Inflammation and Wnt signaling: target for immunomodulatory therapy?

Wnt proteins comprise a large family of highly conserved glycoproteins known for their role in development, cell fate specification, tissue regeneration, and tissue homeostasis. Aberrant Wnt signaling is linked to developmental defects, malignant transformation, and carcinogenesis as well as to inflammation. Mounting evidence from recent research suggests that a dysregulated activation of Wnt signaling is involved in the pathogenesis of chronic inflammatory diseases, such as neuroinflammation, cancer-mediated inflammation, and metabolic inflammatory diseases. Recent findings highlight the role of Wnt in the modulation of inflammatory cytokine production, such as NF-kB signaling and in innate defense mechanisms as well as in the bridging of innate and adaptive immunity. This sparked the development of novel therapeutic treatments against inflammatory diseases based on Wnt modulation. Here, we summarize the role and function of the Wnt pathway in inflammatory diseases and focus on Wnt signaling as underlying master regulator of inflammation that can be therapeutically targeted.Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Forced degradation of cell-based medicinal products guided by flow imaging microscopy: explorative studies with Jurkat cells

Cell-based medicinal products (CBMPs) offer ground-breaking opportunities to treat diseases with limited or no therapeutic options. However, the intrinsic complexity of CBMPs results in great challenges with respect to analytical characterization and stability assessment. In our study, we submitted Jurkat cell suspensions to forced degradation studies mimicking conditions to which CBMPs might be exposed from procurement of cells to administration of the product. Flow imaging microscopy assisted by machine learning was applied for determination of cell viability and concentration, and quantification of debris particles. Additionally, orthogonal cell characterization techniques were used. Thawing of cells at 5 °C was detrimental to cell viability and resulted in high numbers of debris particles, in contrast to thawing at 37 °C or 20 °C which resulted in better stability. After freezing of cell suspensions at -18 °C in presence of dimethyl sulfoxide (DMSO), a DMSO concentration of 2.5% (v/v) showed low stabilizing properties, whereas 5% or 10% was protective. Horizontal shaking of cell suspensions did not affect cell viability, but led to a reduction in cell concentration. Fetal bovine serum (10% [v/v]) protected the cells during shaking. In conclusion, forced degradation studies with application of orthogonal analytical characterization methods allow for CBMP stability assessment and formulation screening.Drug Delivery Technolog

Combining mobilizing agents with busulfan to reduce chemotherapy-based conditioning for hematopoietic stem cell transplantation

In the context of hematopoietic stem cell (HSC) transplantation, conditioning with myelo- and immune-ablative agents is used to eradicate the patient's diseased cells, generate space in the marrow and suppress immune reactions prior to the infusion of donor HSCs. While conditioning is required for effective and long-lasting HSC engraftment, currently used regimens are also associated with short and long-term side effects on extramedullary tissues and even mortality. Particularly in patients with severe combined immunodeficiency (SCID), who are generally less than 1-year old at the time of transplantation and often suffer from existing comorbidities. There is a pressing need for development of alternative, less toxic conditioning regimens. Hence, we here aimed to improve efficacy of currently used myeloablative protocols by combining busulfan with stem-cell niche-directed therapeutic agents (G-CSF or plerixafor) that are approved for clinical use in stem cell mobilization. T, B and myeloid cell recovery was analyzed in humanized NSG mice after different conditioning regimens. Increasing levels of human leukocyte chimerism were observed in a busulfan dose-dependent manner, showing comparable immune recovery as with total body irradiation in CD34-transplanted NSG mice. Notably, a better T cell reconstitution compared to TBI was observed after busulfan conditioning not only in NSG mice but also in SCID mouse models. Direct effects of reducing the stem cell compartment in the bone marrow were observed after G-CSF and plerixafor administration, as well as in combination with low doses of busulfan. Unfortunately, these direct effects on the stem population in the bone marrow were not reflected in increased human chimerism or immune recovery after CD34 transplantation in NSG mice. These results indicate moderate potential of reduced conditioning regimens for clinical use relevant for all allogeneic transplants.Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Particulate impurities in cell-based medicinal products traced by flow imaging microscopy combined with deep learning for image analysis

Cell-based medicinal products (CBMPs) are rapidly gaining importance in the treatment of life-threatening diseases. However, the analytical toolbox for characterization of CBMPs is limited. The aim of our study was to develop a method based on flow imaging microscopy (FIM) for the detection, quantification and characterization of subvisible particulate impurities in CBMPs. Image analysis was performed by using an image classification approach based on a convolutional neural network (CNN). Jurkat cells and Dynabeads were used in our study as a representation of cellular material and non-cellular particulate impurities, respectively. We demonstrate that FIM assisted with CNN is a powerful method for the detection and quantification of Dynabeads and cells with other process related impurities, such as cell agglomerates, cell-bead adducts and debris. By using CNN, we achieved a more than 50-fold lower misclassification rate compared with the use of output parameters from the FIM software. The limit of detection was ~15 000 beads/mL in the presence of ~500 000 cells/mL, making this approach suitable for the detection of these particulate impurities in CBMPs. In conclusion, CNN-assisted FIM is a powerful method for the detection and quantification of cells, Dynabeads and other subvisible process impurities potentially present in CBMPs.Drug Delivery Technolog

Functional definition of a transcription factor hierarchy regulating T cell lineage commitment

T cell factor 1 (Tcf1) is the first T cell-specific protein induced by Notch signaling in the thymus, leading to the activation of two major target genes, Gata3 and Bcl11b. Tcf1 deficiency results in partial arrests in T cell development, high apoptosis, and increased development of B and myeloid cells. Phenotypically, seemingly fully T cell-committed thymocytes with Tcf1 deficiency have promiscuous gene expression and an altered epigenetic profile and can dedifferentiate into more immature thymocytes and non-T cells. Restoring Bcl11b expression in Tcf1-deficient cells rescues T cell development but does not strongly suppress the development of non-T cells; in contrast, expressing Gata3 suppresses their development but does not rescue T cell development. Thus, T cell development is controlled by a minimal transcription factor network involving Notch signaling, Tcf1, and the subsequent division of labor between Bcl11b and Gata3, thereby ensuring a properly regulated T cell gene expression program.Molecular Technology and Informatics for Personalised Medicine and Healt

Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

<p>Abstract</p> <p>Background</p> <p>Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of <it>in vivo </it>expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells.</p> <p>Results</p> <p>We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus.</p> <p>Conclusions</p> <p>These findings indicate that the p53-responsive pCLPG retrovirus did offer expression <it>in vivo </it>and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.</p

Murine Leukemias with Retroviral Insertions at Lmo2 Are Predictive of the Leukemias Induced in SCID-X1 Patients Following Retroviral Gene Therapy

Five X-linked severe combined immunodeficiency patients (SCID-X1) successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2. Genetic evidence also suggests a role for IL2RG in tumor formation, although this remains controversial. Here, we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are similar to those deregulated in human leukemias with high LMO2 expression and are highly predictive of the leukemias induced in SCID-X1 patients. We also provide additional evidence supporting the notion that IL2RG and LMO2 cooperate in leukemia induction but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations

Introduction: Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation.Methods: In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1(F971L/F971L) and Rag1(R972Q/R972Q)), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter.Results and discussion: Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.Transplantation and immunomodulatio

Therapeutic effect of suicide gene-transferred mesenchymal stem cells in a rat model of glioma

We evaluated a new therapeutic strategy for malignant glioma, which combines intratumoral inoculation of mesenchymal stem cells (MSCs) expressing cytosine deaminase gene with 5-fluorocytosine (5-FC) administration. For in vitro and in vivo experiments, MSCs were transfected with adenovirus carrying either enhanced green fluorescent protein gene (AdexCAEGFP) or cytosine deaminase gene (AdexCACD), to establish MSC-expressing EGFP (MSC-EGFP) or CD (MSC-CD). Co-culture of 9L glioma cells with MSC-CD in a medium containing 5-FC resulted in a remarkable reduction in 9L cell viability. The migratory ability of MSC-EGFP toward 9L cells was demonstrated by double-chamber assay. For the in vivo study, rats harboring 9L brain tumors were inoculated with MSC-EGFP or MSC-CD. Immunohistochemistry of rat brain tumors inoculated with MSC-EGFP showed intratumoral distribution of MSC-EGFP. Survival analysis of rats bearing 9L gliomas treated with intratumoral MSC-CD and intraperitoneal 5-FC resulted in significant prolongation of survival compared with control animals. In conclusion, molecular therapy combining suicide gene therapy and MSCs as a targeting vehicle represents a potential new therapeutic approach for malignant glioma, both with respect to the antitumor potential of this system and its neuroprotective effect on normal brain tissue

Autonomous Targeting of Infectious Superspreaders Using Engineered Transmissible Therapies

Infectious disease treatments, both pharmaceutical and vaccine, face three universal challenges: the difficulty of targeting treatments to high-risk ‘superspreader’ populations who drive the great majority of disease spread, behavioral barriers in the host population (such as poor compliance and risk disinhibition), and the evolution of pathogen resistance. Here, we describe a proposed intervention that would overcome these challenges by capitalizing upon Therapeutic Interfering Particles (TIPs) that are engineered to replicate conditionally in the presence of the pathogen and spread between individuals — analogous to ‘transmissible immunization’ that occurs with live-attenuated vaccines (but without the potential for reversion to virulence). Building on analyses of HIV field data from sub-Saharan Africa, we construct a multi-scale model, beginning at the single-cell level, to predict the effect of TIPs on individual patient viral loads and ultimately population-level disease prevalence. Our results show that a TIP, engineered with properties based on a recent HIV gene-therapy trial, could stably lower HIV/AIDS prevalence by ∼30-fold within 50 years and could complement current therapies. In contrast, optimistic antiretroviral therapy or vaccination campaigns alone could only lower HIV/AIDS prevalence by <2-fold over 50 years. The TIP's efficacy arises from its exploitation of the same risk factors as the pathogen, allowing it to autonomously penetrate superspreader populations, maintain efficacy despite behavioral disinhibition, and limit viral resistance. While demonstrated here for HIV, the TIP concept could apply broadly to many viral infectious diseases and would represent a new paradigm for disease control, away from pathogen eradication but toward robust disease suppression