Longitudinal fragility phenotyping contributes to the prediction of lifespan and age-associated morbidity in C57BL/6 and Diversity Outbred mice.

Aging studies in mammalian models often depend on natural lifespan data as a primary outcome. Tools for lifespan prediction could accelerate these studies and reduce the need for veterinary intervention. Here, we leveraged large-scale longitudinal frailty and lifespan data on two genetically distinct mouse cohorts to evaluate noninvasive strategies to predict life expectancy in mice. We applied a modified frailty assessment, the Fragility Index, derived from existing frailty indices with additional deficits selected by veterinarians. We developed an ensemble machine learning classifier to predict imminent mortality (95% proportion of life lived [95PLL]). Our algorithm represented improvement over previous predictive criteria but fell short of the level of reliability that would be needed to make advanced prediction of lifespan and thus accelerate lifespan studies. Highly sensitive and specific frailty-based predictive endpoint criteria for aged mice remain elusive. While frailty-based prediction falls short as a surrogate for lifespan, it did demonstrate significant predictive power and as such must contain information that could be used to inform the conclusion of aging experiments. We propose a frailty-based measure of healthspan as an alternative target for aging research and demonstrate that lifespan and healthspan criteria reveal distinct aspects of aging in mice.

Longitudinal Fragility Phenotyping Predicts Lifespan and Age-Associated Morbidity in C57BL/6 and Diversity Outbred Mice.

Aging studies in mammalian models often depend on natural lifespan data as a primary outcome. Tools for lifespan prediction could accelerate these studies and reduce the need for veterinary intervention. Here, we leveraged large-scale longitudinal frailty and lifespan data on two genetically distinct mouse cohorts to evaluate noninvasive strategies to predict life expectancy in mice. We applied a modified frailty assessment, the Fragility Index, derived from existing frailty indices with additional deficits selected by veterinarians. We developed an ensemble machine learning classifier to predict imminent mortality (95% proportion of life lived [95PLL]). Our algorithm represented improvement over previous predictive criteria but fell short of the level of reliability that would be needed to make advanced prediction of lifespan and thus accelerate lifespan studies. Highly sensitive and specific frailty-based predictive endpoint criteria for aged mice remain elusive. While frailty-based prediction falls short as a surrogate for lifespan, it did demonstrate significant predictive power and as such must contain information that could be used to inform the conclusion of aging experiments. We propose a frailty-based measure of healthspan as an alternative target for aging research and demonstrate that lifespan and healthspan criteria reveal distinct aspects of aging in mice.

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.

Evaluation of tarsal injuries in C57BL/6J male mice.

Tarsal joint abnormalities have been observed in aged male mice on a C57BL background. This joint disease consists of calcaneal displacement, inflammation, and proliferation of cartilage and connective tissue, that can progress to ankylosis of the joint. While tarsal pathology has been described previously in C57BL/6N substrains, as well as in STR/ort and B10.BR strain, no current literature describes this disease occurring in C57BL/6J mice. More importantly the behavioral features that may result from such a change to the joint have yet to be evaluated. This condition was observed in older male mice of the C57BL/6J lineage, around the age of 20 weeks or older, at a frequency of 1% of the population. To assess potential phenotypic sequela, this study sought to evaluate body weight, frailty assessment, home cage wheel running, dynamic weight bearing, and mechanical allodynia with and without the presence of pain relief with morphine. Overall mice with tarsal injuries had significantly higher frailty scores (p< 0.05) and weighed less (p<0.01) compared to unaffected mice. Affected mice had greater overall touch sensitivity (p<0.05) and they placed more weight on their forelimbs (p<0.01) compared to their hind limbs. Lastly, when housed with a running wheel, affected mice ran for a shorter length of time (p<0.01) but tended to run a greater distance within the time they did run (p<0.01) compared to unaffected mice. When tested just after being given morphine, the affected mice performed more similarly to unaffected mice, suggesting there is a pain sensation to this disease process. This highlights the importance of further characterizing inbred mouse mutations, as they may impact research programs or specific study goals.

Human antibodies induce arthritis in mice deficient in the low-affinity inhibitory IgG receptor Fc gamma RIIB.

Rheumatoid arthritis (RA) is a complex autoimmune disease with a poorly understood pathogenesis. The disease is associated with polyclonal B cell activation and the production of autoantibodies (autoAbs), but there is a longstanding controversy as to whether such Abs contribute to, or are secondary to, the pathogenesis of RA. To address the potential pathogenicity of human RA-associated Abs, we developed a passive transfer model involving mice deficient in the low-affinity inhibitory Fc receptor, FcgammaRIIB. We report that plasma or serum from patients with active RA can induce inflammation and histological lesions in FcgammaRIIB-/- mice consistent with arthritis, and that this pathogenic activity is caused by the immunoglobulin G-rich fraction. Our results suggest that humoral autoimmunity can contribute directly to autoimmune arthritis, and that FcgammaRIIB-/- mice are a promising model to evaluate the arthritogenic potential of human autoAbs.

The mouse mutation "thrombocytopenia and cardiomyopathy" (trac) disrupts Abcg5: a spontaneous single gene model for human hereditary phytosterolemia/sitosterolemia.

The spontaneous mouse mutation "thrombocytopenia and cardiomyopathy" (trac) causes macrothrombocytopenia, prolonged bleeding times, anemia, leukopenia, infertility, cardiomyopathy, and shortened life span. Homozygotes show a 20-fold decrease in platelet numbers and a 3-fold increase in platelet size with structural alterations and functional impairments in activation and aggregation. Megakaryocytes in trac/trac mice are present in increased numbers, have poorly developed demarcation membrane systems, and have decreased polyploidy. The thrombocytopenia is not intrinsic to defects at the level of hematopoietic progenitor cells but is associated with a microenvironmental abnormality. The trac mutation maps to mouse chromosome 17, syntenic with human chromosome 2p21-22. A G to A mutation in exon 10 of the adenosine triphosphate (ATP)-binding cassette subfamily G, member 5 (Abcg5) gene, alters a tryptophan codon (UGG) to a premature stop codon (UAG). Crosses with mice doubly transgenic for the human ABCG5 and ABCG8 genes rescued platelet counts and volumes. ABCG5 and ABCG8 form a functional complex that limits dietary phytosterol accumulation. Phytosterolemia in trac/trac mice confirmed a functional defect in the ABCG5/ABCG8 transport system. The trac mutation provides a new clinically significant animal model for human phytosterolemia and provides a new means for studying the role of phytosterols in hematologic diseases and testing therapeutic interventions.

Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region.

Acute myelogenous leukemia (AML) is the most common adult leukemia, characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem (LS) cells. To understand the functional properties of human LS cells, we developed a primary human AML xenotransplantation model using newborn nonobese diabetic/severe combined immunodeficient/interleukin (NOD/SCID/IL)2r gamma(null) mice carrying a complete null mutation of the cytokine gamma c upon the SCID background. Using this model, we demonstrated that LS cells exclusively recapitulate AML and retain self-renewal capacity in vivo. They home to and engraft within the osteoblast-rich area of the bone marrow, where AML cells are protected from chemotherapy-induced apoptosis. Quiescence of human LS cells may be a mechanism underlying resistance to cell cycle-dependent cytotoxic therapy. Global transcriptional profiling identified LS cell-specific transcripts that are stable through serial transplantation. These results indicate the potential utility of this AML xenograft model in the development of novel therapeutic strategies targeted at LS cells.

The occurrence of tarsal injuries in male mice of C57BL/6N substrains in multiple international mouse facilities.

Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in group-housed adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneus-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed.

Humanized NOD/LtSz-scid IL2 receptor common gamma chain knockout mice in diabetes research.

There are many rodent models of autoimmune diabetes that have been used to study the pathogenesis of human type 1 diabetes (T1D), including the non-obese diabetic (NOD) mouse, the biobreeding (BB) rat, and the transgenic mouse models. However, mice and rats are not humans, and these rodent models do not completely recapitulate the autoimmune pathogenesis of the human disease. In addition, many of the reagents, tools, and therapeutics proposed for use in humans may be species specific and cannot be investigated in rodents. Researchers have used nonhuman primates to more closely mimic the human immune system and, to study species-specific therapeutics, but these studies are associated with additional ethical and economic constraints and, to date, no model of autoimmune diabetes in this species has been described. New animal models are needed that will permit the in vivo investigation of human immune systems and analyses of the pathogenesis of human T1D without putting individuals at risk. To fill this need, we are developing humanized mouse models for the in vivo study of T1D. These models are based on our newly generated stock of NOD-scid IL2rgamma(null) mice, which engraft at higher levels with human hematolymphoid cells and exhibit enhanced function of the engrafted human immune systems compared with previous humanized mouse models. Overall, development of these new generations of humanized mice should facilitate in vivo studies of the human immune system as well as permit the investigation of the pathogenesis and effector phases of human T1D.

Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion.

To obtain insights into the cardiomyogenic potential of hematopoietic tissue, we intravenously (i.v.) injected purified hematopoietic stem/progenitor cells into newborn recipients that may fully potentiate the developmental plasticity of stem cells. Transplantation of mouse bone marrow (BM) lineage antigen-negative (Lin-) cells resulted in the generation of the cells that displayed cardiomyocyte-specific antigenic profiles and contractile function when transplanted into syngeneic newborn recipients. To clarify the mechanism underlying the cardiomyogenic potential, green fluorescent protein (GFP)-labeled BM Lin-ScaI+ hematopoietic progenitors were transplanted into neonatal mice constitutively expressing cyan fluorescence protein (CFP). Lambda image acquisition and linear unmixing analysis using confocal microscopy successfully separated GFP and CFP, and revealed that donor GFP+ cardiomyocytes coexpressed host-derived CFP. We further reconstituted human hemopoietic- and immune systems in mice by injecting human cord blood (CB)-derived Lin-CD34+CD38- hematopoietic stem cells (HSCs) into neonatal T cell(-)B cell(-)NK cell- immune-deficient NOD/SCID/IL2rgamma(null) mice. Fluoroescence in situ hybridization analysis of recipient cardiac tissues demonstrated that human and murine chromosomes were colocalized in the same cardiomyocytes, indicating that cell fusion occurred between human hematopoietic progeny and mouse cardiomyocytes. These syngeneic- and xenogeneic neonatal transplantations provide compelling evidence that hematopoietic stem/progenitor cells contribute to the postnatal generation of cardiomyocytes through cell fusion, not through transdifferentiation.

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.

Continuous Glucose Monitoring in Female NOD Mice Reveals Daily Rhythms and a Negative Correlation With Body Temperature.

Previous studies with continuous glucose monitoring in mice have been limited to several days or weeks, with the mouse's physical attachment to the equipment affecting behavior and measurements. In the current study, we measured blood glucose and body temperature at 10-second intervals for 12 weeks in a cohort of NOD/ShiLtJ female mice using wireless telemetry. This allowed us to obtain a high-resolution profile of the circadian rhythm of these two parameters and the onset of hyperglycemic development in real time. The most striking observations were the elevated nocturnal concentrations of glucose into the diabetic range days before elevations in diurnal glucose (when glucose concentrations are historically measured) and the strong, negative correlation between elevated blood glucose concentrations and body temperature with a steady decline of the body temperature with diabetes development. Taken together, this technological advancement provides improved resolution in the study of the disease trajectory of diabetes in mouse models, including relevant translatability to the current technologies of continuous glucose monitoring now regularly used in patients.

Antibiotic-associated Manipulation of the Gut Microbiota and Phenotypic Restoration in NOD Mice.

Segmented filamentous bacterium (SFB) a gram-positive, anaerobic, and intestinal commensal organism directly influences the development of Th17 helper cells in the small intestine of mice. In NOD mice, SFB colonization interferes with the development of type 1 diabetes (T1D), a T-cell-mediated autoimmune disease, suggesting that SFB may influence Th17 cells to inhibit Th1 populations associated with the anti-ß-cell immune response. This effect is a serious concern for investigators who use NOD mice for diabetes research because the expected incidence of disease decreases markedly when they are colonized by SFB. A room housing mice for T1D studies at The Jackson Laboratory was determined by fecal PCR testing to have widespread SFB colonization of multiple NOD strains after a steady decline in the incidence of T1D was noted. Rederivation of all NOD-related mouse strains was not feasible; therefore an alternative treatment using antibiotics to eliminate SFB from colonized mice was undertaken. After antibiotic treatment, soiled bedding from NOD mouse strains housed in SFB-free high-health-status production barrier rooms was used to reintroduce the gastrointestinal microbiota. Over the past 16 mo since treating the mice and disinfecting the mouse room, regular PCR testing has shown that no additional SFB colonization of mice has occurred, and the expected incidence of T1D has been reestablished in the offspring of treated mice.

Deficiency of SHP-1 protein-tyrosine phosphatase in "viable motheaten" mice results in retinal degeneration.

PURPOSE: Viable motheaten mutant mice (abbreviated allele symbol me(v)) are deficient in Src-homology 2-domain phosphatase (SHP)-1, a critical negative regulator of signal transduction in hematopoietic cells. These mice exhibit immune dysfunction, hyperproliferation of myeloid cells, and regenerative anemia. This study focused on the role of SHP-1 in retinal homeostasis. METHODS: Ophthalmoscopy, histology, transmission electron microscopy (TEM), electroretinography (ERG), immunohistochemistry, Western blot, bone marrow transplantation, and genetic crosses were performed for phenotypic characterization and functional studies of retinal degeneration (RD) in me(v)/me(v) mice. RESULTS: Fundus examinations of me(v)/me(v) mice revealed numerous, small white spots. Histologic examination demonstrated photoreceptor loss beginning at 3 weeks of age, and TEM revealed disorganization and reduction in the number of outer segments, as well as the presence of phagocytic cells in the subretinal space. Rod- and cone-mediated ERGs were abnormal. SHP-1 protein was expressed in mouse and human retinal lysates and was localized to the outer nuclear layer of the retina in me(v)/me(v) and control mice. Autoantibodies are not necessary for RD, as B-cell-deficient me(v)/me(v) Igh-6(tm1Cgn) mice had no attenuation of photoreceptor cell loss compared with age-matched me(v)/me(v) mice. Histologic examination of lungs and retinas from normal recipients of me(v)/me(v) marrow revealed the classic acidophilic macrophage pneumonia of me(v)/me(v) mice, but no retinal degeneration. CONCLUSIONS: me(v)/me(v) mice exhibit normal retinal development with the onset of RD at 3 weeks of age and a rapidly progressive loss of photoreceptors. These findings support the hypothesis that SHP-1 plays a critical role in retinal homeostasis.

Accelerated wound healing of alkali-burned corneas in MRL mice is associated with a reduced inflammatory signature.

PURPOSE: The present study was conducted to investigate healing of alkali-burned corneas in MRL/MpJ (MRL) mice. METHODS: Gross, clinical, and histologic criteria were used to compare healing of alkali-burned corneas in MRL and control C57BL/6J (B6) mice. Effects of neutrophil depletion of B6 mice and allogeneic reconstitution of B6 mice with MRL bone marrow on wound healing were evaluated. Gene expression patterns in normal and wounded corneas were surveyed with array-based quantitative real-time RT-PCR (AQPCR). RESULTS: MRL mice showed accelerated reepithelialization and decreased corneal opacity compared with B6 mice after alkali wounding. Marked inflammatory cell infiltration and fibrosis were evident in the corneas and anterior chambers of B6 mice. MRL mice showed less severe lesions, except for stromal edema. Rapid reepithelialization and reduced keratitis/iritis were also observed in neutrophil-depleted B6 mice, but not in B6 mice reconstituted with MRL bone marrow. AQPCR showed transcriptional changes of fewer genes associated with inflammation and corneal tissue homeostasis in alkali-burned corneas from MRL mice. Increased expression of an anti-inflammatory gene, Socs1, and a gene associated with healing, Mmp1a, were evident in MRL corneas. CONCLUSIONS: Alkali-burned corneas heal faster and more completely in MRL mice than in B6 mice, by means of rapid reepithelialization, reduced inflammation, and reduced fibrosis. Reduced inflammation, including decreased neutrophil infiltrates and the lack of a robust proinflammatory gene expression signature correlates with the rapid healing. However, the rapid-healing phenotype is not intrinsic to MRL hematopoietic progenitor cells.

The Tetratricopeptide repeat domain 7 gene is mutated in flaky skin mice: a model for psoriasis, autoimmunity, and anemia.

The flaky skin (fsn) mutation in mice causes pleiotropic abnormalities including psoriasiform dermatitis, anemia, hyper-IgE, and anti-dsDNA autoantibodies resembling those detected in systemic lupus erythematosus. The fsn mutation was mapped to an interval of 3.9 kb on chromosome 17 between D17Mit130 and D17Mit162. Resequencing of known and predicted exons and regulatory sequences from this region in fsn/fsn and wild-type mice indicated that the mutation is due to the insertion of an endogenous retrovirus (early transposon class) into intron 14 of the Tetratricopeptide repeat (TPR) domain 7 (Ttc7) gene. The insertion leads to reduced levels of wild-type Ttc7 transcripts in fsn mice and the insertion of an additional exon derived from the retrovirus into the majority of Ttc7 mRNAs. This disrupts one of the TPRs within TTC7 and may affect its interaction with an as-yet unidentified protein partner. The Ttc7 is expressed in multiple types of tissue including skin, kidney, spleen, and thymus, but is most abundant in germinal center B cells and hematopoietic stem cells, suggesting an important role in the development of immune system cells. Its role in immunologic and hematologic disorders should be further investigated.

Mechanisms of anemia in SHP-1 protein tyrosine phosphatase-deficient "viable motheaten" mice.

OBJECTIVE: Viable motheaten mice (abbreviated gene symbol me(v)) are deficient in SHP-1, a critical negative regulator of signal transduction in hematopoietic cells. These mice exhibit severe immune dysfunction accompanied by hyperproliferation of myeloid cells, widespread inflammatory lesions, and regenerative anemia. The aim of this study was to investigate the mechanisms underlying anemia in me(v)/me(v) mice. MATERIALS AND METHODS: Multiple hematologic parameters, osmotic fragility, and erythropoietin levels were measured to characterize the anemia in me(v)/me(v) mice. B-cell-deficient me(v)/me(v) Igh-6(null) mice were generated to assess the role of anti-erythrocyte antibodies. Coombs assays and flow cytometry were carried out for detection of anti-erythrocyte antibodies. Oxidant production by macrophages, glutathione levels, and lipid peroxidation products in erythrocytes were measured, as was the impact of oxidant on the ultrastructure of me(v)/me(v) erythrocytes. Erythroid maturation and erythrocyte plasma membrane integrity were assessed with flow cytometry by evaluating CD71 expression and annexin V labeling. RESULTS: The regenerative anemia of me(v)/me(v) mice was associated with erythrocyte changes that were independent of the presence of anti-erythrocyte antibodies. Erythrocytes from me(v)/me(v) mice had increased fragility and heightened susceptibility to oxidant damage. Macrophages from me(v)/me(v) mice demonstrated a higher basal level of oxidant production and enhanced production after stimulation. Oxidant damage in me(v)/me(v) erythrocytes was evidenced by a significant elevation of lipid peroxidation and diminished levels of glutathione. CONCLUSION: Our results support the hypothesis that as a consequence of severe inflammatory disease, me(v)/me(v) erythrocytes are subject to exceptionally high oxidative stress resulting in oxidation of phospholipids in the erythrocyte membrane with subsequent hemolysis.

NOD/LtSz-Rag1nullPfpnull mice: a new model system with increased levels of human peripheral leukocyte and hematopoietic stem-cell engraftment.

BACKGROUND: A critical need exists for effective small-animal models that accept engraftment of human hematopoietic progenitor cells and mature lymphocytes. The purpose of this study was to determine the phenotypic effects of perforin (Pfp) deficiency on nonobese diabetic (NOD)-Rag1null mice and to evaluate the ability of NOD/LtSz-Rag1nullPfpnull recipients to support engraftment with human hematolymphoid cells. METHODS: A new genetic stock of NOD mice doubly homozygous for targeted mutations at the recombination activating gene (Rag)-1 and Pfp genes was developed. NOD/LtSz-Rag1nullPfpnull mice were studied for immunopathologic and hematologic abnormalities. The ability of these mice to support engraftment with human peripheral blood mononuclear cells (PBMC) and umbilical-cord blood hematopoietic progenitor cells was assessed. RESULTS: NOD/LtSz-Rag1nullPfpnull mice lacked mature B cells, T cells, natural killer (NK) cell cytotoxic activity and were devoid of serum immunoglobulin (Ig) throughout a 37-week lifespan. These mice supported heightened engraftment with human PBMC as compared with NOD/LtSz-Rag1null controls as evidenced by a 4- to 5-fold increase in percentages of human lymphocytes and a 7- to 13-fold increase in percentages of CD4+ T cells in the peripheral blood and spleen. Total numbers of human CD4+ T cells were increased approximately 20-fold in the spleens of NOD/LtSz-Rag1nullPfpnull mice. These mice also showed approximately 12-fold higher levels of engraftment with human umbilical-cord blood cells compared with NOD/LtSz-Rag1null mice. CONCLUSIONS: NOD/LtSz-Rag1nullPfpnull mice are devoid of mature B cell, T cell, and NK cell cytotoxic activity, engraft at high levels with human PBMC, and hematopoietic progenitor cells and provide a new NK cell-deficient model for human hematolymphoid cell engraftment.

Alloimmune injury and rejection of human skin grafts on human peripheral blood lymphocyte-reconstituted non-obese diabetic severe combined immunodeficient beta2-microglobulin-null mice.

Small animal models with the capacity to support engraftment of a functional human immune system are needed to facilitate studies of human alloimmunity. In the present investigation, non-obese diabetic (NOD) severe combined immunodeficient (scid) beta2-microglobulin-null (B2mnull) mice engrafted with human peripheral blood lymphocytes (hu-PBL-NOD-scid B2mnull mice) were used as in vivo models for studying human skin allograft rejection. Hu-PBL-NOD-scid B2mnull mice were established by injection of human spleen cells or PBLs and transplanted with full-thickness allogeneic human skin. Human cell engraftment was enhanced by injection of anti-mouse CD122 antibody. The respective contributions of human CD4+ and CD8+ cells in allograft rejection were determined using depleting antibodies. Human skin grafts on unmanipulated NOD-scid B2mnull mice uniformly survived but on chimeric hu-PBL-NOD-scid B2mnull mice exhibited severe immune-mediated injury that often progressed to complete rejection. The alloaggressive hu-PBLs did not require prior in vitro sensitization to elicit targeted effector cell activity. Extensive mononuclear cell infiltration directed towards human-origin endothelium was associated with thrombosis and fibrin necrosis. No evidence of graft-versus-host disease was detected. Either CD4+ or CD8+ T cells may mediate injury and alloimmune rejection of human skin grafts on hu-PBL-NOD-scid B2mnull mice. It is proposed that Hu-PBL-NOD-scid B2mnull mice engrafted with human skin will provide a useful model for analysis of interventions designed to modulate human allograft rejection.

Establishment and characterization of a new osteogenic cell line (MOS-J) from a spontaneous C57BL/6J mouse osteosarcoma.

In this paper we describe the establishment and characterization of a transplantable cell line derived from a spontaneously occurring chondroblastic osteosarcoma in a C57BL/6J mouse. The tumor line, MOS-J, forms solid tumors when injected intramuscularly into immunocompetent syngeneic hosts, mimicking endochondral bone development. These transplantable tumors have the capacity to destroy and invade existing bone and invade vessels in close proximity to the tumor. In culture, MOS-J cells form layers of pleomorphic cells with high mitotic activity. These cells have marked alkaline phosphatase activity and form calcified foci in vitro that stain with alizarin red. MOS-J cells also promote osteoclast development in vitro from normal bone marrow cells. These characteristics indicate the potential utility of the MOS-J osteosarcoma cell line as a model for studies of human osteosarcoma and bone biology.