Phase I Study: Safety and Efficacy of an Ex Vivo-Expanded Allogeneic Natural Killer Cell (MG4101) with Rituximab for Relapsed/Refractory B Cell Non-Hodgkin Lymphoma.

The prognosis of non-Hodgkin lymphoma (NHL) remains poor, with an unmet need for novel therapies. MG4101, an ex vivo-expanded allogeneic natural killer (NK) cell, can enhance rituximab antibody-dependent cytotoxicity in relapsed/refractory (r/r) B cell non-Hodgkin lymphoma. This study assessed the safety and efficacy of MG4101 plus rituximab for patients with r/r NHL. Patients received escalating doses of i.v. MG4101 plus rituximab every 2 weeks. IL-2 was administered s.c. after MG4101 treatment. Fludarabine plus cyclophosphamide was administered i.v. before rituximab treatment in cycles 1, 3, and 5. A 3+3 design was used to determine the maximum tolerated dose (MTD) and maximum feasible dose. Assessments were performed over a 6-cycle period, with an extended maintenance period of up to 8 cycles. Nine patients received 3 different doses of MG4101 and rituximab. MTD could not be determined because of the absence of dose-limiting toxicity. Treatment-related adverse events, mostly grade 1 or 2, occurred in 89% of patients. Only 1 patient experienced grade 1 cytokine release syndrome. MG4101 persisted for at least 7 days in 7 patients. Four patients achieved a partial response and 1 patient attained a complete response, for an overall response rate of 55.6%. Two patients showed prolonged responses and low exhaustion marker levels in T cells. For allogeneic NK cell therapy, strategies including the use of the high-affinity hFcγRIIIaV158 variant of the KIR B/x haplotype with lymphodepleting chemotherapy may be promising options for improving clinical efficacy in the antibody combination therapeutic setting as an off-the-shelf product. MG4101 plus rituximab presented a favorable safety profile and overall response rate in patients with r/r NHL.

Tafasitamab mediates killing of B-cell non-Hodgkin's lymphoma in combination with γδ T cell or allogeneic NK cell therapy.

Tafasitamab is an Fc-modified monoclonal antibody that binds to CD19, a cell-surface antigen that is broadly expressed on various types of B-cell non-Hodgkin's lymphoma (NHL). Antibody-dependent cellular cytotoxicity (ADCC), a key mode of action of tafasitamab, is mediated through the binding of tafasitamab's Fc region to FcγRIIIa receptors on immune effector cells and results in antitumor activity. Despite the proven clinical activity of tafasitamab in combination with lenalidomide in the treatment of diffuse large B-cell lymphoma (DLBCL), a higher number of immune cells in cancer patients may improve the activity of tafasitamab. Here, we characterized two ex vivo-expanded FcγRIIIa receptor-expressing cell types-γδ T and MG4101 natural killer (NK) cells-as effector cells for tafasitamab in vitro, and found that in the presence of these cells tafasitamab was able to induce ADCC against a range of NHL cell lines and patient-derived cells. We also explored the concept of effector cell supplementation during tafasitamab treatment in vivo by coadministering MG4101 NK cells in Raji and Ramos xenograft models of NHL. Combination treatment of tafasitamab and allogeneic MG4101 NK cells in these models demonstrated a survival benefit compared with tafasitamab or MG4101 monotherapy (Raji: 1.7- to 1.9-fold increase in lifespan; Ramos: 2.0- to 4.1-fold increase in lifespan). In conclusion, adoptive cell transfer of ex vivo-expanded allogeneic NK or autologous γδ T cells in combination with tafasitamab treatment may potentially be a promising novel approach to increase the number of immune effector cells and enhance the antitumor effect of tafasitamab.

Abnormal Development of Neural Stem Cell Niche in the Dentate Gyrus of Menkes Disease.

Background and Objectives: Menkes disease (MNK) is a rare X-linked recessive disease, caused by mutations in the copper transporting ATP7A gene that is required for copper homeostasis. MNK patients experience various clinical symptoms including neurological defects that are closely related to the prognosis of MNK patients. Neural stem cells (NSCs) in the hippocampal dentate gyrus (DG) produce new neurons throughout life, and defects in DG neurogenesis are often correlated with cognitive and behavioral problems. However, neurodevelopmental defects in the DG during postnatal period in MNK have not been understood yet. Methods and Results: Mottled-brindled (MoBr/y) mice (MNK mice) and littermate controls were used in this study. In vivo microCT imaging and immunohistochemistry results demonstrate that blood vasculatures in hippocampus are abnormally decreased in MNK mice. Furthermore, postnatal establishment of NSC population and their neurogenesis are severely compromised in the DG of MNK mice. In addition, in vitro analyses using hippocampal neurosphere culture followed by immunocytochemistry and immunoblotting suggest that neurogenesis from MNK NSCs is also significantly compromised, corresponding to defective neurogenic gene expression in MNK derived neurons. Conclusions: Our study is the first reports demonstrating that improper expansion of the postnatal NSC population followed by significant reduction of neurogenesis may contribute to neurodevelopmental symptoms in MNK. In conclusion, our results provide new insight into early neurodevelopmental defects in MNK and emphasize the needs for early diagnosis and new therapeutic strategies in the postnatal central nerve system damage of MNK patients.

Therapeutic Effect of IL1ß Priming Tonsil Derived-Mesenchymal Stem Cells in Osteoporosis.

BACKGROUND: Stem cell therapies can be a new therapeutic strategy that may rebalance anabolic and anti-resorptive effects in osteoporosis patients. Tonsil-derived mesenchymal stem cells (TMSCs) can be an alternative therapeutic source for chronic degenerative diseases including osteoporosis. MSCs acquire immune regulatory function under the inflammatory cytokines. Since interleukin (IL) 1ß is known to be one of inflammatory cytokines involved in osteoporosis progression, treatment of IL1ß with TMSCs may enhance immunomodulatory function and therapeutic effects of TMSCs in osteoporosis. METHODS: For IL1ß priming, TMSCs were cultured in the presence of the medium containing IL1ß for 1 day. Characteristics of IL1ß priming TMSCs such as multipotent differentiation properties, anti-inflammatory potential, and suppression of osteoclast differentiation were assessed in vitro. For in vivo efficacy study, IL1ß priming TMSCs were intravenously infused twice with ovariectomized (OVX) osteoporosis mouse model, and blood serum and bone parameters from micro computed tomography images were analyzed. RESULTS: IL1ß priming TMSCs had an enhanced osteogenic differentiation and secreted factors that regulate both osteoclastogenesis and osteoblastogenesis. IL1ß priming TMSCs also suppressed proliferation of peripheral blood mononuclear cells (PBMCs) and decreased expression of Receptor activator of nuclear factor kappa-Β ligand (RANKL) in PHA-stimulated PBMCs. Furthermore, osteoclast specific genes such as Nuclear factor of activated T cells c1 (NFATc1) were effectively down regulated when co-cultured with IL1ß priming TMSCs in RANKL induced osteoclasts. In OVX mice, IL1ß priming TMSCs induced low level of serum RANKL/osteoprotegerin (OPG) ratio on the first day of the last administration. Four weeks after the last administration, bone mineral density and serum Gla-osteocalcin were increased in IL1ß priming TMSC-treated OVX mice. Furthermore, bone formation and bone resorption markers that had been decreased in OVX mice with low calcium diet were recovered by infusion of IL1ß priming TMSCs. CONCLUSION: IL1ß priming can endow constant therapeutic efficacy with TMSCs, which may contribute to improve bone density and maintain bone homeostasis in postmenopausal osteoporosis. Therefore, IL1ß priming TMSCs can be a new therapeutic option for treating postmenopausal osteoporosis.

Adjuvant therapy using ex vivo-expanded allogenic natural killer cells in hepatectomy patients with hepatitis B virus related solitary hepatocellular carcinoma: MG4101 study.

BACKGROUNDS/AIMS: Fewer reports have been published regarding hepatectomy patients with solitary hepatocellular carcinoma (HCC) who received immunotherapeutic agents as adjuvant therapy. We evaluated the safety and efficacy of ex vivo-expanded allogenic natural killer (NK) cells in those patients with modified International Union Against Cancer (UICC) stage T3. METHODS: From August 2014 to October 2015, five patients who underwent hepatic resection received ex vivo-expanded allogenic NK cells. Patients received five rounds of NK cells (2-3×107 cells/kg) at postoperative 4, 6, 8, 12, and 16 weeks. This study is registered with ClinicalTrials.gov, number NCT02008929. RESULTS: The median age of the five patients (three men and two women) was 44.8 years (range, 36-54 years). All had hepatitis B virus-related HCC, and the median tumor size was 2.2 cm (range, 2.1-8.2 cm). None of the patients had any adverse events. HCC recurrence developed in two patients at one year after hepatic resection, but four patients were alive at 3 years. The two recurrence-free patients showed a higher ratio of CD8+ T lymphocyte populations before and after administration of ex vivo-expanded allogenic NK cells compared with the three patients who experienced recurrence. CONCLUSIONS: Immunotherapy using ex vivo-expanded allogenic NK cells in hepatectomy patients can be used safely. Further studies should be investigated for efficacy.

Erratum: Oh, E., et al. Cryopreserved Human Natural Killer Cells Exhibit Potent Antitumor Efficacy against Orthotopic Pancreatic Cancer through Efficient Tumor-Homing and Cytolytic Ability (Running Title: Cryopreserved NK Cells Exhibit Antitumor Effect). Cancers 2019, 11, 966.

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Safety and immune cell kinetics after donor natural killer cell infusion following haploidentical stem cell transplantation in children with recurrent neuroblastoma.

INTRODUCTION: Under the hypothesis that early natural killer cell infusion (NKI) following haploidentical stem cell transplantation (haplo-SCT) will reduce relapse in the early post-transplant period, we conducted a pilot study to evaluate the safety and feasibility of NKI following haplo-SCT in children with recurrent neuroblastoma who failed previous tandem high-dose chemotherapy and autologous SCT. METHODS: We used the high-dose 131I-metaiodobenzylguanidine and cyclophosphamide/fludarabine/anti-thymocyte globulin regimen for conditioning and infused 3 × 107/kg of ex-vivo expanded NK cells derived from a haploidentical parent donor on days 2, 9, and 16 post-transplant. Interleukin-2 was administered (1 × 106 IU/m2/day) subcutaneously to activate infused donor NK cells on days 2, 4, 6, 9, 11, 13, 16, 18, and 20 post-transplant. RESULTS: Seven children received a total of 19 NKIs, and NKI-related acute toxicities were fever (n = 4) followed by chills (n = 3) and hypertension (n = 3); all toxicities were tolerable. Grade ≥II acute GVHD and chronic GVHD developed in two and five patients, respectively. Higher amount of NK cell population was detected in peripheral blood until 60 days post-transplant than that in the reference cohort. Cytomegalovirus and BK virus reactivation occurred in all patients and Epstein-Barr virus in six patients. Six patients died of relapse/progression (n = 5) or treatment-related mortality (n = 1), and one patient remained alive. CONCLUSION: NKI following haplo-SCT was relatively safe and feasible in patients with recurrent neuroblastoma. Further studies to enhance the graft-versus-tumor effect without increasing GVHD are needed.

Anti­photoaging and anti­oxidative activities of natural killer cell conditioned medium following UV­B irradiation of human dermal fibroblasts and a reconstructed skin model.

Conditioned media from various sources comprise numerous growth factors and cytokines and are known to promote the regeneration of damaged tissues. Among these, natural killer cell conditioned medium (NK­CdM) has been shown to stimulate collagen synthesis and the migration of fibroblasts during the wound healing process. With a long­term aim of developing a treatment for skin photoaging, the ability of NK­CdM to prevent ultraviolet­B (UV­B) damage was assessed in neonatal human dermal fibroblasts (NHDFs) and an in vitro reconstructed skin model. The factors present in NK­CdM were profiled using an antibody array analysis. Protein and mRNA levels in UV­B exposed NHDFs treated with NK­CdM were measured by western blotting and quantitative reverse transcription­PCR, respectively. The total antioxidant capacity of NK­CdM was determined to assess its ability to suppress reactive oxygen species. The anti­photoaging effect of NK­CdM was also assessed in a 3D reconstituted human full skin model. NK­CdM induced proliferation of UV­B­treated NHDFs, increased procollagen expression, and decreased matrix metalloproteinase (MMP)­1 expression. NK­CdM also exhibited a potent antioxidant activity as measured by the total antioxidant capacity. NK­CdM inhibited UV­B­induced collagen degradation by inactivating MAPK signaling. NK­CdM also elicited potential anti­wrinkle effects by inhibiting the UV­B­induced increase in MMP­1 expression levels in a 3D reconstituted human full skin model. Taken together, the suppression of both UV­B­induced MMP­1 expression and JNK activation by NK­CdM suggests NK­CdM as a possible candidate anti­skin aging agent.

Cryopreserved Human Natural Killer Cells Exhibit Potent Antitumor Efficacy against Orthotopic Pancreatic Cancer through Efficient Tumor-Homing and Cytolytic Ability (Running Title: Cryopreserved NK Cells Exhibit Antitumor Effect).

Pancreatic cancer is known to be highly aggressive, and desmoplasia-induced accumulation of extracellular matrix (ECM), which is a hallmark of many pancreatic cancers, severely restricts the therapeutic efficacy of both immunotherapeutics and conventional chemotherapeutics due to the ECM functioning as a major physical barrier against permeation and penetration. In the case of cell-based immunotherapeutics, there are several other bottlenecks preventing translation into clinical use due to their biological nature; for example, poor availability of cell therapeutic in a readily usable form due to difficulties in production, handling, shipping, and storage. To address these challenges, we have isolated allogeneic natural killer (NK) cells from healthy donors and expanded them in vitro to generate cryopreserved stocks. These cryopreserved NK cells were thawed to evaluate their therapeutic efficacy against desmoplastic pancreatic tumors, ultimately aiming to develop a readily accessible and mass-producible off-the-shelf cell-based immunotherapeutic. The cultured NK cells post-thawing retained highly pure populations of activated NK cells that expressed various activating receptors and a chemokine receptor. Furthermore, systemic administration of NK cells induced greater in vivo tumor growth suppression when compared with gemcitabine, which is the standard chemotherapeutic used for pancreatic cancer treatment. The potent antitumor effect of NK cells was mediated by efficient tumor-homing ability and infiltration into desmoplastic tumor tissues. Moreover, the infiltration of NK cells led to strong induction of apoptosis, elevated expression of the antitumor cytokine interferon (IFN)-γ, and inhibited expression of the immunosuppressive transforming growth factor (TGF)-ß in tumor tissues. Expanded and cryopreserved NK cells are strong candidates for future cell-mediated systemic immunotherapy against pancreatic cancer.

Optimization of Large-Scale Expansion and Cryopreservation of Human Natural Killer Cells for Anti-Tumor Therapy.

Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD3+ T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. In vivo antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide 'ready-to-use' cell therapy for cancer patients.

Phase I Study of Random Healthy Donor-Derived Allogeneic Natural Killer Cell Therapy in Patients with Malignant Lymphoma or Advanced Solid Tumors.

Natural killer (NK) cells with mismatched killer cell immunoglobulin-like receptor-ligand pairs have shown efficacy and been proven safe in treatment of cancer patients. Ex vivo-expanded and highly activated NK cells (MG4101) had been generated under good manufacturing practice conditions, which demonstrated potent anticancer activity in vitro and in vivo in preclinical studies. The current phase I clinical trial was designed to evaluate safety and possible clinical efficacy of repetitive administrations of MG4101 derived from random unrelated healthy donors into patients with malignant lymphoma or advanced, recurrent solid tumors. The maximum dose (3 × 10(7) cells/kg, triple infusion) was tolerable without significant adverse events. Of 17 evaluable patients, 8 patients (47.1%) showed stable disease and 9 (52.9%) showed progressive disease. We also evaluated the capacity of MG4101 to influence host immune responses. Administration of MG4101 augmented NKG2D expression on CD8(+) T cells and upregulated chemokines that recruit T cells. In contrast, administration of MG4101 reduced regulatory T cells and myeloid-derived suppressor cells and suppressed TGFß production. In conclusion, administration of a large number of MG4101 cells was not only safe and feasible, but also exhibited efficacy in maintaining the effector arm of the host immune response.

Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain. METHODS: Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgamma(null) (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro. RESULTS: Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects. CONCLUSIONS: Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.

Present and Future of Allogeneic Natural Killer Cell Therapy.

Natural killer (NK) cells are innate lymphocytes that are capable of eliminating tumor cells and are therefore used for cancer therapy. Although many early investigators used autologous NK cells, including lymphokine-activated killer cells, the clinical efficacies were not satisfactory. Meanwhile, human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation revealed the antitumor effect of allogeneic NK cells, and HLA-haploidentical, killer cell immunoglobulin-like receptor ligand-mismatched allogeneic NK cells are currently used for many protocols requiring NK cells. Moreover, allogeneic NK cells from non-HLA-related healthy donors have been recently used in cancer therapy. The use of allogeneic NK cells from non-HLA-related healthy donors allows the selection of donor NK cells with higher flexibility and to prepare expanded, cryopreserved NK cells for instant administration without delay for ex vivo expansion. In cancer therapy with allogeneic NK cells, optimal matching of donors and recipients is important to maximize the efficacy of the therapy. In this review, we summarize the present state of allogeneic NK cell therapy and its future directions.

Enhancing T Cell Immune Responses by B Cell-based Therapeutic Vaccine Against Chronic Virus Infection.

Chronic virus infection leads to the functional impairment of dendritic cells (DCs) as well as T cells, limiting the clinical usefulness of DC-based therapeutic vaccine against chronic virus infection. Meanwhile, B cells have been known to maintain the ability to differentiate plasma cells producing antibodies even during chronic virus infection. Previously, α-galactosylceramide (αGC) and cognate peptide-loaded B cells were comparable to DCs in priming peptide-specific CD8(+) T cells as antigen presenting cells (APCs). Here, we investigated whether B cells activated by αGC can improve virus-specific T cell immune responses instead of DCs during chronic virus infection. We found that comparable to B cells isolated from naïve mice, chronic B cells isolated from chronically infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) after αGC-loading could activate CD1d-restricted invariant natural killer T (iNKT) cells to produce effector cytokines and upregulate co-stimulatory molecules in both naïve and chronically infected mice. Similar to naïve B cells, chronic B cells efficiently primed LCMV glycoprotein (GP) 33-41-specific P14 CD8(+) T cells in vivo, thereby allowing the proliferation of functional CD8(+) T cells. Importantly, when αGC and cognate epitope-loaded chronic B cells were transferred into chronically infected mice, the mice showed a significant increase in the population of epitope-specific CD8(+) T cells and the accelerated control of viremia. Therefore, our studies demonstrate that reciprocal activation between αGC-loaded chronic B cells and iNKT cells can strengthen virus-specific T cell immune responses, providing an effective regimen of autologous B cell-based therapeutic vaccine to treat chronic virus infection.

GMP-compliant, large-scale expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells in vitro and in vivo.

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3(+) T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3(-)CD16(+)CD56(+) NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.

Importance of NKG2D-NKG2D ligands interaction for cytolytic activity of natural killer cell.

In this study, we investigate the relationship between natural killer (NK) cell susceptibility and the surface markers of cancer cells. Through phenotypic analysis, we found evidence that more susceptible cancer cell lines (K562 and Jurkat) express more NKG2D ligands. Major histocompatibility complex (MHC) class I chain-related A/B (MIC-A/B) and UL16 binding protein (ULBP) 1-5 molecules are typical ligands of NKG2D. The high killing activity of NK cells against K562 was abolished through the addition of a NKG2D blocking antibody. Upon in vitro stimulation with quercetin, low susceptible cancer cells increased NKG2D ligand expression, leading to enhancement of NK cell cytolytic activity. These results suggested that the anti-cancer activity of NK cells is not dependent on the origin and growth style of the target cells, but is dependent on the surface markers of the target cells.

Human apolipoprotein(a) kringle V inhibits ischemia-induced retinal neovascularization via suppression of fibronectin-mediated angiogenesis.

Retinal neovascularization is observed in progression of diabetic retinopathy. New vessels grow into the vitreous cavity in proliferative diabetic retinopathy, resulting in traction retinal detachment and vitreous hemorrhage. To overcome the catastrophic visual loss due to these complications, efforts have been focused on the treatment of retinal neovascularization. In this study, we demonstrated the inhibitory effect of recombinant human apolipoprotein(a) kringle V (rhLK8) in an animal model of ischemia-induced retinal neovascularization. rhLK8 induced no definite toxicity on endothelial cells and retinal tissues at the therapeutic dosage. Interestingly, rhLK8 showed antiangiogenic effect, particularly on fibronectin-mediated migration of endothelial cells. Further experiments demonstrated high binding affinity of rhLK8 to α3ß1 integrin, and suppression of it might be the mechanism of antiangiogenic effect of rhLK8. Furthermore, rhLK8 inhibited phosphorylation of focal adhesion kinase, resulting in suppression of activation of consequent p130CAS-Jun NH(2)-terminal kinase. Taken together, our data suggested the possible application of rhLK8 in the treatment of retinal neovascularization by suppression of fibronectin-mediated angiogenesis.

The Effect of CXCR4 Overexpression on Mesenchymal Stem Cell Transplantation in Ischemic Stroke.

There is no doubt that the therapeutic efficacy of mesenchymal stem cells (MSCs) needs improvement. SDF-1 (chemokine for MSC homing) and its receptor CXCR4 play a critical role in the migration of MSCs in ischemia. We investigated the effects of the therapeutic application of MSCs transfected to overexpress CXCR4 using an adenoviral construct in the rat stroke model. Both flow cytometry and Western blot analysis indicated that the level of CXCR4 expression was low in naive hMSCs but was consistently high in CXCR4-hMSCs. In vivo migration test using the transwell system showed that the degree of migration was increased in CXCR4-hMSCs compared with the naive hMSCs and was completely blocked by treatment with AMD3100, an antagonist of the CXCR4 receptor. Compared with rats that received naive MSCs, behavioral recovery was more pronounced in rats that received CXCR4-hMSCs (p = 0.023). An immunohistochemistry study using human nuclear antibody (NuMA) showed that the migration of hMSCs in the ischemic boundary zone was increased after 3 days of injection of CXCR4-hMSCs compared with after injection of naive hMSCs. In addition, polymerase chain reaction was performed to assess the biodistribution of human-specific DNA outside the brain after intravenous injection of hMSCs. The expression of human-specific DNA was increased in the lungs of rats receiving naive MSCs, whereas the human-specific DNA expression was increased in the brain of rats receiving CXCR4-hMSCs. Our results indicate that MSCs transfected with the CXCR4 gene expression cassette may be useful in the treatment of cerebral infarction and may represent a new strategy to enhance the efficacy of MSC therapy.

Overexpression of X-linked inhibitor of apoptosis protein (XIAP) is an independent unfavorable prognostic factor in childhood de novo acute myeloid leukemia.

The overexpression of X-linked inhibitor of apoptosis protein (XIAP), a member of IAP family protein, is intuitively expected to be associated with unfavorable clinical features in malignancies; however, there have been only a very limited number of studies reporting the clinical relevance of XIAP expression. This study was performed to investigate the prognostic relevance of XIAP expression in childhood acute myeloid leukemia (AML). In 53 children with de novo AML, the level of XIAP expression was determined by using quantitative reverse transcriptase-polymerase chain reaction and was analyzed with respect to the clinical characteristics at diagnosis and treatment outcomes. As a result, the XIAP expression was found to be higher in patients with extramedullary disease than in those without (P=0.014). In addition, XIAP overexpression (>or=median expression) was associated with an unfavorable day 7 response to induction chemotherapy and also associated with a worse 3-yr relapsefree survival rate (52.7+/-20.9% vs. 85.9+/-14.8%, P=0.014). Multivariate analyses revealed that XIAP overexpression was an independent unfavorable prognostic factor for relapse-free survival (hazard ratio, 6.16; 95% confidence interval, 1.48-25.74; P=0.013). Collectively, XIAP overexpression may be used as an unfavorable prognostic marker in childhood AML.