Interleukin-12 P40- and Fas Ligand-dependent Apoptotic Pathways Involving STAT-1 Phosphorylation Are Triggered During Infection with a Virulent Strain of Toxoplasma Gondii

Infection and Immunity. May, 2003  |  Pubmed ID: 12704131

Toxoplasma gondii is an opportunistic intracellular parasite. Infection with the high-virulence T. gondii strain RH induces inflammatory cytokine overproduction and uncontrolled apoptosis in lymphoid organs. Here, we show by fluorescent terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and binding of fluorescein isothiocyanate-conjugated VAD-FMK, an irreversible pan-caspase inhibitor, that parasite-triggered apoptosis occurs among CD4(+), CD8(+), B220(+), Gr-1(+), and NK1.1(+) splenic populations. Caspases 8 and 9 were activated during infection, implicating cell surface death receptors and mitochondria in apoptosis. Induction of apoptosis was absent among all cell populations in both interleukin-12 (IL-12) p40- and Fas ligand (FasL)-negative mice. STAT-1 phosphorylation correlated with onset of apoptosis during infection, but in the absence of IL-12 p40 and functional FasL, activation of this transcription factor failed to occur. The results demonstrate T. gondii-induced activation of multiple apoptotic pathways, dependent upon both IL-12 p40 and FasL, that may play a role in the lethal pathology of infection.

Apoptosis and the Balance of Homeostatic and Pathologic Responses to Protozoan Infection

Infection and Immunity. Nov, 2003  |  Pubmed ID: 14573625

STAT1 is Essential for Antimicrobial Effector Function but Dispensable for Gamma Interferon Production During Toxoplasma Gondii Infection

Infection and Immunity. Mar, 2004  |  Pubmed ID: 14977926

The opportunistic protozoan Toxoplasma gondii is a prototypic Th1-inducing pathogen inducing strong gamma interferon (IFN-gamma) cytokine responses that are required to survive infection. Intracellular signaling intermediate STAT1 mediates many effects of IFN-gamma and is implicated in activation of T-bet, a master regulator of Th1 differentiation. Here, we show that T. gondii-infected STAT1-null mice fail to upregulate the IFN-gamma-dependent effector molecules inducible nitric oxide synthase (iNOS), IGTP, and LRG-47, which are required for mice to survive infection. Both T-bet and interleukin-12 receptor beta2 (IL-12Rbeta2) failed to undergo normal upregulation in response to T. gondii. Development of IFN-gamma-producing CD4(+) and CD8(+) T lymphocytes was severely curtailed in the absence of STAT1, but a substantial level of STAT1-independent non-T-cell-derived IFN-gamma was induced. Absence of STAT1 also resulted in increased IL-4, Arg1, Ym1, and Fizz1, markers of Th2 differentiation and alternative macrophage activation. Together, the results show that T. gondii induces STAT1-dependent T-lymphocyte and STAT1-independent non-T-cell IFN-gamma production, but that effector functions of this type 1 cytokine cannot operate in the absence of STAT1, resulting in extreme susceptibility to acute infection.

AMP-activated Protein Kinase is Essential for Survival in Chronic Hypoxia

Biochemical and Biophysical Research Communications. May, 2008  |  Pubmed ID: 18359290

This study was undertaken to interrogate cancer cell survival during long-term hypoxic stress. Two systems with relevance to carcinogenesis were employed: Fully transformed BJ cells and a renal carcinoma cell line (786-0). The dynamic of AMPK activity was consistent with a prosurvival role during chronic hypoxia. This was further supported by the effects of AMPK agonists and antagonists (AICAR and compound C). Expression of a dominant-negative AMPK alpha resulted in a decreased ATP level and significantly compromised survival in hypoxia. Dose-dependent prosurvival effects of rapamycin were consistent with mTOR inhibition being a critical downstream mediator of AMPK in persistent low oxygen.

Murine Retroviral Bone Marrow Transplantation Models for the Study of Human Myeloproliferative Disorders

Current Protocols in Pharmacology / Editorial Board, S.J. Enna (editor-in-chief) ... [et Al.]. Dec, 2008  |  Pubmed ID: 22294220

Human myeloproliferative diseases are common hematologic disorders characterized by clonal overproduction of maturing myeloid or erythroid cells, often caused by expression of a mutant, dysregulated tyrosine kinase (TK). These diseases can be accurately modeled in laboratory mice by the retroviral transfer of a mutant TK gene into murine hematopoietic stem and progenitor cells, followed by transplantation of these cells into irradiated recipient mice. This yields a model system for analyzing the molecular pathophysiology of these conditions and provides a platform for testing therapies, particularly molecularly targeted new chemical entities (NCEs). The Basic Protocol in this unit describes the preparation of mouse bone marrow cells to express the relevant human oncogene before transplanting them into irradiated recipient mice. An alternate protocol describes a similar technique that allows specific induction of lymphoproliferative disease by some TKs. Support protocols for generating and titering retroviral stocks are also included. Curr. Protoc. Pharmacol. 43:14.10.1-14.10.28. © 2008 by John Wiley & Sons, Inc.

GCK is Essential to Systemic Inflammation and Pattern Recognition Receptor Signaling to JNK and P38

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2009  |  Pubmed ID: 19246396

Systemic inflammation arising from the organismal distribution of pathogen-associated molecular patterns is a major cause of clinical morbidity and mortality. Herein we report a critical and previously unrecognized in vivo role for germinal center kinase (GCK, genome nomenclature: map4k2), a mammalian Sterile 20 (STE20) orthologue, in PAMP signaling, and systemic inflammation. We find that disruption of gck in mice strongly impairs PAMP-stimulated macrophage cytokine and chemokine release and renders mice resistant to endotoxin-mediated lethality. Bone marrow transplantation studies show that hematopoietic cell GCK signaling is essential to systemic inflammation. Disruption of gck substantially reduces PAMP activation of macrophage Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) via reduced activation of the MAPK-kinase-kinases (MAP3Ks) mixed lineage kinases (MLKs)-2 and -3. Extracellular signal-regulated kinase (ERK) and nuclear factor-kappaB (NF-kappaB) activation are largely unaffected. Thus, GCK is an essential PAMP effector coupling JNK and p38, but not ERK or NF-kappaB to systemic inflammation.

Conformational Control Inhibition of the BCR-ABL1 Tyrosine Kinase, Including the Gatekeeper T315I Mutant, by the Switch-control Inhibitor DCC-2036

Cancer Cell. Apr, 2011  |  Pubmed ID: 21481795

Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for patients with chronic myeloid leukemia (CML). Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead "switch-control" inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML-resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1(T315I)-expressing cell lines, prolongs survival in mouse models of T315I mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph(+) leukemia.

Distinct Graft-versus-leukemic Stem Cell Effects of Early or Delayed Donor Leukocyte Infusions in a Mouse Chronic Myeloid Leukemia Model

Blood. Jan, 2012  |  Pubmed ID: 22072555

Among hematologic neoplasms, chronic myeloid leukemia (CML) is exquisitely sensitive to graft-versus-leukemia (GVL) because patients relapsing after allogeneic hematopoietic stem-cell transplantation (alloHSCT) can be cured by donor leukocyte infusion (DLI); however, the cellular mechanisms and strategies to separate GVL from GVHD are unclear. We used a BCR-ABL1 transduction/transplantation mouse model to study the mechanisms of DLI in MHC-matched, minor histocompatibility antigen-mismatched allogeneic chimeras with CML-like leukemia, in which DLI can be administered at the time of transplantation (early) or after recovery of hematopoiesis (delayed). After early DLI, CML-like leukemia cannot be transferred into immunocompetent secondary recipients as soon as 4 days after primary transplantation, demonstrating that cotransplantation of T lymphocytes blocks the engraftment of BCR-ABL1-transduced stem cells. In contrast, in allogeneic chimeras with established CML-like leukemia, combined treatment with delayed DLI and the kinase inhibitor imatinib eradicates leukemia with minimal GVHD. The GVL effect is directed against minor histocompatibility antigens shared by normal and leukemic stem cells, and is mediated predominantly by CD8+ T cells, with minor contributions from CD5- splenocytes, including natural killer cells. These results define a physiologic model of adoptive immunotherapy of CML that will be useful for investigating the cellular and molecular mechanisms of GVL.