bluuebunny/crossref_metadata_2025 huggingface dataset:
A Kinetic Model for the Homing and Migration of Prenatally Transplanted Marrow
Authors: Shaaban Aimen F. , Kim Heung Bae , Milner Ross , Flake Alan W.
Published: October 2019

Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (106 LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.
Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins
Authors: Gupta Pankaj , Oegema Theodore R. , Brazil Joseph J. , Dudek Arkaduisz Z. , Slungaard Arne , Verfaillie Catherine M.
Published: October 2019

We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell “niches” in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1 or PF4 (LTC-IC maintenance without GAGs, 32 ± 2%; with GAGs, 95 ± 7%; P < .001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 ± 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)
The Shc adaptor protein forms interdependent phosphotyrosine-mediated protein complexes in mast cells stimulated with interleukin 3
Authors: Velazquez Laura , Gish Gerald D. , van der Geer Peter , Taylor Lorne , Shulman Johanna , Pawson Tony
Published: October 2019

The Shc adaptor protein possesses 2 distinct phosphotyrosine (pTyr) recognition modules—the pTyr binding (PTB) domain and the Src homology 2 (SH2) domain—and multiple potential sites for tyrosine (Tyr) phosphorylation (Tyr residues 239, 240, and 317). On stimulation of hematopoietic cells with interleukin 3 (IL-3), Shc becomes phosphorylated and may therefore contribute to IL-3 signaling. We investigated the interactions mediated by the Shc modular domains and pTyr sites in IL-3–dependent IC2 premast cells. The Shc PTB domain, rather than the SH2 domain, associated both in vitro and in vivo with the Tyr-phosphorylated β subunit of the IL-3 receptor and with the SH2-containing 5′ inositol phosphatase (SHIP), and it recognized specific NXXpY phosphopeptides from these binding partners. In IL-3–stimulated mast cells, Shc phosphorylation occurred primarily on Tyr239 and 317 and was dependent on a functional PTB domain. Phosphorylated Tyr317, and to a lesser extent, Tyr239, bound the Grb2 adaptor and SHIP. Furthermore, a pTyr317 Shc phosphopeptide selectively recognized Grb2, Sos1, SHIP, and the p85 subunit of phosphatidylinositol 3′ kinase from mast cells, as characterized by mass spectrometry. These results indicate that Shc undergoes an interdependent series of pTyr-mediated interactions in IL-3–stimulated mast cells, resulting in the recruitment of proteins that regulate the Ras pathway and phospholipid metabolism.
Vascular release of plasminogen activator inhibitor-1 impairs fibrinolysis during acute arterial thrombosis in mice
Authors: Kawasaki Tomihisa , Dewerchin Mieke , Lijnen Henri R. , Vermylen Jos , Hoylaerts Marc F.
Published: October 2019

The role of plasminogen activator inhibitor-1 (PAI-1) in the plasma, blood platelets, and vessel wall during acute arterial thrombus formation was investigated in gene-deficient mice. Photochemically induced thrombosis in the carotid artery was analyzed via transillumination. In comparison to thrombosis in C57BL/6J wild-type (wt) mice (113 ± 19 × 106 arbitrary light units [AU] n = 15, mean ± SEM), thrombosis in PAI-1−/− mice (40 ± 10 × 106 AU, n = 13) was inhibited (P < .01), indicating that PAI-1 controls fibrinolysis during thrombus formation. Systemic administration of murine PAI-1 into PAI-1−/− mice led to a full recovery of thrombotic response. Occurrence of fibrinolytic activity was confirmed in 2-antiplasmin (2-AP)–deficient mice. The sizes of thrombi developing in wt mice, in 2-AP+/− and 2-AP−/− mice were 102 ± 35, 65 ± 8.1, and 13 ± 6.1 × 106 AU, respectively (n = 6 each) (P < .05), compatible with functional plasmin inhibition by 2-AP. In contrast, thrombi in wt mice, t-PA−/− and u-PA−/−mice were comparable, substantiating efficient inhibition of fibrinolysis by the combined PAI-1/2-AP action. Platelet depletion and reconstitution confirmed a normal thrombotic response in wt mice, reconstituted with PAI-1−/− platelets, but weak thrombosis in PAI-1−/− mice reconstituted with wt platelets. Accordingly, murine (wt) PAI-1 levels in platelet lysates and releasates were 0.43 ± 0.09 ng/109 platelets and plasma concentrations equaled 0.73 ± 0.13 ng/mL. After photochemical injury, plasma PAI-1 rose to 2.9 ± 0.7 ng/mL (n = 9, P < .01). The plasma rise was prevented by ligating the carotid artery. Hence, during acute thrombosis, fibrinolysis is efficiently prevented by plasma 2-AP, but also by vascular PAI-1, locally released into the circulation after endothelial injury.
A Leu262Pro mutation in the integrin β3 subunit results in an αIIb-β3 complex that binds fibrin but not fibrinogen
Authors: Ward Christopher M. , Kestin Anita S. , Newman Peter J.
Published: October 2019

Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, IIbβ3. In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant IIbβ3 may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable IIbβ3, findings consistent with type II GT. Genotyping of LD revealed 2 novel β3 mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among β integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Proβ3 cotransfected with wild-type IIb into COS-7 cells showed delayed intracellular maturation and reduced surface expression of easily dissociable complexes. In human embryonic kidney 293 cells, Leu262Proβ3 formed a complex with endogenous av and retracted fibrin clots similarly to wild-type β3. The same cells, however, were unable to bind immobilized fibrinogen. The molecular requirements for IIbβ3 to interact with fibrin compared with fibrinogen, therefore, appear to differ. The region surrounding β3 Leu262 may maintain β3 in a fibrinogen-binding, competent form, but it appears not to be required for receptor interactions with fibrin.
Interferon-α and -β inhibit the in vitro differentiation of immunocompetent human dendritic cells from CD14+ precursors
Authors: McRae Bradford L. , Nagai Taro , Semnani Roshanak Tolouei , van Seventer Jean Maguire , van Seventer Gijs A.
Published: October 2019

Dendritic cell (DC) precursors and immature DC reside in epithelium where they encounter pathogens and cytokines, which stimulate their differentiation. We hypothesized that type-I interferons (IFN- and -β), cytokines that are produced early in the innate immune response against viruses and some bacteria, may influence DC differentiation and function. To examine this possibility, we used an in vitro model of DC differentiation in which initial culture of human CD14+monocytes with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 generates immature DC, and subsequent culture with tumor necrosis factor (TNF)- drives the final development into mature DC. We found in this model that IFN-/β, added from the initiation of the culture on, significantly reduced the survival and altered the morphology and differentiation of DC. TNF-–dependent maturation of IFN-β–treated immature DC led to cells with reduced expression of CD1a, CD40, CD54, and CD80 when compared with mature DC controls. IFN-/β–treated DC further had a reduced capacity to induce naive Th-cell proliferation through allostimulation or anti-CD3 monoclonal antibody stimulation. In addition, IFN-/β–treated DC secreted less IL-12 upon stimulation with Staphylococcus aureus Cowan strain or with CD4+ T cells, and this decrease correlated directly with their inability to support CD4+ T-cell secretion of IFN-γ, even though T-cell lymphotoxin production was unaffected. These findings indicate that type-I IFNs can influence the generation of acquired immune responses by modifying T-helper cell differentiation through the regulation of DC differentiation and function.
Overexpression of murine fizzy-related (fzr) increases natural killer cell–mediated cell death and suppresses tumor growth
Authors: Wang Chun-Xiang , Fisk Bernard C. , Wadehra Madhuri , Su Helen , Braun Jonathan
Published: October 2019

Fizzy-related (fzr) is a recently identified 7WD domain family member implicated in cell cycle regulation of Drosophila and yeast. In this study, the murine homologue of fzr was isolated by suppression subtractive hybridization as a gene with decreased expression during malignant progression of a murine B-lymphoma cell line. Retroviral overexpression of fzr in B-lymphoma cells reduced tumor formation. Those tumors that did arise had diminished or extinguished retroviral Fzr. Surprisingly, fzr overexpression dramatically increased B-lymphoma cell susceptibility to natural killer cell (NK) cytotoxicity, a host-resistant mechanism for tumor formation in this model system. These findings implicate fzr as a new category of genes suppressing B-cell tumorigenesis and suggest a novel role for fzr in the target cell interaction with NK cells.
Deregulation of the ubiquitin system and p53 proteolysis modify the apoptotic response in B-CLL lymphocytes
Authors: Masdehors Peggy , Merle-Béral Hélène , Maloum Karim , Ömura Satoshi , Magdelénat Henri , Delic Jozo
Published: October 2019

We recently reported increased sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Here, we show that only specific—not nonspecific—proteasomal inhibitors can discriminate between malignant and normal lymphocytes in inducing the apoptotic death response. Indeed, lactacystin and its active metaboliteclasto-lactacystin β-lactone induced apoptotic death in CLL but not in normal lymphocytes. This difference was completely abolished when tripeptide aldehydes such as MG132 or LLnL (which can also inhibit calpains) were used as less specific proteasomal inhibitors. Moreover, B-CLL cells exhibited a constitutive altered ubiquitin-proteasome system, including a threefold higher chymotrypsin-like proteasomal activity and high levels of nuclear ubiquitin-conjugated proteins compared with normal lymphocytes. Interestingly, B-CLL cells also displayed altered proteolytic regulation of wild-type p53, an apoptotic factor reported to be a substrate for the ubiquitin-proteasome system. Nuclear wild-type p53 accumulated after lactacystin treatment used at the discriminating concentration in malignant, but not in normal, lymphocytes. In contrast, p53 was stabilized by MG132 or LLnL in malignant and normal cells undergoing apoptosis, indicating that in normal lymphocytes p53 is regulated mainly by calpains and not by the ubiquitin-proteasome system. This work raises the possibility that two different proteolytic pathways controlling p53 stability may be pathologically imbalanced. This could result in modification of apoptosis control, since in CLL-lymphocytes a highly upregulated ubiquitin-proteasome system, which controls p53 stability among other apoptotic factors, was correlated with an increased propensity of these cells to apoptosis triggered by lactacystin.
Endostatin, an antiangiogenic drug, induces tumor stabilization after chemotherapy or anti-CD20 therapy in a NOD/SCID mouse model of human high-grade non-Hodgkin lymphoma
Authors: Bertolini Francesco , Fusetti Lisa , Mancuso Patrizia , Gobbi Alberto , Corsini Chiara , Ferrucci Pier Francesco , Martinelli Giovanni , Pruneri Giancarlo
Published: October 2019

Both chemotherapy and chimeric anti-CD20 monoclonal antibodies are effective agents against B-cell non-Hodgkin lymphoma (NHL). However, patients achieving remission are at risk of relapse. To evaluate the effect of the antiangiogenic drug endostatin used alone and after the administration of cyclophosphamide (CTX) or the anti-CD20 antibody rituximab, we generated a new model of human NHL by transplanting Namalwa cells intraperitoneally into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. First, we determined the most effective treatment schedule for the drugs assessed. When administered alone, CTX (3 courses of 75 mg/kg of body weight given intraperitoneally), rituximab (3 courses of 25 mg/kg given intraperitoneally), and endostatin (5 courses of 50 μg given subcutaneously) delayed tumor growth, and CTX was the most effective in controlling bulky disease. When given after chemotherapy or immunotherapy, endostatin effectively induced tumor stabilization. When mice given CTX or rituximab on days 3, 5, and 7 after transplantation were randomly assigned to receive endostatin or phosphate-buffered saline on days 15 to 19, tumor growth was prevented in endostatin-treated mice as long as the drug was administered. Furthermore, administration of endostatin on days 25 to 29 after tumor regrowth still induced significant tumor regression, whereas CTX and rituximab were not effective. The specific antiangiogenic action of endostatin was confirmed by in vitro and in vivo studies indicating that the drug inhibited proliferation and induced apoptosis of endothelial (but not of NHL) cells. In conclusion, sequential administration of chemotherapy and endostatin seems promising for treating bulky NHL, and the less toxic sequential administration of rituximab and endostatin is promising for treating limited disease.
Reperfusion injury pathophysiology in sickle transgenic mice
Authors: Osarogiagbon U. Raymond , Choong Stephana , Belcher John D. , Vercellotti Gregory M. , Paller Mark S. , Hebbel Robert P.
Published: October 2019

Reperfusion of tissues after interruption of their vascular supply causes free-radical generation that leads to tissue damage, a scenario referred to as “reperfusion injury.” Because sickle disease involves repeated transient ischemic episodes, we sought evidence for excessive free-radical generation in sickle transgenic mice. Compared with normal mice, sickle mice at ambient air had a higher ethane excretion (marker of lipid peroxidation) and greater conversion of salicylic acid to 2,3-dihydroxybenzoic acid (marker of hydroxyl radical generation). During hypoxia (11% O2), only sickle mice converted tissue xanthine dehydrogenase to oxidase. Only the sickle mice exhibited a further increase in ethane excretion during restitution of normal oxygen tension after 2 hours of hypoxia. Only the sickle mice showed abnormal activation of nuclear factor–κB after exposure to hypoxia-reoxygenation. Allopurinol, a potential therapeutic agent, decreased ethane excretion in the sickle mice. Thus, sickle transgenic mice exhibit biochemical footprints consistent with excessive free-radical generation even at ambient air and following a transient induction of enhanced sickling. We suggest that reperfusion injury physiology may contribute to the evolution of the chronic organ damage characteristic of sickle cell disease. If so, novel therapeutic approaches might be of value.
A randomized, double-blind trial of filgrastim (granulocyte colony-stimulating factor) versus placebo following allogeneic blood stem cell transplantation
Authors: Bishop Michael R. , Tarantolo Stefano R. , Geller Robert B. , Lynch James C. , Bierman Philip J. , Pavletic Z. Steven , Vose Julie M. , Kruse Susan , Dix Suzanne P. , Morris Mary E. , Armitage James O. , Kessinger Anne
Published: October 2019

Blood stem cell transplantation (BSCT) results in rapid hematopoietic recovery in both the allogeneic and autologous transplant settings. Because of the large numbers of progenitor cells in mobilized blood, the administration of growth factors after transplantation may not provide further acceleration of hematopoietic recovery. A randomized, double-blind, placebo-controlled study was performed to determine the effects of filgrastim (granulocyte colony-stimulating factor; G-CSF) administration on hematopoietic recovery after allogeneic BSCT. Fifty-four patients with hematologic malignancies undergoing a related, HLA-matched allogeneic BSCT were randomly assigned to receive daily filgrastim at 10 μg/kg or placebo starting on the day of transplantation. A minimum of 3 × 106 CD34+ cells/kg in the allograft was required for transplantation. All patients received a standard preparative regimen and a standard regimen for the prevention of graft-versus-host disease (GVHD). The median time to achieve an absolute neutrophil count greater than 0.5 × 109/L was 11 days (range, 9-20 days) for patients who received filgrastim compared with 15 days (range, 10-22 days) for patients who received placebo (P = .0082). The median time to achieve a platelet count greater than 20 × 109/L was 13 days (range, 8-35 days) for patients who received filgrastim compared with 15.5 days (range, 8-42 days) for patients who received placebo (P = .79). There were no significant differences for red blood cell transfusion independence, the incidence of acute GVHD, or 100-day mortality between the groups. The administration of filgrastim appears to be a safe and effective supportive-care measure following allogeneic BSCT.
Dendritic cells modified to express CD40 ligand elicit therapeutic immunity against preexisting murine tumors
Authors: Kikuchi Toshiaki , Moore Malcolm A. S. , Crystal Ronald G.
Published: October 2019

CD40 ligand (CD40L) is essential for the initiation of antigen-specific T-cell responses. This study is based on the hypothesis that dendritic cells (DCs) genetically modified ex vivo to express CD40L will enhance in vivo presentation of tumor antigen to the cellular immune system with consequent induction of antitumor immunity to suppress tumor growth. To examine this concept, subcutaneous murine tumors were injected with bone marrow-derived DCs that had been modified in vitro with an adenovirus (Ad) vector expressing murine CD40L (AdmCD40L). In B16 (H-2b, melanoma) and CT26 (H-2d, colon cancer) murine models, intratumoral injection of 2 × 106 AdmCD40L-modified DCs (CD40L-DCs) to established (day 8) subcutaneous tumors resulted in sustained tumor regression and survival advantage. This antitumor effect was sustained when the number of CD40L-DCs were reduced 10-fold to 2 × 105. Analysis of spleens from CD40L-DC–treated animals demonstrated that CD40L-DCs injected into the subcutaneous CT26 flank tumors migrated to the spleen, resulting in activation of immune-relevant processes. Consistent with this concept, intratumoral administration of CD40L-DCs elicited tumor-specific cytotoxic T-lymphocyte responses, and the transfer of spleen cells from CD40L-DC–treated mice efficiently protected naive mice against a subsequent tumor challenge. In a distant 2-tumor model of metastatic disease, an untreated B16 tumor in the right flank regressed in parallel with a left B16 tumor treated with direct injection of CD40L-DCs. These results support the concept that genetic modification of DCs with a recombinant CD40L adenovirus vector may be a useful strategy for directly activating DCs for cancer immunotherapy.
Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells
Authors: Palumbo Joseph S. , Kombrinck Keith W. , Drew Angela F. , Grimes Timothy S. , Kiser John H. , Degen Jay L. , Bugge Thomas H.
Published: October 2019

Detailed studies of tumor cell–associated procoagulants and fibrinolytic factors have implied that local thrombin generation and fibrin deposition and dissolution may be important in tumor growth and dissemination. To directly determine whether fibrin(ogen) or plasmin(ogen) are determinants of the metastatic potential of circulating tumor cells, this study examined the impact of genetic deficits in each of these key hemostatic factors on the hematogenous pulmonary metastasis of 2 established murine tumors, Lewis lung carcinoma and the B16-BL6 melanoma. In both tumor models, fibrinogen deficiency strongly diminished, but did not prevent, the development of lung metastasis. The quantitative reduction in metastasis in fibrinogen-deficient mice was not due to any appreciable difference in tumor stroma formation or tumor growth. Rather, tumor cell fate studies indicated an important role for fibrin(ogen) in sustained adhesion and survival of tumor cells within the lung. The specific thrombin inhibitor, hirudin, further diminished the metastatic potential of circulating tumor cells in fibrinogen-deficient mice, although the inhibitor had no apparent effect on tumor cell proliferation in vitro. The absence of plasminogen and plasmin-mediated fibrinolysis had no significant impact on hematogenous metastasis. The authors concluded that fibrin(ogen) is a critical determinant of the metastatic potential of circulating tumor cells. Furthermore, thrombin appears to facilitate tumor dissemination through at least one fibrin(ogen)-independent mechanism. These findings suggest that therapeutic strategies focusing on multiple distinct hemostatic factors might be beneficial in the containment of tumor metastasis.
High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors
Authors: Salmon Patrick , Kindler Vincent , Ducrey Odile , Chapuis Bernard , Zubler Rudolf H. , Trono Didier
Published: October 2019

Recent experiments point to the great value of lentiviral vectors for the transduction of human hematopoietic stem cells (hHSCs). Vectors used so far, however, have been poorly satisfying in terms of either biosafety or efficiency of transgene expression. Herein is described the results obtained with human immunodeficiency virus–based vectors optimized in both of these aspects. It is thus shown that vectors containing the EF1α and, to a lesser extent, the phosphoglycerate kinase (PGK) promoter, govern high-level gene expression in human hematopoietic progenitors as well as derived hematopoietic lineages of therapeutic relevance, such as erythrocytes, granulocytes, monocytes, dendritic cells, and megakaryocytes. EF1α promoter-containing lentiviral vectors can also induce strong transgene expression in primary T lymphocytes isolated from peripheral blood. A self-inactivating design did not affect the performance of EF1α promoter-based vectors but significantly reduced expression from the PGK promoter. This negative effect could nevertheless be largely rescued by inserting the post-transcriptional regulatory element of woodchuck hepatitis virus upstream of the vector 3′ long terminal repeat. These results have important practical implications for the genetic treatment of lymphohematologic disorders as well as for the study of hematopoiesis via the lentivector-mediated modification of hHSCs.
SCF and G-CSF lead to the synergistic induction of proliferation and gene expression through complementary signaling pathways
Authors: Duarte Rafael F. , Frank David A.
Published: October 2019

Stem cell factor (SCF) is a potent costimulatory molecule for many cytokines. Its synergy with granulocyte colony-stimulating factor (G-CSF) results in important biologic and clinical effects, although the mechanism by which this occurs remains poorly understood. To investigate this interaction, this study used a retroviral vector to transduce the G-CSF receptor into MO7e cells, which are known to express the SCF receptor. The transduced G-CSF receptor is functionally active, and the resultant MO7e-G cells recapitulate the proliferative synergy between SCF and G-CSF. When treated with both cytokines, a marked shortening of the G0/G1 phase of the cell cycle occurs, associated with a suppression of the cyclin-dependent kinase inhibitor p27kip-1. In addition, SCF and G-CSF induce the synergistic activation of c-fos, a proto-oncogene involved in propagation of mitogenic signals in hematopoietic cells. G-CSF, but not SCF, induces the tyrosine phosphorylation of STAT1 and STAT3, transcription factors that can mediate the induction of c-fos. However, SCF induces phosphorylation of STAT3 on serine727 (ser727), which is necessary for maximal STAT transcriptional activity, and the combination of SCF and G-CSF leads to complete STAT3 phosphorylation on ser727. The pathways by which SCF and G-CSF lead to serine phosphorylation of STAT3 are distinct and are partially dependent on phosphatidylinositol-3 kinase and ERKs, pathways that are also necessary for the synergistic effects of SCF and G-CSF on proliferation and c-fos induction. Thus, MO7e-G cells provide a powerful system in which the molecular basis of the synergy between SCF and G-CSF can be dissected.
Interleukin-15 redirects the outcome of a tolerizing T-cell stimulus from apoptosis to anergy
Authors: Dooms Hans , Van Belle Tom , Desmedt Marjory , Rottiers Pieter , Grooten Johan
Published: October 2019

Clonal deletion and anergy are 2 mechanisms used by the immune system to establish peripheral tolerance. In vitro, these mechanisms are induced in T lymphocytes by triggering the T-cell receptor (signal 1) in the absence of costimulation (signal 2). T-cell clones have been shown either to become anergic or to die in response to signal 1 alone; yet the factors that govern this choice remain unknown. This study evaluated the influence of the cytokines interleukin (IL)-2 and IL-15 on the response of the Th1 clone hemagglutinin (T-HA) to signal 1, delivered by stimulation with immobilized anti-CD3 monoclonal antibody (mAb). The response induced by immobilized anti-CD3 mAb was dependent on the cytokine milieu; in the presence of IL-2, T-HA cells were subject to apoptosis, whereas in the presence of IL-15 the cells remained viable but showed proliferative unresponsiveness. After release from the anti-CD3 stimulus, the IL-15-rescued T-HA cells regained responsiveness to IL-2 and IL-15 growth factor activity. However, they were unable to proliferate when stimulated with their cognate antigen presented by professional antigen-presenting cells (signal 1 plus 2) and thus had acquired an anergic phenotype. These data assign a novel function to the previously reported antiapoptotic activity of IL-15, namely, the capacity to redirect the T-cell response to partial stimulation from clonal deletion to anergy. Furthermore, they emphasize that the cytokine environment can critically influence the outcome of a tolerizing stimulus.
Molecular modification of a recombinant anti-CD3ε-directed immunotoxin by inducing terminal cysteine bridging enhances anti-GVHD efficacy and reduces organ toxicity in a lethal murine model
Authors: Vallera Daniel A. , Kuroki David W. , Panoskaltsis-Mortari Angela , Buchsbaum Donald J. , Rogers Buck E. , Blazar Bruce R.
Published: October 2019

Immunotoxin (IT) therapy shows potential for selectively eliminating GVHD-causing T cells in vivo, but the field has been hampered by toxicity. Previously, we showed that a genetically engineered IT consisting of a single-chain protein, including the anti-CD3sFv spliced to a portion of diphtheria-toxin (DT390) has anti-GVHD effects, but pronounced organ toxicity common to this class of agent. A recombinant DT390 anti-CD3sFv protein previously shown to have anti-GVHD activity was modified to reduce its filtration into kidney by genetically inserting a cysteine residue downstream of the sFv moiety at the c-terminus of the protein. This modification produced an intermolecular disulfide bridge, resulting in a bivalent, rather than a monovalent IT, termed SS2, that selectively inhibited T-cell proliferation in vitro. Although monomer and SS2 were similar in in vitro activity, SS2 had a superior therapeutic index in vivo with at least 8-fold more being tolerated with reduced kidney toxicity. Most importantly, in a lethal model of GVHD, 40 μg SS2 given for 1 day, protected 100% of the mice from lethal GVHD for 3 months, whereas the maximum tolerated dose (MTD) of monomer protected only 33%. To our knowledge, this is the first time disulfide bonded ITs have been created in this way and this simple molecular modification may address several problems in the IT field because it (1) markedly increased efficacy curing mice of GVHD after a single daily treatment, (2) markedly decreased organ toxicity, (3) increased the tolerated dosage, and (4) created a therapeutic window where none existed before.
Erythropoietin, iron, and erythropoiesis
Authors: Goodnough Lawrence T. , Skikne Barry , Brugnara Carlo
Published: October 2019

Recent knowledge gained regarding the relationship between erythropoietin, iron, and erythropoiesis in patients with blood loss anemia, with or without recombinant human erythropoietin therapy, has implications for patient management. Under conditions of significant blood loss, erythropoietin therapy, or both, iron-restricted erythropoiesis is evident, even in the presence of storage iron and iron oral supplementation. Intravenous iron therapy in renal dialysis patients undergoing erythropoietin therapy can produce hematologic responses with serum ferritin levels up to 400 μg/L, indicating that traditional biochemical markers of storage iron in patients with anemia caused by chronic disease are unhelpful in the assessment of iron status. Newer measurements of erythrocyte and reticulocyte indices using automated counters show promise in the evaluation of iron-restricted erythropoiesis. Assays for serum erythropoietin and the transferrin receptor are valuable tools for clinical research, but their roles in routine clinical practice remain undefined. The availability of safer intravenous iron preparations allows for carefully controlled studies of their value in patients undergoing erythropoietin therapy or experiencing blood loss, or both.
GATA factor transgenes under GATA-1 locus control rescue germline GATA-1 mutant deficiencies
Authors: Takahashi Satoru , Shimizu Ritsuko , Suwabe Naruyoshi , Kuroha Takashi , Yoh Keigyou , Ohta Jun , Nishimura Shigeko , Lim Kim-Chew , Engel James Douglas , Yamamoto Masayuki
Published: October 2019

GATA-1 germline mutation in mice results in embryonic lethality due to defective erythroid cell maturation, and thus other hematopoietic GATA factors do not compensate for the loss of GATA-1. To determine whether the obligate presence of GATA-1 in erythroid cells is due to its distinct biochemical properties or spatiotemporal patterning, we attempted to rescue GATA-1 mutant mice with hematopoietic GATA factor complementary DNAs (cDNAs) placed under the transcriptional control of the GATA-1gene. We found that transgenic expression of a GATA-1 cDNA fully abrogated the GATA-1–deficient phenotype. Surprisingly, GATA-2 and GATA-3 factors expressed from the same regulatory cassette also rescued the embryonic lethal phenotype of the GATA-1 mutation. However, adult mice rescued with the latter transgenes developed anemia, while GATA-1 transgenic mice did not. These results demonstrate that the transcriptional control dictating proper GATA-1 accumulation is the most critical determinant of GATA-1 activity during erythropoiesis. The results also show that there are biochemical distinctions among the hematopoietic GATA proteins and that during adult hematopoiesis the hematopoietic GATA factors are not functionally equivalent.
Mouse Jagged2 is differentially expressed in hematopoietic progenitors and endothelial cells and promotes the survival and proliferation of hematopoietic progenitors by direct cell-to-cell contact
Authors: Tsai Schickwann , Fero Jutta , Bartelmez Steve
Published: October 2019

To study the regulation of the early stages of hematopoiesis, cDNA representational difference analysis was used to isolate genes that were differentially expressed in primitive hematopoietic progenitors. The reasoning was that such genes were more likely to provide functions important to hematopoietic stem cells and progenitors. One of the genes identified through this approach encodes mouse Jagged2(mJagged2). Using quantitative reverse transcription–polymerase chain reaction, it was shown that mJagged2 was differentially expressed in c-kit+ hematopoietic progenitors, including those with the phenotypes of Lin− c-kit+Rhlo Holo and Lin−c-kit+ Rhhi Holo, and that they have been shown to be highly enriched for long-term and short-term repopulating hematopoietic stem cells, respectively. Western blot analyses showed that endothelial cells also expressed high levels of Jagged2, but stromal fibroblasts did not. Using a coculture system we found that exogenous, full-length mJagged2 promoted the survival and proliferation of hematopoietic progenitors, including the high-proliferative potential colony-forming cells. Direct cell-to-cell contact was required for this effect. Taken together, these findings indicate that both c-kit+ hematopoietic progenitors and endothelial cells express Jagged2 and that exogenous, full-length Jagged2 promotes the survival and proliferation of hematopoietic progenitors.
Pharmacologic properties of P2Z/P2X7receptor characterized in murine dendritic cells: role on the induction of apoptosis
Authors: Nihei Oscar Kenji , de Carvalho Antonio Carlos Campos , Savino Wilson , Alves Luiz Anastacio
Published: October 2019

In the immune system, extracellular adenosine 5′-triphosphate (ATP) mediates a variety of effects mainly through activation of a particular receptor subtype, the pore-forming P2Z/P2X7 purinoceptor. This purinergic receptor has been described chiefly in cells of hemopoietic origin such as T cells, thymocytes, monocytes, macrophages, and phagocytic cells of thymic reticulum. In this study, we characterized the P2Z/P2X7 purinoceptor and the ATP-mediated apoptosis in murine spleen–derived dendritic cells (DCs). Dye uptake and apoptosis were evaluated by flow cytometry. ATP-treated DCs were permeable to different low-molecular-weight fluorescent probes such as ethidium bromide, YO-PRO 1, and lucifer yellow. Such an effect was dose-dependent (EC50: 721 μmol/L); mediated by the fully anionic agonist (ATP4−); and specifically stimulated by ATP, BzATP, and ATPγS. Additionally, an ATP-induced increase in intracellular calcium was detected by microfluorometry. Furthermore, ATP treatment induced a significant increase in apoptotic DCs (64.46% ± 3.8%) when compared with untreated control cells (34% ± 5.8%), as ascertained by the TdT-mediated dUTP nick end labeling technique. Both ATP-induced DC permeabilization and apoptosis were inhibited by oxidized ATP, a P2Z/P2X7-specific antagonist. In conclusion, we characterized the expression of the P2Z/P2X7purinoceptor in murine spleen–derived DCs and described its role on the induction of apoptosis.
Peyer's patch eosinophils: identification, characterization, and regulation by mucosal allergen exposure, interleukin-5, and eotaxin
Authors: Mishra Anil , Hogan Simon P. , Brandt Eric B. , Rothenberg Marc E.
Published: October 2019

The gastrointestinal immune system is traditionally thought to be composed of lymphocytes located within Peyer's patches and the lamina propria. We have recently reported that eosinophils also reside in the gastrointestinal tract during healthy states, in particular, within the lamina propria, and that these cells substantially increase after oral allergen exposure. We now demonstrate the presence of eosinophils in Peyer's patches and characterize the signals that regulate the accumulation of eosinophils in Peyer's patches. In contrast to the lamina propria, intestinal Peyer's patches have very low levels of eosinophils under healthy states. However, elevated levels of interleukin-5 (IL-5), generated by transgenic or pharmacologic approaches, result in a dramatic increase in eosinophil levels in Peyer's patches. Most eosinophils are located in the outer cortex and interfollicular regions of the Peyer's patches. To dissect the mechanism of eosinophil trafficking to Peyer's patches, the role of eotaxin was examined. Mice transgenic for IL-5 and genetically deficient in eotaxin were found to have reduced levels of eosinophils in Peyer's patches compared with IL-5-transgenic mice. To prove that eosinophils also traffic to Peyer's patches in wild-type mice, allergic hypersensitivity was induced and Peyer's patches were examined. Exposure to mucosal allergen promoted marked accumulation of eosinophils in Peyer's patches and this process was attenuated in eotaxin-deficient mice. In summary, these data demonstrate that elevated levels of IL-5 and mucosal allergen exposure promote eotaxin-dependent eosinophil trafficking to Peyer's patches. These studies suggest that eosinophils may cooperate with lymphocytes in the development of mucosal immune responses in the gastrointestinal tract.
B waves: a systematic review of terminology, characteristics, and analysis methods
Authors: Martinez-Tejada Isabel , Arum Alexander , Wilhjelm Jens E. , Juhler Marianne , Andresen Morten
Published: October 2019

Background

Although B waves were introduced as a concept in the analysis of intracranial pressure (ICP) recordings nearly 60 years ago, there is still a lack consensus on precise definitions, terminology, amplitude, frequency or origin. Several competing terms exist, addressing either their probable physiological origin or their physical characteristics. To better understand B wave characteristics and ease their detection, a literature review was carried out.

Methods

A systematic review protocol including search strategy and eligibility criteria was prepared in advance. A literature search was carried out using PubMed/MEDLINE, with the following search terms: B waves + review filter, slow waves + review filter, ICP B waves, slow ICP waves, slow vasogenic waves, Lundberg B waves, MOCAIP.

Results

In total, 19 different terms were found, B waves being the most common. These terminologies appear to be interchangeable and seem to be used indiscriminately, with some papers using more than five different terms. Definitions and etiologies are still unclear, which makes systematic and standardized detection difficult.

Conclusions

Two future lines of action are available for automating macro-pattern identification in ICP signals: achieving strict agreement on morphological characteristics of “traditional” B waveforms, or starting a new with a fresh computerized approach for recognition of new clinically relevant patterns.
The experiences of lay health workers trained in task-shifting psychological interventions: a qualitative systematic review
Authors: Shahmalak Ujala , Blakemore Amy , Waheed Mohammad W. , Waheed Waquas
Published: October 2019

Introduction

The prevalence of common mental disorders, such as depression and anxiety, is high and the demand for psychological interventions and talking therapies is increasing. In order to meet this need, it is necessary to explore alternative methods to deliver talking therapies. Training lay health workers (LHWs) to deliver psychological interventions might be one possible solution to address current gaps in service provision. A number of studies have successfully used this approach to deliver psychological interventions in order to meet the demand for mental health care. Despite increased interest in this area, the evidence has not been synthesised or systematically reviewed.

Methods

Electronic databases (MEDLINE, EMBBASE, PsycINFO and CINHAL) were systematically searched to specifically capture studies on task-shifting psychological interventions for common mental disorders. Data were extracted on the experiences of the lay-workers on training and therapy delivery. Thematic analysis was used to analyse the data. Themes and subthemes of LHWs views on receiving training, barriers and facilitators to therapy delivery, factors required to become a successful therapist and the impact of training and therapy delivery on the therapists are described.

Results

10 studies were eligible for inclusion. Key messages were: LHWs were satisfied with training but wanted more robust supervision; not enough time was given to training on understanding mental health problems; LHWs grew in confidence and this impacted on their personal relationships with others.

Conclusion

This is the first review to explore LHWs experiences in training and therapy delivery by synthesising existing qualitative research. A number of key messages derived out of this review can help in further improving the quality of the training programmes and highlighting the benefits that are available for the LHW in delivering psychological interventions.
Aminoacyl tRNA synthetases as potential drug targets of the Panthera pathogen Babesia
Authors: Chhibber-Goel Jyoti , Joshi Sarthak , Sharma Amit
Published: October 2019

Background

A century ago, pantheras were abundant across Asia. Illegal hunting and trading along with loss of habitat have resulted in the designation ofPantheraas a genus of endangered species. In addition to the onslaught from humans, pantheras are also susceptible to outbreaks of several infectious diseases, including babesiosis. The latter is a hemoprotozoan disease whose causative agents are the eukaryotic parasites of the apicomplexan genusBabesia. Babesiosis affects a varied range of animals including humans (Homo sapiens), bovines (e.g.Bos taurus), pantheras (e.g.Panthera tigris,P. leo,P. pardus) and equines.Babesiaspp. are transmitted by the tick vectorIxodes scapularisor ticks of domestic animals, namelyRhipicephalus(Boophilus)microplusandR.(B.)decoloratus. At the level of protein translation within these organisms, the conserved aminoacyl tRNA synthetase (aaRS) family offers an opportunity to identify the sequence and structural differences in the host (Panthera) and parasites (Babesiaspp.) in order to exploit these for drug targetingBabesiaspp.

Methods

Using computational tools we investigated the genomes ofBabesiaspp. andPanthera tigrisso as to annotate their aaRSs. The sequences were analysed and their subcellular localizations were predicted using Target P1.1, SignalP 3.0, TMHMM v.2.0 and Deeploc 1.0 web servers. Structure-based analysis of the aaRSs fromP. tigrisand its protozoan pathogensBabesiaspp. was performed using Phyre2 and chimera.

Results

We identified 33 (B. bovis), 34 (B. microti), 33 (B. bigemina) and 33 (P. tigris) aaRSs in these respective organisms. Poor sequence identity (~ 20–50%) between aaRSs fromBabesiaspp. andP. tigriswas observed and this merits future experiments to validate new drug targets againstBabesiaspp.

Conclusions

Overall this work provides a foundation for experimental investigation of druggable aaRSs fromBabesiasp. in an effort to control Babesiosis inPanthera.
Plasminogen Activator Inhibitor-1 (PAI-1) deficiency predisposes to depression and resistance to treatments
Authors: Party Hélène , Dujarrier Cléo , Hébert Marie , Lenoir Sophie , Martinez de Lizarrondo Sara , Delépée Raphaël , Fauchon Claudine , Bouton Marie-Christine , Obiang Pauline , Godefroy Olivier , Save Etienne , Lecardeur Laurent , Chabry Joëlle , Vivien Denis , Agin Véronique
Published: October 2019

Major depressive disorder (MDD) is one of the most frequent psychiatric illnesses, leading to reduced quality of life, ability to work and sociability, thus ranking among the major causes of disability and morbidity worldwide. To date, genetic and environmental determinants of MDD remain mostly unknown. Here, we investigated whether and how the Plasminogen Activator Inhibitor-1 (PAI-1) may contribute to MDD. We first examined the phenotype of PAI-1 knockout (PAI-1−/−) and wild-type (PAI-1+/+) male mice with a range of behavioral tests assessing depressive-like behaviors (n = 276). We next investigated the mechanisms relating PAI-1 to MDD using molecular, biochemical and pharmacological analyzes. We demonstrate here that PAI-1 plays a key role in depression by a mechanism independent of the tissue-type Plasminogen Activator (tPA) – Brain-Derived Neurotrophic Factor (BDNF) axis, but associated with impaired metabolisms of serotonin and dopamine. Our data also reveal that PAI-1 interferes with therapeutic responses to selective serotonin reuptake inhibitors (escitalopram, fluoxetine). We thus highlight a new genetic preclinical model of depression, with the lack of PAI-1 as a factor of predisposition to MDD. Altogether, these original data reveal that PAI-1 should be now considered as a key player of MDD and as a potential target for the development of new drugs to cure depressive patients resistant to current treatments.