Therapeutic Effect of Schwann Cell-Like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells on Diabetic Neuropathy in db/db Mice.

BACKGROUND: Diabetic neuropathy (DN) is the most common complication of diabetes, and approximately 50% of patients with this disease suffer from peripheral neuropathy. Nerve fiber loss in DN occurs due to myelin defects and is characterized by symptoms of impaired nerve function. Schwann cells (SCs) are the main support cells of the peripheral nervous system and play important roles in several pathways contributing to the pathogenesis and development of DN. We previously reported that human tonsil-derived mesenchymal stem cells differentiated into SCs (TMSC-SCs), named neuronal regeneration-promoting cells (NRPCs), which cells promoted nerve regeneration in animal models with peripheral nerve injury or hereditary peripheral neuropathy. METHODS: In this study, NRPCs were injected into the thigh muscles of BKS-db/db mice, a commonly used type 2 diabetes model, and monitored for 26 weeks. Von Frey test, sensory nerve conduction study, and staining of sural nerve, hind foot pad, dorsal root ganglia (DRG) were performed after NRPCs treatment. RESULTS: Von Frey test results showed that the NRPC treatment group (NRPC group) showed faster responses to less force than the vehicle group. Additionally, remyelination of sural nerve fibers also increased in the NRPC group. After NRPCs treatment, an improvement in response to external stimuli and pain sensation was expected through increased expression of PGP9.5 in the sole and TRPV1 in the DRG. CONCLUSION: The NRPCs treatment may alleviate DN through the remyelination and the recovery of sensory neurons, could provide a better life for patients suffering from complications of this disease.

Preclinical Efficacy of Peripheral Nerve Regeneration by Schwann Cell-like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells in C22 Mice.

Charcot-Marie-Tooth disease (CMT) is a hereditary disease with heterogeneous phenotypes and genetic causes. CMT type 1A (CMT1A) is a type of disease affecting the peripheral nerves and is caused by the duplication of the peripheral myelin protein 22 (PMP22) gene. Human tonsil-derived mesenchymal stem cells (TMSCs) are useful for stem cell therapy in various diseases and can be differentiated into Schwann cell-like cells (TMSC-SCs). We investigated the potential of TMSC-SCs called neuronal regeneration-promoting cells (NRPCs) for peripheral nerve and muscle regeneration in C22 mice, a model for CMT1A. We transplanted NRPCs manufactured in a good manufacturing practice facility into the bilateral thigh muscles of C22 mice and performed behavior and nerve conduction tests and histological and ultrastructural analyses. Significantly, the motor function was much improved, the ratio of myelinated axons was increased, and the G-ratio was reduced by the transplantation of NRPCs. The sciatic nerve and gastrocnemius muscle regeneration of C22 mice following the transplantation of NRPCs downregulated PMP22 overexpression, which was observed in a dose-dependent manner. These results suggest that NRPCs are feasible for clinical research for the treatment of CMT1A patients. Research applying NRPCs to other peripheral nerve diseases is also needed.

Therapeutic Extracellular Vesicles from Tonsil-Derived Mesenchymal Stem Cells for the Treatment of Retinal Degenerative Disease.

BACKGROUND: Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. METHODS: To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. RESULTS: T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. CONCLUSIONS: Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

Patchmatch-Based Robust Stereo Matching Under Radiometric Changes.

In the real world, the two challenges of stereo vision system include a robust system under various radiometric changes and real-time process. To extract depth information from stereoscopic images, this paper proposes Patchmatch-based robust and fast stereo matching under radiometric changes. For this, a cost function was designed and minimized for estimating an accurate disparity map. Specifically, we used a prior probability to minimize the occlusion region and a smoothness term that considers convexity of objects to extract a fine disparity map. For evaluating the performance of the proposed scheme, we used Middlebury stereo data sets with radiometric changes. The experimental result showed that the proposed method outperforms state-of-the-art methods by up to 3.35 percent better and a range of 4.71 - 27.24 times faster result in terms of bad pixel error and processing time, respectively. Therefore, we believe that the proposed scheme can be a useful tool for computer vision-based applications.

Disparity-selective stereo matching using correlation confidence measure.

Recently, the cost-volume filtering (CVF) methods for local stereo matching have provided fast and accurate results compared to those of the other method. However, CVF still causes incorrect results in the occlusion and texture-free regions. In particular, cost aggregation by pixel units involves complex computation because of its dependence on the image resolution and search range. This paper presents a robust stereo matching method for occluded regions. First, we generate cost volumes using the CENSUS transform and the scale-invariant feature transform (SIFT). Then, label-based cost volumes are aggregated using adaptive support weight and the simple linear iterative clustering (SLIC) scheme from two generated cost volumes. In order to obtain optimal disparity by two label-based cost volumes, we select the disparity corresponding to high confidence similarity of CENSUS or SIFT with minimum cost point. Experimental results show that our method estimates the optimal disparity in occlusion information, which exists only in the scene of one of the stereo pairs.

Systematic Proteogenomic Approach To Exploring a Novel Function for NHERF1 in Human Reproductive Disorder: Lessons for Exploring Missing Proteins.

One of the major goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to fill the knowledge gaps between human genomic information and the corresponding proteomic information. These gaps are due to "missing" proteins (MPs)-predicted proteins with insufficient evidence from mass spectrometry (MS), biochemical, structural, or antibody analyses-that currently account for 2579 of the 19587 predicted human proteins (neXtProt, 2017-01). We address some of the lessons learned from the inconsistent annotations of missing proteins in databases (DB) and demonstrate a systematic proteogenomic approach designed to explore a potential new function of a known protein. To illustrate a cautious and strategic approach for characterization of novel function in vitro and in vivo, we present the case of Na(+)/H(+) exchange regulatory cofactor 1 (NHERF1/SLC9A3R1, located at chromosome 17q25.1; hereafter NHERF1), which was mistakenly labeled as an MP in one DB (Global Proteome Machine Database; GPMDB, 2011-09 release) but was well known in another public DB and in the literature. As a first step, NHERF1 was determined by MS and immunoblotting for its molecular identity. We next investigated the potential new function of NHERF1 by carrying out the quantitative MS profiling of placental trophoblasts (PXD004723) and functional study of cytotrophoblast JEG-3 cells. We found that NHERF1 was associated with trophoblast differentiation and motility. To validate this newly found cellular function of NHERF1, we used the Caenorhabditis elegans mutant of nrfl-1 (a nematode ortholog of NHERF1), which exhibits a protruding vulva (Pvl) and egg-laying-defective phenotype, and performed genetic complementation work. The nrfl-1 mutant was almost fully rescued by the transfection of the recombinant transgenic construct that contained human NHERF1. These results suggest that NHERF1 could have a previously unknown function in pregnancy and in the development of human embryos. Our study outlines a stepwise experimental platform to explore new functions of ambiguously denoted candidate proteins and scrutinizes the mandated DB search for the selection of MPs to study in the future.

Synergistic therapeutic effects of cytokine-induced killer cells and temozolomide against glioblastoma.

The presence of active immunity within the brain supports the possibility of effective immunotherapy for glioblastoma (GBM). To provide a clinically-relevant adoptive immunotherapy for GBM using ex vivo expanded cytokine-induced killer (CIK) cells, the treatment capability of CIK cells, either alone or in combination with temozolomide (TMZ) were evaluated. Human CIK (hCIK) cells were cultured from PBMC using activating anti-CD3 antibody and IL-2, which were 99% CD3+, 91% CD3+CD8+ and 29% CD3+CD56+. In vitro, hCIK cells showed tumor-specific cytotoxicity against U-87MG human GBM cells. When hCIK cells were injected into tail veins of immune-compromised mice bearing U-87MG tumors in their brains, numerous CIK cells infiltrated into the brain tumors. CIK treatments (1x10(5), 1x10(6) or 1x10(7), once a week for four weeks) inhibited the tumor growth significantly in a dose-dependent manner; 44, 54 and 72% tumor volume reduction, respectively, compared with the control group (P<0.05). Moreover, hCIK cells (1x10(7), once a week for four weeks) and TMZ (2.5 mg/kg, daily for 5 days) combination treatment further increased tumor cell apoptosis and decreased tumor cell proliferation and vessel density (P<0.05), creating a more potent therapeutic effect (95% reduction in tumor volume) compared with either hCIK cells or TMZ single therapy (72% for both, P<0.05). Taken together, CIK cell-immunotherapy and TMZ chemotherapy have synergistic therapeutic effects and could be combined for a successful treatment of GBM.

Adoptive immunotherapy of human gastric cancer with ex vivo expanded T cells.

Surgical resection of gastric cancer has made significant progress, but majority of patients with advanced gastric cancer face relapse and die within five years. In this study, the antitumor activity of ex vivo expanded T cells against the human gastric cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 antibody-coated flasks for 5 days, followed by incubation in IL-2-containing medium for 9 days. The resulting populations were mostly CD3(+) T cells (97%) and comprised 1% CD3⁻CD56(+), 36% CD3(+)CD56(+), 11% CD4(+), and 80% CD8(+). This heterogeneous cell population was also called cytokine-induced killer (CIK) cells. CIK cells strongly produced IFN-γ, moderately TNF-α, but not IL-2 and IL-4. At an effector-target cell ratio of 30:1, CIK cells destroyed 58% of MKN74 human gastric cancer cells, as measured by the 5¹Cr-release assay. In addition, CIK cells at doses of 3 and 10 million cells per mouse inhibited 58% and 78% of MKN74 tumor growth in nude mouse xenograft assays, respectively. This study suggests that CIK cells may be used as an adoptive immunotherapy for gastric cancer patients.

Antitumor activity of cytokine-induced killer cells in nude mouse xenograft model.

Malignant glioma is the most common primary brain tumor in adults and the median survival for patients is less than a year. Despite aggressive treatments including surgical resection, radiotherapy, and chemotherapy, only modest improvement has been achieved in the survival of patients with glioma. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against human glioma cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 antibody-coated flasks for 5 days, followed by incubation in IL-2-containing medium for 9 days. The number of cells increased more than 200-fold and the viability was >90%. The resulting populations were consisted of 96% CD3(+), 2% CD3(-)CD56(+), 68% CD3(+)CD56(+), 2% CD4(+), <1% CD4(+)CD56(+), 80% CD8(+), and 49% CD8(+)CD56(+). This heterogeneous cell population was called as CIK cells. At an effector-target cell ratio of 30:1, CIK cells destroyed 43% of U-87 MG human glioma cells, as measured by the (51)Cr-release assay. In addition, CIK cells at doses of 0.3, 1, and 3 million cells per mouse inhibited 23%, 40%, and 50% of U-87 MG tumor growth in nude mouse xenograft assays, respectively. This study suggests that CIK cells may be used as an adoptive immunotherapy for glioma cancer patients.

Inhibition of human cervical carcinoma growth by cytokine-induced killer cells in nude mouse xenograft model.

Cervical cancer is a major cause of cancer mortality in women worldwide and is an important public health problem for adult women in developing countries. Despite aggressive treatment with surgery and chemoradiation, the outcomes for cervical cancer patients remain poor. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against human cervical cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 antibody-coated flasks for 5 days, followed by incubation in IL-2-containing medium for 9 days. The resulting populations of CIK cells comprised 95% CD3(+), 3% CD3(-)CD56(+), 35% CD3(+)CD56(+), 11% CD4(+), <1% CD4(+)CD56(+), 80% CD8(+), and 25% CD8(+)CD56(+). At an effector-target cell ratio of 100:1, CIK cells destroyed 56% of KB-3-1 human cervical cancer cells, as measured by the (51)Cr-release assay. In addition, CIK cells at doses of 3 and 10 million cells per mouse inhibited 34% and 57% of KB-3-1 tumor growth in nude mouse xenograft assays, respectively. This study suggests that CIK cells may be used as an adoptive immunotherapy for cervical cancer patients.

Inhibition of human ovarian tumor growth by cytokine-induced killer cells.

Despite the recent improvement in the treatment of ovarian cancer, this disease is still leading cause of cancer death in women. In this study, the anti-tumor activity of cytokine-induced killer (CIK) cells against human ovarian cancer was evaluated in vitro and in vivo. Although CD3+CD56+ cells were rare in fresh human peripheral blood mononuclear cells, they could expand more than 1,000-fold on day 14 in the presence of anti-CD3 antibody plus IL-2. At an effector-target cell ratio of 30:1, CIK cells destroyed 45% of SK-OV-3 human ovarian cancer cells, which was determined by the 51Cr-release assay. In addition, CIK cells at a dose of 23 million cells per mouse inhibited 73% of SK-OV-3 tumor growth in nude mouse xenograft assay. This study suggests that CIK cells may be used as an adoptive immunotherapy for patients with ovarian cancer.

Anti-tumor activity of ex vivo expanded cytokine-induced killer cells against human hepatocellular carcinoma.

Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with natural killer cell phenotypes and functions. In this study, the anti-tumor activity of CIK cells against hepatocellular carcinoma was evaluated in vitro and in vivo. In the presence of anti-CD3 antibody and IL-2 for 14 days, human peripheral blood mononuclear cell population changed to heterogeneous CIK cell population, which comprised 96% CD3(+), 3% CD3( inverted exclamation mark(c))CD56(+), 32% CD3(+)CD56(+), 11% CD4(+), 75% CD8(+), and 30% CD8(+)CD56(+). CIK cells produced significant amounts of IFN-gamma and TNF-alpha; however, produced only slight amounts of IL-2, IL-4, and IL-5. At an effector-target cell ratio of 30:1, CIK cells destroyed 33% of SNU-354 human hepatocellular carcinoma cells, which was determined by the (51)Cr-release assay. In addition, a dose of 1x10(6) CIK cells per mouse inhibited 60% of SNU-354 tumor growth in irradiated nude mice. This study suggests that CIK cells may be used as an adoptive immunotherapy for patients with hepatocellular carcinoma.

Antitumor activity of cytokine-induced killer cells against human lung cancer.

Lung cancer is the leading cause of cancer-related death among men and women in the world. Despite the aggressive treatment with surgery, radiation and chemotherapy, the long term survival for lung cancer patients remains low. In this study, the anti-tumor activity of cytokine-induced killer (CIK) cells against human lung cancer was evaluated in vitro and in vivo. Although CD3(+)CD56(+) CIK cells were rare in fresh human peripheral blood mononuclear cells, they could expand more than 1000-fold on day 14 in the presence of anti-CD3 antibody plus IL-2. At an effector-target cell ratio of 30:1, CIK cells destroyed 98% of NCI-H460 human lung cancer cells, which was determined by the (51)Cr-release assay. In addition, CIK cells at doses of 3 and 30 million cells per mouse inhibited 57% and 77% of NCI-H460 tumor growth in nude mouse xenograft assay, respectively. This study suggests that CD3(+)CD56(+) CIK cells may be used as an adoptive immunotherapy for patients with lung cancer.

Identification of residues which regulate activity of the STE20-related kinase hMINK.

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues; highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T(191) and Y(193) in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T(187) showed reduced enzymatic activity against exogenous substrates but retained autophosphorylation activity. Phosphorylation was confirmed by the use of a phospho-specific T(187) antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed.