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Background Older adults discharged from the Emergency Department (ED) are at high risk for medication interactions and side effects; examples of practice models addressing this transition of care are lacking. Methods This was a prospective cohort study for adults in one of two urban community EDs. Patients ≥50 years of age discharged with at least one new, non-schedule II prescription medication were included. Patients had the option of three transitions of care services: 1) pharmacist-only with home delivery of discharge medications and full medication reconciliation, 2) pharmacist and home health care, including home delivery, medication reconciliation, and a visit from a home health nurse, or 3) either of the above without home delivery. Results Over seven months, 440 ED patients were screened. Of those, 43 patients were eligible, and three patients elected to join the study. All three patients selected pharmacy-only. Identified barriers to enrollment include the rate of schedule II prescriptions from the ED (53% of potential patients) and high patient loyalty to their community pharmacist. Conclusions A pharmacy and home health care transitions of care program was not feasible at an urban community ED. While the pharmacist team identified and managed multiple medication issues, most patients did not qualify due to prescriptions ineligible for delivery. Patients did not want pharmacist or home health nurse involvement in their post ED visit care, many due to loyalty to their community pharmacy. Multiple barriers must be addressed to create a successful inter-professional transition of care model.

Web-based training is a field that advances rapidly. Rapid advancement leads to the development of industry standards and best practices. This case study will review some of those practices related to assessment design and evaluation and how each was applied to a U.S. Army Web-based training product. These best practices include adaptive learning, immediate and meaningful feedback, and assessment security. In addition, the problems that the army faced and the solutions that the development company designed will also be discussed. Towards the end of the case study, the results of the validation will be examined. The case study concludes with a look at future trends that may become best practices.

Philadelphia chromosome (Ph)-like B cell acute lymphoblastic leukemia (B-ALL) is a high-risk leukemia with a gene expression profile similar to BCR-ABL1+ B-ALL. Approximately 50% of all Ph-like B-ALL is characterized by genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL). CRLF2 B-ALL occurs 5 times more often in Hispanic and Native American children than others and is prevalent in adolescents and young adults. The poor outcomes associated with CRLF2 B-ALL represent a major clinical challenge and an important component of pediatric cancer health disparities. Biologically, CRLF2 acts as a receptor component for the cytokine, TSLP, which induces JAK2-STAT5 and PI3/AKT/mTOR pathway activation downstream of binding to CRLF2. Activating JAK mutations are associated with CRLF2 B-ALL, but overall data indicate that JAK mutations are present in 50% or less of CRLF2 B-ALL. Our data show that normal primary human bone marrow (BM) stromal cells express TSLP, suggesting that TSLP-induced CRLF2 signals could play a role in the initiation, maintenance and progression of CRLF2 B-ALL, particularly in cases without JAK mutations. Consistent with this, TSLP has been reported to increase in vitro production of human fetal B cell precursors. However studies of TSLP in B lymphopoiesis have been conducted almost exclusively in mice which show low homology (~40%) to human TSLP and CRLF2. Further, using phospho flow cytometry we show that mouse TSLP is unable to induce increases in pSTAT5, pAKT and pS6 observed in CRLF2 B-ALL cells stimulated with human TSLP, confirming the species specificity of mouse TSLP. These findings underscore the importance and challenge of developing in vivo systems that can model human TSLP-CRLF2 interactions for evaluating therapies and studying leukemogenesis of CRRLF2 B-ALL. To address this challenge we engineered patient-derived xenograft (PDX) mice to produce human TSLP (hTSLP) by transplanting them with stromal cells transduced to express hTSLP (+T mice). Control (-T) mice were produced by transplanting with stroma transduced with a control vector. Supernatant from engineered +T stroma, but not -T stroma, induced JAK/STAT5 and PI3K/AKT/mTOR pathway activation in CRLF2 B-ALL cells. ELISA assays showed normal serum levels of hTSLP (12-32 pg/ml) in +T mice, while hTSLP was undetectable in -T mice. Since TSLP has been shown to increase in vitro production of human B cell precursors, we evaluated the in vivo functionality of our model by comparing the production of normal B cell precursors in the BM of +T and -T PDX mice generated with human umbilical cord blood CD34+ cells. Data from 3 different cord blood donors showed that production of B cell precursors is 3-5 fold increased in +T as compared to -T mice. TSLP-induced increases were specific to B lineage cells, initiated in the earliest CD19+ B cell precursors, and maintained through later stages of B cell development. Next we evaluate the in vivo functionality of our model using primary leukemia cells. +T and -T PDX mice were produced using primary CRLF2 B-ALL cells. BM was harvested and whole genome microarray was performed on isolated CRLF2 B-ALL cells. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in +T as compared to -T PDX mice, confirming hTSLP activity in the +T PDX mice. Next we tested whether +T PDX mice provide an in vivo model of B-ALL that more closely mirrors patients than -T PDX mice. +T and -T PDX mice were generated from primary high risk B-ALL. RNAseq gene expression profiles from primary patient B-ALL cells were compared to those of the same patient sample expanded in +T and -T PDX mice. The gene expression pattern in +T mice was significantly closer to the primary patient sample than those from -T mice. The +T and -T PDX mice described here provide a novel preclinical model for studying the role of TSLP in the initiation, progression and maintenance of CRLF2 B-ALL and for evaluating drug efficacy in an in vivo model that more closely mirrors the in vivo environment present in patients. Disclosures No relevant conflicts of interest to declare.

The classical model for blood cell replenishment is changing with the unexpected realization that hematopoietic stem and progenitor cells (HSPCs) directly sense and respond to pathogens via toll-like receptors (TLRs). For the first time, we show that human HSPCs are severely compromised by repeated exposure to low dose TLR4 ligand as assessed in vivo in humanized mice. CD34+ HSPCs are reduced 50% with concomitant hyperproliferation. Downstream bone marrow lymphoid precursors and peripheral B cells are virtually ablated to the benefit of the myeloid cells. In the mouse model, reciprocal chimeras reveal that numerical and functional hematopoietic stem cell (HSC) defects following repeated exposure to TLR4 ligand require hematopoietic-derived TLR4. Selective deficiency of TLR4 on hematopoietic cells alleviates HSC hyperproliferation and myeloid skewing while cell non-autonomous TLR4 is largely dispensable. Finally, within uncommitted progenitor compartments, we show that HSC and downstream non-renewing multipotent progenitors (MPP) respond directly to TLR4 ligand. Thus, while transient TLR stimulation is thought to enable replenishment of the innate immune compartments that are rapidly consumed through acute infection, chronic TLR stimulation is detrimental to both murine and human HSPC integrity in vivo. These findings have broad implications as persistent exposure to TLR4 ligand occurs in individuals suffering from each chronic infection and obesity.

The role of TSLP in human B cell development is largely unknown and its function in context of IL7 is unstudied. We found that TSLP can replace IL7 in providing a signal essential for in vitro production and proliferation of human CD19+ Pax-5+ B cell progenitors. To study the role of TSLP in human B cell development, in the context of IL7 stimulation, we developed a novel xenograft model. Mouse IL7 is active on human cells, however, this is not the case for TSLP. We took advantage of this to develop a xenograft model system comprised of mice that provide human TSLP (+T mice) and control mice that do not (-T mice). Using this model we found that TSLP increases proliferation and upregulates Mcl-1 in CD34+CD19-IL7Ra progenitors resulting in a 3-4 fold expansion of the CD34+ pro-B cell compartment in +T as compared to -T mice. This expansion is maintained during subsequent stages of B cell development, partially due to increased protection from apoptosis that occurs independently of Bcl-2 family pro-survival proteins. While TSLP stimulates in vitro proliferation of pro-B cells, in vivo proliferation of B lineage precursors was similar in +T and -T mice suggesting redundancy with IL7. B cell subsets in normal pediatric bone marrow samples show progenitor ratios and Mcl-1 expression that mirrors that in +T xenograft mice. These data provide evidence that TSLP function in human B cell development includes both unique activities as well as activities redundant to those of IL7.

Long-term hematopoietic stem cells (LT-HSCs) must replenish all immune cells lost due to natural turnover, inflammation or injury throughout lifetime. Our recently published data demonstrate that following chronic, low-dose TLR4 agonist stimulation, murine HSCs hyperproliferate, preferentially undergo myeloid-specific differentiation and exhibit compromised self-renewal. However, the impact to human LT-HSC function in vivo has not been established. Here we reconstituted NSG mice with human CD34+ HSCs derived from 3 different donors, and tracked hematopoiesis. Following chronic, low-dose exposure to the TLR4 agonist lipopolysaccharide (LPS), human bone marrow CD34+ HSCs were decreased ~2-fold while downstream CLPs and pro-B cells were dramatically reduced about 90%. In spleen and peripheral blood the percentage of CD19+ B cells was decreased 2-4-fold while CD56+ NK and CD3+ T were proportionately increased. Myeloid cells were largely unaffected. These findings are potentially important because TLR4 on HSCs is activated by two ligands, LPS and dietary fatty acids that are commonly elevated in diseases of chronic inflammation as well as in diet-induced obesity. Our studies may provide a previously unrecognized mechanism of human HSC exhaustion and/or compromised hematopoiesis in patients suffering from these chronic conditions.

Chronic exposure to toll-like receptor 4 (TLR4) ligand drives murine hematopoietic stem cell (HSC) exhaustion and loss of lymphoid potential. This finding is significant because TLR4 is activated by plasma lipopolysaccharide (LPS) that is elevated in patients with chronic infection as well in obese individuals. For the first time, we show that human HSCs are severely compromised by TLR4 stimulation. Mice engrafted with human cord blood cells and then exposed to low-dose LPS have variably reduced human CD34+ HSCs with near complete ablation of downstream common lymphoid progenitor (CLP) and B cell precursor subsets in bone marrow. By contrast, myeloid precursors are grossly intact. Peripheral tissue subsets similarly exhibit myeloid>lymphoid skewing. We have shown that LPS-exhausted murine HSCs have reduced levels of the major transcription factor E47. Our newly published findings demonstrate a mechanistic link between E47 and the p21 cell cycle regulator. CD150+ HSCs from mice doubly haploinsufficient for E47 and p21 exhibit hyperproliferation, poor self-renewal, and selective myeloid bias. Finally, we show that murine CD150+ HSC and multipotent progenitors (MPP) express TLR4, and directly respond to LPS in a TLR4-dependent manner. Together, our findings suggest that chronic activation of TLR4 damages the functional integrity of not only murine HSC but also human HSC in vivo, via a mechanism linked to disruption of the E47-p21 pathway.

The human T cell leukaemia virus type I (HTLV-1) Tax protein is an activator of viral and cellular gene expression. Tax does not bind DNA directly, but does interact with cellular DNA binding proteins. These interactions bring Tax to a specific group of promoters and may help to determine the specificity of Tax transactivation. Previous studies have demonstrated that the activity of Tax, when tethered to a given promoter, is enhanced by the presence of adjacent transcription factor binding sites. To examine the specificity of this augmentation, a series of transcription factor binding sites was tested for the ability to enhance the activity of a Gal-Tax fusion protein. The greatest increase in Gal-Tax activity was observed when an octamer binding site was placed adjacent to the Gal4 binding sites. However, the octamer binding site failed to independently function as a Tax responsive element in the absence of an adjacent Tax-tethering element. Oct-2 was not required for augmentation of Gal-Tax activity as this enhancement was observed in BHK-21 cells, which lack Oct-2. The ability of octamer binding sites to augment transcription was specific for Gal-Tax, as compared to other transactivators. Taken together, these results demonstrate that the degree of Tax transactivation can be influenced by the elemental composition of the target promoter.

IL-7 is critical for mouse B cell development and available data suggest that TSLP has overlapping functions. Our previous in vitro studies show that IL-7 expands human B cell progenitors by ~60 fold (JI 2009 82:4255). Our recent experiments provide evidence that TSLP similarly expands human B cell progenitors in vitro and that primary human bone marrow stroma provides an in vivo source of both cytokines. To study interplay between IL-7 and TSLP in early stages of in vivo human B cell lymphopoiesis, we developed a novel xenograft model system that selectively provides IL-7 and/or TSLP stimulation. While mouse IL-7 can act on human cells, TSLP is species-specific. To overcome this obstacle we coupled a xenograft model system engineered to provide human TSLP (hTSLP) with IL-7 neutralizing antibodies. Preliminary data obtained using this model system transplanted with cord blood CD34+ cells shows that the production of B cell precursors is reduce by ~90% in mice that lack both IL-7 and hTSLP stimulation as compared to mice where both are present. The absence of hTSLP stimulation, alone, reduced B cell production by about 50%, while the effects due IL-7 neutralization, alone, were about half this. These data suggest that IL-7 and TSLP play an essential and at least partially overlapping role in human B cell development. Current studies are aimed at defining the differential stage-specific effects of TSLP and IL-7 on survival and proliferation of human B cell progenitors in vivo.

Ikaros encodes a DNA-binding protein that functions as a master regulator of hematopoiesis and a tumor suppressor in pre-B acute lymphoblastic leukemia (B-ALL). Loss of Ikaros function is associated with impaired hematopoiesis and high-risk B-ALL in humans. The mechanism of Ikaros tumor suppressor activity is unknown. Using quantitative chromatin immunoprecipitation (qChIP) we demonstrate that Ikaros binds to the promoters of genes required for B cell differentiation in primary leukemia cells. Luciferase reporter assays demonstrated that Ikaros activates transcription of these B lineage-specific genes. Increased expression of Ikaros via retroviral transduction in B-ALL cells resulted in increased transcription of these critical B cell differentiation genes, as well as increased binding of Ikaros to their promoters. The inhibition of Casein Kinase II (CK2) resulted in dephosphorylation of Ikaros and enhanced Ikaros-mediated transcriptional activation of the B lineage-specific genes in B-ALL. Treatment of the pre-B ALL cell line, Nalm6, with a CK2 inhibitor, resulted in increased Ikaros binding to the promoters of B lineage genes, increased transcription of B cell differentiation genes, and cell cycle arrest. Results suggest that Ikaros functions as a positive regulator of B cell differentiation by directly upregulating the transcription of B lineage-specific genes in B-ALL. The presented data suggest that CK2 has a critical role in B cell differentiation and in leukemogenesis.

IL7 and TSLP are important in murine B cell development, however their role in human B lymphopoiesis is unclear. In contrast to other cytokines that act on B cell progenitors, TSLP lacks species cross reactivity and activates different intracellular signaling pathways in mice and humans. Thus, human models are essential for understanding the role of TSLP in human B lymphopoiesis. We developed a novel human-only model of in vitro B cell development that allows selective cytokine stimulation and used this model to show that IL-7 is essential for human B cell production where it increases B cell progenitors by over 60 fold (JI 2009 82:4255). Here we use this model and a novel xenograft model system to study human B lymphopoiesis from CD34+ HSCs in umbilical cord blood (CB). BrdU incorporation shows that TSLP induces proliferation of human B lineage cells to produce in vitro expansion of B cell progenitors similar to that observed for IL7. TSLP acts directly on human B cell precursors to induce STAT5 phosphorylation and upregulate PAX5. To evaluate the role of TSLP in vivo we developed a novel xenograft model system consisting of human TSLP+ (hTSLP+) and hTSLP- immune deficient NSG mice. Using the hTSLP+/- model system we show that hTSLP augments existing IL7 effects to expand human B lineage cells (from 37% to 68% total B lineage chimerism) in NSG mice, a strain that facilitates human B cell production. These data suggest that TSLP is important in human B lymphopoiesis.

Expression of the IL-7 receptor alpha (IL-7R) is a distinguishing feature of common lymphoid progenitors (CLP) in mice. Human B cell development has been thought to differ from that in mouse with respect to the requirement for IL-7. Our previous studies show that IL-7 plays a critical role in human B cell production (J Immunol. 2009, 182:4255). Here we use IL-7R expression to identify a human CLP population that can be generated from hematopoietic stem cells (HSCs) in umbilical cord blood (CB). Following two weeks of co-culture with primary human bone marrow (BM) stroma, CB CD34+ cells give rise to a CD34+CD19-IL-7R+ progenitor population that is absent from fresh, uncultured CB. IL-7R+ progenitors make up ~17% of the CD19-CD34+ cells present in culture at 2 weeks. A similar IL-7R+ human progenitor population is generated in the BM of immunodeficient mice following transplant with human CD34+ CB cells. FACS-sorted CD34+CD19-IL-7R+ progenitors produced in co-culture or in the human-mouse xenograft model give rise to CD19+ B cells and CD56+ NK cells in bulk and single cell culture, but lack myeloid potential in CFU assays and when transplanted into immunodeficient mice. Ongoing experiments will evaluate the T lineage potential of IL-7R+ progenitors in the human-mouse xenograft model. These data will be important for developing therapies to accelerate and enhance lymphoid reconstitution from HSCs in CB, a hematopoietic source used increasingly for stem cell transplant.

The goal of our research is to define signal transduction pathways that regulate thymocyte differentiation. We have previously reported that Protein Phosphatase 1 (PP1) directly dephosphorylates and regulates the activity of the Ikaros protein - a master regulator of lymphocyte differentiation (Popescu et al. J Biol Chem 2009 284:13869). We extended our studies to determine whether PP1-mediated dephoshorylation of Ikaros controls Ikaros’ function in regulating the expression of the terminal deoxynucleotidyl transferase (TdT) gene during thymocyte differentiation. An Ikaros mutant that is unable to interact with PP1 (IK 465/7A), and that is subsequently hyperphosphorylated, was compared to the wild type Ikaros for DNA-binding affinity toward the TdT promoter and for ability to repress transcription of the TdT gene using a luciferase reporter assay. Chromatin immunoprecipitation assays showed that the loss of PP1-mediated dephosphorylation of Ikaros leads to decreased Ikaros DNA-binding affinity for the D’ regulatory element of the TdT promoter in vivo. The IK 465/7A mutant also failed to repress transcription of TdT in the luciferase reporter assay, suggesting that Ikaros’ interaction with PP1 and subsequent dephosphorylation is essential for its function as repressor of TdT transcription. Results suggest that the PP1 signal transduction pathway regulates thymocyte differentiation by controlling the function of Ikaros and transcriptional repression of the TdT gene.

Signals induced by IL-7 and Flt ligand (FL) are critical components of the regulatory network that governs early B cell development in mice. The role of these cytokines in human B lymphopoiesis is less clear. We previously reported that IL-7 is required for in vitro human B cell production from adult bone marrow (Parrish et al. JI 2009 182:4255). Here, we use a human-only culture model to examine the effects of IL-7 and FL, separately and together, on the production of developmental subsets of human B lineage cells from hematopoietic stem cells in cord blood. While B cell production was essentially absent in cultures without IL-7 or FL, small populations of CD19+ B lineage cells were produced with either IL-7 or FL alone. The combination of IL-7 and FL increased B cell production up to 75 fold. To identify the developmental targets of IL-7 and FL we assessed 3 subsets that include developmentally sequential populations of human B cell precursors. The production of CD19-IL-7Rα+CD34+ progenitors was dependent on FL, but not IL7, and the addition of IL-7 did not expand this population. CD34+ pro-B cells showed variable responses to both IL-7 and FL while IL-7 primarily targeted CD19+CD34- pro-B cells for expansion. The intensity of CD19 staining was approximately a log higher in all cultures with IL-7. These data suggest that FL targets CD19-CD34+IL-7Rα+ progenitors that respond to IL-7 to upregulate CD19 and then expand as CD34 is lost during human B cell differentiation.