Human IL-6 fosters long-term engraftment of patient-derived disease-driving myeloma cells in immunodeficient mice.

Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned human IL-6-transgenic (hIL-6-transgenic) NSG (NSG+hIL6) mice reliably support the engraftment of malignant and premalignant human plasma cells, including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and postrelapse myeloma, plasma cell leukemia, and amyloid light chain amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single-cell RNA sequencing showed nonmalignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma-engrafted mice given CAR T cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results establish NSG+hIL6 mice as an effective patient-derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.

Human IL-6 fosters long-term engraftment of patient derived disease-driving myeloma cells in immunodeficient mice.

Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned hIL-6 transgenic NSG mice (NSG+hIL6) reliably support the engraftment of malignant and pre-malignant human plasma cells including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and post-relapse myeloma, plasma cell leukemia, and AL amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells, developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single cell RNA sequencing showed non-malignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma engrafted mice given CAR T-cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results establish NSG+hIL6 mice as an effective patient derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.

A human immune/muscle xenograft model of FSHD muscle pathology.

Background: Facioscapulohumeral muscular dystrophy (FSHD) disease progression is associated with muscle inflammation, although its role in FSHD muscle pathology is unknown. Methods: We have developed a novel humanized mouse strain, NSG-SGM3-W41, that supports the co- engraftment of human hematopoietic stem cells (HSCs) and muscle myoblasts as an experimental model to investigate the role of innate immunity in FSHD muscle pathology. Results: The NSG-SGM3-W41 mouse supports the selective expansion of human innate immune cell lineages following engraftment of human HSCs and the co-engraftment and differentiation of patient-derived FSHD or control muscle myoblasts. Immunohistological and NanoString RNA expression assays establish that muscle xenografts from three FSHD subjects were immunogenic compared to those from unaffected first-degree relatives. FSHD muscle xenografts preferentially accumulated human macrophages and B cells and expressed early complement genes of the classical and alternative pathways including complement factor C3 protein, which is a mediator of early complement function through opsonization to mark damaged cells for macrophage engulfment. FSHD muscle xenografts also underwent immune donor dependent muscle turnover as assayed by human spectrin ß1 immunostaining of muscle fibers and by NanoString RNA expression assays of muscle differentiation genes. Conclusions: The NSG-SGM3-W41 mouse provides an experimental model to investigate the role of innate immunity and complement in FSHD muscle pathology and to develop FSHD therapeutics targeting DUX4 and the innate immunity inflammatory responses.

NOD-scid IL2rγnull mice lacking TLR4 support human immune system development and the study of human-specific innate immunity.

Agents that induce inflammation have been used since the 18th century for the treatment of cancer. The inflammation induced by agents such as Toll-like receptor agonists is thought to stimulate tumor-specific immunity in patients and augment control of tumor burden. While NOD-scid IL2rγnull mice lack murine adaptive immunity (T cells and B cells), these mice maintain a residual murine innate immune system that responds to Toll-like receptor agonists. Here we describe a novel NOD-scid IL2rγnull mouse lacking murine TLR4 that fails to respond to lipopolysaccharide. NSG-Tlr4null mice support human immune system engraftment and enable the study of human-specific responses to TLR4 agonists in the absence of the confounding effects of a murine response. Our data demonstrate that specific stimulation of TLR4 activates human innate immune systems and delays the growth kinetics of a human patient-derived xenograft melanoma tumor.

Enhanced development of functional human NK cells in NOD-scid-IL2rgnull mice expressing human IL15.

Human innate immunity plays a critical role in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that have opposing roles in the tumor microenvironment, including NK cell subsets that mediate tumor cell cytotoxicity and subsets with regulatory function that contribute to the tumor immune suppressive environment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However, human NK cell development and survival in humanized NOD-scid-IL2rgnull (NSG) mice are severely limited. To enhance NK cell development, we have developed NSG mice that constitutively expresses human Interleukin 15 (IL15), NSG-Tg(Hu-IL15). Following hematopoietic stem cell engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of functional human CD56+ NK cells are detectable in blood and spleen, as compared to NSG mice. Hematopoietic stem cell (HSC)-engrafted NSG-Tg(Hu-IL15) mice also supported the development of human CD3+ T cells, CD20+ B cells, and CD33+ myeloid cells. Moreover, the growth kinetics of a patient-derived xenograft (PDX) melanoma were significantly delayed in HSC-engrafted NSG-Tg(Hu-IL15) mice as compared to HSC-engrafted NSG mice demonstrating that human NK cells have a key role in limiting the tumor growth. Together, these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells, which limit the growth of PDX tumors.

Inactive rhomboid proteins RHBDF1 and RHBDF2 (iRhoms): a decade of research in murine models.

Rhomboid proteases, first discovered in Drosophila, are intramembrane serine proteases. Members of the rhomboid protein family that are catalytically deficient are known as inactive rhomboids (iRhoms). iRhoms have been implicated in wound healing, cancer, and neurological disorders such as Alzheimer's and Parkinson's diseases, inflammation, and skin diseases. The past decade of mouse research has shed new light on two key protein domains of iRhoms-the cytosolic N-terminal domain and the transmembrane dormant peptidase domain-suggesting new ways to target multiple intracellular signaling pathways. This review focuses on recent advances in uncovering the unique functions of iRhom protein domains in normal growth and development, growth factor signaling, and inflammation, with a perspective on future therapeutic opportunities.

Improved mouse models and advanced genetic and genomic technologies for the study of neutrophils.

Mice have been excellent surrogates for studying neutrophil biology and, furthermore, murine models of human disease have provided fundamental insights into the roles of human neutrophils in innate immunity. The emergence of novel humanized mice and high-diversity mouse populations offers the research community innovative and powerful platforms for better understanding, respectively, the mechanisms by which human neutrophils drive pathogenicity, and how genetic differences underpin the variation in neutrophil biology observed among humans. Here, we review key examples of these new resources. Additionally, we provide an overview of advanced genetic engineering tools available to further improve such murine model systems, of sophisticated neutrophil-profiling technologies, and of multifunctional nanoparticle (NP)-based neutrophil-targeting strategies.

Humanized mouse models of immunological diseases and precision medicine.

With the increase in knowledge resulting from the sequencing of the human genome, the genetic basis for the underlying differences in individuals, their diseases, and how they respond to therapies is starting to be understood. This has formed the foundation for the era of precision medicine in many human diseases that is beginning to be implemented in the clinic, particularly in cancer. However, preclinical testing of therapeutic approaches based on individual biology will need to be validated in animal models prior to translation into patients. Although animal models, particularly murine models, have provided significant information on the basic biology underlying immune responses in various diseases and the response to therapy, murine and human immune systems differ markedly. These fundamental differences may be the underlying reason why many of the positive therapeutic responses observed in mice have not translated directly into the clinic. There is a critical need for preclinical animal models in which human immune responses can be investigated. For this, many investigators are using humanized mice, i.e., immunodeficient mice engrafted with functional human cells, tissues, and immune systems. We will briefly review the history of humanized mice, the remaining limitations, approaches to overcome them and how humanized mouse models are being used as a preclinical bridge in precision medicine for evaluation of human therapies prior to their implementation in the clinic.

Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) null (NSG) strains that lack murine MHC class I and II [NSG-ß-2-microglobulin ( B2M) null ( IA IE)null and NSG -( Kb Db) null ( IAnull)]. We observed rapid human IgG clearance in NSG- B2Mnull ( IA IE) null mice whereas clearance in NSG -( Kb Db) null ( IAnull) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( Kb Db) null ( IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

RHBDF2-Regulated Growth Factor Signaling in a Rare Human Disease, Tylosis With Esophageal Cancer: What Can We Learn From Murine Models?

Tylosis with esophageal cancer syndrome (TOC) is a rare autosomal dominant proliferative skin disease caused by missense mutations in the rhomboid 5 homolog 2 (RHBDF2) gene. TOC is characterized by thickening of the skin in the palms and feet and is strongly linked with the development of esophageal squamous cell carcinoma. Murine models of human diseases have been valuable tools for investigating the underlying genetic and molecular mechanisms of a broad range of diseases. Although current mouse models do not fully recapitulate all aspects of human TOC, and the molecular mechanisms underlying TOC are still emerging, the available mouse models exhibit several key aspects of the disease, including a proliferative skin phenotype, a rapid wound healing phenotype, susceptibility to epithelial cancer, and aberrant epidermal growth factor receptor (EGFR) signaling. Furthermore, we and other investigators have used these models to generate new insights into the causes and progression of TOC, including findings suggesting a tissue-specific role of the RHBDF2-EGFR pathway, rather than a role of the immune system, in mediating TOC; and indicating that amphiregulin, an EGFR ligand, is a functional driver of the disease. This review highlights the mouse models of TOC available to researchers for use in investigating the disease mechanisms and possible therapies, and the significance of genetic modifiers of the disease identified in these models in delineating the underlying molecular mechanisms.

ADAM17 is essential for ectodomain shedding of the EGF-receptor ligand amphiregulin.

The epidermal growth factor (EGF)-receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic epithelial cancers. AREG, synthesized as a propeptide, requires conversion to an active peptide by metalloproteases by a process known as ectodomain shedding. Although (ADAM17) a disintegrin and metalloprotease 17 is a key sheddase of AREG, ADAM8-, ADAM15-, and batimastat (broad metalloprotease inhibitor)-sensitive metalloproteases have also been implicated in AREG shedding. In the present study, using a curly bare (Rhbdf2cub ) mouse model that shows loss-of-hair, enlarged sebaceous gland, and rapid cutaneous wound-healing phenotypes mediated by enhanced Areg mRNA and protein levels, we sought to identify the principal ectodomain sheddase of AREG. To this end, we generated Rhbdf2cub mice lacking ADAM17 specifically in the skin and examined the above phenotypes of Rhbdf2cub mice. We find that ADAM17 deficiency in the skin of Rhbdf2cub mice restores a full hair coat, prevents sebaceous gland enlargement, and impairs the rapid wound-healing phenotype observed in Rhbdf2cub mice. Furthermore, in vitro, stimulated shedding of AREG is abolished in Rhbdf2cub mouse embryonic keratinocytes lacking ADAM17. Thus, our data support previous findings demonstrating that ADAM17 is the major ectodomain sheddase of AREG.

Tissue-specific role of RHBDF2 in cutaneous wound healing and hyperproliferative skin disease.

OBJECTIVE: Gain-of-function (GOF) mutations in RHBDF2 cause tylosis. Patients present with hyperproliferative skin, and keratinocytes from tylosis patients' skin show an enhanced wound-healing phenotype. The curly bare mouse model of tylosis, carrying a GOF mutation in the Rhbdf2 gene (Rhbdf2 cub ), presents with epidermal hyperplasia and shows accelerated cutaneous wound-healing phenotype through enhanced secretion of the epidermal growth factor receptor family ligand amphiregulin. Despite these advances in our understanding of tylosis, key questions remain. For instance, it is not known whether the disease is skin-specific, whether the immune system or the surrounding microenvironment plays a role, and whether mouse genetic background influences the hyperproliferative-skin and wound-healing phenotypes observed in Rhbdf2 cub mice. RESULTS: We performed bone marrow transfers and reciprocal skin transplants and found that bone marrow transfer from C57BL/6 (B6)-Rhbdf2 cub/cub donor mice to B6 wildtype recipient mice failed to transfer the hyperproliferative-skin and wound-healing phenotypes in B6 mice. Furthermore, skin grafts from B6 mice to the dorsal skin of B6-Rhbdf2 cub/cub mice maintained the phenotype of the donor mice. To test the influence of mouse genetic background, we backcrossed Rhbdf2 cub onto the MRL/MpJ strain and found that the hyperproliferative-skin and wound-healing phenotypes caused by the Rhbdf2 cub mutation persisted on the MRL/MpJ strain.

A novel hemolytic complement-sufficient NSG mouse model supports studies of complement-mediated antitumor activity in vivo.

Monoclonal antibodies (mAbs) have emerged as a mainstream therapeutic option against cancer. mAbs mediate tumor cell-killing through several mechanisms including complement-dependent cytotoxicity (CDC). However, studies of mAb-mediated CDC against tumor cells remain largely dependent on in vitro systems. Previously developed and widely used NOD-scid IL2rγnull (NSG) mice support enhanced engraftment of many primary human tumors. However, NSG mice have a 2-bp deletion in the coding region of the hemolytic complement (Hc) gene, and it is not possible to evaluate CDC activity in NSG mice. To address this limitation, we generated a novel strain of NSG mice-NSG-Hc1-that have an intact complement system able to generate the membrane attack complex. Utilizing the Daudi Burkitt's human lymphoma cell line, and the anti-human CD20 mAb rituximab, we further demonstrated that the complement system in NSG-Hc1 mice is fully functional. NSG-Hc1 mice expressed CDC activity against Daudi cells in vivo following rituximab treatment and showed longer overall survival compared with rituximab-treated NSG mice that lack hemolytic complement. Our results validate the NSG-Hc1 mouse model as a platform for testing mechanisms underlying CDC in vivo and suggest its potential use to compare complement-dependent and complement-independent cytotoxic activity mediated by therapeutic mAbs.

Early induction of NRF2 antioxidant pathway by RHBDF2 mediates rapid cutaneous wound healing.

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2cub/cub) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2cub/cub and Rhbdf2+/+ mice at 0h, 15min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds.

SVEP1 plays a crucial role in epidermal differentiation.

SVEP1 is a recently identified multidomain cell adhesion protein, homologous to the mouse polydom protein, which has been shown to mediate cell-cell adhesion in an integrin-dependent manner in osteogenic cells. In this study, we characterized SVEP1 function in the epidermis. SVEP1 was found by qRT-PCR to be ubiquitously expressed in human tissues, including the skin. Confocal microscopy revealed that SVEP1 is normally mostly expressed in the cytoplasm of basal and suprabasal epidermal cells. Downregulation of SVEP1 expression in primary keratinocytes resulted in decreased expression of major epidermal differentiation markers. Similarly, SVEP1 downregulation was associated with disturbed differentiation and marked epidermal acanthosis in three-dimensional skin equivalents. In contrast, the dispase assay failed to demonstrate significant differences in adhesion between keratinocytes expressing normal vs low levels of SVEP1. Homozygous Svep1 knockout mice were embryonic lethal. Thus, to assess the importance of SVEP1 for normal skin homoeostasis in vivo, we downregulated SVEP1 in zebrafish embryos with a Svep1-specific splice morpholino. Scanning electron microscopy revealed a rugged epidermis with perturbed microridge formation in the centre of the keratinocytes of morphant larvae. Transmission electron microscopy analysis demonstrated abnormal epidermal cell-cell adhesion with disadhesion between cells in Svep1-deficient morphant larvae compared to controls. In summary, our results indicate that SVEP1 plays a critical role during epidermal differentiation.

Rhbdf2 mutations increase its protein stability and drive EGFR hyperactivation through enhanced secretion of amphiregulin.

The rhomboid 5 homolog 2 (Rhbdf2) gene encodes an inactive rhomboid (iRhom) protease, iRhom2, one of a family of enzymes containing a long cytosolic N terminus and a dormant peptidase domain of unknown function. iRhom2 has been implicated in epithelial regeneration and cancer growth through constitutive activation of epidermal growth factor receptor (EGFR) signaling. However, little is known about the physiological substrates for iRhom2 or the molecular mechanisms underlying these functions. We show that iRhom2 is a short-lived protein whose stability can be increased by select mutations in the N-terminal domain. In turn, these stable variants function to augment the secretion of EGF family ligands, including amphiregulin, independent of metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) activity. In vivo, N-terminal iRhom2 mutations induce accelerated wound healing as well as accelerated tumorigenesis, but they do not drive spontaneous tumor development. This work underscores the physiological prominence of iRhom2 in controlling EGFR signaling events involved in wound healing and neoplastic growth, and yields insight into the function of key iRhom2 domains.

Chronic proliferative dermatitis in Sharpin null mice: development of an autoinflammatory disease in the absence of B and T lymphocytes and IL4/IL13 signaling.

SHARPIN is a key regulator of NFKB and integrin signaling. Mice lacking Sharpin develop a phenotype known as chronic proliferative dermatitis (CPDM), typified by progressive epidermal hyperplasia, apoptosis of keratinocytes, cutaneous and systemic eosinophilic inflammation, and hypoplasia of secondary lymphoid organs. Rag1(-/-) mice, which lack mature B and T cells, were crossed with Sharpin(-/-) mice to examine the role of lymphocytes in CDPM. Although inflammation in the lungs, liver, and joints was reduced in these double mutant mice, dermatitis was not reduced in the absence of functional lymphocytes, suggesting that lymphocytes are not primary drivers of the inflammation in the skin. Type 2 cytokine expression is increased in CPDM. In an attempt to reduce this aspect of the phenotype, Il4ra(-/-) mice, unresponsive to both IL4 and IL13, were crossed with Sharpin(-/-) mice. Double homozygous Sharpin(-/-) , Il4ra(-/-) mice developed an exacerbated granulocytic dermatitis, acute system inflammation, as well as hepatic necrosis and mineralization. High expression of CHI3L4, normally seen in CPDM skin, was abolished in Sharpin(-/-) , Il4ra(-/-) double mutant mice indicating the crucial role of IL4 and IL13 in the expression of this protein. Cutaneous eosinophilia persisted in Sharpin(-/-) , Il4ra(-/-) mice, although expression of Il5 mRNA was reduced and the expression of Ccl11 and Ccl24 was completely abolished. TSLP and IL33 were both increased in the skin of Sharpin(-/-) mice and this was maintained in Sharpin(-/-) , Il4ra(-/-) mice suggesting a role for TSLP and IL33 in the eosinophilic dermatitis in SHARPIN-deficient mice. These studies indicate that cutaneous inflammation in SHARPIN-deficient mice is autoinflammatory in nature developing independently of B and T lymphocytes, while the systemic inflammation seen in CPDM has a strong lymphocyte-dependent component. Both the cutaneous and systemic inflammation is enhanced by loss of IL4 and IL13 signaling indicating that these cytokines normally play an anti-inflammatory role in SHARPIN-deficient mice.

Retrotransposon insertion in the T-cell acute lymphocytic leukemia 1 (Tal1) gene is associated with severe renal disease and patchy alopecia in Hairpatches (Hpt) mice.

"Hairpatches" (Hpt) is a naturally occurring, autosomal semi-dominant mouse mutation. Hpt/Hpt homozygotes die in utero, while Hpt/+ heterozygotes exhibit progressive renal failure accompanied by patchy alopecia. This mutation is a model for the rare human disorder "glomerulonephritis with sparse hair and telangiectases" (OMIM 137940). Fine mapping localized the Hpt locus to a 6.7 Mb region of Chromosome 4 containing 62 known genes. Quantitative real time PCR revealed differential expression for only one gene in the interval, T-cell acute lymphocytic leukemia 1 (Tal1), which was highly upregulated in the kidney and skin of Hpt/+ mice. Southern blot analysis of Hpt mutant DNA indicated a new EcoRI site in the Tal1 gene. High throughput sequencing identified an endogenous retroviral class II intracisternal A particle insertion in Tal1 intron 4. Our data suggests that the IAP insertion in Tal1 underlies the histopathological changes in the kidney by three weeks of age, and that glomerulosclerosis is a consequence of an initial developmental defect, progressing in severity over time. The Hairpatches mouse model allows an investigation into the effects of Tal1, a transcription factor characterized by complex regulation patterns, and its effects on renal disease.

Inflammatory dilated cardiomyopathy in Abcg5-deficient mice.

Dilated cardiomyopathy (DCM) in A/J mice homozygous for the spontaneous thrombocytopenia and cardiomyopathy (trac) mutation results from a single base pair change in the Abcg5 gene. A similar mutation in humans causes sitosterolemia with high plant sterol levels, hypercholesterolemia, and early onset atherosclerosis. Analyses of CD3+ and Mac-3+ cells and stainable collagen in hearts showed inflammation and myocyte degeneration in A/J-trac/trac mice beginning postweaning and progressed to marked dilative and fibrosing cardiomyopathy by 140 days. Transmission electron microscopy (TEM) demonstrated myocyte vacuoles consistent with swollen endoplasmic reticulum (ER). Myocytes with cytoplasmic glycogen and irregular actinomyosin filament bundles formed mature intercalated disks with normal myocytes suggesting myocyte repair. A/J-trac/trac mice fed lifelong phytosterol-free diets did not develop cardiomyopathy. BALB/cByJ-trac/trac mice had lesser inflammatory infiltrates and later onset DCM. BALB/cByJ-trac/trac mice changed from normal to phytosterol-free diets had lesser T cell infiltrates but persistent monocyte infiltrates and equivalent fibrosis to mice on normal diets. B- and T-cell-deficient BALB/cBy-Rag1(null) trac/trac mice fed normal diets did not develop inflammatory infiltrates or DCM. We conclude that the trac/trac mouse has many features of inflammatory DCM and that the reversibility of myocardial T cell infiltration provides a novel model for investigating the progression of myocardial fibrosis.

Engraftment of human HSCs in nonirradiated newborn NOD-scid IL2rγ null mice is enhanced by transgenic expression of membrane-bound human SCF.

Immunodeficient mice engrafted with human HSCs support multidisciplinary translational experimentation, including the study of human hematopoiesis. Heightened levels of human HSC engraftment are observed in immunodeficient mice expressing mutations in the IL2-receptor common γ chain (IL2rg) gene, including NOD-scid IL2rγ(null) (NSG) mice. Engraftment of human HSC requires preconditioning of immunodeficient recipients, usually with irradiation. Such preconditioning increases the expression of stem cell factor (SCF), which is critical for HSC engraftment, proliferation, and survival. We hypothesized that transgenic expression of human membrane-bound stem cell factor Tg(hu-mSCF)] would increase levels of human HSC engraftment in nonirradiated NSG mice and eliminate complications associated with irradiation. Surprisingly, detectable levels of human CD45(+) cell chimerism were observed after transplantation of cord blood-derived human HSCs into nonirradiated adult as well as newborn NSG mice. However, transgenic expression of human mSCF enabled heightened levels of human hematopoietic cell chimerism in the absence of irradiation. Moreover, nonirradiated NSG-Tg(hu-mSCF) mice engrafted as newborns with human HSCs rejected human skin grafts from a histoincompatible donor, indicating the development of a functional human immune system. These data provide a new immunodeficient mouse model that does not require irradiation preconditioning for human HSC engraftment and immune system development.