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The authors sought to identify causal factors that explain the selective benefit of prehospital administration of thawed plasma (TP) in traumatic brain injury (TBI) patients using mediation analysis of a multiomic database.The Prehospital Air Medical Plasma (PAMPer) Trial showed that patients with TBI and a pronounced systemic response to injury [defined as endotype 2 (E2)], have a survival benefit from prehospital administration of TP. An interrogation of high dimensional proteomics, lipidomics and metabolomics previously demonstrated unique patterns in circulating biomarkers in patients receiving prehospital TP, suggesting that a deeper analysis could reveal causal features specific to TBI patients.A novel proteomic database (SomaLogic Inc., aptamer-based assay, 7K platform) was generated using admission blood samples from a subset of patients (n=149) from the PAMPer Trial. This proteomic dataset was combined with previously reported metabolomic and lipidomic datasets from these same patients. A 2-step analysis was performed to identify factors that promote survival in E2-TBI patients who had received early TP. First, features were selected using both linear and multivariate-latent-factor regression analyses. Then, the selected features were entered into the causal mediation analysis.Causal mediation analysis of observable features identified 16 proteins and 41 lipids with a high proportion of mediated effect (50%) to explain the survival benefit of early TP in E2-TBI patients. The multivariate latent-factor regression analyses also uncovered 5 latent clusters of features with a proportion effect30%, many in common with the observable features. Among the observable and latent features were protease inhibitors known to inhibit activated protein C and block fibrinolysis (SERPINA5 and CPB2), a clotting factor (factor XI), as well as proteins involved in lipid transport and metabolism (APOE3 and sPLA(2)-XIIA).These findings suggest that severely injured patients with TBI process exogenous plasma differently than those without TBI. The beneficial effects of early TP in E2-TBI patients may be the result of improved blood clotting and the effect of brain protective factors independent of coagulation.

Significance: The immunoinflammatory responses that follow trauma contribute to clinical trajectory and patient outcomes. While remarkable advances have been made in trauma services and injury management, clarity on how the immune system in humans responds to trauma is lagging. Recent Advances: Multiplexing platforms have transformed our ability to analyze comprehensive immune mediator responses in human trauma. In parallel, with the establishment of large data sets, computational methods have been adapted to yield new insights based on mediator patterns. These efforts have added an important data layer to the emerging multiomic characterization of the human response to injury. Critical Issues: Outcome after trauma is greatly affected by the host immunoinflammatory response. Excessive or sustained responses can contribute to organ damage. Hence, understanding the pathophysiology behind traumatic injury is of vital importance. Future Directions: This review summarizes our work in the study of circulating immune mediators in trauma patients. Our foundational studies into dynamic patterns of inflammatory mediators represent an important contribution to the concepts and computational challenges that these large data sets present. We hope to see further integration and understanding of multiomics strategies in the field of trauma that can aid in patient endotyping and in potentially identifiying certain therapeutic targets in the future. Antioxid. Redox Signal. 35, 1393–1406.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The fourth Innate Immune Memory meeting was held in the historic city of Nijmegen, in the eastern-central part of the Netherlands, to discuss the basic and translational aspects of innate immune memory, popularly known as ‘trained immunity’.

Summary Trauma is a leading cause of death and morbidity worldwide. Here, we present the analysis of a longitudinal multi-omic dataset comprising clinical, cytokine, endotheliopathy biomarker, lipidome, metabolome, and proteome data from severely injured humans. A “systemic storm” pattern with release of 1,061 markers, together with a pattern suggestive of the “massive consumption” of 892 constitutive circulating markers, is identified in the acute phase post-trauma. Data integration reveals two human injury response endotypes, which align with clinical trajectory. Prehospital thawed plasma rescues only endotype 2 patients with traumatic brain injury (30-day mortality: 30.3 versus 75.0%; p = 0.0015). Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) was identified as the most predictive circulating biomarker to identify endotype 2-traumatic brain injury (TBI) patients. These response patterns refine the paradigm for human injury, while the datasets provide a resource for the study of critical illness, trauma, and human stress responses., Graphical abstract, Highlights An integrated longitudinal multi-omic analysis of the human response to trauma Systemic storm and massive consumption patterns are related to early mortality Unique resolution and non-resolution signatures across multiple “omics” platforms Only endotype 2-TBI patients with high UCHL1 levels benefit from early plasma, Wu et al. report a longitudinal multi-omic analysis of the circulation in trauma patients. Cross-platform data integration reveals a massive systemic release of cellular contents (“systemic storm”) and simultaneous consumption of blood constituents. Also defined are patient endotypes that differ in outcomes and responses to early plasma administration.

IntroductionIntegral membrane proteins and lipids constitute the bilayer membranes that surround cells and sub-cellular compartments, and modulate movements of molecules and information between them. Since membrane protein drug targets represent a disproportionately large segment of the proteome, technical developments need timely review. Areas covered: Publically available resources such as Pubmed were surveyed. Bottom-up proteomics analyses now allow efficient extraction and digestion such that membrane protein coverage is essentially complete, making up around one third of the proteome. However, this coverage relies upon hydrophilic loop regions while transmembrane domains are generally poorly covered in peptide-based strategies. Top-down mass spectrometry where the intact membrane protein is fragmented in the gas phase gives good coverage in transmembrane regions, and membrane fractions are yielding to high-throughput top-down proteomics. Exciting progress in native mass spectrometry of membrane protein complexes is providing insights into subunit stoichiometry and lipid binding, and cross-linking strategies are contributing critical in-vivo information. Expert commentary: It is clear from the literature that integral membrane proteins have yielded to advanced techniques in protein chemistry and mass spectrometry, with applications limited only by the imagination of investigators. Key advances toward translation to the clinic are emphasized.

A well-functioning supply chain is a key to success for every business entity. Having an accurate projection on inventory offers a substantial competitive advantage. There are many internal factors like product introductions, distribution network expansion; and external factors such as weather, extreme seasonality, and changes in customer perception or media coverage that affects the performance of the supply chain. In recent years Artificial Intelligence (AI) has been proved to become an extension of our brain, expanding our cognitive abilities to levels that we never thought would be possible. Though many believe AI will replace humans, it is not true, rather it will help us to unleash our true strategic and creative potential. AI consists of a set of computational technologies developed to sense, learn, reason, and act appropriately. With the technological advancement in mobile computing, the capacity to store huge data on the internet, cloud-based machine learning and information processing algorithms etc. AI has been integrated into many sectors of business and been proved to reduce costs, increase revenue, and enhance asset utilization. AI is helping businesses to get almost 100% accurate projection and forecast the customer demand, optimizing their R&D and increase manufacturing with lower cost and higher quality, helping them in the promotion (identifying target customers, demography, defining the price, and designing the right message, etc.) and providing their customers a better experience. These four areas of value creation are extremely important for gaining competitive advantage. Supply-chain leaders use AI-powered technologies to a) make efficient designs to eliminate waste b) real-time monitoring and error-free production and c) facilitate lower process cycle times. These processes are crucial in bringing Innovation faster to the market.

Understanding developmental biology holds the key to the future of medicine as virtually every disease can be viewed as a failure of development. Broadly put, developmental biologists seek to understand the biology of stem cells to answer the immense complexity in the developmental process. If we can decipher the molecular cues important for the maintenance of stem cells it will boost our ability to understand how these cues may go awry in disease states and create effective therapeutics to fight against them. Moreover, in the era of personalized medicine cell therapy using stem cells hold greatest prospect of changing the face of human diseases and alleviating suffering in the near future. Use of stem cell therapy now hold a promising impact to treat various degenerative and genetic diseases including certain type of cancers, neurological diseases, autoimmune diseases, restoration of sight, wound healing, cardiac diseases, liver diseases, metabolic disorders, spinal cord injury and bone disorders etc. There are currently a number of stem cell sources that are being investigated for use in biomedical applications, including adult stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), wherein each presents its own unique advantages and disadvantages. Stem cells have the unique ability to continuously self-renew and differentiate into intermediate and mature cells of a variety of lineages. Though therapeutic potential of adult stem cells is the main focus of our research group; use of ESCs and iPSCs have also been regarded as promising candidates for future therapies considering their pluripotency and personalized therapeutic possibilities. Over the years gut stem cells has been used in analyzing adult stem cell behavior, identifying niche components, modelling pathogen-epithelia interactions, gene editing, disease modelling and orthotopic transplantation etc. The present review highlights the use of intestinal stem cells in various biomedical applications, and how it has helped us to understand many of the complex diseases including various genetic diseases, obesity and cancer and design effective therapy against them.

Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. Whereas techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM) and with glycogen synthase kinase 3 inhibitor (GSK3i), and their effects were studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry and quantitative polymerase chain reaction. Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Whereas ENR medium supported murine enteroid growth, it did not sustain porcine crypts beyond 5days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. Thus, our method maintains juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced by using lentiviral vectors.

Hepatitis E virus (HEV) is a non-enveloped, single-stranded, positive sense RNA virus, which is a major cause of water-borne hepatitis. RNA interference (RNAi) is a sequence-specific cellular antiviral defence mechanism, induced by double-stranded RNA, which we used to investigate knockdown of several genes and the 3′ cis-acting element (CAE) of HEV. In the present report, shRNAs were developed against the putative helicase and replicase domains and the 3′CAE region of HEV. Production of siRNA was confirmed by northern hybridization. The possible innate response induction due to shRNA expressions was verified by transcript analysis for interferon-β and 2′,5′-oligoadenylate synthetase genes and was found to be absent. Initially, the selected shRNAs were tested for their efficiency against the respective genes/3′CAE using inhibition of fused viral subgenomic target domain–renilla luciferase reporter constructs. The effective shRNAs were studied for their inhibitory effects on HEV replication in HepG2 cells using HEV replicon and reporter replicon. RNAi mediated silencing was demonstrated by reduction of luciferase activity in subgenomic target–reporter constructs and reporter replicon. The real time PCR was used to demonstrate inhibition of native replicon replication in transfected cells. Designed shRNAs were found to be effective in inhibiting virus replication to a variable extent (45–93%).

We are evaluating the immune enhancing activities of cytokines for their optimum utility in augmenting cellular immune responses against lung cancer. In this study, we evaluated the mechanism of antitumor responses following IL-7 administration to mice bearing established Lewis lung cancer. IL-7 decreased tumor burden with concomitant increases in the frequency of CD4 and CD8 T lymphocyte subsets, T cell activation markers CXCR3, CD69, and CD127low, effector memory T cells, and T cell cytolytic activity against parental tumor cells. Accompanying the antitumor responses were increases in IFN-γ, CXCL9, CXCL10, and IL-12. Individual neutralization of CD4, CD8 T lymphocytes, or the CXCR3 ligands CXCL9 and CXCL10 reversed the antitumor benefit of IL-7, indicating their importance for optimal responses in vivo. Furthermore, IL-7 decreased the tumor-induced apoptosis of T cells with subsequent decrease of the proapoptotic marker Bim. We assessed the impact of IL-7 treatment on regulatory T cells that negatively impact antitumor immune responses. IL-7 decreased regulatory T Foxp3 as well as cell suppressive activity with a reciprocal increase in SMAD7. These results indicate that IL-7 induces CXCR3 ligand-dependent T cell antitumor reactivity in lung cancer.

Background and Objectives: Studies emphasizing the genotype distribution among ophthalmic isolates of Streptococcus pneumoniae are scarce. There is no conclusive information on genetic relatedness of the ophthalmic isolates with the commensal and systemic isolates of S. pneumoniae. Methods: To find out the relatedness between the ophthalmic, invasive and commensal isolates of S. pneumoniae, genotyping was carried out by using RAPD and BOX-A PCR assays. The serotyping was carried out by a rapid co-agglutination method using 12-pooled S. pneumoniae antisera. Results: Topological relationship was identified in order of similarity, and phylogenetic tree was drawn employing sequential clustering alogarithm by UPGMA linkage analysis. The 61 isolates were distributed in 17 RAPD types, 11 BOX-A types and 21 serotypes. Conclusions: Both genotyping techniques were useful in terms of intraspecies genomic variation in S. pneumoniae. BOX-A PCR typing was found to be slightly superior to RAPD analysis in terms of reproducibility and standardization. No specific S. pneumoniae serotype or genotype was found to have special predilection to cause ophthalmic infections.

The most important system for correcting replication errors that survive the built in editing system of DNA polymerase is the mismatch repair (MMR) system. We have identified a novel mutator strain yycJ in Bacillus anthracis. Mutations in the yycJ gene result in a spontaneous mutator phenotype with a mutational frequency and specificity comparable to that of MMR-deficient strains such as those with mutations in mutL or mutS. YycJ was annotated as a metallo-β-lactamase (MβL) super family member with unknown activity. In this study we carried out a biochemical characterization of YycJ and demonstrated that a recombinant YycJ protein possesses a 5'-3' exonuclease activity at the 5' termini and at nicks of double-stranded DNA. This activity requires a divalent metal cofactor Mn2+ and is stimulated by 5'-phosphate ends of duplex DNA. The mutagenesis of conserved amino acid residues revealed that in addition to the five MβL family conserved motifs, YycJ appears to have its specific motifs that can be used to distinguish YycJ from other closely related MβL family members. A phylogenetic survey showed that putative YycJ homologs are present in several bacterial phyla as well as in members of the Methanomicrobiales and Thermoplasmales from Archaea. We propose that YycJ represents a new group of MβL fold exonucleases, which is likely to act in the recognition of MMR entry point and subsequent removal of the mismatched base in certain MutH-less bacterial species.

Background Myeloid derived suppressor cells (MDSC) are important regulators of immune responses. We evaluated the mechanistic role of MDSC depletion on antigen presenting cell (APC), NK, T cell activities and therapeutic vaccination responses in murine models of lung cancer. Principal Findings Individual antibody mediated depletion of MDSC (anti-Gr1 or anti-Ly6G) enhanced the antitumor activity against lung cancer. In comparison to controls, MDSC depletion enhanced the APC activity and increased the frequency and activity of the NK and T cell effectors in the tumor. Compared to controls, the anti-Gr1 or anti-Ly6G treatment led to increased: (i) CD8 T cells, (ii) NK cells, (iii) CD8 T or NK intracytoplasmic expression of IFNγ, perforin and granzyme (iv) CD3 T cells expressing the activation marker CD107a and CXCR3, (v) reduced CD8 T cell IL-10 production in the tumors (vi) reduced tumor angiogenic (VEGF, CXCL2, CXCL5, and Angiopoietin1&2) but enhanced anti-angiogenic (CXCL9 and CXCL10) expression and (vii) reduced tumor staining of endothelial marker Meca 32. Immunocytochemistry of tumor sections showed reduced Gr1 expressing cells with increased CD3 T cell infiltrates in the anti-Gr1 or anti-Ly6G groups. MDSC depletion led to a marked inhibition in tumor growth, enhanced tumor cell apoptosis and reduced migration of the tumors from the primary site to the lung compared to controls. Therapeutic vaccination responses were enhanced in vivo following MDSC depletion with 50% of treated mice completely eradicating established tumors. Treated mice that rejected their primary tumors acquired immunological memory against a secondary tumor challenge. The remaining 50% of mice in this group had 20 fold reductions in tumor burden compared to controls. Significance Our data demonstrate that targeting MDSC can improve antitumor immune responses suggesting a broad applicability of combined immune based approaches against cancer. This multifaceted approach may prove useful against tumors where MDSC play a role in tumor immune evasion.

BackgroundModifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier.Methodology and principal findingsWe characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA) encased in vault nanocapsules and liposomes. We measured OVA responsive CD8(+) and CD4(+) memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8(+) memory T cells and production of IFNγ plus CD4(+) memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes.Conclusions/significanceThese experiments show that vault nanocapsules induced strong anti-OVA CD8(+) and CD4(+) T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8(+) and CD4(+) T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer.

BackgroundBased on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer.Principal findingsHere we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3(+)T, CCR7(+)T, IFNγ(+)T lymphocytes, DEC205(+) DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells.SignificanceThis study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an "off the shelf" approach to deliver antitumor cytokines to treat a broad range of malignancies.

Streptococcus pneumoniae is common in ocular and systemic infections and is a part of normal nasopharyngeal flora. Very few studies regarding genetic analysis of S. pneumoniae isolates causing eye infections are available. This study was undertaken to do pulse field gel electrophoresis (PFGE) analysis and ribotyping of S. pneumoniae isolates obtained from eye infections, systemic infections and nasopharyngeal flora.Sixty one well characterized S. pneumoniae isolates (38 from ophthalmic infections, 9 from systemic infections and 14 commensals) were characterized using PFGE of the whole genome after SmaI, restriction enzyme digestion and conventional ribotyping using Escherichia coli rRNA operon as the probe. Phylogenetic tree was drawn using unweighted pair group method analysis (UPGMA).The 38 S. pneumoniae isolates from eye infections belonging to 15 serotypes were placed in to 11 PFGE types and 15 ribotypes. The 9 systemic isolates (7 seotypes) were distributed in 7 PFGE types and 6 ribotypes. The 14 commensal isolates were placed in 11 serotypes, 5 PFGE types and 6 ribotypes. Most of the PFGE types and ribotypes consisting of ocular isolates also contained systemic and commensal isolates.Considerable genetic similarity was observed between the isolates from ocular and systemic infections and those colonized in nasopharynx. PFGE analysis could differentiate majority of the isolates according to site of infections. There was a considerable DNA polymorphism within the studied bacterial population.

Information regarding serotype distributions of Streptococcus pneumoniae causing ophthalmic infections is scanty. This study was therefore undertaken to determine the antimicrobial susceptibility status and serotypes of S. pneumoniae isolated from various ophthalmic infections and to compare with those isolated from systemic infections and commensal nasopharyngeal flora.Thirty eight of S. pneumoniae isolates from ophthalmic infections, 9 from systemic infections and 14 from the nasopharynx of apparently healthy school children were biochemically characterized and tested for in vitro antimicrobial susceptibility to various antibiotics. Serotyping of these 61 isolates was done by a rapid co-agglutination method.All the 61 isolates were sensitive to oxacillin (penicillin) and susceptibility against other antimicrobials was variable. No multidrug resistance was observed. The 38 ophthalmic isolates were distributed in 15 different serotypes. Most prevalent serotypes were 14, followed by 8 and 19F. The 9 systemic and 14 commensal. isolates of S. pneumoniae were distributed in 7 and 11 serotypes respectively. Three of the systemic and six of the commensal serotypes were observed in ophthalmic infections whereas four of the commensal serotypes were observed in systemic infections.Resistance to penicillin was not observed. In ophthalmic infections, a wide range of serotypes of S. pneumoniae were observed. More than half of the commensal serotypes obtained in the study as well as majority of the systemic serotypes were observed in ophthalmic infections.

While most forms of Parkinson's Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice. © 2013 Sarafian et al.

Lung cancer remains the leading cause of cancer death in Unites States. Metastatic spread of tumors is the most common cause of therapy failure and mortality for lung cancer. We are defining genetic programs in lung cancer that modulate immune responses that regulate tumor growth and metastases. Activating immune effectors for tumor destruction has the potential for long-term protection and survival. Cancer cells acquire the ability to progress, invade, and metastasize by undergoing the process of epithelial-mesenchymal transition (EMT), by activating transcription factors (for example, Snail, Twist, Zeb, Slug) that repress E-cadherin, a transmembrane glycoprotein essential for epithelial cell-cell adhesion. These transcriptional repressors are normally active during embryogenesis where they program EMT to enable various morphogenetic steps. EMT is involved in tumor progression. Snail expression in primary NSCLCs has been associated with a shorter overall survival. Recent studies have demonstrated the importance of Snail in tumor EMT-induced metastases in melanoma. In this study we are evaluating the mechanistic role of tumor Snail expression on tumor growth and metastases. Our data demonstrates that tumor Snail expression mediates tumor growth and metastasis by modulating immune responses. Genetic knockdown of endogenous tumor Snail expression in the murine lung cancer cell lines [Lewis Lung Cancer (3LL) that spontaneously arose in the C57BL/6 mice] with lentiviral Snail shRNA was validated by qRTPCR and Western blot analyses. With the Snail shRNA we have achieved stable 80–90% Snail knockdown. No differences were noted in growth kinetics of Snail knockdown or controls in vitro. Flow cytometry was utilized to quantify changes in tumor and spleen leukocytic populations. Lung cancer Snail knockdown cells compared to controls: 1) decreased invasiveness (3-fold) and tumor-derived TGFβ (3.3-fold); 2) reduced subcutaneous tumor growth (4-fold), lung metastases (2-fold), tumor nodule TGFβ by ELISA(5-fold), MMP9, and VEGF (2-fold) by qRTPCR; 3) increased tumor infiltrates of CD4 (2-fold) and CD8 (1.5-fold) T lymphocytes that elaborated enhanced IFNγ≈2.5-fold) but reduced levels of IL-10 (2-fold); 4) increased expression of the CD107 (≈7-fold) cytolytic marker in tumor-infiltrating CD8 T cells; 5) augmented the frequencies of innate NK effectors and dendritic cells (DC) (7-fold); 6) reduced the immune suppressors MDSC (2-fold); and 7) reduced MDSC as well as the non-MDSC populations intracellular expression of arginase in the tumors (≈3–4 fold). Although MDSC from the tumor Snail knockdown group was comparable to the controls in suppressing anti-CD3/CD28 T cell proliferation, its suppressive effects on DC antigen processing and presentation activity (APC) to an antigen-specific CD8 T cell line was reduced (2-fold) in comparison to controls. The suppressive effects of MDSC on T cell proliferation and APC activity was reversed (≈2-fold) by catalase or N-acetyl cysteine, respectively. Further experiments will mechanistically delineate the genetic program(s) induced by tumor Snail knockdown that alter the balance of immune effectors and suppressors in the tumor. An adequate understanding of the genetic signatures in the tumor and tumor-host interactions that induce immune suppression and promote tumor growth, invasion and metastases will be crucial for the development of immune-based therapies for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A17.