Reconstitution of huPBL-NSG Mice with Donor-Matched Dendritic Cells Enables Antigen-Specific T-cell Activation

Humanized mouse models provide a unique opportunity to study human immune cells in vivo, but traditional models have been limited to the evaluation of non-specific T-cell interactions due to the absence of antigen-presenting cells. In this study, immunodeficient NOD/SCID/IL2r-γnull (NSG) mice were engrafted with human peripheral blood lymphocytes alone or in combination with donor-matched monocyte-derived dendritic cells (DC) to determine whether antigen-specific T-cell activation could be reconstituted. Over a period of 3 weeks, transferred peripheral blood lymphocytes reconstituted the spleen and peripheral blood of recipient mice with predominantly human CD45-positive lymphocytes. Animals exhibited a relatively normal CD4/CD8 ratio (average 1.63:1) as well as reconstitution of CD3/CD56 (averaging 17.8%) and CD20 subsets (averaging 4.0%). Animals reconstituted with donor-matched CD11c+ DC also demonstrated a CD11c+ population within their spleen, representing 0.27% to 0.43% of the recovered human cells with concurrent expression of HLA-DR, CD40, and CD86. When immunized with adenovirus, either as free replication-incompetent vector (AdV) or as vector-transduced DC (DC/AdV), there was activation and expansion of AdV-specific T-cells, an increase in Th1 cytokines in serum, and skewing of T-cells toward an effector/memory phenotype. T-cells recovered from animals challenged with AdV in vivo proliferated and secreted a Th1-profile of cytokines in response to DC/AdV challenge in vitro. Our results suggest that engrafting NSG mice with a combination of lymphocytes and donor-matched DC can reconstitute antigen responsiveness and allow the in vivo assessment of human immune response to viruses, vaccines, and other immune challenges

Lentiviral Vector Delivery of Human Interleukin-7 (hIL-7) to Human Immune System (HIS) Mice Expands T Lymphocyte Populations

Genetically modified mice carrying engrafted human tissues provide useful models to study human cell biology in physiologically relevant contexts. However, there remain several obstacles limiting the compatibility of human cells within their mouse hosts. Among these is inadequate cross-reactvitiy between certain mouse cytokines and human cellular receptors, depriving the graft of important survival and growth signals. To circumvent this problem, we utilized a lentivirus-based delivery system to express physiologically relevant levels of human interleukin-7 (hIL-7) in Rag2-/-γc-/- mice following a single intravenous injection. hIL-7 promoted homeostatic proliferation of both adoptively transferred and endogenously generated T-cells in Rag2-/-γc-/- Human Immune System (HIS) mice. Interestingly, we found that hIL-7 increased T lymphocyte numbers in the spleens of HIV infected HIS mice without affecting viral load. Taken together, our study unveils a versatile approach to deliver human cytokines to HIS mice, to both improve engraftment and determine the impact of cytokines on human diseases

Human CD34+ CD133+ Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult NOD-SCID Il2rγ−/− (NSG) Mice

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.Singapore-MIT Alliance for Research and Technology ( Infectious Diseases research grant

Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice

BACKGROUND:The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research. METHODOLOGY/PRINCIPAL FINDINGS:We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor gamma-chain knockout (NOD-scid IL2rgamma(null)) mice engrafted with human hematopoietic stem cells. Human CD45(+) cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rgamma(null) mice with HLA-A2(+) human cord blood hematopoietic stem cells, were able to secrete IFN-gamma, IL-2 and TNF-alpha in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353((111-119)), NS4b 2423((181-189)), and NS4a 2148((56-64)). CONCLUSIONS/SIGNIFICANCE:This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections

Measurements of knee rotation-reliability of an external device in vivo

<p>Abstract</p> <p>Background</p> <p>Knee rotation plays an important part in knee kinematics during weight-bearing activities. An external device for measuring knee rotation (the Rottometer) has previously been evaluated for validity by simultaneous measurements of skeletal movements with Roentgen Stereometric Analysis (RSA). The aim of this study was to investigate the reliability of the device.</p> <p>Method</p> <p>The within-day and test-retest reliability as well as intertester reliability of the device in vivo was calculated. Torques of 3, 6 and 9 Nm and the examiner's apprehension of end-feel were used at 90°, 60° and 30° of knee flexion. Intraclass Correlation Coefficient _2,1(ICC _2,1), 95% confidence interval (CI) of ICC and 95% CI between test trials and examiners were used as statistical tests.</p> <p>Result</p> <p>ICC_2,1ranged from 0.50 to 0.94 at all three flexion angles at 6 and 9 Nm as well as end-feel, and from 0.22 to 0.75 at 3 Nm applied torque.</p> <p>Conclusion</p> <p>The Rottometer was a reliable measurement instrument concerning knee rotation at the three different flexion angles (90°, 60° and 30°) with 6 and 9 Nm applied torques as well as the examiner's apprehension of end-feel. Three Nm was not a reliable torque. The most reliable measurements were made at 9 Nm applied torque.</p

Acellular Bone Marrow Extracts Significantly Enhance Engraftment Levels of Human Hematopoietic Stem Cells in Mouse Xeno-Transplantation Models

Hematopoietic stem cells (HSC) derived from cord blood (CB), bone marrow (BM), or mobilized peripheral blood (PBSC) can differentiate into multiple lineages such as lymphoid, myeloid, erythroid cells and platelets. The local microenvironment is critical to the differentiation of HSCs and to the preservation of their phenotype in vivo. This microenvironment comprises a physical support supplied by the organ matrix as well as tissue specific cytokines, chemokines and growth factors. We investigated the effects of acellular bovine bone marrow extracts (BME) on HSC in vitro and in vivo. We observed a significant increase in the number of myeloid and erythroid colonies in CB mononuclear cells (MNC) or CB CD34+ cells cultured in methylcellulose media supplemented with BME. Similarly, in xeno-transplantation experiments, pretreatment with BME during ex-vivo culture of HSCs induced a significant increase in HSC engraftment in vivo. Indeed, we observed both an increase in the number of differentiated myeloid, lymphoid and erythroid cells and an acceleration of engraftment. These results were obtained using CB MNCs, BM MNCs or CD34+ cells, transplanted in immuno-compromised mice (NOD/SCID or NSG). These findings establish the basis for exploring the use of BME in the expansion of CB HSC prior to HSC Transplantation. This study stresses the importance of the mechanical structure and soluble mediators present in the surrounding niche for the proper activity and differentiation of stem cells

Efficient tumour formation by single human melanoma cells

A fundamental question in cancer biology is whether cells with tumorigenic potential are common or rare within human cancers. Studies on diverse cancers, including melanoma, have indicated that only rare human cancer cells ( 0.1 - 0.0001%) form tumours when transplanted into non- obese diabetic/ severe combined immunodeficiency ( NOD/ SCID) mice. However, the extent to which NOD/ SCID mice underestimate the frequency of tumorigenic human cancer cells has been uncertain. Here we show that modified xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/ SCID interleukin- 2 receptor gamma chain null (Il2rg(-/-)) mice, can increase the detection of tumorigenic melanoma cells by several orders of magnitude. In limiting dilution assays, approximately 25% of unselected melanoma cells from 12 different patients, including cells from primary and metastatic melanomas obtained directly from patients, formed tumours under these more permissive conditions. In single- cell transplants, an average of 27% of unselected melanoma cells from four different patients formed tumours. Modifications to xenotransplantation assays can therefore dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.Howard Hughes Medical Institute ; Allen H. Blondy Research Fellowship ; Lewis and Lillian Becker ; University of Michigan Comprehensive Cancer Center ; National Institutes of Health [CA46592]; University of Michigan Flow Cytometry Core Facility ; N. McAnsh and the University of Michigan Cancer Centre Histology Core ; National Institute of Diabetes, Digestive, and Kidney Diseases [NIH5P60- DK20572]; Michigan Diabetes Research and Training Center ; Spanish Ministry of Education ; Marie Curie Outgoing International Fellowship from the European Commission ; Australian National Health and Medical Research Council ; Human Frontiers Science Program and Australia PostThis work was supported by the Howard Hughes Medical Institute and by the Allen H. Blondy Research Fellowship. The University of Michigan Melanoma Bank was supported by a gift from Lewis and Lillian Becker. Flow cytometry was partly supported by the University of Michigan Comprehensive Cancer Center grant from the National Institutes of Health CA46592. We thank: D. Adams, M. White and the University of Michigan Flow Cytometry Core Facility for support; N. McAnsh and the University of Michigan Cancer Centre Histology Core for histological studies; G. K. Smyth for assistance with statistics; and Z. Azizan for support with tissue collection. Antibody production was supported in part by the National Institute of Diabetes, Digestive, and Kidney Diseases, grant NIH5P60- DK20572 to the Michigan Diabetes Research and Training Center. Some antibodies were provided by Caltag or by eBioscience to screen for cancer stem- cell markers. Human primary melanocyte cultures were provided by M. Soengas. Human mesenchymal stem cells were provided by Z. Wang and P. Krebsbach. E. Q. was supported by the Spanish Ministry of Education and the Marie Curie Outgoing International Fellowship from the European Commission. M. S. was supported by the Australian National Health and Medical Research Council, the Human Frontiers Science Program and Australia Post.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62970/1/nature07567.pd

Human-animal chimeras for vaccine development: an endangered species or opportunity for the developing world?

<p>Abstract</p> <p>Background</p> <p>In recent years, the field of vaccines for diseases such as Human Immunodeficiency Virus (HIV) which take a heavy toll in developing countries has faced major failures. This has led to a call for more basic science research, and development as well as evaluation of new vaccine candidates. Human-animal chimeras, developed with a 'humanized' immune system could be useful to study infectious diseases, including many neglected diseases. These would also serve as an important tool for the efficient testing of new vaccine candidates to streamline promising candidates for further trials in humans. However, developing human-animal chimeras has proved to be controversial.</p> <p>Discussion</p> <p>Development of human-animal chimeras for vaccine development has been slowed down because of opposition by some philosophers, ethicists and policy makers in the west-they question the moral status of such animals, and also express discomfort about transgression of species barriers. Such opposition often uses a contemporary western world view as a reference point. Human-animal chimeras are often being created for diseases which cause significantly higher morbidity and mortality in the developing world as compared to the developed world. We argue in our commentary that given this high disease burden, we should look at socio-cultural perspectives on human-animal chimera like beings in the developing world. On examination, it's clear that such beings have been part of mythology and cultural descriptions in many countries in the developing world.</p> <p>Summary</p> <p>To ensure that important research on diseases afflicting millions like malaria, HIV, Hepatitis-C and dengue continues to progress, we recommend supporting human-animal chimera research for vaccine development in developing countries (especially China and India which have growing technical expertise in the area). The negative perceptions in some parts of the west about human-animal chimeras can be used as an opportunity for nurturing important vaccine development research in the developing world.</p