[Show abstract][Hide abstract] ABSTRACT: Adoptive immunotherapy using natural killer (NK) cells has been a promising treatment for intractable malignancies; however, there remain a number of difficulties with respect to the shortage and limited anticancer potency of the effector cells. We here established a simple feeder-free method to generate purified (>90%) and highly activated NK cells from human peripheral blood-derived mononuclear cells (PBMCs). Among the several parameters, we found that simply 1) CD3-depletion, 2) high dose IL-2, and 3) use of a specific culture medium were sufficient to obtain highly purified, expanded (~200-fold) and activated CD3-/CD56+ NK cells from PBMCs that we named zenithal-NK cells (Z-NK). Almost all Z-NK cells expressed the lymphocyte-activated marker CD69, and showed dramatically high expression of activation receptors (i.e., NKG2D), interferon-γ, perforin and granzyme B. Importantly, only 2 hours of reaction at an effector/target ratio of 1:1 was sufficient to kill almost all K562 cells, and the antitumor activity was also replicated in tumor-bearing mice in vivo. Cytolysis was specific for various tumor cells, but not for normal cells, irrespective of MHC class I expression. These findings strongly indicate that Z-NK cells are purified, expanded, and near-fully activated human NK cells and warrant further investigation in a clinical setting.
Human Gene Therapy Methods 07/2013; · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malignant pleural mesothelioma (MPM) is highly intractable and readily spreads throughout the surface of the pleural cavity, and these cells have been shown to express urokinase-type plasminogen activator receptor (uPAR). We here examined the potential of our new and powerful recombinant Sendai virus (rSeV), which shows uPAR-specific cell-to-cell fusion activity (rSeV/dMFct14 (uPA2), named "BioKnife"), for tumor cell killing in two independent orthotopic xenograft models of human. Multicycle treatment using BioKnife resulted in the efficient rescue of these models, in association with tumor-specific fusion and apoptosis. Such an effect was also seen on both MSTO-211H and H226 cells in vitro; however, we confirmed that the latter expressed uPAR but not uPA. Of interest, infection with BioKnife strongly facilitated the uPA release from H226 cells, and this effect was completely abolished by use of either pyrrolidine dithiocarbamate (PDTC) or BioKnife expressing the C-terminus-deleted dominant negative inhibitor for retinoic acid-inducible gene-I (RIG-IC), indicating that BioKnife-dependent expression of uPA was mediated by the RIG-I/nuclear factor-κB (NF-κB) axis, detecting RNA viral genome replication. Therefore, these results suggest a proof of concept that the tumor cell-killing mechanism via BioKnife may have significant potential to treat patients with MPM that is characterized by frequent uPAR expression in a clinical setting.
[Show abstract][Hide abstract] ABSTRACT: Intestinal damage after ischemia followed by revascularization, referred to as "ischemia-reperfusion (I/R) injury," is a devastating complication that can occur after acute superior mesenteric obstruction, or after both elective and emergent abdominal aortic surgery. Once an entire layer of intestine is involved in severe ischemia, the mortality rate reaches 90%; no effective medical treatment has been reported to date. Here, we demonstrate that a somatostatin analogue, octreotide, but not a free-radical scavenger, MCI-186, prevented death due to surgically induced intestinal I/R injury in rats.
Superior mesenteric artery (SMA) of Male Sprague-Dawley rats, that received MCI-186 or octreotide, was surgically clamped, and then the clips were removed and SMA blood flow restored. Survival was assessed, and blood and small intestine were subjected to cell count, enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemistry.
Of interest, pretreatment with octreotide, but not with MCI-186, just before induced intestinal ischemia prompted the early expression of heme oxygenase-1 (HO-1) protein-associated accumulation of CD68-positive cells, a possible cellular source of HO-1. Inversely, the administration of tin protoporphyrin IX (SnPPN), a specific inhibitor of HO-1, completely abolished the therapeutic effects of octreotide, indicating that the favorable effects of octreotide against intestinal I/R injury is predominantly dependent on the early induction of HO-1.
These results suggest that a somatostatin analogue may be useful in leading to an improvement of the prognosis of patients with intestinal I/R injury in the clinical setting.
Journal of Surgical Research 04/2011; 175(2):350-8. · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) play a crucial role in maintaining the immune system. Though DC-based cancer immunotherapy has been suggested as a potential treatment for various kinds of malignancies, its clinical efficacies are still insufficient in many human trials. Issues that limit the clinical efficacy of DC-based immunotherapy, as well as the difficulty of the industrial production of DCs, are largely due to the limited number of autologous DCs available from each patient. We here established a possible breakthrough, a simple cytokine-based culture method to expand the log-scale order of functional human DCs. Floating cultivation of cord-blood CD34(+) cells under an optimized cytokine cocktail led these progenitor cells to stable log-scale proliferation and to DC differentiation. The expanded DCs had typical features of conventional myeloid DCs in vitro. Therefore, the concept of DC expansion should contribute significantly to the progress of DC immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma multiforme (GM), the most frequent primary malignant brain tumor, is highly invasive due to the expression of proteases, including urokinase-type plasminogen activator (uPA). Here, we show the potential of our new and powerful recombinant Sendai virus (rSeV) showing uPA-specific cell-to-cell fusion activity [rSeV/dMFct14 (uPA2), named "BioKnife"] for GM treatment, an effect that was synergistically enhanced by arming BioKnife with the interferon-β (IFN-β) gene. BioKnife killed human GM cell lines efficiently in a uPA-dependent fashion, and this killing was prevented by PA inhibitor-1. Rat gliosarcoma 9L cells expressing both uPA and its functional receptor uPAR (9L-L/R) exhibited high uPA activity on the cellular surface and were highly susceptible to BioKnife. Although parent 9L cells (9L-P) were resistant to BioKnife and to BioKnife expressing IFN-β (BioKnife-IFNβ), cell-cell fusion of 9L-L/R strongly facilitated the expression of IFN-β, and in turn, IFN-β significantly accelerated the fusion activity of BioKnife. A similar synergy was seen in a rat orthotopic brain GM model with 9L-L/R in vivo; therefore, these results suggest that BioKnife-IFNβ may have significant potential to improve the survival of GM patients in a clinical setting.