Phase I/IIa Feasibility Trial of Autologous Quality- and Quantity-Cultured Peripheral Blood Mononuclear Cell Therapy for Non-Healing Extremity Ulcers.

Non-healing wounds are among the main causes of morbidity and mortality. We recently described a novel, serum-free ex vivo expansion system, the quantity and quality culture system (QQc), which uses peripheral blood mononuclear cells (PBMNCs) for effective and noninvasive regeneration of tissue and vasculature in murine and porcine models. In this prospective clinical study, we investigated the safety and efficacy of QQ-cultured peripheral blood mononuclear cell (MNC-QQ) therapy for chronic non-healing ischemic extremity wounds. Peripheral blood was collected from 9 patients with 10 chronic (>1 month) non-healing wounds (8 males, 1 female; 64-74 years) corresponding to ischemic extremity ulcers. PBMNCs were isolated and cultured using QQc. Within a 20-cm area surrounding the ulcer, 2 × 107 cells were injected under local anesthesia. Wound healing was monitored photometrically every 2 weeks. The primary endpoint was safety, whereas the secondary endpoint was efficacy at 12-week post-injection. All patients remained ambulant, and no deaths, other serious adverse events, or major amputations were observed for 12 weeks after cell transplantation. Six of the 10 cases showed complete wound closure with an average wound closure rate of 73.2% ± 40.1% at 12 weeks. MNC-QQ therapy increased vascular perfusion, skin perfusion pressure, and decreased pain intensity in all patients. These results indicate the feasibility and safety of MNC-QQ therapy in patients with chronic non-healing ischemic extremity wounds. As the therapy involves transplanting highly vasculogenic cells obtained from a small blood sample, it may be an effective and highly vasculogenic strategy for limb salvage.

Quality and Quantity-Cultured Human Mononuclear Cells Improve Human Fat Graft Vascularization and Survival in an In Vivo Murine Experimental Model.

BACKGROUND: Fat graft ischemia impedes us from having satisfying long-term results. The quality and quantity culture is a 1-week cell culture that increases the vasculogenic potential of peripheral blood mononuclear cells (PBMNC). This in vivo murine model investigates whether enrichment with quality and quantity-cultured human mononuclear cells (MNC-QQ) improves the vascularization in the human fat graft and whether this decreases the tissue loss. METHODS: Human adipose tissue, PBMNC, MNC-QQ, and stromal vascular fraction were prepared. First, PBMNC, MNC-QQ, and stromal vascular fraction were compared in vitro for vasculogenic potential by endothelial progenitor cell colony-forming and culture assays. Second, 0.25-g fat grafts were created with 1 × 106 PBMNC (n = 16), 1 × 106 MNC-QQ (n = 16), 1 × 106 stromal vascular fraction (n = 16), or phosphate-buffered saline as control (n = 16) before grafting in BALB/c nude mice. Grafts were analyzed for weight persistence, vessel formation by CD31 immunohistochemistry, and angiogenic markers by quantitative polymerase chain reaction. RESULTS: MNC-QQ develop more definitive endothelial progenitor cell colonies and more functional endothelial progenitor cells compared to PBMNC and stromal vascular fraction. Weight persistence after 7 weeks was significantly higher in grafts with MNC-QQ (89.8 ± 3.5 percent) or stromal vascular fraction (90.1 ± 4.2 percent) compared with control (70.4 ± 6.3 percent; p < 0.05). MNC-QQ-enriched grafts had the highest vessel density (96.6 ± 6.5 vessels/mm2; control, 70.4 ± 5.6 vessels/mm2; p < 0.05). MNC-QQ exerted a direct vasculogenic effect through vascular integration and a potential paracrine vascular endothelial growth factor-mediated effect. CONCLUSION: Quality and quantity-cultured human mononuclear cells containing endothelial progenitor cells stimulate fat graft vascularization and enhance graft survival in a rodent recipient.

Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model.

The transplantation of endothelial progenitor cells (EPCs) is used to promote wound angiogenesis. In patients with chronic wounds and accompanying morbidities, EPCs are often compromised in number and function. To overcome these limitations, we previously developed a quality and quantity controlled (QQ) culture system to enrich peripheral blood mononuclear cells (PBMNCs) in EPCs. To evaluate the wound healing efficacy of mononuclear cells (MNCs) harvested after QQ culture (QQMNCs), preclinical studies were performed on large animals. MNCs harvested from the blood of healthy human subjects were cultured in the presence of angiogenic cytokines and growth factors in a serum-free medium for 7 days. A total of 5 × 106 QQMNCs per full-thickness skin defect or control saline was injected into wounds induced in cyclosporine-immunosuppressed pigs. EPC colony-forming assays revealed a significantly higher number of definitive (partially differentiated) EPC colony-forming units in QQMNCs. Flow cytometry evaluation of QQMNC surface markers showed enrichment of CD34+ and CD133+ stem cell populations, significant reduction in CCR2+ cell percentages, and a greater than 10-fold increase in the percentage of anti-inflammatory M2-type macrophages (CD206+ cells) compared with PBMNCs. Wounds treated with QQMNCs had a significantly higher closure rate. Wounds were harvested, frozen, and sectioned at day 21 postoperatively. Hematoxylin and eosin staining revealed that the epithelization of QQMNC-treated wounds was more advanced than in controls. Treated wounds developed granulation tissue with more mature collagen and larger capillary networks. CD31 and human mitochondrial co-staining confirmed the presence of differentiated human cells within newly formed vessels. Real-time polymerase chain reaction (PCR) showed upregulation of interleukin 6 (IL-6), IL-10, and IL-4 in the wound bed, suggesting paracrine activity of the transplanted QQMNCs. Our data demonstrate for the first time that QQ culture of MNCs obtained from a small amount of peripheral blood yields vasculogenic and therapeutic cells effective in wound healing.

Spontaneous regression of essential thrombocythemia with MPL mutation on menopause.

Essential thrombocythemia (ET) is a subtype of myeloproliferative neoplasms. Approximately half of the patients with ET harbor a gain-of-function mutation in the JAK2 gene (JAK2-V617F), a small percentage have mutations in codon 515 of MPL (thrombopoietin receptor) gene, and the rest have neither mutation. Pregnancy is a rare complication of ET, and it has been reported that the number of blood platelets falls with pregnancy in ET patients and the number of blood platelets increases again after a delivery and this phenomenon is observed in JAK2-V617F-positive and JAK2-V617F-negative patients. We report the first case of an ET patient with MPL mutations, whose platelet count improved with the onset of menopause, not pregnancy, and the MPL mutation also simultaneously disappeared.

The Effect of Control-released Basic Fibroblast Growth Factor in Wound Healing: Histological Analyses and Clinical Application.

BACKGROUND: Basic fibroblast growth factors (bFGFs) play a crucial role in wound healing by promoting fibroblast proliferation and neovascularization. However, drawback of bFGF is short half-life in free form. Gelatin has a capability of sustaining growth factors, which are gradually released while degradation. The purpose of this study is to see whether bFGF-impregnated gelatin sheet is effective in a murine model and whether it could also be available for patients in a safe manner. METHODS: Full-thickness skin defect was created on C57BL/6J mice and covered with bFGF with gelatin sheet (group A), bFGF without gelatin sheet (group B), phosphate buffer saline (PBS) with gelatin sheet (group C), and only PBS (group D). Wound healing was evaluated in terms of percent wound closure, granulation thickness, wound maturity, and vascular density. Clinical trial was conducted for patients who received either acute or chronic ulcers. The sheets were put onto the wounds and covered by hydrocolloid dressing, which was changed weekly. RESULTS: Groups A and B exhibited better wound healing than groups C and D in all aspects. Moreover, group A showed better results than group B at day 7 in terms of wound closure, collagen maturity, and vascularity. Efficacy without any adverse events was found in the clinical series. CONCLUSIONS: These findings suggest that control-released bFGF using gelatin sheet is effective for promoting wound healing. Such therapeutic strategy was considered to offer several clinical advantages including rapid healing and reduction of the dressing change with less patient discomfort.

Expression of the apoptosis inducer gene head involution defective in primordial germ cells of the Drosophila embryo requires eiger, p53, and loki function.

Nanos (Nos) is an evolutionarily conserved protein essential for the maintenance of primordial germ cells (PGCs). In Drosophila, the PGCs or pole cells express head involution defective (hid), which is required for caspase activation, but its translation is repressed by maternal Nos. In the absence of Nos activity, translation of hid mRNA into protein induces apoptosis in pole cells. However, it remains unclear how hid mRNA is regulated in pole cells. Here, we report that hid expression requires eiger (egr), a tumor necrosis factor ligand (TNF) homologue, which is induced in pole cells by decapentaplegic (dpp). In addition, we demonstrate that p53 and loki (lok), a damage-activated kinase known to be required for p53 phosphorylation, are both required for hid expression in pole cells. Since maternal lok mRNA is enriched in pole cells, it is possible that ubiquitously distributed p53 is activated in pole cells by maternal Lok. We propose that hid expression is activated in a pole cell-specific manner by loki/p53 and dpp/egr during embryogenesis.

Expression of meiotic genes in the germline progenitors of Drosophila embryos.

Meiosis is one of the fundamental characteristics of germ cells. In Drosophila, genetic screens have identified many genes required for meiotic division. However, it remains elusive as to when and how these meiotic genes are activated during germline development. To obtain insights into their regulatory mechanisms, we examined the expression of 38 meiotic genes in the germline progenitors, pole cells, during embryogenesis. We found that the transcripts of 12 meiotic genes were enriched in pole cells within the embryonic gonads. Among them, bag of marbles (bam), benign gonial cell neoplasia (bgcn), deadhead (dhd), matotopetli (topi) and twine (twe) were activated only in pole cells within the gonads, whereas the transcripts from grapes (grp), Kinesin-like protein at 3A (Klp3A), pavarotti (pav), lesswright (lwr), mei-P26, Topoisomerase 2 (Top2) and out at first (oaf) were distributed ubiquitously in early embryos and then became restricted to pole cells and to a subset of somatic tissues at later embryonic stages. The remaining meiotic genes were either expressed ubiquitously in the embryos (15 genes) or were undetectable in pole cells within the gonads (11 genes). These observations suggest that pole cells have already acquired the potential to express several meiotic genes. Our data will thus provide a useful basis for analyzing how the germline acquires a potential to execute meiosis.

Mechanisms of enhanced apoptosis in HL-60 cells by UV-irradiated n-3 and n-6 polyunsaturated fatty acids.

We examined the effects of arachidonic acid (AA), eicosapentaenoic acid (EPA), and their ultraviolet (UV)-irradiated products on HL-60 cells and isolated mitochondria to explore the following four obscure points in the mechanism of polyunsaturated fatty acids (PUFAs)-induced apoptosis: (i). the role of reactive oxygen species, (ii). the interaction of PUFAs and their metabolites with mitochondria in situ, (iii). the cyclosporine A (CsA)-sensitivity in PUFA-induced membrane permeability transition, (iv). the specificity of oxidized n-3 PUFAs in the induction of apoptosis in cancer cells. UV-oxidized PUFAs contained conjugated dienes and thiobarbituric acid reactive substances (TBARS). The apoptotic effects of PUFAs on HL-60 cells were increased by UV-irradiation whereas the swelling effect of PUFAs on isolated mitochondria was decreased. Both oxidized n-3 and n-6 PUFAs induced increased depolarization, ferricytochrome c release, the activation of various caspases, and DNA-fragmentation in a CsA-insensitive mechanism concomitant with a slight increase in the value of TBARS in cells. Furthermore, there were no significant differences in the mechanism of apoptosis induced by either oxidized AA or oxidized EPA. On the basis of these results, it was concluded that both oxidized n-3 or n-6 PUFAs induced apoptosis in HL-60 cells by a similar mechanism in a CsA-insensitive manner and also that oxidized products of PUFAs, but not the cellular oxidation process itself, play an important role in the mechanism of apoptosis in HL-60 cells.

The effects of immunosuppressants on FAS-mediated activation-induced cell death in human T lymphocytes.

The effects of cyclosporin A (CsA) and methylprednisolone (MP) on Fas-mediated activation-induced cell death (FMAICD) of T lymphocytes were examined. T lymphocytes were activated with the immobilized anti-CD 3 and CD 28 monoclonal antibodies (MoAbs) (activation phase) and incubated further with the agonistic MoAb against Fas (death phase). Cell proliferation and DNA fragmentation were measured by XTT and diphenylamine assay. CsA in the activation phase inhibited DNA fragmentation mediated by anti-Fas MoAb but not MP. The combination of CsA and MP at the lower concentrations had little effect on FMAICD, although they had similar degrees of suppression on T lymphocyte proliferation as the maximum obtained by CsA or MP alone. In the death phase, MP induced apoptosis without 7C11 and CsA had no effects. These results indicate that the combination of CsA and MP at low concentrations could maintain FMAICD with the suppression on T lymphocyte proliferation.

Cholesteryl-hemisuccinate-induced apoptosis of promyelocytic leukemia HL-60 cells through a cyclosporin A-insensitive mechanism.

We reported previously that alpha-tocopheryl-succinate (VES) induced apoptosis of cultured human promyelocytic leukemia cells (HL-60) (Free Radic Res 2000;33:407-18). We have now studied the effect of cholesteryl-hemisuccinate (CS) on the fate of HL-60 cells to clarify whether CS has an effect similar to that of VES. CS inhibited the growth of HL-60 cells without differentiation to granulocytes and induced DNA fragmentation and ladder formation. CS inhibited the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt) and initiated the activation of a caspase cascade. CS triggered the reaction leading to the cleavage of Bid and also released cytochrome c (Cyt. c) from mitochondria. In addition, CS induced mitochondrial membrane depolarization and translocation of Bax to mitochondria in HL-60 cells. However, CS did not induce an increase in the concentration of intracellular calcium ions in HL-60 cells. The membrane depolarization, Cyt. c release, and DNA fragmentation were inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor, but not by cyclosporin A, an inhibitor of membrane permeability transition. These results suggested that CS-induced apoptosis of HL-60 cells might be caused by inhibiting Akt phosphorylation following cleavage of Bid through caspase-8 activation and subsequently via an Apaf complex-caspase cascade pathway.