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Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I(−), MIC-A(+)/B(+) K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

During daily fishing operations, spanner crab Ranina ranina catch rates can fluctuate substantially, but the environmental drivers responsible for these fluctuations largely remain unresolved. Earlier research suggests that spanner crab catchability increases with strengthening currents, but uncertainties surround the magnitude of the measured current speeds and, consequently, their relationship with catch rates. Here, we explore the effects of bottom currents on spanner crab catch rates in South East Queensland, Australia. Using generalized additive mixed modeling, our results indicated that strengthening current speeds increased catch rates until reaching approximately 0.15 m/s, at which point the catch rates began to gradually decline. Results from a general linear regression model also showed that between fishing periods carried out on the same day, catch rates increased or decreased concurrently with current speeds. We conclude that bottom current speed should be considered in future stock assessment models. Better understanding the processes responsible for changes in bottom current speed will enable more accurate estimates of spanner crab population densities in the Australian fishery and will benefit the economic efficiency of commercial crabbing operations. Furthermore, future studies that investigate the effects of current speed on catch rates for other crab species should consider differences in locomotory characteristics and how they may impact the foraging efficiency of crabs under different flow conditions.

Species catchability is an important parameter used to help optimise stock assessment modelling and the economic efficiency of commercial fishing operations. Previous studies have shown several physical oceanographic parameters, including ambient temperature, waves and currents, affect the catchability of spanner crabs (Ranina ranina) throughout the Indo-Pacific. Most notably in the Australian fishery, where oceanographic processes vary over space and time, a positive relationship between bottom boundary layer temperature (BBLT) and catch rates was observed. Here, we aimed to better understand how localised oceanographic processes affected this relationship in the southernmost South-East Queensland (SEQ) sector of the Australian fishery at seasonal and short temporal scales. Our results show cooler BBLT, upwelling-favourable alongshore wind stress and increased catch rates occurred during mating season in austral spring. At the end of austral summer, BBLT began warming, downwelling-favourable winds were dominant, and catch rates declined around the post-moult period. Outputs from the generalised linear models (GLMs) that separated these effects in each season show that, at shorter temporal scales, daily catch rates also increased with episodic BBLT cooling and upwelling-favourable alongshore wind stress, but only during austral autumn and winter. These new findings suggest that region-specific, short-term and seasonal variability of oceanographic processes responsible for changes in BBLT play an important role in influencing the catchability of spanner crabs. We suggest that the effects of region-specific physical oceanographic processes must be considered in future work when investigating the catchability of commercially important fisheries species fished over large spatial domains.

This phase I trial reports the safety and activity of BPX101, a second-generation antigen-targeted autologous antigen presenting cell (APC) vaccine in men with metastatic castration-resistant prostate cancer (mCRPC). To manufacture BPX101, APCs collected in a single leukapheresis were transduced with adenoviral vector Ad5f35 encoding inducible human (ih)-CD40, followed by incubation with protein PA001, which contains the extracellular domain of human prostate-specific membrane antigen. The ih-CD40 represents a modified chimeric version of the dendritic cell (DC) co-stimulatory molecule, CD40, which responds to a bioinert membrane-permeable activating dimerizer drug, rimiducid (AP1903), permitting temporally controlled, lymphoid-localized, DC-specific activation. Eighteen men with progressive mCRPC following ≤1 prior chemotherapy regimen were enrolled to evaluate three doses of BPX101 (4 × 106, 12.5 × 106 and 25 × 106 cells) administered intradermally every 2–4 weeks followed by rimiducid (0.4 mg/kg) intravenous (IV) infusion 24 h after each BPX101 dose. There were no dose-limiting toxicities. Immune upregulation as well as anti-tumor activity was observed with PSA declines, objective tumor regressions and robust efficacy of post-trial therapy. This novel antigen-targeted and in vivo activated immunotherapy platform may warrant further development as monotherapy and as a component of rational combinations.

This review examines the influence of physical oceanographic processes on catchability of spanner crab (Ranina ranina) in northeast Australia. Physical oceanographic processes may affect crab catchability by influencing their activity levels and ability to detect bait. Bottom temperature, current velocity, and swell intensity appear to influence catches of spanner crab. At this stage, it appears warmer temperatures enhance catchability of spanner crab. Spanner crabs were more catchable in stronger currents, and crabs were observed to arrive from down-current of baited traps. However, a decline in catch was observed following periods of intense swell. Data derived from Waverider buoys suggest that occasionally these periods create strong wave-induced seabed current velocities, lead to at depths of 70 m. The oscillatory motion of wave-induced seabed velocities may cause higher suspended sediment concentrations. These observations corroborate the views of local fishermen that spanner crabs avoid ‘murky’ water. The effect of turbidity on catchability requires further research. Overall, we advocate that studies employ robust methodologies to measure physical oceanographic processes to accurately predict catchability. Moreover, large-scale physical oceanographic processes may also play an important role in catchability of spanner crab; including upwelling, eddies, and the East Australian Current. Integrating physical oceanography and fisheries interactions will considerably benefit commercial fishermen as well as provide valuable information for evidence-based management of these valuable resources.

Hydrodynamic dispersion has a significant impact on the mass transport of sediments and contaminants within coastal waters. In this study apparent horizontal dispersion in a tidally-dominated shallow estuary was investigated using field observations and a numerical model. A cluster of four Lagrangian drifters was released in two shallow regions inside Moreton Bay, Australia: between two small islands and in an open water area. During a 16-h tracking period, the drifters generally showed similar behaviour, initially moving with the dominant current and remaining together before spreading apart at the change of tide. Two dispersion regimes were identified, a slow dispersion during the earlier stage and a rapid dispersion during the latter stage of deployment. Such change in regime typically occurred during the succeeding ebb or flow tides, which may be attributable to residual eddies breaking down during reversal of tidal direction. In addition, a power function of the squared separation distance over the apparent dispersion coefficient produced an R2 exceeding 0.7, indicating a significant relationship between them. By applying a three-dimensional hydrodynamic model, the trajectories of artificial particles spreading in the bay were simulated, which allowed the calculation of dispersion coefficients throughout the entire bay. The study results demonstrate that the tidal effects on dispersion were dependent on the effect of tidal excursion and residual current. The tide was found to be the most dominant driver of dispersion in the bay when unobstructed by land; however, bathymetric and shoreline characteristics were also significant localised drivers of dispersion between the two islands as a result of island wake.

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892.

Spencer, D., Lemckert, C.J., Yu, Y., Gustafson, J., Lee, S.Y., Zhang, H., 2014. Quantifying Dispersion in an Estuary: A Lagrangian Drifter Approach. In: Green, A.N. and Cooper, J.A.G. (eds.), Proceedings 13th International Coastal Symposium (Durban, South Africa), Journal of Coastal Research, Special Issue No. 70, pp. 029–034, ISSN 0749-0208. Describing the physical drivers of water bodies plays an integral role in the marine environment. Lagrangian drifters are used to describe hydrodynamics, where tracking groups (clusters) of drifting floats is performed to determine the dispersion behaviour in the water. Using two types of Lagrangian drifters released in Moreton Bay, Australia, the dispersion behaviour is determined over the duration of a semi-diurnal spring tide cycle. A Self-Locating Datum Marker Buoy (SLDMB) was designed as the first drifter type. The second drifter (Small Drifter) was designed to be approximately half the size of the SLDMB. SLDMB and Small Drifter clusters were deployed f...

In the Australian spanner crab (Ranina ranina) fishery, management and industry are looking for improvements to the existing indicators of stock abundance. Prior research linked several oceanographic indices to the catchability of spanner crabs; however, it was unclear whether nearshore (e.g. river-runoff) or region-specific oceanographic features (e.g. eddies and the East Australian Current) are responsible for these effects on catch rates. Using satellite remote sensing and fishery-independent survey data, we analysed the influence of oceanographic and environmental indices on spanner crab catch rates in southern Queensland. Outputs from Generalised Additive Models (GAM) show that catch rates exhibit a large amount of variability between different regions of the fishery, with highest catch rates at fishing grounds within 40 km from the shelf break. Offshore oceanic waters, transported into various regions by different oceanographic processes, were linked to an increase in catch rates. Lower concentrations of surface chlorophyll a were also correlated with higher catch rates, but only in survey regions exposed to the effects of the Fraser Gyre and at the mouth of bays. Overall, results highlighted that the effects of environmental indices on catch rates were not homogeneous across the fishery. Rather, relationships were linked to region-specific (

The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlie the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/β-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found to be coactivated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible β-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-β (TGF-β) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-β signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1–WNT–TGF-β signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets. Cancer Res; 74(2); 609–20. ©2013 AACR.

Recent modest successes in ex vivo dendritic cell (DC) immunotherapy have motivated continued innovation in the area of DC manipulation and activation. Although ex vivo vaccine approaches continue to be proving grounds for new DC manipulation techniques, the intrinsic limits of ex vivo therapy, including high cost, minimal standardization, cumbersome delivery, and poor accessibility, incentivizes the development of vaccines compatible with in vivo DC targeting. We describe here a method to co-deliver both tumor-specific antigen (TSA) and an iMyD88/CD40 adjuvant (iMC), to DCs that combines toll-like receptor (TLR) and CD40 signaling. In this study, we demonstrate that simple TSA delivery via adenoviral vectors results in strong antitumor immunity. Addition of iMC delivered in a separate vector is insufficient to enhance this effect. However, when delivered simultaneously with TSA in a single bicistronic vector (BV), iMC is able to significantly enhance antigen-specific cytotoxic T-cell (CTL) responses and inhibit established tumor growth. This study demonstrates the spatial-temporal importance of concurrent DC activation and TSA presentation. Further, it demonstrates the feasibility of in vivo molecular enhancement of DCs necessary for effective antitumor immune responses.

Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct.We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch.Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence.The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.).

The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c-Cbl--known for its roles in regulating lymphocyte signalling--as a modulator of dendritic cell activation. In c-Cbl-deficient dendritic cells, Toll-like receptor-induced expression of proinflammatory factors, such as interleukin-12, is increased, correlating with a greater potency of dendritic-cell-based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)-κB activity. In addition, c-Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF-κB. Our data indicate that c-Cbl has a crucial, RING-domain-dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50.

Adoptive transfer of antigen-specific cytotoxic T lymphocytes has shown promise for the therapy of cancer. However, tumor-specific T cells are susceptible to diverse inhibitory signals from the tumor microenvironment. The Akt/protein kinase B plays a central role in T-cell proliferation, function, and survival and we hypothesized that expression of constitutively active Akt (caAkt) in T cells could provide resistance to many of these tumor-associated inhibitory mechanisms. caAkt expression in activated human T cells increased proliferation and cytokine production, a likely result of their sustained expression of nuclear factor-κB (NF-κB) and provided resistance to apoptosis by upregulating antiapoptotic molecules. caAkt expressing T cells (caAkt-T-cells) were also relatively resistant to suppression by and conversion into regulatory T cells (Tregs). These characteristics provided a survival advantage to T cells cocultured with tumor cells in vitro; CD3/28-stimulated T cells expressing a chimeric antigen receptor (CAR) specific for disialoganglioside (GD2) that redirected their activity to the immunosuppressive, GD2-expressing neuroblastoma cell line, LAN-1, resisted tumor-induced apoptosis when co-expressing transgenic caAkt. In conclusion, caAkt-transduced T cells showed resistance to several evasion strategies employed by tumors and may therefore enhance the antitumor activity of adoptively transferred T lymphocytes.

SummaryFibroblast Growth Factor Receptor-1 (FGFR1) is commonly overexpressed in advanced prostate cancer (PCa). To investigate causality, we utilized an inducible FGFR1 (iFGFR1) prostate mouse model. Activation of iFGFR1 with chemical inducers of dimerization (CID) led to highly synchronous, step-wise progression to adenocarcinoma that is linked to an epithelial-to-mesenchymal transition (EMT). iFGFR1 inactivation by CID withdrawal led to full reversion of prostatic intraepithelial neoplasia, whereas PCa lesions became iFGFR1-independent. Gene expression profiling at distinct stages of tumor progression revealed an increase in EMT-associated Sox9 and changes in the Wnt signaling pathway, including Fzd4, which was validated in human PCa. The iFGFR1 model clearly implicates FGFR1 in PCa progression and demonstrates how CID-inducible models can help evaluate candidate molecules in tumor progression and maintenance.

Immunotherapy with vaccines represents a novel, targeted nontoxic modality for the therapy of prostate cancer. Systemic immune responses to candidate prostate cancer antigens have been induced in a tumor that has been conventionally viewed as refractory to immunotherapy. Autologous dendritic cell vaccines expressing prostate-specific antigens have shown promising clinical outcomes in a randomized trial in metastatic androgen independent prostate cancer. Other vaccine approaches include granulocyte-macrophage colony-stimulating factor modified tumor cell vaccines and poxvirus vaccines. A combination of vaccines with chemotherapy, radiotherapy, and other biologic agents is also being evaluated. Efforts to optimize vaccine approaches and select ideal patient populations need to continue because there is no evidence supporting a single superior approach.

The expression of fibroblast growth factor receptor (FGFR)-1 correlates with angiogenesis and is associated with prostate cancer (CaP) progression. To more precisely define the molecular mechanisms whereby FGFR1 causes angiogenesis in the prostate we exploited a transgenic mouse model, JOCK-1, in which activation of a conditional FGFR1 allele in the prostate epithelium caused rapid angiogenesis and progressive hyperplasia. By labeling the vasculature in vivo and applying a novel method to measure the vasculature in three dimensions, we were able to observe a significant increase in vascular volume 1 week after FGFR1 activation. Although vessel volume and branching both continued to increase throughout a 6-week period of FGFR1 activation, importantly, we discovered that continued activation of FGFR1 was not required to maintain the new vasculature. Exploring the molecular mediators of the angiogenic phenotype, we observed consistent upregulation of HIF-1alpha, vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2), whereas expression of Ang-1 was lost. Further analysis revealed that loss of Ang-1 expression occurred in the basal epithelium, whereas the increase in Ang-2 expression occurred in the luminal epithelium. Reporter assays confirmed that the Ang-2 promoter was regulated by FGFR1 signaling and a small molecule inhibitor of FGFR activity, PD173074, could abrogate this response. These findings establish a method to follow spontaneous angiogenesis in a conditional autochthonous system, implicate the angiopoietins as downstream effectors of FGFR1 activation in vivo, and suggest that therapies targeting FGFR1 could be used to inhibit neovascularization during initiation and progression of CaP.

Modest clinical outcomes of dendritic-cell (DC) vaccine trials call for the refinement of DC vaccine design. Although many potential antigens have been identified, development of methods to enhance antigen presentation by DCs has lagged. We have engineered a potent, drug-inducible CD40 (iCD40) receptor that permits temporally controlled, lymphoid-localized, DC-specific activation. iCD40 is comprised of a membrane-localized cytoplasmic domain of CD40 fused to drug-binding domains. This allows it to respond to a lipid-permeable, high-affinity dimerizer drug while circumventing ectodomain-dependent negative-feedback mechanisms. These modifications permit prolonged activation of iCD40-expressing DCs in vivo, resulting in more potent CD8(+) T-cell effector responses, including the eradication of previously established solid tumors, relative to activation of DCs ex vivo (P < 0.01), typical of most clinical DC protocols. In addition, iCD40-mediated DC activation exceeded that achieved by stimulating the full-length, endogenous CD40 receptor both in vitro and in vivo. Because iCD40 is insulated from the extracellular environment and can be activated within the context of an immunological synapse, iCD40-expressing DCs have a prolonged lifespan and should lead to more potent vaccines, perhaps even in immune-compromised patients.

Platelet-derived growth factor (PDGF) is a potent mitogen and chemotactic agent which may be involved in the formation of proliferative lesions of the arterial system, such as intimal hyperplasia and atherosclerosis. To examine the regional variation in vessel wall production of this mitogen, PDGF production and PDGF A chain mRNA expression by normal arterial wall was studied as a function of vessel location. PDGF production by canine aortic segments was measured after 72 h in organ culture, revealing significantly more PDGF produced by the distal compared to proximal aorta at 77 +/- 10 versus 14 +/- 6 pg/cm2/72 h (p0.05). Endothelial cells (EC) and smooth muscle cells (SMC), isolated from analogous aortic sites, were grown in tissue culture and the conditioned medium was assayed for PDGF. EC in vitro demonstrated a similar geographic trend in PDGF production (distal=1,501 +/- 389 pg/microgram DNA/72 h, proximal=759 +/- 230 pg/microgram DNA/72 h; p=0.17). PDGF production by SMC in cell culture had a similar pattern with cells from the distal aorta producing 58 +/- 28 pg PDGF/microgram DNA/72 h, compared to cells from the proximal aorta producing 37 +/- 15 pg PDGF/microgram DNA/72 h (p=0.13). Freshly harvested EC and SMC, isolated from the same aortic sites, were subjected to quantitation of PDGF mRNA levels using a coupled reverse transcriptase and polymerase chain reaction amplification method, with glyceraldehyde-phosphate dehydrogenase (GAPDH) as a control. The ratio of PDGF A chain:GAPDH mRNA was significantly greater in distal aortic SMC, 2.30 +/- 0.99, compared to proximal aortic SMC, 1.27 +/- 0.46 (p=0.05), but was not significantly different between proximal and distal aortic EC (p=0.86). These findings demonstrate significant regional differences in PDGF production in the normal canine aorta. Additionally, SMC are implicated as a significant contributor to the regional variation in PDGF production.

Previous studies have shown that Herpes Simplex Virus thymidine kinase (HSV-tk)/ganciclovir (GCV) comprised the most commonly used suicide gene therapy for prostate cancer, with modest results being obtained. However, novel suicide genes, such as Escherichia coli purine nucleoside phosphorylase (PNP), have been utilized to demonstrate more potent tumor killing and an enhanced bystander effect on local, non-expressing cells compared to HSV-tk.PNP/fludarabine (Fludara®; fludarabine phosphate; Berlex Labs, Richmond, CA, USA) was deliveried by prostate-specific, rat probasin-based promoter, ARR2PB. After infection of various cell lines with ADV.ARR(2) PB-PNP and administration of androgen analog, R1881, expression of PNP mRNA was detected; in vivo, the antitumor effect of the ARR(2) PB-PNP/Fludara system was monitored and analyzed, as well as animal survival.After in vitro infection with ADV.ARR(2) PB-PNP (multiplicity of infection = 10), LNCaP cells were more sensitive to a lower concentration Fludara (LD(50) , approximately 0.1 µg/ml) in the presence of R1881. Furthermore, robust bystander effects after R1881/Fludara treatment were observed in LNCaP cells after infection with bicistronic vector ADV.ARR2PB/PNP-IRES-EGFP in contrast to a much weaker effect in cells treated with ADV.CMV-HSV-tk/GCV. In vivo, tumor size in the ADV.ARR2PB-PNP/Fludara treatment group was dramatically smaller than in the control groups, and the mice treated with our system had a significantly prolonged survival, with three of eight mice surviving up to the 160-day termination point, as well as no systemic toxicity.The ARR(2) PB-PNP/Fludara system induced massive tumor cell death and a prolonged life span without systemic cytotoxicity; therefore, it might be a more attractive strategy for suicide gene therapy of prostate cancer.

Dendritic cells (DCs) initiate proinflammatory or regulatory T cell responses, depending on their activation state. Despite extensive knowledge of DC-activating signals, the understanding of DC inhibitory signals is relatively limited. We show that Src homology region 2 domain-containing phosphatase-1 (SHP-1) is an important inhibitor of DC signaling, targeting multiple activation pathways. Downstream of TLR4, SHP-1 showed increased interaction with several proteins including IL-1R–associated kinase-4, and modulated LPS signaling by inhibiting NF-κB, AP-1, ERK, and JNK activity, while enhancing p38 activity. In addition, SHP-1 inhibited prosurvival signaling through AKT activation. Furthermore, SHP-1 inhibited CCR7 protein expression. Inhibiting SHP-1 in DCs enhanced proinflammatory cytokines, IL-6, IL-12, and IL-1β production, promoted survival, and increased DC migration to draining lymph nodes. Administration of SHP-1–inhibited DCs in vivo induced expansion of Ag-specific cytotoxic T cells and inhibited Foxp3+ regulatory T cell induction, resulting in an enhanced immune response against pre-established mouse melanoma and prostate tumors. Taken together, these data demonstrate that SHP-1 is an intrinsic global regulator of DC function, controlling many facets of T cell-mediated immune responses.

Dimerization and oligomerization are general biological control mechanisms contributing to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. Cell permeable, synthetic ligands were devised that can be used to control the intracellular oligomerization of specific proteins. To demonstrate their utility, these ligands were used to induce intracellular oligomerization of cell surface receptors that lacked their transmembrane and extracellular regions but contained intracellular signaling domains. Addition of these ligands to cells in culture resulted in signal transmission and specific target gene activation. Monomeric forms of the ligands blocked the pathway. This method of ligand-regulated activation and termination of signaling pathways has the potential to be applied wherever precise control of a signal transduction pathway is desired.

Studying the effects of the loss of a specific cell type is a powerful approach in biology. Here we present a method based on the controlled activation of the apoptotic machinery. We expressed a modified caspase-3-containing chemical inducer of dimerization (CID)-binding sites in the livers of transgenic mice. In the absence of CID, no liver injury was detectable, underlining the absence of leakage in our system. In contrast, injection of the CID produced activation of the chimeric caspase-3, which led to a dose-dependent pure hepatocyte ablation with subsequent regeneration. This method is effective in both growing and nongrowing cells, and is therefore applicable to a wide range of cells and tissues. Moreover, because apoptosis has been described in numerous pathological circumstances, this system is useful for generating mouse models of human disorders as well as for studying the recovery or regeneration of tissues after cell loss.

Top of pageAbstract Dendritic cell (DC)-based vaccination is a promising strategy to induce cancer immunity. Nevertheless, clinical applications have been almost universally disappointing. One potential limitation of current strategies is the use of maturation cocktails (MC) that include PGE2, which upregulate chemotaxis to lymph nodes, but may inhibit the ultimate activation state of the resulting DC product. Herein, we describe a novel DC-based vaccine that circumvents the use of standard MC and instead utilizes a genetically engineered inducible CD40 (iCD40) receptor 1. Our platform contains human monocyte-derived DCs (MoDCs) transduced with adenovector Ad5/f35-ihCD40 (iCD40-MoDC) and incubated for 2 days in the presence of monophosphoryl lipid A (MPL) and dimerizing drug, AP20187. Here we show that MoDC transduction with Ad5/f35-iCD40 effectively enhances co- stimulatory and migratory functions, including upregulation of IL-6, activation marker CD83, chemokine receptor CCR7 and augmentation of chemotaxis to CCL19/MIP3|[beta]|. Furthermore, the migratory abilities of iCD40-MoDCs were comparable to MoDCs matured with standard maturation cocktail (i.e. TNF-|[alpha]|, IL-1|[beta]|, IL-6 and PGE2). Unlike murine bone marrow-derived DCs expressing iCD40, IL-12 production by human MoDCs required additional TLR-4-ligand agonists, such as LPS or clinical analog MPL, which led to high (ng) levels of IL-12p70 in ihCD40-MoDCs, but not MC-stimulated or control MoDCs. Moreover, iCD40 and TLR4 agonists additively enhanced activation marker expression. We further show that iCD40 and MPL enhanced the immunostimulatory functions of MoDC, such as improving the stimulation and tumor-specific killing activity of MAGE-3 tumor antigen-specific CTL (shown by IFN-|[gamma]| ELISPOT and 51Cr-release assays) and facilitated Th1 polarization of na|[iuml]|ve CD4+ T-helper cells in vitro. Moreover, we have developed a novel in vivo systemof tracking human DCs transduced with adenovirus expressing red-shifted luciferase. MPL-treated iCD40-MoDCs readily migrated to the lymph nodes of SCID mice and were detectable for over 1 week after inoculation. In addition, we appraised the systemic toxicity of the iCD40-DC vaccine in na|[iuml]|ve C57BL/6 male mice. Mouse DCs were pulsed with TRP2 melanoma antigen, transduced with Ad- iCD40 or control Ad-GFP and injected 3 times bi-weekly. Mice were monitored for 3 months and routine CBC and serum chemistry (for liver and kidney functions) was tested regularly. We detected no significant toxicity of the iCD40-DCs vaccine and highly significant long-lived elevation of tumor antigen specific CTLs. This data suggest the potency and safety of this immunotherapeutic approach that combines non-inflammatory TLR stimulation with synthetic activation of iCD40 in human MoDCs. Unlike standard DC maturation approaches, this combination of reagents leads to high IL-12p70 levels, enhanced chemotactic response to lymph node-derived chemokines and augmented tumor antigen-specific CTL and Th1 responses. Inducible CD40-enhanced DCs represent a promising new strategy for cancer immunotherapy.

Background Advances in fluorescence microscopy and mouse transgenesis have made it possible to image molecular events in living animals. However, the generation of transgenic mice is a lengthy process and intravital imaging requires specialized knowledge and equipment. Here, we report a rapid and undemanding intravital imaging method using generally available equipment. Results By DNA tattooing we transfect keratinocytes of living mice with DNA encoding fluorescent biosensors. Subsequently, the behavior of individual cells expressing these biosensors can be visualized within hours and using conventional microscopy equipment. Using this "instant transgenic" model in combination with a corrected coordinate system, we followed the in vivo behavior of individual cells expressing either FRET- or location-based biosensors for several days. The utility of this approach was demonstrated by assessment of in vivo caspase-3 activation upon induction of apoptosis. Conclusion This "instant skin transgenic" model can be used to follow the in vivo behavior of individual cells expressing either FRET- or location-based probes for several days after tattooing and provides a rapid and inexpensive method for intravital imaging in murine skin.

The use of a novel inducible FGF signalling system in the frog Xenopus laevis is reported. We show that the lipophilic, synthetic, dimerizing agent AP20187 is able to rapidly activate signalling through an ectopically expressed mutant form of FGFR1 (iFGFR1) in Xenopus embryos. iFGFR1 lacks an extracellular ligand binding domain and contains an AP20187 binding domain fused to the intracellular domain of mouse FGFR1. Induction of signalling by AP20187 is possible until at least early neurula stages, and we demonstrate that ectopically expressed iFGFR1 protein persists until late neurula stages. We show that activation of signalling through iFGFR1 can mimic a number of previously reported FGF activities, including mesoderm induction, repression of anterior development, and neural posteriorization. We show that competence to morphological posteriorization of the anteroposterior axis by FGF signalling only extends until about stage 10.5. We demonstrate that the competence of neural tissue to express the posterior markers Hoxa7 and Xcad3, in response to FGF signalling, is lost by the end of gastrula stages. We also show that activation of FGF signalling stimulates morphogenetic movements in neural tissue until at least the end of the gastrula stage.