Development of a novel strategy to target CD39 antithrombotic activity to the endothelial-platelet microenvironment in kidney ischemia-reperfusion injury.

Kidney ischemia-reperfusion injury (IRI) is common during transplantation. IRI is characterised by inflammation and thrombosis and associated with acute and chronic graft dysfunction. P-selectin and its ligand PSGL-1 are cell adhesion molecules that control leukocyte-endothelial and leukocyte-platelet interactions under inflammatory conditions. CD39 is the dominant vascular nucleotidase that facilitates adenosine generation via extracellular ATP/ADP-phosphohydrolysis. Adenosine signalling is protective in renal IRI, but CD39 catalytic activity is lost with exposure to oxidant stress. We designed a P-selectin targeted CD39 molecule (rsol.CD39-PSGL-1) consisting of recombinant soluble CD39 that incorporates 20 residues of PSGL-1 that bind P-selectin. We hypothesised that rsol.CD39-PSGL-1 would maintain endothelial integrity by focusing the ectonucleotidase platelet-inhibitory activity and reducing leukocyte adhesion at the injury site. The rsol.CD39-PSGL-1 displayed ADPase activity and inhibited platelet aggregation ex vivo, as well as bound with high specificity to soluble P-selectin and platelet surface P-selectin. Importantly, mice injected with rsol.CD39-PSGL-1 and subjected to renal IRI showed significantly less kidney damage both biochemically and histologically, compared to those injected with solCD39. Furthermore, the equivalent dose of rsol.CD39-PSGL-1 had no effect on tail template bleeding times. Hence, targeting recombinant CD39 to the injured vessel wall via PSGL-1 binding resulted in substantial preservation of renal function and morphology after IRI without toxicity. These studies indicate potential translational importance to clinical transplantation and nephrology.

Evidence of platelet activation at medically used hypothermia and mechanistic data indicating ADP as a key mediator and therapeutic target.

OBJECTIVE: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. METHODS AND RESULTS: Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl(3)-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P(2)Y(12) antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. CONCLUSIONS: The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.

Inflammatory disease and lymphomagenesis caused by deletion of the Myc antagonist Mnt in T cells.

Mnt is a Max-interacting protein that can antagonize the activities of Myc oncoproteins in cultured cells. Mnt null mice die soon after birth, but conditional deletion of Mnt in breast epithelium leads to tumor formation. These and related data suggest that Mnt functions as a tumor suppressor. Here we show that conditional deletion of Mnt in T cells leads to tumor formation but also causes inflammatory disease. Deletion of Mnt caused increased apoptosis of thymic T cells and interfered with T-cell development yet led to spleen, liver, and lymph node enlargement. The proportion of T cells in the spleen and lymph nodes was reduced, and the numbers of cells in non-T-cell immune cell populations were elevated. The disruption of immune homeostasis is linked to a strong skewing toward production of T-helper 1 (Th1) cytokines and enhanced proliferation of activated Mnt-deficient CD4+ T cells. Consistent with Th1 polarization in vivo, extensive intestinal inflammation and liver necrosis developed. Finally, most mice lacking Mnt in T cells ultimately succumbed to T-cell lymphoma. These results strengthen the argument that Mnt functions as a tumor suppressor and reveal a critical and surprising role for Mnt in the regulation of T-cell development and in T-cell-dependent immune homeostasis.

Functions of myc:max in the control of cell proliferation and tumorigenesis.

Deregulation and elevated expression of members of the Myc family of bHLHZip transcription factors are observed in a high percentage of tumors. This close association with human cancers has led to a tremendous effort to define their biological and biochemical activities. Although Myc family proteins have the capacity to elicit a wide range of cell behaviors, their principal function appears to be to drive cells into the cell cycle and to keep them there. However, forced expression of Myc profoundly sensitizes normal cells to apoptosis. Therefore, tumor formation caused by deregulated Myc expression requires cooperating events that disrupt pathways that mediate apoptosis. Myc-dependent tumor formation may also be impeded by a set of related bHLHZip proteins with the demonstrated potential to act as Myc antagonists in cell culture experiments. In this review, we examine the complex activities of Myc family proteins and how their actions might be regulated in the context of a network of bHLHZip proteins.

Enhancing CTL responses to melanoma cell vaccines in vivo: synergistic increases obtained using IFNgamma primed and IFNbeta treated B7-1+ B16-F10 melanoma cells.

Sequentially treating human melanoma cell lines by priming with interferon-gamma before adding interferon-beta was previously found to be the most efficient protocol for producing concurrently increased expression of the three surface antigens B7-1, intercellular adhesion molecule-1 and human histocompatibility leucocyte antigens Class I. The present study describes similar outcomes when the same sequential intercellular adhesion molecule-based protocol is applied to murine B16-F10 melanoma cells as well as preclinical studies using the B16-F10 model as a poorly immunogenic melanoma. Thus, treating B16-F10 cells or a highly expressing B7-1 transfected subline (B16-F10/B7-1 hi) by priming with interferon-gamma for 24 h before adding interferon-beta for a further 48 h (interferon-gamma 72/beta 48) increased expression of all three surface antigens, particularly major histocompatibility complex class I whose increased expression was sustained for several days. As a whole tumour cell vaccine, interferon-gamma 72/beta 48 treated B16-F10 cells produced greater levels of cytoxic T lymphocyte response compared to vaccines prepared from cells treated with a single type of interferon. Furthermore, B16-F10 cells expressing high levels of B7-1 and treated using the interferon-gamma 72/beta 48 protocol (interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi) produced substantially increased cytoxic T lymphocyte responses with a fivefold greater synergy than the combined results of either interferon treated or B7-1 expressing cells tested individually. The resulting CD8+ cytoxic T lymphocyte showed greater specificity for B16-F10 cells with tenfold higher killing than for syngeneic EL-4 lymphoma cells. Killing proceeded via the perforin-mediated pathway. CTL responses were induced independent of CD4+ T helper cells. The majority of mice receiving interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi vaccine in vivo remained tumour free after challenge with 5 x 105 live B16-F10 cells expressing intermediate B7-1 levels. The novel strategy described will help enhance vaccine potency when applied clinically to prepare whole cell based cancer vaccine therapies.