Innovations, challenges, and minimal information for standardization of humanized mice

Mice xenotransplanted with human cells and/or expressing human gene products (also known as humanized mice ) recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called Minimal Information for Standardization of Humanized Mice (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice

Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy

Establishment of an in vivo small animal model of human tumor and human immune system interaction would enable preclinical investigations into the mechanisms underlying cancer immunotherapy. To this end, nonobese diabetic (NOD).Cg- Prkdc(scid)IL2rg(tm1Wjl)/Sz (null; NSG) mice were transplanted with human (h)CD34(+) hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems [humanized NSG (HuNSG)]. HuNSG mice received human leukocyte antigen partially matched tumor implants from patient-derived xenografts [PDX; non-small cell lung cancer (NSCLC), sarcoma, bladder cancer, and triple-negative breast cancer (TNBC)] or from a TNBC cell line-derived xenograft (CDX). Tumor growth curves were similar in HuNSG compared with nonhuman immune-engrafted NSG mice. Treatment with pembrolizumab, which targets programmed cell death protein 1, produced significant growth inhibition in both CDX and PDX tumors in HuNSG but not in NSG mice. Finally, inhibition of tumor growth was dependent on hCD8(+) T cells, as demonstrated by antibody-mediated depletion. Thus, tumor-bearing HuNSG mice may represent an important, new model for preclinical immunotherapy research

In vivo correction of anaemia in beta-thalassemic mice by gammaPNA-mediated gene editing with nanoparticle delivery

The blood disorder, beta-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the gamma position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human beta-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing gammaPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% beta-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation gammaPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration

The Difficulty of Making Reparations Affects the Intensity of Collective Guilt

We examined how the difficulty of making reparations for the harm done to another group affects the intensity of collective guilt. Men were confronted with information documenting male privilege and were told that they would have a chance to help women and reduce patriarchy by collecting signatures on a petition. We manipulated the difficulty of making reparations by asking participants to collect 5, 50, or 100 signatures. As predicted by Brehm's (1999) theory of emotional intensity, collective guilt was a non-monotonic function of the difficulty of making reparations. Men in the moderate difficulty (50 signatures) condition expressed greater collective guilt than participants in the low (5) or high (100) difficulty conditions. Results are discussed in terms of the implications for the theory of emotional intensity, collective guilt, and collective emotions more generally

Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics

There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an allogenized mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts

Verification and Validation Studies for the LAVA CFD Solver

The verification and validation of the Launch Ascent and Vehicle Aerodynamics (LAVA) computational fluid dynamics (CFD) solver is presented. A modern strategy for verification and validation is described incorporating verification tests, validation benchmarks, continuous integration and version control methods for automated testing in a collaborative development environment. The purpose of the approach is to integrate the verification and validation process into the development of the solver and improve productivity. This paper uses the Method of Manufactured Solutions (MMS) for the verification of 2D Euler equations, 3D Navier-Stokes equations as well as turbulence models. A method for systematic refinement of unstructured grids is also presented. Verification using inviscid vortex propagation and flow over a flat plate is highlighted. Simulation results using laminar and turbulent flow past a NACA 0012 airfoil and ONERA M6 wing are validated against experimental and numerical data

Rapid quantification of naive alloreactive T cells by TNF-alpha production and correlation with allograft rejection in mice

Allograft transplantation requires chronic immunosuppression, but there is no effective strategy to evaluate the long-term maintenance of immunosuppression other than assessment of graft function. The ability to monitor naive alloreactive T cells would provide an alternative guide for drug therapy at early, preclinical stages of graft rejection and for evaluating tolerance-inducing protocols. To detect and quantify naive alloreactive T cells directly ex vivo, we used the unique ability of naive T cells to rapidly produce TNF-alpha but not IFN-gamma. Naive alloreactive T cells were identified by the production of TNF-alpha after a 5-hour in vitro stimulation with alloantigen and were distinguished from effector/memory alloreactive T cells by the inability to produce IFN-gamma. Moreover, naive alloreactive T cells were not detected in mice tolerized against specific alloantigens. The frequency of TNF-alpha-producing cells was predictive for rejection in an in vivo cytotoxicity assay and correlated with skin allograft rejection. Naive alloreactive T cells were also detected in humans, suggesting clinical relevance. We conclude that rapid production of TNF-alpha can be used to quantify naive alloreactive T cells, that it is abrogated after the induction of tolerance, and that it is a potential tool to predict allograft rejection

Discovery and Development of Human Monoclonal Antibodies to Block RhD Alloimmunization During Pregnancy

Exposure of an Rh negative mother to red blood cells (RBCs) of an Rh positive fetus results in alloimmunization and development of anti-RhD antibodies. The anti-RhD antibodies cause hemolytic disease of the new born babies during subsequent pregnancies. Current prophylactic treatment involves polyclonal anti-RhD IgG purified from plasma of humans and is administered in approximately 20% of pregnancies. While the current prophylaxis is effective, it involves the use of human plasma and non-RhD specific antibodies, thus posing a risk of transmitting infections and undesired antibody reactions. Moreover, there is a serious scarcity of plasma donors to meet the requirement of anti-RhD antibodies. In this study we propose to discover and develop anti-RhD monoclonal human antibodies to replace the current polyclonal prophylaxis. We are using humanized BLT mice (fetal CD34+ stem cells, liver and thymus) reconstituted with RhD negative donor material and were immunized by using adenovirus containing RhD transgene. Serum samples were collected after 4-6 weeks of immunization. Our results show that the RhD immunized mice had considerably higher titer of IgG and IgA antibodies in the serum compared to the control, suggesting an immune response developed upon immunization. Splenocytes from antibody producing mice will be fused with a human fusion partner for the isolation of hybridomas producing human monoclonal antibodies. The immunoreactivity and functional activity of these antibodies will be discussed