Recombinant human granulocyte-colony stimulating factor and recombinant human macrophage-colony stimulating factor synergize in vivo to enhance proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in mice

Combinations of low dosages of purified recombinant human (rh) macrophage-colony stimulating factor (M-CSF; also termed CSF-1) and rh granulocyte-colony stimulating factor (G-CSF) were compared alone and in combination for their influence on the cycling rates and numbers of bone marrow and splenic granulocyte-macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with iron-saturated human lactoferrin (LF). LF was used to enhance detection of the stimulating effects of exogenously added CSFs. Concentrations of each CSF that were not active in vivo when given alone were active when given together, with the other CSF. The concentrations of rhM-CSF and rhG-CSF needed to increase progenitor cell cycling in the marrow and spleen were reduced by factors of 40-200 when these CSFs were administered in combination with low dosages of the other CSF. At the concentrations of rhM-CSF and rhG-CSF tested, synergism was not noted on absolute numbers of progenitor cells or total nucleated cell counts per organ or circulating in the blood. These findings may have potential relevance when considered in a clinical setting where the CSFs might be used in combination with other biotherapy and/or chemotherapy.     

Transgenic expression of stromal cell-derived factor-1/CXC chemokine ligand 12 enhances myeloid progenitor cell survival/antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo

Hemopoiesis is regulated in part by survival/apoptosis of hemopoietic stem/progenitor cells. Exogenously added stromal cell-derived factor-1 ((SDF-1)/CXC chemokine ligand (CXCL)12) enhances survival/antiapoptosis of myeloid progenitor cells in vitro. To further evaluate SDF-1/CXCL12 effects on progenitor cell survival, transgenic mice endogenously expressing SDF-1/CXCL12 under a Rous sarcoma virus promoter were produced. Myeloid progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte) from transgenic mice were studied for in vitro survival in the context of delayed addition of growth factors. SDF-1-expressing transgenic myeloid progenitors were enhanced in survival and antiapoptosis compared with their wild-type littermate counterparts. Survival-enhancing effects were due to release of low levels of SDF-1/CXCL12 and mediated through CXCR4 and G(alpha)i proteins as determined by ELISA, an antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin. Transgenic effects of low SDF-1/CXCR4 may be due to synergy of SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes with low concentrations of other cytokines to enhance survival of normal mouse myeloid progenitors. Consistent with in vitro results, progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow and splenic myelopoiesis: greatly increased progenitor cell cycling and significant increases in progenitor cell numbers. These results substantiate survival effects of SDF-1/CXCL12, now extended to progenitors engineered to endogenously produce low levels of this cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition to cell trafficking.     

Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34+ progenitor cells

CD26/dipeptidylpeptidase IV (DPPIV) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains. The N terminus of chemokines is known to interact with the extracellular portion of chemokine receptors, and removal of these amino acids in many instances results in significant changes in functional activity. CD26/DPPIV has the ability to cleave the chemokine CXCL12/stromal cell-derived factor 1alpha (SDF-1alpha) at its position two proline. CXCL12/SDF-1alpha induces migration of hemopoietic stem and progenitor cells, and it is thought that CXCL12 plays a crucial role in homing/mobilization of these cells to/from the bone marrow. We found that CD26/DPPIV is expressed by a subpopulation of CD34(+) hemopoietic cells isolated from cord blood and that these cells have DPPIV activity. The involvement of CD26/DPPIV in CD34(+) hemopoietic stem and progenitor cell migration has not been previously examined. Functional studies show that the N-terminal-truncated CXCL12/SDF-1alpha lacks the ability to induce the migration of CD34(+) cord blood cells and acts to inhibit normal CXCL12/SDF-1alpha-induced migration. Finally, inhibiting the endogenous CD26/DPPIV activity on CD34(+) cells enhances the migratory response of these cells to CXCL12/SDF-1alpha. This process of CXCL12/SDF-1alpha cleavage by CD26/DPPIV on a subpopulation of CD34(+) cells may represent a novel regulatory mechanism in hemopoietic stem and progenitor cells for the migration, homing, and mobilization of these cells. Inhibition of the CD26/DPPIV peptidase activity may therefore represent an innovative approach to increasing homing and engraftment during cord blood transplantation.     

Positioning of anchor groups in protein loop prediction: the importance of solvent accessibility and secondary structure elements

The prediction of loop regions in the process of protein structure prediction by homology is still an unsolved problem. In an earlier publication, we could show that the correct placement of the amino acids serving as an anchor group to be connected by a loop fragment with a predicted geometry is a highly important step and an essential requirement within the process (Lessel and Schomburg, Proteins 1999; 37:56-64). In this article, we present an analysis of the quality of possible loop predictions with respect to gap length, fragment length, amino acid type, secondary structure, and solvent accessibility. For 550 insertions and 544 deletions, we test all possible positions for anchor groups with an inserted loop of a length between 3 and 12 amino acids. We could show that approximately 80% of the indel regions could be predicted within 1.5 A RMSD from a knowledge-based loop data base if criteria for the correct localization of anchor groups could be found and the loops can be sorted correctly. From our analysis, several conclusions regarding the optimal placement of anchor groups become obvious: (1) The correct placement of anchor groups is even more important for longer gap lengths, (2) medium length fragments (length 5-8) perform better than short or long ones, (3) the placement of anchor groups at hydrophobic amino acids gives a higher chance to include the best possible loop, (4) anchor groups within secondary structure elements, in particular beta-sheets are suitable, (5) amino acids with lower solvent accessibility are better anchor group. A preliminary test using a combination of the anchor group positioning criteria deduced from our analysis shows very promising results.     

Alternating Host Cell Tropism Shapes the Persistence,Evolution and Coexistence of Epstein–Barr Virus Infections in Human

Epstein–Barr virus (EBV) infects and can persist in a majority of people worldwide. Within an infected host, EBV targets two major cell types, B cells and epithelial cells, and viruses emerging from one cell type preferentially infect the other. We use mathematical models to understand why EBV infects epithelial cells when B cells serve as a stable refuge for the virus and how switching between infecting each cell type affects virus persistence and shedding. We propose a mathematical model to describe the regulation of EBV infection within a host. This model is used to study the effects of parameter values on optimal viral strategies for transmission, persistence, and intrahost competition. Most often, the optimal strategy to maximize transmission is for viruses to infect epithelial cells, but the optimal strategy for maximizing intrahost competition is for viruses to mainly infect B cells. Applying the results of the within-host model, we derive a model of EBV dynamics in a homogeneous population of hosts that includes superinfection. We use this model to study the conditions necessary for invasion and coexistence of various viral strategies at the population level. When the importance of intrahost competition is weak, we show that coexistence of different strategies is possible.     

The opposing actions in vivo on murine myelopoiesis of purified preparations of lactoferrin and the colony stimulating factors

The actions of purified iron-saturated human lactoferrin (LF), purified preparations of human MiaPaCa colony stimulating factor-1 (CSF-1), and recombinant murine interleukin-3 (IL-3) were evaluated in vivo in mice. Studies in vitro were compared at lowered (5%), as well as at normal incubator (20%), oxygen (O2) tension because of the potentially greater physiologic relevance of in vitro studies performed at lowered O2 tension. The results demonstrate that 1) increased release of granulocyte-macrophage colony stimulating factor (GM-CSF) in vitro from pokeweed mitogen stimulated mouse spleen cells and from human mononuclear blood cells occurred at lowered O2 tension, and that human mononuclear blood leukocytes were more sensitive to the LF-induced suppression of GM-CSF release when cells were cultured at 5%, compared to 20%, O2 tension; 2) LF administered intravenously (IV) to mice pretreated with sublethal intraperitoneal dosages of Cytoxan decreased the cycling status of marrow and spleen granulocyte-macrophage (CFU-GM), erythroid (BFU-E-2 and BFU-E-1) and multipotential (CFU-GEMM) progenitor cells and the absolute numbers of these progenitors; these effects were most noticeable if care was taken to deplete endotoxin from the LF samples prior to testing LF in vivo and if the control medium was endotoxin free; 3) endotoxin-depleted LF decreased the cycling status of marrow and spleen CFU-GM, BFU-E, and CFU-GEMM and the numbers of these progenitors in the marrows of mice previously untreated with Cytoxan; these effects were most apparent when assessment of progenitor cells and their cycling rates were evaluated in vitro at lowered (5%) O2 tension; 4) purified natural human CSF-1 increased the absolute numbers of marrow CFU-GM and the cycling status of marrow CFU-GM and CFU-GEMM in mice pretreated with LF; and 5) purified recombinant murine IL-3 stimulated proliferation of day 8 and day 12 CFU-S (colony forming unit-spleen) in mice not previously treated with Cytoxan. These results substantiate the in vivo myelosuppressive effects of LF on CFU-GM and extend these effects to erythroid and multipotential progenitor cells, provide evidence that human CSF-1 has an in vivo action in mice, and confirm the studies of others showing that IL-3 stimulates the proliferation of CFU-S in vivo.     

Constitutive expression of CIITA directs CD4 T cells to produce Th2 cytokines in the thymus

We generated mice expressing a human type III CIITA transgene (CIITA Tg) under control of the CD4 promoter to study the role of CIITA in CD4 T cell biology. The transgene is expressed in peripheral CD4 and CD8 T cells, as well as in thymocytes. When CD4 T cells were differentiated towards the Th2 lineage, both control and CIITA Tg Th2 cells expressed similar levels of Th2 cytokines. Th1 cells from control and CIITA Tg mice cells produced comparable levels of IFN-gamma. CIITA Tg Th1 cells also expressed IL-4, IL-5, and IL-13 in the absence of Stat6. There was an approximate 10-fold increase in the number of peripheral na?ve CD4 T cells and NK1.1- thymocytes producing IL-4 from CIITA Tg mice compared to control mice. Finally, Th1 cells from irradiated control mice reconstituted with CIITA Tg bone marrow displayed the same cytokine production profiles as Th1 cells from CIITA Tg mice. Together, our data demonstrate that CIITA expression pre-disposes CD4 T cells to produce Th2 type cytokines. Moreover, phenotypic similarities between Th1 cells expressing the CIITA transgene and CIITA deficient Th1 cells suggest that the role of CIITA in cytokine regulation is complex and may reflect both direct and indirect mechanisms of T cell development and differentiation.