid surfaces and independent of the angiogenic properties of these cells.

Osteopathic Manipulative Therapy Induces Early Plasma Cytokine Release and Mobilization of a Population of Blood Dendritic Cells

It has been claimed that osteopathic manipulative treatment (OMT) is able to enhance the immune response of individuals. In particular, it has been reported that OMT has the capability to increase antibody titers, enhance the efficacy of vaccination, and upregulate the numbers of circulating leukocytes. Recently, it has been shown in human patients suffering chronic low back pain, that OMT is able to modify the levels of cytokines such as IL-6 and TNF-α in blood upon repeated treatment. Further, experimental animal models show that lymphatic pump techniques can induce a transient increase of cytokines in the lymphatic circulation. Taking into account all these data, we decided to investigate in healthy individuals the capacity of OMT to induce a rapid modification of the levels of cytokines and leukocytes in circulation. Human volunteers were subjected to a mixture of lymphatic and thoracic OMT, and shortly after the levels of several cytokines were evaluated by protein array technology and ELISA multiplex analysis, while the profile and activation status of circulating leukocytes was extensively evaluated by multicolor flow cytometry. In addition, the levels of nitric oxide and C-reactive protein (CRP) in plasma were determined. In this study, our results show that OMT was not able to induce a rapid modification in the levels of plasma nitrites or CRP or in the proportion or activation status of central memory, effector memory or naïve CD4 and CD8 T cells. A significant decrease in the proportion of a subpopulation of blood dendritic cells was detected in OMT patients. Significant differences were also detected in the levels of immune molecules such as IL-8, MCP-1, MIP-1α and most notably, G-CSF. Thus, OMT is able to induce a rapid change in the immunological profile of particular circulating cytokines and leukocytes.

Abstract 1659: Characterizing cytokine secretion in response to dsRNA treatment in ovarian cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ovarian cancer exhibits the lowest long-term survival rates amongst all gynecological cancers, warranting more research directed at finding novel therapies, especially for late-stage disease. Inflammation is known to affect cancer progression, with tumor-promoting or repressive effects possible in different contexts, where the types of leukocytes at the tumor site are heavily implicated in disease prognosis. Tumor-associated macrophages and T-regulatory cells, for example, are known to promote tumor growth by inhibiting the anti-cancer response and by secreting pro-angiogenic factors. Importantly, the specific processes and cellular signaling pathways leading up to leukocyte infiltration in different cancer types remain to be fully elucidated. We hypothesize that dsRNA in the vicinity of ovarian cancer cells (such as from a viral infection or cellular debris) can activate pro-inflammatory regulators in tumor cells, in particular the NF-KB transcription factor, the aberrant over-activation of which is implicated in many cancers. NF-KB activation results in increased production and secretion of numerous cytokines that can in turn recruit pro-angiogenic leukocytes to the tumor microenvironment, thus favoring cancer progression. Herewith, we evaluated the production of numerous cytokines after dsRNA treatment in mouse and human ovarian cancer cells. Additionally, we determined which of the four known dsRNA receptors contributes most to this process. Consistently with our hypothesis, we have shown an increase in several pro-inflammatory cytokines (eg. CCL5 and IL6) in response to dsRNA treatment in both human and mouse ovarian cancer cell lines. We have also determined that the dsRNA receptor MDA5 contributes the most to dsRNA-induced cytokine secretion in both human and mouse ovarian cancer cell lines employing siRNA-directed knock-down of each receptor. Altogether, these data suggest that dsRNA-induced signaling in ovarian cancer cells may facilitate a tumor-favoring environment; thus dsRNA-sensing receptors may constitute attractive therapeutic targets for cancer immunotherapy. Further studies in our lab will determine the specific signaling pathways responsible for the upregulated cytokines as well as use a mouse model to examine the effect of dsRNA receptor knock-down in the cancer cells on tumor growth, angiogenesis, and the leukocyte infiltration profile. Citation Format: Maria Muccioli, Michelle Pate, Fabian Benencia. Characterizing cytokine secretion in response to dsRNA treatment in ovarian cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1659. doi:10.1158/1538-7445.AM2014-1659

Abstract A65: Toll-like receptor 3 signaling in ovarian cancer.

The purpose of this study is to examine the cytokine secretion (in vitro) and leukocyte infiltration profiles (in vivo) in response to Toll-Like Receptor 3 (TLR3) activation in murine epithelial ovarian carcinoma. Ovarian cancer is the most deadly gynecological neoplasm, typically treated with surgery and chemotherapy, and has a less than a 50% 5-year survival rate. Drug resistance develops frequently, and it is necessary to investigate more targeted therapies. Chronic inflammation, which is seen in many cancers, can render an immunosuppressive tumor microenvironment characterized by cytokines that promote leukocyte infiltration, which can potentiate angiogenesis, and subsequent tumor growth and metastasis. Pathways that can generate such inflammation are important to characterize as they may be targeted in the development of novel cancer therapeutics. One regulator of inflammation in some types of epithelial ovarian cancer is TLR3, a protein that senses dsRNA and activates NF-κB, a potent initiator of cytokine secretion. Although typically expressed in immune cells, TLR3 is also present in cancer cells, and may function to direct inflammation at the tumor site towards an immunosuppressive state capable of recruiting pro-angiogenic leukocytes. Using PCR, ELISA, western blot, and microarray experiments on siRNA-generated TLR3-knock-downs and control samples, we are characterizing the cytokine secretion profiles following TLR3 activation with synthetic dsRNA ligands. Furthermore, we will employ this ovarian cancer cell line (ID8) with C57BL/6 mice to generate transplantation tumors and determine the effects of TLR3 signaling on the leukocyte population and presence of angiogenic molecules at the tumor site using flow cytometry and immunohistochemistry. We have determined that treatment of ID8 epithelial ovarian carcinoma cells with TLR3 ligands poly (I:C) or poly (A:U) results in increased secretion of pleiotropic cytokines, such as CCL5 and IL6, that may potentiate leukocyte migration to the tumor site. In vivo studies with C57BL/6 mice will determine whether this TLR3-induced cytokine secretion in the tumor cells translates to the physiological environment and promotes leukocyte infiltration and angiogenesis. By elucidating the contribution of TLR3 to the recruitment of pro-angiogenic leukocytes in ovarian epithelial carcinoma, the results of this project may be useful in the development of anti-angiogenic therapies for certain ovarian tumors. Citation Format: Maria Muccioli, Michelle Pate, Fabian Benencia. Toll-like receptor 3 signaling in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A65.

Fine-Tuning the Activity of Linear Amphipathic Beta-Sheet Antimicrobial Peptides

It is relatively simple to design highly amphipathic linear cationic beta-sheet peptides containing 10-to-11 amino acids that possess potent antimicrobial activity. Usually, however, these peptides also are quite hemolytic, so that there is insufficient selectivity between bacterial and human cells. Peptides with little or no hemolytic (or other toxic) activity toward host cells at 100 or more times the minimum inhibitory concentrations toward bacterial cells might be potential candidates for clinical use as antimicrobials. We have used two strategies to separately attenuate lytic activity toward host cells while maintaining potent antimicrobial activity. Both strategies involve introducing a structural perturbation in the amphipathic beta sheet. First, a hydrophobic amino acid residue can be substituted by proline. Depending upon the location of the substitution within the peptide, it is possible to nearly eliminate hemolytic activity while retaining potent antimicrobial activity. A similar outcome can be achieved by replacing a hydrophobic amino acid residue with a D-amino acid. Here again, the location of the substitution within the peptide is critical for the desired balance of activities. We show here 10- and 11-residue peptides consisting of alternating lysine and leucine in which a single leucine has been replaced by either proline or a D-amino acid. The effects of these substitutions on antimicrobial and hemolytic activities, secondary structure, and ability to induce leakage in lipid vesicles and bacterial cells are compared. The most promising peptides will be tested in vivo to determine their suitability as either topical or systemic antimicrobial agents.