Stat5 is critical for the development and maintenance of myeloproliferative neoplasm initiated by Nf1 deficiency.

Juvenile myelomonocytic leukemia is a rare myeloproliferative neoplasm characterized by hyperactive RAS signaling. Neurofibromin1 (encoded by the NF1 gene) is a negative regulator of RAS activation. Patients with neurofibromatosis type 1 harbor loss-of-function mutations in NF1 and have a 200- to 500-fold increased risk of juvenile myelomonocytic leukemia. Leukemia cells from patients with juvenile myelomonocytic leukemia display hypersensitivity to certain cytokines, such as granulocyte-macrophage colony-stimulating factor. The granulocyte-macrophage colony-stimulating factor receptor utilizes pre-associated JAK2 to initiate signals after ligand binding. JAK2 subsequently activates STAT5, among other downstream effectors. Although STAT5 is gaining recognition as an important mediator of growth factor signaling in myeloid leukemias, the contribution of STAT5 to the development of hyperactive RAS-initiated myeloproliferative disease has not been well described. In this study, we investigated the consequence of STAT5 attenuation via genetic and pharmacological approaches in Nf1-deficient murine models of juvenile myelomonocytic leukemia. We found that homozygous Stat5 deficiency extended the lifespan of Nf1-deficient mice and eliminated the development of myeloproliferative neoplasm associated with Nf1 gene loss. Likewise, we found that JAK inhibition with ruxolitinib attenuated myeloproliferative neoplasm in Nf1-deficient mice. Finally, we found that primary cells from a patient with KRAS-mutant juvenile myelomonocytic leukemia displayed reduced colony formation in response to JAK2 inhibition. Our findings establish a central role for STAT5 activation in the pathogenesis of juvenile myelomonocytic leukemia and suggest that targeting this pathway may be of clinical utility in these patients.

Using online instruction and virtual laboratories to teach hemostasis in a medical laboratory science program.

Hemostasis laboratory testing methods have changed significantly over the past decades, from totally manual, to fully automated methodologies. Most medical laboratory educators prefer to use manual or semiautomated methods to teach hemostasis so that students can "see" what is occurring during the testing method, but many semi-automated instruments are no longer commercially available or are not cost-effective for education programs. In consideration of these factors and due to programmatic expansion to a coordinate campus, the CLS program explored new ways to teach hemostasis methods equitably and affordably across two distant locations. Working with an instructional design team versed in online education, five virtual hemostasis laboratory exercises were created that mimic the manual methodologies. Web-based didactic instruction was also developed to teach the testing theory and pathophysiology related to patient results. The efficacy of the virtual instruction was evaluated through assessment of student performance on exam questions, professional certification scores for the platelet/hemostasis sub-category, student satisfaction surveys, and evaluation of student performance during their clinical experience. Results showed that students in the virtual delivery format performed significantly better on exam questions compared to the traditional delivery method group, but there was no significant difference in their performance on the professional certification exam. Both student and preceptor feedback have been positive on the value of the exercises for students' understanding of hemostasis.

Development of online conferencing and web-based in-service modules for preceptor training.

Over the past five years our clinical laboratory sciences (CLS) program more than doubled student enrollment to help address the workforce shortages in our state. At the same time, medical laboratory technician programs were also increasing enrollment, putting significant pressure on the already limited number of clinical training spaces. To help alleviate the impact on clinical sites, major changes were made to the traditional clinical rotation model; shifting from a clinical training to a clinical experience and a simultaneous decrease in length from 22 to 12 weeks. This change required extensive in-service training for clinical preceptors including rationale for the new clinical experience model, review of student campus education, expectations for preceptors and students, as well as developing and implementing new evaluation methods of student performance. Through funding from the Department of Labor, online interactive modules introducing the concepts of new clinical experience module for both preceptors and for students were developed. To provide a more in-depth and specific in-service for the preceptors, web-conferences were held to describe the students' didactic and laboratory educational background and to discuss the expectations and evaluations of students. Two course management system sites were developed, one for preceptors and one for students, so they could access clinical experience information and materials at any time and from any location. These course sites also allowed us to convert evaluation processes to an online format. Survey feedback from preceptors indicates that this system was well received providing a cost-effective method for preceptor education and communication.

Implementing virtual microscopy improves outcomes in a hematology morphology course.

In this study, we evaluated the efficacy of virtual microscopy as the primary mode of laboratory instruction in undergraduate level clinical hematology teaching. Distance education (DE) has become a popular option for expanding education and optimizing expenses but continues to be controversial. The challenge of delivering an equitable curriculum to distant locations along with the need to preserve our slide collection directed our effort to digitize the slide sets used in our teaching laboratories. Students enrolled at two performance sites were randomly assigned to either traditional microscopy (TM) or virtual microscopy (VM) instruction. The VM group performed significantly better than the TM group. We anticipate that this approach will play a central role in the distributed delivery of hematology through distance education as new programs are initiated to address workforce shortage needs.

Nf1 mutant mice with p19ARF gene loss develop accelerated hematopoietic disease resembling acute leukemia with a variable phenotype.

Juvenile Myelomonocytic Leukemia (JMML) is a relentlessly progressive myeloproliferative/myelodysplastic (MPD/MDS) hematopoietic disorder more common in patients with any one of at least three distinct genetic lesions, specifically NF1 gene loss and PTPN11 and NRAS mutations. NF1 and PTPN11 are molecular lesions associated with Neurofibromatosis Syndrome Type I (NF1 Syndrome) and Noonan's Syndrome, respectively. The occurrence of JMML is rare; even among those predisposed with these syndromes to development of disease, and secondary genetic events likely contribute to the development and progression of disease. In NF1 syndrome, loss of p53 function is a common event in solid tumors, but uncommon in JMML, suggesting that the p53 pathway may be modified by other events in this hematopoietic disorder. The work presented here investigates the possible role of the p19(Arf) (p19) tumor suppressor in development of MPD associated with Nf1 gene loss in mice. We find that Nf1 mutant hematopoietic cells with loss of p19 develop accelerated hematopoietic disease similar to acute leukemia with a variable phenotype. This suggests that p19 may play a role in development of JMML and evaluation of the human p19 homolog (p14(ARF)) in JMML may be informative.

Persistence of CD133+ cells in human and mouse glioma cell lines: detailed characterization of GL261 glioma cells with cancer stem cell-like properties.

The concept of cancer stem cells suggests that there are malignant stem-like cells within a tumor that are responsible for tumor renewal and resistance to cytotoxic therapies. Studies have identified glioma stem-like cells that extrude Hoechst 33342 dye, representing a double-negative "side population" (SP) thought to be selectively resistant to drug therapy. A CD133+ stem cell-like subpopulation has been isolated from a human glioma that was enriched for tumor-initiating cells. It is unknown whether CD133+ cells with similar phenotype persist in established glioma cell lines, or if CD133 is a marker of glioma stem-like cells in rodents. We investigated whether CD133+ and SP cells existed in the GL261 cell line, a syngeneic mouse glioma model that is widely used for preclinical and translational research. Intracerebral injection of less than 100 CD133+ GL261 cells formed tumors, whereas it required 10,000 CD133(-) cells to initiate a tumor. CD133+ GL261 cells expressed nestin, formed tumor spheres with high frequency, and differentiated into glial and neuronal-like cells. Similar to GL261, seven human glioma cell lines analyzed also contained a rare CD133+ population. Surprisingly, we found that CD133+ GL261 cells did not reside in the SP, nor did the majority ( approximately 94%) of CD133+ human glioma cells. These results demonstrate that the expression of CD133 in murine glioma cells is associated with enhanced tumorigenicity and a stem-like phenotype. This study also reveals a previously unrecognized level of heterogeneity in glioma cell lines, exposing several populations of cells that have characteristics of cancer stem cells.

Vav promoter-tTA conditional transgene expression system for hematopoietic cells drives high level expression in developing B and T cells.

OBJECTIVE: We previously showed that Vav promoter-tetracycline transactivator (Vav-tTA)-driven tetracycline-regulated element (TRE)-NRAS(V12) expression resulted in mastocytosis development in mice. To investigate which hematopoietic cells express TRE-driven transgenes when combined with Vav-tTA, we assayed hematopoietic cells, including bone marrow-derived mast cells (BMMC) and CD34-positive hematopoietic progenitor cells (HPC) as well as myeloid and lymphoid lineages. To determine if suppression of NRAS(V12) expression early in life would delay mastocytosis we treated developing and juvenile mice with doxycycline (Dox). MATERIALS AND METHODS: Vav-tTA-driven luciferase expression was assayed by live mouse imaging and relative light unit measurement before or after treating Vav-tTA and TRE-luciferase (TRE-Luc) cotransgenic mice with Dox. Magnetic cell sorting and fluorescence-activating cell sorting methods were used to sort hematopoietic cells. To suppress TRE-mediated luciferase or NRAS(V12) expression in Vav-tTA cotransgenic mice, we added Dox to the drinking water. RESULTS: B cells in the bone marrow and T cells in the thymus expressed Vav-tTA-driven luciferase at much higher levels than in myeloid cells, BMMC, and CD34-positive HPC, which showed relatively low levels. Dox treatment completely eliminated the luciferase expression from all hematopoietic cells. Repression of TRE-NRAS(V12) expression early in life was sufficient to increase the latency of mastocytosis development. CONCLUSION: The Vav-tTA transgenic line will be very useful for conditional transgene expression in developing B and T cells. Vav-tTA-driven NRAS(V12) expression is sufficient for mastocytosis development, but not for myeloid leukemia. Lymphoid cells are resistant to NRAS(V12) transformation despite high level of expression.

The GAP-related domain of neurofibromin attenuates proliferation and downregulates N- and K-Ras activation in Nf1-negative AML cells.

Inactivation of the NF1 tumor suppressor causes myeloproliferative diseases. NF1 encodes a GTPase activating protein (GAP) for Ras. Myeloid cells with loss of NF1 have high levels of Ras-GTP, functionally equivalent to the effects of RAS oncogenes. We investigated the effects of the NF1 GAP-related domain (GRD) in proliferation, apoptosis and Ras-GTP levels in Nf1-negative acute myeloid leukemia (AML) cells. In AML cells, with cooperating mutations, the expression of the neurofibromin GRD causes significant reductions of N- and K-Ras-GTP levels, which is not incompatible with AML cell survival, but which is strongly selected against due to suppression of proliferation.

Improving Critical Thinking Using a Web-Based Tutorial Environment.

With a broad range of subject matter, students often struggle recognizing relationships between content in different subject areas. A scenario-based learning environment (SaBLE) has been developed to enhancing clinical reasoning and critical thinking among undergraduate students in a medical laboratory science program and help them integrate their new knowledge. SaBLE incorporates aspects of both cognitive theory and instructional design, including reduction of extraneous cognitive load, goal-based learning, feedback timing, and game theory. SaBLE is a website application that runs in most browsers and devices, and is used to develop randomly selected scenarios that challenge user thinking in almost any scenario-based instruction. User progress is recorded to allow comprehensive data analysis of changes in user performance. Participation is incentivized using a point system and digital badges or awards. SaBLE was deployed in one course with a total exposure for the treatment group of approximately 9 weeks. When assessing performance of SaBLE participants, and controlling for grade point average as a possible confounding variable, there was a statistically significant correlation between the number of SaBLE levels completed and performance on selected critical-thinking exam questions addressing unrelated content.

Emerging concepts in glioma biology: implications for clinical protocols and rational treatment strategies.

Glioblastoma multiforme (GBM), the most common primary central nervous system neoplasm, is a complex, heterogeneous disease. The recent identification of stem cells in murine tumor xenografts that were capable of recapitulating the tumor phenotype adds a new dimension of complexity to the already challenging treatment of patients with GBMs. Although specific cellular and genetic changes are commonly associated with GBM, the mechanism by which those changes occur may have a significant impact on treatment outcome. Of the many bioinformatics techniques developed in recent years, gene expression profiling has become a commonly used research tool for investigating tumor characteristics, and the development of rationally targeted molecular therapies has also accelerated following the initial success of specifically designed inhibitors in the treatment of malignancies. Despite these advances in research techniques and targeted molecular therapies, however, limited clinical impact has been achieved in the treatment of infiltrative malignancies such as GBMs. Thus, further extension in survival of patients with GBMs may require use of multiple analyses of tumors to develop tailored therapies that reflect the inter- and intratumoral heterogeneity of this disease. In this review, the authors briefly consider the potential use of expression profiling combined with mutation analysis in the development of treatment modalities to address the heterogeneity of this complex tumor phenotype.

De novo induction of genetically engineered brain tumors in mice using plasmid DNA.

Spontaneous mouse models of cancer show promise to more accurately recapitulate human disease and predict clinical efficacy. Transgenic mice or viral vectors have been required to generate spontaneous models of glioma, a lethal brain tumor, because nonviral gene transfer is typically transient. To overcome this constraint, we used the Sleeping Beauty transposable element to achieve chromosomal integration of human oncogenes into endogenous brain cells of immunocompetent mice. Genetically engineered, spontaneous brain tumors were induced with plasmid DNA in a matter of weeks in three separate mouse strains. The phenotype of tumors was influenced by the combination of oncogenes delivered, resembling human astrocytoma or glioblastoma in the majority of cases. At least five different genes can be cotransfected simultaneously including reporters, allowing measurement of tumor viability by in vivo imaging. This model can accelerate brain tumor research in a variety of ways such as generation of "humanized" models for high throughput drug screening and candidate gene validation with exceptional speed and flexibility.

Beta common receptor inactivation attenuates myeloproliferative disease in Nf1 mutant mice.

Neurofibromatosis type 1 (NF1) syndrome is caused by germline mutations in the NF1 tumor suppressor, which encodes neurofibromin, a GTPase activating protein for Ras. Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and lethally irradiated mice given transplants with homozygous Nf1 mutant (Nf1-/-) hematopoietic stem cells develop a fatal myeloproliferative disorder (MPD) that models JMML. We investigated the requirement for signaling through the GM-CSF receptor to initiate and sustain this MPD by generating Nf1 mutant hematopoietic cells lacking the common beta chain (Beta c) of the GM-CSF receptor. Mice reconstituted with Nf1-/-, beta c-/- stem cells did not develop evidence of MPD despite the presence of increased number of immature hematopoietic progenitors in the bone marrow. Interestingly, when the Mx1-Cre transgene was used to inactivate a conditional Nf1 mutant allele in hematopoietic cells, concomitant loss of beta c-/- reduced the severity of the MPD, but did not abrogate it. Whereas inhibiting GM-CSF signaling may be of therapeutic benefit in JMML, our data also demonstrate aberrant proliferation of Nf1-/-myeloid progenitors that is independent of signaling through the GM-CSF receptor.

Repressible transgenic model of NRAS oncogene-driven mast cell disease in the mouse.

To create a model in which to study the effects of RAS dysregulation in hematopoietic disease, we developed separate founder lines of transgenic mice, with the tetracycline transactivator (tTA) driven by the Vav hematopoietic promoter in one line and NRASV12 driven by the tetracycline responsive element (TRE2) in the other. When these lines are crossed, doubly transgenic animals uniformly develop a disease similar to human aggressive systemic mastocytosis (ASM) or mast cell leukemia (MCL) when they are between 2 and 4 months of age. Disease is characterized by tissue infiltrates of large, well-differentiated mast cells in the spleen, liver, skin, lung, and thymus. Analysis of bone sections shows small to large foci of similarly well-differentiated mast cells. Results also show that transgene expression and diseases are repressible through the administration of doxycycline in the drinking water of affected animals, indicating that NRASV12 expression is required to initiate and maintain disease in doubly transgenic mice. Our inducible system of transgenes, developed as a model of mutant NRASV12 oncogene-driven myeloid disease, will be useful for studying the role of RAS dysregulation in hematopoietic disease in general and in discrete human diseases, specifically ASM and MCL.

Cecal colonization and systemic spread of Candida albicans in mice treated with antibiotics and dexamethasone.

Infections with Candida albicans have become a significant problem among very low birth weight infants in the neonatal intensive care unit. Risk factors are multiple and include administration of antibiotics and glucocorticoids, such as dexamethasone. Experiments were designed to study the combined effect of oral broad-spectrum antibiotics and parenteral dexamethasone on cecal colonization and extraintestinal dissemination of C. albicans in separate groups of mice that were orally inoculated with one of four C. albicans strains that were either wild-type INT1/INT1 or had one or more disruptions of the INT1 gene. Intestinal colonization was monitored by quantitative culture of the mouse cecum, and extraintestinal invasion was monitored by quantitative culture of the draining mesenteric lymph nodes and kidneys. At sacrifice, the average numbers of cecal C. albicans differed from 7.7 log(10)/g to 6.7 log(10)/g (p < 0.01) in mice orally inoculated with C. albicans containing two functional copies of INT1 and no functional copies of INT1, respectively. The incidence of extraintestinal dissemination to mesenteric lymph nodes and kidneys correspondingly varied from 57 to 13% (p < 0.01) and 83 to 4% (p < 0.01) in mice inoculated with these two C. albicans strains. Mice orally inoculated with C. albicans containing one functional copy of INT1 had intermediate levels of cecal colonization and extraintestinal dissemination. Thus, cecal colonization and extraintestinal dissemination of C. albicans was facilitated in antibiotic-treated mice given dexamethasone. In addition, the presence of two functional copies of the INT1 gene was associated with the greatest levels of cecal colonization and extraintestinal dissemination of C. albicans.

Adherence of yeast and filamentous forms of Candida albicans to cultured enterocytes.

OBJECTIVE: Systemic candidiasis is a major cause of complicating infections in intensive care units. Morbidity and mortality are high, even in those who receive appropriate antifungal therapy. Because the intestinal tract is considered a major portal of entry for systemic candidiasis, experiments were designed to clarify the ability of yeast and filamentous forms, as well as the INT1 gene product, to influence adherence of Candida albicans to the intestinal epithelium. DESIGN: Controlled. SETTING: University teaching hospital research laboratory. SUBJECTS: Mature Caco-2 and HT-29 cultured enterocytes. INTERVENTIONS: C. albicans INT1 mutant strains, defective in filament production, were used to observe the ultrastructural surface interactions of C. albicans with cultured intestinal epithelial cells, namely Caco-2 and HT-29 cells. These mutant strains also were used to quantify the effect of the INT1 gene product on C. albicans adherence (yeast and filamentous forms) to cultured enterocytes. Ultrastructural surface interactions of C. albicans with cultured enterocytes were observed with high resolution scanning electron microscopy. C. albicans adherence to cultured enterocytes was quantified by using a colorimetric enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Both yeast and filamentous forms of C. albicans appeared tightly adherent to the apical surface of cultured enterocytes, and INT1 appeared to have little, if any, effect on these ultrastructural surface interactions. The distal ends of C. albicans filaments appeared to mediate adherence to enterocyte apical microvilli, and thigmotropism (contact guidance) appeared to play a role in C. albicans adherence. The absence of functional INT1 was associated with decreased adherence of C. albicans yeast forms to cultured enterocytes. CONCLUSIONS: Although functional INT1 appeared to facilitate adherence of C. albicans yeast forms to cultured enterocytes, the role of INT1 in adherence of filamentous forms was unclear, and both yeast and filamentous forms could adhere to, and perhaps invade, the apical surface of cultured enterocytes.