Individuals with neurofibromatosis type 1 (NF1) are predisposed to certain cancers including juvenile chronic myelogenous leukaemia (JCML). The NF1 tumour-suppressor gene encodes a protein (neurofibromin) that accelerates GTP hydrolysis on Ras proteins. Here we show that primary leukaemic cells from children with NF1 show a selective decrease in NF1-like GTPase activating protein (GAP) activity for Ras but retain normal cellular GAP activity. Leukaemic cells also show an elevated percentage of Ras in the GTP-bound conformation. JCML cells are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF), and we observed a similar pattern of aberrant growth in haematopoietic cells from Nf1-/- mouse embryos. These data define a specific role for neurofibromin in negatively regulating GM-CSF signaling through Ras in haematopoietic cells and they suggest that hypersensitivity to GM-CSF may be a primary event in the development of JCML.

Both alleles of the NF1 gene are inactivated in leukemic cells in some patients with neurofibromatosis type 1. NF1 appears to function as a tumor-suppressor gene in immature myeloid cells.

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Activated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation, and has more recently been shown to be a potent anti-inflammatory mediator. Using cultured human cells, we show here that APC up-regulates the angiogenic promoters matrix metalloproteinase-2 in skin fibroblasts and umbilical vein endothelial cells, vascular endothelial growth factor in keratinocytes and fibroblasts, and monocyte chemoattractant protein-1 in fibroblasts. In the chick embryo chorioallantoic membrane assay, APC promoted the granulation/remodeling phases of wound healing by markedly stimulating angiogenesis as well as promoting reepithelialization. In a full-thickness rat skin-healing model, a single topical application of APC enhanced wound healing compared to saline control. APC-treated wounds had markedly more blood vessels on day 7 and a significantly lower infiltration of neutrophils at days 4 and 7. The broad spectrum matrix metallo-proteinase, GM6001, prevented the ability of APC to promote wound healing. In summary, our results show that APC promotes cutaneous wound healing via a complex mechanism involving stimulation of angiogenesis and inhibition of inflammation. These unique properties of APC make it an attractive therapeutic agent to promote the healing of chronic wounds.

No abstract

E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein–encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein–driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1–induced leukemia.

The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified.

IntroductionHemophilia A is the most common severe inherited bleeding disorder. Patients with this disease are treated with recombinant or plasma-derived factor VIII (FVIII), which allows them to lead relatively normal lives. 1 In approximately 25% of treated patients, however, the development of anti-FVIII antibodies (FVIII inhibitors) severely complicates FVIII replacement therapy and significantly increases morbidity within the hemophilia population. [2][3][4][5] These antibodies neutralize the procoagulant cofactor activity of FVIII or enhance its clearance from plasma. 5 In economically developed countries, there are 2 approaches to the clinical management of FVIII inhibitors: the treatment or prevention of bleeding and long-term immune tolerance induction (ITI). Bleeding is controlled with variably effective and expensive FVIII-bypassing agents, such as recombinant (r) FVIIa and FEIBA (FVIII-inhibitor bypassing agent). In contrast, ITI is usually attempted through the administration of FVIII at a dose and frequency that depends on the ITI protocol. 6 This treatment approach is practically challenging, costly, and can take months to years to become effective. In light of the significant limitations of the current treatment options, the development of effective, rapid, and economical ITI strategies is a clinical priority.Currently, the most consistent model to study FVIII inhibitors is the hemophilia A mouse (FVIII Ϫ/Ϫ ). [7][8][9] Repeated intravenous infusion of human FVIII into hemophilia A mice results in high titer inhibitor formation. This is a CD4 ϩ T cell-dependent process that requires costimulation. [9][10][11][12] The dependence on CD4 ϩ T cells for inhibitor formation also occurs in humans. Evidence of this first came from hemophilia A patients with FVIII inhibitors who were also HIV ϩ : as patient CD4 ϩ levels declined, there was concomitant disappearance of FVIII inhibitors. 13 Therefore, therapies that blocked T-cell activation seemed to be promising candidates to prevent inhibitor formation.Indeed, Qian et al demonstrated that FVIII Ϫ/Ϫ B7.2 Ϫ/Ϫ doubleknockout mice will not develop anti-FVIII antibodies (Abs) after repeated immunization with FVIII, and that blocking the CD80-CD28 costimulatory interaction with soluble cytotoxic T lymphocyte antigen-4 (CTLA-4)-immunoglobulin (Ig) in FVIII Ϫ/Ϫ mice also prevented inhibitor formation. 10 Additional studies in FVIII Ϫ/Ϫ mice showed that blockade of the CD40-CD40L interaction with anti-CD40L monoclonal Ab (mAb) also protects against FVIII inhibitor formation. 11,12 However, costimulatory blockade must be applied with each FVIII administration to maintain tolerance, and once the blockade is removed, the protective effect is lost. As the potential health risks of long-term costimulatory blockade have not yet been determined and because many hemophilia A patients are treated frequently with FVIII and would most likely need to coadminister blockade with each infusion, this therapy is not a viable option.To reach the clinic, a therapy that induces tole...