Efficacy of Neonatal Porcine Bone Marrow-Derived Mesenchymal Stem Cell Xenotransplantation for the Therapy of Hind Limb Lymphedema in Mice.

Lymphedema is an intractable disease with few effective therapeutic options. Autologous mesenchymal stem cell (MSC) transplantation is a promising therapy for this disease. However, its use is limited by the cost and time for preparation. Recently, xenotransplantation of porcine MSCs has emerged as an alternative to autologous MSC transplantation. In this study, we aimed to clarify the usefulness of neonatal porcine bone marrow-derived MSC (NpBM-MSC) xenotransplantation for the treatment of lymphedema. One million NpBM-MSCs were xenotransplanted into the hind limbs of mice with severe lymphedema (MSC transplantation group). The therapeutic effects were assessed by measuring the femoral circumference, the volume of the hind limb, the number and diameter of lymphatic vessels in the hind limb, and lymphatic flow using a near-infrared fluorescence (NIRF) imaging system. We compared the effects using mice with lymphedema that did not undergo NpBM-MSC transplantation (negative control group). The condition of the transplanted NpBM-MSCs was also evaluated histologically. The femoral circumference and volume of the hind limb had been normalized by postoperative day (POD) 14 in the MSC transplantation group, but not in the negative control group (P = 0.041). NIRF imaging revealed that lymphatic flow had recovered in the MSC transplantation group by POD 14, as shown by an increase in luminance in the hind limb. Histological assessment also showed that the xenotransplantation of NpBM-MSC increased the proliferation of lymphatic vessels, but they had been rejected by POD 14. The xenotransplantation of NpBM-MSCs is an effective treatment for lymphedema, and this is mediated through the promotion of lymphangiogenesis.

Safety and Potential Effect of Intrauterine Infusion of Autologous Adipose Tissue-Derived Regenerative Cells in Patients With Implantation Failure: A Pilot Study.

BACKGROUND: Implantation failure due to thin endometrium has emerged as a major cause of infertility. In this study, we aimed to assess the safety and preliminary efficacy of adipose tissue-derived regenerative cells (ADRCs), a source of adipose-derived stem cells, in infertility patients with implantation failure. METHODS: Five infertile women with implantation failure despite artificial reproductive technology were enrolled in this study and treated with ADRCs via the intrauterine route. The primary outcome was the incidence of adverse events. Additional outcomes were endometrial thickness after ADRC treatment and pregnancy success after embryo transfer. RESULTS: There were no adverse events in any patient. There was no elevation of white blood cell count, C-reactive protein, or D-dimer levels. There was a significant difference in endometrial thickness in the secretory phase before versus after intrauterine transplantation of ADRCs (3.8 ± 1.3 mm versus 8.8 ± 2.8 mm, respectively; p<0.05). A gestational sac and fetal heartbeat were detected on transvaginal ultrasound in two of five patients. CONCLUSION: Intrauterine infusion of autologous ADRCs is a simple and safe procedure that may ameliorate the endometrial microenvironment in infertile women with implantation failure.

The porcine islet-derived organoid showed the characteristics as pancreatic duct.

Organoid is a tissue-engineered organ-like structure that resemble as an organ. Porcine islet-derived organoid might be used as an alternative donor of porcine islet xenotransplantation, a promising therapy for severe diabetes. In this study, we elucidated the characteristics of porcine islet organoids derived from porcine islets as a cell source for transplantation. Isolated porcine islets were 3D-cultured using growth factor-reduced matrigel in organoid culture medium consist of advanced DMEM/F12 with Wnt-3A, R-spondin, EGF, Noggin, IGF-1, bFGF, nicotinamide, B27, and some small molecules. Morphological and functional characteristics of islet organoids were evaluated in comparison with 2D-cultured islets in advanced DMEM/F12 medium. Relatively short-term (approximately 14 days)-cultured porcine islet organoids were enlarged and proliferated, but had an attenuated insulin-releasing function. Long-term (over a month)-cultured islet organoids could be passaged and cryopreserved. However, they showed pancreatic duct characteristics, including cystic induction, strong expression of Sox9, loss of PDX1 expression, and no insulin-releasing function. These findings were seen in long-term-cultured porcine islets. In conclusion, our porcine islet organoids showed the characteristics of pancreatic ducts. Further study is necessary for producing porcine islet-derived organoids having characteristics as islets.

Establishment of a Simple, Reproducible, and Long-lasting Hind Limb Animal Model of Lymphedema.

Background: Lymphedema is an intractable disease for which there is currently no established curative therapy. A reliable and long-lasting lymphedema model is essential for development of better treatments. In this study, we aimed to establish a simple, reproducible and long-lasting mouse model of lymphedema. Methods: Our model is characterized by a combination of a circumferential skin incision in the femoral region, complete dissection of regional lymph nodes, and ablation of the inguinal route in the femoral region. The characteristics of the lymphedema were evaluated and compared with those of two other models. One of these models involved dissection of the subiliac, popliteal, and sciatic lymph nodes (model A) and the other excision of the subiliac, popliteal, and sciatic lymph nodes with cauterization of lymphatic vessels and closure without a skin excision (model B). Results: Although the lymphedema in models A and B resolved spontaneously, that in the new model lasted for a month with increases in femoral circumference and hind limb volume, thickening of the skin, especially subcutaneous tissue, and congestion of peripheral lymphatic vessels. Furthermore, this model could be used for assessing the therapeutic effects of syngeneic mesenchymal stem cell transplantation. The average operation time for the new model was 14.4 ±â€…1.3 minutes. Conclusion: Long-lasting lymphedema can be achieved by our new model, making it suitable for assessing therapies for lymphedema.

Elevation in white blood cell count and development of hyper LDL cholesterolemia.

To investigate the relationship between white blood cell (WBC) count and incidence of hyper-low-density lipoprotein (LDL) cholesterolemia in a population-based longitudinal study. This is a retrospective study using data of annual health check-ups for residents of Iki City, Japan. A total of 3312 residents (≥ 30 years) without hyper-LDL cholesterolemia at baseline were included in this analysis. Primary outcome was incidence of hyper-LDL cholesterolemia (LDL cholesterol levels ≥ 3.62 mmol/L and/or use of lipid lowering drugs). During follow-up (average 4.6 years), 698 participants development of hyper-LDL cholesterolemia (incidence 46.8 per 1000 person-years). Higher incidence of hyper-LDL cholesterolemia was observed among participants with higher leukocyte count (1st quartile group: 38.5, 2nd quartile group: 47.7, 3rd quartile group: 47.3, and 4th quartile group: 52.4 per 1,000 person-years, P = 0.012 for trend). Statistically significant relation was observed even after adjustment for age, gender, smoking, alcohol intake, leisure-time exercise, obesity, hypertension and diabetes: hazard ratio 1.24 (95% confidence interval 0.99 to 1.54) for 2nd quartile group, 1.29 (1.03-1.62) for 3rd quartile group and 1.39 (1.10-1.75) for 4th quartile group, compared with 1st quartile group (P for trend = 0.006). Increased WBC count was related to incidence of hyper-LDL cholesterolemia in general Japanese population.

Differentiation of human adipose tissue-derived mesenchymal stromal cells into steroidogenic cells by adenovirus-mediated overexpression of NR5A1 and implantation into adrenal insufficient mice.

BACKGROUND AIMS: Cell therapy for adrenal insufficiency is a potential method for physiological glucocorticoid and mineralocorticoid replacement. We have previously shown that mouse mesenchymal stromal cells (MSCs) differentiated into steroidogenic cells by the viral vector-mediated overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1), an essential regulator of steroidogenesis, and their implantation extended the survival of bilateral adrenalectomized (bADX) mice. METHODS: In this study, we examined the capability of NR5A1-induced steroidogenic cells prepared from human adipose tissue-derived MSCs (MSC [AT]) and the therapeutic effect of the implantation of human NR5A1-induced steroidogenic cells into immunodeficient bADX mice. RESULTS: Human NR5A1-induced steroidogenic cells secreted adrenal and gonadal steroids and exhibited responsiveness to adrenocorticotropic hormone and angiotensin II in vitro. In vivo, the survival time of bADX mice implanted with NR5A1-induced steroidogenic cells was significantly prolonged compared with that of bADX mice implanted with control MSC (AT). Serum cortisol levels, which indicate hormone secretion from the graft, were detected in bADX mice implanted with steroidogenic cells. CONCLUSIONS: This is the first report to demonstrate steroid replacement by the implantation of steroid-producing cells derived from human MSC (AT). These results indicate the potential of human MSC (AT) to be a source of steroid hormone-producing cells.

Induction of potassium channel regulator KCNE4 in a submandibular lymph node metastasis model.

Cancer cells often metastasize to the lymph nodes (LNs) before disseminating throughout the body. Clinically, LN metastasis correlates with poor prognosis and influences treatment options. Many studies have shown that cancer cells communicate with immune and stromal cells to prepare a suitable niche for metastasis. In this study, mice were injected with B16-F10 murine melanoma cells to generate a tongue submandibular lymph node (SLN) metastasis model in which genes of interest could be investigated. Microarray analyses were performed on SLNs, identifying 162 upregulated genes, some of which are known metastasis genes. Among these upregulated genes, Kcne4, Slc7a11, Fscn1, and Gadd45b were not associated with metastasis, and increased expression of Kcne4 and Slc7a11 was confirmed by real-time PCR and immunohistochemistry. The roles of KCNE4 in chemokine production and cell adhesion were examined using primary lymphatic endothelial cells, and demonstrated that Ccl17 and Ccl19, which are involved in melanoma metastasis, were upregulated by KCNE4, as well as Mmp3 matrix metalloproteinase. Expression of KCNE4 was detected in human LNs with metastatic melanoma. In conclusion, we found that LN metastatic melanoma induces KCNE4 expression in the endothelium of LNs.

Xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells improves diabetic wound healing by promoting angiogenesis and lymphangiogenesis.

BACKGROUND: Some clinical trials have shown the usefulness of stem cell therapy for diabetic foot ulcers. However, the donor supply is limited, and the process is time consuming and expensive. This study assessed the therapeutic effects of neonatal porcine bone marrow-derived mesenchymal stem cell (npBM-MSC) xenotransplantation using diabetic wound model mice. METHODS: All layers of back skin were removed from streptozotocin-induced diabetic mice. In the npBM-MSCs group, npBM-MSCs were transplanted to the wound, and syngeneic mouse bone marrow-derived mesenchymal stem cells (mBM-MSCs) were transplanted to the wound in the mBM-MSCs group. The control group comprised diabetic mice that did not receive cellular therapy. The therapeutic effects of the transplantation were evaluated according to the rate of wound closure and the promotion of neovascularization in the wound. RESULTS: The wound closure rate was significantly improved in the npBM-MSCs group compared with the control group (p < .001 at postoperative day [POD] 4 and p < .01 at POD 7) and mBM-MSCs groups (p < .05 at POD 4). Prominent promotion of both angiogenesis and lymphangiogenesis was observed in the npBM-MSCs group. Furthermore, the expression of murine Prox1 and both porcine and murine Vegfs and Tgfb1 in the wounds was enhanced until POD 4 by npBM-MSCs transplantation. The amounts of vascular endothelial growth factor (VEGF) A, VEGFC, and transforming growth factor ß1 secreted from npBM-MSCs were higher than those from mBM-MSCs (p < .05). CONCLUSION: Xenotransplantation of npBM-MSCs improved diabetic wound healing by promoting both angiogenesis and lymphangiogenesis.

CCN2-induced lymphangiogenesis is mediated by the integrin αvß5-ERK pathway and regulated by DUSP6.

Lymphangiogenesis is essential for the development of the lymphatic system and is important for physiological processes such as homeostasis, metabolism and immunity. Cellular communication network factor 2 (CCN2, also known as CTGF), is a modular and matricellular protein and a well-known angiogenic factor in physiological and pathological angiogenesis. However, its roles in lymphangiogenesis and intracellular signaling in lymphatic endothelial cells (LECs) remain unclear. Here, we investigated the effects of CCN2 on lymphangiogenesis. In in vivo Matrigel plug assays, exogenous CCN2 increased the number of Podoplanin-positive vessels. Subsequently, we found that CCN2 induced phosphorylation of ERK in primary cultured LECs, which was almost completely inhibited by the blockade of integrin αvß5 and partially decreased by the blockade of integrin αvß3. CCN2 promoted direct binding of ERK to dual-specific phosphatase 6 (DUSP6), which regulated the activation of excess ERK by dephosphorylating ERK. In vitro, CCN2 promoted tube formation in LECs, while suppression of Dusp6 further increased tube formation. In vivo, immunohistochemistry also detected ERK phosphorylation and DUSP6 expression in Podoplanin-positive cells on CCN2-supplemented Matrigel. These results indicated that CCN2 promotes lymphangiogenesis by enhancing integrin αvß5-mediated phosphorylation of ERK and demonstrated that DUSP6 is a negative regulator of excessive lymphangiogenesis by CCN2.

Lymphangiogenesis and angiogenesis rescue murine ischemic hindlimb via transient receptor potential vanilloid 4.

In this study, we assessed the regulation of transient receptor potential vanilloid 4 (TRPV4) promoting lymphangio/angiogenesis to improve the ischemic hindlimb animal model, and revealed that (1) a TRPV4 agonist improved the blood flow of ischemic hindlimbs by inducing both angiogenesis and lymphangiogenesis; (2) excessive TRPV4 expression was detected on lymphatic endothelial cells (LECs) in the ischemic hindlimb; and (3) hypoxic conditions promoted Ca2+ influx into LECs via TRPV4. It is considered that the upregulation of both lymphatic and blood vessels by activating TRPV4 would be a promising therapeutic strategy for peripheral artery disease.

Xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells improves murine hind limb ischemia through lymphangiogenesis and angiogenesis.

BACKGROUND: The clinical utility of stem cell therapy for peripheral artery disease has not been fully discussed, and one obstacle is limited donor supplies. In this study, we attempted to rescue mouse ischemic hind limb by xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs). METHODS: Neonatal porcine bone marrow-derived mesenchymal stem cells were transplanted to ischemic hind limbs of male C57BL/6J mice (npBM-MSCs group). Mice with syngeneic transplantation of mouse BM-MSCs (mBM-MSCs group) were also prepared for comparison. The angiogenic effects were evaluated by recovery of blood flow on laser Doppler imaging, histologic findings, and genetic and protein levels of angiogenic factors. RESULTS: Regarding laser Doppler assessments, blood flow in the hind limb was rapidly recovered in the npBM-MSCs group, compared with that in the mBM-MSCs group (P = .016). Compared with the mBM-MSCs group, the npBM-MSCs group had early and prominent lymphangiogenesis [P < .05 on both post-operative days (PODs) 3 and 7] but had similar angiogenesis. Regarding genomic assessments, xenotransplantation of npBM-MSCs enhanced the expressions of both porcine and murine Vegfc in the hind limbs by POD 3. Interestingly, the level of murine Vegfc expression was significantly higher in the npBM-MSCs group than in the mBM-MSCs group on PODs 3 and 7 (P < .001 for both). Furthermore, the secreted VEGFC protein level was higher from npBM-MSCs than from mBM-MSCs (P < .001). CONCLUSION: Xenotransplantation of npBM-MSCs contributed to the improvement of hind limb ischemia by both angiogenesis and lymphangiogenesis, especially promotion of the latter. npBM-MSCs may provide an alternative to autologous and allogeneic MSCs for stem cell therapy of critical limb ischemia.

Changes in Immunological Status in Patients With Metastatic Colorectal Cancer Treated With First-line Chemoimmunotherapy.

BACKGROUND/AIM: Chemoimmunotherapy is a promising treatment for various malignant diseases. In this study, we examined whether first-line chemoimmunotherapy using adoptive immune-cell therapy was effective for metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: The therapeutic efficacy and safety of the standard first-line chemoimmunotherapy with adoptive αß T cell therapy and bevacizumab were assessed using thirty-two patients with mCRC in our hospital. Immunological status after this chemoimmunotherapy was also evaluated. RESULTS: The response and disease control rates were 68.8% and 87.5%, respectively. Further, median progression-free and overall survival were 14.2 and 35.3 months. Immunotherapy-associated toxicity was minimal. Significant decrease in the change of monocyte number (p=0.006) and increase in the change of rate of lymphocyte-to-monocyte ratio (p=0.039) were seen in the complete response group. CONCLUSION: First-line chemoimmunotherapy with adoptive αß T cell therapy may be useful for mCRC.

Extension of Survival in Bilaterally Adrenalectomized Mice by Implantation of SF-1/Ad4BP-Induced Steroidogenic Cells.

Mesenchymal stroma/stem cells (MSCs) exist in adult tissues, such as adipose tissue and bone marrow, and differentiate into cells of multiple lineages. In previous studies, we found that MSCs differentiate into steroidogenic cells by forced expression of steroidogenic factor 1 (SF-1)/adrenal 4 binding protein (Ad4BP), the master regulator of steroidogenesis and differentiation of pituitary gonadotrophs, adrenal glands, and gonads. In this study, SF-1/Ad4BP-induced steroidogenic cells derived from mouse adipose tissue-derived MSCs (ADSCs) were implanted under the kidney capsule of bilateral adrenalectomized (bAdx) mice. bAdx mice did not survive after 7 days. However, 4 of 9 bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells, 1 of 10 bAdx mice transplanted with control ADSCs, and bAdx mice transplanted with an adrenal gland survived for 30 days. Plasma corticosterone levels in bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells and control ADSCs were 5.41 ±â€…2.26 ng/mL (mean ±â€…SEM) and undetectable at 7 days after implantation, respectively. After removal of the kidney bearing the graft from the surviving mice at 30 days after implantation, plasma corticosterone was not detected in any of the samples. Immunohistochemical staining revealed SF-1/Ad4BP-positive cells under the capsule of the kidney. Although we performed an adrenocorticotropin (ACTH) loading test on bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells, ACTH responsiveness was not observed. Implantation of steroidogenic cells derived from ADSCs into bAdx mice increased the basal plasma corticosterone level and extended the survival of bAdx mice, suggesting the capability of restoring steroidogenic cells by cell transplantation therapy for adrenal insufficiency.

Evaluation of vascular pain in patients with colorectal cancer receiving peripheral venous chemotherapy with or without oxaliplatin.

Oxaliplatin is a key chemotherapy drug in patients with colorectal cancer. Administration of oxaliplatin via a peripheral vein often causes vascular pain. However, no studies have evaluated vascular pain in patients with colorectal cancer in relation to peripheral venous administration of chemotherapy with or without oxaliplatin. We evaluated oxaliplatin-induced vascular pain using subjective and objective methods. We determined if oxaliplatin induced vascular pain in patients with colorectal cancer using a Visual Analog Scale (VAS) and a PainVision PS-2100 device. We compared VAS score between chemotherapy regimens with or without oxaliplatin, and between genders. We also examined the correlations of VAS score with pain intensity examined by the PainVision PS-2100, and with age and vessel diameter. A total of 98 patients with colorectal cancer were enrolled in this study, including 78 patients who received oxaliplatin via peripheral venous administration and 20 who received chemotherapy without oxaliplatin. The median VAS scores in patients with and without oxaliplatin were 36.5 (interquartile range 9.0-60.0) and 0 (0-4.0), respectively (P < 0.001), and the median pain intensities according to PainVision were 43.5 (14.3-98) and 36.5 (9.3-58.5), respectively (P < 0.001). There was a positive correlation between VAS and pain intensity (r = 0.584), but no correlation between VAS score and age (r = -0.174) or vessel diameter (r = -0.107). Peripheral venous administration of oxaliplatin induced vascular pain, measured both subjectively and objectively, in patients with colorectal cancer, regardless of vessel diameter.

The Spleen as an Optimal Site for Islet Transplantation and a Source of Mesenchymal Stem Cells.

This review demonstrates the unique potential of the spleen as an optimal site for islet transplantation and as a source of mesenchymal stem cells. Islet transplantation is a cellular replacement therapy used to treat severe diabetes mellitus; however, its clinical outcome is currently unsatisfactory. Selection of the most appropriate transplantation site is a major factor affecting the clinical success of this therapy. The spleen has long been studied as a candidate site for islet transplantation. Its advantages include physiological insulin drainage and regulation of immunity, and it has recently also been shown to contribute to the regeneration of transplanted islets. However, the efficacy of transplantation in the spleen is lower than that of intraportal transplantation, which is the current representative method of clinical islet transplantation. Safer and more effective methods of islet transplantation need to be established to allow the spleen to be used for clinical transplantation. The spleen is also of interest as a mesenchymal stem cell reservoir. Splenic mesenchymal stem cells contribute to the repair of damaged tissue, and their infusion may thus be a promising therapy for autoimmune diseases, including type 1 diabetes mellitus and Sjogren’s syndrome.

Hepatectomy for Liver Metastases of Colorectal Cancer After Adoptive Chemoimmunotherapy Using Activated αß T-cells.

BACKGROUND/AIM: Various types of chemoimmunotherapies for malignant tumors have been reported. However, there are few reports on hepatectomy after chemoimmunotherapy. We evaluated the safety and efficacy of hepatectomy for patients with stage IV colorectal liver metastases (CLM) after chemoimmunotherapy using activated αß T-cells. PATIENTS AND METHODS: From June 2012 to December 2016, five patients who underwent hepatectomy after receiving capecitabine and oxaliplatin (XELOX) plus bevacizumab and ex vivo-expanded αß T-lymphocytes as first-line chemoimmunotherapy were included. RESULTS: The median age of the five patients (two men, three women) was 61.4 (range=56-75) years. The surgical procedure was partial hepatectomy in two, laparoscopic partial hepatectomy in two, and one case of partial hepatectomy with subsegmentectomy. There was no postoperative complication of Clavien-Dindo grade 3A or higher. One patient had multiple lung metastases. CONCLUSION: Hepatectomy after chemoimmunotherapy using activated αß T-cells for CLM can be performed safely.

Clinical Study on the Medical Value of Combination Therapy Involving Adoptive Immunotherapy and Chemotherapy for Stage IV Colorectal Cancer (COMVI Study).

BACKGROUND: Adoptive immunotherapy for cancer has evolved through development of novel technologies for generating a large number of activated killer cells, such as αß T-cells, γδ T-cells, and natural killer cells. There has been no prospective trial of combination therapy involving adoptive immunotherapy and first-line chemotherapy for stage IV colorectal cancer. The present pilot study aimed to evaluate the safety and feasibility of combination therapy involving adoptive immunotherapy and chemotherapy for stage IV colorectal cancer (COMVI study). PATIENTS AND METHODS: The COMVI study was a prospective, single-arm pilot trial. Therapy in each 21-day treatment cycle involved XELOX (130 mg/m2 of oxaliplatin on day 1 plus 1,000 mg/m2 of capecitabine twice daily on days 1-14), bevacizumab (7.5 mg/kg on day 1), and αß T-lymphocytes (over 5×109 on day 18) cultured ex vivo with an immobilized antibody to CD3 and interleukin-2. RESULTS: The study included six patients (two men and four women) between June 2013 and September 2014. The median patient age was 68 years (range=55-75 years). The overall response rate was 83.3% [complete response in two (33.3%); partial response in three (50.0%); stable disease in one (16.7%); no cases of progressive disease]. The tumor volume reduction rate was 53% (range=38.0-100%). The median progression-free and overall survival durations were 567 and 966 days, respectively. Most adverse events were mild-to-moderate in intensity, and no grade 4 adverse events occurred in the six patients. Only one patient experienced grade 3 hypertension and ileus. Immunotherapy-associated toxicity was minimal in this study. CONCLUSION: Combination therapy involving adoptive immunotherapy and chemotherapy for stage IV colorectal cancer is feasible and safe. Phase II prospective studies are needed to confirm the safety and efficacy of such chemoimmunotherapy.

A Modified Adenovirus Vector-Mediated Antibody Screening Method Identifies EphA2 as a Cancer Target.

BACKGROUND: We constructed a genetically modified adenovirus vector incorporating an IgG Fc-binding motif from staphylococcal protein A, Z33 (Adv-FZ33). Adv-FZ33 allows an antibody to redirect the vector to a target molecule on the cell surface. We attempted to search for target antigen candidates and antibodies that allowed highly selective gene transduction into malignant tumors. METHODS: Hybridoma libraries producing monoclonal antibodies (mAbs) were screened that increased transduction efficiency in cancer cell lines after cross-linking with Adv-FZ33. Target antigens of the mAbs were identified by immunoprecipitation and mass spectrometry. Of these mAbs, we noted a clone, F2-27, that recognized the receptor tyrosine kinase EphA2. Next, we generated an adenovirus vector, Ax3CMTK-FZ33, that expressed a herpes simplex virus thymidine kinase (HSV-TK). The therapeutic efficacy of F2-27-mediated HSV-TK gene transduction, followed by ganciclovir (GCV) administration, was studied in vitro. The inhibitory effect of F2-27 on cancer cell invasion was investigated by a three-dimensional spheroid formation assay. RESULTS: In vitro reporter gene expression after Adv-FZ33 infection via F2-27 was 146 times higher than with control mAb in EphA2-expressing cancer cell lines. F2-27-mediated Ax3CMTK-FZ33 infection induced the HSV-TK gene in an F2-27-dependent manner and had a highly effective cytotoxic effect in a GCV-dependent manner. Additionally, F2-27 independently inhibited migration of EphA2-positive breast cancer cell lines in three-dimensional culture. CONCLUSION: Our modified adenovirus and hybridoma screening system is useful for the development of targeted cancer therapy, and F2-27 has the potential to be an antibody-based therapy for various EphA2-positive cancers.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

OBJECTIVE: Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. APPROACH AND RESULTS: AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 µm; P=0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P=0.0015; interleukin-10: 7.6-fold, P=0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 µm; P=0.0306), increased collagen density (2.4-fold; P=0.0432), and increased number of M2 macrophages (2.1-fold; P=0.0335). CONCLUSIONS: CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation.

The Spleen Is an Ideal Site for Inducing Transplanted Islet Graft Expansion in Mice.

Alternative islet transplantation sites have the potential to reduce the marginal number of islets required to ameliorate hyperglycemia in recipients with diabetes. Previously, we reported that T cell leukemia homeobox 1 (Tlx1)+ stem cells in the spleen effectively regenerated into insulin-producing cells in the pancreas of non-obese diabetic mice with end-stage disease. Thus, we investigated the spleen as a potential alternative islet transplantation site. Streptozotocin-induced diabetic C57BL/6 mice received syngeneic islets into the portal vein (PV), beneath the kidney capsule (KC), or into the spleen (SP). The marginal number of islets by PV, KC, or SP was 200, 100, and 50, respectively. Some plasma inflammatory cytokine levels in the SP group were significantly lower than those of the PV group after receiving a marginal number of islets, indicating reduced inflammation in the SP group. Insulin contents were increased 280 days after islet transplantation compared with those immediately following transplantation (p<0.05). Additionally, Tlx1-related genes, including Rrm2b and Pla2g2d, were up-regulated, which indicates that islet grafts expanded in the spleen. The spleen is an ideal candidate for an alternative islet transplantation site because of the resulting reduced inflammation and expansion of the islet graft.