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The fate of invariant NKT (iNKT) cells following activation remains controversial and unclear. We systemically examined how iNKT cells are regulated following TCR-dependent and -independent activation with α-galactosylceramide (αGC) or IL-18 plus IL-12, respectively. Our studies reveal activation by αGC or IL-18 plus IL-12 induced transient depletion of iNKT cells exclusively in the liver that was independent of caspase 3-mediated apoptosis. The loss of iNKT cells was followed by repopulation and expansion of phenotypically distinct cells via different mechanisms. Liver iNKT cell expansion following αGC, but not IL-18 plus IL-12, treatment required an intact spleen and IFN-γ. Additionally, IL-18 plus IL-12 induced a more prolonged expansion of liver iNKT cells compared with αGC. iNKT cells that repopulate the liver following αGC had higher levels of suppressive receptors PD-1 and Lag3, whereas those that repopulate the liver following IL-18 plus IL-12 had increased levels of TCR and ICOS. In contrast to acute treatment that caused a transient loss of iNKT cells, chronic αGC or IL-18 plus IL-12 treatment caused long-term systemic loss requiring an intact thymus for repopulation of the liver. This report reveals a previously undefined role for the liver in the depletion of activated iNKT cells. Additionally, TCR-dependent and -independent activation differentially regulate iNKT cell distribution and phenotype. These results provide new insights for understanding how iNKT cells are systemically regulated following activation.

Publisher Summary Tissue inflammation induced by antigens and pathogens varies considerably, but many diseases have a common pathology including stress and activation of epithelial cells that promotes innate immune responses, providing the host with its first line of defense against infections. Signals generated by subsets of innate lymphocytes, including natural killer (NK) cells, natural killer T (NKT) cells, and antigen presenting cells (APCs) during this early host response determine the nature of downstream adaptive immune responses. NK cells represent a population of specialized lymphocytes capable of recognizing and eliminating a wide range of cancer and virus-infected cells but not normal cells. The function of NK cells is regulated by a fine balance of inhibitory and activating signals, which are mediated by a diverse array of cell-surface receptors. Natural killer (NK) and natural killer T (NKT) cells represent unique lymphoid subsets of the innate immune system that are both critical for some aspects of initiating and directing host adaptive immune responses.These cells share numerous phenotypic markers and functional features, yet are lymphocytes of distinct developmental lineages.As first responders, these innate cells rapidly produce cytokines that modulate various activities of other leukocytes which subsequently influence the ensuing inflammatory response.Moreover, through the expression of effector molecules such as Fas and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) these cells are capable of directly targeting infected or transformed cells for elimination through cytotoxic pathways.Furthermore, both NK and NKT cells polarize T (helper) cell responses and enhance antigen presentation to cytotoxic T-lymphocytes.

Treatment of mice bearing orthotopic, metastatic tumors with anti-CD40 antibody resulted in only partial, transient anti-tumor effects whereas combined treatment with IL-2/anti-CD40, induced tumor regression. The mechanisms for these divergent anti-tumor responses were examined by profiling tumor-infiltrating leukocyte subsets and chemokine expression within the tumor microenvironment after immunotherapy. IL-2/anti-CD40, but not anti-CD40 alone, induced significant infiltration of established tumors by NK and CD8 + T cells. To further define the role of chemokines in leukocyte recruitment into tumors, we evaluated anti-tumor responses in mice lacking the chemokine receptor, CCR2. The anti-tumor effects and leukocyte recruitment mediated by anti-CD40 alone, were completely abolished in CCR2 −/− mice. In contrast, IL-2/anti-CD40-mediated leukocyte recruitment and reductions in primary tumors and metastases were maintained in CCR2 −/− mice. Treatment of mice with IL-2/anti-CD40, but not anti-CD40 alone, also caused an IFN-γ-dependent increase in the expression of multiple Th1 chemokines within the tumor microenvironment. Interestingly, although IL-2/anti-CD40 treatment increased Tregs in the spleen, it also caused a coincident IFN-γ-dependent reduction in CD4 + /FoxP3 + Tregs, myeloid-derived suppressor cells and Th2 chemokine expression specifically within the tumor microenvironment that was not observed after treatment with anti-CD40 alone. Similar effects were observed using IL-15 in combination with anti-CD40. Taken together, our data demonstrate that IL-2/anti-CD40, but not anti-CD40 alone, can preferentially reduce the overall immunosuppressive milieu within the tumor microenvironment. These results suggest that the use of anti-CD40 in combination with IL-2 or IL-15 may hold substantially more promise for clinical cancer treatment than anti-CD40 alone.

Natural killer (NK) and NKT cells are a first line of defense against pathogens and transformed cells. However, dysregulation of their function can lead to autoimmune disease. A better understanding of the mechanisms controlling NK and NKT effector function should lead to the development of improved strategies for the treatment of many diseases. The site in which NK and NKT cells reside should be taken into account, because accumulating evidence suggests that the tissue microenvironment strongly influences their function. In this regard, the liver represents a unique immunologic organ in which the balance between the need for tolerance and the ability to respond rapidly to pathogens and tissue injury is tightly regulated. NK cells in the liver have augmented cytolytic activity as compared to other organs, which is consistent with a role for liver-associated NK cells in being critical effector cells for inhibiting tumor metastasis in the liver. Several studies also suggest that hepatic NKT cells have different functions than those in other organs. Whereas splenic and thymic NKT cells have been shown to suppress diabetes development, facilitate the induction of systemic tolerance and are regulated by IL-4 and other Th2 cytokines, certain subsets of NKT cells in the liver are important sources of Th1 cytokines such as Interferon gamma, and are the primary mediators of anti-tumor responses. The unique properties and roles as critical effector cells make NK and NKT cells within the liver microenvironment attractive targets of immunotherapeutic approaches that have the goal of controlling tumor metastasis in the liver.

Recently, our laboratory reported that secondary CD8+ T cell-mediated antitumor responses were impaired following successful initial antitumor responses using various immunotherapeutic approaches. Although immunotherapy stimulated significant increases in CD8+ T cell numbers, the number of CD4+ T cells remained unchanged. The current investigation revealed a marked differential expansion of CD4+ T cell subsets. Successful immunotherapy surprisingly resulted in an expansion of CD4+Foxp3+ regulatory T (Treg) cells concurrent with a reduction of conventional CD4+ T (Tconv) cells, despite the marked antitumor responses. Following immunotherapy, we observed differential up-regulation of PD-1 on the surface of CD4+Foxp3+ Treg cells and CD4+Foxp3− Tconv cells. Interestingly, it was the ligand for PD-1, B7-H1 (PDL-1), that correlated with Tconv cell loss after treatment. Furthermore, IFN-γ knockout (IFN-γ−/−) and IFN-γ receptor knockout (IFN-γR−/−) animals lost up-regulation of surface B7-H1 even though PD-1 expression of Tconv cells was not changed, and this correlated with CD4+ Tconv cell increases. These results suggest that subset-specific expansion may contribute to marked shifts in the composition of the T cell compartment, potentially influencing the effectiveness of some immunotherapeutic approaches that rely on IFN-γ.

Pancreatic Ductal Adenocarcinoma (PDAC) is among the most malignant cancers, with 5-year survival of 6%. Chronic inflammation is associated with increased risk of PDAC. Nitric oxide (NO) is an important mediator of inflammatory responses and plays a role in tumorigenesis. The principal source of an increased and sustained level of NO is inducible nitric oxide synthase (NOS2). In this study, we’ve tested the hypothesis that NO enhances tumor progression in pancreatic cancer. We found that higher NOS2 expression in tumors was associated with poor survival in resected PDAC patients (p = 0.011). We then investigated the role of NO by deleting NOS2 gene in a genetically engineered mouse model of pancreatic cancer (KPC mice) with pancreas-specific activation of mutant KRAS and P53, which faithfully recapitulates the development and progression of human pancreatic cancer. NOS2-deficient KPC mice showed longer survival compared to the KPC littermates with wildtype NOS2 (p Citation Format: Jian Wang, Peijun He, Matthias M. Gaida, Shouhui Yang, Aaron Schetter, Jochen Gaedcke, Michael Ghadimi, Thomas Ried, Harris G. Yfantis, Dong H. Lee, Jonathan M. Weiss, Nadar Hanna, H. Richard Alexander, S. Perwez Hussain. Nitric oxide signaling pathway as a pathogenic driver in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 923. doi:10.1158/1538-7445.AM2015-923

Pro-inflammatory IL-17-producing T cells termed Th17 are actively involved in the pathogenesis of GVHD. The development and function of Th17 cells is dependent on activation of STAT3, RORgt and IRF4 transcription factors. Aberrant activation of Rho-associated kinase 2 (ROCK2) leads to induction of IL-17 and IL-21 secretion via IRF4-dependent mechanism. KD025, is a potent and selective ROCK2 inhibitor, which when given to healthy human subjects down-regulated the ability of T cells to secrete IL-21 and IL-17, but not interferon (IFN)-g, in response to TCR stimulation in vitro (Figure 1). KD025 inhibits STAT3 phosphorylation which supports RORgt, Th17 generation, and IL-21 production. Concurrently, KD025 increases STAT5 phosphorylation and Treg suppressor function in a dose-responsive fashion. KD025 treatment therefore shifts Th17/Treg balance. Th17 cells have been linked to in vivo pro-inflammatory responses, antibody production, and fibrosis. Conversely, Tregs can offset these pathogenic responses. Given the profile of KD025, we tested the effects of this inhibitor on cGVHD pathogenesis in a multi-organ system rodent model of disease that is driven by IL-21 responses and is associated with lung, liver and intestinal fibrosis. We observed that Th17/Rorc deficient T cells are unable to mediate cGVHD pathogenesis. In mice with established cGVHD, therapy was initiated with 30, 100, or 150 mg/kg/dose of KD025 daily from d28-56. Treated mice had a dose dependent decrease in the development of pathogenic pulmonary function as determined by whole body plethysmography (Figure 2) which correlated with a marked reduction of antibody deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. KD025 administration also resulted in a 2-fold decrease in collagen deposition in the lungs of mice treated with the highest dose of KD025. The spleens of mice treated with 150 mg/kg dose of KD025 had a decrease in the frequency of germinal centers compared to the vehicle treated mice. To determine the selective role of STAT3 on T cells, mice were transplanted with wildtype (WT) bone marrow (BM) and WT or inducible STAT3 deficient T cells. In parallel cohorts, the role of STAT3 in BM-derived B cells, precursors of germinal center B cells, was examined using WT vs inducible STAT3 deficient BM cells + WT T cells. We demonstrate here that mice transplanted with inducible STAT3 deficient T cells or BM cells had pulmonary function comparable to the healthy negative controls, suggesting that STAT3 is a potential therapeutic target in both T and B cells is necessary for the development of cGVHD and providing mechanistic insight into how KD025 may ameliorate active cGVHD. Studies are in progress to test KD025 administration in a murine scleroderma model using a minor histocompatibility antigen disparate donor-recipient strain that we have shown to be dependent upon STAT3 expressing donor T cells and a STAT3 inhibitor in both cGVHD models described here. Together, these data demonstrate that KD025 is effective at decreasing STAT3-dependent production of IL-21 and IL-17 and the use of KD025 is a potentially novel therapeutic intervention for the treatment of cGVHD. Fig 1 Oral administration of KD025 down-regulates the IL-17 and IL-21 secretion in human PBMCs upon stimulation ex vivo. Human PBMCs were purified from healthy human subjects before and after oral administration of KD025 at doses 40, 120, 240, 320 and stimulated ex vivo. Cytokine secretion was determined after 48 hours by ELISA. Fig 1. Oral administration of KD025 down-regulates the IL-17 and IL-21 secretion in human PBMCs upon stimulation ex vivo. Human PBMCs were purified from healthy human subjects before and after oral administration of KD025 at doses 40, 120, 240, 320 and stimulated ex vivo. Cytokine secretion was determined after 48 hours by ELISA. Fig 2 KD025 is an effective therapy for established murine cGVHD. Mice were given KD025 (150 mg/kg) d.28-56. PFTs indicate normal resistance, elastance and better compliance. Lung Ig deposition and fibrosis were comparable to BM controls. Fig 2. KD025 is an effective therapy for established murine cGVHD. Mice were given KD025 (150 mg/kg) d.28-56. PFTs indicate normal resistance, elastance and better compliance. Lung Ig deposition and fibrosis were comparable to BM controls. Disclosures No relevant conflicts of interest to declare.

Electroporation of dendritic cells (DCs) with tumor lysate elicited greater antitumor responses in vitro and in vivo, using less lysate than standard coincubation. Electroloaded DCs had normal surface marker expression and matured into competent antigen-presenting cells. In a renal carcinoma (RENCA) model, mice were pretreated with lysate-loaded DCs before tumor challenge. Mice that received DCs electroloaded with RENCA lysate had significantly smaller tumors (9 ± 6 mm 2 ) than mice given DCs coincubated with the same lysate (23 ± 5 mm 2 ). To evaluate a metastatic therapeutic tumor model, mice were first injected with Lewis lung carcinoma (LLC) and then given 2 doses of cryopreserved LLC lysate-loaded DCs. Mice treated with electroloaded DCs had a 50% reduction in lung metastases compared with control mice that received no DCs or DCs loaded with liver lysate. In contrast, DCs coincubated with LLC lysate were indistinguishable from controls. Tumor lysate-electroloaded but not-coincubated DCs also primed syngeneic mouse splenocytes in vitro to produce interferon-γ and, specifically, lyse tumor cells. The electroloaded DCs elicited specific T-cell responses with less lysate than the amount reported in standard coincubation procedures. This approach may be particularly useful when small amounts of tumor material are available.

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.

Microtubules play critical roles in a variety of cell processes, including mitosis, organelle transport, adhesion and migration, and the maintenance of cell polarity [1–3]. Microtubule-associated proteins (MAPs) regulate the dynamic organization and stability of microtubules, often through either cell-specific or cell division stage-specific interactions [4, 5]. To identify novel cytoskeletal-associated proteins and peptides that regulate microtubules and other cytoskeletal and adhesive structures, we have developed a GFP cDNA screening strategy based on identifying gene products that localize to these structures. Using this approach, we have identified a novel MAP, GLFND, that shows homology to the Opitz syndrome gene product [6], localizes to a subpopulation of microtubules that are acetylated, and protects microtubules from depolymerization with nocodazole. Expression of an N-terminal deletion binds microtubules but alters their organization. During the cell cycle, GLFND dissociates from microtubules at the beginning of mitosis and then reassociates at cytokinesis. Furthermore, ectopic expression of GLFND inhibits cell division and cytokinesis in CHO cells. These observations make GLFND unique among MAPs characterized thus far.

Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.

Pancreatic Ductal Adenocarcinoma (PDAC) is among the most malignant cancer, with 5-year survival of a mere 6%. Chronic inflammation is associated with an increased risk of PDAC. Nitric oxide (NO•) is an important mediator of immune and inflammatory responses and plays a role in tumorigenesis. The principal source of an increased and sustained level of NO• is inducible nitric oxide synthase (NOS2). In this study, we have tested the hypothesis that NO• enhances tumor progression in pancreatic cancer. We found that a higher NOS2 expression in tumors was associated with poor survival in resected PDAC patients (p=0.011). We then used a genetic strategy to investigate the role of NO• in the development and progression of PDAC by deleting NOS2 gene in a genetically engineered mouse model of pancreatic cancer (KPC mice) with pancreas specific activation of mutant KRAS and P53, which faithfully recapitulates the development and progression of human pancreatic cancer. NOS2-deficient KPC mice showed a longer survival as compared to the KPC littermates with wild type NOS2 (p Citation Format: Jian Wang, Peijun He, Matthias M. Gaida, Shouhui Yang, Aaron Schetter, Jochen Gaedcke, Michael Ghadimi, Thomas Ried, Harris G. Yfantis, Dong H. Lee, Jonathan M. Weiss, Jim Stauffer, Nader Hanna, H. Richard Alexander, S. Perwez Hussain. Nitric oxide enhances tumor progression and disease aggressiveness in pancreatic cancer. [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 926. doi:10.1158/1538-7445.AM2014-926

Ninety-seven healthy male and female subjects, 18–70 years of age, entered a randomized, double-blind, placebo-controlled, multicenter study conducted over a 24-week treatment period and a 72-week follow-up (study week 96) period without treatment. Subjects received: (1) terbinafine 250 mg for 12 weeks followed by placebo for 12 weeks; (2) terbinafine 250 mg for 24 weeks; or (3) placebo for 24 weeks. Clinical and mycologic evaluations of a target toenail were performed before, during, and after the treatment period. Evaluations consisted of measurements of nail growth, unaffected nail length, and estimated percentage of nail involvement. Cultures and KOH microscopy were used for mycologic evaluations. The primary efficacy variable was effective treatment, defined as negative mycology and either no nail involvement or at least 5 mm of new unaffected nail growth at 48 weeks. Complete cure, the secondary variable, was defined as negative mycology and zero nail involvement at 48 weeks. Overall efficacy was assessed after 12, 18, and 24 weeks of therapy and every 6 weeks up to week 48 by both investigator and subject using a five-point scale (excellent, very good, good, fair, and poor). Adverse events were assessed after 4, 8, 12, 18, and 24 weeks of treatment and after a 6-week follow-up period with no treatment. Adverse events were graded by severity (mild, moderate, or severe) and relationship to the study drug (not related, remote or unlikely, possibly, probably, or definitely).

The expression of chemokines, including monocyte chemoattractant protein (MCP)-1, by many cell types contributes to the pathogenesis of inflammatory diseases. We examined MCP-1 expression in human umbilical vein endothelial cells (EC) following cytokine treatment. We specifically compared the effect of TGF-beta 1 on this cytokine-induced expression, as TGF-beta has been shown to have immunosuppressive effects on EC. EC expressed MCP-1 mRNA and protein in response to TNF alpha, IFN gamma or IL-1beta, but not TGF-beta1. TGF-beta1 in cotreatment with either TNF alpha or IL-1beta, but not IFN gamma, significantly decreased MCP-1 mRNA and protein expression, as compared to TNF alpha or IL-1beta treatment alone. Pretreatment with TGF-beta had no effect on any cytokine-induced MCP-1 expression. TGF-beta had no effect on MCP-mRNA stability. Examination of TNF receptor expression by flow cytometry revealed that TNF alpha treatment caused a decrease of p75 expression on the cell surface. The p55 receptor was not detected at the cell surface, but was localized intracellularly by confocal microscopy. Treatment of EC with TGF-beta alone decreased p75 surface expression and in cotreatment with TNF alpha, caused an additive decrease in p75 surface expression, as compared to TNF alpha treatment alone. Whereas mRNA expression for both receptors was increased with TNF alpha treatment, this was decreased with TGF-beta/TNF alpha cotreatment, as compared to TNF alpha treatment alone. Thus, the expression of TNF receptors was also down-modulated by TGF-beta. These findings indicate additional mechanisms by which TGF-beta exerts immunosuppressive properties on EC.

The pathophysiology of central nervous system (CNS) inflammatory disease is dependent, in part, on leukocyte recruitment across the blood-brain barrier. The expression of cytokines and chemokines by astrocytes may contribute to this process. Astrocytes express monocyte chemoattractant protein-1 (MCP-1), an activator of monocytes and a chemoattractant for monocytes and activated T cells. We examined the regulation of MCP-1 expression in human fetal astrocytes following cytokine treatment in the presence and absence of transforming growth factor beta (TGF-beta). TGF-beta, TNFalpha and IL-1beta, but not IFNgamma, induced MCP-1 mRNA and protein. TGF-beta, in cotreatment with TNFalpha caused an additive increase in MCP-1 mRNA, but not protein. In combination with IFNgamma, TGF-beta significantly increased MCP-1 mRNA and protein, as compared to either untreated, TGF-beta- or IFNgamma-treated astrocytes. However, TGF-gamma in cotreatment with IL-1beta decreased MCP-1 mRNA and protein, as compared to IL-1beta alone. Treatment of astrocytes with TGF-beta prior to TNFalpha, IFNgamma or IL-1beta treatment significantly increased MCP-1 expression. The kinetics of cytokine expression in the CNS may differentially regulate astrocyte-derived MCP-1 expression and subsequent recruitment and activation of leukocytes.

Meeting abstracts To investigate the relationship between tumor-associated CD40 expression, RCC patient survival and tumor stage. The expression of CD40, TUNEL and CD8 in human renal cell carcinomas was analyzed by immunohistochemistry. Computer-assisted quantitation of protein expression was used

The liver is an immunologically unique organ containing tolerogenic dendritic cells (DC) that maintain an immunosuppressive microenvironment. Although systemic IL-12 administration can improve responses to tumors, the effects of IL-12-based treatments on DC, in particular hepatic DC, remain incompletely understood. In this study, we demonstrate systemic IL-12 administration induces a 2–3 fold increase in conventional, but not plasmacytoid, DC subsets in the liver. Following IL-12 administration, hepatic DC became more phenotypically and functionally mature, resembling the function of splenic DC, but differed as compared to their splenic counterparts in the production of IL-12 following co-stimulation with toll-like receptor (TLR) agonists. Hepatic DCs from IL-12 treated mice acquired enhanced T cell proliferative capabilities similar to levels observed using splenic DCs. Furthermore, IL-12 administration preferentially increased hepatic T cell activation and IFNγ expression in the RENCA mouse model of renal cell carcinoma. Collectively, the data shows systemic IL-12 administration enables hepatic DCs to overcome at least some aspects of the inherently suppressive milieu of the hepatic environment that could have important implications for the design of IL-12-based immunotherapeutic strategies targeting hepatic malignancies and infections.

Mutant KRAS in lung cancers induce molecular pathways that regulate cellular proliferation, survival and inflammation, which enhance tumorigenesis. Inducible nitric oxide synthese (NOS2) up-regulation and sustained nitric oxide (NO•) generation are induced during the inflammatory response and correlate positively with lung tumorigenesis. To explore the mechanistic contribution of NOS2 to KRAS-induced lung tumorigenesis and inflammation, we used a genetic strategy of crossing NOS2 knockout (NOS2KO) C57BL6 inbred mice with a KRASG12D-driven mouse lung cancer model. KRASG12D/NOS2KO mice exhibited delayed lung tumorigenesis, resulting in a longer overall survival time compared with that of KRASG12D/NOS2WT (wildtype) controls. Correspondingly, tumors in KRASG12D/NOS2KO mice had reduced tumor cell proliferation in adenomas and carcinomas. NOS2-deficiency also led to dramatically suppressed inflammatory response by attenuation of macrophage recruitment into alveoli and within tumor foci. In contrast, FOXP3+ regulatory T cells were increased in tumors from KRASG12D/NOS2KO mice. We further analyzed the expression of microRNA-21 (miR-21), an oncogenic non-coding RNA involved in oncogenic Ras signaling, by quantitative reverse transcription PCR and in situ hybridization. Lung carcinomas dissected from KRASG12D/NOS2KO mice showed a significantly reduced miR-21 expression along with decreased tumor cell proliferation, suggesting that NOS2-deficiency could attenuate RAS signaling pathways that transactivate miR-21 expression. Therefore, deletion of NOS2 decreases lung tumor growth as well as inflammatory responses initiated by oncogenic KRAS, suggesting that both KRAS and NOS2 cooperate in driving lung tumorigenesis and inflammation. Inhibition of NOS2 may have a therapeutic value in lung cancers with oncogenic KRAS mutations.

A computational model is employed to study the reacting flow within the engine and near-field plumes of several small gaseous hydrogen-oxygen thrusters. The model solves the full Navier-Stokes equations coupled with species diffusion equations for a hydrogen-oxygen reaction kinetics system and includes a two-equation q-omega model for turbulence. Predictions of global performance parameters and localized flowfield variables are compared with experimental data in order to assess the accuracy with which these flowfields are modeled and to identify aspects of the model which require improvement. Predicted axial and radial velocities 3 mm downstream of the exit plane show reasonable agreement with the measurements. The predicted peak in axial velocity in the hydrogen film coolant along the nozzle wall shows the best agreement; however, predictions within the core region are roughly 15 percent below measured values, indicating an underprediction of the extent to which the hydrogen diffuses and mixes with the core flow. There is evidence that this is due to three-dimensional mixing processes which are not included in the axisymmetric model.

mTOR signaling, which plays a central role in controlling cell cell proliferation, survival, mobility and angiogenesis, is considered an important target for new anticancer drugs development. The involvement of the CD40/CD154 signaling pathway has also been reported to play an important role in regulating anti-tumor immune responses. In this study, using a first-in-class orally bioavailable mTOR kinase inhibitor, AZD8055, which, unlike rapamycin, can inhibit both mTORC1 and mTORC2, we evaluated the anti-tumor efficacy of mTOR kinase inhibitor treatment alone in a murine model of metastatic renal cell carcinoma (Renca) and compared biological responses achieved by administering AZD8055 as a single agent to those obtained in combination with agonistic anti-CD40 antibody. In vitro survival assays showed that Renca cell apoptosis could be induced by AZD8055. In vivo, although both agents administered alone had some anti-tumor efficacy, the combination of AZD8055 and CD40 Ab induced a significant anti-tumor response in both intra-renal and intra-splenic inoculations model of renal cell carcinoma, as compared to single agent treatments. The combination treatment, also resulted in an increase and activation of tumor- and liver-associated macrophages, NK cells and CD8 T cells in vivo. AZD8055/anti-CD40 treatment also increased the level of systemic Th1 cytokines, including IL-12, IFN-γ, TNFα. IFN-γ production by CD8+ T cells and TNFα production by macrophages were also found to be elevated in the AZD8055/anti-CD40 combination treated-group, compared with any single treatment group. Furthermore, the expression of Th1-associated chemokines, RANTES and IP10, were significantly induced in the combination treated-group. These data suggest that the combination of mTOR kinase targeting agents with immunotherapy could be an area of further development in renal cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5596.

Using an orthotopic mouse model of renal cell carcinoma, we showed previously that IL-2/αCD40 combination immunotherapy resulted in synergistic anti-tumor responses. In contrast, the use of either IL-2 or αCD40 as single agents mediated only partial, transient anti-tumor effects. We now show that treatment of tumor-bearing mice with IL-2/αCD40, but not either IL-2 or αCD40 alone, induces significant nitric oxide synthase (NOS2) expression within the tumor microenvironment through an interferon gamma-dependent pathway. In saline-control treated mice (low NO levels), NO inhibition reduced lung metastases by itself. However, in the context of immunotherapy (high NO levels), NOS2 inhibition completely abrogated the ability of IL-2/αCD40 treatment to reduce lung metastases but had no effect on the ability of IL-2/αCD40 to reduce primary kidney tumor burden. We further show that IL-2/αCD40 induces the NO-dependent decrease in matrix metalloproteinase (MMP) expression and activity, concomitant with an increase in the expression of tissue-inhibitor of metalloproteinase (TIMP)-1 within the tumor microenvironment. Finally, the treatment of tumor-bearing mice with the NO donor, JS-K significantly reduced lung metastases but had no effect on primary tumor size. These data distinguish the primary anti-tumor effects of IL-2/αCD40 immunotherapy, which are independent of NO expression, from the important ability of IL-2/αCD40 to inhibit lung metastases through the NO-dependent regulation of MMP and TIMP functions. Furthermore, the reduced MMP9 activity may be indicative of M1 polarized macrophages within the tumor microenvironment after IL-2/αCD40 treatment. Our data demonstrate the mechanistic basis for the IL-2/αCD40-mediated control of lung metastases and indicate that the context-dependent application of NO-inducing agents may hold considerable promise for the treatment of metastastic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5594.

Validation of Computational Fluid Dynamics (CFD) codes developed for prediction and evaluation of rocket performance is hampered by a lack of experimental data. Non-intrusive laser based diagnostics are needed to provide spatially and temporally resolved gas dynamic and fluid dynamic measurements. This paper reports the first non-intrusive temperature and species measurements in the plume of a 110 N gaseous hydrogen/oxygen thruster at and below ambient pressures, obtained with spontaneous Raman spectroscopy. Measurements at 10 mm downstream of the exit plane are compared with predictions from a numerical solution of the axisymmetric Navier-Stokes and species transport equations with chemical kinetics, which fully model the combustor-nozzle-plume flowfield. The experimentally determined oxygen number density at the centerline at 10 mm downstream of the exit plane is four times that predicted by the model. The experimental number density data fall between those numerically predicted for the exit and 10 mm downstream planes in both magnitude and radial gradient. The predicted temperature levels are within 10 to 15 percent of measured values. Some of the discrepancies between experimental data and predictions result from not modeling the three dimensional core flow injection mixing process, facility back pressure effects, and possible diffuser-thruster interactions.

A previous time-derivative preconditioning procedure for solving the Navier-Stokes is extended to the chemical species equations. The scheme is implemented using both the implicit ADI and the explicit Runge-Kutta algorithms. A new definition for time-step is proposed to enable grid-independent convergence. Several examples of both reacting and non-reacting propulsion-related flowfields are considered. In all cases, convergence that is superior to conventional methods is demonstrated. Accuracy is verified using the example of a backward facing step. These results demonstrate that preconditioning can enhance the capability of density-based methods over a wide range of Mach and Reynolds numbers.

A Doppler-resolved laser Rayleigh scattering diagnostic was applied to a 111 N thrust, regenerative and fuel-film cooled, gaseous hydrogen/gaseous oxygen rocket engine. The axial and radial mean gas velocities were measured from the net Doppler shifts observed for two different scattering angles. Translational temperatures and number densities were estimated from the Doppler widths and scattered intensities, respectively, by assuming that water was the dominant scattering species in the exhaust. The experimental results are compared with theoretical predictions from a full Navier-Stokes code (RD/RPLUS) and the JANNAF Two-Dimensional Kinetics (TDK) and Standardized Plume Flowfield (SPF-II) codes. Discrepancies between the measured and predicted axial velocities, temperatures, and number densities are evident. Radial velocity measurements, however, show excellent agreement with predictions. The discrepancies are attributed primarily to inefficient mixing and combustion caused by the injection of excessive oxidizer along one side of the thrust chamber. Thrust and mass flow rate estimates obtained from the Rayleigh measurements show excellent agreement with the globally measured values.

Natural Killer (NK) cells are lymphocytes of the innate immune system that are able to kill a variety of tumors and pathogen-infected cells without prior sensitization. NK cells are the first lymphoid cells to repopulate following bone marrow transplantation (BMT). The present study shows that murine NK cells developing in vivo after BMT are phenotypically and functionally distinguishable from mature adult NK cells. Using a congeneic BMT mouse model (CD45.1/H-2b → C57BL/6, CD45.2/H-2b) we demonstrated here that donor derived BM-NK progenitors (CD122+NK1.1-CD3-)were present in the BM and periphery (spleen), at day 14 after BMT, and developed into NK cells expressing high levels of CD122, NKG2D, CD94, DX5 and CD43. However, surface expression of Ly49 inhibitory receptors was altered with Ly49C/I being under-expressed (9.8 %± 3.2 %), and Ly49G2 being highly expressed (62 %± 3.1 %) when compared to NK cells from non-transplanted adult mice (Ly49C/I, 43.1 %± 7.6 % and Ly49G2, 44.3 %± 13.5 %). At 28 days after BMT, there was a normalization with regard to Ly49 phenotype. The developing NK cells manifested functional deficits compared to non-transplanted adult NK, even when stimulated in vitro with Interleukin (IL)-2, as evidenced by decreased interferongamma (IFN-g) production and cytolytic activity. In order to examine the effects of major histocompatibility complex (MHC) molecules on NK development independently of allelic variation of Ly49s, we performed syngeneic BMT using B10D2 mice (H-2d). We observed the same pattern of Ly49s expression during NK development demonstrating that the acquisition of these receptors early after BMT is not determined by the MHC haplotype. Because IL-15 plays a crucial role in NK cell development, we administrated human (h) IL-15, via hydrodynamic injection of the respective gene, at day 11 after BMT, to accelerate NK maturation. This method resulted in a high but transient production of hIL-15 (736 ± 41 pg/ml) in the serum for up to 2 weeks. A subsequent but temporary increase in the numbers of donor derived NK cells (3 fold) occurred within 72 hours, in both BM and periphery, following hIL-15 gene delivery. Despite these augmenting effects, the developing NK cells still exhibited poor functionality compared to non-transplanted adult mice. These studies describe the possibility of NK development in the spleen, and indicate that normal surface expression of Ly49C/I and Ly49G2 early after BMT is time dependent, but MHC independent. Moreover, administration of hIL-15 is not sufficient to induce NK functional maturation early after BMT despite increased NK numbers.

IL-18 + IL-12 treatment elicited significant antitumor activity in the liver via an increase in NK cells and a concurrent decrease in the detectable number of NKT cells. To determine both the fate of NKT cells and the origins of NK influx into the liver following IL-18 + IL-12 treatment, we optimized liver leukocyte isolation techniques and determined that IL-18 + IL-12 was not causing NKT cells to down-modulate their TCR, as was previously reported with anti-CD3 or α-GalCer activation. Also, no discernable apoptosis was detected at 6 or 24 hours. However, IL-18 + IL-12 treatment caused an increase in NKT cells in the spleen that coincided with a decrease in these cells in the liver. We also observed a decrease in NK cells in the spleen that coincided with an increase of these cells in the liver. These findings suggest that IL-18 + IL-12 induces a well coordinated but opposing relocation of selected subsets. To identify potential genes involved in cell migration, we used a chemokine array and identified the differential gene expression in the spleen and liver. In the spleen, we found that IL-18 + IL-12 induced CCL19 in the spleen which is the ligand for CCR7 on NKT cells. In contrast, in the liver this treatment induced CCL9 and CCL20 which are respective ligands for CCR1 and CCR6 on NK cells. These data delineate potentially novel, redistribution mechanisms whereby proinflammatory cytokines may modulate the liver leukocyte repertoire.

Several immunotherapeutic models have shown that interferon gamma (IFNγ) is required for tumor regression. Treatment with IFNγ as a sole therapy, however, has resulted in disappointing outcomes. Cytokine therapies, such as IL-2/IL-12 and IL-12/IL-18, each involves IFN-γ mediated reduction in primary tumors but they can have an unanticipated increase in secondary metastases. Therefore, tumor responsiveness to IFNγ may alter its progression and metastasic potential. Mouse renal carcinoma cells expressing a dominant negative IFNγ-receptor had a reduced ability to metastasize, suggesting that IFNγ may exert potentially pro-metastatic effects in the tumor microenvironment. The goal of this study is to identify changes in gene expression in the tumor microenvironment in response to IFNγ treatment. Using qPCR gene profiling, we identified the increased expression of several pro-metastatic genes specifically in the tumor microenvironment after IFNγ treatment, namely heparanase, CD44, s100a4, and matrix metalloproteinase 7. We are now investigating whether the upregulation of these genes is caused by host and/or tumor responses to IFNγ. Our results suggest that IFNγ regulates extra-cellular matrix remodeling protein expression in the local microenvironment, which may contribute to tumor metastases.

Top of pageAbstract There is considerable interest in the use of genetically modified activated or malignant B cells as antigen presenting cells for immunotherapy applications. However, these cells are difficult targets for gene transfer by both viral and non-viral methodologies. Under optimal conditions, we previously demonstrated approximately 50% transfection efficiency by electroporating chronic lymphocytic leukemia (CLL) patients' malignant B cells with DNA plasmid encoding the eGFP marker gene. We next investigated whether transgene expression could be further increased by transfecting mRNA reasoning that in vitro transcribed RNA can be translated simultaneously when it is delivered to the cytosol. Here, we report that transfecting CLL-B cells with mRNA resulted in superior transgene expression. We first electroporated CLL -B cells with 5′-end capped mRNA encoding for the marker gene, eGFP, which was obtained by in vitro transcription of the full length cDNA (on pCI backbone) with a commercially available T7 polymerase kit (Ambion). The transfected cells were analyzed by FACS for transfection efficiency measuring eGFP expression and for cell viability by PI exclusion. Data showed that both cell viability and transfection efficiency were 90% at 3 hr post transfection. CLL patients' malignant B are ineffective antigen-presenting cells, due to the decreased cell surface expression of the co-stimulatory molecules needed to activate T cells after the engagement of T-cell receptor. Forced expression of hCD40L has been shown to up-regulate the expression of co-stimulatory and adhesion molecules and render them to be more efficient antigen-presenting cells, however, its expression has been difficult to achieve in human CLL-B cells. Full length hCD40L mRNA was obtained by the same procedure as described for eGFP and electroporated into CLL-B cells. The transfected cells were immunostained using a FITC-conjugated monoclonal antibody to hCD40L (BD Pharmingen) and analyzed by FACS. The cell surface expression of hCD40L was achieved in greater than 50% of the CLL-B cells as early as 2 hrs post transfection and persisted for at least 48 hrs. Cell viability, when normalized against the control cells was 90%. FACS analysis of the co-stimulatory molecules revealed that hCD40L expression correlated with an up-regulation of both CD80/CD86. Significant up-regulation of CD86 was detected as early as 2 – 4 hr post transfection. The mean fluorescence intensity of CD86 expression was increased approximately 10 fold vs. control cells. mRNA transfection eliminates the concern of genomic modification, gives rise to superior gene expression, safety and cell viability, and offers significant advantages over other gene transfer methodology. Transient gene expression by mRNA transfection should be sufficient and possibly preferable for both in vivo and ex vivo (CTL generation, adoptive) immunotherapy. A scalable, electroporation based system has been developed which will be a useful tool for clinical applications that require large volume mRNA transfection.

Ce que trouve J. Poulin chez Hemingway, c'est une ethique de l'ecriture implicant la liberation d'une partie de soi-meme, du cote androgyne qui se cache derriere l'agressivite masculine des protagonistes. Inscription du texte hemingwayen du Jardin d'Eden dans Le Vieux Chagrin

Immune deficiency immediately following bone marrow transplantation (BMT) increases susceptibility to opportunistic infections as well as tumor relapse. Natural Killer (NK) cells play important roles in the resistance to virally infected and transformed cells. Interleukin (IL)-15 has been shown to be essential for NK cell development and survival. We administered human (h) IL-15 cDNA (pIL-15) via hydrodynamic delivery to murine recipients undergoing congenic BMT to determine its effects on NK cell reconstitution. Hydrodynamic pIL-15 delivery resulted in high levels of hIL-15 protein in the serum that lasted for several days and then quickly declined. The appearance of hIL-15 was followed by a significant increase of mature donor-derived NK cells within the bone marrow, spleens, and livers of the treated recipients. No accumulation of immature NK cell progenitors was observed. The NK cells from IL-15-treated recipients displayed an activated phenotype and were lytically active toward tumor targets in vitro to a similar degree as did those cells from recipients treated with control plasmid. This suggests that the predominant effect of IL-15 was a quantitative increase in total NK cell numbers and not qualitative changes in NK cell functions. No toxicities or adverse effects were observed. Studies performed in transplanted mice bearing renal carcinoma tumors demonstrated that this mode of hIL-15 gene delivery resulted in increased antitumor responses. These results support the use of cytokine gene transfer-based regimens as a platform to augment NK cell recovery after BMT.

Roman du voyage, roman de la quete, " W. B. " est surtout une aventure litteraire, une exploration du continent americain a travers ses textes. Filiation entre ce roman et " Travels with Charley " de Steinbeck et " De quoi t'ennuies-tu, Eveline " de G. Roy