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Clinical data elements (CDEs) (e.g., age, smoking history), blood markers and chest computed tomography (CT) structural features have been regarded as effective means for assessing lung cancer risk. These independent variables can provide complementary information and we hypothesize that combining them will improve the prediction accuracy. In practice, not all patients have all these variables available. In this paper, we propose a new network design, termed as multi-path multi-modal missing network (M3Net), to integrate the multi-modal data (i.e., CDEs, biomarker and CT image) considering missing modality with multiple paths neural network. Each path learns discriminative features of one modality, and different modalities are fused in a second stage for an integrated prediction. The network can be trained end-to-end with both medical image features and CDEs/biomarkers, or make a prediction with single modality. We evaluate M3Net with datasets including three sites from the Consortium for Molecular and Cellular Characterization of Screen-Detected Lesions (MCL) project. Our method is cross validated within a cohort of 1291 subjects (383 subjects with complete CDEs/biomarkers and CT images), and externally validated with a cohort of 99 subjects (99 with complete CDEs/biomarkers and CT images). Both cross-validation and external-validation results show that combining multiple modality significantly improves the predicting performance of single modality. The results suggest that integrating subjects with missing either CDEs/biomarker or CT imaging features can contribute to the discriminatory power of our model (p < 0.05, bootstrap two-tailed test). In summary, the proposed M3Net framework provides an effective way to integrate image and non-image data in the context of missing information., RFW all-conference best paper finalist, SPIE2021 Medical Imaging

Type II diabetes mellitus (T2DM) is a significant public health concern with multiple known risk factors (e.g., body mass index (BMI), body fat distribution, glucose levels). Improved prediction or prognosis would enable earlier intervention before possibly irreversible damage has occurred. Meanwhile, abdominal computed tomography (CT) is a relatively common imaging technique. Herein, we explore secondary use of the CT imaging data to refine the risk profile of future diagnosis of T2DM. In this work, we delineate quantitative information and imaging slices of patient history to predict onset T2DM retrieved from ICD-9 codes at least one year in the future. Furthermore, we investigate the role of five different types of electronic medical records (EMR), specifically 1) demographics; 2) pancreas volume; 3) visceral/subcutaneous fat volumes in L2 region of interest; 4) abdominal body fat distribution and 5) glucose lab tests in prediction. Next, we build a deep neural network to predict onset T2DM with pancreas imaging slices. Finally, motivated by multi-modal machine learning, we construct a merged framework to combine CT imaging slices with EMR information to refine the prediction. We empirically demonstrate our proposed joint analysis involving images and EMR leads to 4.25% and 6.93% AUC increase in predicting T2DM compared with only using images or EMR. In this study, we used case-control dataset of 997 subjects with CT scans and contextual EMR scores. To the best of our knowledge, this is the first work to show the ability to prognose T2DM using the patients’ contextual and imaging history. We believe this study has promising potential for heterogeneous data analysis and multi-modal medical applications.

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, is the target for more than 50 commercially available herbicides, and is a promising target for antimicrobial drug discovery. Herein, we have expressed and purified AHAS from

Purpose: Deep learning is a promising technique for spleen segmentation. Our study aims to validate the reproducibility of deep learning-based spleen volume estimation by performing spleen segmentation on clinically acquired computed tomography (CT) scans from patients with myeloproliferative neoplasms. Approach: As approved by the institutional review board, we obtained 138 de-identified abdominal CT scans. A sum of voxel volume on an expert annotator’s segmentations establishes the ground truth (estimation 1). We used our deep convolutional neural network (estimation 2) alongside traditional linear estimations (estimation 3 and 4) to estimate spleen volumes independently. Dice coefficient, Hausdorff distance, [Formula: see text] coefficient, Pearson [Formula: see text] coefficient, the absolute difference in volume, and the relative difference in volume were calculated for 2 to 4 against the ground truth to compare and assess methods’ performances. We re-labeled on scan–rescan on a subset of 40 studies to evaluate method reproducibility. Results: Calculated against the ground truth, the [Formula: see text] coefficients for our method (estimation 2) and linear method (estimation 3 and 4) are 0.998, 0.954, and 0.973, respectively. The Pearson [Formula: see text] coefficients for the estimations against the ground truth are 0.999, 0.963, and 0.978, respectively (paired [Formula: see text]-tests produced [Formula: see text] between 2 and 3, and 2 and 4). Conclusion: The deep convolutional neural network algorithm shows excellent potential in rendering more precise spleen volume estimations. Our computer-aided segmentation exhibits reasonable improvements in splenic volume estimation accuracy.

Although graphene oxide (GO) membranes show great potential for nanofiltration, there is always a trade-off between the permeation flux and rejection rate. In this study, a covalent organic framework (COF-TpPa) was incorporated into a GO membrane for efficient dye separation. The good bonding interactions between two species was facilitated via a facile hot-pressing (HP) method, mitigating the issue of the solubility of GO in water and preventing the interface void. The as-prepared composite membrane (HP-COF-TpPa/GO) exhibited greatly enhanced stability in universal pH conditions with good retention of its original morphology. The COF-TpPa provided additional water passage in the membrane, leading to the water permeance of 166.8 L m−2 h−1 bar−1, which was much higher than that of most membranes. Furthermore, owing to the molecular sieving, electrostatic repulsion and improved adhesion brought by introducing the COF-TpPa with HP step, the HP-COF-TpPa/GO exhibited a more efficient dye rejection rate (97.05%) of methylene blue as compared to the pristine GO membrane (91.87%) and COF-TpPa/GO without the HP process (68.57%). This study can provide an alternative approach of fabricating high-performance COF/GO membranes for nanofiltration and extend the application of COF materials in the separation field.

In this paper, we present the automatic labeling framework for sulci in the human lateral prefrontal cortex (PFC). We adapt an existing spherical U -Net architecture with our recent surface data augmentation technique to improve the sulcal labeling accuracy in a developmental cohort. Specifically, our framework consists of the following key components: (1) augmented geometrical features being generated during cortical surface registration, (2) spherical U -Net architecture to efficiently fit the augmented features, and (3) post-refinement of sulcal labeling by optimizing spatial coherence via a graph cut technique. We validate our method on 30 healthy subjects with manual labeling of sulcal regions within PFC. In the experiments, we demonstrate significantly improved labeling performance (0.7749) in mean Dice overlap compared to that of multi-atlas (0.6410) and standard spherical U-Net (0.7011) approaches, respectively (p < 0.05). Additionally, the proposed method achieves a full set of sulcal labels in 20 seconds in this developmental cohort.

This study centers around virtual teaching assistants (“VTA”) and the potential application of VTAs to reduce the burden on teachers across secondary schools in China exacerbated by the Double Reduction Policy. It seeks to understand how VTAs could be implemented in the classroom while ensuring student and teacher well-being, success, and happiness. Through interviews with fourteen different experts spanning industry, academia, and education, combined with extensive review of the existing literature and ecosystem of VTAs, research shows that VTAs are potentially a major aid in the context of exam review, after school tutoring, automated grading, and student performance reporting. However, they need to be created carefully with an attentive eye towards student support to ensure that the learning experience is not diminished. It must be clearly communicated to all stakeholders that the goal of a VTA is not to replace teachers, but rather to support them such that they can spend more time with students and less time on mundane tasks.

Splenomegaly segmentation on computed tomography (CT) abdomen anatomical scans is essential for identifying spleen biomarkers and has applications for quantitative assessment in patients with liver and spleen disease. Deep convolutional neural network automated segmentation has shown promising performance for splenomegaly segmentation. However, manual labeling of abdominal structures is resource intensive, so the labeled abdominal imaging data are rare resources despite their essential role in algorithm training. Hence, the number of annotated labels (e.g., spleen only) are typically limited with a single study. However, with the development of data sharing techniques, more and more publicly available labeled cohorts are available from different resources. A key new challenging is to co-learn from the multi-source data, even with different numbers of labeled abdominal organs in each study. Thus, it is appealing to design a co-learning strategy to train a deep network from heterogeneously labeled scans. In this paper, we propose a new deep convolutional neural network (DCNN) based method that integrates heterogeneous multi-resource labeled cohorts for splenomegaly segmentation. To enable the proposed approach, a novel loss function is introduced based on the Dice similarity coefficient to adaptively learn multi-organ information from different resources. Three cohorts were employed in our experiments, the first cohort (98 CT scans) has only splenomegaly labels, while the second training cohort (100 CT scans) has 15 distinct anatomical labels with normal spleens. A separate, independent cohort consisting of 19 splenomegaly CT scans with labeled spleen was used as testing cohort. The proposed method achieved the highest median Dice similarity coefficient value (0.94), which is superior (p-value

Human in-the-loop quality assurance (QA) is typically performed after medical image segmentation to ensure that the systems are performing as intended, as well as identifying and excluding outliers. By performing QA on large-scale, previously unlabeled testing data, categorical QA scores can be generatedIn this paper, we propose a semi-supervised multi-organ segmentation deep neural network consisting of a traditional segmentation model generator and a QA involved discriminator. A large-scale dataset of 2027 volumes are used to train the generator, whose 2-D montage images and segmentation mask with QA scores are used to train the discriminator. To generate the QA scores, the 2-D montage images were reviewed manually and coded 0 (success), 1 (errors consistent with published performance), and 2 (gross failure). Then, the ResNet-18 network was trained with 1623 montage images in equal distribution of all three code labels and achieved an accuracy 94% for classification predictions with 404 montage images withheld for the test cohort. To assess the performance of using the QA supervision, the discriminator was used as a loss function in a multi-organ segmentation pipeline. The inclusion of QA-loss function boosted performance on the unlabeled test dataset from 714 patients to 951 patients over the baseline model. Additionally, the number of failures decreased from 606 (29.90%) to 402 (19.83%). The contributions of the proposed method are threefold: We show that (1) the QA scores can be used as a loss function to perform semi-supervised learning for unlabeled data, (2) the well trained discriminator is learnt by QA score rather than traditional true/false, and (3) the performance of multi-organ segmentation on unlabeled datasets can be fine-tuned with more robust and higher accuracy than the original baseline method., 7 pages, 5 figures, Accepted by SPIE 2020: Medical Imaging

The combination of cytotoxic treatment modalities, including oncolytic viral gene therapies and immunotherapy, usually yields a synergistic effect. In the current study, a bicistronic adenoviral vector, Ad-CD-GMCSF, carrying the cytosine deaminase (CD) and granulocyte-macrophage colony-stimulating factor (GM-CSF) transcription units driven by a cytomegalovirus promoter was constructed, and the

Dynamic contrast enhanced computed tomography (CT) is an imaging technique that provides critical information on the relationship of vascular structure and dynamics in the context of underlying anatomy. A key challenge for image processing with contrast enhanced CT is that phase discrepancies are latent in different tissues due to contrast protocols, vascular dynamics, and metabolism variance. Previous studies with deep learning frameworks have been proposed for classifying contrast enhancement with networks inspired by computer vision. Here, we revisit the challenge in the context of whole abdomen contrast enhanced CTs. To capture and compensate for the complex contrast changes, we propose a novel discriminator in the form of a multi-domain disentangled representation learning network. The goal of this network is to learn an intermediate representation that separates contrast enhancement from anatomy and enables classification of images with varying contrast time. Briefly, our unpaired contrast disentangling GAN(CD-GAN) Discriminator follows the ResNet architecture to classify a CT scan from different enhancement phases. To evaluate the approach, we trained the enhancement phase classifier on 21060 slices from two clinical cohorts of 230 subjects. Testing was performed on 9100 slices from 30 independent subjects who had been imaged with CT scans from all contrast phases. Performance was quantified in terms of the multi-class normalized confusion matrix. The proposed network significantly improved correspondence over baseline UNet, ResNet50 and StarGAN performance of accuracy scores 0.54. 0.55, 0.62 and 0.91, respectively. The proposed discriminator from the disentangled network presents a promising technique that may allow deeper modeling of dynamic imaging against patient specific anatomies., Comment: 8 pages, 4 figures

Annual low dose computed tomography (CT) lung screening is currently advised for individuals at high risk of lung cancer (e.g., heavy smokers between 55 and 80 years old). The recommended screening practice significantly reduces all-cause mortality, but the vast majority of screening results are negative for cancer. If patients at very low risk could be identified based on individualized, image-based biomarkers, the health care resources could be more efficiently allocated to higher risk patients and reduce overall exposure to ionizing radiation. In this work, we propose a multi-task (diagnosis and prognosis) deep convolutional neural network to improve the diagnostic accuracy over a baseline model while simultaneously estimating a personalized cancer-free progression time (CFPT). A novel Censored Regression Loss (CRL) is proposed to perform weakly supervised regression so that even single negative screening scans can provide small incremental value. Herein, we study 2287 scans from 1433 de-identified patients from the Vanderbilt Lung Screening Program (VLSP) and Molecular Characterization Laboratories (MCL) cohorts. Using five-fold cross-validation, we train a 3D attention-based network under two scenarios: (1) single-task learning with only classification, and (2) multi-task learning with both classification and regression. The single-task learning leads to a higher AUC compared with the Kaggle challenge winner pre-trained model (0.878 v. 0.856), and multi-task learning significantly improves the single-task one (AUC 0.895, p, Finalist for the 2020 Robert F. Wagner All Conference Best Student Paper Award, SPIE 2020 Medical Imaging. (Highest award in Image Processing section)

Recently, multi-task networks have shown to both offer additional estimation capabilities, and, perhaps more importantly, increased performance over single-task networks on a "main/primary" task. However, balancing the optimization criteria of multi-task networks across different tasks is an area of active exploration. Here, we extend a previously proposed 3D attention-based network with four additional multi-task subnetworks for the detection of lung cancer and four auxiliary tasks (diagnosis of asthma, chronic bronchitis, chronic obstructive pulmonary disease, and emphysema). We introduce and evaluate a learning policy, Periodic Focusing Learning Policy (PFLP), that alternates the dominance of tasks throughout the training. To improve performance on the primary task, we propose an Internal-Transfer Weighting (ITW) strategy to suppress the loss functions on auxiliary tasks for the final stages of training. To evaluate this approach, we examined 3386 patients (single scan per patient) from the National Lung Screening Trial (NLST) and de-identified data from the Vanderbilt Lung Screening Program, with a 2517/277/592 (scans) split for training, validation, and testing. Baseline networks include a single-task strategy and a multi-task strategy without adaptive weights (PFLP/ITW), while primary experiments are multi-task trials with either PFLP or ITW or both. On the test set for lung cancer prediction, the baseline single-task network achieved prediction AUC of 0.8080 and the multi-task baseline failed to converge (AUC 0.6720). However, applying PFLP helped multi-task network clarify and achieved test set lung cancer prediction AUC of 0.8402. Furthermore, our ITW technique boosted the PFLP enabled multi-task network and achieved an AUC of 0.8462 (McNemar test, p < 0.01)., Accepted by Medical Imaging, SPIE2020

Tissue window filtering has been widely used in deep learning for computed tomography (CT) image analyses to improve training performance (e.g., soft tissue windows for abdominal CT). However, the effectiveness of tissue window normalization is questionable since the generalizability of the trained model might be further harmed, especially when such models are applied to new cohorts with different CT reconstruction kernels, contrast mechanisms, dynamic variations in the acquisition, and physiological changes. We evaluate the effectiveness of both with and without using soft tissue window normalization on multisite CT cohorts. Moreover, we propose a stochastic tissue window normalization (SWN) method to improve the generalizability of tissue window normalization. Different from the random sampling, the SWN method centers the randomization around the soft tissue window to maintain the specificity for abdominal organs. To evaluate the performance of different strategies, 80 training and 453 validation and testing scans from six datasets are employed to perform multi-organ segmentation using standard 2D U-Net. The six datasets cover the scenarios, where the training and testing scans are from (1) same scanner and same population, (2) same CT contrast but different pathology, and (3) different CT contrast and pathology. The traditional soft tissue window and nonwindowed approaches achieved better performance on (1). The proposed SWN achieved general superior performance on (2) and (3) with statistical analyses, which offers better generalizability for a trained model.

Dielectric elastomer actuators (DEAs) exhibit interesting muscle-like attributes including large voltage-induced deformation and high energy density, thus can function as artificial muscles for soft robots/devices. This paper focuses on soft planar DEAs, which have extensive applications such as artificial muscles for jaw movement, stretchers for cell mechanotransduction, and vibration shakers for tactile feedback, etc. Specifically, we develop a soft planar DEA, in which compression springs are employed to make the entire structure freestanding. This soft freestanding actuator can achieve both linear actuation and turning without increasing the size, weight, or structural complexity, which makes the actuator suitable for driving a soft crawling robot. Furthermore, its simple structure and homogeneous deformation allow for analytic modeling, which can be used to interpret the large voltage-induced deformation and interesting mechanics phenomenon (i.e., wrinkling and electromechanical instability). A preliminary demonstration showcases that this soft planar actuator can be employed as an artificial muscle to drive a soft crawling robot.

Cyclin-dependent kinase 8 (CDK8), a gene encoding the cyclin-dependent kinase (CDK) component of the Mediator complex, is known as a colon cancer oncogene. Our recent study showed that CDK8 plays an important role in the formation of pancreatic cancer, but the CDK8 expression levels were not completely identical in different pancreatic cancer samples. The level of CDK8 expression depended on whether the K-ras gene was mutated; its expression was much higher in samples carrying a K-ras mutation than in wild-type K-ras samples. Moreover, CDK8 expression was reduced following mutated K-ras knockdown in K-ras-mutated pancreatic cancer cells, whereas CDK8 expression was increased following expression of mutated K-ras in wild-type K-ras cells. Our study demonstrates that mutated K-ras stimulates CDK8 expression, possibly by regulating HIF-1α, and both CDK8 and mutated K-ras were confirmed to promote cell proliferation and prevent apoptosis in vitro. Additionally, we found that both CDK8 and mutated K-ras promote the invasion and migration of pancreatic cancer cells via the positive regulation of the Wnt/β-catenin signaling pathway, thereby increasing the expression of Snail1 and ZEB1, which act as important stimulating factors of the epithelial-to-mesenchymal transition (EMT). Finally, knockdown of either CDK8 or mutated K-ras contributed to attenuated pancreatic cancer growth in BALB/c nude mice. In conclusion, these findings demonstrate that mutated K-ras promotes CDK8 expression and that the regulatory effects of CDK8 on the EMT are partially attributed to the Wnt/β-catenin signaling pathway.

A vaccine platform has been created by attaching the target-associated antigen (TAA) for the vaccine to the extracellular domain (ecd) of the potent immunostimulatory signal CD40 ligand (CD40L). Attachment of the TAA to the CD40L promotes uptake of the TAA into dendritic cells (DCs), binding to Class I as well as Class II MHC leading to presentation of the TAA on the DCs, expansion of the TAA-specific B cell and CD8 effector T-cell lymphocytes, and induction of a memory response. In addition, the TAA/ecdCD40L vaccine can overcome anergy, induce regressions of pre-existing subcutaneous (SC) nodules of cancer cells, and induce high titers of neutralizing antibodies against viral antigens. This vaccine, which can be administered SC as a TAA/ecdCD40L fusion protein, or as expression vectors (viral or plasmid) or as a vector prime-protein boost strategy, is applicable to the development of vaccine for a wide range of cancers and infectious agents.

Background: Combination of cytotoxic treatments with immunotherapeutic agents seem more efficacious than using either strategy alone. Oncolytic viral vectors carrying immunostimulatory genes like GM-CSF have emerged as treatment options in various tumors. Previously, we have shown that combination of suicide gene therapy either with GM-CSF or vector-induced dendritic cells has significant anti-tumor efficacy. In the current study, we aimed to combine the cytotoxic effect of the suicide gene cytosine deaminase and the immunostimulatory effect of granulocyte macrophage colony stimulating factor in a single vector construct. Methods: We have constructed a bicistronic adenoviral vector carrying cytosine deaminase (CD) and granulocyte macrophage-colony stimulating factor (GM-CSF) genes combined with an IRES element and driven by CMV promoter (Ad-CMV-CDiresGMCSF). We tested the in vitro efficacy of the vector in various tumor cell lines and in a mouse model of colon cancer. Results: Our bicistronic vector Ad-CMV-CDiresGMCSF showed significant in-vitro cytotoxic effects on tumor cell lines similar to the vector carrying CD gene alone when 5-FC added. Likewise, the GM-CSF producing efficacy of the bicistronic vector was comparable to the vector carrying GM-CSF gene alone. In the syngeneic colon cancer model, the bicistronic vector carrying CD and GM-CSF genes yielded significant tumor shrinkage and overall survival when compared to the control suicide vector carrying CD gene alone. Accordingly, anti-tumor immune response parameters including CTL assay and immune cell infiltration of tumor tissue were significantly improved in the Ad-CMV-CDiresGMCSF vector treated group (p Citation Format: Hakan Akbulut, Arzu Coleri, Gunce Sahin, Yucheng Tang, Albert Deisseroth, Fikri Icli. A bicistronic adenoviral vector carrying cytosine deaminase and GM-CSF genes significantly improves antitumor immunity and overall survival in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5911.

To target chemotherapy to tumor vascular endothelial cells (TVECs), we created the AdTie2RprCDFib(knob-RGD+) vector by inserting into an AdEasy adenoviral vector (Ad) backbone: (i) the cytosine deaminase (CD) gene driven by the Tie2 receptor promoter (Tie2Rpr) into the E1 region of Ad; (ii) mutations that reduce binding of the fiber knob to the Coxsackie adenovirus receptor (CAR); and (iii) the RGD peptide into the H1 loop of fiber for binding to the alpha(V)beta(3) integrin receptors on TVECs. To reduce uptake of the AdTie2RprCDFib(knob-RGD+) by reticuloendothelial (RE) and liver cells, we intravenously (i.v.) injected Hetastarch and low-dose Ad (one million vector particles (VPs)) prior to i.v. injection of a therapeutic dose (one billion VPs) of the AdTie2RprCDFib(knob-RGD+) vector. This treatment induced regressions of N202 breast cancer and B16 melanoma without toxicity to normal tissues. We showed that the tumor regression was induced by infection of the TVECs and not by the infection of tumor cells by the AdTie2RprCDFib(knob-RGD+) vector.

Soft robots have shown great potential for surveillance applications due to their interesting attributes including inherent flexibility, extreme adaptability, and excellent ability to move in confined spaces. High mobility combined with the sensing systems that can detect obstacles plays a significant role in performing surveillance tasks. Extensive studies have been conducted on movement mechanisms of traditional hard-bodied robots to increase their mobility. However, there are limited efforts in the literature to explore the mobility of soft robots. In addition, little attempt has been made to study the obstacle-detection capability of a soft mobile robot. In this paper, we develop a soft mobile robot capable of high mobility and self-sensing for obstacle detection and avoidance. This robot, consisting of a dielectric elastomer actuator as the robot body and four electroadhesion actuators as the robot feet, can generate 2D mobility, i.e. translations and turning in a 2D plane, by programming the actuation sequence of the robot body and feet. Furthermore, we develop a self-sensing method which models the robot body as a deformable capacitor. By measuring the real-time capacitance of the robot body, the robot can detect an obstacle when the peak capacitance drops suddenly. This sensing method utilizes the robot body itself instead of external sensors to achieve detection of obstacles, which greatly reduces the weight and complexity of the robot system. The 2D mobility and self-sensing capability ensure the success of obstacle detection and avoidance, which paves the way for the development of lightweight and intelligent soft mobile robots.

Multiple investigators have reported the presence of defects in the immune response of the elderly [Castle In: Clin Infect Dis 31:578, 2000; Ortqvist et al. In: Eur Respir J 30:414-422, 2007; Saurwein-Teissl et al. In: J Immunol 168:5893, 2002; Haynes et al. In: Proc Natl Acad Sci USA 100:15053-15058, 2003]. These defects reduce the magnitude of the immune response to infection and to vaccination. In individuals greater than 55 years of age, the probability of developing a fully protective neutralizing antibody response to the yearly multivalent particle inactivated influenza vaccine is less than 20% [Jefferson et al. In: Lancet 264:1165-1174, 2005; Goodwin et al. In: Vaccine 24:1159-1169, 2006; Jackson et al. In: Lancet 372:398-405, 2008; Simonsen and Taylor In: Lancet 7:658-666, 2007]. The defects in the aged immune system that are responsible for this limited response to vaccination in the older age groups include functional defects of the antigen presenting cells, functional defects in CD4 helper CD4 T cells and monocytes, and an altered microenvironment [Eaton et al. In: J Exp Med 200:1613-1622, 2004; Dong et al. In: J Gen Virol 84:1623-1628, 2003; Deng et al. In: Immunology 172:3437-3446, 2004; Cella et al. In: J Exp Med 184:747-752, 1996]. Starting at puberty, the involution of the thymus and the consequent reduction of the export of naïve T cells specific to neo-antigens leads to the reduction of the ratio of antigen naïve to memory cells as chronological age advances [Prelog In: Autoimmun Rev 5:136-139, 2006; McElhaney et al. In: J Immunology 176:6333-6339, 2006]. Changes in glycosylation of T cells and target antigens acquired during the aging process and the antibodies to these new glycopeptides and glycoproteins may also contribute to a reduction in the functioning of the adaptive immune response [Ishii et al. In: J Clin Neurosci 14:110-115, 2007; Shirai et al. In: Clin Exp Immunol 12:455-464, 1972; Adkins and Riley In: Mech Ageing Dev 103:147-164, 1998; Ben-Yehuda and Weksler In: Cancer Investigation 10:525-531, 1992]. One of the more interesting examples of the functional defects in the cells of the adaptive immune response is a reduced level of expression in the surface cytoadhesion and activation receptor molecules on CD4 helper T cells undergoing activation during vaccination. Upon infection or vaccination, CD40L is typically increased on the surface of CD4 helper T cells during activation, and this increased expression is absolutely essential to the CD40L promotion of expansion of antigen-specific B cells and CD 8 effector T cells in response to infection or vaccination [Singh et al. In: Protein Sci 7:1124-1135, 1998; Grewal and Flavell In: Immunol Res 16: 59-70, 1997; Kornbluth In: J Hematother Stem Cell Res 11:787-801, 2002; Garcia de Vinuesa et al. In: Eur J Immunol 29:3216-3224, 1999]. In aged human beings and mice, the reduced levels of expression of CD40 ligand (CD40L) in activated CD4 helper T cells is dramatically reduced [Eaton et al. In: J Exp Med 200:1613-1622, 2004; Dong et al. In: J Gen Virol 84:1623-1628, 2003]. To circumvent the reduction in CD40L expression and the subsequent reduction in immune response in the elderly, we have developed a chimeric vaccine comprised of the CD40L linked to the target antigen, in a replication incompetent adenoviral vector and in booster protein. This review will discuss the implementation the potential use of this approach for the vaccination of the older populations for cancer and infection.

Optimization methods have been widely applied in sheet metal forming area. However, the existence of variations during manufacturing processes significantly may influence final product quality, rendering non-robust optimal solutions. In this paper, experiments were conducted to investigate how a stochastic behavior of noise factors affected stamping quality. Robust design models for sheet metal forming process integrated adaptive importance sampling with response surface method, in order to minimize impact of the variations and achieve reliable process parameters. Support Vector Machine with nonlinear capability in both pattern recognition and regression was adopted to map the relation between input process parameters and part quality. A cup drawing example was employed to verify the feasibility of the proposed method. Comparisons were conducted between different optimization models to demonstrate robustness of the adaptive importance sampling method. Final results showed that the stamping part quality was optimized under the specified constraint requirements.

Energy harvesting (EH) from ambient environment is an attractive and green solution to the problem of limited lifetime of wireless and mobile devices such as cognitive relay nodes. In this paper, we extend the previous works on cognitive two-way relaying network by introducing energy harvesting transceiver nodes that can accumulate energy to improve the lifetime of the relay network. We investigate an optimal offline joint relay selection and power allocation scheme where secondary transceivers and relays are energy harvesting nodes. We put forward an offline optimization problem of the maximization of the amplify-and-forward (AF) cooperative communication system throughput which can be solved by Lagrange function. Numerical results show that the proposed scheme performs better than random relay selection scheme on the restriction of transmit power and primary user (PU) interference.

We showed that the Ad-sig-TAA/ecdCD40L vaccine induces a tumor suppressive immune response to the hMUC-1 and rH2N tumor-associated self Ags (TAA) and to the Annexin A1 tumor vascular Ag, even in mice in which anergy exists to these Ags. When the TAA/ecdCD40L protein is given s.c. as a boost following the Ad-sig-TAA/ecdCD40L vector, the levels of the TAA-specific CD8 T cells and Abs increase dramatically over that seen with vector alone, in young (2-mo-old) as well as old (18-mo-old) mice. The Abs induced against hMUC-1 react with human breast cancer. This vaccine also induces a 4-fold decrement of negative regulatory CD4CD25FOXP3-T cells in the tumor tissue of 18-mo-old mice. These results suggest that the Ad-sig-TAA/ecdCD40L vector prime-TAA/ecdCD40L protein boost vaccine platform may be valuable in reducing postsurgery recurrence in a variety of epithelial neoplasms.

To develop a method to overcome the anergy that exists in tumor hosts to cancer, we have designed an adenoviral vector for thein vivoactivation and tumor antigen loading of dendritic cells. This adenoviral vector encodes a fusion protein composed of an amino-terminal tumor-associated antigen fragment fused to the CD40 ligand (CD40L). Subcutaneous injection of an adenoviral vector encoding a fusion protein of the human papillomavirus E7 foreign antigen linked to the CD40L generates CD8+T cell-dependent immunoresistance to the growth of the E7-positive syngeneic TC-1 cancer cells in C57BL/6 mice for up to 1 year. We also studied the s.c. injection of a vector carrying the gene for the human MUC-1 (hMUC-1) self-antigen fused to the CD40L. When this vector was injected into hMUC-1.Tg mice, which are transgenic for the hMUC-1 antigen, the growth of syngeneic hMUC-1-positive LL1/LL2hMUC-1 mouse cancer cells was suppressed in 100% of the injected animals. The hMUC-1.Tg mice are anergic to the hMUC-1 antigen before the injection of the vector. These experimental results show that it is possible to use vector injection to activate a long-lasting cellular immune response against self-antigens in anergic animals. The vector-mediatedin vivoactivation, and tumor-associated antigen loading of dendritic cells does not require additional cytokine boosting to induce the immune response against the tumor cells. This vector strategy may therefore be of use in the development of immunotherapy for the many carcinomas in which the hMUC-1 antigen is overexpressed.

Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and the E1a gene are driven by the L-plastin tumor-specific promoter (AdLpCDIRESE1a). A similar vector driven by the CMV promoter has also been constructed (AdCMVCDIRESE1a) as a control. We have carried out in vitro cytotoxicity in carcinomas of the breast, ovary and colon, and in vivo efficacy studies with these vectors in an animal model of colon cancer. While the addition of the AdLpCDIRESE1a vector to established cancer cell lines showed significant cytotoxicity in tumor cells derived from carcinomas of the breast (MCF-7), colon (HTB-38) and ovary (Ovcar 5), no significant toxicity was seen in explant cultures of normal human mammary epithelial cells (HMEC) exposed to this vector. The addition of 5-fluorocytosine (5FC) significantly increased the cytotoxicity in an additive fashion of both the AdLpCDIRESE1a and AdCMVCDIRESE1a vectors as well as that of the AdLpCD replication incompetent vector to established tumor cell lines. However, no significant cytotoxicity was observed with the addition of 5FC to explant cultures of normal human mammary epithelial cells that had been exposed to the L-plastin-driven vectors. Studies with mixtures of infected and uninfected tumor cell lines showed that the established cancer cell lines infected with the AdLpCDIRESE1a vector generated significant toxicity to surrounding uninfected cells (the "bystander effect") even at a ratio of 0.25 of infected cells to infected + uninfected cells in the presence of 5FC. The injection of the AdLpCDIRESE1a vector into subcutaneous deposits of human tumor nodules in the nude mice was potentiated by administering 5-FC by intraperitoneal injection. This treatment resulted in a decreased tumor size and a decreased tumor cell growth rate. The mice treated with a combination of the AdLpCDIRESE1a vector intratumoral injection and intraperitoneal 5FC injections lived much longer than the other experimental groups exposed to the viral vector alone or to the combination of the intratumoral AdLpCD replication incompetent vector injections plus intraperitoneal 5-FC injections. These encouraging results with our newly constructed AdLpCDIRESE1a vector suggest a need for further study of its utility in a preclinical model of intracavitary therapy of pleural or peritoneal carcinomatosis.

We have previously demonstrated that a truncated form of the L-plastin promoter can confer tumor-specific patterns of expression on replication-incompetent adenoviral vector reporter and therapeutic transcription units. In this report, a 2.5-kb truncated version of the L-plastin promoter was placed 5' to the E1A gene of a wild-type adenovirus. The vector generated (Ad-Lp-E1A) was directly cytotoxic to established breast and ovarian cancer cell lines and to primary explant cultures derived from ovarian cancer, but was not cytotoxic to explant cultures of normal mammary epithelial cells. This vector was not cytotoxic to cell lines in which the L-plastin E1A transcription unit was not expressed, whereas the same cell lines were sensitive to the cytotoxic effect of a replication-competent adenoviral vector in which the cytomegalovirus (CMV) promoter drove E1A expression. When the tyrosinase promoter/enhancer was placed 5' to the E1A gene in the adenoviral backbone, the resulting vector (Ad-Tyr-E1A) was selectively toxic to melanoma cells and one percent as toxic to explants of ovarian cancer cells as the Ad-Lp-E1A vector. Injection of these vectors (Ad-Lp-E1A and Ad-Tyr-E1A) into nodules derived from the MCF-7 and MDA-MB-468 human breast cancer cell lines and the TF-2 human melanoma cell line, respectively, which were growing subcutaneously in severe combined immunodeficiency (SCID) mice, induced regression of these tumors. Such vectors may therefore be useful in cancer treatment.

High mobility group protein A2 (HMGA2) and octamer-binding transcription factor 4 (Oct4) are transcription factors that play major roles in the acquisition of cancer stemness phenotypes and tumorigenicity of malignant neoplasms. The aim of this study was to analyze the association between HMGA2 and Oct4 expression and various clinicopathologic features in gastric cancer patients including invasion, metastasis, and clinical prognosis, in addition to overall survival. Immunohistochemistry was performed to explore the expression of HMGA2 and Oct4 in 158 gastric cancer and surrounding non-tumor tissues. Moreover, HMGA2 and Oct4 mRNA and protein levels were also detected by qRT-PCR and Western blotting, respectively, in 86 clinical tissue specimens and various gastric epithelial cell lines (GES-1, SGC7901, MKN45, and MKN27). Finally, associations between HMGA2 and Oct4 expression and clinicopathological features were analyzed by Pearson correlation coefficient. Survival analysis was performed by univariate and multivariate analyses. Taken together, we found that HMGA2 and Oct4 expression was significantly higher in gastric cancer tissues compared with non-cancerous tissues (P < 0.01), and HMGA2 and Oct4 protein levels were significantly higher in poorly differentiated gastric cancer cell lines (MKN45), moderately differentiated cell lines (SGC7901), and well-differentiated cell lines (MKN28) compared with human immortalized gastric epithelial cell lines (GES-1) (P < 0.01). Elevated HMGA2 and Oct4 levels were significantly associated with poor clinical prognosis (P < 0.05). Further conclusion showed that coexpression of HMGA2 and Oct4 in gastric cancer correlated with tumor invasion, metastasis, and clinical prognosis and predicted an unfavorable clinical outcome. These transcription factors may represent useful biomarkers to identify patients at high risk of postoperative recurrence.

This paper presents an integrated Bayesian probabilistic methodology to calibrate parameters of a predictive model and quantitatively evaluate its validity and predictive capacity with non-normality data, considering uncertainties. Bayes network is developed to graphically represent the relationships of all model variables. Bayesian regression theory associated with Markov Chain Monte Carlo simulation and Gibbs sampling is presented to calibrate model parameters to improve model accuracy. The Bayesian method is compared to maximum likelihood and nonlinear optimisation approaches. A generic procedure is presented to integrate the model calibration and quantitative validation. Anderson-Darling goodness-of-fit test and Box-Cox transformation are employed respectively to perform normality hypothesis test of difference data and normality conversion. The confidence of calibrated model is quantified via Bayesian inference method. The integrated methodology and procedure is demonstrated with a nonlinear analytical model for pressure loss prediction in a gas turbine and five sets of different measurement data.

Fierce competition, fluctuating market and opportunities in emerging market are forcing companies to structure product design globally to reduce cost and improve efficiency. However, the solutions to old issues are always accompanied by new challenges. Traditional design flow is struggling to catch up with the pace of customer requirement. And mixed global design teams, composed of senior and junior designers, are trying to design with uniform quality. A real case occurred was that an experienced designer provides a DIN solution when the manufacturing requirement was ASTM, which results additional round of product design. In order to reduce those risks and overcome the challenges, a multiobjective optimization framework for gas turbine engine support was developed to shorten the product development and find an optimal design solution. To manage the risks arising from sourcing, a concept of design for global sourcing (DfGs) was proposed and implemented by incorporating design standards database and supply chain interface during preliminary design stage. Three practical design cases were performed to validate the proposed method. The results demonstrated: 1) Cost and product development cycle was sharply reduced; 2) Design flexibility was achieved by DfGs concept; 3) Trade-off among conflicting design objectives was accomplished by cost estimation model.

The vasculature of mouse breast tumor spheroids grown on mammary fat pad tissue in an intravital microscopy (IVM) viewing chamber was shown to derive from infiltrating angiogenic mammary vessels. The receptors tissue factor (TF), alpha V beta 3 integrin and Tie-2 were expressed on the vascular endothelium in the periphery but not in the center of the tumor spheroids nor in the mammary tissue nor in smooth muscle tissue, whereas Tie-1 and PCAM-1 were expressed extensively in the entire tumor and in the vascular endothelium of the entire tumor nodule and in normal mammary tissue. TF is a specific target for adenoviral vector-mediated cancer immunotherapy. Subcutaneous injection of the AdfVII/IgG(1)Fc vector leads to the release into the system circulation of a fVII/IgG(1)Fc immunoconjugate molecule that binds specifically and tightly to TF on vascular endothelial cells and tumor cells, activating a cytolytic immune response against the targeted cells. We show that a single administration of the AdfVII/IgG(1)Fc vector destroys the peripheral but not the central vasculature of a tumor spheroid, causing partial tumor regression; additional administrations prevent regeneration of the peripheral vasculature and regrowth of the tumor. These findings indicate that a critical parameter for optimizing tumor damage is the schedule for successive administrations of the AdfVII/IgG(1)Fc, which should coincide with the regeneration of the peripheral vasculature and continue until the tumor is destroyed.

S.c. injection of the Ad-sig-tumor-associated antigen (TAA)/ecdCD40L vector vaccine has been shown to induce a CD8 immune response against TAA for up to 1 year. The first goal of this article is to test if the injection of autologous dendritic cells infected ex vivo with the Ad-sig-TAA/ecdCD40L can increase the immune response induced against TAA. The second goal is to test the effect of adding local chemotherapy in the form of i.t. injection of the AdCDIRESE1A vector-directed chemotherapy on the immune response induced by i.t. injection of adenoviral vector-activated dendritic cells. The results show that the i.t. injection of the AdCDIRESE1A chemotherapy sensitization vector, which encodes the cytosine deaminase chemotherapy sensitization transcription unit, to the i.t. injection of Ad-sig-ecdCD40L vector-infected dendritic cells increased the level of suppression of the growth of the CCL-51 breast cancer cells. The combination of i.t. injection of the AdCDIRESE1A chemotherapy sensitization vector and Ad-sig-ecdCD40L vector-infected dendritic cells into s.c. CCL-51 breast cancer nodules suppressed the growth of uninjected metastatic tumor nodules in the lung. Finally, adding the i.t. injection of the AdCDIRESE1A chemotherapy sensitization vector to the i.t. administration of dendritic cells infected with a rat HER-2/neu (rH2N)–expressing vector (Ad-sig-rH2N/ecdCD40L) led to the induction of rH2N-specific antitumoral immunity in rH2N transgenic mice (which are anergic to the rH2N antigen). This anti-rH2N immune response suppressed the growth of established H2N-positive NT2 breast cancer more efficiently than did the vector-targeted chemotherapy or Ad-sig-rH2N/ecdCD40L-infected dendritic cell vaccine alone. [Mol Cancer Ther 2006;5(8):1975–85]

Purpose: 5-Fluorouracil (5-FU) has been combined in the past with other drugs for the combination chemotherapy for cancers of the breast, ovary, and colon. These drug regimens were limited by the fact that 5-FU fails to kill nondividing cancer cells at the doses that are safe to deliver. The goal of the present study is to test the feasibility of replacing 5-FU in established 5-FU combination chemotherapy with the Ad-LpCDIRESE1A/5-fluorocytosine (5-FC) system for the purpose of reducing toxicity and increasing efficacy. Experimental Design: We have replaced 5-FU in the weekly combination of CPT-11, folinic acid (FA) and 5-FU chemotherapy by 5-FC and an adenoviral vector that carries the L-plastin (Lp) tumor-specific promoter-driven transcription unit encoding the cytosine deaminase gene linked to the E1A gene by an internal ribosomal entry site element. This combination is called “genetic combination therapy.” The goal of using the vector was to decrease the toxicity to normal tissue and to increase the efficacy of therapy in the cancer cells by increasing the concentration of 5-FU sufficiently high that even nondividing cancer cells would be killed by 5-FU through its incorporation into mRNA and consequent inhibition of synthesis of functional proteins. We compared the in vivo efficacy of the genetic combination therapy with the conventional combination chemotherapy in a mouse colon cancer model. Results: Both replication-competent and -noncompetent adenoviral vectors carrying an L-plastin–driven cytosine deaminase transcription unit when combined with 5-FC, CPT-11, and FA showed increased in vitro therapeutic activity that was significantly higher than that of the conventional chemotherapy combination. Tumor-bearing mice treated with the genetic combination therapy showed a statistically significant advantage in terms of increased response rate, response duration, survival, and reduced toxicity when compared with tumor-bearing mice treated with the conventional combination chemotherapy. Conclusions: Replacement of 5-FU in 5-FU–based combination chemotherapy with the Ad-LpCDIRESE1A vector and 5-FU reduces toxicity and increases efficacy. This is a concept that could be potentially applied widely for many forms of cancer treatment.

Our goal in the present work was to characterize the multiple steps involved in overcoming the anergy that exists in tumor hosts to tumor-associated antigen (TAA). Our studies showed that the subcutaneous injection of the Ad-sig-TAA/ecdCD40L vector resulted in secretion of the TAA/ecdCD40L protein for at least 10 days from infected cells. Binding of the TAA/ecdCD40L protein to dendritic cells (DCs) resulted in the induction of CCR-7 chemokine receptor expression and cytokine release. This was followed by migration of the DCs to regional lymph nodes. Tetramer staining, enzyme-linked immunospot (ELISPOT) assay, and cytotoxicity assay all showed that the Ad-sig-TAA/ecdCD40L vector increased the levels of splenic CD8+ T cells specific for the 2 TAAs (human MUC1 [hMUC1] and HPV E7) tested. Vaccination with the Ad-sighMUC1/ecdCD40L vector suppressed the growth of hMUC1 antigen-positive tumor cells in 100% of the test mice that were previously anergic to the hMUC1 antigen. These data suggest that Ad-sig-TAA-ecd/ecdCD40L vector injections may be of value in treating the many epithelial malignancies in which TAA-like hMUC1 is overexpressed. (Blood. 2004;104:2704-2713)

Vascular endothelial growth factor (VEGF)-C is a lymphangiogenic factor implicated in lymphatic metastasis. In this study, we investigated the role of the type I insulin-like growth factor receptor (IGF-IR) in the regulation of VEGF-C expression. We used Lewis lung carcinoma subline M-27 cells transfected with human IGF-IR cDNA. These cells, but not the wild-type cells, expressed VEGF-C mRNA, produced a M(r) 58,000 VEGF-C precursor protein, and secreted a M(r) 29,000 processed form in response to IGF-I. In vivo, they acquired a lymph node metastasizing potential. VEGF-C induction was abolished in cells expressing an IGF-IR with tyrosine-phenylalanine substitutions in the kinase domain, but not in the COOH-terminal domain. The induction was phosphatidylinositol 3'-kinase dependent and, to a lesser extent, mitogen-activated protein kinase signaling dependent, as determined by the use of the respective inhibitors LY294002 (84.6% reduction) and PD98059 (38% reduction). The results identify the IGF-IR as a positive regulator of VEGF-C expression and implicate it in the control of lymphatic metastasis.

To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope.Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22/Sal was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypeptide. Then the single- chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Psi 2 cells with retroviral plasmid and the fusion gene expressing plasmid. To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed.The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen.These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.

In order to overcome defects in the immune response in older individuals, we have designed an adenoviral vector (Ad-sig-TAA/ecdCD40L) for the in vivo activation and tumor antigen loading of dendritic cells (DCs). This adenoviral vector encodes a fusion protein composed of an aminoterminal tumor associated antigen (TAA) fragment fused to the extracellular domain (ecd) of the CD40 ligand (CD40L) at the carboxyl terminal end. Two sc injections of this vector have broken tolerance to specific tumor associated antigens in two separate TAA.Tg transgenic mouse models (mice transgenic for rat Her-2-Neu (rH2N) and human MUC-1 (hMUC-1)). The immunoprotection extends for over a year and is independent of CD4 cells. We showed that the sc injection of the hMUC-1/ecdCD40L protein following the sc injection of the Ad-sig-TAA/ecdCD40L vector (this vector prime-protein boost is called VPP) increases the levels of both the cellular and humoral immune response over that achievable with just the vector injections alone in the hMUC-1.Tg mice. The antibodies from hMUC-1 VPP vaccinated mice bind to biopsy specimens of human cancers of the breast and prostate. Importantly, the VPP vaccination can induce an immune response even in 18 month old mice which completely suppresses tumor growth in old (18 month old) mice. The Ad-sig-rH2N/ecdCD40L vaccination suppresses the evolution of spontaneous breast cancer in the rH2N.Tg mouse model. The Ad-sig-HA/ecdCD40L vector prime-HA/ecdCD40L protein boost which is directed to the H5N1 avian influenza hemagglutinin (HA) antigen induces neutralizing antibodies against the H5N1 486 avian influenza virus at 1/4000 dilution of the serum.

Abstract #902 Background: There are at least three types of cells in tumor tissue which express the Tie2 receptor (Tie2R): dividing vascular endothelial cells (TVECs), Tie2R-expressing monocytes (TEMs) and Tie2R-expressing mesenchymal progenitor cells (TMPCs). We tested if the targeting of chemotherapy to these classes of Tie2R positive cells could suppress tumor cell growth. Material and Methods: We constructed replication incompetent adenoviral vectors carrying the cytosine deaminase (CD) suicide gene driven by the mouse Tie2R transcriptional promoter and enhancer sequences. In these viruses, we inserted an RGD-4C peptide into the HI loop of the fiber knob domain of the vector to increase its transduction efficiency in TVECs. At the same time, we added two mutations (S408E and P409A) in the AB loop of the fiber, which ablated the CAR-binding ability of the vector. The resulting vector was named AdTie2RCD(MRGD). Results: In vitro viral infection assays involving the human umbilical vein endothelial cells (HUVECs) showed that the RGD-modified vectors have a higher transduction efficiency as compared with their adenoviral vector counterparts which have a wild type fiber protein. Mice carrying the rat Her-2-Neu positive N202 mouse breast cancer cells and the mouse B16 melanoma cells were treated with intravenous injections of Hetastarch and the AdTie2RCD(MRGD) vector followed by intraperitoneal injections of 5-Fluorocytosine (5-FC). The Hetastarch was given before the intraveinous infusion of the adenoviral vector in order to reduce the uptake of the adenoviral vector by the reticuloendothelial cell system. The Tie2R targeted chemotherapy sensitization vectors induced greater degrees of suppression of tumor cell growth than did the control group, achieved the similar treatment effect to its CMV promoter counterpart, but with less side effect. Histological analysis showed that these vectors specifically targeted the TVECs, TEMs and TMPCs through which they may have exerted cytotoxic effects in the presence of 5-FC on the tumor cells. Discussion: Our results showed that N202 breast cancer cell line is more responsive to the Tie2R-targeting therapy compared to B16 mouse melanoma cell line and the Tie2R-directed anti-angiogenic therapy deserves further study, in combination with other types of therapy to pave the way for future clinic trails. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 902.

Since a significant portion (30%) of individuals diagnosed with epithelial neoplasms have highly chemotherapy resistant CD44+CD24−/low cancer stem cells in the peripheral blood as well as in solid tumor nodules at diagnosis, we have been developing methods of cancer treatment which depend on methods of killing the cancer stem cells which depend on mechanisms other than chemotherapy for treatment. Vaccination has been one of these approaches. Unfortunately, it has been known for a long time that the immune response is diminished in older individuals due to decreased numbers of CD4 and CD8 T cells and due to acquisition of functional defects in CD4 cells. An example is the diminished expression of the CD40L in activated CD4 T cells in older individuals which limits the cellular and humoral response to vaccines in the older age groups. The presence of the CD40L on the CD4 helper T cells is important for vaccine induced expansion of antigen specific T cells and B cells. In order to overcome these problems, we have developed an Ad-sig-TAA/ecdCD40L vector prime-TAA/ecdCD40L protein boost vaccine strategy. The Ad-sig-TAA/ecdCD40L carries a transcription unit encoding the extracellular domain (ecd) of the CD40 ligand (CD40L) linked to tumor associated antigen (TAA). The TAA are in turn linked to a secretory signal peptide (sig) so that the TAA/ecdCD40L protein will be secreted from the vector infected cells at the injection site of the vector continuously over a 10–14 day period. The CD40L targets the TAA to the DCs, activates the DCs, and carries the TAA into the DC so that the TAA fragments are presented on Class I MHC. The sc injection of the TAA/ecdCD40L protein booster following the sc administration of the Ad-sig-TAA/ecdCD40L (we call this the TAA/ecdCD40L VPP vaccine) induces complete regressions of pre-existing tumor even in aged mice (18 months old). We then chose human MUC-1 as the TAA for testing the efficacy of our vaccine platform for cancer stem cells. Over 90% of the late stage patients with cancers of the breast, ovary, prostate, and lung have been reported to overexpress a hypoglycosylated form of MUC-1 which correlates with decreased time to progression. The hypoglycosylation on MUC-1 in cancer cells creates a tumor specific target for vaccines. Kufe’s lab has shown that overexpression of MUC-1 in the cancer cell promotes resistance to therapy by reducing p53 (Cancer Cell7: 167, 2005) and induces proliferation by activation of the NFkappaB (Nature Cell Biology9: 1419, 2007). Finn’s lab has shown that MUC-1 is a marker present on >90% of the “tumor stem cell” population as well as the more mature cells in breast cancer (Ca Res68: 2419, 2008). In order to test the efficacy of the hMUC-1/ecdCD40L VPP vaccine on tumor stem cells, we first isolated hMUC-1 positive Lewis Lung carcinoma cells by stably transfecting the hMUC-1 cDNA into these cells. We showed that the Ad-sig-hMUC-1/ecdCD40L vector prime-hMUC-1/ecdCD40L protein boost vaccine overcomes anergy to hMUC-1 antigen (hMUC-1) in hMUC-1.Tg mice. We then injected 100,000 human MUC- 1 positive Lewis Lung mouse carcinoma cells (LL2/LL1hMUC-1) mouse cancer cells into the syngeneic C57BL/6J mice. ELISPOT assay showed that the hMUC-1/ecdCD40L VPP vaccine increased the level of antigen specific CD8 effector cells in the lymph node draining the site of injection of the tumor to 900 hMUC-1 specific CD8 T cells/100,000 total cells and to 325 hMUC-1 specific CD8 T cells/100,000 cells in the spleen. The level of cytotoxicity of spleen cells increased 6 fold following vaccination (spleen cells from control and vaccinated mice were exposed in vitro to mitomycin C treated hMUC positive LL2/LL1hMUC-1 cells for 5 days before testing). Measurement of the growth of the sc tumor nodule at the sc injection site showed a 20 fold decrease in the total size of the tumor in the vaccinated vs the control mice and the size of the tumor was decreasing in the vaccinated mice vs the control group at the time of sacrifice at 24 days. Finally, the tumor nodule from the vaccinated and unvaccinated mice were excised post mortem, minced, treated with collagenase and DNAse I and strained through gauze. FACS analysis showed that in the vaccinated mice, the number of the MUC-1 positive tumor stem cells with the CD44+CD24− immunophenotype decreased over 20 fold during the treatment period. These results suggest that the hMUC-1/ecdCD40L VPP vaccine could suppress the levels of hMUC-1 cancer stem cells in pre-existing tumor nodules. This vaccine platform has entered clinical phase I testing.

Individuals of advanced chronological age exhibit an impaired immune response to vaccines. This may be due to a reduction in the ratio of antigen naïve/memory CD4 and CD8 T cells and acquisition of functional defects in activated “helper” CD4 T cells (eg diminished CD40 ligand (CD40L) expression) during the aging process. The absence of the CD40L on activated CD4 helper T cells reduces the magnitude of expansion of antigen specific T and B cells induced by vaccination. In order to circumvent this defective response to vaccines among individuals in the fifth and sixth decades of life, our laboratory has developed an adenoviral vector (Ad-sig-TAA/ecdCD40L) vaccine which is designed to overcome the absence of CD40L expression in activated CD4 helper T cells in older individuals. The subcutaneous (sc) injection of this vector leads to the release of a fusion protein composed of a TAA linked to the extracellular domain (ecd) of the CD40L, which binds to the CD40 receptor on DCs, activates the DCs, and leads to the presentation of TAA fragments on Class I MHC. Two sc injections of the TAA/ecdCD40L protein as a booster following the sc administration of the Ad-sig-TAA/ecdCD40L vector (we call this the TAA/ecdCD40L VPP vaccine) expands the magnitude of the cellular and humoral immune response induced by the vector in 18 month old aged mice as well as in younger mice. In order to explore ways of further amplifying the immune response induced by this vaccine, we decided to test the feasibility of using this vaccine following treatments which reduce the number of T cells in the body of the test subject. We hypothesized that during states of chemotherapy or radiation induced lymphopenia, the number of negative regulatory CD4CD25FoxP3 T cells would be reduced, and all of the regulatory signals in the T cell compartment would be promoting expansion of T cells, thus creating an ideal state for vaccination. To test this hypothesis, we injected 100,000 cells from an established neoplastic cell line sc. Three days later, we administered myeloablative doses of total body irradiation (TBI) followed by a T cell depleted syngeneic bone marrow transplant (TCDBMT) to reconstitute neutrophil and platelet production. Three days following the TBI and TCDBMT, we intravenously infused donor lymphocytes (DLI) from a TAA/ ecdCD40L VPP vaccinated syngeneic donor. Four weeks later, we vaccinated the recipient mouse further with TAA/ecdCD40L sc injections. We tested this for a TAA composed of a junctional peptide from the p210Bcr-Abl protein of chronic myelogenous leukemia (CML) and for the E7 protein of the human papilloma virus (HPV). We found that in the case of the BcrAbl/ecdCD40L VPP vaccine, 50% of the mice treated with TBI, TCDBMT, ten million lymphocytes (DLI) from BcrAbl/ecdCD40L VPP vaccinated syngeneic donors followed in 4 weeks by 3 BcrAbl/ecdCD40L protein sc injections of the recipient test mouse, developed a complete response with the vaccination and that these mice remained disease free beyond 250 days after injection of the P210Bcr-Abl positive 32D leukemia cells, whereas C56BL/6J test mice treated with TBI and TCDBMT without DLI from vaccinated donors nor sc BcrAbl/ecdCD40L sc booster vaccination following injection with the p210Bcr-Abl positive 32D myeloid leukemia cell line all died by day 32. Mice treated with TBI, TCDBMT, DLI from unvaccinated donors followed by vaccination of the recipient with 3 sc BcrAbl/ecdCD40L protein injections exhibited a degree of leukemia suppression that was equal to mice receiving TBI, TCDBMT, DLI from a BcrAbl/ecdCD40L VPP vaccinated donor and BcrAbl/ecdCD40L vaccination. To test the effect of the TAA/ecdCD40L VPP vaccine against an antigen associated with an epithelial neoplasm, we injected 100,000 E7 positive TC-1 mouse cancer cells into syngeneic C57BL6J mice followed in 3–5 days by myeloablative doses of TBI and engrafting doses of TCDBMT. Three days later, the mice received 10 million spleen cells from syngeneic donor mice previously vaccinated with the E7/ecdCD40L VPP vaccine. Finally, 4 weeks later, the test mice received sc E7/ecdCD40L protein booster injections. The vaccinated mice achieved much greater degrees of tumor suppression than was seen following TBI and TCDBMT without DLI from vaccinated donors. These studies show that it is possible to induce a robust adaptive immune response by vaccination with the TAA/ecdCD40L VPP vaccine even in severely lymphopenic individuals.

In order to develop a method to overcome the immune tolerance of cancer, we have designed an Ad-sig-TAA/ecdCD40L adenoviral vector vaccine for the in vivo activation and tumor antigen loading of dendritic cells (DCs). Subcutaneous (sc) injection of the Ad-sig-TAA/ecdCD40L adenoviral vector results in the secretion for 10 days from the vector infected cells of a fusion protein composed of a fragment of a tumor associated antigen (TAA) fused to the extracellular domain (ecd) of the CD40 ligand (CD40L). CD40L is a homo-trimeric protein normally found on B cells and helper CD4+ T cell lymphocytes. All of the sequences necessary to stabilize this trimeric structure of the protein are contained within the ecd of the CD40L protein. The binding of the TAA/ecdCD40L protein to DCs induces migration of these DCs to the regional lymph nodes. These DCs carry fragments of TAA bound to surface MHC Class I molecules. We have shown that the Ad-sig-TAA/ecdCD40L vector strategy can induce a cellular and humoral immune that persists for over a year indicating that a durable memory response is generated. We showed that sc injections of the Ad-sig-rH2N/ecdCD40L vector in rH2N.Tg mice induces a cellular and humoral immune response against the rat Her-2-Neu (rH2N) antigen which is associated with breast cancer. We showed that the sc injection of the Ad-sig-rH2N/ecdCD40L adenoviral vector in a rH2N.Tg transgenic mouse induced resistance to the growth of rH2N positive cancer cells in mice previously anergic to the rH2N antigen. We demonstrated that the sc injection of the Ad-sig-hMUC-1/ecdCD40L vector suppressed the growth of tumor cells positive for the human MUC-1 (hMUC-1) antigen in hMUC-1.Tg mice which were previously anergic to the hMUC-1 antigen. The sc injection of the Ad-sig-hMUC-1/ecdCD40L vector followed by sc injection of two booster injections of the hMUC-1/ecdCD40L protein induced high levels of hMUC-1 specific tumor infiltrating effector CD8 positive T cells and hMUC-1 antibodies which bound to human breast and prostate cancer cells. In addition, we recently showed that the Ad-sig-TAA/ecdCD40L strategy could be used to activate a cellular and humoral immune response against Annexin A1 (AnxA1), which is a marker uniquely displayed on the luminal membrane of tumor vascular endothelial cells but not on the luminal membrane of vascular endothelial cells of normal tissue. The subcutaneous injection of the Ad-sig-AnxA1/ecdCD40L vector suppressed the growth of AnxA1 negative tumor cells in a syngeneic mouse tumor model. This vector prime/protein boost vaccination was found to induce increased levels of effector CD8 positive T cells in the target tumor. These effector T cells were shown express increased levels of the genes encoding the CCR5 chemokine receptor and the CCL3 chemokine ligand which promote the infiltration of antigen specific effector T cells in the target tumor tissues. The response to cancer vaccines is often reduced in older individuals in part due to an intrinsic functional defect in CD4 cells. The Ad-sig-TAA/ecdCD40L vaccine may circumvent this defect because we have shown that the induction of the immune response is CD4 independent. These data suggest that this vector prime-protein boost vaccination strategy will be useful in the reduction of the frequency of recurrence following initial therapy for a wide variety of neoplastic diseases.

In order to develop a method to overcome the immune tolerance of cancer, we have designed an adenoviral vector, designated Ad-sig-TAA/ecdCD40L, which carries a transcription unit encoding the extracellular domain (ecd) of the CD40 ligand (CD40L) linked to TAA. The TAA are in turn linked to a secretory signal peptide (sig). This work was based on earlier DNA vaccines reported by Ralph Reisfeld. The binding of the CD40L to the CD40 receptor on DCs activates them. The binding of the TAA/CD40L chimeric protein leads to the internalization of the TAA and its ultimate presentation of Class I MHC on the DCs. In vivo and in vitro analysis showed that the subcutaneous injection of the Ad-sig-TAA/ecdCD40L adenoviral vector led to the development of a CD8+ T cell lymphocyte systemic immune response against TAA for up to a year. One of the TAA tested is MUC-1, which is overexpressed in epithelial cancer cells. We have shown that the Ad-sig-ecdhMUC-1/ecdCD40L vector makes the hMUC-1.Tg mice (which otherwise are killed by the growth of hMUC-1 positive cancer cell lines) resistant to the engraftment and growth of a hMUC-1 positive cancer cell line. In contrast, the cancer cell line which lacks the hMUC-1 antigen engrafts and grows well in these transgenic mice. We have also shown that subcutaneous injections of the hMUC-1/ecdCD40L protein following the vector injection increase the level of the hMUC-1 specific T cells, and generates high levels of serum antibodies against the hMUC-1 antigen. Importantly, these antibodies bind to breast and prostate cancer cancer cells in biopsy specimens from human subjects. The target of these antibodies is a 20 amino acid repeat in the MUC-1 extracellular domain which can completely block the binding of the antibodies to the human breast cancer epithelial cells in biopsy specimens. The schedule of one subcutaneous (sc) injection of the Ad-sig-MUC-1/ecdCD40L vector followed in 7 and 14 days by a sc injection of the MUC-1/ecdCD40L protein is the best schedule among many other combinations and schedules tested. In addition to the MUC-1 self antigen, we have tested the Ad-sig-rH2N/ecdCD40L vector which contains the rat her-2-neu (rH2N) antigen. We tested this vaccine in rH2N.Tg mice which are anergic to the rH2Nantigen. We found that one sc injection of the Ad-sig-rH2N/ecdCD40L vector followed by 2 sc injections of the rH2N/ecdCD40L protein breaks tolerance which exists in the rH2N.Tg mice which are otherwise tolerant of the rH2N TAA. This Ad-sig-TAA/ecdCD40L vector strategy outperforms both corresponding DNA vaccines as well as cellular based cytokine modified tumor cell vaccines. We plan to complete the preclinical testing necessary to bring this new strategy into phase I toxicity testing in the near future.

Adenoviral vectors have been used to deliver therapeutic transcription units to epithelial malignancies for short term modification of these cells for therapy. The adenoviral vector is an attractive choice since it infects most epithelial cells (normal and neoplastic), and expresses its transgenes in cells which are non-dividing as well as dividing. The expression of the therapeutic transgenes in the normal cells can generate toxicity and the expression of the CAR receptor, which is necessary for infection, on the surface of the tumor cells is variable in cancer. In addition, the use of replication incompetent adenoviral vector has the theoretical limitation of incomplete infection of the target tumor cells. In order to create an adenoviral vector which is more selective and more potent, I. Chung in our laboratory truncated the L-plastin promoter and showed that when it is introduced into the adenoviral backbone, that it drives the expression of therapeutic transgenes in a tumor specific manner. X.Y. Peng, L. Zhang, and H. Akbulut then showed that when the L-plastin promoter is placed 5′ of the viral replication gene EIA or the chemotherapy sensitization gene, cytosine deaminase (CD) in an adenoviral vector, infected breast or ovarian cancer cells are destroyed, whereas normal breast or ovarian epithelial explant cultures are resistant to the toxic effect of these vectors. L. Zhang and H. Akbulut showed that the L-plastin promoter driving the expression of a bicistronic transcription unit composed of the CD gene linked to the ElA gene was more toxic than adenoviral vectors carrying only a single therapeutic gene under the control of the L-plastin promoter. We are currently studying the effect of adding chemotherapy to the infection of tumor cells with these vectors, and also engineering the fibrillar protein of the vector so as to direct its infection more specifically to the tumor cells.

BANGKOK (AP) — The son of two Spanish film stars has been arrested in Thailand on suspicion of murdering and dismembering a Colombian surgeon on a tourist island, police said.Daniel Sancho Bronchalo was taken from Koh Phagnan, where the remains of Edwin Arrieta Arteaga were found, to another island, Samui, where he is being held, Surat Thani provincial police commander Saranyu Chamnanrat said Tuesday. [ABSTRACT FROM PUBLISHER]