ALBERT

Description: In this paper, a fixed-point iterative filter developed from the classical extended Kalman filter (EKF) was proposed for general nonlinear systems. As a nonlinear filter developed from EKF, the state estimate was obtained by applying the Kalman filter to the linearized system by discarding the higher-order Taylor series items of the original nonlinear system. In order to reduce the influence of the discarded higher-order Taylor series items and improve the filtering accuracy of the obtained state estimate of the steady-state EKF, a fixed-point function was solved though a nested iterative method, which resulted in a fixed-point iterative filter. The convergence of the fixed-point function is also discussed, which provided the existing conditions of the fixed-point iterative filter. Then, Steffensen’s iterative method is presented to accelerate the solution of the fixed-point function. The final simulation is provided to illustrate the feasibility and the effectiveness of the proposed nonlinear filtering method.

Description: Background: In plants, the growth of an aerial organ to its characteristic size relies on the coordination of cell proliferation and expansion. These two different processes occur successively during organ development, with a period of overlap. However, the mechanism underlying the cooperative and coordinative regulation of cell proliferation and expansion during organ growth remains poorly understood. Results: This study characterized a new Arabidopsis ORGAN SIZE RELATED (OSR) gene, OSR2, which participates in the regulation of cell expansion process during organ growth. OSR2 was expressed primarily in tissues or organs undergoing growth by cell expansion, and the ectopic expression of OSR2 resulted in enlarged organs, primarily through enhancement of cell expansion. We further show that OSR2 functions redundantly with ARGOS-LIKE (ARL), another OSR gene that regulates cell expansion in organ growth. Moreover, morphological and cytological analysis of triple and quadruple osr mutants verified that the four OSR members differentially but cooperatively participate in the regulation of cell proliferation and cell expansion and thus the final organ size. Conclusions: Our results reveal that OSR2 is functional in the regulation of cell expansion during organ growth, which further implicates the involvement of OSR members in the regulation of both cell proliferation and expansion and thus the final organ size. These findings, together with our previous studies, strongly suggest that OSR-mediated organ growth may represent an evolutionary mechanism for the cooperative regulation of cell proliferation and expansion during plant organogenesis.

Description: Abstract 2109 In addition to maintaining steady state hematopoiesis in the adult life, bone marrow (BM) derived cells contain endothelial progenitor cell (EPC) activity and may play a role in promoting vascular regeneration upon injury or angiogenesis during tumor progression. Whether hematopoietic stem/progenitor cells (HSPCs) in the BM can give rise to functional EPCs and if they contribute significantly to endothelial cell regeneration remain controversial questions. Cdc42 is a member of the Rho GTPase family that has been shown to play essential and unique roles in multiple lineages of blood cell regulation including HSPC proliferation and cytoskeleton organization. In addition, Cdc42 is found to regulate blood vessel permeability and endothelial lumen formation [1, 7]. By using a conditional knockout (KO) and BM transplant mouse model (i.e. Mx1-cre; Cdc42loxP/loxP mice and transplant recipients) that allows specific, inducible deletion of cdc42 gene in the bone marrow compartment and a small molecule Cdc42 activity-specific inhibitor (CASIN), we have examined the hypothesis that Cdc42 critically regulates the BM-derived EPC and/or angiogenic supporting cell production and function. First, we found that inducible Cdc42 KO in BM cells inhibited colony-forming activity of total BM cells by ~10-fold and ~40-fold in two CFU-EPC assays that have been used in the published literature - in EGM2 medium containing VEGF, FGF2, IGF1 and EGF [6] and a modified EGM2 medium containing VEGF, FGF2 and IGF1 [8], respectively. The colonies formed from Cdc42 KO BM gave a distinct small round morphology. Concomitantly, Cdc42 deletion resulted in a ~3-fold enhancement of hematopoietic colony-forming CFU-C activity of the BM cells, consistent with our previous report on hematopoietic regulation by Cdc42. Both effects on EPC and HSPC activities can be attributed to hematopoietic cell regulation by Cdc42, since similar reduction of CFU-EPC activity and increase in CFU-C activity were observed in Mx-cre; Cdc42lox/lox transplant recipient mice after polyIC induction. Second, given the controversies in the literature about cell markers of BM EPCs, FACS analysis using 5 different sets of putative EPC markers was carried out to examine the effect of Cdc42 knockout on BM EPC compartment. PolyIC induced Cdc42 deletion led to ~4-fold reduction of CD45lo/CD11b-/VE-Cad+ cells [2], ~3-fold reduction of CD45-/PDGFRa+ [5], ~3 fold reduction of CD45-/Lin-/Flk1+ [3, 4], 2-fold reduction of CD45+/Lin-/Flk1+ and 3-fold decrease of CD45-/CD31+ EC population [9]. Similar results were also obtained from the BM Cdc42 deleted transplant recipients. Third, to further distinguish EPCs of blood lineage from those of non-blood lineage in the BM cells, purified CD45+ or CD45- cells were isolated from BM cells by double FACS sorting, and their abilities to produce EC-like cells and to express endothelial markers CD31, PDGFRa and vWF in a matrigel culture, were analyzed. Cdc42 deletion led to a drastic reduction of the tube-forming and CD31+/PDGFRa+/vWF+ cells by over 100-fold, in both the CD45+ and CD45- BM cell culture. Fourth, treatment of WT BM cells with CASIN abolished colony-forming activity of BM cells in the CFU-EPC assays, mimicking that of Cdc42 knockout. Upon CASIN withdrawn, the cells could continue differentiation into adherent EC-like colonies. Finally, Cdc42 knockout resulted in a 3-fold reduction of VEGFR2 cell surface presentation, but not expression, in BM cells, suggesting Cdc42 may regulate VEGFR2 localization to impact endothelial lineage commitment. While experiments to dissect the signaling mechanisms and the requirement of Cdc42 for angiogenesis and endothelial or endothelial cell-supporting function are ongoing, these results indicate that Cdc42 is essential for BM derived EPC or EPC-like activities. Targeting Cdc42 may provide a novel strategy in modulating BM-EPC and EC regeneration. References: 1. Broman et al. Circ Res 98, 73–80 (2006) 2. Gao et al. Science 319, 195–8 (2008) 3. Chakroborty et al. JCI 118, 1380–9 (2008) 4. Ciarrocchi et al. PloS One. 2, e1338 (2007) 5. Morikawa et al. JEM 206, 2483–96 (2009) 6. Pitchford et al. Cell Stem Cell 4, 62–72 (2009) 7. Sacharidou et al. Blood 115, 5259–69 (2010) 8. Wary et al. Stem Cells 27, 3112–20 (2009) 9. Yoder et al. Blood 109, 1801–9(2007) Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 508 Adult hematopoietic stem/progenitor cells (HSPCs) are maintained in the unique microenvironment of bone marrow (BM) niche to sustain life-long blood production of mammals. Several types of cells in the BM, including endothelial cells (ECs), have been shown to contribute to the niche components in regulating the homeostasis of HSPCs as well as leukemia stem cells in the BM. However, because of the current technical limitations in studying BM ECs in mice, particularly the lack of an EC-specific inducible gene targeting method in adult mice and controversies surrounding the suitable murine EC culture conditions in vitro, the signaling pathways that regulate the putative BMEC niche remain poorly understood. Here we have utilized several complementary mouse models to achieve an inducible, BMEC-specific deletion of Cdc42 to unveil an essential role of this Rho family GTPase in regulating BMEC morphogenesis and proliferation, and in maintaining hematopoietic homeostasis of adult mice. First, by using the interferon-inducible Mx1-Cre;Cdc42lox/lox and the tamoxifen inducible Rosa26-CreER;Cdc42lox/lox mice engrafted with syngeneic WT HSPCs, we found that both conditions of inducible deletion of Cdc42 in the BM microenvironment, including BMECs, caused sinusoidal dilation, a drastic decline of endothelial capillary density, and a loss of endothelial progenitor activity and mature endothelial cells, in the bone marrow. Significantly, the BMEC phenotypes of these non-specific Cre-driver genetic models could be recapitulated by using a mouse model of Tie2-CreER;Cdc42lox/lox that allows inducible endothelial lineage-specific deletion of Cdc42 in the BM in response to tamoxifen, which we have shown rigorously by lineage tracing to be a suitable model for BMEC characterizations (Feng et al., ASH abstract, 2012). Second, we have established an in vitro culture system that expands and maintains primary BMECs in culture, allowing a direct assessment of the proliferation, survival, adhesion, migration, tube formation, as well as capillary assembly in 3D, activities of the BMECs. Deletion of Cdc42 by in vivo tamoxifen induction or in vitro Cre transduction of the BM cells of Tie2-CreER; Cdc42lox/lox genotype resulted in a complete loss of angiogenesis activity of BMECs that was associated with a drastic reduction of adhesion, tube-formation, and 3D capillary assembly capabilities of BMECs. In addition, endothelial cell proliferation was also compromised, as seen by a decrease of CyclinD1 expression and a diminished BrdU incorporation. These genetic results were phenocopied by findings using a Cdc42 specific inhibitor, CASIN, as CASIN treatment inhibited BMEC adhesion, diminished BM endothelial colony-forming activity, and suppressed BMEC tube-formation. A rescue experiment using retroviral expression of a panel of effector-coupling defective Cdc42 mutants and/or constitutively active PAK or AKT mutant in the Cdc42 knockout BMECs identified AKT and p21 PAK as the major downstream effectors of Cdc42 in BMEC maintenance. Third, using the three inducible mouse models, we found that Cdc42 in BMECs and microenvironment is required for maintaining hematopoietic homeostasis. Deletion of Cdc42 in BMECs caused a mobilization of HSPCs to the peripheral organs and a subsequent reduction of wild-type HSPCs in the BM, as assayed by genotypic FACS analysis, colony-forming activity and competitive transplantation of BM cells from endothelial Cdc42-null or control mice. Taken together, our results demonstrate that the Rho GTPase Cdc42 in BMECs mediates critical signaling cascade through PAK and Akt in maintaining the BMEC angiogenesis and morphogenesis. Cdc42 in BMECs could serve as a potential therapeutic target for bone marrow diseases including leukemia and lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2618 Rho family small GTPases are critical regulators of multiple functions of blood cells, including adhesion, migration, proliferation, survival and gene expression. Elucidation of their roles in hematopoiesis could be fundamental for understanding the mechanisms of various blood diseases and improving therapeutic outcomes for hematopoietic abnormalities. The Rho GTPases Rac1, Rac2 and Cdc42 have been found essential in maintaining hematopoietic stem cell (HSC) niche residency and regulating myelopoiesis and lymphopoiesis in previous mouse gene targeting studies. Using a dominant-negative mutant overexpression approach, an earlier study showed that suppression of RhoA activity enhanced HSC proliferation and engraftment potential; however, the bone fide role of RhoA in blood development remains unknown given limitations of such a mutant overexpression approach in specificity, dosage effect, and physiological relevance. Here, we stringently define the function of RhoA in HSC maintenance and hematopoiesis using an interferon inducible RhoA conditional knockout mouse model (Mx-cre+; RhoAloxp/loxp). Systematic deletion of RhoA caused lethality of the mice 7 days post polyIC induction due to hematopoietic failure that was accompanied by drastically decreased bone marrow (BM) cellularity (to ∼1/3 of wild type controls), a loss of splenocytes, and a significant reduction of cell counts of most cell lineages in peripheral blood, suggesting RhoA is required for multiple blood cell lineage differentiation and production. Syngenic transplant experiments yielded similar results, demonstrating that these effects are intrinsic to the hematopoietic compartment. The observed cytopenia resulting from RhoA loss was associated with the exhaustion of BM phenotypic HSPC (Lin−Sca1+c-kit+, LSK) and the hierarchical progenitor cells (Lin−c-kit+, LK) in number and frequency, a complete loss of colony forming activities, and a total engraftment failure. In addition, BrdU chase labeling and Annexin V/7-AAD staining revealed that RhoA deletion caused a transient increase of proliferation (1.7 fold increase in S phase) and reduction of survival (16.8 fold reduction in Annexin V− 7-AAD−) of remaining LSK in the BM. These results indicate that RhoA plays an indispensible, cell autonomous role in HSPC maintenance and hematopoiesis. In a competitive transplantation model where Mx-cre+;RhoAloxp/loxp or Mx-cre−;RhoAloxp/loxp CD45.2+ BM cells and WT CD45.1+ competitor cells were transplanted at 1:1 ratio into syngenic CD45.1+ recipients prior to polyIC induction, RhoA deletion also caused a complete extinction of donor derived (CD45.2+) Mac1+Gr1+ myeloid cells, B220+ B cells, as well as CD3+ T cells, in the peripheral. Interestingly, distinct from these more differentiated lineages, BM CD45.2+ LSK population was only marginally affected (88.9 % of pre-polyIC induction level) while the CD45.2+ LK cells and later hierarchical lineages were rapidly eliminated after RhoA deletion in this competitive transplant model. This was associated with increased apoptosis in CD45.2+ RhoA−/− LK, but not LSK, cells, suggesting a specific requirement of RhoA in the myeloid progenitor cell survival. Further, the CD45.2+ RhoA−/− LSK and LSKCD150+ cell populations, not differentiated donor-derived progenitors, from the primary competitive transplant BM, were able to be maintained in secondary transplant recipients 5 months post-transplantation, indicating that RhoA serves as a key regulator at an early progenitor differentiation step. Interestingly, RhoA deletion did not affect lin− cell p-MLC and p-cofilin contents and in vitro expansion in response to SCF stimulation, suggesting that RhoA is not required for actomyosin signaling nor SCF induced proliferation. Taken together, our results implicate RhoA as a unique and essential regulator of multipotent progenitor differentiation and survival that controls multi-lineage hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model. Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value 〈 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4. Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice. Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. Disclosures Al-Homsi: Millennium Pharmaceuticals: Research Funding.