MYCN induces neuroblastoma in primary neural crest cells.

Neuroblastoma (NBL) is an embryonal cancer of the sympathetic nervous system (SNS), which causes 15% of pediatric cancer deaths. High-risk NBL is characterized by N-Myc amplification and segmental chromosomal gains and losses. Owing to limited disease models, the etiology of NBL is largely unknown, including both the cell of origin and the majority of oncogenic drivers. We have established a novel system for studying NBL based on the transformation of neural crest cells (NCCs), the progenitor cells of the SNS, isolated from mouse embryonic day 9.5 trunk neural tube explants. Based on pathology and gene expression analysis, we report the first successful transformation of wild-type NCCs into NBL by enforced expression of N-Myc, to generate phenotypically and molecularly accurate tumors that closely model human MYCN-amplified NBL. Using comparative genomic hybridization, we found that NCC-derived NBL tumors acquired copy number gains and losses that are syntenic to those observed in human MYCN-amplified NBL including 17q gain, 2p gain and loss of 1p36. When p53-compromised NCCs were transformed with N-Myc, we generated primitive neuroectodermal tumors with divergent differentiation including osteosarcoma. These subcutaneous tumors were metastatic to regional lymph nodes, liver and lung. Our novel experimental approach accurately models human NBL and establishes a new system with potential to study early stages of NBL oncogenesis, to functionally assess NBL oncogenic drivers and to characterize NBL metastasis.

Novel MYC-driven medulloblastoma models from multiple embryonic cerebellar cells.

Group3 medulloblastoma (MBG3) that predominantly occur in young children are usually associated with MYC amplification and/or overexpression, frequent metastasis and a dismal prognosis. Physiologically relevant MBG3 models are currently lacking, making inferences related to their cellular origin thus far limited. Using in utero electroporation, we here report that MBG3 mouse models can be developed in situ from different multipotent embryonic cerebellar progenitor cells via conditional expression of Myc and loss of Trp53 function in several Cre driver mouse lines. The Blbp-Cre driver that targets embryonic neural progenitors induced tumors exhibiting a large-cell/anaplastic histopathology adjacent to the fourth ventricle, recapitulating human MBG3. Enforced co-expression of luciferase together with Myc and a dominant-negative form of Trp53 revealed that GABAergic neuronal progenitors as well as cerebellar granule cells give rise to MBG3 with their distinct growth kinetics. Cross-species gene expression analysis revealed that these novel MBG3 models shared molecular characteristics with human MBG3, irrespective of their cellular origin. We here developed MBG3 mouse models in their physiological environment and we show that oncogenic insults drive this MB subgroup in different cerebellar lineages rather than in a specific cell of origin.

Transgenic mouse model of IgM+ lymphoproliferative disease mimicking Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a low-grade incurable immunoglobulin M+ (IgM+) lymphoplasmacytic lymphoma for which a genetically engineered mouse model of de novo tumor development is lacking. On the basis of evidence that the pro-inflammatory cytokine, interleukin 6 (IL6), and the survival-enhancing oncoprotein, B cell leukemia 2 (BCL2), have critical roles in the natural history of WM, we hypothesized that the enforced expression of IL6 and BCL2 in mice unable to perform immunoglobulin class switch recombination may result in a lymphoproliferative disease that mimics WM. To evaluate this possibility, we generated compound transgenic BALB/c mice that harbored the human BCL2 and IL6 transgenes, EµSV-BCL2-22 and H2-Ld-hIL6, on the genetic background of activation-induced cytidine deaminase (AID) deficiency. We designated these mice BCL2+IL6+AID- and found that they developed-with full genetic penetrance (100% incidence) and suitably short latency (93 days median survival)-a severe IgM+ lymphoproliferative disorder that recapitulated important features of human WM. However, the BCL2+IL6+AID- model also exhibited shortcomings, such as low serum IgM levels and histopathological changes not seen in patients with WM, collectively indicating that further refinements of the model are required to achieve better correlations with disease characteristics of WM.

Rodent immunohistochemistry: pitfalls and troubleshooting.

Immunohistochemistry (IHC) is a common adjunct in pathology for morphologic diagnosis, research pathology, and studying the pathogenesis of the disease. Proper technique and interpretation of an immunohistochemistry assay is of utmost importance. A variety of problems, including the presence of artifacts (nonspecific background or other staining problems) and the differentiation between nonspecific and specific staining, commonly occur. It is essential that antibody quality and IHC technique be optimized. We review the histologic patterns of specific and nonspecific staining after using IHC techniques, as well as basic troubleshooting procedures, and provide some examples of nonspecific staining and other artifacts especially in formalin-fixed, paraffin-embedded tissues (FFPE) of mice.

Cryptogenic organizing pneumonia in Tomm5(-/-) mice.

Almost all mitochondrial proteins are encoded in the nuclear DNA and synthesized in the cytosol as pre-proteins. There is a protein translocase located in the mitochondrial outer membrane that transports mitochondrial pre-proteins into mitochondria. The central component of this translocase of the outer mitochondrial membrane (TOMM) complex is TOMM40, and TOMM5 is one of three small subunits associated with TOMM40. Translocase of outer mitochondrial membrane 5 homolog (Tomm5(-/-)) knockout mice demonstrated an unexpected lung-specific phenotype characterized by widespread intra-alveolar fibrosis. Although TOMM5-deficient mice tested normal in a very broad range of phenotyping assays, they displayed histopathological lesions in the lung that were consistent with those reported in humans with cryptogenic organizing pneumonia (COP), which is also known as bronchiolitis obliterans organizing pneumonia (BOOP). The lesions had a patchy distribution in the lung and were characterized by the presence of intraluminal fibrogenic buds consisting of fibroblasts and myofibroblasts embedded in a loose connective tissue matrix that occupied the lumina of alveoli and alveolar ducts, with preservation of underlying alveolar architecture. In addition to macrophages, which were numerous in affected and surrounding alveoli, eosinophils comprised the most common and widespread inflammatory cell. Taken together, the findings in Tomm5(-/-) mice provide yet another example of the value of histopathology as a baseline assay in high-throughput phenotyping systems.

Genetically defining the mechanism of Puma- and Bim-induced apoptosis.

Using genetically modified mouse models, we report here that p53 upregulated modulator of apoptosis (Puma) and Bcl-2 interacting mediator of cell death (Bim), two pro-apoptotic members of the B-cell lymphoma protein-2 (Bcl-2) family of proteins, cooperate in causing bone marrow and gastrointestinal tract toxicity in response to chemo and radiation therapy. Deletion of both Puma and Bim provides long-term survival without evidence of increased tumor susceptibility following a lethal challenge of carboplatin and ionizing radiation. Consistent with these in vivo findings, studies of primary mast cells demonstrated that the loss of Puma and Bim confers complete protection from cytokine starvation and DNA damage, similar to that observed for Bax/Bak double knockout cells. Biochemical analyses demonstrated an essential role for either Puma or Bim to activate Bax, thereby leading to mitochondrial outer membrane permeability, cytochrome c release and apoptosis. Treatment of cytokine-deprived cells with ABT-737, a BH3 mimetic, demonstrated that Puma is sufficient to activate Bax even in the absence of all other known direct activators, including Bim, Bid and p53. Collectively, our results identify Puma and Bim as key mediators of DNA damage-induced bone marrow failure and provide mechanistic insight into how BH3-only proteins trigger cell death.

Morphological and immunohistochemical characterization of sarcomatous tumors in wild-type and genetically engineered mice.

Malignant soft tissue tumors are commonly observed in wild-type and gene-targeted mice. These tumors have different degrees of differentiation, cellularity, cellular atypia, nuclear pleomorphism, normal and abnormal mitosis, and giant tumor cells with enlarged polylobulated nuclei. They are often diagnosed as pleomorphic sarcoma, undifferentiated sarcoma, fibrosarcoma, malignant fibrous histiocytoma, sarcoma, or sarcoma, not otherwise specified. Pleomorphic sarcomas have no morphological differentiation toward a differentiated mesenchymal or other tumor type in hematoxylin and eosin-stained sections. With the use of immunohistochemistry, human and mouse, tumors associated with these broad nonspecific diagnoses can often be demonstrated to be of a specific cellular lineage. With mouse models being used to delineate the molecular mechanisms, pathogenesis, and cellular origin of human sarcomas, it will be necessary to correlate the morphological and cellular lineage and the molecular profiles of the pleomorphic tumors associated with these mouse models. The results presented here show that with the use of immunohistochemistry, the cellular lineage of many mouse tumors with pleomorphic features can be determined.

Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines.

BACKGROUND: In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics. METHODS: We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays. FINDINGS: A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs. INTERPRETATION: The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.

Dmp1 is haplo-insufficient for tumor suppression and modifies the frequencies of Arf and p53 mutations in Myc-induced lymphomas.

Loss of Dmp1, an Arf transcriptional activator, leads to spontaneous tumorigenesis in mice, causing death from various forms of cancer by two years of age. Retention and expression of the wild-type Dmp1 allele in tumors arising in Dmp1(+/-) mice demonstrate that Dmp1 can be haplo-insufficient for tumor suppression. The mean latency of E(mu)-Myc-induced B-cell lymphomas is halved on a Dmp1(-/-) or Dmp1(+/-) genetic background. Although p53 mutations or Arf deletion normally occur in approximately 50% of E(mu)-Myc-induced lymphomas, Dmp1 loss obviates selection for such mutations, indicating that Dmp1 is a potent genetic modifier of the Arf-p53 pathway in vivo.

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d).

Male mice lacking both the Ink4c and Ink4d genes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18(Ink4c) and p19(Ink4d) are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking both Ink4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lacking Ink4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast, Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed in Ink4c/Ink4d double-null males. Our data indicate that p18(Ink4c) and p19(Ink4d) are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.

Persistent transmission of mouse hepatitis virus by transgenic mice.

Variation in susceptibility to viral infection is well documented across mouse strains. Specific combinations of viral strains and murine hosts may favor viral infection and disease, and could potentially allow the unexpected development of chronic, persistent, or latent infections. In some genetically modified strains of mice, immune function and perhaps other physiologic or metabolic systems may be substantially or marginally impaired. In the case study reported here, we document the apparent persistent transmission of mouse hepatitis virus (MHV) over a two-year period by MHV-seropositive transgenic mice. Transmission occurred via direct contact with seropositive mice and exposure to contaminated bedding. However, MHV was not detected at diagnostic laboratories by use of viral isolation or reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of tissues from MHV-seropositive animals. Our observation, together with the constantly expanding varieties of immune-impaired or poorly characterized murine hosts and the burgeoning dissemination of these animals throughout the biomedical research community, indicate that unexpected pathophysiologic presentations of common murine viral diseases may present new challenges to the biomedical research community in the future.

p53-independent functions of the p19(ARF) tumor suppressor.

The p19(ARF) tumor suppressor antagonizes Mdm2 to induce p53-dependent cell cycle arrest. Individual TKO (triple knock out) mice nullizygous for ARF, p53, and Mdm2 develop multiple tumors at a frequency greater than those observed in animals lacking both p53 and Mdm2 or p53 alone, demonstrating that p19(ARF) can act independently of the Mdm2-p53 axis in tumor surveillance. Reintroduction of ARF into TKO mouse embryo fibroblasts (MEFs), but not into those lacking both p53 and ARF, arrested the cell division cycle in the G1 phase. Inhibition of the retinoblastoma protein had no effect on the ability of ARF to arrest TKO MEFs. Thus, in the absence of Mdm2, p19(ARF) interacts with other targets to inhibit cell proliferation.

Effect of CD40 ligand and other immunomodulators on Pneumocystis carinii infection in rat model.

The corticosteroid-treated animal is well established as an experimental model for the study of Pneumocystis carinii pneumonitis (PCP). Latent or acquired infection with P. carinii in the murine lung progresses to fatal pneumonitis when the host is profoundly immunocompromized. In this study the effects of five immunomodulators; recombinant CD40 ligand (CD40L), bryostatin 1, recombinant FLT3 ligand (FLT3L), recombinant granulocyte colony-stimulating factor (G-CSF) and recombinant interleukin-15 (IL-15) were investigated against PCP in a dexamethasone immunosuppressed Sprague-Dawley rat model. The majority of rats (70%) treated with CD40L at the onset of dexamethasone immunosuppression were protected against PCP. When CD40L was given after 10 days of immunosuppression, only 40% of the rats resolved the infection. However, 95% of the control animals developed PCP. Immunosuppressed rats treated with bryostatin 1, an immune activator had a partial (50%) protection against P. carinii infection. In contrast, daily administration of FLT3L, IL-15 or G-CSF provided no protection against P. carinii infection.

Disruption of the ARF transcriptional activator DMP1 facilitates cell immortalization, Ras transformation, and tumorigenesis.

The DMP1 transcription factor induces the ARF tumor suppressor gene in mouse fibroblasts, leading to cell cycle arrest in a p53-dependent manner. We disrupted sequences encoding the DNA-binding domain of DMP1 in mouse embryonic stem cells and derived animals lacking the functional protein. DMP1-null animals are small at birth, and males develop more slowly than their wild-type littermates. Some adult animals exhibit seizures and/or obstuctive uropathy, each of unknown cause. The growth of explanted DMP1-null mouse embryo fibroblasts (MEFs) is progressively retarded as cells are passaged in culture on defined transfer protocols; but, unlike the behavior of normal cells, p19(ARF), Mdm2, and p53 levels remain relatively low and DMP1-null MEFs do not senesce. Whereas the establishment of cell lines from MEFs is usually always accompanied by either p53 or ARF loss of function, continuously passaged DMP1-null cells readily give rise to established 3T3 and 3T9 cell lines that retain wild-type ARF and functional p53 genes. Early-passage DMP1-null cells, like MEFs from either ARF-null or p53-null mice, can be morphologically transformed by oncogenic Ha-Ras (Val-12) alone. Splenic lymphocytes harvested from both DMP1-null and ARF-null mice exhibit enhanced proliferative responses in long-term cultures when stimulated to divide with antibody to CD3 and interleukin-2. Although only 1 of 40 DMP1-null animals spontaneously developed a tumor in the first year of life, neonatal treatment with dimethylbenzanthracene or ionizing radiation induced tumors of various histologic types that were not observed in similarly treated DMP1(+/+) animals. Karyotypic analyses of MEFs and lymphomas from DMP1-null animals revealed pseudodiploid chromosome numbers, consistent with the retention of wild-type p53. Together, these data suggest that ARF function is compromised, but not eliminated, in animals lacking functional DMP1.

Protection from Cryptosporidium parvum infection by gammadelta T cells in mice that lack alphabeta T cells.

BACKGROUND AND PURPOSE: Cryptosporidium parvum establishes a parasitic relationship with epithelial cells of the intestine. Infection with this protozoan is resolved in the immunocompetent host, but persistent life-threatening infection develops in the immunocompromised host. We propose that gammdelta T cells in the intestinal mucosa play a role in immunity to C. parvum. METHODS: Intestinal intra-epithelial lymphocyte and lamina propria T-cell subsets were examined in mice infected with C. parvum. The mice are homozygous for a deletion of the TCRalpha chain gene, TCRalpha(-/-) and, therefore, lack conventional alphabeta T cells, but retain a population of T cells with gammadelta T-cell receptors. To examine the contribution of gammadelta T cells to immunity, these mice were treated with monoclonal antibody GL3-3A, specific for this T-cell receptor, then were inoculated with C. parvum oocysts. Lymphocyte subsets and hematoxylin and eosin (H&E)-stained intestinal sections from untreated mice were compared with those from mice treated with either a low dose of GL3-3A for 6 weeks, or a high dose of GL3-3A for 16 weeks. RESULTS: The proportion of gammadelta T cells in the lamina propria increased in infected mice. In mice treated with a low dose of GL3-3A, a population of gammadelta T cells that had characteristics of activated cells, was still evident 6 weeks after inoculation. No C. parvum developmental forms were identified in the intestinal sections of mice under these conditions. However, TCRalpha(-/-) mice treated with a high dose of GL3-3A were depleted of gammadelta T cells, and 50% of the mice were infected with C. parvum. CONCLUSIONS: The gammadelta T cells contribute to protection against C. parvum infection. In the absence of conventional T cells, activation of intestinal gammadelta T cells may prevent infection with this organism.

Toxicity evaluation of prophylactic treatments for mites and pinworms in mice.

The detection of external and internal parasites in laboratory mice is a particularly problematic aspect of animal health evaluation. Because these organisms must be detected by direct examination of the feces or hair coat, low-level infestation or sporadic shedding can make them difficult to detect, thereby undermining confidence that negative reports are truly negative. Prophylactic treatment of suspect colonies with anthelminthics and/or insecticides may therefore be indicated under some circumstances. However, when considering the use of prophylactic treatments, the potential for toxicity is an important factor, especially in genetically modified strains of mice. To evaluate the potential toxicity of prophylactic anti- parasitic treatments on strains of mice that are commonly used as experimental models and in genetic engineering in our facility, we surveyed a number of strains and ages of mice for toxic reactions during treatment regimens that combine anthelminthic and anti-acaricidal agents. Three experimental protocols (ivermectin, piperazine, and dichlorvos in combination; ivermectin alone; and fenbendazole/permethrin or fenbendazole/dichlorvos) were evaluated. Our data suggest a potential for toxicity associated with these treatments and indicate to us that prophylactic treatment regimens should be initiated with caution.

Effects of sleep deprivation and other stressors on the immune and inflammatory responses of influenza-infected mice.

Many stressors have well-documented effects on host immune competence. However, two important stressors that have not been extensively characterized in terms of their immune-modulatory properties are sleep deprivation and alterations in light:dark cycles. We therefore evaluated the effects of these stressors on the immune and inflammatory responses of mice inoculated intranasally with influenza virus. In contrast to a previous report, sleep deprivation did not significantly alter viral clearance or antibody titers of either virus-naive or immunized mice. Exposure to constant light also failed to affect these variables. However, repeated overnight restraint, a well-characterized stressor, reduced the pulmonary inflammatory response elicited by influenza virus, as previously reported. The data indicate that sleep deprivation and altered light cycles do not markedly influence selected host defense responses to influenza infection under the conditions tested.

Rodent quarantine programs: purpose, principles, and practice.

In animal research, validity and reproducibility of data are critically influenced by the microbial status of the experimental animals. One of the most crucial aspects of assuring quality in animal research is providing research personnel with confidence that experimental results will not be invalidated due to interference caused by infectious disease. An effective quarantine program is essential to providing this assurance. Quarantine programs are generally instituted to prevent the introduction of rodent pathogens into established specific-pathogen-free colonies in a facility. Therefore, programs should be designed to isolate newly acquired rodents until their health status can be determined and to maximize the probability that microorganisms of interest will be detected before the animals are introduced into (and thus, could potentially contaminate) established colonies. Important principles that are critical to designing an effective quarantine program will be discussed here, as will the practical implementation of these principles. Although quarantine programs may be costly in terms of time and effort, these costs must be balanced against the potential costs of disease outbreaks that could invalidate long-term studies, alter normal biological baselines, and cause the loss or necessitate re-derivation of rare or valuable strains of rodents. Reducing the incidence of quarantine failures through appropriate program design and implementation helps to maintain the confidence of research personnel in the value of quarantine programs and in our competence as specialists in laboratory animal management and as partners in the research process.

Disruption of T-cell differentiation precedes T-cell tumor formation in LMO-2 (rhombotin-2) transgenic mice.

Transgenic mice expressing LMO-2 (rhombotin-2) were constructed by placing the LMO-2 gene under control of the metallothionein promoter. Thymic tumors developed in approximately 15% of the transgenic mice between 37 and 71 weeks. Only T-cell tumors were found in the transgenic mice despite high expression of LMO-2 in all tissues. The thymic tumors were aggressive and were invariably associated with metastasis to non-lymphoid organs. In approximately 50% of apparently healthy transgenic mice there was up to a 10-fold expansion of CD4-CD8- double negative (DN) cells. Expansion of the DN cells was accompanied by the compensatory decrease in CD4+CD8+ double positive (DP) cells, indicating that breach of homeostasis within the thymus had not occurred in these animals. The increase in DN cells was associated with a clonal expansion of thymocytes, and increased proliferation within the thymus. Our data indicate that the ectopic expression of LMO-2 in T-cells disrupts normal T-cell differentiation by selectively expanding the DN thymocyte population prior to breach of homeostasis and overt leukemia/lymphoma.