An Elementary Model of Focal Adhesion Detachment and Reattachment During Cell Reorientation Using Ideas from the Kinetics of Wiggly Energies

A simple, transparent, two-dimensional, nonlinear model of cell reorientation is constructed in this paper. The cells are attached to a substrate by “focal adhesions” that transmit the deformation of the substrate to the “stress fibers” in the cell. When the substrate is subjected to a deformation, say an in-plane bi-axial deformation with stretches λ1 and λ2, the stress fibers deform with it and change their length and orientation. In addition, the focal adhesions can detach from the substrate and reattach to it at new nearby locations, and this process of detachment and reattachment can happen many times. In this scenario the (varying) fiber angle Θ in the reference configuration plays the role of an internal variable. In addition to the elastic energy of the stress fibers, the energy associated with the focal adhesions is accounted for by a wiggly energy ϵacos Θ / ϵ, 0 < ϵ≪ 1. Each local minimum of this energy corresponds to a particular configuration of the focal adhesions. The small amplitude ϵa indicates that the energy barrier between two neighboring configurations is relatively small, and the small distance 2 πϵ between the local minima indicates that a focal adhesion does not have to move very far before it reattaches. The evolution of this system is studied using a gradient flow kinetic law, which is homogenized for ϵ→ 0 using results from weak convergence. The results determine (a) a region of the λ1, λ2-plane in which the (referential) fiber orientation remains stuck at the angle Θ and does not evolve, and (b) the evolution of the orientation when the stretches move out of this region as the fibers seek to minimize energy

Immune-mediated loss of transgene expression from virally transduced brain cells is irreversible, mediated by IFNγ, perforin, and TNFα, and due to the elimination of transduced cells

The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4 and CD8 T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cellsFil: Zirger, Jeffrey M.. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Puntel, Mariana. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bergeron, Josee. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Wibowo, Mia. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Moridzadeh, Rameen. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Bondale, Niyati. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Barcia, Carlos. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Kroeger, Kurt M.. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Liu, Chunyan. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Castro, Maria Graciela. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos. University of Michigan; Estados UnidosFil: Lowenstein, Pedro R.. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados Unidos. University of Michigan; Estados Unido

How digital is agriculture in a subset of countries from South America? Adoption and limitations

Digital agriculture (DA) can contribute solutions to meet an increase in healthy, nutritious, and affordable food demands in an efficient and sustainable way. South America (SA) is one of the main grain and protein producers in the world but the status of DA in the region is unknown. A systematic review and case studies from Brazil, Argentina, Uruguay, and Chile were conducted to address the following objectives: (1) quantify adoption of existing DA technologies, (2) identify limitations for DA adoption; and (3) summarise existing metrics to benchmark DA benefits. Level of DA adoption was led by Brazil and Argentina followed by Uruguay and at a slower rate, Chile. GPS guidance systems, mapping tools, mobile apps and remote sensing were the most adopted DA technologies in SA. The most reported limitations to adoption were technology cost, lack of training, limited number of companies providing services, and unclear benefits from DA. Across the case studies, there was no clear definition of DA. To mitigate some of these limitations, our findings suggest the need for a DA educational curriculum that can fulfill the demand for job skills such as data processing, analysis and interpretation. Regional efforts are needed to standardise these metrics. This will allow stakeholders to design targeted initiatives to promote DA towards sustainability of food production in the region

Safety Profile of Gutless Adenovirus Vectors Delivered into the Normal Brain Parenchyma: Implications for a Glioma Phase 1 Clinical Trial

Abstract Adenoviral vectors (Ads) have been evaluated in clinical trials for glioma. However, systemic immunity against the vectors can hamper therapeutic efficacy. We demonstrated that combined immunostimulation and cytotoxic gene therapy provides long-term survival in preclinical glioma models. Because helper-dependent high-capacity Ads (HC-Ads) elicit sustained transgene expression, in the presence of antiadenoviral immunity, we engineered HC-Ads encoding conditional cytotoxic herpes simplex type 1 thymidine kinase and immunostimulatory cytokine Fms-like tyrosine kinase ligand-3 under the control of the TetOn system. Escalating doses of combined HC-Ads (1?108, 1?109, and 1?1010 viral particles [VP]) were delivered into the rat brain. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points after vector delivery. Histopathological analysis did not reveal any evidence of toxicity or long-term inflammation at the lower doses tested. Vector genomes were restricted to the injection site. Serum chemistry did not uncover adverse systemic side effects at any of the doses tested. Taken together, our data indicate that doses of up to 1?109 VP of each HC-Ad can be safely administered into the normal brain. This comprehensive toxicity and biodistribution study will lay the foundations for implementation of a phase 1 clinical trial for GBM using HC-Ads.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98452/1/hgtb%2E2012%2E060.pd

Study of the Efficacy, Biodistribution, and Safety Profile of Therapeutic Gutless Adenovirus Vectors as a Prelude to a Phase I Clinical Trial for Glioblastoma

Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor in humans. Systemic immunity against gene therapy vectors has been shown to hamper therapeutic efficacy; however, helper-dependent high-capacity adenovirus (HC-Ad) vectors elicit sustained transgene expression, even in the presence of systemic anti-adenoviral immunity. We engineered HC-Ads encoding the conditional cytotoxic herpes simplex type 1 thymidine kinase (TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Flt3L). Flt3L expression is under the control of the regulatable Tet-ON system. In anticipation of a phase I clinical trial for GBM, we assessed the therapeutic efficacy, biodistribution, and clinical and neurotoxicity with escalating doses of HC-Ad-TetOn-Flt3L + HC-Ad-TK in rats. Intratumoral administration of these therapeutic HC-Ads in rats bearing large intracranial GBMs led to long-term survival in ~70% of the animals and development of antiglioma immunological memory without signs of neuropathology or systemic toxicity. Systemic anti-adenoviral immunity did not affect therapeutic efficacy. These data support the idea that it would be useful to develop HC-Ad vectors further as a therapeutic gene-delivery platform to implement GBM phase I clinical trials

How digital is agriculture in a subset of countries from South America? Adoption and limitations.

Abstract. Digital agriculture (DA) can contribute solutions to meet an increase in healthy, nutritious, and affordable food demands in an efficient and sustainable way. South America (SA) is one of the main grain and protein producers in the world but the status of DA in the region is unknown. A systematic review and case studies from Brazil, Argentina, Uruguay, and Chile were conducted to address the following objectives: (1) quantify adoption of existing DA technologies, (2) identify limitations for DA adoption; and (3) summarise existing metrics to benchmark DA benefits. Level of DA adoption was led by Brazil and Argentina followed by Uruguay and at a slower rate, Chile. GPS guidance systems, mapping tools, mobile apps and remote sensing were the most adopted DA technologies in SA. The most reported limitations to adoption were technology cost, lack of training, limited number of companies providing services, and unclear benefits from DA. Across the case studies, there was no clear definition of DA. To mitigate some of these limitations, our findings suggest the need for a DA educational curriculum that can fulfill the demand for job skills such as data processing, analysis and interpretation. Regional efforts are needed to standardise these metrics. This will allow stakeholders to design targeted initiatives to promote DA towards sustainability of food production in the region.Special issue

Identification and Visualization of CD8+ T Cell Mediated IFN-γ Signaling in Target Cells during an Antiviral Immune Response in the Brain

CD8+ T cells infiltrate the brain during an anti-viral immune response. Within the brain CD8+ T cells recognize cells expressing target antigens, become activated, and secrete IFNγ. However, there are no methods to recognize individual cells that respond to IFNγ. Using a model that studies the effects of the systemic anti-adenoviral immune response upon brain cells infected with an adenoviral vector in mice, we describe a method that identifies individual cells that respond to IFNγ. To identify individual mouse brain cells that respond to IFNγ we constructed a series of adenoviral vectors that contain a transcriptional response element that is selectively activated by IFNγ signaling, the gamma-activated site (GAS) promoter element; the GAS element drives expression of a transgene, Cre recombinase (Ad-GAS-Cre). Upon binding of IFNγ to its receptor, the intracellular signaling cascade activates the GAS promoter, which drives expression of the transgene Cre recombinase. We demonstrate that upon activation of a systemic immune response against adenovirus, CD8+ T cells infiltrate the brain, interact with target cells, and cause an increase in the number of cells expressing Cre recombinase. This method can be used to identify, study, and eventually determine the long term fate of infected brain cells that are specifically targeted by IFNγ. The significance of this method is that it will allow to characterize the networks in the brain that respond to the specific secretion of IFNγ by anti-viral CD8+ T cells that infiltrate the brain. This will allow novel insights into the cellular and molecular responses underlying brain immune responses

Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF.Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-alpha and IFN-gamma. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation.These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells' vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials

T Cells' Immunological Synapses Induce Polarization of Brain Astrocytes In Vivo and In Vitro: A Novel Astrocyte Response Mechanism to Cellular Injury

Astrocytes usually respond to trauma, stroke, or neurodegeneration by undergoing cellular hypertrophy, yet, their response to a specific immune attack by T cells is poorly understood. Effector T cells establish specific contacts with target cells, known as immunological synapses, during clearance of virally infected cells from the brain. Immunological synapses mediate intercellular communication between T cells and target cells, both in vitro and in vivo. How target virally infected astrocytes respond to the formation of immunological synapses established by effector T cells is unknown.Herein we demonstrate that, as a consequence of T cell attack, infected astrocytes undergo dramatic morphological changes. From normally multipolar cells, they become unipolar, extending a major protrusion towards the immunological synapse formed by the effector T cells, and withdrawing most of their finer processes. Thus, target astrocytes become polarized towards the contacting T cells. The MTOC, the organizer of cell polarity, is localized to the base of the protrusion, and Golgi stacks are distributed throughout the protrusion, reaching distally towards the immunological synapse. Thus, rather than causing astrocyte hypertrophy, antiviral T cells cause a major structural reorganization of target virally infected astrocytes.Astrocyte polarization, as opposed to hypertrophy, in response to T cell attack may be due to T cells providing a very focused attack, and thus, astrocytes responding in a polarized manner. A similar polarization of Golgi stacks towards contacting T cells was also detected using an in vitro allogeneic model. Thus, different T cells are able to induce polarization of target astrocytes. Polarization of target astrocytes in response to immunological synapses may play an important role in regulating the outcome of the response of astrocytes to attacking effector T cells, whether during antiviral (e.g. infected during HIV, HTLV-1, HSV-1 or LCMV infection), anti-transplant, autoimmune, or anti-tumor immune responses in vivo and in vitro