STUDIES ON REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY IN A PRIMITIVE TELEOSTEI, THE AMERICAN SHAD (Alosa sapidissima)

American shad is an anadromous fish that displays asynchronous ovarian development. In an effort to enhance our understanding of the reproductive cycle of this primitive Teleostei species, and provide better management tools for the Chesapeake fishery, we have conducted studies of its gonadal and hormonal cycles. We developed various assays to measure reproductive endocrine factors including: 17,20b-dihydroxy-4-pregnen-3-one (DHP) and GnRH. Fish were collected from the Susquehanna River during their spawning migration. One group of fish was sacrificed on site to assess reproductive parameters of wild shad. A second group was transported to a Maryland State hatchery and treated with gonadotropin-releasing hormone agonist (GnRHa) using several delivery systems. These treatments were followed during a two-week period by measurement of various hormonal levels. In addition, fecundity and fertilization of the hatchery groups were measured daily. Our results shed light on the reproductive physiology and endocrinology of the American shad and lay the foundation for usage of GnRHa to induce shad spawning in captivity

The Gonadotropin Releasing Hormone-3 System in Zebrafish: Early Development and Regulation

The objective of this study was to expand our understanding of the early development of forebrain Gonadotropin Releasing Hormone (GnRH) neurons in vertebrates in general and in fish in particular. The correct migration during early development of the hypophysiotropic GnRH neurons from the olfactory region to the hypothalamus is crucial for normal gonadal development and reproduction. We developed a Tg(GnRH3:EGFP) zebrafish line in which EGFP is specifically expressed in GnRH3 neurons. Using this line, we have studied in detail the early spatiotemporal development of the GnRH3 system in vivo. In addition, we have studied various factors, including GnRH3, Netrins and Hedgehog to better understand some of the mechanisms that mediate this complex axophilic neuron migration event. Lastly, we have conducted targeted GnRH3 neuron ablation experiments in view of determining the embryonic origin of POA-hypothalamic GnRH3 neurons and the effect of lack of GnRH3 neurons in the CNS. Our findings show that: 1) GnRH neurons first differentiate and express GnRH3 at 24-26 hours post fertilization (hpf) and immediately thereafter begin to extend fibers. 2) GnRH3 neurons project a complex network of fibers, prior the GnRH3 soma migration, to various CNS regions, and to the pituitary. 3) GnRH3 soma begin migrating towards the hypothalamus at 3 days post fertilization (dpf), passing through the terminal nerve (TN), lateral telencephalon, and reaching the hypothalamus by 12 dpf. 4) expression of GnRH3 itself is necessary for the normal early differentiation and fiber extensions of GnRH3 neurons. 5) Netrin1a is directly involved as a chemoattractant in GnRH3 fiber organization and subsequently, in GnRH3 soma migration to the hypothalamus. 6). Netrin2 is required for normal early ZF embryogenesis. 7). Sonic hedgehog a does not serve as a specific factor in the development of the GnRH3 system. 8). GnRH3 neuron regeneration capacity is temporally limited. 9). Successful ablation of olfactory GnRH3 neurons during development results in lack of GnRH3 neurons in the entire sexually mature brain as well as abnormal gonadal development and inability to reproduce. This study expands our understanding vis-à-vis the early events that occur during GnRH3 system development and that regulate this complex process. In a broader sense these findings augment current knowledge regarding the regulation of long range tangential neuron migration during development

Bespoke cell therapy manufacturing platforms - a contradiction in terms?

Advanced biological therapies, such as cell and viral therapeutics, will have a transformative effect on healthcare. In many cases these therapies are curative rather than palliative and aim to treat a wide range of diseases including malignancies, cardiovascular, immune, and metabolic disorders. As cell therapies begin to enter commercialization stages, some of the bigger challenges that need to be addressed include bottlenecks in production and high cost of goods. At the same time, switching to manufacturing platforms that might allow scale-up (allogeneic) and scale-out (autologous) to allow commercialization at an acceptable cost of goods could result in changes to the cell product critical quality attributes. For the field to move forward, it is imperative to enable the use of production platforms that allow commercialization, yielding high quality and quantity of cells at acceptable costs. Yet, as opposed to the highly commercialized mammalian cell protein manufacturing, in which the same cell types and processes are used to produce many different proteins, the diversity of cell types and processes in cell therapy may require significantly different manufacturing methods. Does this mean that a multitude of different manufacturing platforms is needed and feasible for cell therapy manufacturing? Or is the development of a one-size-fits-all platform a superior and possible approach? How would an optimal bespoke platform approach look like, and would it work for different modalities (allogeneic vs. autologous), and a diversity of cell types and processes? These questions will be addressed and possible considerations and solutions presented

Manufacturing of patient specific novel T cell therapies using the Cocoon® Platform automated system

Engineered T cell therapies, particularly chimeric antigen receptor T cell (CAR-T) immunotherapies, have proved effective against hematologic cancers. However, CAR-T therapies can potentiate immune responses causing cytokine release syndrome (CRS; “cytokine storm”) leading to adverse events in patients. Additionally, CAR-T has shown sporadic success in solid tumor indications. Novel therapies which activate T cells via the native T cell receptors (TCR) have shown greater tumor antigen recognition providing an alternative therapy which may prove effective against solid tumors. Utilizing novel cell immunotherapy modalities is only part of the solution as challenges remain to scale manufacturing to meet commercial demand. Scaling out commercial patient-specific cell therapy manufacturing for large populations using current methods will be expensive (cleanrooms and FTEs) and complex (logistics). Innovative manufacturing solutions will be required to manufacture patient-specific therapies in a robust and cost-effective manner. The Cocoon® Platform is one such innovation, a functionally-closed, automated, scalable cell therapy manufacturing platform. This abstract highlights a therapeutic T cell process translated from an open, manual process to the Cocoon® Platform. During process translation, the functionally-closed Cocoon® Platform was used to automate cell seeding, activation, transduction, feeding, real-time process monitoring, washing, and final product harvest using the single-use Cocoon cassette. During process development and translation, important process parameters were identified, optimized, and programmed enabling multiple process step automation removing the need for manual intervention. For the process, 200 million CD4+ and CD8+ isolated T cells were inoculated with TransActTM activator. The following day, cells were transduced with HER-2 lentivirus vector at various multiplicities of infection (MOI). Cells were expanded with a predefined feeding strategy in media supplemented with IL-2 until final product harvest. Following harvest, cells were assessed for cell yield, viability, transduction efficiency, and VCN. T cell phenotype and functionality was assessed via flow cytometry. The Cocoon manufacturing processes yielded 2.7 x 109 viable cells on average with viability \u3e85%. The Cocoon processes supported both CD4+ and CD8+ T cell expansion with 68% CD4+ T cells and 31% CD8+ T cells on average. The final product exhibited high T cell purity and viability (i.e. \u3e90% abTCR+ and 89% abTCR+, respectively) with transduction efficiencies varied from ~30% to \u3e65% depending on the process MOI. Vector copy number (VCN) was evaluated after each process and found to be ≤5 copies/transduced T cell. In summary, a gene-modified T cell process was successfully translated to the Cocoon and the harvested final products met all pre-defined acceptable criteria. The Cocoon represents a tool for manufacturing cell therapies in a robust manner, while maintaining comparability, and lowering manufacturing costs via increased automation. Ultimately the Cocoon will enable and accelerate development of cell therapies to address solid tumor indications and meet a critical patient need

Enabling stem cell based therapies: Adaptable and scalable manufacturing of human pluripotent stem cells

Enabling stem cell-base therapies requires innovative solutions to close the gaps which exist between research and commercialization. Allogeneic cell therapy indications that target large patient populations will necessitate the use of flexible cell production platforms to meet required cell quantities. Here we will show how moving away from conventional 2D culture platforms and developing a truly scalable, controlled bioreactor platforms for cell expansion enables meeting cell quantity demand for clinical applications while allowing comparability between the various scales. Likewise, it enhances process automation and allows integration of online monitoring systems. These bioreactor platforms are flexible cell production platforms, applicable to various cell types. Utilizing many common components, such as bioreactor controllers and centralized up-stream and down-stream hardware, while being able to quickly and easily change components such as vessels, media and microcarriers. The capability of effectively culturing adherent stem cells, namely pluripotent stem cells, will be presented. Cells are expanded in suspension, in a controlled bioreactor, obtaining high fold expansion without compromising cell quality, and the capacity to be further differentiated. This achieved through avoiding 2D cell culture steps, reduces footprint, labor and cost, while enhancing process control and cell product quality

Networks and Language in the 2010 Election

The midterm (2010) election in the U.S. presented a unique opportunity to study the online social media strategy of various political groups. Although candidates had previously leveraged social media, the prevalence of use during this election allows us to study a significant percentage of candidates and a novel glimpse into their networks and messaging. In combination, the networks and associated content reflect positioning of candidates both structurally and in framing in relation to other politicians. In our work, we study the use of Twitter by House, Senate and gubernatorial candidates during the midterm elections in the U.S. Our data includes almost 700 candidates and over 460k tweets that they produced in the 3.5 years leading to the elections. We utilize graph and text mining techniques to analyze differences between Democrats, Republicans and Tea Party candidates, and suggest a novel use of language modeling for estimating content cohesiveness. Our findings show significant differences in the usage patterns of social media, and suggest conservative candidates used this medium more effectively, conveying a coherent message and maintaining a dense graph of connections. Despite the lack of party leadership, we find Tea Party members display both structural and language‐based cohesiveness. Finally, we investigate the relation between network structure, content and election results by creating a proof‐of‐concept model that extends incumbency models to predict candidate victory

An automated and closed system for patient specific CAR-T cell therapies

Autologous cell therapies, particularly chimeric antigen receptor T-cell (CAR-T) immunotherapies, are becoming a promising treatment option for difficult diseases. Immunotherapies for blood cancers have dominated the pipeline, while treatments for solid tumors have started to become more successful. However, as the market continues to grow and more clinical trials begin globally, the challenge of manufacturing autologous cell therapies remains significant. A greater number of patients will lead to an increase in cost, labor, and the complexity of logistics for scaling out the commercial production of patient specific therapies. To enable clinical and commercial success, novel manufacturing platforms, such as closed and automated systems, will be required to produce cost effective and robust therapies. This abstract highlights a successful CAR-T process translation from a manual process to an automated patient scale system. To accomplish a CAR-T process translation, we utilized a platform that automates cell seeding, activation, transduction, real time process monitoring, feeding, washing and concentration, and harvesting. In order to mimic a therapeutic CAR-T cell process, manual research scale processes were optimized, scaled up, and then programmed to run automatically without manual intervention. In these processes, 100 million peripheral blood mononuclear cells (PBMC) were first inoculated with CD3/CD28 activation beads. The following day, cells were transduced with HER-2 lentivirus vector. Cells were then expanded with a defined feeding strategy and IL-2 supplements until harvested when target yields were reached. After harvest, cells were analyzed for cell yield, viability, transduction efficiency, and an array of cell phenotype, potency and functionality via FACS and killing assays. Specifically, CAR-T cells were analyzed for the presence of naïve T cells, T stem cell memory, T central memory, T effector memory, and T effector cells. We show here how we optimized, scaled up, and automated manual processes to reach clinical requirements. Automated runs using the above process with cells transduced by HER-2 virus yielded an average of 2 x 109 cells post harvest with a viability \u3e 90%. Automated runs and associated controls were able to support the expansion of both CD4+ and CD8+ T cells with 73% CD4+ T cells and 20% CD8+ T cells. Harvested cells yielded approximately 80% NGFR+ cells with a higher detection of NGFR in the CD4+ fraction than in the CD8+ fraction for all samples. Both CD4+ and CD8+ subsets demonstrated T cell phenotype such as naïve T cells, T stem cell memory, T central memory, T effector memory, and T effector cells. Both subsets also only expressed between 15-20% of immunosuppressive regulatory T cells. Cell health was evaluated by the levels of exhaustion marker, PD-1, which was 19% in CD4+ T cells and \u3c 1% in CD8+ T cells. Furthermore, there was a negligible amount of senescent T cells and anergic cells and \u3c 10% expression of the apoptotic marker, Caspase-3. Subsequently, cells from multiple automated runs showed the specific killing of NGFR+ tumor line were correlated with high levels of effector cytokines: TNF-alpha (~34%) and IFN-gamma (20-25%) as compared to a manual control. In summary, automated CAR-T process in the Cocoon system yields a healthy populations of T cell subsets. This system is a viable solution to translate labor-intensive CAR-T process into a fully automated system, thus allowing scalability, high yield, reduction of manufacturing cost, and better process control to yield high quality CAR-T cells

Tuning of Collagen Scaffold Properties Modulates Embedded Endothelial Cell Regulatory Phenotype in Repair of Vascular Injuries In Vivo

Perivascularly implanted matrix embedded endothelial cells (MEECs) are potent regulators of inflammation and intimal hyperplasia following vascular injuries. Endothelial cells (ECs) in collagen scaffolds adopt a reparative phenotype with significant therapeutic potential. Although the biology of MEECs is increasingly understood, tuning of scaffold properties to control cell-substrate interactions is less well-studied. It is hypothesized that modulating scaffold degradation would change EC phenotype. Scaffolds with differential degradation are prepared by cross-linking and predegradation. Vascular injury increases degradation and the presence of MEECs retards injury-mediated degradation. MEECs respond to differential scaffold properties with altered viability in vivo, suppressed smooth muscle cell (SMC) proliferation in vitro, and altered interleukin-6 and matrix metalloproteinase-9 expression. When implanted perivascularly to a murine carotid wire injury, tuned scaffolds change MEEC effects on vascular repair and inflammation. Live animal imaging enables real-time tracking of cell viability, inflammation, and scaffold degradation, affording an unprecedented understanding of interactions between cells, substrate, and tissue. MEEC-treated injuries improve endothelialization and reduce SMC hyperplasia over 14 d. These data demonstrate the potent role material design plays in tuning MEEC efficacy in vivo, with implications for the design of clinical therapies.National Institutes of Health (U.S.) (Grant R01 GM 49039

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Cell therapy manufacturing single use components: Implementation and considerations

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