p38␣, p38␤, p38␥, and p38␦ are four isoforms of p38 mitogenactivated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38␣ and p38␥ transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38␦ mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38␤ is minimally expressed in early CD34 ؉ hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation͞activation of p38␣, MAPK kinase 3͞6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38␣, MAPK kinase kinase 3͞6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38␣ does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38␣ and p38␦ isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation͞ activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor ␣, an inflammatory cytokine that is modulated by p38␣, is expressed by differentiating erythroblasts and inhibition of p38␣ or tumor necrosis factor ␣ results in reduction in differentiation. Taken together, our data demonstrate that both p38␣ and ␦ isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.

The mammalian forkhead transcription factors, FOXO3a (FKHRL1), FOXO1a (FKHR) and FOXO4 (AFX) are negatively regulated by PKB/Akt kinase. In the present study we examined the engagement of forkhead family of transcription factors in erythropoietin (Epo)-and stem cell factor (SCF)-mediated signal transduction. Our data show that all three forkhead family members, FOXO3a, FOXO1a and FOXO4 are phosphorylated in human primary erythroid progenitors. Experiments performed to determine various upstream signaling pathways contributing to phosphorylation of forkhead family members show that only PI-3-kinase pathway is required for inactivation of FOXO3a. Our data also demonstrate that during Epo deprivation FOXO3a interacts with the transcriptional coactivator p300 and such interaction is disrupted by stimulation of cells with Epo. To determine the domains in FOXO3a, mediating its interaction with p300, we performed GST pull-down assays and found that the Nterminus region containing the ®rst 52 amino acids was su cient for binding p300. Finally, our data demonstrate that FOXO3a and FOXO1a are acetylated during growth factor deprivation and such acetylation is reversed by stimulation with Epo. Thus mammalian forkhead transcription factors are involved in Epo and SCF signaling in primary erythroid progenitors and may play a role in the induction of apoptotic and mitogenic signals.

Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug l-asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine dependency of ALL cells. In addition to hydrolyzing the amino acid l-asparagine, all FDA-approved l-asparaginases also have significant l-glutaminase coactivity. Since several reports suggest that l-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced l-glutaminase coactivity might be clinically beneficial if their antileukemic activity would be preserved. Here we show that novel low l-glutaminase variants developed on the backbone of the FDA-approved l-asparaginase were highly efficacious against both T- and B-cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer l-asparaginases without l-glutaminase activity for the treatment of human ALL. A new l-asparaginase-based therapy is less toxic compared with FDA-approved high l-glutaminase enzymes .

The majority of colon cancer patients will develop advanced disease with the liver being the most common site of metastatic disease. Patients with increased numbers of tumor-infiltrating lymphocytes in primary colon tumors and liver metastases have improved outcomes. However, the molecular factors which could empower anti-tumor immune responses in this setting remained to be elucidated. We reported that the immunostimulatory cytokine LIGHT (TNFSF14) in the microenvironment of colon cancer metastases associates with improved patient survival, and here we demonstrate in an immunocompetent murine model that colon tumors expressing LIGHT stimulate lymphocyte proliferation and tumor-cell specific anti-tumor immune responses. In this model, increasing LIGHT expression in the microenvironment of either primary tumors or liver metastases triggered regression of established tumors and slowed the growth of liver metastases, driven by cytotoxic T-lymphocyte mediated anti-tumor immunity. These responses corresponded with significant increases in tumor-infiltrating lymphocytes and increased expression of lymphocyte-homing signals in the metastatic tumors. Further, we demonstrated evidence of durable tumor-specific anti-tumor immunity. In conclusion, increasing LIGHT expression increased T-cell proliferation, activation, and infiltration, resulting in enhanced tumor-specific immune-mediated tumor regressions in primary tumors and colorectal liver metastases. Mechanisms to increase LIGHT in the colon cancer microenvironment warrant further investigation and hold promise as an immunotherapeutic strategy.

Here we examined the addition of intensity-modulated total marrow irradiation (TMI) delivered using a linear accelerator to a myeloablative chemotherapy conditioning regimen before allogeneic hematopoietic stem cell transplantation (HSCT). In this phase I study, we enrolled 14 patients with high-risk hematologic malignancies who received escalating doses of TMI at 3 Gy (n = 3), 6 Gy (n = 3), 9 Gy (n = 6), and 12 Gy (n = 2) in combination with intravenous (i.v.) fludarabine 160 mg/m(2) and targeted busulfan (area under the curve, 4800 μM*minute). Peripheral blood mobilized stem cells were obtained from HLA-matched related (n = 9) or unrelated (n = 4) or 1 antigen-mismatched unrelated (n = 1) donors. All patients rapidly engrafted and recovered their immune cells. Overall, Bearman extrahematologic toxicity were limited to grades 1 or 2, with oral mucositis grade 1 in 64% and grade 2 in 36% of the patients. With a median follow-up of 1126 days (range, 362 to 1469) for living patients, the overall survival was 50% and relapse-free survival was 43%. Of 7 deaths, 3 were due to relapse and 4 to transplantation-related complications. We conclude that 9 Gy TMI can be combined with myeloablative chemotherapy in the design of new preparative regimens for HSCT. This study was registered at clinicaltrials.gov as NCT00988013.

Cholinergic agents are known to affect the epithelial transport of H2O and electrolytes in the kidney. In proximal tubule cells, cholinergic agonists increase basolateral Na-HCO(3) cotransport activity via M(1) muscarinic receptor activation. The signaling intermediates that couple these G protein-coupled receptors to cotransporter activation, however, are not well defined. We therefore sought to identify distal effectors of muscarinic receptor activation that contribute to increased NBC activity in cultured proximal tubule cells. As demonstrated previously for acute CO2-regulated cotransport activity, we found that inhibitors of Src family kinases (SFKs) or the classic mitogen-activated protein kinase (MAPK) pathway prevented the stimulation of NBC activity by carbachol. The ability of carbachol to activate Src, as well as the proximal (Raf) and distal [extracellular signal-regulated kinases 1 and 2 (ERK1/2)] elements of the classic MAPK module, was compatible with these findings. Cholinergic stimulation of ERK1/2 activity was also completely prevented by overexpression of a dominant negative mutant of Ras (N17-Ras). Taken together, these findings suggest a requirement for the sequential activation of SFKs, Ras, and the classic MAPK pathway [Raf-->MAPK/ERK kinase (MEK)-->ERK]. These findings provide important insights into the molecular mechanisms underlying cholinergic regulation of NBC activity in renal epithelial cells. They also suggest a specific mechanism whereby cholinergic stimulation of the kidney can contribute to pH homeostasis.

YFP(+) BMCs in thy1-YFP mice have immunophenotypic features of MDSCs. They secrete NGF and promote neuroregeneration. Their immunosuppressive and neurotrophic actions are preserved after corneal infiltration. These findings increase our understanding of the beneficial roles played by leukocyte trafficking in the cornea and may lead to therapeutic strategies that use NGF-secreting myeloid cells to repair diseased or injured neurons.

IntroductionCD34 ϩ cell populations in human bone marrow and peripheral blood can act as antigen-presenting cells (APCs) and activate allogeneic T cells in vitro due to the presence of committed myeloid progenitors that constitutively express HLA and adhesion and costimulatory molecules. 1,2 These latter molecules are upregulated on CD34 ϩ cells upon binding to T cells or after addition of tumor necrosis factor-␣ (TNF␣) for 24 hours to the culture. 2,3 Similarly, ICOS-L expression on CD34 ϩ cells can be induced by TNF␣ and can act as an alternative costimulatory pathway on stem cells. 4 Moreover, the expression of costimulatory molecules identifies a subpopulation of CD34 ϩ progenitors that differentiate into dendritic cells (DCs) in liquid culture. 3,5 CD34 ϩ cells from cord blood (CB) also generate myeloid DCs in response to granulocytemacrophage colony-stimulating factor (GM-CSF) and TNF␣ in vitro 6,7 and allow the engraftment of functional DCs in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. 8 After hematopoietic stem cell (HSC) transplantation, allogeneic donor T cells respond to host APCs and initiate graft-versus-host disease (GVHD) but also facilitate the engraftment of donor CD34 ϩ cells. 9,10 Experiments in NOD/SCID mice suggested an important role of CD8 ϩ T cells in facilitating the migration, homing, and engraftment of CD34 ϩ cells through alteration of signaling via the chemokine receptor CXCR4. 11 However, while T cells can enhance the stimulatory activity of DCs, 12 the effect of donor T cells on the APC function of CD34 ϩ cells is not known. Multiple clinical studies have shown direct correlations between CD34 ϩ cell dose [13][14][15][16] and the development of GVHD. Mielcarek et al 17 have hypothesized that donor CD34 ϩ APCs, or APCs derived from CD34 ϩ cells, may contribute to the development of GVHD by indirect presentation of host alloantigen.In this study, we show that allogeneic T cells can affect the function of CD34 ϩ cells by inducing the proliferation and rapid differentiation of a subset of progenitors into monocytic-dendritic precursors capable of both direct and indirect alloantigen presentation. Cotransplantation of CD34 ϩ cells and allogeneic T cells into NOD/SCID mice resulted in a better engraftment of human CD45 ϩ cells with dendritic phenotype than cotransplantation with autologous T cells. Among the mechanisms used by T cells to stimulate CD34 ϩ cell proliferation and differentiation, we describe here both the ligation of cellular receptors and the release of cytokines, such as GM-CSF and TNF␣. Materials and methods Flow cytometryFlow cytometric analysis was performed on samples of peripheral blood (PB) and CB cells. Cell separationCB mononuclear cells (MNCs) were obtained by centrifugation over Ficoll/Hypaque (Amersham Biosciences, Piscataway, NJ) gradients. Lightdensity cells were washed twice in PBS (Cambrex, Walkersville, MD) with 1% bovine serum albumin (BSA; Sigma Chemical, St Louis, MO), and CD34 ϩ cells were purified by MidiMACS high-gradient ma...